67. Cellular oncogenes II.
Mechanism of cellular oncogene activation
Summary
Proto-oncogenes are normal genes in the human genome that encode proteins that are positive
regulators of cell proliferation and survival. Proto-oncogenes can be converted into cellular
oncogenes by various genetic mechanisms responsible for the formation of tumor cells. These
may include point mutations, deletions, viral DNA sequence integration, reciprocal
translocations, or gene amplification that affect the proto-oncogene sequence. The exact
mechanism of their formation, possible diagnostic methods, and options for the inhibition of
the synthesized onco-protein are described in this chapter. In addition to genetic mechanisms,
the activation of proto-oncogenes may also be caused by epigenetic changes.
Key words: proto-oncogene, cellular oncogene, retroviral oncogene, point mutation, deletion,
provirus, insertional mutagenesis, reciprocal translocation, gene amplification
The development of human malignant tumors is caused by mutations or epigenetic alterations
of proto-oncogenes (20-30%) or tumor suppressor genes (55-65%) in somatic cells, or less
frequently in germ cells.
Proto-oncogenes are normal genes in the human genome, and their protein products are
involved in regulating important cellular processes. Many proto-oncogene proteins stimulate
cell proliferation, while others stimulate cell survival or inhibit apoptosis. The expression of
these proteins, as well as their activity, is tightly regulated in cells. Products encoded by proto-
oncogenes include many proteins involved in growth factor signaling e.g. growth factors (e.g.
PDGF), growth factor receptors (e.g. NGF receptor, EGF receptor), G-proteins (e.g. Ras),
protein kinases (e.g. Raf, Src, Abl), transcription factors (e.g. Myc, Fos, Jun), cyclins (e.g.
cyclin D1). There are also a number of proto-oncoproteins involved in cell survival signaling,
e.g. PI 3 K, Akt/PKB, and Bcl-2 protein.
Proto-oncogenes are potentially dangerous genes in the human genome, that can be converted
into cellular oncogenes responsible for malignant transformation or retroviral oncogenes by the
oncogene capture mechanisms.
Cellular oncogenes are abnormal genes that are formed by the activation of proto-oncogenes.
These genes are expressed in an unregulated, constitutive fashion, resulting in increased cell
proliferation and/or increased cell survival, which promotes the transformation of cells into
tumor cells.
Retroviral oncogenes are proto-oncogenes found in the genome of strongly oncogenic
retroviruses. These abnormal genes are expressed in an unregulated, constitutive manner and
induce increased proliferation or increased survival of the infected cell.
Mechanisms of proto-oncogene activation
Conversion of proto-oncogenes into cellular oncogenes can occur by several mechanisms.
These can have two main consequences:
• some change in the structure of the proto-oncogene occurs, resulting in the abnormal
structure of the encoded protein. As a consequence, the protein becomes unregulated,
constitutively active, and this leads to tumor formation.
• the regulatory region of the proto-oncogene is affected, resulting in increased expression
of the encoded protein. The structure of the protein is normal in this case, but the amount
becomes many times higher than in a normal cell, sufficient to transform the cell into a
tumor cell.
It is important to note that genetic alterations occur randomly, affecting any region of the
genome. They can lead to the development of tumors if a change in a proto-oncogene results in
increased constitutive activation or expression of the encoded protein.
Mechanisms of proto-oncogene activation:
1. Mutation
a. Point mutation
A point mutation is a change in a single nucleotide. In proto-oncogenes, point mutations
resulting in amino acid substitutions that cause the protein to become constitutively active are
significant. This leads to malignant transformation of the cell. Some examples of proto-
oncogenes activated by point mutations in human tumors are:
• ras family (rasH, rasK, rasN) proto-oncogenes, the most common point mutation in
response to chemical carcinogens results in an amino acid substitution (glycine amino
acid → valine amino acid). This amino acid change occurs in the GTP-binding region
of the monomeric G-protein, resulting in a constitutively active, GTP-bound Ras protein
that cannot be inactivated. This leads to increased stimulation of growth factor signaling
and increased cell proliferation. Point mutations in ras genes can be found in ~20-25%
of all human tumors (e.g. pancreatic, thyroid, colon tumors).
• neu (neuroblastoma)/HER2 (human epidermal growth factor receptor 2) proto-
oncogene encodes a type of EGF receptor. Point mutation resulting in an amino acid
substitution occurs in the region encoding the transmembrane domain of the receptor,
which results in dimerization of the receptor without ligand binding and activation of
the growth factor signaling pathway, leading to increased cell proliferation (e.g. in
neuroblastoma from neural tissue tumors).
• gsp proto-oncogene encodes the subunit of the heterotrimeric Gs protein. Point
mutation resulting in an amino acid substitution results in a constitutively active, GTP-
bound form of the protein. This may result in increased cell proliferation in cells in
which the activation of the cAMP signaling pathway regulates cell proliferation (e.g.
pituitary, thyroid tumors).
• gip proto-oncogene encodes the subunit of the heterotrimeric Gi protein. The point
mutation, which results in an amino acid substitution, causes the protein to become
constitutively active and GTP-bound. This may result in increased proliferation in cells
in which inactivation of the cAMP signaling pathway regulates cell proliferation (e.g.
ovarian, adrenocortical tumors).
• B-raf proto-oncogene encodes a MAPKKK. Point mutation resulting in an amino acid
substitution occurs in the region encoding the protein's kinase domain, resulting in a
constitutively active form of the protein and stimulation of the growth factor signaling
pathway, leading to increased cell proliferation (e.g. in malignant melanoma, a skin
tumor originating from our melanin pigment producing cells).
b. Deletion
Activation of proto-oncogenes can also be caused by a deletion affecting the coding region. In
this case, a break in the sequence of the proto-oncogene is generated, and the region is excised
and lost. Deletions that result in increased constitutive activation of the coded protein can lead
to tumors. The erbB1 proto-oncogene encodes a type of EGF receptor. A deletion affecting this
gene results in the loss of the ligand-binding domain of the receptor, which causes the
synthesized protein to dimerize even in the absence of a ligand. This way it becomes
constitutively active, and continuously stimulates growth factor signaling (e.g. in glioblastoma
brain tumors).
2. Viral DNA integration
Weakly oncogenic retroviruses lack viral oncogenes in their genomes, yet they can induce
tumors in some infected cells. What is the mechanism? The mechanism of infection of weakly
oncogenic retroviruses involves the synthesis of double-stranded cDNA from genomic viral
RNA molecules that enter the cell. This cDNA product is called provirus, which translocates to
the nucleus and integrates randomly into the host cell’s genome. Malignant transformation of
the cell occurs when the provirus is randomly integrated into or directly adjacent to a proto-
oncogene sequence. The first weakly oncogenic retrovirus to be shown to be tumorigenic was
ALV (avian leukaemia virus), a virus that causes lymphoma in chicken cells. Following
infection, chicken cells that had undergone malignant transformation were examined, and it was
found that the site of provirus integration was in the c-myc proto-oncogene sequence in a high
percentage of the cases (Figure 1.). Integration occurred in the majority of cases in the region
between exons 1 and 2 of the c-myc proto-oncogene. Following integration, deletion of part of
the region occurred, resulting in the loss of exon 1 and part of the provirus. The c-Myc mRNA
was synthesized from exons 2 and 3, followed by the synthesis of c-Myc protein.
Figure 1. Mechanism of insertional mutagenesis during ALV-induced lymphoma formation.
This protein has the same structure as the protein synthesized in normal cells. The problem with
this c-Myc protein is that, due to the integration of the provirus, the LTR sequence of the
provirus, rather than the normal promoter, controls the expression of this region. The LTR
sequence is an extremely strong promoter, so the expression of the c-Myc protein in this cell
can be several 100-fold higher than in normal cells. The c-Myc protein is a helix-loop-helix-
type transcription factor involved in growth factor signaling, and its increased levels cause the
malignant transformation of the cell. This mechanism is called insertional mutagenesis. Weakly
oncogenic retroviruses can cause tumors by this mechanism, despite the absence of viral
oncogenes in their genome. Since insertional mutagenesis occurs only in certain infected cells,
the resulting tumor is monoclonal, containing only the progeny of the cell in which the provirus
has been integrated into a proto-oncogene sequence.
3. Chromosomal translocation
Chromosomal breaks can be caused by a variety of factors (e.g. spontaneously, by reactive
oxygen free radicals, ionizing radiation, UV light, chemicals) and are repaired by double-
stranded DNA repair mechanisms after the cell cycle’s arrest. If the repair does not occur, the
deleted DNA sequences can be translocated to other chromosome regions. Reciprocal
translocation is the term used to describe a structural chromosomal abnormality in which
fragments of two non-homologous chromosomes that have been deleted are exchanged. The
resulting fusion chromosomes are diagnosed by PCR or spectral karyotyping/multicolor
fluorescence in situ hybridization (FISH).
Reciprocal translocation can involve any part of the genome, by a random event. It leads to
tumor formation if the affected region contains a protooncogene and the translocation results in
the activation of the protooncogene. There are two types of protooncogene activation due to
reciprocal translocation:
• chromosome breaks and rearrangements alter the structure of protooncogenes, resulting
in the formation of fusion genes that encode a constitutively active fusion protein,
• the rearrangement does not change the structure of the protooncogene, but the
rearrangement results in a change in its regulatory region, resulting in increased
expression.
Some examples of human tumors that develop as a result of chromosome translocation:
a. Chronic myeloid leukaemia (CML)
In chronic myeloid leukaemia (CML), an increased, uncontrolled proliferation of white blood
cells occurs. An abnormally small chromosome 22, called Philadelphia chromosome (Ph1), is
detected in >95% of patients. Ionizing radiation has been described as a risk factor for the
disease. The chromosomes involved in the translocation are the c-abl proto-oncogene-coding
region of submetacentric chromosome 9 and the bcr (breakpoint cluster region) region of
acrocentric chromosome 22 (Figure 2.). Both chromosomes break in these regions and the
broken fragments are then exchanged. This results in the formation of fusion genes on both
chromosomes 9 and 22, but the one on chromosome 9 is not directly involved in the
development of the tumor. Chromosome 22 becomes smaller due to the translocation and the
resulting bcr/abl fusion gene is responsible for the development of the tumor.
Figure 2. Formation of the Philadelphia chromosome in chronic myeloid leukaemia.
From the bcr/abl fusion gene, a bcr/abl fusion mRNA is synthesized, followed by the synthesis
of a Bcr/Abl fusion protein having constitutively active tyrosine protein kinase activity. This
fusion protein can activate several signaling pathways, including, for example, activation of the
Ras/ERK signaling pathway to increase cell proliferation; activation of the PI 3-K signaling
pathway and phosphorylation of STAT proteins to increase cell survival; and phosphorylation
of cytoskeleton proteins to increase cell motility.
Options to diagnose the disease:
• the patient has an elevated white blood cell count in the peripheral blood smear,
• FISH technique can be used to visualize the fusion chromosomes,
• by PCR, one primer is specific for the bcr region and the other primer is specific for the
abl region, therefore amplification is only possible in case of reciprocal translocation.
During the course of the disease, patients are usually diagnosed in the chronic phase, during
which administration of a targeted therapeutic agent causes apoptosis of 95% of tumour cells.
Glivec is a small molecule nonreceptor tyrosine protein kinase inhibitor that specifically binds
to the ATP-binding region of the Bcr/Abl fusion protein, thereby inhibiting phosphorylation of
the fusion protein substrates. If the patient does not receive treatment at this stage, the disease
progresses to an acute phase or blast crisis, during which new mutations accumulate in the
tumor cells and immature white blood cell progenitors (blasts) appear in the peripheral
circulation. At this stage, Glivec is already ineffective in a high percentage of cases and other
targeted therapeutic agents are required.
b. Burkitt's lymphoma
Burkitt's lymphoma is a lymphatic tumor originating from B lymphocytes, it occurs mainly in
childhood and has also been described in immunodeficient patients in adulthood. The reciprocal
translocation underlying the tumor involves the c-myc protooncogene-coding region on
chromosome 8 and the immunoglobulin heavy chain (IgH)-coding region on chromosome 14
(Figure 3.). Expression of the IgH/c-myc fusion gene is regulated by a strong promoter of the
immunoglobulin gene, and therefore the amount of c-Myc protein is significantly increased
compared to the amount of protein synthesized in normal cells. The c-Myc protein is a
transcription factor expressed in growth factor signaling. An increase in its amount leads to
increased cell proliferation.
Figure 3. Reciprocal translocation involved in the pathomechanism of Burkitt's lymphoma.
Epstein-Barr virus (EBV) infection during immunodeficiency due to malaria infection is also
thought to play a role in the development of the disease. The proliferation of infected B cells is
increased, which increases the chances of IgH/c-myc reciprocal translocation.
When the disease is diagnosed, the normal histological structure of a section of the patient's
affected lymph node disappears and tumor cells infiltrate the entire organ. Bright macrophages
can be found among the homogeneously basophilic tumor cells, a histological picture known
as "starry sky". Reciprocal translocation can be identified by PCR and FISH techniques.
c. Human follicular B-cell lymphoma
In human follicular B-cell lymphoma, the anti-apoptotic Bcl-2 protein gene is translocated next
to the gene encoding the immunoglobulin heavy chain. The expression of the IgH/Bcl-2 fusion
gene is regulated by a strong promoter of the immunoglobulin gene, and therefore the amount
of Bcl-2 protein is significantly increased compared to the amount of protein synthesized in
normal cells. As a consequence of reciprocal translocation, cell survival is enhanced.
d. Acute promyelocytic leukaemia
Differentiation of myeloid cells is regulated by retinoic acid through a signal transduction
process activated by the retinoic acid receptor (Figure 4). A fusion gene generated by reciprocal
translocation affecting the retinoic acid receptor gene prevents the normal differentiation
process and increased cell proliferation leads to tumor formation.
Figure 4. Pathomechanism of acute promyelocytic leukaemia.
5. Gene amplification
Gene amplification is the process of multiplying a particular gene in the genome. This leads to
tumor formation if the affected region encodes a protooncogene, as the amount of the encoded
protein is greatly increased by amplification. It is caused by a defect in re-replication block,
whereby a region is replicated several times during the S-phase, and then these regions form a
tandemly repeated gene sequence (Figure 5.).
Figure 5. Mechanism of gene amplification.
Gene amplification results in the development of two types of chromosomal abnormalities,
which can be detected by karyotype analysis. Banding techniques involve a special staining
procedure in which different regions of the chromosomes are stained differently. In case of gene
amplification, a particular region is widened, and called a homogeneously staining region. The
amplified gene fragments may also break out of the DNA molecule to form extra-chromosomal
fragments, so-called double minutes (Figure 6. A). The number of these mini-chromosomes is
proportional to the degree of malignancy of the tumor and also to the worsened prognosis of
the affected patients. Homogeneously staining chromosomal regions, or double minute
chromosomes, can also be visualized by FISH using a gene-specific, fluorescent probe (Figure
6.B).
Figure 6. Chromosomal abnormalities generated by gene amplification (A.) and their
detection by metaphase FISH (B.).
For the diagnosis of gene amplification, an interphase FISH technique can also be performed
using a gene-specific fluorescent probe (Figure 7-), which shows 2 fluorescent signals for a
gene with a normal copy number, and more fluorescent signals per cell for an amplified gene
due to its increased copy number.
Figure 7. Detection of gene amplification by interphase FISH.
A normal gene-specific probe was labelled with a green, fluorescent dye, the amplified gene-
specific probe was labelled with a red, fluorescent dye.
Gene amplification results in an increase in the amount of the encoded protein, which can be
detected by immunohistochemistry in tumor tissue (Figure 8.).
Figure 8. Detection of protein overexpression due to gene amplification by
immunohistochemistry.
The image shows the HER2 protein detected by immunohistochemistry. Immunocomplexes
were visualized by diaminobenzidine (DAB) reaction. In normal breast tissue (A), the EGF
receptor in the cell membrane is identified by a brown signal. In breast carcinoma tissue (B),
the brown color reaction is more pronounced due to the increased amount of the receptor
protein.
Examples of protooncogenes activated by gene amplification:
• N-myc (in neuroblastoma, lung carcinoma), L-myc (in small cell lung carcinoma), c-
myc (in leukaemia) encoding for transcription factors,
• PRAD1/CCND1 encoding cyclin D1 protein (in breast carcinoma),
• neu/HER2 encoding EGFR (in ovarian, breast carcinoma).
For tumors that develop as a result of EGFR-encoding neu/HER2 gene amplification, a targeted
therapy drug, Herceptin, may be effectively used. This is an anti-HER2 antibody that binds to
EGF receptors overexpressed on the surface of tumor cells and prevents their dimerization,
hence the activation of growth factor signaling. It also promotes the endocytosis of EGF
receptors, reducing the number of cell surface receptors and the activity of the cell proliferation
pathway.
Epigenetic mechanisms leading to the activation of protooncogenes
• DNA methylation: during tumor formation, the promoter of some genes may be
hypermethylated, while others are hypomethylated. If the promoter region of a
protooncogene gene is hypomethylated, the expression of that protooncogene gene will
be increased in that tumor cell, which will enhance cell proliferation or survival.
• Histone modifications: post-translational reversible modifications of nucleosomal
histone proteins regulate gene expression positively or negatively due to changes in the
chromatin structure. Increased acetylation or methylation leading to activation of
histone proteins that bind to DNA regions encoding protooncogenes leads to loosening
of chromatin structure and increased expression of the respective protooncogene.
• Non-coding RNA molecules: regulate the translation or degradation of mRNAs. The
expression of mRNAs transcribed from protooncogenes is also regulated by miRNAs.
When their synthesis is inhibited, e.g. due to hypermethylation of the promoter of the
gene encoding the miRNA, the expression of the protooncogene is increased.
