Biology of Cancer notes

Lecture 1 – Origin of cancer

Each cancer has its own pathway and protein that it is depending on.
Treatment must be tailored according to this.

Cancer: genetic disease

●​ Change in DNA/expression of the DNA. There are a few that are viral
related but there could also be mutations.
●​ Multi step process where due to genetic changes normal cells are
transformed into cancer cells with increasing rate
●​ Darwinian evolution based on growth advantage

Benign tumours stay together in a closed environment → remain

localised at the original location
●​ Intact basement membrane
●​ Residual lumen
●​ Denser stroma & cellular crowding
Malignant tumours invade the surrounding tissue and can spread →
metastasize
●​ structural disorganization → no lumen, irregular nuclei
●​ invasive growth → stromal invasion

Cancers originate from diff type of tissues / cell types

1.​ Carcinoma: epithelial tissue
a.​ Adeno = gland epithelium (breast, lung, pancreas)
b.​ Squamous = epithelium (skin, esophagus)
2.​ Sarcoma: muscle, connective tissue, bone
3.​ Leukemia: hematopoietic stem cells
4.​ Gliomas: brain cells
5.​ Melanoma: pigment cells in the skin
→ many subtypes of cancers within these types
World’s most common type of cancers:
●​ epithelial tumors of the lung, colon, breast, stomach and cervix.
●​ Epithelial cancers are most common because epithelial cells divide fast and they are exposed to the
environment (food, light, smoke etc).

I.​ Origin of cancer

1 abnormal cell
Cancers are monoclonal = arise from a single mutated cell that proliferates uncontrollably
●​ Normal cell: heterogeneous expression of G6PD → Mixed X-chromosome activation (both mom’s and
dad’s X are seen in different cells).
●​ Tumor: only 1 expression of G6PD → Only one X-chromosome version (all cells came from the same
"mother" cancer cell).
Somatic Mutation in the cell
●​ SM appear in normal cells in the body and aren't hereditary
●​ SM can arise through chromosomal translocations, carcinogenic substances, radiation and viruses
Germline vs somatic mutation
●​ GL mutations
○​ Happens in germ cells (sperm/egg) pre fertilization
○​ affect the entire organism → passed to offspring.
○​ E.g.,: inheritable mutation → BRCA-1 or 2
●​ Somatic mutations
○​ affect only specific tissues
○​ are not inherited.
○​ Causes cancer in one part of the body (e.g., lung, skin).
○​ E.g.,: UV-induced skin cancer, smoking-induced lung cancer, translocation bn chr 9 and 22
(philadelphia chr)
Some cancers run in families (germ-line), while others happen due to environmental factors (somatic mutations).
Cancer arises from the accumulation of mutations overtime
●​ Spontaneous mutation freq: 1/gene/105-106 cell divisions
●​ The incidence of cancer increases with age → accumulation of cancer-causing mutations
●​ Exponential growth of cancer overtime you age: could be due to evolution

Cancer development – from less abnormal cells (pre-malignant) → malignant

II.​ Cancer development process is accelerated by
1.​ Consecutive cycles of mutations/selections → Cells with harmful mutations outcompete normal cells.
●​ Successive cycles of mutations ultimately lead to cancer
●​ Genetic and epigenetic inactivation of genes
○​ Inactivating mutation (eg TSG mutations: TP53 in many cancers)
○​ Histone modification (eg silencing of TSG)
○​ DNA methylation: promoter can't bind to DNA → protein can’t be transcribed (eg
hypermethylation of BRCA1 in BC)
NOTE: last 2 are epigenetic inactivation → reversible → potential target for therapies
●​ Histone: proteins where DNA wrap around
○​ Tightly wrapped DNA → inactive genes → silenced genes
○​ Loosely wrapped DNA → active genes → transcription
 ●​ DNA methylation: methyl group attached to cytosine nt → gene silencing
Not all mutations are beneficial for cancer cells
●​ Cancer needs a balance of mutations—enough to drive growth, but not so many that the
cells die.
●​ Natural selection favors cancer cells with an optimal level of mutations.
●​ Too little or too much genetic instability prevents cancer from developing successfully.

Cancer may also arise from cancer stem cells (tumor initiating cells)
●​ Genetic or epigenetic changes → cancer cells with self-renewal ability → uncontrolled
growth.
●​ Cancer stem cells drive tumor formation, producing rapidly dividing cells.
●​ Therapy resistant

2.​ Increased genetic instability → More mutations occur as DNA repair mechanisms fail.
Many changes in cancer cells on a chromosomal level
●​ Chromosomal instability (translocations) and changes in a total number of chromosomes
are frequently observed in cancers

3.​ Decreased cell death (apoptosis) and differentiation

→ Mutated cells avoid programmed death.
Decreased apoptosis in abnormal cells
●​ Apoptosis = a process of programmed cell death
●​ Plays an important role in
○​ Embryogenesis
○​ Removal of old epithelial cells and activated
immune cells

4.​ Increased proliferation → Cells divide uncontrollably.

●​ Polyp formation in the gut = consequence of increased proliferation in the intestinal epithelium
●​ Disruption of homeostasis in abnormal cells
○​ Both increased cell division and decreased apoptosis → change in homeostasis (living & dead
cells balance) → tumorigenesis

5.​ Independence from the environment (metastasizing) → Cells become metastatic, spreading to other
organs.
●​ How big can a cancer be before you need a vasculature? Around 3 mm → hypoxic → cells are dying and
it needs oxygen
●​ Formation of a metastasis
○​ Few stage process, tumor cells need to be decoupled from their environment
○​ Crossing the stroma
○​ Vascular growth (angiogenesis) is required

External cancer risk factors

1.​ Epidemiology of cancer
●​ Great differences between countries
●​ Japan: lots of stomach cancer → smoked foods
 2.​ Environmental factors
●​ Mutagens: smoke, asbestos
●​ Radiation induced DNA damage: UV light, radioactive agents
●​ Viruses: HPV, HIV
The Ames test is used to analyse the mutagenicity of substances
●​ Rat liver extract tested with possible mutagen → added to a plate → look at how many colonies are
formed → more colonies, more independence without histidine
●​ Some drugs need to be activated by the liver (due to the metabolites) hence the use of rat liver
increased prevalence of smoking results in a sharp increase of lung cancer cases among women and men

known/suspected causes of human cancers

●​ Asbestos → mesothelioma
●​ UV → Melanoma
●​ HPV → cervical cancer

Identification of cancer-causing genes

1.​ Oncogenes: 1 mutation is sufficient → activating mutation
enables oncogene to stimulate cell proliferation. It will disturb the
angiogenesis. Oncogenes are not heritable.
a.​ Gain of function mutation
b.​ Dominant growth stimulating effect
2.​ Tumor suppressor gene: 2 mutations needed → 2 inactivating
mutations functionally eliminates the tumor suppressor gene,
stimulating cell proliferation.
a.​ Loss of function mutation
b.​ Hereditary cancer
→ Activating oncogene mutations are not inherited because they cause early lethal consequences, while tumor
suppressor gene mutations are inherited because they require an additional mutation to trigger cancer.

Viral infection causes cancer

●​ Retroviruses could transform cells by inserting viral DNA into genes → these were oncogenes
●​ V-onc = viral oncogene, carried by a virus
●​ C-onc = cellular oncogene, resides in a host chromosome → regulate cell growth and cell cyce
Many different v-onc for different types of viruses
A mutation in overexpression of an oncogene (e.g. Ras) results in an increased gene activity, causing continuous
proliferation and overcoming cell-cell contact inhibition
●​ Cancer cells: able to grow on top of each other

3 reasons for increased activity of a proto-oncogene

1.​ Deletions or points mutations (Ras)
2.​ Amplification and resulting overexpression (Myc)
3.​ Chromosomal rearrangement
a.​ Gene under control of an another promoter
b.​ Gene fused with another gene (Bcr-Abl)

1.​ Ras
 ●​ Ras = key signaling protein in the Ras-Raf-MEK-ERK pathway, which
controls cell growth, survival, and proliferation
●​ Normally, Ras is activated by growth factors (e.g., EGF), switching
between an inactive GDP-bound state and an active GTP-bound
state.
●​ Mutations in Ras keep it permanently active (Ras-GTP state) →
constant cell division, even without growth signals.
●​ Uncontrolled Ras activation promotes cancer, making it a dominant
oncogene.
●​ lung, colon, and pancreatic cancers.

2.​ Myc
●​ Myc = transcription factor that regulates cell growth, division, and
metabolism.
●​ Gene amplification causes the normal gene to be overexpressed
●​ Burkitt’s lymphoma, neuroblastoma, and breast cancer.

3.​ Bcr-Abl
●​ Chromosomal translocation in chronic myeloid leukemia (CML) leads to
production of the Bcr-Abl fusion protein
●​ between chromosome 9 (Abl gene) and chromosome 22 (Bcr gene).
●​ This forms the Philadelphia chromosome (Ph¹) → uncontrolled tyrosine kinase
activity.
●​ Bcr-Abl drives constant cell proliferation, causing Chronic Myeloid Leukemia (CML).

Tumor suppressor gene

Knudson’s 2-hit hypothesis
The chance for inactivation of the retinoblastoma (Rb) gene is much
greater in people who have already inherited a mutated form of Rb
gene from their parents
●​ Hereditary cancer syndromes require only one additional
mutation ("second hit"), making cancer much more likely.
●​ Nonhereditary cancers require two independent
mutations, making them far less common.
●​ The "two-hit hypothesis" explains how TSG must be completely inactivated to cause cancer.

Changed signaling cascades in eg cervical and colon cancer due to loss of TSG function
●​ Cell cycle/apoptosis
○​ Inactivation of p53 (50% of all tumors)
○​ Inactivation of Rb/p16
●​ Wnt signaling pathway
○​ Inactivation of APC

Inhibition of tumor suppressor genes by HPV in cervical cancer

●​ HPV infection = first step in the carcinogenesis and origination of the
cervical cancer
 ●​ Viral proteins of HPV inhibit both p53 and RB.
●​ E6 inhibits p53 and E7 inhibits Rb → safety brakes are lost
●​ These inhibitions → cells proliferating more

Altered signaling pathways in cancer

Inactivating p53 mutations in cancers
●​ P53 plays an important role in almost all cancers we know
●​ Binding of a p53 tetramer to DNA → suppresses transcription
●​ P53 functions: integrates various signaling pathways such as
DNA damage, after which it can promote a growth arrest (via
activation of p21) or apoptosis by switching relevant for this
process genes

Loss of TSG →leads to Wnt signaling

●​ Stepwise pattern of mutational activation (order is
important)
●​ E.g., Loss of APC results in an accumulation of
beta-catenin and initiates adenoma-carcinoma
sequence
●​ APC is a TSG that regulates β-catenin degradation to
control Wnt signaling.
●​ APC mutations (TSG) → Wnt signaling in colon
●​ Wnt-beta catenin signaling pathway
●​ APC mutations → uncontrolled Wnt signaling → b catenin activation →
and excessive cell division → colorectal cancer.

Tumor suppressor genes in colon

1.​ FAP: familial adenomatous polyposis. Abundant polyps, moderately
malignant
2.​ HNPCC: hereditary nonpolyposis colorectal cancer. Scarce polyps, very
malignant

Cancer prevention
1.​ Vaccinations young women/men against cervical cancer: HPV16/18
2.​ Chemoprevention for colon cancer: aspirin, celecoxib for adenomas
(inhibitors COX-1 and COX-2)
Creating VIOXX = COX-2 inhibitor:
●​ Removed from the market
●​ Inc heart attack & brain hemorrhage
Challenges in chemoprevention
●​ Must be used for a prolonged period of time
●​ Effectiveness must outweigh toxicity

Conclusions = Cancer is caused by

●​ Activation of oncogenes/inactivation of TSG
●​ Altered signaling pathways
 ●​ Accumulation of mutations (order of mutation acquisition is relevant)

Lecture 2 – Cellular Oncogenes, GFs, receptors, signaling

Main insight: certain genes can cause cancer. Viruses have very small genomes. so they have been very
informative regarding the min # genes that is sufficient to cause cancer: identification of oncogenes and TSG
●​ Experiments: exposure of normal cells to viruses → cancer cells
●​ Only cervical carcinomas and hepatomas can directly be caused by viruses in patients
●​ Infectious retroviral particles produced by cancer cells have not been found

An oncogene is a mutated gene that contributes to the development of cancer.

●​ In their normal unmutated state, oncogenes are called proto oncogenes.
●​ Play roles in the regulation of cell division.
Tumor suppressor: gene in the body that can suppress or block the development of cancer.
●​ A tumor suppressor gene directs the prod of a protein that is part of the system that e.g. regulates cell
division.
●​ The tumor suppressor protein plays a role in keeping cell division in check
●​ When mutated, a TSG is unable to do its job → uncontrolled cell growth may occur
●​ This may contribute to development of a cancer

Oncogenes discovered in human tumor cell lines are related to those carried by transforming retrovirusses
I.​ erbB2/neu/HER2
●​ HER2 (erbB2/neu) is an oncogene that encodes a receptor tyrosine kinase involved in cell growth and
survival.
●​ Amplification of this oncogene in human breast cancer predicts poor prognosis (sruvival)
●​ Leads to more RNA, more protein
●​ Copy-number alterations are frequent in human cancers, presumably resulting in enhanced expression of
growth-promoting genes
II.​ myc
●​ Few copies of myc: high survival rate
●​ N-Myc amplification drives neuroblastoma progression, leading to poor survival outcomes.
●​ It is a key prognostic marker and a potential therapeutic target.

Transfection of DNA = strategy for detecting non-viral oncogenes

Q: if oncogenes exist, either caused by exposure to chemicals, x rays or viruses, how can we identify them amongst
22k normal genes
A: Isolate genes and chop them up into small pieces → transfect in healthy cells → only the cells that have received
an oncogene mutated and is able to give rise to cancer

DNA from human tumor cells also contain oncogenes.

●​ Only a subset of all our 20k genes can act as oncogene
○​ 727 cancer driver genes
■​ 70 germline
■​ 342 somatic
Proto-oncogenes can be activated by genetic change affecting either the protein
expression or structure
In some cases tumors aren't driven by gene amplifications, but by mutations, eg in the
case of bladder cancer driven by H-RAS mutations.
●​ Signal transduction:
○​ Ras off: Ras-GDP
○​ Ras on: Ras-GTP. (glycine →valine): amino acid change
●​ Highly controlled.
●​ In oncogene, (in the absence of signal from the outside), Ras-GTP is constantly
on
●​ This is much more tumor specific and cell type specific.

Variations on a theme: the myc oncogene can arise via at

least 3 additional distinct mechanisms:
1.​ Gene amplifications
2.​ Gene mutations
3.​ Chromosomal translocations resulting in
increased Myc expression via fusion with active
promoters

A diverse array of structural changes in proteins can also lead to oncogene activation
I.​ Chromosomal translocation resulting in loss of negative regulation via
microRNAs
●​ Regulatory elements in the transcript to which microRNAs combine. As soon as
the regulatory elements are lost → protein becomes more stable → more mRNA
degradation.
●​ Chromosomal translocations can remove microRNA regulatory sites, preventing
normal gene repression, and promoting cancer progression through uncontrolled
oncogene expression.

II.​ Chromosomal translocation resulting in altered function of proteins

●​ Bcr-Abl (philadelphia chrom) causing leukemia → 3 breakpoints in the bcr gene.
Abl is actually a kinase, so it can phosphorylate a group down signaling molecule.
So you get a new protein in frame which encodes for a new function of protein bcr.
→ an active kinase with a multitude of function → can phosphorylate (in the
absence of signal from the outside)
●​ Abl gene normally encodes a tyrosine kinase, which regulates cell growth.
●​ The Bcr-Abl fusion protein results in continuous activation of
tyrosine kinase signaling, leading to uncontrolled cell division and
cancer (CML).
●​ Chromosomal translocation (Philadelphia chromosome) fuses Bcr
(Chr 22) and Abl (Chr 9).
●​ Signalling: Bcr-Abl fusion protein activates multiple pathways
(JAK/STAT, PI3K/Akt, MAPK).

Therapy = Gleevec/Imatinib
 ●​ Bcr-abl needs ATP to be phosphorylated → substrate+effector → which leads to leukemia
●​ If the binding part of ATP is inhibited by Imatinib → no substrate + effector → no leukemia
development of drug resistance is a problem
Cancer is often caused by alterations in our DNA (oncogenes/tumor suppressors):
1.​ Mutations in genes, without copy-number changes (Point mutations in RAS)
2.​ Genome amplifications resulting in enhanced expression of e.g. growth Promoting genes (MYC)
3.​ Chromosomal translocations resulting in enhanced expression of oncogenes
​ - fusion to active promoters (MYC)
​ - loss of negative regulation via microRNAs (let7-HMGA2)
​ - fusion proteins with altered function (BCR-ABL)

Cancer incidences vary dramatically between subtypes, why?

Lifetime risk for diagnosed with
●​ lung cancer = 6.9%
●​ Thyroid cancer = 1.08%
●​ Brain cancer = 0.6%​
Pelvic bone = 0.003%
Cancer can be caused by:
●​ Environmental factors (UV, chemicals, irradiation etc) →do not explain cancer risk freq in all cases
●​ Inherited mutations in DNA →do not explain cancer risk freq
●​ Spontaneous mutations in DNA that occur during replication

3 causes of mutations
1.​ External factors
2.​ Repair mechanism is not perfect: with every cell division, there are always going to be mutations.
3.​ Inheritable cancers

Classification of stem cells

1.​ Embryonic stem cells: pluripotent stem cells
a.​ Derived from the inner cell mass of a
blastocyst.
b.​ Can differentiate into any cell type in the
body (except extra-embryonic tissues).
2.​ Adult stem cells: multipotent stem cells
a.​ Found in various tissues of the body
(e.g., brain, heart, bone marrow).
b.​ Can differentiate into a limited range of
cell types related to their tissue of origin.
→ Variations in cancer risk among tissues can be explained by the number of stem cell divisions

Both cancer and evolution are driven by mutation and selection:

●​ In cancer, mutations lead to dysfunctional cells taking over the body.
●​ In evolution, mutations lead to adaptive traits improving species survival.
→ dual role of mutations— either detrimental (cancer) or advantageous (evolution), depending on the context.

Q: How can oncogenes transform cells?

A: Signal transduction

Growth factors, receptors and cancers

Normal metazoan (=multicellular organism) cells control each other’s lives

Appropriate tissue homeostasis depends on

●​ Generation of new cells when needed
●​ Discarding extra/unneeded/old cells
●​ Wound repair
●​ Appropriate immune response
Cells often cannot/should not decide themselves
●​ Key instructors: growth factors → they decide if cells divide, differentiate or die

Normal versions of oncogene-encoded proteins often serve as important machinery

HER signaling pathway, crucial for cell growth, survival,
migration, adhesion, and differentiation.
●​ Growth factors (EGF, TGF-α, NRGs) activate HER
receptors (HER1, HER2, HER3, HER4) on the cell surface.
●​ Signaling cascades:
○​ RAS-RAF-MAPK → Cell growth &
differentiation
○​ PI3K-AKT → Survival & apoptosis inhibition
○​ JAK-STAT → Differentiation
●​ Transcription factors (MYC, JUN, STAT) regulate gene
expression for cancer-related processes.
Relevance:
●​ HER2 overexpression drives cancers like breast cancer,
targeted by drugs like trastuzumab (Herceptin).
●​ Dysregulation leads to tumor progression and metastasis.

●​ Oncogenes drive cancer by enhancing proliferation, invasion, and survival.

●​ Tumor Suppressor Genes contribute to cancer when inactivated, affecting growth suppression and
immune evasion.
These genes could be TFs, receptors, kinases, phosphatases

The EGF receptor functions as a tyrosine kinase

Epidermal growth factor (EGF) drives cell proliferation by activating its receptor:
EGFR

Tyrosine kinase domain: Phosphorylates downstream targets on a tyrosine

(Y) residue
●​ Receptor tyrosine kinases (59) share clear homologies in the
intracellular part: EGFR, NGFR, PDGFR, VEGFR, FGFR, EphR, IGF-1RF
●​ In the extracellular part RTKs are quite distinct → can be activated
by all different kinds of molecules on the outside
●​ But the downstream signaling is the same
 ●​ tyrosine kinase receptors often involved in tumor pathogenesis

All receptor tyrosine kinases have their own sets of ligands

●​ Importantly: many of these RTK-ligand pairs are absent in single cell eukaryotes
●​ but already present in the first simple (multicellular) metazoans such
as worms and flies! → invented just before metazoan life emerged
●​ TKs evolved to regulate complex cell communication needed for
multicellular organisms.

How can EGFR become an oncogene?

●​ Altered GFR can function as an oncoprotein
●​ Avian erythroblastosis virus (AEV) can induce erythroleukemia.
●​ AEV encodes the erbB oncogene → is the EGFR minus ectodomain (extracellular
ligand-binding domain)
●​ Tyrosine kinase domain remains permanently active

How can receptor alterations induce cancer?

●​ Normal cell: Ligand binding brings 2 molecules of receptor together
●​ Cancer cell: ligand independent firing. 2 ways:
○​ Mutations affecting structure:
■​ mutations resulting in constitutive dimer without ligand
■​ and/or absence of an extracellular part of the signaling
domain. Intracellular part remains → signaling
continues.
○​ Overexpression of receptors

cKit receptor mutations

●​ c-Kit is an RTK activated by SCF, regulating cell growth.
●​ Different mutation sites in the c-Kit → diff cancers
●​ Mutations in the juxtamembrane domain prevent normal regulation.

Hyperactivation of receptors can also be caused by aberrant autocrine signaling.

●​ Normal signaling: GF (e.g., TGF-α, PDGF) are released by one cell and act
on another cell (paracrine signaling).
●​ Aberrant autocrine signaling: Cancer cells produce and respond to their
own growth factors, leading to constant receptor activation.
●​ Example: EGFR & PDGFR hyperactivation drives epithelial & mesenchymal
cancers.

v-sis (PDGF)
●​ Autocrine signaling in cancer cells: cancer cell produces and responds to its own
ligand/GF → constant activation independent of other cells
●​ Paracrine signaling: GF acts on other cells
●​ Endocrine signaling: GF acts on other tissues

Transphosphorylation underlies the operations of receptor tyrosine kinases

Q: how do tyrosine kinase receptors emit signals from outside the cell to the inside?
A: Dimerization model

Mechanism of RTK signaling

1.​ Ligand (e.g., EGF) binds to the receptor.
2.​ Receptor dimerization occurs (two receptors pair up).
3.​ Transphosphorylation → Each receptor phosphorylates the other on tyrosine residues, activating
intracellular signaling.
4.​ Cell growth, survival, and proliferation.

Summary
●​ Multicellular organisms make use of ligand receptor interactions to convey extracellular signals to the
inside (autocrine, paracrine, endocrine)
●​ Receptor activation often involves ligand binding, transphosphorylation,and phosphorylation of
intracellular proteins
●​ Cancer can develop due to
○​ High expression of autocrine GF/ligands
○​ High expression of receptors
○​ Mutation in receptors

Not all receptors are tyrosine kinases (RTK):

1.​ Serine, threonine kinase domains (in TGF-β receptors)
●​ TGF-β receptors have serine/threonine kinase domains
Activation steps:
●​ TGF-β ligand binds to the Type-II receptor.
●​ Type-II receptor phosphorylates Type-I receptor, activating it.
●​ Type-I receptor propagates signaling inside the cell.

2.​Cytokine receptors
●​ e.g., α-interferon receptors lack intrinsic kinase activity.
●​ Instead, they recruit Janus kinases (JAKs) (e.g., Jak1, Tyk2) to transmit signals.
●​ Ligand binding (e.g., α-interferon) → receptor dimerization → JAK
transphosphorylation and activation.
●​ Activated JAKs phosphorylate downstream targets (STAT protein), to regulate
gene expression.
●​ No TK domain, cytokine receptors rely on JAK kinases for signaling.

3.​ NOTCH signaling

●​ Intracellular NOTCH acts as transcription factor (TF)
●​ Notch receptors regulate cell fate, differentiation, and cancer.
●​ Activation steps:
1.​ Delta ligand (from a neighboring cell) binds Notch receptor.
2.​ 1st cleavage releases the extracellular part.
3.​ 2nd cleavage releases the Notch intracellular domain (NICD = TF)
4.​ NICD enters the nucleus, acting as a transcription factor to control
gene expression.
 ●​ Notch mutations drive leukemia and solid tumors.
4.​ Patched-smoothened (hedgehog) signaling
●​ Without hedgehog: patched receptor inhibits smoothened → no signal → Gli (TF) repressed → blocked
gene activation
●​ With Hedgehog: bind to patch protein → Smoothened released → activation of Gli = TF → drives gene
expression

5.​ Wnt signaling

Without Wnt (left):
●​ Β-catenin (TF) degraded by a destruction complex (GSK-3β, APC, Axin, Wtx).
●​ No gene activation.
With Wnt (right):
●​ Wnt binds Frizzled & LRP, activating Dishevelled → actin recruited to
cytoplasm
●​ GSK-3β is inhibited, preventing β-catenin degradation.
●​ β-catenin enters the nucleus to drive gene expression, promoting cell
proliferation & stem cell maintenance.
Wnt signaling stabilizes β-catenin to drive gene expression; its dysregulation
promotes cancer.

Integrin receptors sense association between the cell and ECM

●​ ECM is composed of glycoproteins, hyaluronan and proteoglycans
supporting cells
●​ Cells can bind to the ECM via Integrin receptors
Integrins come in dimers
●​ Alpha and beta subunit
●​ Heterodimers
●​ Integrins cluster to form focal adhesions
Focal adhesions = clusters of integrins that link actin cytoskeleton to ECM, enabling
cell movement and cancer metastasis.
●​ ECM induces dimerization →Intracellular signaling to the actin
cytoskeleton
Constitutive activation of RAS results in → integrin-independent growth without
focal adhesions: anchorage independent growth hallmark of cancer

Summary:
receptors/GF associated with cancer =
●​ 59 tyrosine kinase receptors
●​ >50 Cytokine receptors (recruit kinases)
●​ 11 TGBb-like receptors (serine/threonine kinases)
●​ 4 Notch receptors: intracellular NOTCH as TF
●​ 2 Patched receptors: GLI as TF
●​ 10 Frizzled receptors: b-Catenin as ​TF
●​ 24 integrins: adhesion-mediated signaling (RAS mut: anchorage-independent growth)

Cytoplasmic signaling circuitry programs many of the traits of cancer

tyrosine phosphorylation controls the location/actions of cytoplasmic proteins

SRC protein = tyrosine kinase

●​ Tyrosine kinase with many different targets, explains multiplicity in phenotypes
○​ Inactive Src: Held in a closed conformation via phosphorylation at Y527.
○​ Active Src: Y527 dephosphorylation & Y416 phosphorylation activate kinase function, promoting
cell growth & migration
●​ Src phosphorylate proteins on other tyrosine residues
●​ One activation of Src → multiple phosphorylations of other downstream signaling
pathways
●​ Cytoplasmic protein
●​ Molecular signal integration domains

Q: How can phosphorylation activate proteins? Or activate signaling pathways?

A: Phosphate adds a negative charge, causing:
1.​ Conformational change → altering protein function.
2.​ Electrostatic interactions with positively charged residues (e.g., in SH1/2/3 domains of Src
kinase)
The example = SRC protein

SH2 domain is a common feature in many proteins which mediates communications between them
●​ creating a negatively charged binding point for arginine (positively charged)
●​ 110 distinct SH2 domain containing proteins encoded in human genome
Phosphorylated tyrosine residues form binding platforms for other proteins
●​ Phosphorylated tyrosine (pY) residues serve as docking sites for signaling
proteins via SH2 domains.
●​ SH2 domains bind phosphotyrosine, enabling tyrosine kinase signaling and
cancer progression
A multiplicity of protein-protein interaction modules exist:
●​ Signaling proteins use modular domains (e.g., SH2→ phosphotyrosine, 14-3-3 →

😊
phosphoserine/threonine) to recognize phosphorylated targets and relay
signals.
The PI3K-PKB/Akt pathway
Activation
●​ GF (insulin, T-cell activators) activate PI3-Kinase (PI3K) via IRS (insulin
receptor substrate).
●​ PI3K produces PtdIns(3,4,5)P₃, recruiting PKB/Akt via its PH domain.
●​ PKB/Akt is activated by PDK1 (T308) & PDK2 (S473) phosphorylation.
Downstream Effects: PKB/Akt phosphorylates multiple targets, binding 14-3-3 proteins
to regulate metabolism, survival, and growth.

Histone methylation is an active epigenetic mark that can drive (oncogenic) gene
transcription
●​ E.g., H3K27-ac → BET proteins (Bromodomain and Extra-Terminal domain)
recognize acetylated histones and promote transcription.
 ✅
🚦
●​ Acetylation = "Opens" chromatin → More transcription
●​ Methylation = "Marks" genes for activation OR repression

AP-1
●​ AP-1 (Activator Protein-1) is a TF complex that regulates gene expression in response to stimuli like growth
factors, stress, and cytokines.
●​ It consists of c-Jun and c-Fos, which form a heterodimer via the leucine zipper motif.
●​ Leucine zipper is a structural motif that facilitates protein dimerization and DNA
binding.
●​ The AP-1 complex binds to DNA at specific TGA(C/G)TCA motifs in the major groove,
influencing cell proliferation, differentiation, and apoptosis.
Why is this important in cancer?
●​ AP-1 is often deregulated in cancer and contributes to oncogenic signaling
●​ c-Jun and c-Fos can be phosphorylated, which enhances their transcriptional
activity, linking them to cancer progression.

SH2 and SH3 groups explain how GF receptors activate RAS

●​ SH2 binds p-Tyr on the TK receptor.
●​ SH3 recruits Sos (GEF).
●​ Sos swaps GDP for GTP on Ras.
●​ Active Ras → downstream signaling → cell growth.

Summary
2 outcomes of signaling:
●​ signal transduction: extracellular ligand → activated receptor →
intracellular signaling cascade → TF activation → gene expression
or:
●​ signal transduction: extracellular ligand → activated receptor → intracellular signaling cascade → eg
morphological changes → adhesion/migration
PTMs: post-translational modifications such as phosphorylation (Y, S, T) allow signal transduction from one
protein to another
●​ phosphorylation = neg charge, allows dimerization of SH2 domains, conformational changes
●​ many other PTMs exist: acetylation, ubiquitination, sumoylation, methylation
●​ many protein-protein interaction domains, protein-DNA interaction domains etc

A large series of different types of signaling cascades exist

●​ Ras (GDP/GTP):
○​ MAPK/ERK pathway (phosphorylation)
○​ PI3K pathway (PIP3)
○​ Rac/Cdc42: philopodia/lamelipodia
●​ JAK-STAT pathway: hematopoiesis, cytokines
●​ Integrin pathway: adhesion/migration/cytoskeletal rearrangements
●​ Wnt pathway: inactivation of GSK3b results in stabilization of bCatenin (TF)
●​ GPCR signaling (7 membrane spanning receptors)
●​ NFkB pathway: degradation of IkB results in release of NFkB (TF)
●​ Notch pathway: cleavage of Notch receptor, intracellular notch (TF)
 ●​ Hedgehog-Patched-Smoothened-Gli pathway
●​ TGFb pathway: Smads
→ mutations in any of these pathways can contribute to cancer development

Mechanisms of drug resistance.

1.​ BCR-ABL resistance to gleevec
a.​ Mutations in the kinase domain disrupt drug binding.
b.​ T315I mutation prevents Imatinib from fitting into ATP-binding site.
c.​ Other mutations (G250, E255, M351) affect activation loops.
d.​ Result: BCR-ABL remains active → uncontrolled signaling & leukemia progression.
2.​ NPMcyt
a.​ NPM1 wt is a nucleolar protein but mutation results in mislocalization to the cytoplasm, but also
the chromatin where it drive oncogene gene transcription (HOXA locus)
b.​ Acute myeloid Leukemia: mutation in NPM1 → impaired differentiation, shortage of functional
mature blood cells. Still incurable in majority
c.​ Resistance to Menin inhibitors (targeting NPMcyt-Menin interaction) and FLT3 inhibitors (many
AML also have FLT3 mutations) arises through MEN1 mutations and compensatory pathways.
Targeting multiple pathways simultaneously could improve treatment outcomes.

Lecture 3 – TSG

GFR: proteins involved in signal transduction pathway in the cell membrane

Tumor suppressor genes counterbalance oncogenes
●​ Proto Oncogenes & oncogenes drive proliferation of cells
●​ Proto Oncogenes products participate in growth-stimulatory signals, mutations in these genes lead to
continuous growth stimulating signals, and proliferation of cancer cells
●​ TSG counterbalance proto-oncogene activity → cancer pathogenesis that is as important to cancer as the
activation of oncogenes
●​ TSG → genes that in one way or another normally function to reduce the likelihood that a clinically
detectable tumor will appear in one of the body’s tissues

Dominance and recessiveness of the tumorigenic phenotype

Cell fusion exp: When normal and cancer cells are fused → cancer has usually a recessive phenotype because TSG
from the normal cells will give a brake to the oncogenes
2 phenotype options:
●​ Tumorigenic (Cancer alleles dominant) → The fused cell becomes cancerous, meaning oncogenes drive
cancer even in the presence of normal genes. (common in virus-induced cancers)
●​ Non-tumorigenic (Cancer alleles recessive) → The hybridccell remains normal, meaning TSG prevent
cancer.
Cancer induced by a virus should lead to a dominant phenotype → virus inactivates the TSG
●​ Without the involvement of a tumor virus, its malignant phenotype would
be recessive upon fusion of tumor cell with a normal cell
●​ In case of recessive TSG, both wild type copies would need to be
eliminated to lose its activity
 ●​ Most cancers usually has a recessive phenotype

How to lose two gene copies?

Retinoblastoma (Rb) = TSG
●​ tumor of the retina;
●​ arising in the precursors of photoreceptor cells
●​ (1 in 20k children)
●​ The probability to lose a TSG is much greater than gaining oncogenes
○​ Inherited mutations require only one additional "hit", making cancer more likely.
○​ Sporadic cases need two independent mutations, making them rarer.
●​ Hereditary: increases the chance to get Rb because of the lost TSG
Rb protein regulates cell cycle progression
●​ RB is a tumor suppressor that acts as a checkpoint in the cell cycle.
Mechanism Rb:
●​ Inactivated = Cyclin E-CDK2 complex → Rb is phosphorylated → E2F (TF) is
released → G1 → S transition → DNA replication → proliferation
●​ Activated = p21 (CDK inhibitor) → Rb not phosphorylated → E2F binds to
Rb → no genes transcribed for the S phase → differentiation/senescence
(aging)

Gatekeepers and caretakers

●​ TSG can also encode microRNAs in addition to proteins
●​ Well known TSG: TP53, and Rb, BRCA
●​ Gatekeepers: TSGs that function to directly control the biology of cells by affecting how they proliferate,
differentiate, or die (TP53)
●​ Caretakers: DNA maintenance TSG affect cell biology only indirectly by controlling the rate at which cells
accumulate mutant genes (BRCA1)

Elimination of the second wild type Rb gene copy

1.​ Loss of heterozygosity (LOH) by mitotic
recombination
●​ Once you've lost one copy, losing 2nd gene copy:
occurs at higher freq compared to random mutation
●​ Genes nearby of Rb on chrom 13 will also lose heterozygosity and become homozygous
●​ This genetic alteration of a gene or a chromosomal region = loss of heterozygosity (LOH)
●​ Heterozygosity at Rb locus (one normal and one mutant RB allele).
●​ Mitotic recombination occurs during G2/M phase, leading to chromosome exchange.
Daughter cells outcome:
●​ Retention of heterozygosity (normal function preserved).
●​ Loss of heterozygosity (LOH) → Both copies now mutant → No functional RB
protein → Uncontrolled cell division → Cancer
.
2.​ LOH by gene conversion
●​ Temporal switch in template during DNA replication (S phase)
●​ More frequent than LOH by mitotic recombination
●​ During DNA replication (S phase), cells copy their DNA to prepare for division.
 ●​ Normally, DNA polymerase (the enzyme that copies DNA) follows one template.
●​ Gene conversion happens when DNA polymerase jumps to a different template (homologous
chromosome) and then jumps back.
●​ If the normal RB gene is replaced by a mutated copy, the cell loses its functional RB gene, leading to Loss
of Heterozygosity (LOH).

3.​ LOH by hemizygosity, chromosomal loss by nondisjunction, promoter methylation

●​ Hemizygosity: loss (breakage) of chrom region → loss of genes on a chrom
arm
●​ Nondisjunction: loss of an entire chrom due to inappropriate chromosomal
segregation at mitosis (similar to down syndrome’s extra chromosome)
●​ CpG methylation: shutting down the expression of a gene by methylation
of promoter by DNA methyltransferases (epigenetic silencing)

Identifying possible TSG:

1.​ Measurement of deleted chromosomal segments carrying TSG
●​ Chromosomal region flanking a TSG → undergoes LOH together w the TSG itself
●​ The existence of a still uncloned TSG is possible using anonymous genetic marker lying nearby the
unknown TSG = linkage analysis
●​ Chromosome: p arm is the small arm (petit)
●​ If a tumor repeatedly shows LOH in a specific chromosome region, it likely contains a TSG.

2.​ Linkage analysis in APC gene (familial adenomatous polyposis coli)

●​ Assembly of multigenerational pedigrees for APC → checking the heterozygosity in parents both healthy
and diseased
●​ APC gene associated with FAP (hereditary colorectal cancer)

3.​ Methylation of the RASSF1A promoter

●​ RASSF1A (Ras association domain family 1 isoform A)
●​ Repression of gene expression by CpG island methylation in promoter region by DNA methyltransferases
(=epigenetic silencing)
●​ Promoter: where transcription starts, if methylated, RNA polymerase cant transcribe the DNA
●​ This means → looking for deletion of genes is not always reliable because the gene can still be intact but
just not expressed due to the methylation

Best way to test if a gene is a TSG: introduce wild type gene expression in cancer cells
What criteria can be used to define a TSG?
Not good enough → simply lacking gene expression
●​ Some genes are naturally turned off in certain tissues as part of normal differentiation.
●​ This does not automatically mean it is a TSG

1.​ Genetic criteria

●​ Repeated detection of LOH of a gene in tumor cell genomes
●​ Homozygous inactivation with clear mutations: Gene is TSG = If it undergoes LOH in many tumor cell
genomes and if the resulting homozygous alleles bear clear and obvious inactivating mutations
 ●​ Promoter methylation can silence the gene: Consider the fact that the activity of many TSG can be
eliminated by promoter methylation
2.​ Functional tests:
●​ complement normal gene and test if cancer props are reduced = reintroduced wild
type (normal) genes into cancer cells
●​ Gene knockout; delete gene function in normal cells and test for enhanced
tumorigenicity

Therapeutic opportunity
●​ Gene therapy strategies
●​ epigenetic therapy (reversing methylation status of genes)
●​ In some cases: targeted therapy
○​ PTEN loss (TSG) → hyperactivity of Akt/PKB and PI3K → Cancer cells depend
on for their continued viability.
○​ So if we target this pathway we could regulate the cancer cells

P53 and apoptosis: master guardian and executioner

Guardian of the genome: TP53
●​ P53 mutations are found in over 50% human cancers
●​ These mutations result in a functionally inactive p53 protein
●​ Can be inactivated by viral proteins eg HPV E6
Function
●​ A TF that can activate cell cycle arrest to allow DNA repair or activate apoptosis
●​ Mutated p53 interferes w ability to halt the cell cycle or induce apoptosis

Effect of p53 on cell transformation

●​ P53 is not a typical TSG = mutant p53 has a dominant effect?
●​ Losing one copy of p53 = cells will proliferate more
●​ Mutant p53 = even more cell proliferation
Pick me girl – p53
●​ ras + p53 deletion → Many transformed colonies
○​ No p53 = No tumor suppression → Cancer grows.
●​ ras + p53 mutant → Even more transformed colonies
○​ Mutant p53 enhances transformation (dominant-negative
effect).
●​ ras + wild-type p53 → No transformed colonies
○​ Wild-type p53 blocks tumor formation (classic TSG function).
→ Unlike typical TSGs, mutant p53 not only loses function but also promotes cancer, explaining its dominant effect
in tumors.
Understanding the p53 phenotype:
Dominant-negative allele
●​ P53 functions as a tetramer (4 subunits form an active protein) → overexpression of p53
can cause it to have a dominant phenotype
●​ A single mutated allele can block normal p53 activity
●​ Differences in outcome:
○​ P53 point mutation → dominant negative → almost complete loss of function
 ○​ P53 deletion → null mutation → some tumor suppression remains
●​ Dominant negative = mutant protein interferes with the normal (wild-type) protein,
preventing it from working properly. (one broken leg in a chair → useless chair)

P53 forms a homotetramer and acts as a TF

●​ Specialised domains in p53
○​ Mdm2-binding region → Regulates p53 degradation via the
proteasome.
○​ DNA-binding domain → Controls tumor suppression by
activating target genes (where most mutations occur)
○​ Tetramerization domain → Forms active p53 complexes.
○​ Nuclear Localization Signals (NLS) → Ensures p53 enters the
nucleus.
●​ Most mutations occur in DNA-binding domain of p53 → indicating that it’s a
very important function of p53

P53 activating signals and p53’s downstream effects

●​ P53 monitors the integrity/proper functioning of cellular processes
●​ Diverse surveillance systems signal to p53 leading to a halt to cell proliferation
or triggering of apoptosis
●​ This is why cancer cells want to get rid of the p53 alarm response
●​ function p53: angiogenesis, cell cycle arrest, apoptosis, cell repair

P53 and radiation response → p21 dependent cell cycle arrest and apoptosis
●​ The lack of p53 stimulates cancer development → makes cells more resistant to radiation-induced
apoptosis
●​ p21 mediates cell cycle arrest, allowing repair.

Control of p53 levels by Mdm2

●​ P53 is a TF regulating exp of multiple target genes, incl MDM2
(E3-ubiquitin ligase=labels protein to become degraded).
●​ In our cells we have garbage containers called proteasomes and they break
down proteins that are not needed anymore
●​ MDM2 binds p53 leading to degradation by the ubiquitin-proteasome
system
●​ Negative feedback loop by p53-MDM2
●​ Many positive and negative signals affect MDM2-p53 interactions (various
stress-induced signals affect MDM2-p53 interactions (various
stress-induced kinases and proliferation signals)
Negative Feedback Loop
●​ More p53 → More Mdm2 → More p53 degradation (keeps levels in check).
●​ If p53 is overactive, Mdm2 lowers it to prevent unnecessary apoptosis.
●​ If DNA damage occurs, stress signals inhibit Mdm2, allowing p53 to rise and trigger cell cycle arrest or
apoptosis.
Apoptosis: regulated and orderly destruction of a cell through a genetically encoded process also known as
programmed cell death (PCD)
●​ Doesn’t induce inflammation
●​ As important and intrinsic to the cell as eg cell division
●​ Conserved in evolution
●​ In development of multicellular organisms from worms to humans (metazoans) maintaining cell numbers
●​ Maintaining tissue homeostasis (tissue size and removal of aberrant cells)
●​ Important role in immune system
○​ Elimination of self-reactive T-cells
○​ NK cells elimination of virally infected cells.
●​ Dysregulated Apoptosis Causes(human) disease → carcinogenesis

Diverse manifestations of the apoptotic program:

●​ Membrane blebbing, chromatin condensation, cell shrinkage(pyknosis), DNA
fragmentation, fragmented golgi, fragmented nuclei, phagocytosis of apoptotic
bodies by MP/neighbor cells

Different forms of cell death:

1.​ Anoikis: loss of anchorage to ECM → apoptosis (prevent metastasis)
2.​ Necroptosis: Regulated necrosis → triggers inflammation (viral infection)
3.​ Autophagy: (self eat), basic catabolic mechanism that involves the cellular
degradation of unnecessary or dysfunctional cellular components through the action
of lysosomes

P53 and apoptosis

●​ Cytochrome c normally located in the intermembrane space of
the mitochondria
●​ Cytochrome c release in the cytosol from mitochondria during
apoptosis
●​ It is a strong trigger for the apoptotic process

Checkpoints for apoptosis in the mitochondrial pathway

●​ Bak and Bax: pore formation → pro apoptotic
●​ Bcl2 anti apoptotic

🔹
●​ BH3 only → inactivates Bcl2
Healthy Cells
●​ Bcl-2 inhibits Bax and Bak, keeping mitochondria intact.

🔹
●​ Cytochrome c stays inside the mitochondria → No apoptosis.
Apoptotic Cells
1.​ BH3-only proteins (red triangles) inactivate Bcl-2.
2.​ Bax and Bak oligomerize, forming pores in the
mitochondrial membrane → Cyt c is released into
cytosol.
3.​ Cytochrome c binds Apaf-1 → Forms the apoptosome.
4.​ Apoptosome activates caspase-9 → Activates caspase-3
→ Apoptosis is executed.
Bcl2 and related proteins: preventing and promoting apoptosis
●​ Human genome is known to encode 24 Bcl2 related proteins
●​ 6 are anti apoptotic (pro-survival)
●​ 18 are pro apoptotic: Bax, Bak, BH3

Pro-apoptotic signals acting through various Bcl-2-related proteins

I.​ The apoptotic caspase cascade (intrinsic apoptosis)

Caspases = cysteine-proteases synthesized as inactive zymogens (procaspases)
●​ Cleave target proteins at aspartic acid residues at the P1 position
●​ Caspase 2, 3, 6, 7, 8, 9 and 10 are involved in apoptosis.
●​ Divided into
○​ Intiators: 2, 8, 9 and 10 (8 and 9 involved in cancer) →
activate downstream caspases
○​ Effectors: 3, 6, 7 → execute apoptosis by cleaving proteins
Cytochrome c leaking out triggers the apoptosome formation by Apaf-1 →
recruiting caspase 9 → caspase 9 is activated in the apoptosome protein
complex → activation of the executioner caspases (3, 6, 7) → death substrates cleaved → apoptotic fragments
Mechanism:
1️⃣ Pro-apoptotic signals (e.g., DNA damage, hypoxia) activate Bax/Bak.​
2️⃣ Bax/Bak form mitochondrial pores → Cytochrome c is released.​
3️⃣ Cytochrome c binds Apaf-1 (Apoptotic protease activating factor-1) → forming the apoptosome.​
4️⃣ Apoptosome activates initiator caspase-9 → which in turn activates effector caspases (3, 6, 7).​
5️⃣ Effector caspases cleave key death substrates (e.g., ICAD, lamin, actin), leading to cell dismantling and death.
Apoptotic regulation
●​ Inhibitors of apoptosis (IAPs) → regulate the apoptotic pathway
→ normally block the executioner caspases
●​ Smac/DIABLO = a protein that can block the IAPs → open up the
caspase cascade (mitochondrial apoptotic pathway)

II.​ Death receptors (extrinsic apoptosis)

●​ triggers apoptosis via external signals, activating death receptors
on the cell surface.
●​ TRAIL receptors on the membrane and TNFR1
●​ Upon binding to ligand → trimerization inside the cell (receptors
come close together) → achieve confirmation to recruit FADD
(death domains)
●​ FADD binds to caspase 8 → Caspase cascade
Mechanism
1️⃣ Death Ligand binds Death Receptor (e.g., FasL binds Fas).
2️⃣ Receptor clustering recruits FADD, forming DISC.
3️⃣ DISC activates initiator caspase-8/10.
4️⃣ Caspase-8/10 cleaves executioner caspases (3, 6, 7), leading to apoptosis.

III.​ Convergence of intrinsic and extrinsic apoptotic pathways

 ●​ Initial stages of death receptor activated leads to Bid protein (BH3 only protein) activated → release of
cytochrome c from mitochondria
●​ Leads to downstream activation of executioner caspases
●​ Often you need this crosstalk between intrinsic and extrinsic pathways depending on how the cell is wired.

✅
Extrinsic & Intrinsic Pathway Link – tBid Activation

✅
Caspase-8 can activate the intrinsic (mitochondrial) pathway by cleaving Bid → tBid.​
tBid translocates to mitochondria, activating Bax/Bak, leading to cytochrome c
release.

P53 activates apoptosis via multiple signals

●​ Activation of p53 activates one of the death receptor
●​ P53 could also upregulate Bad, PUMA, and NOXA (BH3-only) → also
activate the mitochondrial pathway through the Bcl-2 family
●​ Indirect way of p53 apoptosis = Blocking IGF-1R → PI3K → Akt/PKB →

✔️
cell survival signals inactivated
Intrinsic (Mitochondrial) Pathway: p53 activates Bax, leading to cytochrome c

✔️
release, apoptosome formation, and caspase activation.
Extrinsic (Death Receptor) Pathway: p53 upregulates Fas, activating
caspase-8, which either directly activates executioner caspases or links to

✔️
mitochondria via tBid.
Inhibits Survival Signals: p53 upregulates IGFBP-3, blocking PI3K/AKT survival
signaling and activating FOXO3, promoting apoptosis.

Cancer cell: a lot of stress → hallmark of cancer = evading apoptosis in various ways
●​ Acting directly on caspases = promoter methylation → can’t be transcribed
anymore
●​ etc

Therapeutic proapoptotic strategies

●​ FLIP blocks the death receptor pathway by binding FADD preventing it from
activating caspases.
●​ Inhibiting FLIP could induce apoptosis
●​ Providing death receptor ligands could also be a nice approach
●​ Restoring functional p53 into cancer patient is effective in tumor regression

Lecture 4 – invasion and metastasis

Disseminated tumors
●​ Metastatic disease = responsible for 90% of cancer associated mortality
●​ Metastases = cells from primary tumor mass → entered blood and lymphatic vessels (lymph nodes) →
spread throughout the body where they may form new colonies
●​ Micrometastases may be missed
Systemic therapy: immunotherapy is the only effective way to treat cancer that has metastasised to the brain and
other parts of the body
Seed and soil hypothesis
●​ Ability of a disseminated cancer cell to successfully found a metastasis depends on
whether a distant tissue offers it a hospitable environment to survive and proliferate
●​ Cancer cells (seed) can only colonise tissues with favourable conditions (soil) that
enables outgrowth

Primary tumors and their metastatic tropisms

●​ Width of the arrows is relative measure for metastasis formation in organs
●​ Mechanism for metastasis preferences not yet clear

Metastatic tropisms
●​ Metastatic cells follow the layout of the vessels/bloodstream.
●​ Eg colon carcinoma cells metastasize to the liver which may reflect transportations of these cancer cells via
the portal vein that connects the lower GI tract and the spleen ending up in the capillary beds of the liver
that are fed by this vein
●​ Colonisation of distant tissues by cancer cells is supported by normal physiological processes such as
localised wounding
●​ Areas chronic inflammation within the body of a cancer patient provide a spectrum of mitogenic and
trophic signal (chemoattractants) facilitating colonization and formation of secondary tumors

I.​ Early stages of metastasis

●​ Invasion through the basement membrane
●​ Degradation of the ECM
●​ Metastatic cells are anoikis resistant (=epithelial cells loosen from membrane) = a
form of apoptosis that's triggered by detachment of a cell from a solid substrate
such as an ECM

II.​ Invasion-metastasis cascade

●​ Metastasis is an inefficient process particularly the
colonization step is rate limiting
●​ Role of the tumor microenvironment during dissemination
and colonization
Primary tumor formation → localised invasion → intravasation →
transport through circulation → arrest of microvessels of various
organs → extravasation → micrometastasis → macrometastasis

III.​ Intravasation = entering the blood stream

●​ Help for intravasation: a triad of cells = 3 cellular components involved
○​ Mena⁺ carcinoma cells: These cancer cells have high motility due to the Mena protein, which
regulates the actin cytoskeleton.
○​ Macrophages: help cancer cells by creating pathways for entry into blood vessels.
○​ Endothelial cells: These line blood vessels and allow cancer cells to pass through.
●​ Mena actin cytoskeleton-regulating protein is associated with cell motility of cancer cell

IV.​ Tumor cells in circulation

 ●​ Circulating tumor cells (CTC) are often found in the circulation as multicellular aggregates
●​ Many CTCs are trapped in lung microvessels (dense microvascular network)

V.​ Extravasation = from vessel to tissue

1.​ Metastasizing cell (brown) trapped physically in capillary
2.​ Large number of platelets (blue) attach to cancer cell →
microthrombus formation
3.​ Cancer cell pushes aside endothelial cell (green)→ direct contact
with underlying capillary basement membrane (orange)
4.​ Microthrombus dissolved by proteases responsible for dissolving
clots
5.​ Cancer cell proliferates in the lumen capillary
6.​ Cancer cells break through the capillary BM and invade the surrounding tissue parenchyma (gray area)

Detection of micrometastases in the marrow or lymph node

●​ Disseminated TC in the marrow, often termed DTCs, seems to represent a better prognostic marker than
CTCs in the blood
●​ Cytokeratin positive cells in marrows of a cohort of breast cancer patients were counted
●​ Patients w cytokeratin positive or negative marrows are grouped
●​ Cytokeratin positive marrows had a worse prognosis after initial diagnosis
●​ DTC (metastases in BM/LN)= better prognostic marker than CTC (metastases in circulation)

Genetic heterogeneity of micrometastases and the evolution of colonizing ability

●​ Genetically heterogeneous primary tumor cell population seeds equally = heterogenous micrometastases
●​ Removal of the primary (surgery) leaves behind micrometastases (minimal residual disease)
●​ Overtime one of these micrometastatic cell clones (blue) acquires the ability to colonize ie to grow into
macroscopic metastasis
●​ Even though primary tumor is removed, there still will be a secondary shower of metastatic dissemination
●​ Model: genetic basis for metastasesf

Nongenetic metastases: Epithelial mesenchymal Transition (EMT)

●​ Epithelial cell acquire mesenchymal properties
●​ Primary carcinomas = organised in epithelial cell layers as seen in normal tissues and is incompatible with
the motility and the invasiveness
●​ Carcinoma cells shed their epithelial phenotypes by EMT
●​ Cells lose epithelial morphology and gene expression and acquire characteristic of mesenchymal cells
●​ EMT = occurring during embryogenesis & wound healing
●​ EMT is induced by extracellular signals from the TME
●​ Microenvironment dependent signals may be temporally: EMT is reversible
●​ Model: non genetic basis for metastases

EMT during embryological development: eg motile and invasive neural cells from the PNS
●​ where neural crest cells undergo EMT migrate from the neural tube (NT) to form different tissues.
●​ Cells from the upper region of the neural tube
●​ Migrate and invade in the embryonic neural crest of chordates by undergoing EMT
 ●​ These cells give rise to melanocytes, the peripheral nervous system, and facial bones
●​ → EMT is not just in cancer—it’s crucial for normal development too.

Adherens junctions = keep epithelial (tumor) cells in tight linked structures

●​ Cadherins (e-cadherin) connect epithelial cells
●​ Actin filament bundles are anchored to cadherins, making the cells tied together
●​ These structures must be broken during the metastatic process
●​ Normal cells die if detached (anoikis), but cancer cells resist anoikis → metastasis
●​ E-cadherin expression is lost at the invasive front
●​ At the core of the tumor e cadherin localises to the cell membranes while this expression is lost at the
invasive front of the tumor

Cellular changes during EMT

●​ Loss of epithelial cell properties
○​ Tight junctions and epithelial adherens junctions involving E-cadherin
○​ Cell polarity
○​ Cytokeratin (intermediate filament) expression
○​ Epithelial gene expression program
●​ Gain mesenchymal properties → more aggressive phenotype
○​ protease secretion (MMP2/9) → Degrade the ECM facilitating tumor cell spreading
○​ Increased resistance to apoptosis
○​ Motility
○​ Invasiveness
○​ Stem cell like traits
○​ Fibroblast like cells

E-cadherin and beta-catenin (Wnt signaling induces

EMT)
●​ B catenin is recruited inside the cell and
binds to cell membrane (e cadherin)
●​ If e cadherin is downregulated (part of
EMT) then beta catenin can't bind anymore
●​ In This case b catenin is the known
activator
●​ Losing e cadherin → beta catenin can
induce Wnt signaling pathway → more
stem cell like traits
●​ Beta catenin: from membrane/cytoplasm
(tumor center) to nucleus at the invasive
front → activating genes for EMT
This transition promotes cell detachment, invasion, and metastasis.

Biochemical changes regulating/accompanying EMT

●​ Epithelial markers: E cadherin, b catenin, y catenin → when downregulated → weakened cell adhesion
●​ Mesenchymal markers: fibronectin, vimentin → upregulated → cell motility and invasion
EMT is reversible = EMT and MET
●​ EMT allows tumor cells to escape
●​ while MET helps them re-establish at new sites.

Control of the EMT by TGF-b

●​ Autocrine TGFβ signaling maintains mesenchymal state; cells transduced with a non functional TGFβ
receptor become epithelial
●​ TGFβ from stroma induces EMT (αVβ6 integrin (mesenchymal marker) positive tumor cells)
●​ TGF-β drives EMT but is reversible.
●​ Blocking TGF-β signaling could prevent metastasis by maintaining the epithelial state.

During (EMT), β-catenin = driving the transition from an E → M phenotype

1.​ Transcriptional Activation: β-catenin translocates to the nucleus and activates Wnt/β-catenin signaling,
inducing the expression of EMT-related transcription factors like Snail, Slug, Twist, and ZEB1, which
repress E-cadherin and promote mesenchymal characteristics.
2.​ Loss of Cell-Cell Adhesion: In epithelial cells, β-catenin is part of adherens junctions, binding to
E-cadherin. During EMT, E-cadherin is downregulated, freeing β-catenin to enhance Wnt signaling and
promote motility.
3.​ Cytoskeletal Remodeling & Migration: β-catenin upregulates mesenchymal markers like N-cadherin and
vimentin, enhancing cytoskeletal reorganization, cellular plasticity, and invasiveness.

Signals regulating EMT

●​ In the epithelial state activity of secreted TGF-beta and canonical Wnts is
blocked by the concomitant secretion of inhibitors – BMPs, DKK1, SFRP1
●​ When Cell shuts down secretion of BMPs & wnt inhibitors → EMT is triggered
also helped by the secretion of non-canonical Wnt proteins
●​ Reversible process

Signals triggering EMT

Stromal Cells Induce EMT:
●​ Fibroblasts, macrophages, and inflammatory cells secrete signals
like TGF-β, Wnts, TNF-α, FGF, and HGF, promoting EMT.
●​ Myofibroblasts and mesenchymal stem cells enhance EMT
through PGE₂ and cathepsin.
Key Signaling Pathways:
●​ TGF-β downregulates E-cadherin, weakening cell adhesion.
●​ Wnt signaling stabilizes β-catenin, activating EMT-related genes.
●​ PI3K/Akt and NF-κB enhance cell survival, motility, and invasion.
●​ Ras and Raf oncogenes suppress apoptosis and drive tumor
progression.

Transcription factors that regulate EMT

●​ These EMT TFs act as transcriptional repressors and activators
controlling EMT
●​ EMT inducing TFs confer stem cell properties on epithelial cells
 ●​ Cell has CD44 high and less CD24 → more stem cell population
●​ EMT TFs (snail/twist) generate CSC (cancer stem cells) → making tumors more aggressive and
therapy-resistant.

ZEB1/2 and miR20: alternation between the E and M states

●​ ZEB1/2 are keys in regulating the decision in carcinoma cells to activate an EMT
program or not
●​ They form a bistable switch with miR-200

EMT-inducing TF (Snail, Twist) worsen prognosis by driving relapse and metastasis.

Silencing EMT factors (like Twist) can suppress metastasis without affecting

Conclusions
●​ Nongenetic changes–heterotypic signals of stromal origin–rather than genetic
changes contribute to malignancy in carcinoma cells
○​ In other words, malignancy in carcinoma cells is influenced more by stromal signals rather than
genetic mutations
●​ EMT TFs are regulated by stimuli derived from both tumor and normal stromal microenvironments and
induce the mesenchymal state at the tumor site and reversal to an epithelial state when encountering the
normal stromal microenvironment at metastatic sites
EMT at the Primary Tumor Site:
●​ Tumor microenvironment signals (e.g., TGF-β, Wnts, TNF-α) activate EMT-inducing transcription factors
(Snail, Twist, ZEB).
●​ This induces a mesenchymal state, promoting invasion and intravasation into the bloodstream.
MET at Metastatic Sites:
●​ Once carcinoma cells reach a new (normal) microenvironment, they can revert to an epithelial state
(MET).
●​ This transition allows colonization and secondary tumor formation.

Lecture 5 – Bioinformatics in Oncology

Simplified model how cancer develops

1.​ Genetic and environmental factors
2.​ defects in DNA repair mechanisms
3.​ Accumulation of somatic genomic alterations
4.​ Altering gene expression levels or altered protein function
5.​ Tumor behavior and treatment response
Cancer immunity cycle → Utilising this cycle for creating rational
immunotherapy

Targeting the cancer immunity cycle

●​ Priming phase (LN): inhibition signals (antibody/CTLA-4)
○​ Costimulatory signal: B7 (DC) binds to CD28 (TC) →
activation.
 ○​ Inhibitory signal: CTLA-4 competes with CD28 for B7 binding, downregulating T cell activation.
○​ Checkpoint Blockade Therapy:
■​ Antibodies against CTLA-4 block this inhibitory
interaction, enhancing T cell activation.
■​ Example: Ipilimumab (anti-CTLA-4)
●​ Effector phase (peripheral tissues): negative regulation (PDL1/PD1)
○​ Inhibitory checkpoint: PD-1 (on T cells) interacts with PD-L1 (on
cancer cells), suppressing T cell activity and allowing immune
evasion.
○​ Checkpoint Blockade Therapy: Antibodies targeting PD-1 (e.g.,
Nivolumab, Pembrolizumab) or PD-L1 (e.g., Atezolizumab,
Durvalumab)prevent immune suppression and restore T
cell-mediated tumor killing.

Pathological complete responses with immune checkpoint inhibitors

●​ At day 84: tumor lesions grow in size → pseudo progression: increase of
lesion size related to treatment which simulates progressive disease (=
inflamed immune cells)
●​ At day 112: tumor lesions reduce
●​ Day 503: complete tumor regression

Cancer immune set point

●​ Is the threshold that must be exceeded in a patient to trigger an
anti-cancer immune response

Factors influencing the cancer immune setpoint

●​ Tumor mutational burden
●​ Protein expression of immune checkpoint (e.g. PD-L1)
●​ Composition of the tumor microenvironment, for example CD8+ T-cells
●​ mRNA signatures (mostly interferon gamma)

Tumor neoantigens: mutated DNA → tumor proteins → neoantigens (foreign protein recognised by APC)
Per patient, the mutational load could be different
Question: Would a high mutational burden be better for ICI (immunotherapy) response?
Answer: yes → more mutational burden → more tumor neoantigens → better immune response

1.​ Tumor mutational burden

●​ Certain cancers have higher tumor mutational burden such
as melanoma
●​ Immunotherapy works better with the cancers that has high
mutational burden → higher neoantigens

2.​ Somatic copy number alterations

●​ Mutations could happen as point mutations
 ●​ Or mutations could also be that a huge chunk of gene gets duplicated → whole chromosomes or smaller
portions of chromosomes → segments of DNA / gene amplified or deleted
●​ More genomic instability
Relations with ICI response
●​ Mutational load higher = higher response rate
●​ Copy number load higher = lower response rate

Question: Would a high copy number burden be better for ICI response?
Answer: no it wouldn't be better
→ How are copy number alterations (CNA) involved in the rewiring of the cancer-immune set point?

How does the somatic copy number alterations alter gene expression levels?

Expression profiles in the public domain

●​ In every year, there are new samples put to the
genomic profile
●​ Change in DNA can’t be related to the samples
●​ A lot of expression profiles but not paired to the
genomic profile

Example of an expression profile of complex biopsy

●​ Complex biopsy = diagnostic purposes → thick needle in tumor lesions → not only tumor cells in there,
cells from the immune microenvironment also present
●​ Looking at the gene expression profile of cancer samples allow us to figure out how the CNA affect gene
expression levels and how that is associated with response to immunotherapy over the cancer-immune
setpoint
Visualising the expression profile from biopsy
●​ Destroy cells in the complex biopsy → extract mRNA and measure → expression profile
●​ Each dot represents a gene, y axis = amount of expression, x axis = where these genes are mapped into
our genome from chrom 1 - 22
Limitation:
●​ Expression profile represent the average expression pattern of genes over all cells present in biopsy,
including experimental/batch effects (skin, lung, normal, TME, tumor cells, handling of biopsy can also
influence gene expression levels)
●​ Problem to solve: disentangle them → aim: exploring how the SCNA (somatic copy number alterations)
occur in tumor cells affect all kinds of gene expression levels & its effects on cancer-immune setpoint.
Specifically, to see the effect of SCNA downstream of expression and composition of TME → important
factor in determining response chance to immunotherapy
●​ Figure out SCNA, how they affect expression levels, and its relation to the TME

Effect of CNAs on gene expression levels is often overshadowed

●​ Cancer cell line = has for certain chromosomes (highlighted) an
extra copy
●​ Due to this extra copy, you'd expect the genes mapped there
would have a higher expression levels visible in expression
profiling but sadly not
 ●​ Reason: we imagine that the effect of CNA on gene expression level is overshadowed by other things
(other cells present in biopsy)
●​ Solution: expression profiles in the public domain from different repositories.

Computational biology/bioinformatics
●​ Pulling apart the average expression profiles in
separate expression patterns that can be associated
with cell types/biological processes/batch
effects/CNA
●​ Mathematical model to pull this complex biopsies
(avg expression profiles) in separate building blocks
●​ These building blocks = most genes are not expressed,
but at specific parts of the genome, a lot of genes are
either down/up regulated → these building blocks
represent the CNA occurring in the cancer
genome of that sample, and this is the effect of
the CN on the expression
●​ Signals found: at the genomic region, signals have
a really high rate
●​ The signals matched the copy number levels
●​ Showing the effect of copy number alterations

Transcriptional adaptation to copy number alterations

(TACNA)
●​ Identified building blocks → regenerate the
regional samples back again → give you
expression profile where only the downstream effect of the CNA is shown

Correlations between TACNA (downstream effect of CNA) and SNP data (measured
CNA)
→ Certain tumor types have would have a better correlation between downstream
effect of CNA gene expression profiling in TACNA and the measured copy number
alteration (SNP)
The expression profiling helps differentiate tumor types into 2:
1.​ Copy number driven (top)
○​ Ovarian, breast cancer etc are very copy number driven
○​ High correlation bn profiles & measured genomic data w/ copy
numbers → prototypical examples of cancers with a lot of CNA, in contrast to AML
2.​ Mutation driven (somatic point mutation) (bottom=blue)
○​ AML = they're not copy number driven, rather they are single nucleotide point mutations driven
○​ Low correlation bn CNA measured and profiling → due to AML having a limited number of SCNA,
AML is a mutation driven tumor type
○​ Not many copy numbers, but a lot of somatic point mutations

No perfect correlation between TACNA and SNP data

●​ Some genes aren’t influenced by CNA
 ●​ Some genes are highly influenced by CNA

Why are certain genes on certain CNA not highly expressed? While some are highly expressed?
Answer → Biology behind adaptation to CNA
●​ Other mechanisms in tumor cells that even if
there is a CNA, and the expression of that
gene mapping into that region should be
highly expressed, other cancer mechanisms
would make sure that it goes down → gene
suppression
○​ Those genes suppressed have to do
with mechanisms that can activate
the immune system (immune related gene sets)
○​ Genes on CNA that are highly influenced (CNA → exp levels go high) → genes involved in
proliferation & cell growth
●​ If the CNA is there, this tumor cell shouldnt activate the immune system bc it would destroy itself
●​ Whereas the proliferation-related gene sets are beneficial for the tumors, therefore they’d want to highly
express those
●​ Evolutionary process of the carcinogenesis = despite the CNA, other mechanisms will ensure that immune
activating signals will be suppressed, and gene related to proliferation won't be suppressed → hallmark of
cancer in immune evasion

Transcriptional adaptation to CNA = how is transcriptionally different genomic regions getting adapted to CNA
●​ Low degree of transcriptional adaptation = genes related to low CNA
●​ More copy number alterations = genes related to immune got a higher degree of transcriptional
adaptation

TACNA landscape in large set of cancers

●​ Inspecting the profiling which depicts the downstream effect of the CNA on the mRNA expression levels
●​ certain genomic areas are more affected than other ones
●​ specific tumor types have their own specific patterns

Transcriptional adaptation = when DNA mutations occur (CNA), either up or down, transcriptionally, genes aren't
translated in the transcriptome level

Change in copy number alterations = change in treatment

Infer composition of the tumor microenvironment

Correlation between TACNA signals and CD8 T-cell infiltration
●​ More copy number burden, there's less CD8 T cell infiltration
●​ Gene enrichment per cytoband on correlations between TACNA expression levels and CD8+ T cell
abundance
Translating to lab
●​ patients = diagnosed = certain threshold of anti-cancer immune response
●​ immunotherapy —> treat people above this threshold
●​ certain people exceed this threshold —> immune system activated
problem: there are people that are still unresponsive to immunotherapy (87%)
solution: give immunotherapy + drug targeting IRF2 (due to the association in IRF2 activation —> bc they lead to
less CD8 T cell infiltration
●​ this drug = suppress IRF2 in hopes that this mechanism is activated, more cD8 T cell infiltration —>
increased chance of immunotherapy response

Lecture 6 – Epigenetics

Def: the study of heritable changes in gene function that occur without a change in the sequence of the DNA
●​ Histone modification: acetylation, methylation, phosphorylation, ubiquitination, etc
●​ DNA methylation
●​ Nucleosome remodeling

DNA packaging – highly optimised

Every mitotic cell has a 23 pair chromosomes
●​ App 30k genes
●​ App 2m DNA per cell
Each individual consists of 10 billion cells
500k trips around the equator – 3.5 trips from the sun to pluto

I.​ Histone modification

●​ A combo of diff molecules can attach to the tails of proteins (histones) →
altering the activity of the DNA wrapped around them
●​ Nucleosome is consisting of 8 histones
●​ DNA wrapped around these histones
●​ Depending on the DNA modification
○​ you can have more open chromatin structure
○​ or more closed chromatin structure
●​ To make an inactive or closed chromatin structure, you need to make a
few complexes
PRC1 complexes and PRC2 core subunits: making chromatin more
inaccessible (epigenetic silencing)
●​ PRC1.1 is ubiquinating it
●​ PRC2.1 adds methyl groups to the H3K27 → trimethylated:
inactive mark of chromatin
●​ PRC2.2: ubiquitination from PCR1.1 then PRC2.2 can add
the methyl group

Function in Histone Modifications:

●​ H2A Ubiquitination: The primary function of PRC1,
mediated by RING1A/B, represses transcription.
●​ Histone Deacetylation: In ncPRC1.6, HDAC1/2 contributes to transcriptional repression by
removing acetyl groups from histones.
●​ Histone Methylation Cross-talk: PRC1 interacts with PRC2 (which methylates H3K27) through CBX
proteinsin cPRC1.
MECHANISM
PRC1 (Polycomb Repressive Complex 1)
●​ Function: Ubiquitinates H2A at K119 → represses transcription.
●​ Core subunits: RING1A/B, PCGF proteins.
●​ Types:
○​ Canonical PRC1 (cPRC1): Recognizes H3K27me3 (via CBX
proteins).
○​ Non-canonical PRC1 (ncPRC1): Independent of PRC2,
RYBP/YAF2 enhances H2A ubiquitination.
PRC2 (Polycomb Repressive Complex 2)
●​ Function: Methylates H3K27 (H3K27me3) → silences genes.
●​ Core subunits: EZH1/2 (methyltransferase), SUZ12, EED,
RbAp46/48.
●​ Types:
○​ PRC2.1: Contains PCL1-3, EPOP, PALI1/2 → regulates
PRC2 activity.
○​ PRC2.2: Includes JARID2, AEBP2 → enhances PRC2
recruitment.
How They Work Together
●​ PRC2 adds H3K27me3 → PRC1 binds via CBX → H2A
ubiquitination → chromatin compaction & gene silencing.
●​ PRC2 (H3K27me3) → PRC1 (H2AK119Ub) → Gene repression.
●​ ncPRC1 can repress genes independently of PRC2.

Methyl group added to a lysine

●​ Methyl group on K4 = open chromatin structure
●​ K9 & K27 = closed chromatin structure
Mechanism of methylation
●​ First there is a writer that adds the methyl group
●​ And then it is read by all different kinds of protein if the gene is turned off or on
○​ NURF: Nucleosome remodeling factor
○​ HMT: Histone methyltransferase (writers)
○​ HDMT: Histone demethylase (erasers)

Normal cell: You have the nucleosomes to which DNA are wrapped around →
activating histone modification → making gene accessible for transcription →
transcription factors can access the promoters

Tumor cell: Tumor suppressor genes have closed chromatin structure and you also
have DNA methylations in the CpG islands. Closed and compact cell = protected from
dangers from the outside

Repressive histone modification lead to closed chromatin structure, no

transcriptional activity

Nucleosome remodeling: the SWI/SNF remodeling complex

SWI/SNF: SWItch/ Sucrose Non-Fermentable remodels nucleosomes
2 complexes
1.​ BAF (SWI/SNF-A)
2.​ PBAF(SWI/SNF-B)
Each with their own unique complexes, but they share evolutionary conserved cores and
variant subunits
●​ Can either move away: the gene should not be active so there’s a nucleosome
→ not accessible
●​ Nucleosome remodeling refers to the process by which chromatin structure is
altered to regulate DNA accessibility for transcription, replication, and repair.
●​ The SWI/SNF remodeling complex is an ATP-dependent chromatin remodeler
that repositions, ejects, or modifies nucleosomes to facilitate gene expression.
●​ This complex plays a crucial role in transcriptional regulation by making specific
genes more accessible to transcription factors and RNA polymerase.
●​ It is also involved in DNA repair and cell differentiation, and mutations in its
components (mostly conserved cores or unique complexes) are linked to
various cancers.

DNA methylation occurs at the 5th position of a cytosine

●​ Cytosine that Precede Guanine can become methylated → called CpG dinucleotides
●​ Methylation is mediated by DNA methyltransferases (DNMTs)
○​ De novo methylation: DNMT-3a and DNMT-3b
○​ Maintenance: DNMT-1
●​ CpG dinucleotides often found in clusters = CpG islands
○​ A region w at least 200 base pairs
○​ G-C percentage of >50%
○​ CpG ratio Obs-Exp>0.6 (more CpG instead of GC)
●​ Promoter hypermethylation can silence genes
DNA replication and methylation
●​ Methylated CpG on both strands
●​ Replication: parents strands are methylated, whereas
the newly synthesized daughter strands are not.
●​ DNMT-1 → daughter strands are methylated → both
strands are then now methylated

Upregulation of DNMT-3b during Carcinogenesis

●​ DNMT-3b: De novo methylation
●​ During carcinogenesis: more malignant cancers → more DNMT-3b →
more de novo methylation occurring in promoter sites

Altered DNA methylation and chromatin status in cancer

In normal cells
●​ the CpG islands near gene promoters are unmethylated = more
open chromatin structure → accessible transcription start sites
for RNA polymerase. Important for housekeeping and TSG
 ●​ Rest of DNA = methylated = not on the C-G region = closed chromatin structure → important for DNA
stability
In cancer:
●​ DNA methylation of the promoter → histone modifications are in a closed chromatin structure → gene of
interest, mostly TSG, not accessible anymore → gene silencing
●​ the CpG islands are highly methylated
●​ Histone modification is closed
●​ Rest of the genome = no DNA methylation and histone modification is more open → translocation of DNA
during replication → more vulnerable for DNA breakage → can activate oncogenes and promote genomic
instability

DNA demethylation
●​ TET: Ten Eleven Translocation enzyme
●​ TDG: Thymine DNA Glycosylase
●​ BER: Base Excision Repair
Steps
1.​ TET enzymes (TET1/2/3)
○​ Oxidize 5mC → 5hmC → 5fC → 5caC using O₂ and α-KG.
2.​ TDG (Thymine DNA Glycosylase)
○​ Recognizes and removes 5fC/5caC, creating an abasic
site.
3.​ Base Excision Repair (BER)

🔹
○​ Fills the gap with unmethylated cytosine (C), completing demethylation.
TET oxidizes, TDG excises, BER restores cytosine.

Cancer epigenome
Mutations in
1.​ Histone (modifications and variants): MLL, EZH2, UTX
2.​ DNA methylation (TET2, IDH1, IDH2, DNMT3A)
3.​ Nucleosome (remodelling and positioning) = 5NF5, AR1D1A, PBRM1

Question: a mutation in TET2 results in more DNA methylation

TET2 is responsible for demethylation so a mutation in it would lead to no more demethylation →
more DNA methylation

Hypermethylation of gene promoters can be detected by PCR

●​ Methylation specific PCR: bisulfite treatment → unmethylated cytosine converted to uracil
→ primers detecting methylated DNA
●​ Differential DNA methylation in normal vs cancer cells:
○​ Hypermethylation in tumors silences tumor suppressor genes.
○​ DNA methylation changes can be used as biomarkers for cancer detection.
○​ Adjacent tissues may exhibit early methylation changes, indicating a pre-cancerous state.
●​ Prevalence of DNA methylation in tumors
○​ A lot of genes hypermethylated genes found in human tumor cell genomes
○​ Advantage for cancer cell → cancer is less responsive to chemotherapy due to better DNA repair
etc
 ○​ A lot of pathways affected by DNA methylation
○​ It is not a specific chr affected by DNA methylation → it occurs in all of the chr

Usage of epigenetics
A.​ Cervical cancer screening
I.​ Cytomorphological assessment of the cervix
●​ Different levels of Pap staining
●​ Higher than CIN2 → treat premalignant cancers to prevent cancer
II.​ DNA methylation as a diagnostic tool
Scraping from patients with an abnormal smear
The level of both genes is upregulated with the severity of the underlying region especially in the patients that
should not be referred to the hospital = methylation is 0
●​ Specificity is high
●​ Premalignant: 20-25%
●​ cancer lesions: can detect it all

B. Prognosis/prediction: methylated MGMT in glioblastomas (brain tumor)

●​ Normally: treated with radiotherapy
●​ Unmethylated MGMT = worse overall survival compared to patients that have methylated MGMT → if you
have unmethylated MGMT it can metabolise
●​ If you have methylated MGMT = no more MGMT gene = cant metabolise
●​ Methylated MGMT → better overall survival

C. Therapeutics:
1.​ DNA demethylation agents
Nucleoside analogue: 5-azacytidine, 5-aza2’-deoxycytidine
2 types of DNA demethylation agents:
a.​ Enzyme trapping: DNA replication needed
i.​ replication of tumor cell
ii.​ nucleoside analogue incorporated into DNA
iii.​ Incorporated at a site where the former
methyalted DNA was but now in daughter
strand
iv.​ DNMT trapped in cytosine, preventing them
from functioning
v.​ Cannot methylate the azacytidine
vi.​ Passively demethylated DNA
vii.​ Reactivates TSG
b.​ Enzyme blocking: don't need tumor cell replication
i.​ Non nucleoside analogue → wont be incorporated into DNA
ii.​ Go into pocket of the DNMT
iii.​ DNMT cannot methylate newly synthesized DNA anymore
iv.​ Gradual reactivating of genes

2.​ histone deacetylase inhibitors

 ●​ Normally when histones are deacetylated → more closed chromatin structure
●​ Now with the histone deacetylase inhibitors → more open chromatin structure
●​ Examples: vorinostat

3.​ PCR2 inhibitors

●​ EZH2 inhibitors (Tazemetostat) are clinically approved for cancers.
●​ EED inhibitors disrupt PRC2 complex formation.
●​ Protein degradation (PROTAC) approaches offer a long-lasting effect.
●​ Potential cancer therapy by reactivating tumor suppressor genes silenced by PRC2.

Effects of epigenetic therapy

●​ Less metastasis
●​ Decreases cell proliferation and survival
●​ More apoptosis or chemotherapy sensitivity
●​ More immune responsiveness
●​ Less chemoresistance
●​ Less stem like behaviour

Key concepts
●​ DNA promoter hypermethylation often results in transcriptional silencing.
●​ TSG´s often affected by epigenetic regulation.
●​ During carcinogenesis more DNA promoter hypermethylation of TSG’s.
●​ Histone modification/DNA methylation is reversible.
●​ DNA methylation can be used as a diagnostic marker.

Lecture 7 – Cell cycle

Cell cycle clock

●​ G1: cell grows
●​ Late G1 phase: R point → is the cell fit
enough to divide?
●​ S-phase: replication of genome
●​ G2: cell prepares to divide
●​ Mitosis: division of cell and genome

Mitosis
The primary purpose of a cell cycle: equal distribution of
genomic material between two daughter cells
●​ All the individual chromosomes aligned at metaphase
and anaphase they're being pulled away

Cell cycle: 3 outcomes

 1.​ Ideal: 2n → replication (4n) → mitosis (2 cells of 2n)
2.​ Aberrant: 2n→ replication (4n) → replication (8n) → mitosis (2 cells of 4n)
3.​ Aberrant: 2n → replication (4n) → mitosis (2 cells of 2n) →
mitosis (2 cells of 1n)

How cells maintain an ordered cell cycle

Cyclin: protein that goes up and down everytime cell divides →
regulatory subunit
Cdk: cyclin-dependent kinase → can only function if it is bound to cyclin
●​ Cyclin-Cdk prevents replication (S-phase)
●​ no Cyclin-Cdk = no mitosis

cyclin-Cdk regulation
1.​ Cyclin production: 2 neg phosphate groups inhibiting CDK
2.​ CDK phosphorylation: 1 positive phosphate added → activating it
3.​ Cyclin phosphorylation: 1 additional phosphate on cyclin
4.​ Nuclear import: taken to nucleus where cell division takes place
5.​ CDK dephosphorylation: 2 inhibitor phospho groups from (1) removed →
active cyclin CDK w/ 1 phospho group on each Cyclin & CDK
6.​ Inhibitor binding: in case of DNA damage → CKI(Cyclin kinase inhibitor)
7.​ Cyclin degradation
→ many levels of controls

Each phase of a cell cycle is characterized by a unique profile of CDK-cyclin activity

●​ G1 = cyclin D – CDK4/6
●​ Pass R point: cell starts to prepare for S phase = cyclin E – CDK2
●​ S phase = cyclin A - CDK2
●​ G2 = cyclin A – CDC2 (or CDK1)
●​ M = cyclin B – CDC2
Restriction-point (R-point)
●​ until the R-point, cells are sensitive to growth factors
●​ thereafter: ‘point of no return’
●​ first activation of Cdk4/6, then non-reversible commitment
●​ Cdk4/6 needs signals from the outside
●​ G1 until R point: period during which cells are responsive to mitogenic GFs and to TGF-b

Signaling from the outside

●​ Growth factors
●​ Extracellular matrix
●​ Cytokines

Signal from the outside world in this case GF will connect to receptor —> activating cascade of
events —> RAS, Raf —> Cyclin CDK complex that activates the G1

Cyclin D production is the key to activation of the cell cycle

After the R-point: the cell cycle is autonomous:
●​ Only Cdk4/6 is controlled by extracellular signals
●​ After that it is a cell autonomous program
●​ Chain of activating each other reactions
●​ Mainly via cyclin production

CKI proteins: CDK inhibitors

●​ INK4 proteins: inhibit Cdk4/Cdk6
●​ P21/p27/p57: can inhibit all other CDKs
●​ Inhibition occurs due to a steric hindrance

How does cyclin/CDKs start the cell cycle?

●​ Pass the R point, how does the cell cycle start?
●​ E2F is a family of TF
●​ They bind at the promoter region of genes required for cell division
●​ Multiple family members: 1,2,3 = activating; 4,5,6,7,8 = inhibitory
●​ In non-dividing cells, E2F transcription factors are bound by the Retinoblastoma proteins
(RB1) → brake for E2F
●​ E2Fs when bound to RB1 are inactive

Activation of E2F
1.​ RB1 becomes (hyper)phosphorylated by CDK4/6 and CDK2
2.​ Phosphorylated RB no longer binds E2Fs

RB1 phosphorylation during the cell cycle

●​ RB1 phosphorylation occurs from R point to end of mitosis
●​ Pass R point of no return: E2Fs TF activated producing other cyclin D and CDK
●​ where cells commit to cell cycle progression
RB also related to eye cancer: carrier of one mutation gives rise to increased cell growth

Amplification-loop at the start of the cell cycle

positive feedback loop, lil bit of activity of CDK4/6 phosphorylates Rb → E2F released → more cyclin E –
CDK2 → more phosphorylation of RB → making sure cells don't go back and don't return in the cell cycle

Oncogenes and tumor suppressors regulate the R-point

●​ Red: oncogenes → mitogens → pathways → cyclin D → inactivate Rb → R point transition
●​ Blue: TSG → CKI proteins: CDK inhibitors (p21, p15, p27) → block cell cycle by inhibiting CDK
Frequent amplifications of oncogenes involved in regulation of the R-point

Key points:
●​ Cell cycle is an ordered sequence of cell cycle phases
●​ It is controlled by Cyclin-CDK complexes
●​ Cyclin-CDKs themselves are also highly regulated
●​ Cyclin D initiates the cell cycle
●​ After the R-point, cell cycle is irreversible
 ●​ Retinoblastoma protein (RB1) ensures that the cell cycle does not start automatically
●​ Many oncogenes/tumor suppressors play a role in this pathway

How often can cells divide and which mechanisms control it?
Senescence
●​ keratinocyte layer (in purple) is thinner in an old skin
●​ keratinocyte stem cells have limited amount of divisions → gives rise in these cells that move up to the
outside of the skin
●​ When we age, cells lose the ability to proliferate

Number of cell divisions is limited

●​ After reaching this limit, cell division is inhibited (cell cycle arrest)
●​ This phenomenon: Senescence → eternal sleeping test

Senescence:
●​ You want to keep CDK down by inhibitors of CDK/cyclin kinases: P21 and p16 (CKI proteins)
●​ As cells age, CKI proteins increase

The amount of cell divisions can be affected by stress:

●​ DNA damage
●​ oxidative damage ( 20% oxygen vs. 3% oxygen)
●​ defects in DNA repair

Telomeres: determines the limited amount of cell divisions

●​ young: long telomeres
●​ telomeres shorten as cell divides
●​ CRISIS: signal that the rest cells
●​ GT-rich repeats: (TTAGGG)n
●​ conserved in all eukaryotes
●​ thousands of repeats
●​ become shorter per division
●​ too short: no chromosome protection

Why do telomeres become shorter per cell division?

→ the piece where primer is bound to cannot be reproduced
●​ replication of the ‘chromosome body’
●​ lagging strand needs a ‘primer’
●​ replication of telomere
●​ primer can’t bind at chromosome ends
●​ without a primer: no replication at final part of chromosome
DNA replication: DNA opens up
●​ leading strand: polymerase continuously prod DNA → some point,
●​ lagging strand does this in a discontinuous way, polymerase starts when DNA opens
up, then RNA primers need to be added constantly, done in batches.
 ○​ At some point, blue strand becomes too short → polymerase has no space to bind to DNA → last
piece of lagging strand cannot be duplicated –> shortened telomeres

Telomeres protect the ends of chromosomes

●​ Telomeres erosion: telomeres are critically short → removed
→ cells fuse them together (end-end fusion)
●​ Without telomeres: DNA ends are being repaired
●​ Abnormal structures
●​ Give rise to problems with cell division
If this happens repeatedly: fusion-breakage-fusion cycles
●​ Chromosomes with eroded telomeres fused together → this
will lead to breakage
●​ Break wont be exactly at the 2 telomeres used to be →
uneven breakage
●​ Different chromosome (non-homologous) could also be fused
to it
●​ Result: chromosomal deletions and amplifications

Telomeres are protected by creating a loop

●​ At the end of the telomere: final piece will go back to the
chromosome → loop → there’s no end
●​ Overhang of the telomere will have the G-rich DNA strand → fall
back into itself, pushes a
●​ ‘T-Loops’ which mask DNA termini
●​ Come up with crazy chromosome figures that prevent cell
division
2 types of loops:
a.​ D (displacement loop): the overhang part of the G rich strand
b.​ T loop: G rich strand plus C rich strand

Different proteins are needed to fold telomeres into Loops

●​ These proteins recognize and bind to telomere
repeats

In normal cells, telomeres get shorter per division and cells

cease to divide → form a loop
→ but then why don't cancer cells stop dividing?????

Telomerase enzyme (TERT)

●​ Normally needed in stem cells: they need to
continuously have telomeres (skin etc)
●​ Can extend the length of telomeres
●​ Consists of a DNA polymerase and the RNA template
●​ Can add telomere repeats to the DNA end
●​ Brings its own RNA primer
→ this is why cancer cells don't stop dividing despite the telomeres shortening.
→ Tumors: expression of hTert (80-90% cancers) confers unlimited growth
→ potential therapy for cancer??

Key points
●​ Telomeres become shorter with each division cycle
●​ Too short telomeres inhibit cell growth
●​ Telomeres are being folded into ‘T-Loops’
●​ Tumors can endlessly divide thanks to a high expression of telomerase

Genomic integrity
DNA damage and cancer:
1.​ DNA damage causes cancer
a.​ Epidemiology
b.​ model systems
2.​ DNA damage is used to treat cancer
a.​ Chemotherapy
b.​ radiotherapy
3.​ DNA damage in healthy tissue is responsible for side effects of cancer
therapy
a.​ Malnutrition
b.​ immune failure

Chemically-induced DNA damage

Carcinogens
1.​ Genotoxic
a.​ Direct acting carcinogens
i.​ dimethyl sulfate
ii.​ cycophosphamide
b.​ Indirect acting carcinogens
i.​ polycyclic aromatic
hydrocarbons → present in cigs
ii.​ nitrosamine

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
iii.​ insecticides
2.​ Non-genotoxic

Cigarette smoke contains >60 carcinogens

●​ Many are genotoxic
●​ Smoking accounts for 80-90% of all lung cancer cases

How do we keep our DNA healthy?

●​ Organization of our body
●​ DNA repair
 1.​ Through organization of our body:
a.​ Stem cells
i.​ keep these cells as far away from danger as possible
ii.​ occasionally divide
iii.​ distant from possibly toxic agents
b.​ Transit-amplifying cells
i.​ frequently divide
c.​ Differentiated cells
i.​ do not divide
ii.​ in contact with toxic agents
Example: cells in the gut
●​ The stem cells are as far away from the lumen of the intestine as possible
●​ Villus is in contact with food
●​ Stem cells in the bottom give rise to transit amplifying cell and thereby differentiated villi

2.​ DNA repair

For every type of DNA damage, there is an appropriate DNA repair
pathway
a.​ DNA ss break → base-excision repair
b.​ Replication error → mismatch repair
c.​ DNA ds break → recombination, end joining
d.​ Bulky adducts → nucleotide excision repair

I.​ Mismatch repair

●​ During duplication: wrong nucleotide
Repair: DNA polymerase can proofread
not repairing gives 1 daughter cell w mutation
repairing it without knowing which nucleotide is the
mutated one gives rise to 2 daughter cells with
mutations
●​ But sometimes it is not enough → mismatch

Scenario 1: mismatch, unrepaired

●​ At the next DNA replication
○​ Template strand: Normal
sequence same with original
○​ New strand with the
mismatched nucleotide:
mutated daughter cell
Scenario 2: mismatch, repaired in template
strand
At the next DNA replication
●​ Template strand: mutation
●​ New strand: mutation
Question: how to detect the mismatch in the newly synthesized DNA strand?
1.​ Nick is used to identify the new strand. Nicks are harmless and will be filled up
eventually
2.​ Bases around mismatch are removed from new strand
3.​ Gap in new strand is polymerized
Replication error frequency reduced from 1:107 to 1:109

II. Non-homologous end joining (NHEJ)

●​ Error prone
●​ Occurs during the G1 phase of cell cycle
●​ In G1 cell: diploid cell (for each chr, 2 diff copies)
●​ Copies are at diff sides of the nucleus
●​ In S: replication
●​ G2: exact copies of chromosomes attached at 1 centromere → sister chromatids
●​ Mitosis: separation of chromosomes
●​ G1 and G2 cells are completely different → in the opoortunies they offer for DNA repair

DNA break repair by end-joining (NHEJ)

DSB: 2 BP overhang, and only 1 on the other break
1.​ Cleaning ends: removal of phosphate
2.​ Base removal
3.​ End joining
The repair is:
●​ Non-homologous: It doesn't use any other DNA sequence (no template
used)
●​ Non conservative: end product is not the same as starting product
●​ With Base loss (mutation)

III. Homologous recombination

A perfect copy of the exact same DNA molecule
●​ Alignment of DNA with newly synthesized sister chromatid
●​ Broken DNA needs to ‘pair’ with intact DNA
Challenge: DNA info is not on
the outside. Real information is
on the inside of the double
helix. You need to somehow
make that the DNA break interacts and reads what the DNA sequence is on the
inside. We need to then modify the DNA break. Enzymes nucleases eat DNA and
they chop up the DNA from 5’ to 3’ direction and this can be done on both sides of
the DNA break. → single strand overhangs → can read the nucleotides on the base
sequences. This needs to be protected
●​ Using a template: make sure that the blue one is repaired, that we use the
green template as reference
1.​ Nucleases chop away one of the 2 strands, done in both ends of DNA
breaks (5-3)
2.​ Helicase opens up the green strand
 3.​ RPA binds to blue DNA → Invasion into intact homologue and subsequent (semi) conservative repair
4.​ RAD51 recombinase: find homologous sequence
5.​ Polymerase invades and produces new DNA
Error free

What happens if DNA cant be repaired → accumulate DNA mutations

ATM: master regulator

●​ Make sure cell stop dividing: cel cycle arrest (p21: CDK inhibitor)
●​ DNA repair: BRCA1/2
●​ Apoptosis: puma, noxa, p53
I.​ ATM mutation: Ataxia Telangiectasia (A-T)
(Border-Sedgwick syndrome or Louis-Bar syndrome)
Features:
●​ both copies of the ATM gene are mutated
●​ rare disease (1:40.000-1:100.000)
●​ progressive difficulty with coordination of movement (Ataxia) and clusters of enlarged blood vessels
(Telangiectasia)
●​ Radiosensitivity
●​ increased risk of cancer (40%) (mostly lymphoma/leukemia)
NB: also in non-hereditary tumors
→ indicating that if you dont have ATM protein → chance of getting cancer is high
II.​ Mre11 mutation: Ataxia Telangiectasia-like disease (ATLD)
Characteristics:
●​ similar to AT, but milder
●​ both copies of Mre11 mutated
●​ 30% of patients develop cancer
●​ radiosensitivity
III.​ NBS1 mutation: Nijmegen Breakage syndrome
Characteristics:
●​ both copies of NBS1 are mutated
●​ microcephaly/distinct facial appearance
●​ strong tendency to develop lymphoma
●​ Radiosensitivity
IV.​ BRCA1/BRCA2 mutations: hereditary early onset breast/ovarian cancer
Characteristics:
●​ only one copy of BRCA1/BRCA2 is mutated in affected individuals
●​ repair through HR is defective
●​ tumors have lost normal/wildtype BRCA1/2 allele
●​ high chance of cancer development
if cell loses one allele, it will accumulate mutations, as it does not have the repair mechanism

Shared characteristics
●​ Extremely sensitive to irradiation
●​ Predisposition to various cancer types
●​ illustrates the importance for DNA maintenance to keep healthy
Dependency on the tissue type and the type of cancer that rises to it
●​ Cell needs DNA repair, otherwise DNA damage → onset of tumor growth
●​ Mutation in one of these repair pathways → specific cancer

Defects in tumors → give therapeutic opportunities

●​ Table with one broken leg → useless
●​ The broken leg Eg. defect in homologous recombination → BRCA1 mutation
●​ These tumors become very dependent on BER, since BRCA1 doesnt work anymore

Single stranded DNA break: BER(BASE EXCISION REPAIR)

●​ These tumors dependent on BER
●​ PARP1: puts ribose sugar on DNA, mark it for repair
●​ BER (Base-excision repair) requires Parp1 → puts a lot of ribose rings on
top of the the broken DNA → forming christmas tree of sugar molecules
that can recruit this single stranded DNA break
Base-Excision Repair (BER) and PARP1:
●​ PARP1 is essential for the (BER) pathway, which fixes single-strand DNA
breaks (SSBs).
●​ If BER is functional, SSBs are efficiently repaired, maintaining genomic
integrity.

PARP1 inhibitors→ in dividing cells, Parp1 inhibition leads to DNA DSBs →

repair can’t happen. Single stranded break remains → DNA replicated → single
stranded break converted into double stranded break. This is not necessarily bad
for cells if we have pathways required to fix this.
●​ In BRCA1 mutation carriers, inhibition of PARP makes BER inactive, and
BRCA1 mutation carrier can't repair the DSB from the pARP inhibition
●​ Parp1 inhibition is very toxic in BRCA1 or BRCA2 mutant tumors
●​ 2 defects together make a lethal event
PARP Inhibition and Genomic Instability:
●​ consequence of PARP inhibition: unrepaired SSBs persist, and during DNA replication, these SSBs convert
into double-strand breaks (DSBs).
●​ DSBs are more severe and require repair via homologous recombination (HR).
Relation to Synthetic Lethality:
●​ Synthetic lethality occurs when the simultaneous loss of two genes or pathways leads to cell death,
whereas the loss of just one does not.
●​ Cancer therapy using PARP inhibitors exploits this concept:
○​ BRCA1/BRCA2-deficient cancer cells already have defective homologous recombination repair
(HRR), making them highly dependent on BER and PARP1 for DNA repair.
○​ PARP inhibitors block BER, leading to accumulation of DNA breaks and genomic instability,
ultimately killing BRCA-deficient cancer cells.
○​ Normal cells with functional HRR survive because they can still repair DNA damage.
Key Takeaway: PARP inhibitors are used in cancer therapy (e.g., in BRCA-mutated cancers) to induce synthetic
lethality by blocking the repair of DNA damage, leading to selective tumor cell death while sparing normal cells.
Key points:
●​ DNA is kept healthy by the organization of our body …and by DNA repair
●​ Different types of DNA damage have distinct type of repair mechanism
●​ Sometimes error-free (HR), sometimes with errors (NHEJ)
●​ Defects in DNA repair increase the risk of cancer
●​ And offers opportunities for targeted treatment of cancer

Lecture 8 – Tumor Immunology and Immunotherapy

How does a T cell know how to attack?

Antigens and presentation by cells of the body
●​ Ongoing process of proteins generation and breaking down
●​ Proteins loaded in proteasome → broken down into peptides →
ER
●​ Peptides presented on MHC on APC → recognised by TCR
●​ Self proteins are constantly being presented on APC for
recognition by T cells → T cells ignore (tolerance)

Diversity of the T cell receptor repertoire = random

●​ TCR forms a perfectly complementary structure to allow it to recognise MHC and specific peptide
●​ TCR generation is random
●​ TCR genome: alpha and beta chain → random reshuffling of genome +
different combo of different alpha & beta chain
●​ Incredibly diverse
●​ During the maturation of T cells from hematopoietic stem cells, these
gene segments are randomly rearranged → unique TCR
●​ Sequence of TCR determine which peptide
it recognises

Neo-antigens and presentation by cancer cells

●​ Mutation associated neo peptide
sequence: Mutations on cancer cell on
DNA level → causes a change in the
protein structure
●​ Normally T cell ignores normal proteins
●​ But they will respond to peptides derived
from mutation (neoantigens)

What if T cells recognise the wrong target? → Autoimmune diseases

How do T cells kill their target?

2 ways:
A.​ Lysis: T cells polarise lytic granules in cancer cells → from cytochrome
of T cells into the cancer cells → making holes in cancer cells →
release of lytics granules
 B.​ Apoptosis:
a.​ T cell contact with target cell
b.​ Fas/FasL
c.​ FADD forming death domains
d.​ Pro caspase 8
e.​ Pro caspase 3: active caspase 3 → scissors
f.​ Apoptosis

The immune cancer arms race

●​ Cancer cells try to evade this process
●​ Subclones develop in any cancer overtime
●​ Tumor cells develop → second mutation → second tumor populations occur
●​ Different clones with different mutations in a single tumor
●​ Some mutations maybe shared initially, but the mutations occurring later on will be
different

This difference in mutation can be visualised in a tree

●​ Trunk mutations: occur early on, found in each and every cancer cell
●​ Branches: clonal trees that exist in individual clone
Contributes to diversity in mutations of different clonal groups
Ubiquitous → shared → private

Post therapy/immune response, most clones die off.

●​ Any clone that develops resistance will survive this therapy / immune response and
restart this process again

3 phases
1.​ Elimination phase: immune system responds
2.​ Equilibrium phase: tumor cells with mutations
that allow them to survive this initial killing →
preventing the tumor from growing further but
also preventing the immune cells from killing it
3.​ Escape phase

Evidence supporting immune:cancer arms race

I.​ Immune mediated rejection/selection of tumors in
RAG2-/- MICE
●​ Wild type: normal mice
●​ Transgenic mice: lost RAG2-/- protein
RAG2 → essential for genetic recombination that creates TCR.
RAG2 absent: no TCR
Treating RAG2 absent mice with carcinogen:
●​ Wild Type mice: only a couple of them generate tumor
→ weakly immunogenic
●​ T cell deficient mice: all of them develop tumor (both
strongly and weakly immunogenic)
Transplant tumors
●​ Taking strongly immunogenic tumors from RAG-/- mice → WT mice rejects them: bc tumors were formed
in mice without T cell response, and WT mice has a functioning immune system
●​ Taking weakly immunogenic tumors → WT mice develops tumor
Takeaway: Only tumors that form in the presence of immune selection pressure can reform in immunocompetent
mice. immune system actively sculpts tumor characteristics, favoring the survival of less immunogenic tumors.
This is a key concept in cancer immunoediting and has direct implications for immunotherapy design.
→ T cells are crucial for the rejection

II.​ Immune mediated rejection of tumors

(antigen-mediated rejection)
●​ 2 different kinds of tumors injected into mice: CMS5 and
MethA
●​ immunized mouse with irradiated tumor cells, lets say
MethA
●​ Immune system recognise this
●​ Then inject the same tumor cell line, MethA, the host
will reject this
●​ If we inject blue tumor, CMS5, tumor grows
Key takeaway: Tumor rejection is antigen-specific:
●​ The immune system only eliminates tumors expressing
recognized antigens.
●​ If the tumor expresses a different antigen, immune escape occurs, leading to tumor growth.

Isolate T cells from mice that rejected this tumor cells (immunised mice) → 24D3
Tumor specific antigens (TSA): highly immunogenic, only found in cancer cells
tumor associated antigens (TAA): weakly immunogenic, found in both cancer and normal cells
●​ T cells only respond to the tumor that it was initially raised against (MethA)
●​ Only giving the mice clone specific T cells is enough to suppress tumor progression
●​ T cell response is critical for tumor rejection and for forming memory t cells that prevent further
progression of tumor
2 types of tumors
1.​ Weakly immunogenic: tumor cells too similar to self antigens → cancer cells can evade immune detection
2.​ Strongly immunogenic: amount of mutations fairly high, a lot of neoantigens presented → T cells
effectively cancer cells

T cells discriminate cancer cells from 'normal' cells by:

a.​ Recognizing changes in the levels of cell surface antigen-presenting molecules on cancer cells
b.​ Recognizing mutation-induced changes in the amino acid sequence of proteins
c.​ Changing their T cell receptor to recognize mutations in cancer cells
d.​ Recognizing self-antigens underexpressed by cancer cells
Note: Level of expression is not that important, mutation induced chances in the AA sequence is more important

T cell receptors are:

a.​ Generated through mRNA splicing of receptor transcripts
b.​ Produced when a T cell recognizes a cancer cell
 c.​ Produced through genomic recombination during T cell development
d.​ Only capable of recognizing non-self antigens

Why is cancer not contagious?

MHC-I and MHC-II (HLA) diversity
●​ Is different in everyone
●​ MHC-I: 6 HLA alleles per person (HLA-A/B/C)
●​ Peptide: 6-12 AA long from any protein
→ T cell recognises not only the peptide, but also a specific MHC
It doesn't have anything to do with the antigens, simply the difference in
antigen presenting cells will cause rejection in tumor transplantations

Rejection of HLA mismatched tumors

●​ 2 diff mice species: BALB/c and C57BL/6
●​ Induce tumors in these different strains of mice
●​ And then transplant these tumors (strain specific MHC-I molecules)
●​ BALB/c tumors only grow in another BALB/c mice
●​ C57BL/6 mice only grow in another C57BL/6 mice
○​ Tumors only grow in syngeneic hosts (same genetic
background) → because the immune system recognizes the
tumors as "self." → immune evasion
○​ No tumors in allogeneic host (diff genetic background) → Tumors are rejected in allogeneic
hosts because of MHC class I mismatching, which triggers an immune response against foreign
MHC molecules

When can cancer be contagious?

Immunosuppressed individuals: cancer can be contagious

Loss of antigen presenting molecules:

●​ If the cells lack antigen presentation → no HLA molecules → T cells cant recognise the antigen
●​ There is also a transmissible cancer even with presence of antigen presenting molecules. Unknown reason

Does the immune system stop tumours from forming in humans?

Immune mediated suppression of tumors in humans
●​ For cancers that are caused by infectious agents, as well as non viral cancers, there is an increased risk
●​ Immune system controls the formation of cancers

Does the immune system only control cancers related to infectious diseases?
Immune mediated suppression of tumors (T cell pressure)
●​ T cells are key for cancers also unrelated to infectious diseases
●​ Simple surgery: TILs help cancer suppression

Neo-antigens and presentation by cancer cells

●​ Depending on what type of tumor you have, you’ll also have different mutations
●​ Tumors with defective dna repair very often accumulate way more mutations
●​ More mutations you have, stronger immune response
 ●​ Very paradoxically, from the immune perspective, if tumor cant fix its dna, it will accumulate more
mutations which triggers stronger t cell response, patients survive disease
●​ Tumor triggers such a strong immune response, that removing the mass is enough
●​ Total survival of a patient is 15% determined by immune response, on top of the tumor types, and tumor
cells
●​ Patients with a tumor that has a lot of mutations appear to do better (when treated obv)
●​ Total number of mutations make a difference

Tumors form anywhere

If immune response is enough, why do these patients develop tumors in the first place? Loss of antigen
presentation

Resistance mechanisms
Loss of antigen presentation
●​ If you treat patients with chemotherapy, they use antigen presentation
●​ So you're killing cancer cells with chemotherapy
●​ But the cancer cells that are left, have lost antigen presentation
●​ The chemotherapy makes the cells more sensitive to the immune system
●​ After chemotherapy you're left with cells that don't have antigen presentation
●​ Chemotherapy: selection pressure
●​ What's not recognised is not removed
●​ Remaining cells cant be recognised by the immune system = so whether or not u have t cells it doesn't
matter anymore
●​ This poses a problem to treatments = everytime you treat a clonal mutations, there are more that arises

Immune evasion by checkpoint molecules

1.​ First checkpoint: CD80/86-CTLA4 inhibits T cell from becoming
active (at lymph node: when DC interacts w T cell)
2.​ Second checkpoint: PDL1-PD1 also inhibits T cell (T cell gets
into tumor - at peripheral tissues)
anti-CTLA4 and anti-PD1 → checkpoint inhibitors that induce immune
response against cancer

In addition to this, combining checkpoint inhibitors and

immunosuppressants
●​ Number of mutations matter, as well as the quality of the
mutations
●​ Mutatogram: x axis = cancer types, y axis = mutation rate
●​ Melanoma, lung, stomach cancers = exposed to external
carcinogenic factors → high mutation rate

Heterogeneity of neoantigens
Subclones in tumor mutation types
●​ Immune response against truncal mutations is better than immune response
against private mutations / shared mutations
 ●​ The patients that do well not necessarily have more mutations, but they have more clonal mutations than
subclonal mutations
Quality over quantity
●​ More heterogeneity of neoantigens = less effective treatment

Pathologic response: in NICHE-2

●​ Colorectal cancer
●​ Regression of tumor: 98% of 111 patients in efficacy analysis
○​ Major pathologic response: 95%
○​ Pathologic complete response: 68%
●​ 3yrs: 100% DFS
●​ Monotherapy: also tumor regression

How can cancers evade immune response / immunotherapy?

a.​ By mutating and losing targeted neoantigens
Which of the following factors determine how well tumors
respond to immune checkpoint inhibitors?
a.​ Immunogenicity of neo-antigenic epitope
b.​ PDL-1 expression
c.​ Mutational burden and clonal diversity of tumors
d.​ All of the above
When does cancer become contagious
●​ Immunosuppressed
●​ Loss of antigen presentation cell

Take home messages

●​ How do T cells recognise their target?
○​ Antigen presentation
○​ TCR
●​ How does the immune system distinguish cancer from healthy tissue? → mutated neoantigens
○​ NK cells detect lack of MHC-I → kill cells
○​ TSA → activate C8 T cell killing
●​ How does cancer evade immune destruction?
○​ Downregulation of antigen presentation (loss of MHC-I and/or mutated neoantigens)
○​ Immune checkpoint activation (overexpression of PDL-1 and CTLA-4)
○​ T cell exhaustion
○​ Immunosuppressive TME (recruitment of suppressive cells)
●​ How can we target cancer immune-evasion in patients?
1.​ Checkpoint Inhibitors (Reactivating T Cells)
●​ Anti-PD-1/PD-L1 (e.g., Pembrolizumab, Nivolumab): Block PD-1 interactions, reactivating exhausted T cells.
●​ Anti-CTLA-4 (e.g., Ipilimumab): Prevents CTLA-4 from inhibiting T cell activation.
2.​ Adoptive T cell therapy
●​ CAR-T cells: engineered T cells to kill tumor antigens
●​ TIL therapy
3.​ Cancer vaccines
●​ Neo antigen vaccines: patient specific tumor antigens to train the immune system
 4.​ TME modulation

Lecture 9 – Noncoding RNA

The central dogma

●​ DNA → RNA → Protein
●​ Replication → transcription → translation

Human genome contains about 20k protein coding genes

The G-value paradox

●​ Coding sequences don't reflect complexity of
organism
●​ If we are so complex, why do we have so little
protein coding genes? (<2% of RNA)

The noncoding genome

Noncoding sequences correlate with complexity of organisms

Noncoding RNA: miRNA, lncRNA, circRNA

microRNA
I.​ miRNA biogenesis
●​ Characteristics
○​ 22 nucleotide single stranded RNA
○​ 2800 known human
○​ Highly conserved
●​ Biogenesis
○​ Transcribed by RNA-polymerase II
○​ Processed from stem loop like structure (secondary structure) ie primary
microRNA transcripts
○​ 2 enzymatic steps: from pri to premiRNA, and from pre to mature
miRNAs
■​ Drosha = in nucleus → cutting away the flanking sequences →
pri-miRNA → pre-miRNA
■​ Dicer = in cytoplasm→ one strand is neglected, and one is selected → mature miRNA
○​ Loading into the RNA induced silencing complex (RISC)
●​ Function: inhibit protein translation

II.​ Genomic organisation of microRNAs

●​ Coding gene
○​ Intronic
○​ Exonic
●​ Noncoding
○​ Intronic
 ○​ Exonic
Monocistronic: one longer transcript encodes for one standard RNA (one single stem loop segment)
Poly-cystronic: one transcript multiple precursors contained → efficient bc one rNA codes for multiple microRNAs
(multiple stem loop segments) (e.g.m miR-17-92 → oncogenic)

III.​ Sequence homology drivers miRNA target gene recognition

RNA induced silencing complex: RISC
●​ miRNA is the guide
●​ Binds RNA transcripts based on partial sequence
complementarity
●​ Usually in 3’-UTR (miRNA responsive element(MRE)) = high
complementarity region
Target recognition
●​ Nucleotide 2-8: seed sequence
○​ High homology to its targets
○​ Limited sequence homology in other part
One microRNA binding to multiple target genes → specificity is determined by only a few nucleotides

IV.​ Flanking sequences affect binding efficiency

●​ Calculate the dissociation constant for a lot of microRNAs
present in human genome
●​ Calculate the binding energy and associate it with dissociation
energy
●​ Competition in the cell to which type of sequence it will bind
●​ And the energy needed for each transcript is different
●​ Abundance also matters
Flanking sequences (extra nucleotides around a miRNA binding site)
affect how strongly miRNA binds to mRNA. Some sequences increase
binding efficiency (high occupancy), leading to stronger gene regulation, while others reduce binding (low
occupancy). Even with the same core binding site, surrounding nucleotides can make a big difference in miRNA
function.

V.​ miRNA nomenclature

●​ Sequential numbering (miR-1. miR-2, …)
○​ Exceptions:
■​ Homologous miRNAs in diff organisms get the same number
■​ Let-7 and Lin-4 families
●​ Addition of -1 or -2 to the miRNA number refers to diff genomic loci that encode mature mirNAs with
identical sequence
●​ Addition of a, b, c, d etc is used for miRNAs w the same seed sequence but have at least 1 nucleotide diff
in the other regions
●​ miRNAs derived from one stem-loop fragment
○​ -5p or -3p to indicate the least abundant strand

VI.​ Complex microRNA – target gene interactions

30% if all genes are regulated by miRNAs
 ●​ Each mirNA has multiple cell type specific target genes
●​ Target genes can b e targeted buy multiple miRNAs
Complex interaction

VII.​ Competing endogenous (ce)RNA networks define miRNA:

target gene interactions
●​ Regulation of target genes
●​ The cell itself is dependent on the endogenous level of all
the transcripts that can potentially interact with the
miRNA
●​ miRNA Regulation
○​ miRNAs bind to mRNAs at specific sites (MRE –
miRNA response elements) in the 3' UTR to
suppress gene expression.
○​ A pool of miRNAs is available to bind to different RNA molecules.
●​ Competing Endogenous RNAs (ceRNAs)
○​ Other non-coding RNAs (like lncRNAs, pseudogenes, and circRNAs) can also bind to miRNAs,
reducing their availability to bind and suppress mRNA.
○​ This competition influences gene expression by "sponging" miRNAs away from their target
mRNAs.
●​ Effect on Gene Expression
○​ If ceRNAs bind more miRNAs, less repression of mRNA occurs → higher gene expression.
○​ If there are fewer ceRNAs, more miRNAs can bind mRNA → lower gene expression.
Note: ceRNAs compete for miRNAs, affecting how much miRNA is available to regulate target genes. This
competition plays a crucial role in gene expression and cellular processes.

Regulation of target gene mRNA determined by:

●​ #quality of binding sites
●​ Availability of binding sites
●​ miRNA levels
●​ RNA levels

Long noncoding RNAs

●​ Transcribed by rNA polymerase II (like protein coding genes)
●​ >200 nt long
●​ Lack protein coding potential
●​ >100k know in in the human genome
●​ Conservation
○​ Limited conservation in transcript
○​ High positional conservation: if there's a lncRNA close to TP53 in human, then there's also a
lncRNA close to TP53 in mouse
 ○​ High conservation in promoter regions: different from miRNA

I.​ How do lncRNAs exert their

functions?
Bind to
●​ RNA:RNA homoduplex
●​ RNA-DNA heteroduplex
●​ RNA:DNA:DNA triplex
●​ Chromatin binding
Serve as
●​ Scaffold: attaches to other molecules that aren’t together
●​ Decoy: catch away some proteins/RNA molecules from their
normal functions
●​ Signaling molecules
●​ Guiding molecules
→ depends on where they are in the cell

II.​ Regulatory roles of nuclear and cytoplasmic lncRNAs

●​ In nucleus:
○​ regulation conformation of chromosomes,
○​ transcription regulation,
○​ recruitment of chromatin modifier
●​ In cytoplasm:
○​ modulate splicing,
○​ mRNA degradation,
○​ translation inhibition,
○​ microRNA sponge

III.​ Cis and trans-acting nucleus lncRNAs

●​ Trans-acting: lncRNA located at one locus of a genome → acting at one or
multiple distant loci
●​ Cis-acting: lncRNA located at a locus → acting at a neighbouring locus

IV.​ lncRNA nomenclature

●​ Position with respect to protein coding gene
○​ Exonic: lncRNA overlapping with the
protein coding genes
○​ Intronic: not overlapping with exons,
but w introns
○​ Intergenic: in between 2 protein coding genes
○​ Antisense: lncRNA gene transcribed from the other strand than the protein codon gene
○​ Overlapping: in the neighbourhood of the gene, but its not fitting the others
●​ Long intergenic noncoding (linc)RNA with numerical suffix

Circular RNAs
I.​ Circular RNA biogenesis
 ●​ Formed by back splicing of linear coding and noncoding transcripts
●​ 3 main types: circular exonic RNAs (ecircRNA), circular intronic RNA (ciRNA), and exon-intron circRNA
(EIciRNA)
●​ Backspliced fusion parts is unique: unique regions can bind proteins or encode for proteins
●​ BACKSPLICING:
○​ the end of exon 2 ligated to beginning of exon 2
○​ Exon 2 and 3 ligated together
●​ Normally the end of exon 1 is spliced and linked to the beginning of
exon 2, intron is cleaved, the end of exon 2 is linked to beginning of
exon 3

II.​ Circular RNA characteristics

●​ Mainly derived from protein coding exons (pre-mRNA) → UNLIKE
miRNA and lncRNA
○​ 1 up to 8 exons
○​ Most circles consist of 2 exons
●​ 300-2000 nt long
●​ Tissue specific expression

III.​ Circular RNA: diverse regulatory functions

1.​ miRNA sponges or decoys: circle RNAs are very stable bc RNA degradation enzymes usually start from the
ends. So its circularity protects it from RNA degradation enzymes. circRNA bind to miRNA → gene
translation.
2.​ Splicing and transcription: be part of the transcription machinery and serve as scaffold to bring other
proteins to start transcription. Competition between linear and circular transcripts
3.​ Protein scaffolding
4.​ Protein sponges or decoys
5.​ Translation: templates for short proteins

Characteristics of different classis of ncRNAs

Conclusion
●​ Noncoding genome correlates with
complexity of an organism
●​ >50% of the noncoding genome is
transcribed into noncoding RNAs
●​ 3 major classes of regulatory ncRNAs:
miRNA, lncRNA, circRNA
●​ Noncoding RNA play regulatory roles at
different levels via a variety of different
mechanisms and play crucial regulatory roles in almost all cellular processes

Elucidating the role of ncRNAs in cancer pathogenesis

●​ miRNAs peak 22k
●​ lncRNAs peak 10k
●​ circRNAs peak 3k
Evidence supporting a role in oncogenesis
Based on expression profiling studies (using microarrays, small RNA seq, RNA in situ hybridization)
●​ Altered expression compared to normal counterparts
●​ Cancer type specific expression profiles

Altered expression levels in cancer cells

I.​ Specific microRNA expression patterns in cancer
●​ Each Malignancy has its own specific pattern
●​ Unique patterns: highly expressed in breast cancer, intermediate in colon and lung, but really low in
pancreas and stomach
●​ It is not just background noise → functionally involved
II.​ Tumor cell specific lncRNA (3 diff lncRNAs) expression in Hodgkin lymphoma
III.​ Cancer type specific expression patterns of circRNA
●​ RNA seq
●​ Compare number of circRNA in different types of breast cancer eg LA vs TNBC
●​ Some circRNAs upregulated, some downregulated

Genomic aberrations at ncRNA loci

●​ CNA = copy number alterations
●​ Structural aberrations
I.​ Chromosomal aberrations resulting in altered miRNA expression
levels
●​ Burkitt lymphoma: translocation → MYC overexpression →
miR-17-92 increased
●​ Other lymphoma: amplification → miR-19-92 increased
●​ Chronic lymphocytic leukemia: deletion → miR-15 & miR-16
decreased
●​ miR-19-92: oncogenic, polycistronic

II.​ Noncoding RNAs at the MYC locus

●​ Neighbouring MYC’s locus = PVT1 (lncRNA)
○​ Stem loops that read codes for several miRNAs
○​ Many different PVT1 isoforms.
●​ Chromosomal translocation: when occurring here → linked to MYC,
or may also target PVT1
●​ Viral integration
●​ Chromosomal deletion
●​ Amplification
→ at this lncRNA locus (PVT1) which also contains a lot of miRNA, there's a lot of chromosomal aberrations
observed in cancer → suggesting that these ncRNA may also be the target of chromosomal aberrations

III.​ Oncogenic role of fusion-circRNAs derived from cancer associated chromosomal translocations
●​ Promote transformation and cell survival
●​ Tumor promoting properties in in vivo models
●​ The fusion circRNA and protein collaborate in oncogenesis
altered/novel ncRNA levels affect tumor cell growth by modulating:
●​ Proliferation
●​ Apoptosis
●​ Invasive growth
●​ Metabolism
I.​ Let-7 inhibits growth of osteosarcoma cells
●​ Let-7: Tumor suppressor miRNA
II.​ miR-21 and miR-34 affect growth of breast cancer cells
●​ These mir-34 mir-21 Strong reduction of BC cells
III.​ Knockdown of lncrNA EWSAT1 reduces growth of osteosarcoma
●​ Inhibition of this lncRNA → reduction
IV.​ Targeting circRNA in colon cancer affects growth (circ_0079993)
●​ Strong reduction of tumor

Induction/inhibition of ncRNAs induce cancer in mice models

I.​ Causal link between microRNAs and B-cell lymphoma
●​ Dicer: critical step in processing miRNA
●​ If dicer is knocked out + myc overexpression → no miRNA → reduced lymphomagenesis
●​ Dicer knockout reduces miRNA levels
●​ microRNAs contribute to B-cell lymphoma development
II.​ Tumor Suppressor role of lncRNA XIST in hematopoietic cells
●​ XIST -/- mice
○​ Impaired inactivation of X chr in females
○​ Genome wide copy number changes
●​ Develop highly aggressive myeloproliferative neoplasm
III.​ lncRNA PVT1 enhances oncogenic potential of MYC
●​ MYC + PVT → more tumors

Regulate/target cancer associated genes/pathways

I.​ onco-miRNAs and tumor suppressor miRNAs
●​ Tumor suppressor miRNA work on oncogenic proteins
→ loss of TS miRNA → loss of suppression of
oncogenes → leads to cancer
○​ Let-7 → MYC
○​ miR-15 → BCL2
●​ Oncogenic miRNA target protein codons of the tumor
suppressor genes → overexpression of oncogenic miRNA → more suppression → no TSG → cancer
○​ miR-155 → NIAM
○​ miR-21 → PTEN
Loss of TS miRNA = loss of suppression = more oncogenes = cancer
Overexpression of oncogenic miRNA = more suppression = no TSG = cancer

II.​ ncRNAs play crucial roles in regulatory network of MYC

●​ Some of lncRNAs act on MYC, directly or indirectly
●​ Transcription of MYC is then affected
APPLICATIONS OF ncRNAs in clinical oncology
●​ Diagnostic: classification, disease marker
●​ Prognostic: Depending on the levels of expression of the RNAs, you can classify how well the patients will
respond to a specific treatment
●​ Therapeutic: induction/inhibition of ncRNAs

Novel cancer treatment strategies: targeting RNA

●​ Targeting cancer associated RNAs
●​ Using RNAs to inhibit cancer associated survival
mechanisms

Inhibition of miR-10b reduces metastatic potential of breast

cancer cells

Summary
●​ ncRNA play essential roles in (almost) all cellular processes
●​ ncRNAs play significant roles in pathogenesis of cancer based on:
○​ Altered expression
○​ Located at cancer associated chromosomal loci
○​ Affect growth of cancer cells
○​ Regulate cancer-associated genes / pathways
○​ Induce cancer in mouse models
○​ Targeting ncRNAs has therapeutic potential

Lecture 10 – targeted therapy

Sensitive to chemotherapy
●​ Combination therapy is more effective than consecutive
therapies
●​ Problem: toxicity
●​ Chemotherapy targets the proliferating cells
●​ Enough normal-cells that can repopulate epithelium
●​ Capacity of cells to repair is higher in normal-cells than in
cancer cells

Chemotherapy on testicular cancer has high cure rate (80-90%)

Sensitivity/Resistance to chemotherapy
Treatment of a disseminated disease
A.​ Induction of remission after 2 cycles
B.​ Non sustained remission (relapse)
C.​ Ideal curative treatment
D.​ Adjuvant treatment after surgery
Resistance to chemotherapy
●​ Life extension by chemotherapy is disseminated (metastatic) disease
●​ But no cure in general
●​ Tumor type: leukemia, multiple myeloma, NSCLC, ovarian cancer, colon cancer

Onset of cancer is accelerated by:

●​ Consecutive cycles of mutations-selections
●​ Increased genetic instability
●​ Less cell death and differentiation
●​ Increased proliferation
●​ Gaining autonomy from the environment (metastasis)
Spontaneous mutations: 1/gene 10^5 - 10^6 cell divisions

Why is chemotherapy still very effective in many cases?

●​ Cancer cells divide more often
●​ normal cells repair dna damage better
●​ normal tissue recovers better

Tumor suppressor gene p53 has an important role in response

●​ Mutations in p53 or inactivated p53 by HPV E6 protein (cervical cancer)

Resistance to chemotherapy
Tumor cell intrinsic mechanisms of resistance →

Ideal targets for molecular therapy

●​ Chemotherapy/radiotherapy: inhibit dividing cells
●​ Molecular marker for targeted therapy → more optimal
○​ Present in most patients with a certain disease
○​ Plays a crucial role in the development of the disease
○​ Has a unique activity
■​ Disease is depending on this activity
■​ Activity is replaceable in normal cells

Tumors are not self-standing entities (bag full of tumor cells)

●​ Tumors consist of tumor cells and the microenvironment of diverse normal cells (including fibroblasts and
immune cells, endothelial cells)
●​ Neurons are innervating these tumors by signaling

Neurons interactions facilitate cancer hallmarks

●​ Sustaining proliferative signaling
●​ Resisting cell death
●​ Activating invasion

Stromal cells interact with tumor cells

Interactions between tumor cells and fibroblasts stimulates growth

 ●​ Transformed human mammary epithelial cells (txHMEC)
●​ Faster tumor growth due to
○​ Fibroblasts (excrete growth factors)
○​ Matrigel (ECM made by mouse sarcoma cells)
●​ Look at the tumor not only as single cells, but you should also take a look at the tumor microenvironment

New Hallmarks of cancer

●​ Genome instability and mutation
●​ Deregulation
●​ Immune evasion

Low molecular weight tyrosine kinase inhibitors vs Anti receptor antibodies as anti cancer agents
1.​ Small molecules can be used as a drug against certain proteins
→ used to target inside the cell
a.​ Intracellular
b.​ Low molecular weight
c.​ Serum half life: short
d.​ -nib (Imatinib)
2.​ Or antibodies → targeting outside of cell ​
a.​ High molecular weight
b.​ Serum half life: long
c.​ -mab (Trastuzumab)

Oncogene addiction: tumor cells get addicted to oncogenes (pathway that helps them to grow as fast as possible)
●​ Targeted therapy against oncogenes:
○​ tumor cell apoptosis (death)
○​ Tumor cell senescence (not growing anymore but also not die, aging)
○​ Tumor cell stasis (proliferative arrest)

Many receptors are proto-oncogenes

●​ Ligand binding to receptor and the intracellular part

Why are there many different receptors?

They are active in different tissues

Oncogenes:
●​ EGFR pathway
●​ HER-2 pathway
●​ MAPK pathway

Members of the HER family can bind each other and trigger signal transduction by
activating mutations/overexpression
●​ Ligand binding induces dimerization of EGFR, HER3, HER4
●​ Receptor dimer is active
 ●​ HER2 dimerizes ligand-independently
Due to the binding → change in confirmation of the kinase domain within the cell →
protein becomes active → downstream signal

Mechanism:
Receptor tyrosine kinases activate PI3K/AKT/mTOR and RAS/MAPK signaling pathways

Inhibitions of signal transduction

1.​ Intracellularly: Tyrosine kinase inhibitor goes into the pocket of the intracellular
part of receptors → blocked signaling pathway
2.​ Extracellularly: Using antibody that blocks either binding of ligand or prevents
the receptors from dimerizing
I.​ Example of intracellular signal transduction inhibition:
TKIs for mutated EGFR in lung cancer, colon (intracellular targeting)
●​ Many tumors: EGFR domain is mutated and it’s constantly active, irrespective
of ligands presence
●​ So if we make a drug that binds to EGFR kinase domain → block signaling,
competition with ATP
●​ If we target extracellularly, using mab, cancer can still induce secondary
mutation → drug can no longer bind to it
●​ So new drug has to be invented to be specific to this secondary mutation
●​ Heterogeneity of mutations occurring in tumor cells → resistant tumors
●​ Drugs are specific to the active domain
●​ In normal tissue: these receptors also play a role → some toxicity in cells dependent on this
EGFR pathway
●​ Therefore this drug could also affect non cancer cells

Skin toxicity/effect is a predictor of EGFR inhibitor effectiveness

●​ Drug is often activated/inactivated by the liver enzymes
●​ So drug could be metabolised by the liver enzymes more or less in different patients
●​ Give rise to different toxicity and efficacy
●​ Therapeutic window = treat patients while maintaining controlled toxicity and efficacy
●​ Using Toxicity as a readout

II.​ Example of extracellular signal transduction inhibition

HER2 overexpression and activation
●​ Overexpression in many tumor types: Breast cancer (20%), Ovarian cancer, Non small cell lung cancer
●​ overexpression/activation by
○​ Increased gene copy number
○​ Increased mRNA transcription
○​ Increased cell membrane expression
○​ Loss of the extracellular domain
→ Humanized anti-HER2 antibody

Blocking HER2 in Breast Cancer

 ●​ Dimerization → MAPK + PI3K pathway → cell cycle progression & proliferation + protein synthesis and cell
growth
●​ Humanized anti-HER2 antibody → prevents dimerization of protein

anti-HER2 antibody: trastuzumab in HER2 positive breast cancers

●​ Mono Treatment: increased survival
●​ Combination treatment: antibody and chemotherapy → increased
survival

This drug is not effective in ovarian cancer, even though her2 mutations are
also present in ovarian cancer.

HER2 imaging for cancer diagnosis: PET machine

●​ Cancer diagnosed
●​ Targeting medication injected in patients
●​ Homing on tumor
Problem:
●​ HER2+, but antibodies may not reach the tumor.
●​ There could also be subpopulations of the tumor that is not HER2
positive
●​ Tumor likes to be in the bones with breast cancer

HER2 overexpression happens in many tumors

However: Non breast tumors often insensitive to trastuzumab
Reason: HER2 signaling not essential, other tumors rely on other pathways
to drive its cancer progression
III.​ Example of drug resistance
50% melanomas have mutated BRAF protein (V600E)
●​ Mutated BRAF V600E is sensitive to vemurafenib
●​ BRAF : part of RAS/MAPK pathway
●​ Inhibitor blocks kinase domain (RAF inhibitor)
●​ Vemurafenib improves disease free survival in melanomas, but
tumors quickly become resistant
Vemurafenib resistance occur due to RAS mutation/kinome activation
A)​ BRAF inhibitor: results in more BRAF-CRAF binding, and CRAF
and ERK become highly active (usually together with oncogenic
RAS)
B)​ BRAF/MEK inhibitors: cause other kinase receptors to become overexpressed as a compensation
mechanism, and ERK becomes highly active

How to overcome/prevent resistance to targeted drugs?

●​ Combination of targeted drugs
●​ Combination of targeted therapy (fast response) plus immune checkpoint inhibitors (long term response)
●​ Chemo: induces damage + immune checkpoint inhibitor

Apoptosis
Tumor suppressor gene p53 (wildtype in 50% tumors)
●​ Target gene transcription
●​ MDMX: Works alongside MDM2 to inhibit p53 activity.
●​ MDM2: Functions as an E3 ubiquitin ligase, leading to p53 degradation.
●​ core regulation of p53 → inhibits it
●​ P53 / MDMX & MDM2 = negative feedback loop
●​ Removal of these inhibition → p53 activation → apoptosis

Which protein function is easier to inhibit?

a.​ Kinase (enzymatic) activity → easier (small molecules)
b.​ Protein-protein interaction → harder = no conformational change, no active site

Reactivation of the wild type p53 in drug combo with cisplatin → more apoptosis
●​ Stapled peptide → avoid movement of wild type peptide
●​ Peptides can be taken up
●​ Peptide inhibitor → bigger → easier to block protein protein
interaction

A.​ Monoclonal Antibodies (mAbs):

●​ Bind very specifically to their targets.
●​ Act extracellularly (outside the cell).
●​ High specificity but limited to surface or secreted proteins.
B.​ Small Molecules:
●​ Less specific than mAbs.
●​ Can inhibit enzymatic activity inside cells (intracellular action).
●​ However, they are less effective at blocking protein-protein
interactions (e.g., BCL2 and MDM2 inhibitors).
C.​ Small Peptides:
●​ Can be designed to be more selective and stronger blockers of protein-protein interactions inside cells.
●​ An example of this approach is stapled peptides, which are designed to have increased stability,
specificity, and cell permeability.

Summary
●​ Tumors consist of tumor cells and a microenvironment of various normal cells (including immune cells)
●​ Tumor cells and interaction with their environment may cause drug resistance and are therapeutic targets
●​ These processes are NOT (or not specifically) addressed by chemotherapy
●​ Many targeted therapeutics have proven themselves in the clinic (imatinib=CML,lung cancer,
trastuzumab=HER2+ BC, vemurafenib=melanoma)
●​ Resistance to targeted drugs occurs fast
●​ Big challenge: which drug combinations for which type of tumor

1.​ Imatinib
Tyrosine Kinase Inhibitor
●​ Target: kinase domain of e,g, EGFR (lung, colon cancer)
●​ Cancer type: CML, gastrointestinal stromal tumors (GIST), lung
●​ Mechanism:
 ○​ Inhibits the BCR-ABL tyrosine kinase, which is an abnormal fusion protein driving uncontrolled
proliferation.
○​ Also blocks c-KIT and PDGFR (important in GIST).
2.​ Trastuzumab
●​ Target: HER2 (Human Epidermal Growth Factor Receptor 2)
●​ Cancer type: HER2-positive breast cancer, gastric cancer
●​ Mechanism:
○​ Binds to HER2 receptors, preventing signaling that promotes cell growth.
○​ Enhances immune-mediated destruction of HER2+ cancer cells
3.​ Vemurafenib
●​ Target: Mutant BRAF V600E kinase
●​ Cancer type: Metastatic melanoma with BRAF V600E mutation
●​ Mechanism: Blocks the hyperactive BRAF kinase, which drives uncontrolled cell division.
●​ Clinical Impact:
○​ Led to rapid tumor shrinkage and prolonged survival in melanoma patients.
○​ However, resistance can develop (e.g., via MEK reactivation), leading to combination strategies
with MEK inhibitors (e.g., cobimetinib).

Lecture 11 – anti-cancer therapies: classical and modern approaches

Prevention and early diagnosis are critical (more so than therapy) for patient survival

PDGF-receptor: high expression in i.a. metastases of colon carcinoma

Future trends
Stratification (= division of patients into groups) of patients with breast tumors based on functional genomics
●​ Personalised precision medicine
●​ Different groups of patients, different target receptors, different treatments

The classical anticancer therapies

Mustard gas: a medicine from WW1
●​ Observation: WBC counts decrease when exposed to mustard gas → BM cell growth is suppressed by gas
●​ WBC rapidly divides, RBC doesn't.
●​ Conclusion: the mustard gas affects the rapidly dividing cells → basis for alkylating cytostatics
●​ Alkylating agents are still extensively used today

Groups of anticancer drugs

I.​ Alkylating cytostatic agents: disrupt DNA replication by binding to base pairs
Mechanism of action:
First: activation of drugs in the body (eg cyclophosphamide):
1.​ Enzymatic (cytochrome P450 = once activated, generate free radicals)
a.​ detoxify products that are toxic to the body (in liver)
b.​ IMPORTANT ROLE IN DRUG METABOLISM.
c.​ Some drugs aren’t detoxified by cyt P450, but activated
2.​ Spontaneous (non-enzymatic)
 a.​ End product binds to nucleotide (mainly to guanylate) → crosslinks in DNA
b.​ Very positively charged product
c.​ Removal of electron → Reactive product arises
d.​ Increase in very positive nuclear / chemical center → reacts with the most neg side of the body
(guanylate)
e.​ DNA is very negatively charged (most negative=guanylate)
f.​ Another guanylate with the neighbour crosslink
g.​ Guanylate-activated drug-guanylate
End product binds to nt (mainly guanylate) → cross-links in DNA
→ Alkylating agents attack DNA, preferred reaction is the crosslink, but there are many other targets, other adverse
effects
→ in the end DNA replication is disturbed
→ cell with highest DNA replication activity stops dividing
→ explains hair loss (rapidly dividing cells)

II. Antimetabolites
●​ Interfere with DNA (and RNA) synthesis by the insertion of wrong building blocks
●​ Nucleotide analogs
●​ Molecules that resemble nucleotides (A,T,C,G) with slight differences
●​ Similar enough that DNA polymerase can recognise them, but different enough so that a different building
block is taken up
●​ Also no DNA replication in the end

III. Topoisomerase inhibitor

●​ Topo-isomerases are essential for DNA transcription
●​ DNA transcription occurs in multiple spots (thousands) simultaneously
●​ There's an incredible amount of tensions in DNA-strain = due to supercoiling
●​ DNA polymerase can only use single stranded DNA
●​ So first DNA needs to be unwinded
Topoisomerase I and II: in cells
●​ When DNA double helix is read (during cell division or RNA synthesis) everything gets tangled up
●​ Topoisomerase enzymes (I and II) make a reversible break in DNA, act as a swivel, and repair the break
●​ Cuts DNA in 2 → unwinds the knot → restores the binding
Topoisomerase inhibitors : anti tumor agents
●​ Topoisomerase inhibitors bind to topoisomerases
Mechanism:
●​ They either prevent topoisomerase from binding to DNA or
●​ they cause irreversible binding of topoisomerases to DNA
Effect: breaks in DNA strands

E.g., Doxorubicin (adriamycin)

●​ One of the most frequently used anticancer drugs
●​ Multiple mechanisms of action → doxo is a very powerful cytostatic drug
1.​ Topoisomerase inhibitor
2.​ Binds to DNA (alkylating properties) = activated by cytochrome p450
3.​ Generates oxygen radicals (post activation by cyt p450) → O2-/H2O2/OH-
IV. Plant alkaloids: anti tumor agents
Impact on microtubules
●​ Microtubules in a normal cell
●​ Microtubules during cell division
We are 70% water, if we dont have these microtubules, we wouldve been
flat on the floor lol. Microtubules give 3D shape to cell and to us. They
serve as railroads for proteins. Proteins produced in the cell, synthesised
in the cytoplasm, transported to the otherside of the cell using these
microtubules. Also play an important role in mitosis. Bringing
chromosomes to each side of the cell during cell division → even number
of chromosomes in each daughter cell.

Plant alkaloids are derived from many plants

Mechanism of action
A.​ At steady state: microtubules are made up of tubulin (monomer) → assembly → disassembly
B.​ Polymerization blocked by vincristine or vinblastine → Continuous disassembly
C.​ Polymerization stabilized by paclitaxel → stable microtubule
Interfering with assembly/disassembly of dimeric components → problem for microtubules

V. Hormone drugs
●​ Some tumors highly express estrogen receptors (ER), androgen (testosterone) receptors (AR), eg breast
cancer, prostrate cancer
●​ In steroid sensitive tumors:
○​ Breast cancer: binding to ER
■​ Estrogen analogue = tamoxifen (blocking estrogen production)
○​ Prostate cancer: binding to AR:
■​ non steroidal anti androgen = flutamide / or
■​ cease testosterone production

Most often combo of cytostatic agents are being used: w/ different modes of action
→ prevent the induction of drug resistance
→ synergistic effects = lower the combo of most toxic components → reduce adverse effects

Obstacle in anti cancer treatments: Tumor Resistance

P glycoprotein: ATP dependent drug efflux pump →. Encoded by MDR1 genes
●​ Its physiological function is to protect cells from toxins.
●​ But it pumps out drugs as well.

Tumor resistance factors

●​ Downreg of transporters → drug doesnt get into cell
●​ Upreg of multidrug resistance (MDR) genes→ drug pumped out
●​ Downreg of cyt p450 enzymes → lack of drug activation
●​ Inc exp of drug metabolizing enzymes → acc degrad of drugs
 ●​ Recovery from dmg or induction of protective enzymes → dna repair enzyme, inc exp of target enzyme
(methotrexate), oxygen radical scavengers
Outcomes of resistance
a.​ MDR cells → therapy → survival of the fittest
b.​ Tumor stem cell —> therapy → stem cell survival
c.​ Tumor stem cell → therapy → mutation → drug-induced mutation

New therapies: magic bullets (biological) → antibodies and angiogenesis inhibitors

●​ Development of tumor specific antibodies (esp against GF receptors)
●​ Antibodies: role in immune defense → used as medicine

Next to tumor cells: many diff cell types

●​ Endothelial
●​ Pericytes
●​ MP
●​ Mast cells
●​ NP
●​ Fibroblasts
●​ Stem cells
●​ ECM
Origin of these cells
●​ Influx from blood
●​ Local proliferation
●​ Recruitment from BM
Cells in tumor communicate with each other → in order for tumor to stimulate its own growth → through growth
factor receptors and the production of the GF receptors that are neighbours

Cells migrate out of the tumor: metastasis → growth in other tissues

●​ Not enough to just inhibit mitosis, you’d need to inhibit complex processes as well to fully inhibit tumors
(metastasis)

Blood vessel wall and ECM: important for metastasis

ECM: around blood vessel (gel like structure)
1.​ Tumor cells stick to wall of blood vessel
2.​ Migrate to a keyhole
3.​ Dissolve the ECM (collagen, firm, solid material → enzymes needed to get through)
4.​ Find proper tissue and grow there
●​ If cells wouldn't be firmly attached to a basement membrane (=ECM) the bloodvessel would get damaged
at every heartbeat
●​ Tumor cells migrate through the matrix by release of collagenases (=matrix metalloproteinases, MMP).
this dissolves ECM.

Tumor growth and metastasis: barriers

ECM/basement membrane
●​ All cells are firmly attached to the ECM (containing many molecules)
●​ ECM is also a storage pool for GF (FGF, VEGF) and cytokines = VEGF → new blood vessels
 ●​ When ECM is damaged, these GFs may be released
●​ The matrix is dissolved
●​ Tumors grow further
●​ This also occurs in chronic inflammation, which could induce tumor formation

Matrix degradation stimulates growth of surrounding cells

●​ Tumor cells produce matrix degrading enzymes to metastasize
●​ MMP (matrix metalloproteinases) = break cell cell contacts and activate bioactive molecules in the
basement membrane
●​ Post secretion MMP are deactivated by Tissue inhibitors of MetalloProteinases (TIMPs)

Neo-angiogenesis in tumors: vascular endothelial growth factor (VEGF)

●​ Induces formation of new blood vessels
●​ tumor>0.2 mm: lack of O2 → prod of extra blood vessels (=angiogenesis) is needed → promotes further
growth and enables metastases
Angiogenesis (=growth of blood vessels) → tumor growth
Insufficient neo angiogenic activity → tumor necrosis
●​ Normal vessel = Closed tight endothelium
●​ Tumor associated vessels = diminished pericyte coverage → leaky, permeable, hasty, sloppy vessels →
problems

Tumor itself provides for production of extra blood vessels and for the
breakdown of the matrix
●​ Recruiting signals from BM→ mast cells, MP → MMP →
soluble, active VEGF → angiogenesis
●​ Balance between activators and inhibitors of angiogenesis.
Imbalance of these pro and anti angiogenesis factors in tumors

Targeting of the vascular endothelium

Anti-angiogenesis: targeting VEGF-R
●​ avastin(=bevacizumab): monoclonal antibody
●​ Conclusion: anti angiogenic factors not so curative on their own but effective in combination therapies
●​ It didn't work on its own because once treated with the MAB, tumor reduces in size, but they produce
even more VEGF-R

Future targets for therapies:

1.​ stroma (=ECM + cells producing ECM) → the
surrounding tissue “nurtures” the tumor
●​ Endothelial cells, pericytes, fibroblasts, lymphatic
cells, NP, MP, Mast, basement membrane/ECM
●​ Not rapidly dividing → possible new targets for
intervention → not able to produce factors that are
resistance for particular drug (less resistance)
2.​ Tumor mediated nerve neurogenesis: axonogenesis
and reprogramming in the TME
●​ Axonogenesis
 ●​ Neurogenesis
●​ Neuronal reprogramming

Why do most drugs fail in the clinic?

●​ 700 mab → 99% fail
●​ Drug development: ​studies in cell lines (in vitro) and in mouse models (in vivo)
●​ Answer: tumor resistance, heterogeneity in tumor mutations, model mice dont have an immune system,
no angiogenesis
●​ Lots of drugs work in vitro, and in vivo (mice) but not in complex situation
Situations in patients is much more complex than in most experiments

Biological therapy: injected intravenously (can’t be taken orally bc it'll be digested in the stomach)
Chemotherapy: ingested orally

Summary
●​ Classical anticancer drugs: There are 5 different groups, all with different mechanisms of actions. They are
mostly used in combinations.
●​ Therapy resistance is a key problem. Use of a combination of drugs with different mechanisms of actions
reduces the risk of resistance.
●​ A tumor is not 1 cel type that simply grows
●​ Metastasis is a complex process involving many steps.
●​ Angiogenesis is an important step in tumor growth and metastasis.
●​ Drugs inhibiting angiogenesis are innovative, but can not act alone.
Lecture 12 – animal models for improved prediction of tumor sensitivity to targeted therapy

Screening for targeted drugs

●​ Cell lines and their xenografts
●​ NCI panel of 60 human tumor cell lines for screening
○​ mutations/amplification
○​ Rna exp
○​ Protein exp
●​ Disadvantage:
○​ screen based on human depends on cell lines
○​ Cancer cells are injected subcutaneously = no metastases due to rapid growth
○​ Mice are immunocompromised otherwise human cancer cells would be rejected

TRAIL receptor ligand interaction boosts apoptosis in cancer cell lines (via the extrinsic pathway)
●​ TRAIL Effective in mice w a human xenograft (COLO205)
●​ Also in combo w chemotherapy (5FU/CPT)
Problems
a.​ Resistance to targeted drugs in vivo rapidly observed
b.​ Promising drugs in vitro/in vivo showed no clinical efficacy
i.​ Targeting TRAIL death receptors + chemotherapy is very effective in in vitro and in vivo models
ii.​ TRAIL receptor is expressed in patient-derived tumors
New drugs are being clinically tested (phase 1-3)
1.​ Phase 1: Max tolerated dose
2.​ Phase 2: Which tumor type/whcih stage
3.​ Phase 3: Comparison w other therapies (standard therapy)
Only a small amount of drug reaches the clinic

Lack of good models, use of wrong models and publication bias

Models for targeted therapy

1.​ Cancer cell lines
Pros: easy to use for mechanistic studies (drug resistance/driver mutation) or as xenografts
Cons:
●​ Simplistic cell population: no stromal cells etc → no predictive value for an activity in patients
●​ Limited amount of models exist per tumor type
●​ Cell line origin is often questionable, mycoplasma-free (solve w STR
profiling)
STR=short tandem repeat analysis
15-20 primers sets used for unique identification
●​ Mycoplasma-positive (bacteria can live in cells) (mycoplasma
testing)
●​ Cell lines can easily lose or gain chromosomes (or part of a chrom)
depending on culturing etc → make them less homogenous than
before

2.​ Ex vivo ovarian cancer tissue cut into slices (krumdieck slicer)
●​ 300 micrometers slices of tumors
●​ Crosstalk bn drugs targeting ex/intrinsic apoptotic pathway
○​ Trail: extrinsic pathway activated
○​ Chemotherapy, irradiation, DDR inhibitors: intrinsic
Eg Cisplatin/TRAIL induce apoptosis in an ex vivo ovarian cancer tissue
derived from patients
Pros:
●​ Viable for >72 hrs
●​ TME intact
●​ Possible to inhibit signaling pathways (monitor effect on
normal&cancer cells)
Cons:
●​ Variation in tissue slices (same as in tumors)
●​ Possible to observe only short term effects
●​ You're missing half life of the drugs → its not metabolised in kidneys/livers
Slices are continuously exposed to TRAIL
●​ In vivo trail has a short half life (<40min post IV injection and<2 hrs post IP injection)

3.​ Mouse models

Pros:
 ●​ Some medicines need to be activated/converted
in the liver
●​ Half-life of drugs in the blood and tissues can
only be monitored in animal models
Cons: no contribution of the immune system

1.​ Syngeneic Model (Left)

a.​ Uses immunocompetent mice with
transplanted tumors.
b.​ Allows metastasis since the immune
system is intact.
Injecting GEMM into syngeneic model (genetically identical mouse strain) → metastases
Good bc GEMM: no metastasis
Pro:
●​ Immune system is present (immunocompetent)
●​ tumors grow at the original site
Con:
●​ limited amount of mouse models is available for drug testing (they are expensive)
●​ often, it is unknown which genes are cancer “drivers”
●​ or many different genes can cause the same cancer

2.​ Subcutaneous xenograft: under the skin

a.​ Uses immunodeficient mice with human tumor cells injected under the skin.
b.​ No metastasis due to rapid tumor growth.
c.​ Key disadvantage: lacks a functional immune system, no metastases

3.​ Orthotopic xenograft: placed where tumor normally grows

Bioluminescent subcutaneous & orthotopic xenografts for an easy quantification of the tumor amount
●​ Emits light → penetrate skin/muscle (tumor visible until 1cm deep) → visualising an orthotopic tumor
growth of luc positive cancer cell line
●​ Note: trail+chemo effective in vitro, tumor slice, and in vivo models. But no clear clinical effect
●​ Allows metastasis, mimicking real tumor progression.

4.​ GEMM: genetically engineered mouse models: knockout of gene TP53KO → nonmetastatic
Cre-lox system used to create GEMM
●​ KNOCKOUT P53/brca
●​ Results GFP exp
●​ Cre = bacterial recombinant protein
●​ Specifically recognises loxP site
Making it cell type specific
●​ Activating mutant KRAS eg only in lung cells, CRE must only be exp in lung cells
●​ Tissue specific promoter for CRE gene (promoter exp in lung tissue)
●​ Eg CRE controlled by heart specific promoter, activation of CRE is followed by emission of light(luc) and
blue stain (b-gal)

4. Patient derived xenografts (PDX)

 ●​ Small pieces of tumor directly transplanted from patients into immunodeficient mice (NSG)
●​ In PDX: a mix of tumor cells, stromal cells, immune cells from the patient
●​ Interaction bn epithelial cells and other cell types can occur
●​ Facilitation of receptor ligand interaction
○​ In testis
■​ PDGF-A positive epithelial cells
■​ PDGF-Ra positive mesenchymal cells
●​ PDX are made in special immunodeficient mice (otherwise tumor rejected)
●​ Long time before PDX tumors grow
Mouse stroma and mouse endothelial cells infiltrate into PDX
●​ Human tumor w normal cells from mice
●​ Still looks like patients tumor
PDX model of the ovarian cancer presents the same sensitivity to chemotherapy as the patient

Future therapies:
●​ Use PDX for screening drug activity
●​ Characterize genetic profile of sensitive/resistant PDX tumors

Pros
●​ TME
●​ Many models per tumpr type
●​ Improved prediction of sensitivity to classical chemotherapy & targeted drugs
●​ Maintenance of heterogeneity of cell populations
Cons
●​ Immunodeficient mice
●​ Stromal cells and endothelial cells of mouse origin
●​ High costs of the mode
●​ Ethical concerns

5. Cancer stem cells

●​ Used for making organoids (miniature tissue) by treating them w specific GF
●​ Per tissue or tumor type different combo of GF needed
●​ Organoids made from cancer stem cells from patient tumor tissue
●​ Cancer organoids: biobanking/cryopreservation, drug screening, genetic screening
Pros
●​ Many models
●​ Biobanking
●​ Easier drug screening
●​ Genetic screens
●​ Cancer stem cell based
●​ Heterogeneity bn organoids
Cons
●​ Lack of stromal / endothelial cells
●​ GF dependent
●​ Genetic drift
 ●​ No drug pharmacokinetics (liver metabolism)

inter/intratumor genetic heterogeneity

●​ Inter patient population subtyoes
●​ Intra patient spatial, temporal
●​ Intra tumor tissue
●​ Intra tumor genetic

Intrinsic and acquired resistance

Additional strategies
●​ Drug holiday
●​ Collateral sensitivity
●​ Blocking resistance mechanism
●​ Novel drug targeting the driver protein
●​ Combinations
Can be tested in the various models

Matching patients to therapy

1.​ Molecular profiling: FISH, IHC, qPCR
2.​ Prognostic markers, markers predictive of drug sensitivity/resistance, markers predictive of adverse events
3.​ Personalised cancer therapy

Summary
●​ Need to increase success rate in bringing drug to clinic (more treatment options/lower costs)
●​ Use of cell lines remains useful for first steps of functional testing and for cell line based xenograft
●​ CAM model and tissue slices provide an insight into the short time effects
●​ In GEMM/syngeneic tumor models, immune system functional but unsuitable for species specific drug
testing
●​ Xenografts (PDX models) are suitable for drug testing in mice, and better reflect the situation in patients
than cell line-based xenografts
●​ Organoids (patient-derived cancer stem cells) are suitable for genetic and drug screening