REVIEW

Hallmarks of Cancer: New Dimensions

Douglas Hanahan

ABSTRACT The hallmarks of cancer conceptualization is a heuristic tool for distilling the vast
complexity of cancer phenotypes and genotypes into a provisional set of underly-
ing principles. As knowledge of cancer mechanisms has progressed, other facets of the disease have
emerged as potential refinements. Herein, the prospect is raised that phenotypic plasticity and dis-
rupted differentiation is a discrete hallmark capability, and that nonmutational epigenetic reprogram-
ming and polymorphic microbiomes both constitute distinctive enabling characteristics that facilitate
the acquisition of hallmark capabilities. Additionally, senescent cells, of varying origins, may be added

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to the roster of functionally important cell types in the tumor microenvironment.

Significance: Cancer is daunting in the breadth and scope of its diversity, spanning genetics, cell and
tissue biology, pathology, and response to therapy. Ever more powerful experimental and computa-
tional tools and technologies are providing an avalanche of “big data” about the myriad manifestations
of the diseases that cancer encompasses. The integrative concept embodied in the hallmarks of cancer
is helping to distill this complexity into an increasingly logical science, and the provisional new dimen-
sions presented in this perspective may add value to that endeavor, to more fully understand mecha-
nisms of cancer development and malignant progression, and apply that knowledge to cancer medicine.

INTRODUCTION steps of tumor pathogenesis. Certainly, the diversity of malig-

nant pathogenesis spanning multiple tumor types and an
The Hallmarks of Cancer were proposed as a set of func-
increasing plethora of subtypes includes various aberrations
tional capabilities acquired by human cells as they make
(and hence acquired capabilities and characteristics) that are
their way from normalcy to neoplastic growth states, more
the result of tissue-specific barriers necessarily circumvented
specifically capabilities that are crucial for their ability to
during particular tumorigenesis pathways. While appreciat-
form malignant tumors. In these articles (1, 2), Bob Weinberg
ing that such specialized mechanisms can be instrumental,
and I enumerated what we imagined were shared commonali-
we limited the hallmarks designation to parameters having
ties that unite all types of cancer cells at the level of cellular
broad engagement across the spectrum of human cancers.
phenotype. The intent was to provide a conceptual scaffold
The eight hallmarks currently comprise (Fig.  1, left) the
that would make it possible to rationalize the complex phe-
acquired capabilities for sustaining proliferative signaling,
notypes of diverse human tumor types and variants in terms
evading growth suppressors, resisting cell death, enabling
of a common set of underlying cellular parameters. Initially
replicative immortality, inducing/accessing vasculature,
we envisaged the complementary involvement of six distinct
activating invasion and metastasis, reprogramming cellular
hallmark capabilities and later expanded this number to
metabolism, and avoiding immune destruction. In the most
eight. This formulation was influenced by the recognition
recent elaboration of this concept (2), deregulating cellular
that human cancers develop as products of multistep pro-
metabolism and avoiding immune destruction were segre-
cesses, and that the acquisition of these functional capabili-
gated as “emerging hallmarks,” but now, eleven years later, it
ties might be mapped in some fashion to the distinguishable
is evident that they, much like the original six, can be consid-
ered core hallmarks of cancer, and are included as such in the
current depiction (Fig. 1, left).
Ludwig Institute for Cancer Research – Lausanne Branch, Lausanne, Swit- As we noted at the time, these hallmark traits, on their
zerland. The Swiss Institute for Experimental Cancer Research (ISREC)
own, fail to address the complexities of cancer pathogenesis,
within the School of Life Sciences at the Swiss Federal Institute of Tech-
nology Lausanne (EPFL), Lausanne, Switzerland. The Swiss Cancer Center that is, the precise molecular and cellular mechanisms that
Leman (SCCL), Lausanne, Switzerland. allow evolving preneoplastic cells to develop and acquire
Corresponding Author: Douglas Hanahan, Agora Translational Cancer these aberrant phenotypic capabilities in the course of tumor
Research Center, Rue du Bugnon 25A, Lausanne CH-1011, Switzerland. development and malignant progression. Accordingly, we
Phone: 41-21-545-1119; E-mail: douglas.hanahan@epfl.ch added another concept to the discussion, portrayed as “ena-
Cancer Discov 2022;12:31–46 bling characteristics,” consequences of the aberrant condi-
doi: 10.1158/2159-8290.CD-21-1059 tion of neoplasia that provide means by which cancer cells
©2021 American Association for Cancer Research and tumors can adopt these functional traits. As such, the

JANUARY 2022 CANCER DISCOVERY | 31

REVIEW Hanahan

Sustaining Evading Emerging hallmarks &

proliferative signaling growth suppressors enabling characteristics
Unlocking Nonmutational
phenotypic epigenetic
Deregulating Avoiding plasticity reprogramming
cellular immune
metabolism destruction

Resisting Enabling
cell death replicative
immortality

Genome
instability & Tumor-promoting
mutation inflammation
Senescent Polymorphic

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cells microbiomes
Inducing or accessing Activating invasion
vasculature & metastasis

Figure 1.  In essence: the Hallmarks of Cancer, circa 2022. Left, the Hallmarks of Cancer currently embody eight hallmark capabilities and two enabling
characteristics. In addition to the six acquired capabilities—Hallmarks of Cancer—proposed in 2000 (1), the two provisional “emerging hallmarks” intro-
duced in 2011 (2)—cellular energetics (now described more broadly as “reprogramming cellular metabolism”) and “avoiding immune destruction”—have
been sufficiently validated to be considered part of the core set. Given the growing appreciation that tumors can become sufficiently vascularized either
by switching on angiogenesis or by co-opting normal tissue vessels (128), this hallmark is also more broadly defined as the capability to induce or oth-
erwise access, principally by invasion and metastasis, vasculature that supports tumor growth. The 2011 sequel further incorporated “tumor-promoting
inflammation” as a second enabling characteristic, complementing overarching “genome instability and mutation,” which together were fundamentally
involved in activating the eight hallmark (functional) capabilities necessary for tumor growth and progression. Right, this review incorporates additional
proposed emerging hallmarks and enabling characteristics involving “unlocking phenotypic plasticity,” “nonmutational epigenetic reprogramming,” “poly-
morphic microbiomes,” and “senescent cells.” The hallmarks of cancer graphic has been adapted from Hanahan and Weinberg (2).

enabling characteristics reflected upon molecular and cel- UNLOCKING PHENOTYPIC PLASTICITY
lular mechanisms by which hallmarks are acquired rather
During organogenesis, the development, determination,
than the aforementioned eight capabilities themselves.
and organization of cells into tissues in order to assume
These two enabling processes were genome instability and
homeostatic functions is accompanied by terminal differen-
tumor-promoting inflammation.
We further recognized that the tumor microenvironment tiation, whereby progenitor cells—sometimes irrevocably—
(TME), herein defined to be composed of heterogeneous and stop growing upon culmination of these processes. As such,
interactive populations of cancer cells and cancer stem cells the end result of cellular differentiation is in most cases
along with a multiplicity of recruited stromal cell types—the antiproliferative and constitutes a clear barrier to the con-
transformed parenchyma and the associated stroma—is now tinuing proliferation that is necessary for neoplasia. There
widely appreciated to play an integral role in tumorigenesis is increasing evidence that unlocking the normally restricted
and malignant progression. capability for phenotypic plasticity in order to evade or
Given the continued interest in these formulations and our escape from the state of terminal differentiation is a criti-
enduring intent to encourage ongoing discussion and refine- cal component of cancer pathogenesis (3). This plasticity
ment of the Hallmarks scheme, it is appropriate to consider can operate in several manifestations (Fig. 2). Thus, nascent
a frequently posed question: are there additional features of cancer cells originating from a normal cell that had advanced
this conceptual model that might be incorporated, respecting down a pathway approaching or assuming a fully differenti-
the need to ensure that they are broadly applicable across the ated state may reverse their course by dedifferentiating back
spectrum of human cancers? Accordingly, I present several to progenitor-like cell states. Conversely, neoplastic cells
prospective new hallmarks and enabling characteristics, ones arising from a progenitor cell that is destined to follow a
that might in due course become incorporated as core com- pathway leading to end-stage differentiation may short-
ponents of the hallmarks of cancer conceptualization. These circuit the process, maintaining the expanding cancer cells in
parameters are “unlocking phenotypic plasticity,” “nonmu- a partially differentiated, progenitor-like state. Alternatively,
tational epigenetic reprogramming,” “polymorphic microbi- transdifferentiation may operate, in which cells that were
omes,” and “senescent cells” (Fig. 1, right). Importantly, the initially committed into one differentiation pathway switch
examples presented in support of these propositions are illus- to an entirely different developmental program, thereby
trative but by no means comprehensive, as there is a growing acquiring tissue-specific traits that were not preordained by
and increasingly persuasive body of published evidence in their normal cells-of-origin. The following examples sup-
support of each vignette. port the argument that differing forms of cellular plasticity,

32 | CANCER DISCOVERY JANUARY 2022 AACRJournals.org

Hallmarks of Cancer: New Dimensions REVIEW

Unlocking
phenotypic Progenitor cell Differentiated cell
plasticity
Normal differentiation

Dedifferentiation

Blocked
differentiation

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Transdifferentiation

Figure 2.  Unlocking phenotypic plasticity. Left, phenotypic plasticity is arguably an acquired hallmark capability that enables various disruptions of
cellular differentiation, including (i) dedifferentiation from mature to progenitor states, (ii) blocked (terminal) differentiation from progenitor cell states,
and (iii) transdifferentiation into different cell lineages. Right, depicted are three prominent modes of disrupted differentiation integral to cancer patho-
genesis. By variously corrupting the normal differentiation of progenitor cells into mature cells in developmental lineages, tumorigenesis and malignant
progression arising from cells of origin in such pathways is facilitated. The hallmarks of cancer graphic has been adapted from Hanahan and Weinberg (2).

when taken together, constitute a functionally distinct the strict definition of this provisional hallmark as separable
hallmark capability. and independent.
Another line of evidence involves suppressed expression
Dedifferentiation of the MITF master regulator of melanocyte differentiation,
Colon carcinogenesis exemplifies disrupted differentiation, which is evidently involved in the genesis of aggressive forms
in that there is a teleological necessity for incipient cancer of malignant melanoma. Loss of this developmental TF is
cells to escape from the conveyer belt of terminal differen- associated with the reactivation of neural crest progenitor
tiation and exfoliation, which could in principle occur via genes and the downregulation of genes that characterize
dedifferentiation of not yet irrevocably terminally differenti- fully differentiated melanocytes. The reappearance of the
ated colonic epithelial cells, or via blocked differentiation neural crest genes indicates that these cells revert to the pro-
of progenitor/stem cells in the crypts that spawn these dif- genitor state from which melanocytes arise developmentally.
ferentiating cells. Both differentiated cells and stem cells Moreover, a lineage tracing study of BRAF-induced melano-
have been implicated as cell-of-origin for colon cancer (4–6). mas established mature pigmented melanocytes as the cells
Two developmental transcription factors (TF), the homeobox of origin, which undergo dedifferentiation during the course
of tumorigenesis (9). Of note, the mutant BRAF oncogene,
protein HOXA5 and SMAD4, the latter involved in BMP
which is found in more than half of cutaneous melanomas,
signal transmission, are highly expressed in differentiating
induces hyperproliferation that precedes and hence is mecha-
colonic epithelial cells, and typically lost in advanced colon
nistically separable from the subsequent dedifferentiation
carcinomas, which characteristically express markers of stem
arising from downregulation of MITF. Another study func-
and progenitor cells. Functional perturbations in mouse
tionally implicated upregulation of the developmental TF
models have shown that forced expression of HOXA5 in ATF2, whose characteristic expression in mouse and human
colon cancer cells restores differentiation markers, suppresses melanomas indirectly suppresses MITF1, concomitant with
stem cell phenotypes, and impairs invasion and metastasis, malignant progression of the consequently dedifferentiated
providing a rationale for its characteristic downregulation melanoma cells (10). Conversely, expression in melanomas
(7, 8). SMAD4, by contrast, both enforces differentiation and of mutant forms of ATF2 that fail to repress MITF results in
thereby suppresses proliferation driven by oncogenic WNT well-differentiated melanomas (11).
signaling, revealed by the engineered loss of SMAD4 expres- Additionally, a recent study (12) has associated lineage
sion, providing an explanation for its loss of expression so as dedifferentiation with malignant progression from pancre-
to enable dedifferentiation and, subsequently, WNT-driven atic islet cell neoplasias into metastasis-prone carcinomas;
hyperproliferation (5). Notably, the loss of both of these “dif- these neuroendocrine cells and derivative tumors arise from
ferentiation suppressors” with consequent dedifferentiation a developmental lineage that is distinct from the one gen-
is associated with acquisition of other hallmark capabilities, erating the far larger number of adjacent cells that form
as are other hallmark-inducing regulators, which complicates the exocrine and pancreas and the ductal adenocarcinomas

JANUARY 2022 CANCER DISCOVERY | 33

REVIEW Hanahan

that arise therefrom. Notably, the multistep differentiation Other examples of differentiation modulators involve the
pathway of islet progenitor cells into mature  β cells has been metabolite alpha-ketoglutarate (αKG), a necessary cofactor
thoroughly characterized (13). Comparative transcriptome for a number of chromatin-modifying enzymes, which is
profiling reveals that adenoma-like islet tumors are most sim- demonstrably involved in stimulating certain differentiated
ilar to immature but differentiated insulin-producing β cells, cell states. In pancreas cancer, the tumor suppressor p53
whereas the invasive carcinomas are most similar to embry- stimulates the production of αKG and maintenance of a more
onic islet cell precursors. The progression toward poorly well-differentiated cell state, whereas prototypical loss of p53
differentiated carcinomas involves a first step of dedifferen- function results in reductions in  αKG levels and consequent
tiation that does not initially involve increased proliferation dedifferentiation associated with malignant progression (20).
or reduced apoptosis when compared with the well-differen- In one form of liver cancer, mutation of an isocitrate dehy-
tiated adenomas, both of which rather occur later. Thus, the drogenase gene (IDH1/2) results in the production not of
discrete step of dedifferentiation is not driven by observable differentiation-inducing  αKG but rather a related “onco-
alterations in the hallmark traits of sustained proliferation metabolite,” D-2-hydroxygluterate (D2HG), which has been
and resistance to apoptosis. Rather, upregulation of a miRNA shown to block hepatocyte differentiation from liver progeni-
previously implicated in specifying the islet progenitor state, tor cells by D2HG-mediated repression of a master regulator
one that is downregulated during terminal differentiation of hepatocyte differentiation and quiescence, HNF4a. The
of  β cells, has been shown to orchestrate the observed dedif- D2HG-mediated suppression of HNF4a function elicits a

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ferentiation occurring during malignant progression (12). proliferative expansion of the hepatocyte progenitor cells in
the liver, which become susceptible to oncogenic transforma-
Blocked Differentiation tion upon subsequent mutational activation of the KRAS
While the above examples illustrate how suppression of oncogene that drives malignant progression to liver cholan-
differentiation factor expression can facilitate tumorigenesis giocarcinoma (21). Mutant IDH1/2 and their oncometabolite
by enabling more well-differentiated cells to dedifferentiate D2HG are also operative in a variety of myeloid and other
into progenitors, in other cases incompletely differentiated solid tumor types, where D2HG inhibits  αKG-dependent
progenitor cells can suffer regulatory changes that actively dioxygenases necessary for histone and DNA methylation
block their continued advance into fully differentiated, typi- events that mediate alterations in chromatin structure dur-
cally nonproliferative states. ing developmental lineage differentiation, thereby freezing
Acute promyelocytic leukemia (APL) has long been docu- incipient cancer cells in a progenitor state (22, 23).
mented to result from a chromosomal translocation that An additional, related concept is “circumvented differen-
fuses the PML locus with the gene encoding the retinoic tiation,” wherein partially or undifferentiated progenitor/
acid  α  nuclear receptor (RARα). Myeloid progenitor cells stem cells exit the cell cycle and become dormant, residing in
bearing such translocations are evidently unable to continue protective niches, with the potential to reinitiate proliferative
their usual terminal differentiation into granulocytes, result- expansion (24), albeit still with the selective pressure to dis-
ing in cells trapped in a proliferative, promyelocytic progeni- rupt their programmed differentiation in one way or another.
tor stage (14). Proof-of-concept of this scheme comes from
treating cultured APL cells, mouse models of this disease, Transdifferentiation
as well as afflicted patients, with retinoic acid, the ligand of The concept of transdifferentiation has long been rec-
RARα; this therapeutic treatment causes the neoplastic APL ognized by pathologists in the form of tissue metaplasia,
cells to differentiate into ostensibly mature nonproliferating wherein cells of a particular differentiated phenotype mark-
granulocytes, short-circuiting their continuing proliferative edly change their morphology to become clearly recognizable
expansion (14–16). as elements of another tissue, of which one prominent exam-
A variation on this theme involves another form of acute ple is Barrett’s esophagus, where chronic inflammation of
myeloid leukemia, this one carrying the t(8;21) translocation, the stratified squamous epithelium of the esophagus induces
which produces the AML1–ETO fusion protein. This protein transdifferentiation into a simple columnar epithelium that
can, on its own, transform myeloid progenitors, at least in is characteristic of the intestine, thereby facilitating the sub-
part by blocking their differentiation. Therapeutic interven- sequent development of adenocarcinomas, and not the squa-
tion in mouse models and in patients with a pharmaco- mous cell carcinomas that would be anticipated to arise from
logic inhibitor of a chromatin-modifying histone deacetylase this squamous epithelium (3). Now, molecular determinants
(HDAC) causes the myeloid leukemia cells to recommence are revealing mechanisms of transdifferentiation in various
their differentiation into cells with a more mature myeloid cancers, both for cases where gross tissue metaplasia is evi-
cell morphology. Concomitant with this response is a reduc- dent and for others where it is rather more subtle, as the
tion in proliferative capacity, thereby impairing the progres- following examples illustrate.
sion of this leukemia (17, 18). One illuminating case for transdifferentiation as a dis-
A third example, in melanoma, involves a developmental crete event in tumorigenesis involves pancreatic ductal ade-
TF, SOX10, which is normally downregulated during mel- nocarcinoma (PDAC), wherein one of the implicated cells
anocyte differentiation. Gain- and loss-of-function studies of origin, the pancreatic acinar cell, can become transdiffer-
in a zebrafish model of BRAF-induced melanoma have dem- entiated into a ductal cell phenotype during the initiation
onstrated that aberrantly maintained expression of SOX10 of neoplastic development. Two TFs—PTF1a and MIST1—
blocks differentiation of neural progenitor cells into melano- govern, via their expression in the context of self-sustaining,
cytes, enabling BRAF-driven melanomas to form (19). “feed-forward” regulatory loops, the specification and

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Hallmarks of Cancer: New Dimensions REVIEW

maintenance of the differentiated pancreatic acinar cell oncogenic signaling pathway known to drive the neoplastic
state (25). Both of these TFs are frequently downregulated growth of these cells (33). Drug-resistant cancer cells switch,
during neoplastic development and malignant progression via broad epigenetic shifts in specific chromatin domains and
of human and mouse PDAC. Functional genetic studies in the altered accessibility of two superenhancers, to a devel-
mice and cultured human PDAC cells have demonstrated opmentally related but distinct cell type. The newly gained
that experimentally forced expression of PTF1a impairs phenotypic state of the BCC cells enables them to sustain
KRAS-induced transdifferentiation and proliferation, and expression of the WNT oncogenic signaling pathway, which
can also force the redifferentiation of already neoplastic in turn imparts independence from the drug-suppressed HH/
cells into a quiescent acinar cell phenotype (26). Conversely, SMO signaling pathway (34). As might be anticipated from
suppression of PTF1a expression elicits acinar-to-ductal this transdifferentiation, the transcriptome of the cancer cells
metaplasia, namely transdifferentiation, and thereby sensi- shifts from a gene signature reflecting the implicated cell-of-
tizes the duct-like cells to oncogenic KRAS transformation, origin of BCCs, namely the stem cells of hair follicle bulge,
accelerating subsequent development of invasive PDAC to one indicative of the basal stem cells that populate the
(27). Similarly, forced expression of MIST1 in KRAS-express- interfollicular epidermis. Such transdifferentiation to enable
ing pancreas also blocks transdifferentiation and impairs drug resistance is being increasingly documented in different
the initiation of pancreatic tumorigenesis otherwise facili- forms of cancer (35).
tated by the formation of premalignant duct-like (PanIN) Developmental lineage plasticity also appears to be

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lesions, whereas genetic deletion of MIST1 enhances their prevalent among the major subtypes of lung carcinomas,
formation and the initiation of KRAS-driven neoplastic that is, neuroendocrine carcinomas [small-cell lung cancer
progression (28). Loss of either PTF1 or MIST1 expression (SCLC)] and adenocarcinomas + squamous cell carcinomas
during tumorigenesis is associated with elevated expression [collectively non–small cell lung cancer (NSCLC)]. Single-
of another developmental regulatory TF, SOX9, which is cell RNA sequencing has revealed remarkably dynamic
normally operative in the specification of ductal cells (27, and heterogeneous interconversion among these subtypes
28). Forced upregulation of SOX9, obviating the need to as well as distinct variations thereof during the stages in
downregulate PTF1a and MIST1, has also been shown to lung tumorigenesis, subsequent malignant progression,
stimulate transdifferentiation of acinar cells into a ductal and responses to therapy (36–38). Thus, rather than the
cell phenotype that is sensitive to KRAS-induced neoplasia simple conceptualization of a pure clonal switch from
(29), implicating SOX9 as a key functional effector of their one lineage into another, these studies paint a much more
downregulation in the genesis of human PDAC. Thus, complex picture, of dynamically interconverting subpopu-
three TFs that regulate pancreatic differentiation can be lations of cancer cells exhibiting characteristics of multiple
variously altered to induce a transdifferentiated state that developmental lineages and stages of differentiation, a
facilitates—in the context of mutational activation of KRAS— sobering realization in regard to lineage-based therapeutic
oncogenic transformation and the initiation of tumorigen- targeting of human lung cancer. Regulatory determinants
esis and malignant progression. of this dynamic phenotypic plasticity are beginning to be
Additional members of the SOX family of chromatin- identified (37, 39, 40).
associated regulatory factors are on the one hand broadly
associated both with cell fate specification and lineage switch- Synopsis
ing in development (30), and on the other with multiple The three classes of mechanism described above highlight
tumor-associated phenotypes (31). Another salient example selective regulators of cellular plasticity that are separable—at
of SOX-mediated transdifferentiation involves a mechanism least in part—from core oncogenic drivers and other hallmark
of therapeutic resistance in prostate carcinomas. In this case, capabilities. Beyond these examples lies a considerable body
loss of the RB and p53 tumor suppressors—whose absence of evidence associating many forms of cancer with disrupted
is characteristic of neuroendocrine tumors—in response to differentiation concomitant with the acquisition of tran-
antiandrogen therapy is necessary but not sufficient for the scriptome signatures and other phenotypes—for example,
frequently observed conversion of well-differentiated pros- histologic morphology—associated with progenitor or stem
tate cancer cells into carcinoma cells that have entered a dif- cell stages observed in the corresponding normal tissue-of-
ferentiation lineage with molecular and histologic features origin or in other more distantly related cell types and line-
of neuroendocrine cells, which notably do not express the ages (41–43). As such, these three subclasses of phenotypic
androgen receptor. In addition to loss of RB and p53, the plasticity—dedifferentiation of mature cells back to progeni-
acquired resistance to antiandrogen therapy requires upregu- tor states, blocked differentiation to freeze developing cells
lated expression of the SOX2 developmental regulatory gene, in progenitor/stem cell states, and transdifferentiation to
which is demonstrably instrumental in inducing transdif- alternative cell lineages—appear to be operative in multiple
ferentiation of the therapy-responsive adenocarcinoma cells cancer types during primary tumor formation, malignant
into derivatives that reside in a neuroendocrine cell state that progression, and/or response to therapy. There are, however,
is refractory to the therapy (32). two conceptual considerations. First, dedifferentiation and
A third example also reveals transdifferentiation as a strat- blocked differentiation are likely intertwined, being indis-
egy employed by carcinoma cells to avoid elimination by tinguishable in many tumor types where the cell-of-origin—
a lineage-specific therapy, in this case involving basal cell differentiated cell or progenitor/stem cell—is either unknown
carcinomas (BCC) of the skin treated with a pharmaco- or alternatively involved. Second, the acquisition or mainte-
logic inhibitor of the Hedgehog-Smoothened (HH/SMO) nance of progenitor cell phenotypes and loss of differentiated

JANUARY 2022 CANCER DISCOVERY | 35

REVIEW Hanahan

Nonmutational
epigenetic reprogramming

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Figure 3.  Nonmutational epigenetic reprogramming. Much as during embryogenesis and tissue differentiation and homeostasis, growing evidence
makes the case that instrumental gene-regulatory circuits and networks in tumors can be governed by a plethora of corrupted and co-opted mechanisms
that are independent from genome instability and gene mutation. The hallmarks of cancer graphic has been adapted from Hanahan and Weinberg (2).

features is in most cases an imprecise reflection of the normal including metastatic lesions, and during the development
developmental stage, being immersed in a milieu of other of adaptive resistance to therapy. One result is the now
hallmark-enabling changes in the cancer cell that are not widespread appreciation that mutations in genes that organ-
present in naturally developing cells. In addition, yet another ize, modulate, and maintain chromatin architecture, and
form of phenotypic plasticity involves cell senescence, dis- thereby globally regulate gene expression, are increasingly
cussed more generally below, wherein cancer cells induced detected and functionally associated with cancer hallmarks
to undergo ostensibly irreversible senescence are instead able (46–48).
to escape and resume proliferative expansion (44). Finally, There is, in addition, a case to be made for another appar-
as with other hallmark capabilities, cellular plasticity is not ently independent mode of genome reprogramming that
a novel invention or aberration of cancer cells, but rather involves purely epigenetically regulated changes in gene
the corruption of latent but activatable capabilities that expression, one that might be termed “nonmutational epi-
various normal cells use to support homeostasis, repair, and genetic reprogramming” (Fig.  3). Indeed, the proposition
regeneration (45). of mutation-less cancer evolution and purely epigenetic
Collectively, these illustrative examples encourage consid- programming of hallmark cancer phenotypes was raised
eration of the proposition that unlocking cellular plasticity almost a decade ago (49) and is increasingly discussed
to enable various forms of disrupted differentiation consti- (46, 50–52).
tutes a discrete hallmark capability, distinguishable in regu- The concept of nonmutational epigenetic regulation of
lation and cellular phenotype from the well-validated core gene expression is of course well established as the central
hallmarks of cancer (Fig. 2). mechanism mediating embryonic development, differentia-
tion, and organogenesis (53–55). In the adult, for example,
long-term memory involves changes in gene and histone
NONMUTATIONAL EPIGENETIC
modification, in chromatin structure, and in the triggering of
REPROGRAMMING gene expression switches that are stably maintained over time
The enabling characteristic of genome (DNA) instabil- by positive and negative feedback loops (56, 57). Growing
ity and mutation is a fundamental component of cancer evidence supports the proposition that analogous epigenetic
formation and pathogenesis. At present, multiple interna- alterations can contribute to the acquisition of hallmark
tional consortia are cataloging mutations across the genome capabilities during tumor development and malignant pro-
of human cancer cells, doing so in virtually every type of gression. A few examples are presented below in support of
human cancer, at different stages of malignant progression, this hypothesis.

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Microenvironmental Mechanisms within, has broad effects on the invasive and other phe-
of Epigenetic Reprogramming notypic characteristics of cancer cells. Compared with the
If not solely by consequence of oncogenic mutations, how normal tissue ECM from which tumors originate, the tumor
then is the cancer cell genome reprogrammed? A growing ECM is typically characterized by increased cross-linking and
body of evidence indicates that the aberrant physical proper- density, enzymatic modifications, and altered molecular com-
position, which collectively orchestrate—in part via integrin
ties of the tumor microenvironment can cause broad changes
receptors for ECM motifs—stiffness-induced signaling and
in the epigenome, from which changes beneficial to the phe-
gene-expression networks that elicit invasiveness and other
notypic selection of hallmark capabilities can result in clonal
hallmark characteristics (71).
outgrowth of cancer cells with enhanced fitness for prolif-
In addition to such regulatory mechanisms endowed by
erative expansion. One common characteristic of tumors (or
the physical tumor microenvironment, paracrine signaling
regions within tumors) is hypoxia, consequent to insufficient
involving soluble factors released into the extracellular milieu
vascularization. Hypoxia, for example, reduces the activity of
by the various cell types populating solid tumors can also con-
the TET demethylases, resulting in substantive changes in
tribute to the induction of several morphologically distinct
the methylome, in particular hypermethylation (58). Insuf-
invasive growth programs (72), only one of which—dubbed
ficient vascularization likely also limits the bioavailability of
“mesenchymal”—seems to involve the aforementioned EMT
critical blood-borne nutrients, and nutrient deprivation has
epigenetic regulatory mechanism.

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been shown for example to alter translational control and
consequently enhance the malignant phenotype of breast Epigenetic Regulatory Heterogeneity
cancer cells (59).
A growing knowledge base is heightening appreciation of
A persuasive example of hypoxia-mediated epigenetic regu-
the importance of intratumoral heterogeneity in generating
lation involves a form of invariably lethal pediatric epend- the phenotypic diversity where the fittest cells for prolif-
ymoma. Like many embryonic and pediatric tumors, this erative expansion and invasion outgrow their brethren and
form lacks recurrent mutations, in particular a dearth of hence are selected for malignant progression. Certainly, one
driver mutations in oncogenes and tumor suppressors. facet of this phenotypic heterogeneity is founded in chronic
Rather, the aberrant growth of these cancer cells is demon- or episodic genomic instability and consequent genetic het-
strably governed by a gene regulatory program induced by erogeneity in the cells populating a tumor. In addition, it
hypoxia (60, 61). Notably, the putative cell-of-origin of this is increasingly evident that there can be non–mutationally
cancer resides in a hypoxic compartment, likely sensitizing based epigenetic heterogeneity. A salient example involves
cells resident therein to the initiation of tumorigenesis by as the linker histone H1.0, which is dynamically expressed and
yet unknown cofactors. repressed in subpopulations of cancer cells within a number
Another persuasive line of evidence for microenvironmen- of tumor types, with consequent sequestration or accessibil-
tally mediated epigenetic regulation involves the invasive ity, respectively, of megabase-sized domains, including ones
growth capability of cancer cells. A classic example involves conveying hallmark capabilities (73). Notably, the population
the reversible induction of invasiveness of cancer cells at the of cancer cells with repressed H1.0 were found to have stem-
margins of many solid tumors, orchestrated by the devel- like characteristics, enhanced tumor-initiating capability, and
opmental regulatory program known as the epithelial-to- an association with poor prognosis in patients.
mesenchymal transition (EMT; refs. 62–64). Notably, a mas- Another example of epigenetically regulated plasticity has
ter regulator of the EMT, ZEB1, has been recently shown to been described in human oral squamous cell carcinomas
induce expression of a histone methyltransferase, SETD1B, (SCC), wherein cancer cells at the invasive margins adopt a
that in turn sustains ZEB1 expression in a positive feedback partial EMT (p-EMT) state lacking the aforementioned mes-
loop that maintains the (invasive) EMT regulatory state enchymal TFs but expressing other EMT-defining genes that
(65). A previous study similarly documented that induction are not expressed in the central core of the tumors (74). The
of EMT by upregulated expression of a related TF, SNAIL1, p-EMT cells evidently do not represent a clonal compartmen-
caused marked alterations in the chromatin landscape con- talization of mutationally altered cells: cultures of primary
sequent to induction of a number of chromatin modifiers, tumor-derived cancer cells contain dynamic mixtures of both
whose activity was demonstrably necessary for the mainte- p-EMThi and p-EMTlo cells, and when p-EMThi/lo cells were
nance of the phenotypic state (66). Furthermore, a roster of FACS-purified and cultured, both reverted to mixed popula-
conditions and factors to which cancer cells at the margins of tions of p-EMThi and p-EMTlo cells within 4 days. Moreover,
tumors are exposed, including hypoxia and cytokines secreted although paracrine signals from the adjacent stroma could
by stromal cells, can evidently induce the EMT and in turn be envisaged as deterministic for the p-EMThi state, the sta-
invasiveness (67, 68). ble presence and regeneration of the two epigenetic states in
A distinctive example of microenvironmental program- culture argues for a cancer cell–intrinsic mechanism. Notably,
ming of invasiveness, ostensibly unrelated to the EMT pro- this conclusion is supported by analysis of 198 cell lines rep-
gram, involves autocrine activation, in pancreas cancer cells resenting 22 cancer types, including SCC, wherein 12 stably
and others, via interstitial pressure–driven fluid flow, of a heterogeneous epigenetic states (including the p-EMT in
neuronal signaling circuit involving secreted glutamate and SCC) were variously detected in the cell line models as well
its receptor NMDAR (69, 70). Notably, the prototypical stiff- as their cognate primary tumors (75). Again, the heteroge-
ness of many solid tumors, embodied in extensive alterations neous phenotypic states could not be linked to detectable
to the extracellular matrix (ECM) that envelop the cells genetic differences, and in several cases FACS-sorted cells of

JANUARY 2022 CANCER DISCOVERY | 37

REVIEW Hanahan

a particular state were shown to dynamically reequilibrate and mutation. Notably, it can be anticipated that nonmuta-
upon culture, recapitulating a stable balance among the het- tional epigenetic reprogramming will prove to be integrally
erogeneous states seen in the original cell lines. involved in enabling the provisional new hallmark capability
Additionally, technologies for genome-wide profiling of of phenotypic plasticity discussed above, in particular being
diverse attributes—beyond DNA sequence and its mutational a driving force in the dynamic transcriptomic heterogeneity
variation—are illuminating influential elements of the cancer that is increasingly well documented in cancer cells populat-
cell genome’s annotation and organization that correlate ing malignant TMEs. The advance of single cell multi-omic
with patient prognosis, and increasingly with hallmark capa- profiling technologies is envisaged to illuminate the respec-
bilities (76–78). Epigenomic heterogeneity is being revealed tive contributions of and interplay between mutation-driven
by increasingly powerful technologies for profiling genome- versus nonmutational epigenetic regulation to the evolution
wide DNA methylation (79, 80), histone modification (81), of tumors during malignant progression and metastasis.
chromatin accessibility (82), and posttranscriptional modifi-
cation and translation of RNA (83, 84). A challenge in regard
to the postulate being considered herein will be to ascertain
POLYMORPHIC MICROBIOMES
which epigenomic modifications in particular cancer types An expansive frontier in biomedicine is unfolding via
(i) have regulatory significance and (ii) are representative of illumination of the diversity and variability of the plethora
purely nonmutational reprogramming, as opposed to being of microorganisms, collectively termed the microbiota, that

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mutation-driven and thus explainable by genome instability. symbiotically associate with the barrier tissues of the body
exposed to the external environment—the epidermis and the
Epigenetic Regulation of the Stromal Cell Types internal mucosa, in particular the gastrointestinal tract, as
Populating the Tumor Microenvironment well as the lung, the breast, and the urogenital system. There
In general, the accessory cells in the tumor microenvi- is growing appreciation that the ecosystems created by resi-
ronment that functionally contribute to the acquisition dent bacteria and fungi—the microbiomes—have profound
of hallmark capabilities are not thought to suffer genetic impact on health and disease (87), a realization fueled by
instability and mutational reprogramming to enhance their the capability to audit the populations of microbial species
tumor-promoting activities; rather it is inferred that these using next-generation sequencing and bioinformatic tech-
cells—cancer-associated fibroblasts, innate immune cells, and nologies. For cancer, the evidence is increasingly compelling
endothelial cells and pericytes of the tumor vasculature— that polymorphic variability in the microbiomes between
are epigenetically reprogrammed upon their recruitment by individuals in a population can have a profound impact on
soluble and physical factors that define the solid tumor micro- cancer phenotypes (88, 89). Association studies in human
environment (2, 85). It can be anticipated the multi-omic pro- and experimental manipulation in mouse models of can-
filing technologies currently being applied to cancer cells will cer are revealing particular microorganisms, principally but
increasingly be used to interrogate the accessory (stromal) not exclusively bacteria, which can have either protective
cells in tumors to elucidate how normal cells are corrupted or deleterious effects on cancer development, malignant
to functionally support tumor development and progression. progression, and response to therapy. So too can the global
For example, a recent study (86) suggests that such reprogram- complexity and constitution of a tissue microbiome at large.
ming can involve modifications of the epigenome in addition Indeed, while the gut microbiome has been the pioneer of
to the inductive interchange of cytokines, chemokines, and this new frontier, multiple tissues and organs have associ-
growth factors that alter intracellular signaling networks in ated microbiomes, which have distinctive characteristics in
all of these cell types: when mouse models of metastasis to regard to population dynamics and diversity of microbial
lung were treated with a combination of a DNA methyltrans- species and subspecies. This growing appreciation of the
ferase inhibitor (5-azacytidine) and an inhibitor of histone importance of polymorphically variable microbiomes in
modification (an HDAC), the infiltrating myeloid cells were health and disease posits the question: is the microbiome
found to have switched from an immature (tumor-promot- a discrete enabling characteristic that broadly affects, both
ing) progenitor state into cells resembling mature interstitial positively and negatively, the acquisition of hallmark capa-
(tumor-antagonizing) macrophages, which, in contrast to bilities for cancer? I reflect on this possibility below, illustrat-
their counterparts in untreated tumors, were incapable of ing evidence for some of the prominent tissue microbiomes
supporting the hallmark capabilities necessary for efficient implicated in cancer hallmarks (Fig.  4), beginning with the
metastatic colonization (86). It can be envisaged that multi- most prominent and evidently impactful microbiome, that
omic profiling and pharmacologic perturbation will serve to of the intestinal tract.
elucidate the reprogrammed epigenetic state in such myeloid
cells as well as other hallmark-enabling accessory cell types
populating tumor microenvironments. Diverse Modulatory Effects of the Gut Microbiome
It has long been recognized that the gut microbiome is fun-
Synopsis damentally important for the function of the large intestine
Collectively, these illustrative snapshots support the prop- (colon) in degrading and importing nutrients into the body
osition that nonmutational epigenetic reprograming will as part of metabolic homeostasis, and that distortions in the
come to be accepted as a bona fide enabling characteristic microbial populations—dysbiosis—in the colon can cause a
that serves to facilitate the acquisition of hallmark capa- spectrum of physiologic maladies (87). Among these has
bilities (Fig. 3), distinct from that of genomic DNA instability been the suspicion that the susceptibility, development, and

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Hallmarks of Cancer: New Dimensions REVIEW

Gut

Skin Lung
Modulating
tumor
Growth
Inflammation
Immune
evasions
Genome
instability
Vaginal/ Therapy
Oral resistance
cervical

Polymorphic
microbiomes Tumor

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Figure 4.  Polymorphic microbiomes. Left, while intersecting with the enabling characteristics of tumor-promoting inflammation and genomic instabil-
ity and mutation, there is growing reason to conclude that polymorphic microbiomes in one individual versus another, being resident in the colon, other
mucosa and connected organs, or in tumors themselves, can diversely influence—by either inducing or inhibiting—many of the hallmark capabilities,
and thus are potentially an instrumental and quasi-independent variable in the puzzle of how cancers develop, progress, and respond to therapy. Right,
multiple tissue microbiomes are implicated in modulating tumor phenotypes. In addition to the widely studied gut microbiome, other distinctive tissue
microbiomes, as well as the tumor microbiome, are implicated in modulating the acquisition—both positively and negatively—of the illustrated hallmark
capabilities in certain tumor types. The hallmarks of cancer graphic has been adapted from Hanahan and Weinberg (2).

pathogenesis of colon cancer is influenced by the gut micro- bacteria; the connection between butyrate-induced senes-
biome. In recent years, persuasive functional studies, involv- cence and enhanced colon tumorigenesis was demonstrated
ing fecal transplants from colon tumor–bearing patients and by the use of a senolytic drug that kills senescent cells,
mice into recipient mice predisposed to develop colon cancer which impaired tumor growth (92). In addition, bacterial-
has established a principle: there are both cancer-protective produced butyrate has pleiotropic and paradoxical effects on
and tumor-promoting microbiomes, involving particular differentiated cells versus undifferentiated (stem) cells in the
bacterial species, which can modulate the incidence and colonic epithelium in conditions where the intestinal barrier
pathogenesis of colon tumors (90). is disrupted (dysbiosis) and the bacteria are invasive, affect-
The mechanisms by which microbiota impart these modu- ing, for example, cellular energetics and metabolism, histone
latory roles are still being elucidated, but two general effects modification, cell-cycle progression, and (tumor-promoting)
are increasingly well established for tumor-promoting micro- innate immune inflammation that is immunosuppressive of
biomes and in some cases for specific tumor-promoting adaptive immune responses (93).
bacterial species. The first effect is mutagenesis of the colonic Indeed, a broad effect of polymorphic microbiomes
epithelium, consequent to the production of bacterial tox- involves the modulation of the adaptive and innate immune
ins and other molecules that either damage DNA directly, systems via multifarious routes, including the production by
or disrupt the systems that maintain genomic integrity, or bacteria of “immunomodulatory” factors that activate dam-
stress cells in other ways that indirectly impair the fidel- age sensors on epithelial or resident immune cells, resulting
ity of DNA replication and repair. A case in point is E. coli in the expression of a diverse repertoire of chemokines and
carrying the PKS locus, which demonstrably mutagenizes cytokines that can sculpt the abundance and characteristics
the human genome and is implicated in conveying hallmark- of immune cells populating the colonic epithelia and its
enabling mutations (91). underlying stroma and draining lymph nodes. In addition,
Additionally, bacteria have been reported to bind to the certain bacteria can breach both the protective biofilm and
surface of colonic epithelial cells and produce ligand mimet- the mucus lining the colonic epithelia and proceed to dis-
ics that stimulate epithelial proliferation, contributing in rupt the epithelial cell–cell tight junctions that collectively
neoplastic cells to the hallmark capability for proliferative maintain the integrity of the physical barrier that normally
signaling (88). Another mechanism by which specific bacterial compartmentalizes the intestinal microbiome. Upon invad-
species promote tumorigenesis involves butyrate-producing ing the stroma, bacteria can trigger both innate and adap-
bacteria, whose abundance is elevated in patients with colo- tive immune responses, eliciting secretion of a repertoire of
rectal cancer (92). The production of the metabolite butyrate cytokines and chemokines. One manifestation can be the
has complex physiologic effects, including the induction of creation of tumor-promoting or tumor-antagonizing immune
senescent epithelial and fibroblastic cells. A mouse model microenvironments, consequently protecting against or
of colon carcinogenesis populated with butyrate-producing facilitating tumorigenesis and malignant progression. Con-
bacteria developed more tumors than mice lacking such cordantly, the modulation by distinctive microbiomes in

JANUARY 2022 CANCER DISCOVERY | 39

REVIEW Hanahan

individual patients of the intertwined parameters of (i) elicit- organ/tissue-specific differences in the constitution of the
ing (innate) tumor promoting inflammation and (ii) escap- associated microbiomes in homeostasis, aging, and cancer,
ing (adaptive) immune destruction can be associated not with both overlapping and distinctive species and abundan-
only with prognosis, but also with responsiveness or resist- cies to that of the colon (104, 105). Moreover, association
ance to immunotherapies involving immune checkpoint studies are providing increasing evidence that local tumor-
inhibitors and other therapeutic modalities (89, 94–96). antagonizing/protective versus tumor-promoting tissue
Provisional proof-of-concept has come from recent stud- microbiomes, similarly to the gut microbiome, can modulate
ies demonstrating restored efficacy to immunotherapy susceptibility and pathogenesis to human cancers arising in
following transplants of fecal microbiota from therapy- their associated organs (106–109).
responsive patients into patients with melanoma who had
progressed during prior treatment with immune checkpoint Impact of Intratumoral Microbiota?
blockade (97, 98). Finally, pathologists have long recognized that bacteria can
An ongoing mystery has involved the molecular mecha- be detected within solid tumors, an observation that has now
nisms by which particular and variable constituents of the been substantiated with sophisticated profiling technologies.
gut microbiome systemically modulate the activity of the For example, in a survey of 1,526 tumors encompassing seven
adaptive immune system, either enhancing antitumoral human cancer types (bone, brain, breast, lung, melanoma,
immune responses evoked by immune checkpoint blockade, ovary, and pancreas), each type was characterized by a distinc-

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or rather eliciting systemic or local (intratumoral) immu- tive microbiome that was largely localized inside cancer cells
nosuppression. A recent study has shed some light: certain and immune cells, and within each tumor type, variations in
strains of Enterococcus (and other bacteria) express a pepti- the tumor microbiome could be detected and inferred to be
doglycan hydrolyase called SagA that releases mucopeptides associated with clinicopathologic features (110). Microbiota
from the bacterial wall, which can then circulate systemi- have been similarly detected in genetically engineered de novo
cally and activate the NOD2 pattern receptor, which in turn mouse models of lung and pancreas cancer, and their absence
can enhance T-cell responses and the efficacy of checkpoint in germ-free mice and/or their abrogation with antibiotics can
immunotherapy (99). Other immunoregulatory molecules demonstrably impair tumorigenesis, functionally implicating
produced by specific bacterial subspecies are being identi-
the tumor microbiome as an enabler of tumor-promoting
fied and functionally evaluated, including bacteria-produced
inflammation and malignant progression (111, 112). Associa-
inosine, a rate-limiting metabolite for T-cell activity (100).
tion studies in human pancreatic ductal adenocarcinoma and
These examples and others are beginning to chart the molec-
functional tests via fecal transplants into tumor-bearing mice
ular mechanisms by which polymorphic microbiomes are
have established that variations in the tumor microbiome—
indirectly and systemically modulating tumor immunobiol-
and the associated gut microbiome—modulate immune phe-
ogy, above and beyond immune responses consequent to
notypes and survival (113). An important challenge for the
direct physical interactions of bacteria with the immune
future will be to extend these implications to other tumor
system (101, 102).
types, and to delineate the potentially separable contribu-
Beyond the causal links to colon cancer and melanoma, the
tions of constitution and variation in the tumor microbiome
gut microbiome’s demonstrable ability to elicit the expres-
to that of the gut (and local tissue of origin) microbiome,
sion of immunomodulatory chemokines and cytokines that
enter the systemic circulation is evidently also capable of potentially by identifying specific microbial species that are
affecting cancer pathogenesis and response to therapy in functionally influential in one location or the other.
other organs of the body (94, 95). An illuminating example Synopsis
involves the development of cholangiocarcinomas in the
liver: gut dysbiosis allows the entry and transport of bacteria Among the fascinating questions for the future is whether
and bacterial products through the portal vein to the liver, microbiota resident in different tissues or populating incipi-
where TLR4 expressed on hepatocytes is triggered to induce ent neoplasias have the capability to contribute to or interfere
expression of the chemokine CXCL1, which recruits CXCR2- with the acquisition of other hallmark capabilities beyond
expressing granulocytic myeloid cells (gMDSC) that serve to immunomodulation and genome mutation, thereby influ-
suppress natural killer cells so as to evade immune destruc- encing tumor development and progression. There are clues
tion (103), and likely convey other hallmark capabilities (85). that particular bacterial species can directly stimulate the
As such, the gut microbiome is unambiguously implicated as hallmark of proliferative signaling, for example, in colonic
an enabling characteristic that can alternatively facilitate or epithelium (88), and modulate growth suppression by alter-
protect against multiple forms of cancer. ing tumor suppressor activity in different compartments of
the intestine (114), whereas direct effects on other hallmark
Beyond the Gut: Implicating Distinctive capabilities, such as avoiding cell death, inducing angiogene-
Microbiomes in Other Barrier Tissues sis, and stimulating invasion and metastasis, remain obscure,
Virtually all tissues and organs exposed, directly or indi- as does the generalizability of these observations to multiple
rectly, to the outside environment are also repositories for com- forms of human cancer. Irrespective, there is an increasingly
mensal microorganisms (104). Unlike the intestine, where the compelling case to be made that polymorphic variation in
symbiotic role of the microbiome in metabolism is well recog- microbiomes of the intestine and other organs constitutes
nized, the normal and pathogenic roles of resident microbiota a distinctive enabling characteristic for the acquisition of
in these diverse locations is still emerging. There are evidently hallmark capabilities (Fig.  4), albeit intersecting with and

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Hallmarks of Cancer: New Dimensions REVIEW

complementing those of genome instability and mutation, from their SASP-expressing, nonproliferative condition, and
and tumor-promoting inflammation. resume cell proliferation and manifestation of the associ-
ated capabilities of fully viable oncogenic cells (44). Such
transitory senescence is most well documented in cases of
SENESCENT CELLS
therapy resistance (44), representing a form of dormancy
Cellular senescence is a typically irreversible form of pro- that circumvents therapeutic targeting of proliferating can-
liferative arrest, likely evolved as a protective mechanism for cer cells, but may well prove to be more broadly operative in
maintaining tissue homeostasis, ostensibly as a complemen- other stages of tumor development, malignant progression,
tary mechanism to programmed cell death that serves to and metastasis.
inactivate and in due course remove diseased, dysfunctional, Moreover, the hallmark-promoting capabilities of senes-
or otherwise unnecessary cells. In addition to shutting down cent cells are not limited to senescent cancer cells. Cancer-
the cell division cycle, the senescence program evokes changes associated fibroblasts (CAF) in tumors have been shown to
in cell morphology and metabolism and, most profoundly, undergo senescence, creating senescent CAFs that are demon-
the activation of a senescence-associated secretory phenotype strably tumor-promoting by virtue of conveying hallmark
(SASP) involving the release of a plethora of bioactive pro- capabilities to cancer cells in the TME (115, 116, 121). More-
teins, including chemokines, cytokines, and proteases whose over, senescent fibroblasts in normal tissues produced in
identity is dependent on the cell and tissue type from which a part by natural aging or environmental insults have similarly

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senescent cell arises (115–117). Senescence can be induced in been implicated in remodeling tissue microenvironments via
cells by a variety of conditions, including microenvironmen- their SASP so as to provide paracrine support for local inva-
tal stresses such as nutrient deprivation and DNA damage, as sion (so-called “field effects”) and distant metastasis (116) of
well as damage to organelles and cellular infrastructure, and neoplasias developing in proximity. Additionally, senescent
imbalances in cellular signaling networks (115, 117), all of fibroblasts in aging skin have been shown to recruit—via their
which have been associated with the observed increase in the SASP—innate immune cells that are both immunosuppres-
abundance of senescent cells in various organs during aging sive of adaptive antitumoral immune responses anchored
(118, 119). by CD8 T cells, and stimulatory of skin tumor growth (123),
Cellular senescence has long been viewed as a protective with the latter effect potentially reflecting paracrine contribu-
mechanism against neoplasia, whereby cancerous cells are tions of such innate immune cells (myeloid cells, neutrophils,
induced to undergo senescence (120). Most of the afore- and macrophages) to other hallmark capabilities.
mentioned instigators of the senescent program are asso- While less well established, it seems likely that other abun-
ciated with malignancy, in particular DNA damage as a dant stromal cells populating particular tumor microen-
consequence of aberrant hyperproliferation, so-called onco- vironments will prove to undergo senescence, and thereby
gene-induced senescence due to hyperactivated signaling, modulate cancer hallmarks and consequent tumor phe-
and therapy-induced senescence consequent to cellular and notypes. For example, therapy-induced senescent tumor
genomic damage caused by chemotherapy and radiotherapy. endothelial cells can enhance proliferation, invasion, and
Indeed, there are well-established examples of the protective metastasis in breast cancer models (124, 125).
benefits of senescence in limiting malignant progression (118,
Certainly, such clues warrant investigation in other tumor
119). To the contrary, however, an increasing body of evidence
types to assess generality of fibroblastic, endothelial, and
reveals quite the opposite: in certain contexts, senescent cells
other stromal cell senescence as a driving force in tumor
variously stimulate tumor development and malignant pro-
evolution. Also currently unresolved are the regulatory
gression (119, 121). In one illuminating case study, senescent
mechanisms and functional determinants through which
cells were pharmacologically ablated in aging mice, in par-
a particular senescent cell type in a given TME evokes a
ticular depleting senescent cells characteristically expressing
tumor-promoting versus a tumor-antagonizing SASP, which
the cell-cycle inhibitor p16−INK4a: in addition to delaying mul-
can seeming be alternatively induced in the same senescing
tiple age-related symptoms, the depletion of senescent cells
cell type, perhaps by different instigators when immersed in
in aging mice resulted in reduced incidences of spontaneous
distinctive physiologic and neoplastic microenvironments.
tumorigenesis and cancer-associated death (122).
The principal mechanism by which senescent cells pro-
mote tumor phenotypes is thought to be the SASP, which is Synopsis
demonstrably capable of conveying, in paracrine fashion to The concept that tumors are composed of genetically
viable cancer cells in proximity, as well as to other cells in the transformed cancer cells interacting with and benefiting from
TME, signaling molecules (and proteases that activate and/ recruited and epigenetically/phenotypically corrupted acces-
or desequester them) so as to convey hallmark capabilities. sory (stromal) cells is well established as instrumental to the
Thus, in different experimental systems, senescent cancer pathogenesis of cancer. The considerations discussed above
cells have been shown to variously contribute to prolifera- and described in the reviews and reports cited herein (and
tive signaling, avoiding apoptosis, inducing angiogenesis, elsewhere) make a persuasive case for the proposition that
stimulating invasion and metastasis, and suppressing tumor senescent cells (of whatever cellular origin) should be consid-
immunity (116, 118, 120, 121). ered for addition to the roster of functionally significant cells
Yet another facet to the effects of senescent cancer cells in the tumor microenvironment (Fig.  5). As such, senescent
on cancer phenotypes involves transitory, reversible senes- cells warrant being factored into the quest for deep knowl-
cent cell states, whereby senescent cancer cells can escape edge of cancer mechanisms. Furthermore, the realization of

JANUARY 2022 CANCER DISCOVERY | 41

REVIEW Hanahan

Senescent cells
(multiple origins)

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Senescent cells

Figure 5.  Senescent cells. Heterogeneous cancer cell subtypes as well as stromal cell types and subtypes are functionally integrated into the manifes-
tations of tumors as outlaw organs. Clues are increasingly implicating senescent cell derivatives of many of these cellular constituents of the TME, and
their variable SASPs, in modulating hallmark capabilities and consequent tumor phenotypes. The hallmarks of cancer graphic has been adapted from
Hanahan and Weinberg (2).

their importance motivates the ancillary goal to therapeuti- (ii) MYC (https://cancer.sanger.ac.uk/cosmic/census-page/
cally target tumor-promoting senescent cells of all constitu- MYC),
tions, be it by pharmacologic or immunologic ablation, or by (iii) NOTCH (https://cancer.sanger.ac.uk/cosmic/census-
reprogramming the SASP into tumor-antagonizing variants page/NOTCH1; ref. 127), and
(115, 121, 126). (iv) TP53 (https://cancer.sanger.ac.uk/cosmic/census-page/
TP53),
CONCLUDING REMARKS highlighting the important challenge to more fully elu-
cidate the regulatory networks governing these acquired
While the eight hallmarks of cancer and their two ena-
capabilities.
bling characteristics have proved of enduring heuristic value in
In addition to adding cellular plasticity to the roster,
the conceptualization of cancer, the considerations presented
nonmutational epigenetic reprogramming and polymorphic
above suggest that there may be new facets of some general-
variations in organ/tissue microbiomes may come to be incor-
ity and hence of relevance to more fully understanding the
porated as mechanistic determinants—enabling characteris-
complexities, mechanisms, and manifestations of the disease.
tics—by which hallmark capabilities are acquired, along with
By applying the metric of discernable if not complete inde- tumor-promoting inflammation (itself partially intercon-
pendence from the 10 core attributes, it is arguable that these nected to the microbiome), above and beyond the mutations
four parameters may well—pursuant to further validation and and other aberrations that manifest the afore-mentioned
generalization beyond the case studies presented—become inte- oncogenic drivers.
grated into the hallmarks of cancer schematic (Fig. 6). Thus, cel- Finally, senescent cells of different origins—including can-
lular plasticity may come to be added to the roster of hallmark cer cells and various stromal cells—that functionally contrib-
capabilities. Notably, while the eight core and this nouveau ute to the development and malignant progression of cancer,
capability are each, by their definition as a hallmark, concep- albeit in markedly distinctive ways to those of their nonsenes-
tually distinguishable, aspects of their regulation are at least cent brethren, may become incorporated as generic compo-
partially interconnected in some and perhaps many cancers. nents of the TME. In conclusion, it is envisaged that raising
For example, multiple hallmarks are coordinately modulated these provisional “trial balloons” will stimulate debate, dis-
in some tumor types by canonical oncogenic drivers, including cussion, and continuing experimental investigation in the
(i) KRAS (https://cancer.sanger.ac.uk/cosmic/census-page/ cancer research community about the defining conceptual
KRAS), parameters of cancer biology, genetics, and pathogenesis.

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Hallmarks of Cancer: New Dimensions REVIEW

Sustaining Evading
proliferative signaling growth suppressors

Unlocking Nonmutational
phenotypic plasticity epigenetic reprogramming

Deregulating Avoiding immune

cellular destruction
metabolism

Resisting cell Enabling

death replicative
immortality

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Genome
Tumor-promoting
instability &
inflammation
mutation

Polymorphic
Senescent cells
microbiomes

Inducing or accessing Activating invasion &

vasculature metastasis

Figure 6.  Hallmarks of Cancer—new additions. Depicted are the canonical and prospective new additions to the “Hallmarks of Cancer.” This treatise
raises the possibility, aiming to stimulate debate, discussion, and experimental elaboration, that some or all of the four new parameters will come to be
appreciated as generic to multiple forms of human cancer and hence appropriate to incorporate into the core conceptualization of the hallmarks of cancer.
The hallmarks of cancer graphic has been adapted from Hanahan and Weinberg (2).

Author’s Disclosures 5. Perekatt AO, Shah PP, Cheung S, Jariwala N, Wu A, Gandhi V,

et  al. SMAD4 suppresses WNT-driven dedifferentiation and onco-
No disclosures were reported. genesis in the differentiated gut epithelium. Cancer Res 2018;78:
4878–90.
Acknowledgments 6. Shih IM, Wang TL, Traverso G, Romans K, Hamilton SR, Ben-Sasson S,
First and foremost, I profoundly thank Bob Weinberg for an et  al. Top-down morphogenesis of colorectal tumors. Proc Natl
exceptional tradition of insightful and formative discussions, and Acad Sci U S A 2001;98:2640–5.
for excellent comments and suggestions to the first vignette of this 7. Ordóñez-Morán P, Dafflon C, Imajo M, Nishida E, Huelsken J.
manuscript. Additionally, I wish to thank: Ben Stanger; Bradley HOXA5 counteracts stem cell traits by inhibiting Wnt signaling in
colorectal cancer. Cancer Cell 2015;28:815–29.
Bernstein, Giovanni Ciriello, and William Flavahan; Jennifer Wargo;
8. Tan SH, Barker N. Stemming colorectal cancer growth and metas-
and Sheila Stewart for their valuable comments and suggestions
tasis: HOXA5 forces cancer stem cells to differentiate. Cancer Cell
on the four vignettes, respectively, and SayoStudio for assistance in 2015;28:683–5.
crafting the figures. 9. Köhler C, Nittner D, Rambow F, Radaelli E, Stanchi F, Vandamme N,
et  al. Mouse cutaneous melanoma induced by mutant BRaf arises
Received August 4, 2021; revised November 17, 2021; accepted from expansion and dedifferentiation of mature pigmented mel-
November 18, 2021; published first January 12, 2022. anocytes. Cell Stem Cell 2017;21:679–93.
10. Shah M, Bhoumik A, Goel V, Dewing A, Breitwieser W, Kluger H,
et al. A role for ATF2 in regulating MITF and melanoma develop-
REFERENCES ment. PLoS Genet 2010;6:e1001258.
11. Claps G, Cheli Y, Zhang T, Scortegagna M, Lau E, Kim H, et al. A
1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100: transcriptionally inactive ATF2 variant drives melanomagenesis.
57–70. Cell Rep 2016;15:1884–92.
2. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. 12. Saghafinia S, Homicsko K, Di Domenico A, Wullschleger S,
Cell 2011;144:646–74. Perren A, Marinoni I, et  al. Cancer cells retrace a stepwise differ-
3. Yuan S, Norgard RJ, Stanger BZ. Cellular plasticity in cancer. Cancer entiation program during malignant progression. Cancer Discov
Discov 2019;9:837–51. 2021;11:2638–57.
4. Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, 13. Yu X-X, Qiu W-L, Yang L, Zhang Y, He M-Y, Li L-C, et al. Defining
van den Born M, et al. Crypt stem cells as the cells-of-origin of intes- multistep cell fate decision pathways during pancreatic develop-
tinal cancer. Nature 2009;457:608–11. ment at single-cell resolution. EMBO J 2019;38:e100164.

JANUARY 2022 CANCER DISCOVERY | 43

REVIEW Hanahan

14. de Thé H. Differentiation therapy revisited. Nat Rev Cancer 2018; 37. LaFave LM, Kartha VK, Ma S, Meli K, Del Priore I, Lareau C, et al.
18:117–27. Epigenomic state transitions characterize tumor progression in
15. He LZ, Merghoub T, Pandolfi PP. In vivo analysis of the molecular mouse lung adenocarcinoma. Cancer Cell 2020;38:212–28.
pathogenesis of acute promyelocytic leukemia in the mouse and its 38. Marjanovic ND, Hofree M, Chan JE, Canner D, Wu K, Trakala M,
therapeutic implications. Oncogene 1999;18:5278–92. et  al. Emergence of a high-plasticity cell state during lung cancer
16. Warrell RP, de Thé H, Wang ZY, Degos L. Acute promyelocytic leu- evolution. Cancer Cell 2020;38:229–46.
kemia. N Engl J Med 1993;329:177–89. 39. Drapkin BJ, Minna JD. Studying lineage plasticity one cell at a time.
17. Bots M, Verbrugge I, Martin BP, Salmon JM, Ghisi M, Baker A, et al. Cancer Cell 2020;38:150–2.
Differentiation therapy for the treatment of t(8;21) acute myeloid leu- 40. Inoue Y, Nikolic A, Farnsworth D, Liu A, Ladanyi M, Somwar  R,
kemia using histone deacetylase inhibitors. Blood 2014;123:1341–52. et  al. Extracellular signal-regulated kinase mediates chromatin
18. Ferrara FF, Fazi F, Bianchini A, Padula F, Gelmetti V, Minucci S, rewiring and lineage transformation in lung cancer [Internet]. Can-
et  al. Histone deacetylase-targeted treatment restores retinoic acid cer Biology; 2020 Nov. Available from: http://biorxiv.org/lookup/
signaling and differentiation in acute myeloid leukemia. Cancer Res doi/10.1101/2020.11.12.368522.
2001;61:2–7. 41. Dravis C, Chung C-Y, Lytle NK, Herrera-Valdez J, Luna G, Trejo CL,
19. Kaufman CK, Mosimann C, Fan ZP, Yang S, Thomas AJ, Ablain J, et  al. Epigenetic and transcriptomic profiling of mammary gland
et al. A zebrafish melanoma model reveals emergence of neural crest development and tumor models disclose regulators of cell state
identity during melanoma initiation. Science 2016;351:aad2197. plasticity. Cancer Cell 2018;34:466–82.
20. Morris JP, Yashinskie JJ, Koche R, Chandwani R, Tian S, Chen C-C, 42. Malta TM, Sokolov A, Gentles AJ, Burzykowski T, Poisson L,
et al. α-Ketoglutarate links p53 to cell fate during tumour suppres- Weinstein JN, et  al. Machine learning identifies stemness features

Downloaded from http://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdf by guest on 08 September 2023

sion. Nature 2019;573:595–9. associated with oncogenic dedifferentiation. Cell 2018;173:338–54.
21. Saha SK, Parachoniak CA, Ghanta KS, Fitamant J, Ross KN, 43. Miao Z-F, Lewis MA, Cho CJ, Adkins-Threats M, Park D, Brown JW,
Najem MS, et al. Mutant IDH inhibits HNF-4α to block hepatocyte et  al. A dedicated evolutionarily conserved molecular network
differentiation and promote biliary cancer. Nature 2014;513:110–4. licenses differentiated cells to return to the cell cycle. Dev Cell
22. Dang L, Su S-SM. Isocitrate dehydrogenase mutation and (R)-2-hy- 2020;55:178–94.
droxyglutarate: from basic discovery to therapeutics development. 44. De Blander H, Morel A-P, Senaratne AP, Ouzounova M, Puisieux A.
Annu Rev Biochem 2017;86:305–31. Cellular plasticity: a route to senescence exit and tumorigenesis.
23. Waitkus MS, Diplas BH, Yan H. Biological role and therapeutic Cancers 2021;13:4561.
potential of IDH mutations in cancer. Cancer Cell 2018;34:186–95. 45. Merrell AJ, Stanger BZ. Adult cell plasticity in vivo: de-differentia-
24. Phan TG, Croucher PI. The dormant cancer cell life cycle. Nat Rev tion and transdifferentiation are back in style. Nat Rev Mol Cell Biol
Cancer 2020;20:398–411. 2016;17:413–25.
25. Jiang M, Azevedo-Pouly AC, Deering TG, Hoang CQ, DiRenzo D, 46. Baylin SB, Jones PA. Epigenetic determinants of cancer. Cold Spring
Hess DA, et al. MIST1 and PTF1 collaborate in feed-forward regula- Harb Perspect Biol 2016;8:a019505.
tory loops that maintain the pancreatic acinar phenotype in adult 47. Flavahan WA, Gaskell E, Bernstein BE. Epigenetic plasticity and the
mice. Mol Cell Biol 2016;36:2945–55. hallmarks of cancer. Science 2017;357:eaal2380.
26. Krah NM, Narayanan SM, Yugawa DE, Straley JA, Wright CVE, 48. Jones PA, Issa J-PJ, Baylin S. Targeting the cancer epigenome for
MacDonald RJ, et  al. Prevention and reversion of pancreatic tum- therapy. Nat Rev Genet 2016;17:630–41.
origenesis through a differentiation-based mechanism. Dev Cell 49. Huang S. Tumor progression: Chance and necessity in Darwinian
2019;50:744–54. and Lamarckian somatic (mutationless) evolution. Prog Biophys
27. Krah NM, De La O J-P, Swift GH, Hoang CQ, Willet SG, Chen Pan F, Mol Biol 2012;110:69–86.
et al. The acinar differentiation determinant PTF1A inhibits initia- 50. Darwiche N. Epigenetic mechanisms and the hallmarks of cancer:
tion of pancreatic ductal adenocarcinoma. eLife 2015;4:e07125. an intimate affair. Am J Cancer Res 2020;10:1954–78.
28. Shi G, DiRenzo D, Qu C, Barney D, Miley D, Konieczny SF. Main- 51. Feng Y, Liu X, Pauklin S. 3D chromatin architecture and epigenetic
tenance of acinar cell organization is critical to preventing Kras- regulation in cancer stem cells. Protein Cell 2021;12:440–54.
induced acinar-ductal metaplasia. Oncogene 2013;32:1950–8. 52. Nam AS, Chaligne R, Landau DA. Integrating genetic and non-
29. Kopp JL, von Figura G, Mayes E, Liu F-F, Dubois CL, Morris JP, genetic determinants of cancer evolution by single-cell multi-omics.
et al. Identification of Sox9-dependent acinar-to-ductal reprogram- Nat Rev Genet 2021;22:3–18.
ming as the principal mechanism for initiation of pancreatic ductal 53. Bitman-Lotan E, Orian A. Nuclear organization and regulation of
adenocarcinoma. Cancer Cell 2012;22:737–50. the differentiated state. Cell Mol Life Sci CMLS 2021;78:3141–58.
30. Julian LM, McDonald AC, Stanford WL. Direct reprogramming with 54. Goldberg AD, Allis CD, Bernstein E. Epigenetics: a landscape takes
SOX factors: masters of cell fate. Curr Opin Genet Dev 2017;46:24–36. shape. Cell 2007;128:635–8.
31. Grimm D, Bauer J, Wise P, Krüger M, Simonsen U, Wehland M, et al. 55. Zeng Y, Chen T. DNA methylation reprogramming during mam-
The role of SOX family members in solid tumours and metastasis. malian development. Genes 2019;10:257.
Semin Cancer Biol 2020;67:122–53. 56. Hegde AN, Smith SG. Recent developments in transcriptional and
32. Mu P, Zhang Z, Benelli M, Karthaus WR, Hoover E, Chen C-C, et al. translational regulation underlying long-term synaptic plasticity
SOX2 promotes lineage plasticity and antiandrogen resistance in and memory. Learn Mem 2019;26:307–17.
TP53- and RB1-deficient prostate cancer. Science 2017;355:84–8. 57. Kim S, Kaang B-K. Epigenetic regulation and chromatin remodeling
33. Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, in learning and memory. Exp Mol Med 2017;49:e281.
Tibes  R, et  al. Inhibition of the hedgehog pathway in advanced 58. Thienpont B, Van Dyck L, Lambrechts D. Tumors smother their
basal-cell carcinoma. N Engl J Med 2009;361:1164–72. epigenome. Mol Cell Oncol 2016;3:e1240549.
34. Biehs B, Dijkgraaf GJP, Piskol R, Alicke B, Boumahdi S, Peale F, 59. Gameiro PA, Struhl K. Nutrient deprivation elicits a transcriptional
et al. A cell identity switch allows residual BCC to survive Hedgehog and translational inflammatory response coupled to decreased pro-
pathway inhibition. Nature 2018;562:429–33. tein synthesis. Cell Rep 2018;24:1415–24.
35. Boumahdi S, de Sauvage FJ. The great escape: tumour cell plasticity in 60. Lin GL, Monje M. Understanding the deadly silence of posterior
resistance to targeted therapy. Nat Rev Drug Discov 2020;19:39–56. fossa A ependymoma. Mol Cell 2020;78:999–1001.
36. Groves SM, Ireland A, Liu Q, Simmons AJ, Lau K, Iams WT, 61. Michealraj KA, Kumar SA, Kim LJY, Cavalli FMG, Przelicki D,
et al. Cancer Hallmarks Define a Continuum of Plastic Cell States Wojcik JB, et al. Metabolic regulation of the epigenome drives lethal
between Small Cell Lung Cancer Archetypes [Internet]. Systems infantile ependymoma. Cell 2020;181:1329–45.
Biology; 2021 Jan. Available from: http://biorxiv.org/lookup/doi/10. 62. Bakir B, Chiarella AM, Pitarresi JR, Rustgi AK. EMT, MET, plasticity,
1101/2021.01.22.427865. and tumor metastasis. Trends Cell Biol 2020;30:764–76.

44 | CANCER DISCOVERY JANUARY 2022 AACRJournals.org

Hallmarks of Cancer: New Dimensions REVIEW

63. Gupta PB, Pastushenko I, Skibinski A, Blanpain C, Kuperwasser C. 87. Thomas S, Izard J, Walsh E, Batich K, Chongsathidkiet P, Clarke G,
Phenotypic plasticity: driver of cancer initiation, progression, and et al. The host microbiome regulates and maintains human health:
therapy resistance. Cell Stem Cell 2019;24:65–78. a primer and perspective for non-microbiologists. Cancer Res
64. Lambert AW, Weinberg RA. Linking EMT programmes to normal 2017;77:1783–812.
and neoplastic epithelial stem cells. Nat Rev Cancer 2021;21:325–38. 88. Dzutsev A, Badger JH, Perez-Chanona E, Roy S, Salcedo R, Smith CK,
65. Lindner P, Paul S, Eckstein M, Hampel C, Muenzner JK, Erlenbach- et al. Microbes and cancer. Annu Rev Immunol 2017;35:199–228.
Wuensch K, et  al. EMT transcription factor ZEB1 alters the epige- 89. Helmink BA, Khan MAW, Hermann A, Gopalakrishnan V, Wargo JA.
netic landscape of colorectal cancer cells. Cell Death Dis 2020;11:147. The microbiome, cancer, and cancer therapy. Nat Med 2019;25:
66. Javaid S, Zhang J, Anderssen E, Black JC, Wittner BS, Tajima K, 377–88.
et  al. Dynamic chromatin modification sustains epithelial-mesen- 90. Sears CL, Garrett WS. Microbes, microbiota, and colon cancer. Cell
chymal transition following inducible expression of Snail-1. Cell Host Microbe 2014;15:317–28.
Rep 2013;5:1679–89. 91. Pleguezuelos-Manzano C, Puschhof J, Rosendahl Huber A, van
67. Serrano-Gomez SJ, Maziveyi M, Alahari SK. Regulation of epithelial- Hoeck A, Wood HM, Nomburg J, et  al. Mutational signature in
mesenchymal transition through epigenetic and post-translational colorectal cancer caused by genotoxic pks+ E. coli. Nature 2020;580:
modifications. Mol Cancer 2016;15:18. 269–73.
68. Skrypek N, Goossens S, De Smedt E, Vandamme N, Berx G. 92. Okumura S, Konishi Y, Narukawa M, Sugiura Y, Yoshimoto S,
Epithelial-to-mesenchymal transition: epigenetic reprogramming Arai  Y, et  al. Gut bacteria identified in colorectal cancer patients
driving cellular plasticity. Trends Genet TIG 2017;33:943–59. promote tumourigenesis via butyrate secretion. Nat Commun 2021;
69. Li L, Hanahan D. Hijacking the neuronal NMDAR signaling circuit 12:5674.

Downloaded from http://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdf by guest on 08 September 2023

to promote tumor growth and invasion. Cell 2013;153:86–100. 93. Salvi PS, Cowles RA. Butyrate and the intestinal epithelium: modu-
70. Li L, Zeng Q, Bhutkar A, Galván JA, Karamitopoulou E, Noordermeer lation of proliferation and inflammation in homeostasis and dis-
D, et al. GKAP acts as a genetic modulator of NMDAR signaling to ease. Cells 2021;10:1775.
govern invasive tumor growth. Cancer Cell 2018;33:736–51. 94. Fessler J, Matson V, Gajewski TF. Exploring the emerging role of
71. Mohammadi H, Sahai E. Mechanisms and impact of altered tumour the microbiome in cancer immunotherapy. J Immunother Cancer
mechanics. Nat Cell Biol 2018;20:766–74. 2019;7:108.
72. Odenthal J, Takes R, Friedl P. Plasticity of tumor cell invasion: 95. Gopalakrishnan V, Helmink BA, Spencer CN, Reuben A, Wargo JA.
governance by growth factors and cytokines. Carcinogenesis 2016; The influence of the gut microbiome on cancer, immunity, and
37:1117–28. cancer immunotherapy. Cancer Cell 2018;33:570–80.
73. Torres CM, Biran A, Burney MJ, Patel H, Henser-Brownhill T, 96. Zitvogel L, Ma Y, Raoult D, Kroemer G, Gajewski TF. The micro-
Cohen A-HS, et al. The linker histone H1.0 generates epigenetic and biome in cancer immunotherapy: diagnostic tools and therapeutic
functional intratumor heterogeneity. Science 2016;353:aaf1644. strategies. Science 2018;359:1366–70.
74. Puram SV, Tirosh I, Parikh AS, Patel AP, Yizhak K, Gillespie S, et al. 97. Baruch EN, Youngster I, Ben-Betzalel G, Ortenberg R, Lahat A, Katz L,
Single-cell transcriptomic analysis of primary and metastatic tumor et  al. Fecal microbiota transplant promotes response in immuno-
ecosystems in head and neck cancer. Cell 2017;171:1611–24. therapy-refractory melanoma patients. Science 2021;371:602–9.
75. Kinker GS, Greenwald AC, Tal R, Orlova Z, Cuoco MS, McFarland 98. Davar D, Dzutsev AK, McCulloch JA, Rodrigues RR, Chauvin J-M,
JM, et  al. Pan-cancer single-cell RNA-seq identifies recurring pro- Morrison RM, et  al. Fecal microbiota transplant overcomes resist-
grams of cellular heterogeneity. Nat Genet 2020;52:1208–18. ance to anti–PD-1 therapy in melanoma patients. Science 2021;
76. Murtha M, Esteller M. Extraordinary cancer epigenomics: think- 371:595–602.
ing outside the classical coding and promoter box. Trends Cancer 99. Griffin ME, Espinosa J, Becker JL, Luo J-D, Carroll TS, Jha JK,
2016;2:572–84. et al. Enterococcus peptidoglycan remodeling promotes checkpoint
77. Nebbioso A, Tambaro FP, Dell’Aversana C, Altucci L. Cancer epige- inhibitor cancer immunotherapy. Science 2021;373:1040–6.
netics: moving forward. PLoS Genet 2018;14:e1007362. 100. Mager LF, Burkhard R, Pett N, Cooke NCA, Brown K, Ramay H,
78. Tavernari D, Battistello E, Dheilly E, Petruzzella AS, Mina M, et  al. Microbiome-derived inosine modulates response to check-
Sordet-Dessimoz J, et al. Non-genetic evolution drives lung adeno- point inhibitor immunotherapy. Science 2020;369:1481–9.
carcinoma spatial heterogeneity and progression. Cancer Discov 101. Ansaldo E, Belkaid Y. How microbiota improve immunotherapy.
2021;11:1490–507. Science 2021;373:966–7.
79. Heyn H, Vidal E, Ferreira HJ, Vizoso M, Sayols S, Gomez A, et  al. 102. Ansaldo E, Farley TK, Belkaid Y. Control of immunity by the micro-
Epigenomic analysis detects aberrant super-enhancer DNA methyla- biota. Annu Rev Immunol 2021;39:449–79.
tion in human cancer. Genome Biol 2016;17:11. 103. Zhang Q, Ma C, Duan Y, Heinrich B, Rosato U, Diggs LP, et al. Gut
80. Saghafinia S, Mina M, Riggi N, Hanahan D, Ciriello G. Pan-cancer microbiome directs hepatocytes to recruit MDSCs and promote
landscape of aberrant DNA methylation across human tumors. Cell cholangiocarcinoma. Cancer Discov 2021;11:1248–67.
Rep 2018;25:1066–80. 104. Ding T, Schloss PD. Dynamics and associations of microbial com-
81. Audia JE, Campbell RM. Histone modifications and cancer. Cold munity types across the human body. Nature 2014;509:357–60.
Spring Harb Perspect Biol 2016;8:a019521. 105. Byrd AL, Liu M, Fujimura KE, Lyalina S, Nagarkar DR, Charbit B,
82. Corces MR, Granja JM, Shams S, Louie BH, Seoane JA, Zhou W, et  al. Gut microbiome stability and dynamics in healthy donors
et al. The chromatin accessibility landscape of primary human can- and patients with non-gastrointestinal cancers. J Exp Med 2021;
cers. Science 2018;362:eaav1898. 218:e20200606.
83. Esteve-Puig R, Bueno-Costa A, Esteller M. Writers, readers and 106. Healy CM, Moran GP. The microbiome and oral cancer: more ques-
erasers of RNA modifications in cancer. Cancer Lett 2020;474: tions than answers. Oral Oncol 2019;89:30–3.
127–37. 107. Swaney MH, Kalan LR. Living in your skin: microbes, molecules and
84. Janin M, Coll-SanMartin L, Esteller M. Disruption of the RNA mechanisms. Infect Immun 2021;89:e00695–20.
modifications that target the ribosome translation machinery in 108. Willis JR, Gabaldón T. The human oral microbiome in health
human cancer. Mol Cancer 2020;19:70. and disease: from sequences to ecosystems. Microorganisms 2020;
85. Hanahan D, Coussens LM. Accessories to the crime: functions 8:308.
of cells recruited to the tumor microenvironment. Cancer Cell 109. Xu J, Peng J-J, Yang W, Fu K, Zhang Y. Vaginal microbiomes and
2012;21:309–22. ovarian cancer: a review. Am J Cancer Res 2020;10:743–56.
86. Lu Z, Zou J, Li S, Topper MJ, Tao Y, Zhang H, et al. Epigenetic ther- 110. Nejman D, Livyatan I, Fuks G, Gavert N, Zwang Y, Geller LT, et al.
apy inhibits metastases by disrupting premetastatic niches. Nature The human tumor microbiome is composed of tumor type-specific
2020;579:284–90. intracellular bacteria. Science 2020;368:973–80.

JANUARY 2022 CANCER DISCOVERY | 45

REVIEW Hanahan

111. Jin C, Lagoudas GK, Zhao C, Bullman S, Bhutkar A, Hu B, et  al. 120. Lee S, Schmitt CA. The dynamic nature of senescence in cancer. Nat
Commensal microbiota promote lung cancer development via γδ T Cell Biol 2019;21:94–101.
cells. Cell 2019;176:998–1013. 121. Wang B, Kohli J, Demaria M. Senescent cells in cancer therapy:
112. Pushalkar S, Hundeyin M, Daley D, Zambirinis CP, Kurz E, friends or foes? Trends Cancer 2020;6:838–57.
Mishra A, et al. The pancreatic cancer microbiome promotes onco- 122. Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, et al.
genesis by induction of innate and adaptive immune suppression. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifes-
Cancer Discov 2018;8:403–16. pan. Nature 2016;530:184–9.
113. McAllister F, Khan MAW, Helmink B, Wargo JA. The tumor micro- 123. Ruhland MK, Loza AJ, Capietto A-H, Luo X, Knolhoff BL,
biome in pancreatic cancer: bacteria and beyond. Cancer Cell Flanagan KC, et al. Stromal senescence establishes an immunosup-
2019;36:577–9. pressive microenvironment that drives tumorigenesis. Nat Com-
114. Kadosh E, Snir-Alkalay I, Venkatachalam A, May S, Lasry A, Elyada mun 2016;7:11762.
E, et  al. The gut microbiome switches mutant p53 from tumour- 124. Hwang HJ, Lee Y-R, Kang D, Lee HC, Seo HR, Ryu J-K, et  al.
suppressive to oncogenic. Nature 2020;586:133–8. Endothelial cells under therapy-induced senescence secrete CXCL11,
115. Birch J, Gil J. Senescence and the SASP: many therapeutic avenues. which increases aggressiveness of breast cancer cells. Cancer Lett
Genes Dev 2020;34:1565–76. 2020;490:100–10.
116. Faget DV, Ren Q, Stewart SA. Unmasking senescence: context- 125. Wang D, Xiao F, Feng Z, Li M, Kong L, Huang L, et  al. Sunitinib
dependent effects of SASP in cancer. Nat Rev Cancer 2019;19: facilitates metastatic breast cancer spreading by inducing endothe-
439–53. lial cell senescence. Breast Cancer Res 2020;22:103.
117. Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, 126. Amor C, Feucht J, Leibold J, Ho Y-J, Zhu C, Alonso-Curbelo D, et al.

Downloaded from http://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdf by guest on 08 September 2023

Bishop C, et  al. Cellular senescence: defining a path forward. Cell Senolytic CAR T cells reverse senescence-associated pathologies.
2019;179:813–27. Nature 2020;583:127–32.
118. He S, Sharpless NE. Senescence in health and disease. Cell 2017; 127. Aster JC, Pear WS, Blacklow SC. The varied roles of notch in cancer.
169:1000–11. Annu Rev Pathol 2017;12:245–75.
119. Kowald A, Passos JF, Kirkwood TBL. On the evolution of cellular 128. Kuczynski EA, Vermeulen PB, Pezzella F, Kerbel RS, Reynolds AR.
senescence. Aging Cell 2020;19:e13270. Vessel co-option in cancer. Nat Rev Clin Oncol 2019;16:469–93.

46 | CANCER DISCOVERY JANUARY 2022 AACRJournals.org