JANUARY 2022 CANCER DISCOVERY | 31
INTRODUCTION
The Hallmarks of Cancer were proposed as a set of functional
capabilities acquired by human cells as they make
their way from normalcy to neoplastic growth states, more
specifically capabilities that are crucial for their ability to
form malignant tumors. In these articles (1, 2), Bob Weinberg
and I enumerated what we imagined were shared commonalities
that unite all types of cancer cells at the level of cellular
phenotype. The intent was to provide a conceptual scaffold
that would make it possible to rationalize the complex phenotypes
of diverse human tumor types and variants in terms
of a common set of underlying cellular parameters. Initially
we envisaged the complementary involvement of six distinct
hallmark capabilities and later expanded this number to
eight. This formulation was influenced by the recognition
that human cancers develop as products of multistep processes,
and that the acquisition of these functional capabilities
might be mapped in some fashion to the distinguishable
steps of tumor pathogenesis. Certainly, the diversity of malignant
pathogenesis spanning multiple tumor types and an
increasing plethora of subtypes includes various aberrations
(and hence acquired capabilities and characteristics) that are
the result of tissue-specific barriers necessarily circumvented
during particular tumorigenesis pathways. While appreciating
that such specialized mechanisms can be instrumental,
we limited the hallmarks designation to parameters having
broad engagement across the spectrum of human cancers.
The eight hallmarks currently comprise (Fig.  1, left) the
acquired capabilities for sustaining proliferative signaling,
evading growth suppressors, resisting cell death, enabling
replicative immortality, inducing/accessing vasculature,
activating invasion and metastasis, reprogramming cellular
metabolism, and avoiding immune destruction. In the most
recent elaboration of this concept (2), deregulating cellular
metabolism and avoiding immune destruction were segregated
as “emerging hallmarks,” but now, eleven years later, it
is evident that they, much like the original six, can be considered
core hallmarks of cancer, and are included as such in the
current depiction (Fig. 1, left).
As we noted at the time, these hallmark traits, on their
own, fail to address the complexities of cancer pathogenesis,
that is, the precise molecular and cellular mechanisms that
allow evolving preneoplastic cells to develop and acquire
these aberrant phenotypic capabilities in the course of tumor
development and malignant progression. Accordingly, we
added another concept to the discussion, portrayed as “enabling
characteristics,” consequences of the aberrant condition
of neoplasia that provide means by which cancer cells
and tumors can adopt these functional traits. As such, the
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 underlying
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 disrupted
differentiation is a discrete hallmark capability, and that nonmutational epigenetic reprogramming
and polymorphic microbiomes both constitute distinctive enabling characteristics that facilitate
the acquisition of hallmark capabilities. Additionally, senescent cells, of varying origins, may be added
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 computational
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 dimensions
presented in this perspective may add value to that endeavor, to more fully understand mechanisms
of cancer development and malignant progression, and apply that knowledge to cancer medicine.
Ludwig Institute for Cancer Research – Lausanne Branch, Lausanne, Switzerland.
The Swiss Institute for Experimental Cancer Research (ISREC)
within the School of Life Sciences at the Swiss Federal Institute of Technology
Lausanne (EPFL), Lausanne, Switzerland. The Swiss Cancer Center
Leman (SCCL), Lausanne, Switzerland.
Corresponding Author: Douglas Hanahan, Agora Translational Cancer
Research Center, Rue du Bugnon 25A, Lausanne CH-1011, Switzerland.
Phone: 41-21-545-1119; E-mail: douglas.hanahan@epfl.ch
Cancer Discov 2022;12:31–46
doi: 10.1158/2159-8290.CD-21-1059
©2021 American Association for Cancer Research
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
32 | CANCER DISCOVERY JANUARY 2022	AACRJournals.org
HanahanREVIEW
enabling characteristics reflected upon molecular and cellular
mechanisms by which hallmarks are acquired rather
than the aforementioned eight capabilities themselves.
These two enabling processes were genome instability and
tumor-promoting inflammation.
We further recognized that the tumor microenvironment
(TME), herein defined to be composed of heterogeneous and
interactive populations of cancer cells and cancer stem cells
along with a multiplicity of recruited stromal cell types—the
transformed parenchyma and the associated stroma—is now
widely appreciated to play an integral role in tumorigenesis
and malignant progression.
Given the continued interest in these formulations and our
enduring intent to encourage ongoing discussion and refinement
of the Hallmarks scheme, it is appropriate to consider
a frequently posed question: are there additional features of
this conceptual model that might be incorporated, respecting
the need to ensure that they are broadly applicable across the
spectrum of human cancers? Accordingly, I present several
prospective new hallmarks and enabling characteristics, ones
that might in due course become incorporated as core components
of the hallmarks of cancer conceptualization. These
parameters are “unlocking phenotypic plasticity,” “nonmutational
epigenetic reprogramming,” “polymorphic microbiomes,”
and “senescent cells” (Fig. 1, right). Importantly, the
examples presented in support of these propositions are illustrative
but by no means comprehensive, as there is a growing
and increasingly persuasive body of published evidence in
support of each vignette.
UNLOCKING PHENOTYPIC PLASTICITY
During organogenesis, the development, determination,
and organization of cells into tissues in order to assume
homeostatic functions is accompanied by terminal differentiation,
whereby progenitor cells—sometimes irrevocably—
stop growing upon culmination of these processes. As such,
the end result of cellular differentiation is in most cases
antiproliferative and constitutes a clear barrier to the continuing
proliferation that is necessary for neoplasia. There
is increasing evidence that unlocking the normally restricted
capability for phenotypic plasticity in order to evade or
escape from the state of terminal differentiation is a critical
component of cancer pathogenesis (3). This plasticity
can operate in several manifestations (Fig. 2). Thus, nascent
cancer cells originating from a normal cell that had advanced
down a pathway approaching or assuming a fully differentiated
state may reverse their course by dedifferentiating back
to progenitor-like cell states. Conversely, neoplastic cells
arising from a progenitor cell that is destined to follow a
pathway leading to end-stage differentiation may shortcircuit
the process, maintaining the expanding cancer cells in
a partially differentiated, progenitor-like state. Alternatively,
transdifferentiation may operate, in which cells that were
initially committed into one differentiation pathway switch
to an entirely different developmental program, thereby
acquiring tissue-specific traits that were not preordained by
their normal cells-of-origin. The following examples support
the argument that differing forms of cellular plasticity,
Sustaining
proliferative signaling
Deregulating
cellular
metabolism
Resisting
cell death
Genome
instability &
mutation
Inducing or accessing
vasculature
Activating invasion
& metastasis
Tumor-promoting
inflammation
Evading
growth suppressors
Emerging hallmarks &
enabling characteristics
Avoiding
immune
destruction
Unlocking
phenotypic
plasticity
Senescent
cells
Polymorphic
microbiomes
Nonmutational
epigenetic
reprogramming
Enabling
replicative
immortality
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” introduced
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 otherwise
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,” “polymorphic
microbiomes,” and “senescent cells.” The hallmarks of cancer graphic has been adapted from Hanahan and Weinberg (2).
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
	 JANUARY 2022 CANCER DISCOVERY | 33
Hallmarks of Cancer: New Dimensions REVIEW
when taken together, constitute a functionally distinct
hallmark capability.
Dedifferentiation
Colon carcinogenesis exemplifies disrupted differentiation,
in that there is a teleological necessity for incipient cancer
cells to escape from the conveyer belt of terminal differentiation
and exfoliation, which could in principle occur via
dedifferentiation of not yet irrevocably terminally differentiated
colonic epithelial cells, or via blocked differentiation
of progenitor/stem cells in the crypts that spawn these differentiating
cells. Both differentiated cells and stem cells
have been implicated as cell-of-origin for colon cancer (4–6).
Two developmental transcription factors (TF), the homeobox
protein HOXA5 and SMAD4, the latter involved in BMP
signal transmission, are highly expressed in differentiating
colonic epithelial cells, and typically lost in advanced colon
carcinomas, which characteristically express markers of stem
and progenitor cells. Functional perturbations in mouse
models have shown that forced expression of HOXA5 in
colon cancer cells restores differentiation markers, suppresses
stem cell phenotypes, and impairs invasion and metastasis,
providing a rationale for its characteristic downregulation
(7, 8). SMAD4, by contrast, both enforces differentiation and
thereby suppresses proliferation driven by oncogenic WNT
signaling, revealed by the engineered loss of SMAD4 expression,
providing an explanation for its loss of expression so as
to enable dedifferentiation and, subsequently, WNT-driven
hyperproliferation (5). Notably, the loss of both of these “differentiation
suppressors” with consequent dedifferentiation
is associated with acquisition of other hallmark capabilities,
as are other hallmark-inducing regulators, which complicates
the strict definition of this provisional hallmark as separable
and independent.
Another line of evidence involves suppressed expression
of the MITF master regulator of melanocyte differentiation,
which is evidently involved in the genesis of aggressive forms
of malignant melanoma. Loss of this developmental TF is
associated with the reactivation of neural crest progenitor
genes and the downregulation of genes that characterize
fully differentiated melanocytes. The reappearance of the
neural crest genes indicates that these cells revert to the progenitor
state from which melanocytes arise developmentally.
Moreover, a lineage tracing study of BRAF-induced melanomas
established mature pigmented melanocytes as the cells
of origin, which undergo dedifferentiation during the course
of tumorigenesis (9). Of note, the mutant BRAF oncogene,
which is found in more than half of cutaneous melanomas,
induces hyperproliferation that precedes and hence is mechanistically
separable from the subsequent dedifferentiation
arising from downregulation of MITF. Another study functionally
implicated upregulation of the developmental TF
ATF2, whose characteristic expression in mouse and human
melanomas indirectly suppresses MITF1, concomitant with
malignant progression of the consequently dedifferentiated
melanoma cells (10). Conversely, expression in melanomas
of mutant forms of ATF2 that fail to repress MITF results in
well-differentiated melanomas (11).
Additionally, a recent study (12) has associated lineage
dedifferentiation with malignant progression from pancreatic
islet cell neoplasias into metastasis-prone carcinomas;
these neuroendocrine cells and derivative tumors arise from
a developmental lineage that is distinct from the one generating
the far larger number of adjacent cells that form
the exocrine and pancreas and the ductal adenocarcinomas
Unlocking
phenotypic
plasticity
Progenitor cell Differentiated cell
Normal differentiation
Dedifferentiation
Blocked
differentiation
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 pathogenesis.
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).
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
34 | CANCER DISCOVERY JANUARY 2022	AACRJournals.org
HanahanREVIEW
that arise therefrom. Notably, the multistep differentiation
pathway of islet progenitor cells into mature β cells has been
thoroughly characterized (13). Comparative transcriptome
profiling reveals that adenoma-like islet tumors are most similar
to immature but differentiated insulin-producing β cells,
whereas the invasive carcinomas are most similar to embryonic
islet cell precursors. The progression toward poorly
differentiated carcinomas involves a first step of dedifferentiation
that does not initially involve increased proliferation
or reduced apoptosis when compared with the well-differentiated
adenomas, both of which rather occur later. Thus, the
discrete step of dedifferentiation is not driven by observable
alterations in the hallmark traits of sustained proliferation
and resistance to apoptosis. Rather, upregulation of a miRNA
previously implicated in specifying the islet progenitor state,
one that is downregulated during terminal differentiation
of β cells, has been shown to orchestrate the observed dedifferentiation
occurring during malignant progression (12).
Blocked Differentiation
While the above examples illustrate how suppression of
differentiation factor expression can facilitate tumorigenesis
by enabling more well-differentiated cells to dedifferentiate
into progenitors, in other cases incompletely differentiated
progenitor cells can suffer regulatory changes that actively
block their continued advance into fully differentiated, typically
nonproliferative states.
Acute promyelocytic leukemia (APL) has long been documented
to result from a chromosomal translocation that
fuses the PML locus with the gene encoding the retinoic
acid  α  nuclear receptor (RARα). Myeloid progenitor cells
bearing such translocations are evidently unable to continue
their usual terminal differentiation into granulocytes, resulting
in cells trapped in a proliferative, promyelocytic progenitor
stage (14). Proof-of-concept of this scheme comes from
treating cultured APL cells, mouse models of this disease,
as well as afflicted patients, with retinoic acid, the ligand of
RARα; this therapeutic treatment causes the neoplastic APL
cells to differentiate into ostensibly mature nonproliferating
granulocytes, short-circuiting their continuing proliferative
expansion (14–16).
A variation on this theme involves another form of acute
myeloid leukemia, this one carrying the t(8;21) translocation,
which produces the AML1–ETO fusion protein. This protein
can, on its own, transform myeloid progenitors, at least in
part by blocking their differentiation. Therapeutic intervention
in mouse models and in patients with a pharmacologic
inhibitor of a chromatin-modifying histone deacetylase
(HDAC) causes the myeloid leukemia cells to recommence
their differentiation into cells with a more mature myeloid
cell morphology. Concomitant with this response is a reduction
in proliferative capacity, thereby impairing the progression
of this leukemia (17, 18).
A third example, in melanoma, involves a developmental
TF, SOX10, which is normally downregulated during melanocyte
differentiation. Gain- and loss-of-function studies
in a zebrafish model of BRAF-induced melanoma have demonstrated
that aberrantly maintained expression of SOX10
blocks differentiation of neural progenitor cells into melanocytes,
enabling BRAF-driven melanomas to form (19).
Other examples of differentiation modulators involve the
metabolite alpha-ketoglutarate (αKG), a necessary cofactor
for a number of chromatin-modifying enzymes, which is
demonstrably involved in stimulating certain differentiated
cell states. In pancreas cancer, the tumor suppressor p53
stimulates the production of αKG and maintenance of a more
well-differentiated cell state, whereas prototypical loss of p53
function results in reductions in αKG levels and consequent
dedifferentiation associated with malignant progression (20).
In one form of liver cancer, mutation of an isocitrate dehydrogenase
gene (IDH1/2) results in the production not of
differentiation-inducing  αKG but rather a related “oncometabolite,”
D-2-hydroxygluterate (D2HG), which has been
shown to block hepatocyte differentiation from liver progenitor
cells by D2HG-mediated repression of a master regulator
of hepatocyte differentiation and quiescence, HNF4a. The
D2HG-mediated suppression of HNF4a function elicits a
proliferative expansion of the hepatocyte progenitor cells in
the liver, which become susceptible to oncogenic transformation
upon subsequent mutational activation of the KRAS
oncogene that drives malignant progression to liver cholangiocarcinoma
(21). Mutant IDH1/2 and their oncometabolite
D2HG are also operative in a variety of myeloid and other
solid tumor types, where D2HG inhibits  αKG-dependent
dioxygenases necessary for histone and DNA methylation
events that mediate alterations in chromatin structure during
developmental lineage differentiation, thereby freezing
incipient cancer cells in a progenitor state (22, 23).
An additional, related concept is “circumvented differentiation,”
wherein partially or undifferentiated progenitor/
stem cells exit the cell cycle and become dormant, residing in
protective niches, with the potential to reinitiate proliferative
expansion (24), albeit still with the selective pressure to disrupt
their programmed differentiation in one way or another.
Transdifferentiation
The concept of transdifferentiation has long been recognized
by pathologists in the form of tissue metaplasia,
wherein cells of a particular differentiated phenotype markedly
change their morphology to become clearly recognizable
as elements of another tissue, of which one prominent example
is Barrett’s esophagus, where chronic inflammation of
the stratified squamous epithelium of the esophagus induces
transdifferentiation into a simple columnar epithelium that
is characteristic of the intestine, thereby facilitating the subsequent
development of adenocarcinomas, and not the squamous
cell carcinomas that would be anticipated to arise from
this squamous epithelium (3). Now, molecular determinants
are revealing mechanisms of transdifferentiation in various
cancers, both for cases where gross tissue metaplasia is evident
and for others where it is rather more subtle, as the
following examples illustrate.
One illuminating case for transdifferentiation as a discrete
event in tumorigenesis involves pancreatic ductal adenocarcinoma
(PDAC), wherein one of the implicated cells
of origin, the pancreatic acinar cell, can become transdifferentiated
into a ductal cell phenotype during the initiation
of neoplastic development. Two TFs—PTF1a and MIST1—
govern, via their expression in the context of self-sustaining,
“feed-forward” regulatory loops, the specification and
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
	 JANUARY 2022 CANCER DISCOVERY | 35
Hallmarks of Cancer: New Dimensions REVIEW
maintenance of the differentiated pancreatic acinar cell
state (25). Both of these TFs are frequently downregulated
during neoplastic development and malignant progression
of human and mouse PDAC. Functional genetic studies in
mice and cultured human PDAC cells have demonstrated
that experimentally forced expression of PTF1a impairs
KRAS-induced transdifferentiation and proliferation, and
can also force the redifferentiation of already neoplastic
cells into a quiescent acinar cell phenotype (26). Conversely,
suppression of PTF1a expression elicits acinar-to-ductal
metaplasia, namely transdifferentiation, and thereby sensitizes
the duct-like cells to oncogenic KRAS transformation,
accelerating subsequent development of invasive PDAC
(27). Similarly, forced expression of MIST1 in KRAS-expressing
pancreas also blocks transdifferentiation and impairs
the initiation of pancreatic tumorigenesis otherwise facilitated
by the formation of premalignant duct-like (PanIN)
lesions, whereas genetic deletion of MIST1 enhances their
formation and the initiation of KRAS-driven neoplastic
progression (28). Loss of either PTF1 or MIST1 expression
during tumorigenesis is associated with elevated expression
of another developmental regulatory TF, SOX9, which is
normally operative in the specification of ductal cells (27,
28). Forced upregulation of SOX9, obviating the need to
downregulate PTF1a and MIST1, has also been shown to
stimulate transdifferentiation of acinar cells into a ductal
cell phenotype that is sensitive to KRAS-induced neoplasia
(29), implicating SOX9 as a key functional effector of their
downregulation in the genesis of human PDAC. Thus,
three TFs that regulate pancreatic differentiation can be
variously altered to induce a transdifferentiated state that
facilitates—in the context of mutational activation of KRAS—
oncogenic transformation and the initiation of tumorigenesis
and malignant progression.
Additional members of the SOX family of chromatinassociated
regulatory factors are on the one hand broadly
associated both with cell fate specification and lineage switching
in development (30), and on the other with multiple
tumor-associated phenotypes (31). Another salient example
of SOX-mediated transdifferentiation involves a mechanism
of therapeutic resistance in prostate carcinomas. In this case,
loss of the RB and p53 tumor suppressors—whose absence
is characteristic of neuroendocrine tumors—in response to
antiandrogen therapy is necessary but not sufficient for the
frequently observed conversion of well-differentiated prostate
cancer cells into carcinoma cells that have entered a differentiation
lineage with molecular and histologic features
of neuroendocrine cells, which notably do not express the
androgen receptor. In addition to loss of RB and p53, the
acquired resistance to antiandrogen therapy requires upregulated
expression of the SOX2 developmental regulatory gene,
which is demonstrably instrumental in inducing transdifferentiation
of the therapy-responsive adenocarcinoma cells
into derivatives that reside in a neuroendocrine cell state that
is refractory to the therapy (32).
A third example also reveals transdifferentiation as a strategy
employed by carcinoma cells to avoid elimination by
a lineage-specific therapy, in this case involving basal cell
carcinomas (BCC) of the skin treated with a pharmacologic
inhibitor of the Hedgehog-Smoothened (HH/SMO)
oncogenic signaling pathway known to drive the neoplastic
growth of these cells (33). Drug-resistant cancer cells switch,
via broad epigenetic shifts in specific chromatin domains and
the altered accessibility of two superenhancers, to a developmentally
related but distinct cell type. The newly gained
phenotypic state of the BCC cells enables them to sustain
expression of the WNT oncogenic signaling pathway, which
in turn imparts independence from the drug-suppressed HH/
SMO signaling pathway (34). As might be anticipated from
this transdifferentiation, the transcriptome of the cancer cells
shifts from a gene signature reflecting the implicated cell-oforigin
of BCCs, namely the stem cells of hair follicle bulge,
to one indicative of the basal stem cells that populate the
interfollicular epidermis. Such transdifferentiation to enable
drug resistance is being increasingly documented in different
forms of cancer (35).
Developmental lineage plasticity also appears to be
prevalent among the major subtypes of lung carcinomas,
that is, neuroendocrine carcinomas [small-cell lung cancer
(SCLC)] and adenocarcinomas + squamous cell carcinomas
[collectively non–small cell lung cancer (NSCLC)]. Singlecell
RNA sequencing has revealed remarkably dynamic
and heterogeneous interconversion among these subtypes
as well as distinct variations thereof during the stages in
lung tumorigenesis, subsequent malignant progression,
and responses to therapy (36–38). Thus, rather than the
simple conceptualization of a pure clonal switch from
one lineage into another, these studies paint a much more
complex picture, of dynamically interconverting subpopulations
of cancer cells exhibiting characteristics of multiple
developmental lineages and stages of differentiation, a
sobering realization in regard to lineage-based therapeutic
targeting of human lung cancer. Regulatory determinants
of this dynamic phenotypic plasticity are beginning to be
identified (37, 39, 40).
Synopsis
The three classes of mechanism described above highlight
selective regulators of cellular plasticity that are separable—at
least in part—from core oncogenic drivers and other hallmark
capabilities. Beyond these examples lies a considerable body
of evidence associating many forms of cancer with disrupted
differentiation concomitant with the acquisition of transcriptome
signatures and other phenotypes—for example,
histologic morphology—associated with progenitor or stem
cell stages observed in the corresponding normal tissue-oforigin
or in other more distantly related cell types and lineages
(41–43). As such, these three subclasses of phenotypic
plasticity—dedifferentiation of mature cells back to progenitor
states, blocked differentiation to freeze developing cells
in progenitor/stem cell states, and transdifferentiation to
alternative cell lineages—appear to be operative in multiple
cancer types during primary tumor formation, malignant
progression, and/or response to therapy. There are, however,
two conceptual considerations. First, dedifferentiation and
blocked differentiation are likely intertwined, being indistinguishable
in many tumor types where the cell-of-origin—
differentiated cell or progenitor/stem cell—is either unknown
or alternatively involved. Second, the acquisition or maintenance
of progenitor cell phenotypes and loss of differentiated
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
36 | CANCER DISCOVERY JANUARY 2022	AACRJournals.org
HanahanREVIEW
features is in most cases an imprecise reflection of the normal
developmental stage, being immersed in a milieu of other
hallmark-enabling changes in the cancer cell that are not
present in naturally developing cells. In addition, yet another
form of phenotypic plasticity involves cell senescence, discussed
more generally below, wherein cancer cells induced
to undergo ostensibly irreversible senescence are instead able
to escape and resume proliferative expansion (44). Finally,
as with other hallmark capabilities, cellular plasticity is not
a novel invention or aberration of cancer cells, but rather
the corruption of latent but activatable capabilities that
various normal cells use to support homeostasis, repair, and
regeneration (45).
Collectively, these illustrative examples encourage consideration
of the proposition that unlocking cellular plasticity
to enable various forms of disrupted differentiation constitutes
a discrete hallmark capability, distinguishable in regulation
and cellular phenotype from the well-validated core
hallmarks of cancer (Fig. 2).
NONMUTATIONAL EPIGENETIC
REPROGRAMMING
The enabling characteristic of genome (DNA) instability
and mutation is a fundamental component of cancer
formation and pathogenesis. At present, multiple international
consortia are cataloging mutations across the genome
of human cancer cells, doing so in virtually every type of
human cancer, at different stages of malignant progression,
including metastatic lesions, and during the development
of adaptive resistance to therapy. One result is the now
widespread appreciation that mutations in genes that organize,
modulate, and maintain chromatin architecture, and
thereby globally regulate gene expression, are increasingly
detected and functionally associated with cancer hallmarks
(46–48).
There is, in addition, a case to be made for another apparently
independent mode of genome reprogramming that
involves purely epigenetically regulated changes in gene
expression, one that might be termed “nonmutational epigenetic
reprogramming” (Fig.  3). Indeed, the proposition
of mutation-less cancer evolution and purely epigenetic
programming of hallmark cancer phenotypes was raised
almost a decade ago (49) and is increasingly discussed
(46, 50–52).
The concept of nonmutational epigenetic regulation of
gene expression is of course well established as the central
mechanism mediating embryonic development, differentiation,
and organogenesis (53–55). In the adult, for example,
long-term memory involves changes in gene and histone
modification, in chromatin structure, and in the triggering of
gene expression switches that are stably maintained over time
by positive and negative feedback loops (56, 57). Growing
evidence supports the proposition that analogous epigenetic
alterations can contribute to the acquisition of hallmark
capabilities during tumor development and malignant progression.
A few examples are presented below in support of
this hypothesis.
Nonmutational
epigenetic reprogramming
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).
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
	 JANUARY 2022 CANCER DISCOVERY | 37
Hallmarks of Cancer: New Dimensions REVIEW
Microenvironmental Mechanisms
of Epigenetic Reprogramming
If not solely by consequence of oncogenic mutations, how
then is the cancer cell genome reprogrammed? A growing
body of evidence indicates that the aberrant physical properties
of the tumor microenvironment can cause broad changes
in the epigenome, from which changes beneficial to the phenotypic
selection of hallmark capabilities can result in clonal
outgrowth of cancer cells with enhanced fitness for proliferative
expansion. One common characteristic of tumors (or
regions within tumors) is hypoxia, consequent to insufficient
vascularization. Hypoxia, for example, reduces the activity of
the TET demethylases, resulting in substantive changes in
the methylome, in particular hypermethylation (58). Insufficient
vascularization likely also limits the bioavailability of
critical blood-borne nutrients, and nutrient deprivation has
been shown for example to alter translational control and
consequently enhance the malignant phenotype of breast
cancer cells (59).
A persuasive example of hypoxia-mediated epigenetic regulation
involves a form of invariably lethal pediatric ependymoma.
Like many embryonic and pediatric tumors, this
form lacks recurrent mutations, in particular a dearth of
driver mutations in oncogenes and tumor suppressors.
Rather, the aberrant growth of these cancer cells is demonstrably
governed by a gene regulatory program induced by
hypoxia (60, 61). Notably, the putative cell-of-origin of this
cancer resides in a hypoxic compartment, likely sensitizing
cells resident therein to the initiation of tumorigenesis by as
yet unknown cofactors.
Another persuasive line of evidence for microenvironmentally
mediated epigenetic regulation involves the invasive
growth capability of cancer cells. A classic example involves
the reversible induction of invasiveness of cancer cells at the
margins of many solid tumors, orchestrated by the developmental
regulatory program known as the epithelial-tomesenchymal
transition (EMT; refs. 62–64). Notably, a master
regulator of the EMT, ZEB1, has been recently shown to
induce expression of a histone methyltransferase, SETD1B,
that in turn sustains ZEB1 expression in a positive feedback
loop that maintains the (invasive) EMT regulatory state
(65). A previous study similarly documented that induction
of EMT by upregulated expression of a related TF, SNAIL1,
caused marked alterations in the chromatin landscape consequent
to induction of a number of chromatin modifiers,
whose activity was demonstrably necessary for the maintenance
of the phenotypic state (66). Furthermore, a roster of
conditions and factors to which cancer cells at the margins of
tumors are exposed, including hypoxia and cytokines secreted
by stromal cells, can evidently induce the EMT and in turn
invasiveness (67, 68).
A distinctive example of microenvironmental programming
of invasiveness, ostensibly unrelated to the EMT program,
involves autocrine activation, in pancreas cancer cells
and others, via interstitial pressure–driven fluid flow, of a
neuronal signaling circuit involving secreted glutamate and
its receptor NMDAR (69, 70). Notably, the prototypical stiffness
of many solid tumors, embodied in extensive alterations
to the extracellular matrix (ECM) that envelop the cells
within, has broad effects on the invasive and other phenotypic
characteristics of cancer cells. Compared with the
normal tissue ECM from which tumors originate, the tumor
ECM is typically characterized by increased cross-linking and
density, enzymatic modifications, and altered molecular composition,
which collectively orchestrate—in part via integrin
receptors for ECM motifs—stiffness-induced signaling and
gene-expression networks that elicit invasiveness and other
hallmark characteristics (71).
In addition to such regulatory mechanisms endowed by
the physical tumor microenvironment, paracrine signaling
involving soluble factors released into the extracellular milieu
by the various cell types populating solid tumors can also contribute
to the induction of several morphologically distinct
invasive growth programs (72), only one of which—dubbed
“mesenchymal”—seems to involve the aforementioned EMT
epigenetic regulatory mechanism.
Epigenetic Regulatory Heterogeneity
A growing knowledge base is heightening appreciation of
the importance of intratumoral heterogeneity in generating
the phenotypic diversity where the fittest cells for proliferative
expansion and invasion outgrow their brethren and
hence are selected for malignant progression. Certainly, one
facet of this phenotypic heterogeneity is founded in chronic
or episodic genomic instability and consequent genetic heterogeneity
in the cells populating a tumor. In addition, it
is increasingly evident that there can be non–mutationally
based epigenetic heterogeneity. A salient example involves
the linker histone H1.0, which is dynamically expressed and
repressed in subpopulations of cancer cells within a number
of tumor types, with consequent sequestration or accessibility,
respectively, of megabase-sized domains, including ones
conveying hallmark capabilities (73). Notably, the population
of cancer cells with repressed H1.0 were found to have stemlike
characteristics, enhanced tumor-initiating capability, and
an association with poor prognosis in patients.
Another example of epigenetically regulated plasticity has
been described in human oral squamous cell carcinomas
(SCC), wherein cancer cells at the invasive margins adopt a
partial EMT (p-EMT) state lacking the aforementioned mesenchymal
TFs but expressing other EMT-defining genes that
are not expressed in the central core of the tumors (74). The
p-EMT cells evidently do not represent a clonal compartmentalization
of mutationally altered cells: cultures of primary
tumor-derived cancer cells contain dynamic mixtures of both
p-EMThi
and p-EMTlo
cells, and when p-EMThi/lo
cells were
FACS-purified and cultured, both reverted to mixed populations
of p-EMThi
and p-EMTlo
cells within 4 days. Moreover,
although paracrine signals from the adjacent stroma could
be envisaged as deterministic for the p-EMThi
state, the stable
presence and regeneration of the two epigenetic states in
culture argues for a cancer cell–intrinsic mechanism. Notably,
this conclusion is supported by analysis of 198 cell lines representing
22 cancer types, including SCC, wherein 12 stably
heterogeneous epigenetic states (including the p-EMT in
SCC) were variously detected in the cell line models as well
as their cognate primary tumors (75). Again, the heterogeneous
phenotypic states could not be linked to detectable
genetic differences, and in several cases FACS-sorted cells of
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
38 | CANCER DISCOVERY JANUARY 2022	AACRJournals.org
HanahanREVIEW
a particular state were shown to dynamically reequilibrate
upon culture, recapitulating a stable balance among the heterogeneous
states seen in the original cell lines.
Additionally, technologies for genome-wide profiling of
diverse attributes—beyond DNA sequence and its mutational
variation—are illuminating influential elements of the cancer
cell genome’s annotation and organization that correlate
with patient prognosis, and increasingly with hallmark capabilities
(76–78). Epigenomic heterogeneity is being revealed
by increasingly powerful technologies for profiling genomewide
DNA methylation (79, 80), histone modification (81),
chromatin accessibility (82), and posttranscriptional modification
and translation of RNA (83, 84). A challenge in regard
to the postulate being considered herein will be to ascertain
which epigenomic modifications in particular cancer types
(i) have regulatory significance and (ii) are representative of
purely nonmutational reprogramming, as opposed to being
mutation-driven and thus explainable by genome instability.
Epigenetic Regulation of the Stromal Cell Types
Populating the Tumor Microenvironment
In general, the accessory cells in the tumor microenvironment
that functionally contribute to the acquisition
of hallmark capabilities are not thought to suffer genetic
instability and mutational reprogramming to enhance their
tumor-promoting activities; rather it is inferred that these
cells—cancer-associated fibroblasts, innate immune cells, and
endothelial cells and pericytes of the tumor vasculature—
are epigenetically reprogrammed upon their recruitment by
soluble and physical factors that define the solid tumor microenvironment
(2, 85). It can be anticipated the multi-omic profiling
technologies currently being applied to cancer cells will
increasingly be used to interrogate the accessory (stromal)
cells in tumors to elucidate how normal cells are corrupted
to functionally support tumor development and progression.
For example, a recent study (86) suggests that such reprogramming
can involve modifications of the epigenome in addition
to the inductive interchange of cytokines, chemokines, and
growth factors that alter intracellular signaling networks in
all of these cell types: when mouse models of metastasis to
lung were treated with a combination of a DNA methyltransferase
inhibitor (5-azacytidine) and an inhibitor of histone
modification (an HDAC), the infiltrating myeloid cells were
found to have switched from an immature (tumor-promoting)
progenitor state into cells resembling mature interstitial
(tumor-antagonizing) macrophages, which, in contrast to
their counterparts in untreated tumors, were incapable of
supporting the hallmark capabilities necessary for efficient
metastatic colonization (86). It can be envisaged that multiomic
profiling and pharmacologic perturbation will serve to
elucidate the reprogrammed epigenetic state in such myeloid
cells as well as other hallmark-enabling accessory cell types
populating tumor microenvironments.
Synopsis
Collectively, these illustrative snapshots support the proposition
that nonmutational epigenetic reprograming will
come to be accepted as a bona fide enabling characteristic
that serves to facilitate the acquisition of hallmark capabilities
(Fig. 3), distinct from that of genomic DNA instability
and mutation. Notably, it can be anticipated that nonmutational
epigenetic reprogramming will prove to be integrally
involved in enabling the provisional new hallmark capability
of phenotypic plasticity discussed above, in particular being
a driving force in the dynamic transcriptomic heterogeneity
that is increasingly well documented in cancer cells populating
malignant TMEs. The advance of single cell multi-omic
profiling technologies is envisaged to illuminate the respective
contributions of and interplay between mutation-driven
versus nonmutational epigenetic regulation to the evolution
of tumors during malignant progression and metastasis.
POLYMORPHIC MICROBIOMES
An expansive frontier in biomedicine is unfolding via
illumination of the diversity and variability of the plethora
of microorganisms, collectively termed the microbiota, that
symbiotically associate with the barrier tissues of the body
exposed to the external environment—the epidermis and the
internal mucosa, in particular the gastrointestinal tract, as
well as the lung, the breast, and the urogenital system. There
is growing appreciation that the ecosystems created by resident
bacteria and fungi—the microbiomes—have profound
impact on health and disease (87), a realization fueled by
the capability to audit the populations of microbial species
using next-generation sequencing and bioinformatic technologies.
For cancer, the evidence is increasingly compelling
that polymorphic variability in the microbiomes between
individuals in a population can have a profound impact on
cancer phenotypes (88, 89). Association studies in human
and experimental manipulation in mouse models of cancer
are revealing particular microorganisms, principally but
not exclusively bacteria, which can have either protective
or deleterious effects on cancer development, malignant
progression, and response to therapy. So too can the global
complexity and constitution of a tissue microbiome at large.
Indeed, while the gut microbiome has been the pioneer of
this new frontier, multiple tissues and organs have associated
microbiomes, which have distinctive characteristics in
regard to population dynamics and diversity of microbial
species and subspecies. This growing appreciation of the
importance of polymorphically variable microbiomes in
health and disease posits the question: is the microbiome
a discrete enabling characteristic that broadly affects, both
positively and negatively, the acquisition of hallmark capabilities
for cancer? I reflect on this possibility below, illustrating
evidence for some of the prominent tissue microbiomes
implicated in cancer hallmarks (Fig. 4), beginning with the
most prominent and evidently impactful microbiome, that
of the intestinal tract.
Diverse Modulatory Effects of the Gut Microbiome
It has long been recognized that the gut microbiome is fundamentally
important for the function of the large intestine
(colon) in degrading and importing nutrients into the body
as part of metabolic homeostasis, and that distortions in the
microbial populations—dysbiosis—in the colon can cause a
spectrum of physiologic maladies (87). Among these has
been the suspicion that the susceptibility, development, and
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
	 JANUARY 2022 CANCER DISCOVERY | 39
Hallmarks of Cancer: New Dimensions REVIEW
pathogenesis of colon cancer is influenced by the gut microbiome.
In recent years, persuasive functional studies, involving
fecal transplants from colon tumor–bearing patients and
mice into recipient mice predisposed to develop colon cancer
has established a principle: there are both cancer-protective
and tumor-promoting microbiomes, involving particular
bacterial species, which can modulate the incidence and
pathogenesis of colon tumors (90).
The mechanisms by which microbiota impart these modulatory
roles are still being elucidated, but two general effects
are increasingly well established for tumor-promoting microbiomes
and in some cases for specific tumor-promoting
bacterial species. The first effect is mutagenesis of the colonic
epithelium, consequent to the production of bacterial toxins
and other molecules that either damage DNA directly,
or disrupt the systems that maintain genomic integrity, or
stress cells in other ways that indirectly impair the fidelity
of DNA replication and repair. A case in point is E. coli
carrying the PKS locus, which demonstrably mutagenizes
the human genome and is implicated in conveying hallmarkenabling
mutations (91).
Additionally, bacteria have been reported to bind to the
surface of colonic epithelial cells and produce ligand mimetics
that stimulate epithelial proliferation, contributing in
neoplastic cells to the hallmark capability for proliferative
signaling (88). Another mechanism by which specific bacterial
species promote tumorigenesis involves butyrate-producing
bacteria, whose abundance is elevated in patients with colorectal
cancer (92). The production of the metabolite butyrate
has complex physiologic effects, including the induction of
senescent epithelial and fibroblastic cells. A mouse model
of colon carcinogenesis populated with butyrate-producing
bacteria developed more tumors than mice lacking such
bacteria; the connection between butyrate-induced senescence
and enhanced colon tumorigenesis was demonstrated
by the use of a senolytic drug that kills senescent cells,
which impaired tumor growth (92). In addition, bacterialproduced
butyrate has pleiotropic and paradoxical effects on
differentiated cells versus undifferentiated (stem) cells in the
colonic epithelium in conditions where the intestinal barrier
is disrupted (dysbiosis) and the bacteria are invasive, affecting,
for example, cellular energetics and metabolism, histone
modification, cell-cycle progression, and (tumor-promoting)
innate immune inflammation that is immunosuppressive of
adaptive immune responses (93).
Indeed, a broad effect of polymorphic microbiomes
involves the modulation of the adaptive and innate immune
systems via multifarious routes, including the production by
bacteria of “immunomodulatory” factors that activate damage
sensors on epithelial or resident immune cells, resulting
in the expression of a diverse repertoire of chemokines and
cytokines that can sculpt the abundance and characteristics
of immune cells populating the colonic epithelia and its
underlying stroma and draining lymph nodes. In addition,
certain bacteria can breach both the protective biofilm and
the mucus lining the colonic epithelia and proceed to disrupt
the epithelial cell–cell tight junctions that collectively
maintain the integrity of the physical barrier that normally
compartmentalizes the intestinal microbiome. Upon invading
the stroma, bacteria can trigger both innate and adaptive
immune responses, eliciting secretion of a repertoire of
cytokines and chemokines. One manifestation can be the
creationoftumor-promotingortumor-antagonizingimmune
microenvironments, consequently protecting against or
facilitating tumorigenesis and malignant progression. Concordantly,
the modulation by distinctive microbiomes in
Skin
Vaginal/
cervical
Polymorphic
microbiomes Tumor
Oral
Gut
Lung
Modulating
tumor
Growth
Inflammation
Immune
evasions
Genome
instability
Therapy
resistance
Figure 4.  Polymorphic microbiomes. Left, while intersecting with the enabling characteristics of tumor-promoting inflammation and genomic instability
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).
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
40 | CANCER DISCOVERY JANUARY 2022	AACRJournals.org
HanahanREVIEW
individual patients of the intertwined parameters of (i) eliciting
(innate) tumor promoting inflammation and (ii) escaping
(adaptive) immune destruction can be associated not
only with prognosis, but also with responsiveness or resistance
to immunotherapies involving immune checkpoint
inhibitors and other therapeutic modalities (89, 94–96).
Provisional proof-of-concept has come from recent studies
demonstrating restored efficacy to immunotherapy
following transplants of fecal microbiota from therapyresponsive
patients into patients with melanoma who had
progressed during prior treatment with immune checkpoint
blockade (97, 98).
An ongoing mystery has involved the molecular mechanisms
by which particular and variable constituents of the
gut microbiome systemically modulate the activity of the
adaptive immune system, either enhancing antitumoral
immune responses evoked by immune checkpoint blockade,
or rather eliciting systemic or local (intratumoral) immunosuppression.
A recent study has shed some light: certain
strains of Enterococcus (and other bacteria) express a peptidoglycan
hydrolyase called SagA that releases mucopeptides
from the bacterial wall, which can then circulate systemically
and activate the NOD2 pattern receptor, which in turn
can enhance T-cell responses and the efficacy of checkpoint
immunotherapy (99). Other immunoregulatory molecules
produced by specific bacterial subspecies are being identified
and functionally evaluated, including bacteria-produced
inosine, a rate-limiting metabolite for T-cell activity (100).
These examples and others are beginning to chart the molecular
mechanisms by which polymorphic microbiomes are
indirectly and systemically modulating tumor immunobiology,
above and beyond immune responses consequent to
direct physical interactions of bacteria with the immune
system (101, 102).
Beyond the causal links to colon cancer and melanoma, the
gut microbiome’s demonstrable ability to elicit the expression
of immunomodulatory chemokines and cytokines that
enter the systemic circulation is evidently also capable of
affecting cancer pathogenesis and response to therapy in
other organs of the body (94, 95). An illuminating example
involves the development of cholangiocarcinomas in the
liver: gut dysbiosis allows the entry and transport of bacteria
and bacterial products through the portal vein to the liver,
where TLR4 expressed on hepatocytes is triggered to induce
expression of the chemokine CXCL1, which recruits CXCR2expressing
granulocytic myeloid cells (gMDSC) that serve to
suppress natural killer cells so as to evade immune destruction
(103), and likely convey other hallmark capabilities (85).
As such, the gut microbiome is unambiguously implicated as
an enabling characteristic that can alternatively facilitate or
protect against multiple forms of cancer.
Beyond the Gut: Implicating Distinctive
Microbiomes in Other Barrier Tissues
Virtually all tissues and organs exposed, directly or indi-
rectly,totheoutsideenvironmentarealsorepositoriesforcommensal
microorganisms (104). Unlike the intestine, where the
symbiotic role of the microbiome in metabolism is well recognized,
the normal and pathogenic roles of resident microbiota
in these diverse locations is still emerging. There are evidently
organ/tissue-specific differences in the constitution of the
associated microbiomes in homeostasis, aging, and cancer,
with both overlapping and distinctive species and abundancies
to that of the colon (104, 105). Moreover, association
studies are providing increasing evidence that local tumorantagonizing/protective
versus tumor-promoting tissue
microbiomes, similarly to the gut microbiome, can modulate
susceptibility and pathogenesis to human cancers arising in
their associated organs (106–109).
Impact of Intratumoral Microbiota?
Finally, pathologists have long recognized that bacteria can
be detected within solid tumors, an observation that has now
been substantiated with sophisticated profiling technologies.
For example, in a survey of 1,526 tumors encompassing seven
human cancer types (bone, brain, breast, lung, melanoma,
ovary, and pancreas), each type was characterized by a distinctive
microbiome that was largely localized inside cancer cells
and immune cells, and within each tumor type, variations in
the tumor microbiome could be detected and inferred to be
associated with clinicopathologic features (110). Microbiota
have been similarly detected in genetically engineered de novo
mouse models of lung and pancreas cancer, and their absence
in germ-free mice and/or their abrogation with antibiotics can
demonstrably impair tumorigenesis, functionally implicating
the tumor microbiome as an enabler of tumor-promoting
inflammation and malignant progression (111, 112). Association
studies in human pancreatic ductal adenocarcinoma and
functional tests via fecal transplants into tumor-bearing mice
have established that variations in the tumor microbiome—
and the associated gut microbiome—modulate immune phenotypes
and survival (113). An important challenge for the
future will be to extend these implications to other tumor
types, and to delineate the potentially separable contributions
of constitution and variation in the tumor microbiome
to that of the gut (and local tissue of origin) microbiome,
potentially by identifying specific microbial species that are
functionally influential in one location or the other.
Synopsis
Among the fascinating questions for the future is whether
microbiota resident in different tissues or populating incipient
neoplasias have the capability to contribute to or interfere
with the acquisition of other hallmark capabilities beyond
immunomodulation and genome mutation, thereby influencing
tumor development and progression. There are clues
that particular bacterial species can directly stimulate the
hallmark of proliferative signaling, for example, in colonic
epithelium (88), and modulate growth suppression by altering
tumor suppressor activity in different compartments of
the intestine (114), whereas direct effects on other hallmark
capabilities, such as avoiding cell death, inducing angiogenesis,
and stimulating invasion and metastasis, remain obscure,
as does the generalizability of these observations to multiple
forms of human cancer. Irrespective, there is an increasingly
compelling case to be made that polymorphic variation in
microbiomes of the intestine and other organs constitutes
a distinctive enabling characteristic for the acquisition of
hallmark capabilities (Fig.  4), albeit intersecting with and
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
	 JANUARY 2022 CANCER DISCOVERY | 41
Hallmarks of Cancer: New Dimensions REVIEW
complementing those of genome instability and mutation,
and tumor-promoting inflammation.
SENESCENT CELLS
Cellular senescence is a typically irreversible form of proliferative
arrest, likely evolved as a protective mechanism for
maintaining tissue homeostasis, ostensibly as a complementary
mechanism to programmed cell death that serves to
inactivate and in due course remove diseased, dysfunctional,
or otherwise unnecessary cells. In addition to shutting down
the cell division cycle, the senescence program evokes changes
in cell morphology and metabolism and, most profoundly,
the activation of a senescence-associated secretory phenotype
(SASP) involving the release of a plethora of bioactive proteins,
including chemokines, cytokines, and proteases whose
identity is dependent on the cell and tissue type from which a
senescent cell arises (115–117). Senescence can be induced in
cells by a variety of conditions, including microenvironmental
stresses such as nutrient deprivation and DNA damage, as
well as damage to organelles and cellular infrastructure, and
imbalances in cellular signaling networks (115, 117), all of
which have been associated with the observed increase in the
abundance of senescent cells in various organs during aging
(118, 119).
Cellular senescence has long been viewed as a protective
mechanism against neoplasia, whereby cancerous cells are
induced to undergo senescence (120). Most of the aforementioned
instigators of the senescent program are associated
with malignancy, in particular DNA damage as a
consequence of aberrant hyperproliferation, so-called oncogene-induced
senescence due to hyperactivated signaling,
and therapy-induced senescence consequent to cellular and
genomic damage caused by chemotherapy and radiotherapy.
Indeed, there are well-established examples of the protective
benefits of senescence in limiting malignant progression (118,
119). To the contrary, however, an increasing body of evidence
reveals quite the opposite: in certain contexts, senescent cells
variously stimulate tumor development and malignant progression
(119, 121). In one illuminating case study, senescent
cells were pharmacologically ablated in aging mice, in particular
depleting senescent cells characteristically expressing
the cell-cycle inhibitor p16−INK4a
: in addition to delaying multiple
age-related symptoms, the depletion of senescent cells
in aging mice resulted in reduced incidences of spontaneous
tumorigenesis and cancer-associated death (122).
The principal mechanism by which senescent cells promote
tumor phenotypes is thought to be the SASP, which is
demonstrably capable of conveying, in paracrine fashion to
viable cancer cells in proximity, as well as to other cells in the
TME, signaling molecules (and proteases that activate and/
or desequester them) so as to convey hallmark capabilities.
Thus, in different experimental systems, senescent cancer
cells have been shown to variously contribute to proliferative
signaling, avoiding apoptosis, inducing angiogenesis,
stimulating invasion and metastasis, and suppressing tumor
immunity (116, 118, 120, 121).
Yet another facet to the effects of senescent cancer cells
on cancer phenotypes involves transitory, reversible senescent
cell states, whereby senescent cancer cells can escape
from their SASP-expressing, nonproliferative condition, and
resume cell proliferation and manifestation of the associated
capabilities of fully viable oncogenic cells (44). Such
transitory senescence is most well documented in cases of
therapy resistance (44), representing a form of dormancy
that circumvents therapeutic targeting of proliferating cancer
cells, but may well prove to be more broadly operative in
other stages of tumor development, malignant progression,
and metastasis.
Moreover, the hallmark-promoting capabilities of senescent
cells are not limited to senescent cancer cells. Cancerassociated
fibroblasts (CAF) in tumors have been shown to
undergo senescence, creating senescent CAFs that are demonstrably
tumor-promoting by virtue of conveying hallmark
capabilities to cancer cells in the TME (115, 116, 121). Moreover,
senescent fibroblasts in normal tissues produced in
part by natural aging or environmental insults have similarly
been implicated in remodeling tissue microenvironments via
their SASP so as to provide paracrine support for local invasion
(so-called “field effects”) and distant metastasis (116) of
neoplasias developing in proximity. Additionally, senescent
fibroblasts in aging skin have been shown to recruit—via their
SASP—innate immune cells that are both immunosuppressive
of adaptive antitumoral immune responses anchored
by CD8 T cells, and stimulatory of skin tumor growth (123),
with the latter effect potentially reflecting paracrine contributions
of such innate immune cells (myeloid cells, neutrophils,
and macrophages) to other hallmark capabilities.
While less well established, it seems likely that other abundant
stromal cells populating particular tumor microenvironments
will prove to undergo senescence, and thereby
modulate cancer hallmarks and consequent tumor phenotypes.
For example, therapy-induced senescent tumor
endothelial cells can enhance proliferation, invasion, and
metastasis in breast cancer models (124, 125).
Certainly, such clues warrant investigation in other tumor
types to assess generality of fibroblastic, endothelial, and
other stromal cell senescence as a driving force in tumor
evolution. Also currently unresolved are the regulatory
mechanisms and functional determinants through which
a particular senescent cell type in a given TME evokes a
tumor-promoting versus a tumor-antagonizing SASP, which
can seeming be alternatively induced in the same senescing
cell type, perhaps by different instigators when immersed in
distinctive physiologic and neoplastic microenvironments.
Synopsis
The concept that tumors are composed of genetically
transformed cancer cells interacting with and benefiting from
recruited and epigenetically/phenotypically corrupted accessory
(stromal) cells is well established as instrumental to the
pathogenesis of cancer. The considerations discussed above
and described in the reviews and reports cited herein (and
elsewhere) make a persuasive case for the proposition that
senescent cells (of whatever cellular origin) should be considered
for addition to the roster of functionally significant cells
in the tumor microenvironment (Fig. 5). As such, senescent
cells warrant being factored into the quest for deep knowledge
of cancer mechanisms. Furthermore, the realization of
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
42 | CANCER DISCOVERY JANUARY 2022	AACRJournals.org
HanahanREVIEW
their importance motivates the ancillary goal to therapeutically
target tumor-promoting senescent cells of all constitutions,
be it by pharmacologic or immunologic ablation, or by
reprogramming the SASP into tumor-antagonizing variants
(115, 121, 126).
CONCLUDING REMARKS
While the eight hallmarks of cancer and their two enabling
characteristics have proved of enduring heuristic value in
the conceptualization of cancer, the considerations presented
above suggest that there may be new facets of some generality
and hence of relevance to more fully understanding the
complexities, mechanisms, and manifestations of the disease.
By applying the metric of discernable if not complete independence
from the 10 core attributes, it is arguable that these
four parameters may well—pursuant to further validation and
generalization beyond the case studies presented—become integrated
into the hallmarks of cancer schematic (Fig. 6). Thus, cellular
plasticity may come to be added to the roster of hallmark
capabilities. Notably, while the eight core and this nouveau
capability are each, by their definition as a hallmark, conceptually
distinguishable, aspects of their regulation are at least
partially interconnected in some and perhaps many cancers.
For example, multiple hallmarks are coordinately modulated
in some tumor types by canonical oncogenic drivers, including
(i)	 KRAS (https://cancer.sanger.ac.uk/cosmic/census-page/
KRAS),
(ii)	 MYC (https://cancer.sanger.ac.uk/cosmic/census-page/
MYC),
(iii)	 NOTCH (https://cancer.sanger.ac.uk/cosmic/censuspage/NOTCH1;
ref. 127), and
(iv)	 TP53 (https://cancer.sanger.ac.uk/cosmic/census-page/
TP53),
highlighting the important challenge to more fully elucidate
the regulatory networks governing these acquired
capabilities.
In addition to adding cellular plasticity to the roster,
nonmutational epigenetic reprogramming and polymorphic
variations in organ/tissue microbiomes may come to be incorporated
as mechanistic determinants—enabling characteristics—by
which hallmark capabilities are acquired, along with
tumor-promoting inflammation (itself partially interconnected
to the microbiome), above and beyond the mutations
and other aberrations that manifest the afore-mentioned
oncogenic drivers.
Finally, senescent cells of different origins—including cancer
cells and various stromal cells—that functionally contribute
to the development and malignant progression of cancer,
albeit in markedly distinctive ways to those of their nonsenescent
brethren, may become incorporated as generic components
of the TME. In conclusion, it is envisaged that raising
these provisional “trial balloons” will stimulate debate, discussion,
and continuing experimental investigation in the
cancer research community about the defining conceptual
parameters of cancer biology, genetics, and pathogenesis.
Senescent cells
(multiple origins)
Senescent cells
Figure 5.  Senescent cells. Heterogeneous cancer cell subtypes as well as stromal cell types and subtypes are functionally integrated into the manifestations
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).
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
	 JANUARY 2022 CANCER DISCOVERY | 43
Hallmarks of Cancer: New Dimensions REVIEW
Author’s Disclosures
No disclosures were reported.
Acknowledgments
First and foremost, I profoundly thank Bob Weinberg for an
exceptional tradition of insightful and formative discussions, and
for excellent comments and suggestions to the first vignette of this
manuscript. Additionally, I wish to thank: Ben Stanger; Bradley
Bernstein, Giovanni Ciriello, and William Flavahan; Jennifer Wargo;
and Sheila Stewart for their valuable comments and suggestions
on the four vignettes, respectively, and SayoStudio for assistance in
crafting the figures.
Received August 4, 2021; revised November 17, 2021; accepted
November 18, 2021; published first January 12, 2022.
REFERENCES
	 1.	 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:
57–70.
	 2.	 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.
Cell 2011;144:646–74.
	 3.	 Yuan S, Norgard RJ, Stanger BZ. Cellular plasticity in cancer. Cancer
Discov 2019;9:837–51.
	 4.	 Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H,
van den Born M, et al. Crypt stem cells as the cells-of-origin of intestinal
cancer. Nature 2009;457:608–11.
	 5.	Perekatt AO, Shah PP, Cheung S, Jariwala N, Wu A, Gandhi V,
et al. SMAD4 suppresses WNT-driven dedifferentiation and oncogenesis
in the differentiated gut epithelium. Cancer Res 2018;78:
4878–90.
	 6.	 ShihIM,WangTL,TraversoG,RomansK,HamiltonSR,Ben-Sasson S,
et  al. Top-down morphogenesis of colorectal tumors. Proc Natl
Acad Sci U S A 2001;98:2640–5.
	 7.	Ordóñez-Morán P, Dafflon C, Imajo M, Nishida E, Huelsken J.
HOXA5 counteracts stem cell traits by inhibiting Wnt signaling in
colorectal cancer. Cancer Cell 2015;28:815–29.
	 8.	 Tan SH, Barker N. Stemming colorectal cancer growth and metastasis:
HOXA5 forces cancer stem cells to differentiate. Cancer Cell
2015;28:683–5.
	 9.	 Köhler C, Nittner D, Rambow F, Radaelli E, Stanchi F, Vandamme N,
et al. Mouse cutaneous melanoma induced by mutant BRaf arises
from expansion and dedifferentiation of mature pigmented melanocytes.
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 development.
PLoS Genet 2010;6:e1001258.
	 11.	 Claps G, Cheli Y, Zhang T, Scortegagna M, Lau E, Kim H, et al. A
transcriptionally inactive ATF2 variant drives melanomagenesis.
Cell Rep 2016;15:1884–92.
	12.	Saghafinia S, Homicsko K, Di Domenico A, Wullschleger S,
Perren A, Marinoni I, et  al. Cancer cells retrace a stepwise differentiation
program during malignant progression. Cancer Discov
2021;11:2638–57.
	 13.	 Yu X-X, Qiu W-L, Yang L, Zhang Y, He M-Y, Li L-C, et al. Defining
multistep cell fate decision pathways during pancreatic development
at single-cell resolution. EMBO J 2019;38:e100164.
Sustaining
proliferative signaling
Unlocking
phenotypic plasticity
Deregulating
cellular
metabolism
Resisting cell
death
Genome
instability &
mutation
Senescent cells
Inducing or accessing
vasculature
Activating invasion &
metastasis
Polymorphic
microbiomes
Tumor-promoting
inflammation
Enabling
replicative
immortality
Avoiding immune
destruction
Nonmutational
epigenetic reprogramming
Evading
growth suppressors
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).
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
44 | CANCER DISCOVERY JANUARY 2022	AACRJournals.org
HanahanREVIEW
	 14.	 de Thé H. Differentiation therapy revisited. Nat Rev Cancer 2018;
18:117–27.
	 15.	 He LZ, Merghoub T, Pandolfi PP. In vivo analysis of the molecular
pathogenesis of acute promyelocytic leukemia in the mouse and its
therapeutic implications. Oncogene 1999;18:5278–92.
	 16.	 Warrell RP, de Thé H, Wang ZY, Degos L. Acute promyelocytic leukemia.
N Engl J Med 1993;329:177–89.
	 17.	 Bots M, Verbrugge I, Martin BP, Salmon JM, Ghisi M, Baker A, et al.
Differentiation therapy for the treatment of t(8;21) acute myeloid leukemia
using histone deacetylase inhibitors. Blood 2014;123:1341–52.
	 18.	 Ferrara FF, Fazi F, Bianchini A, Padula F, Gelmetti V, Minucci S,
et al. Histone deacetylase-targeted treatment restores retinoic acid
signaling and differentiation in acute myeloid leukemia. Cancer Res
2001;61:2–7.
	 19.	 Kaufman CK, Mosimann C, Fan ZP, Yang S, Thomas AJ, Ablain J,
et al. A zebrafish melanoma model reveals emergence of neural crest
identity during melanoma initiation. Science 2016;351:aad2197.
	 20.	 Morris JP, Yashinskie JJ, Koche R, Chandwani R, Tian S, Chen C-C,
et al. α-Ketoglutarate links p53 to cell fate during tumour suppression.
Nature 2019;573:595–9.
	21.	Saha SK, Parachoniak CA, Ghanta KS, Fitamant J, Ross KN,
Najem MS, et al. Mutant IDH inhibits HNF-4α to block hepatocyte
differentiation and promote biliary cancer. Nature 2014;513:110–4.
	 22.	 Dang L, Su S-SM. Isocitrate dehydrogenase mutation and (R)-2-hydroxyglutarate:
from basic discovery to therapeutics development.
Annu Rev Biochem 2017;86:305–31.
	23.	Waitkus MS, Diplas BH, Yan H. Biological role and therapeutic
potential of IDH mutations in cancer. Cancer Cell 2018;34:186–95.
	 24.	 Phan TG, Croucher PI. The dormant cancer cell life cycle. Nat Rev
Cancer 2020;20:398–411.
	 25.	 Jiang M, Azevedo-Pouly AC, Deering TG, Hoang CQ, DiRenzo D,
Hess DA, et al. MIST1 and PTF1 collaborate in feed-forward regulatory
loops that maintain the pancreatic acinar phenotype in adult
mice. Mol Cell Biol 2016;36:2945–55.
	26.	Krah NM, Narayanan SM, Yugawa DE, Straley JA, Wright CVE,
MacDonald RJ, et al. Prevention and reversion of pancreatic tumorigenesis
through a differentiation-based mechanism. Dev Cell
2019;50:744–54.
	 27.	 Krah NM, De La O J-P, Swift GH, Hoang CQ, Willet SG, Chen Pan F,
et al. The acinar differentiation determinant PTF1A inhibits initiation
of pancreatic ductal adenocarcinoma. eLife 2015;4:e07125.
	 28.	 Shi G, DiRenzo D, Qu C, Barney D, Miley D, Konieczny SF. Maintenance
of acinar cell organization is critical to preventing Krasinduced
acinar-ductal metaplasia. Oncogene 2013;32:1950–8.
	 29.	 Kopp JL, von Figura G, Mayes E, Liu F-F, Dubois CL, Morris JP,
et al. Identification of Sox9-dependent acinar-to-ductal reprogramming
as the principal mechanism for initiation of pancreatic ductal
adenocarcinoma. Cancer Cell 2012;22:737–50.
	 30.	 Julian LM, McDonald AC, Stanford WL. Direct reprogramming with
SOX factors: masters of cell fate. Curr Opin Genet Dev 2017;46:24–36.
	 31.	 Grimm D, Bauer J, Wise P, Krüger M, Simonsen U, Wehland M, et al.
The role of SOX family members in solid tumours and metastasis.
Semin Cancer Biol 2020;67:122–53.
	 32.	 Mu P, Zhang Z, Benelli M, Karthaus WR, Hoover E, Chen C-C, et al.
SOX2 promotes lineage plasticity and antiandrogen resistance in
TP53- and RB1-deficient prostate cancer. Science 2017;355:84–8.
	33.	Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL,
Tibes  R, et  al. Inhibition of the hedgehog pathway in advanced
basal-cell carcinoma. N Engl J Med 2009;361:1164–72.
	 34.	 Biehs B, Dijkgraaf GJP, Piskol R, Alicke B, Boumahdi S, Peale F,
et al. A cell identity switch allows residual BCC to survive Hedgehog
pathway inhibition. Nature 2018;562:429–33.
	 35.	 Boumahdi S, de Sauvage FJ. The great escape: tumour cell plasticity in
resistance to targeted therapy. Nat Rev Drug Discov 2020;19:39–56.
	36.	Groves SM, Ireland A, Liu Q, Simmons AJ, Lau K, Iams WT,
et al. Cancer Hallmarks Define a Continuum of Plastic Cell States
between Small Cell Lung Cancer Archetypes [Internet]. Systems
Biology; 2021 Jan. Available from: http://biorxiv.org/lookup/doi/10.
1101/2021.01.22.427865.
	 37.	 LaFave LM, Kartha VK, Ma S, Meli K, Del Priore I, Lareau C, et al.
Epigenomic state transitions characterize tumor progression in
mouse lung adenocarcinoma. Cancer Cell 2020;38:212–28.
	 38.	 Marjanovic ND, Hofree M, Chan JE, Canner D, Wu K, Trakala M,
et al. Emergence of a high-plasticity cell state during lung cancer
evolution. Cancer Cell 2020;38:229–46.
	 39.	 Drapkin BJ, Minna JD. Studying lineage plasticity one cell at a time.
Cancer Cell 2020;38:150–2.
	 40.	 Inoue Y, Nikolic A, Farnsworth D, Liu A, Ladanyi M, Somwar  R,
et  al. Extracellular signal-regulated kinase mediates chromatin
rewiring and lineage transformation in lung cancer [Internet]. Cancer
Biology; 2020 Nov. Available from: http://biorxiv.org/lookup/
doi/10.1101/2020.11.12.368522.
	 41.	 Dravis C, Chung C-Y, Lytle NK, Herrera-Valdez J, Luna G, Trejo CL,
et al. Epigenetic and transcriptomic profiling of mammary gland
development and tumor models disclose regulators of cell state
plasticity. Cancer Cell 2018;34:466–82.
	42.	Malta TM, Sokolov A, Gentles AJ, Burzykowski T, Poisson L,
Weinstein JN, et al. Machine learning identifies stemness features
associated with oncogenic dedifferentiation. Cell 2018;173:338–54.
	 43.	 Miao Z-F, Lewis MA, Cho CJ, Adkins-Threats M, Park D, Brown JW,
et  al. A dedicated evolutionarily conserved molecular network
licenses differentiated cells to return to the cell cycle. Dev Cell
2020;55:178–94.
	 44.	 De Blander H, Morel A-P, Senaratne AP, Ouzounova M, Puisieux A.
Cellular plasticity: a route to senescence exit and tumorigenesis.
Cancers 2021;13:4561.
	 45.	 Merrell AJ, Stanger BZ. Adult cell plasticity in vivo: de-differentiation
and transdifferentiation are back in style. Nat Rev Mol Cell Biol
2016;17:413–25.
	 46.	 Baylin SB, Jones PA. Epigenetic determinants of cancer. Cold Spring
Harb Perspect Biol 2016;8:a019505.
	 47.	 Flavahan WA, Gaskell E, Bernstein BE. Epigenetic plasticity and the
hallmarks of cancer. Science 2017;357:eaal2380.
	 48.	 Jones PA, Issa J-PJ, Baylin S. Targeting the cancer epigenome for
therapy. Nat Rev Genet 2016;17:630–41.
	 49.	 Huang S. Tumor progression: Chance and necessity in Darwinian
and Lamarckian somatic (mutationless) evolution. Prog Biophys
Mol Biol 2012;110:69–86.
	 50.	 Darwiche N. Epigenetic mechanisms and the hallmarks of cancer:
an intimate affair. Am J Cancer Res 2020;10:1954–78.
	 51.	 Feng Y, Liu X, Pauklin S. 3D chromatin architecture and epigenetic
regulation in cancer stem cells. Protein Cell 2021;12:440–54.
	52.	Nam AS, Chaligne R, Landau DA. Integrating genetic and nongenetic
determinants of cancer evolution by single-cell multi-omics.
Nat Rev Genet 2021;22:3–18.
	 53.	 Bitman-Lotan E, Orian A. Nuclear organization and regulation of
the differentiated state. Cell Mol Life Sci CMLS 2021;78:3141–58.
	 54.	 Goldberg AD, Allis CD, Bernstein E. Epigenetics: a landscape takes
shape. Cell 2007;128:635–8.
	 55.	 Zeng Y, Chen T. DNA methylation reprogramming during mammalian
development. Genes 2019;10:257.
	 56.	 Hegde AN, Smith SG. Recent developments in transcriptional and
translational regulation underlying long-term synaptic plasticity
and memory. Learn Mem 2019;26:307–17.
	 57.	 Kim S, Kaang B-K. Epigenetic regulation and chromatin remodeling
in learning and memory. Exp Mol Med 2017;49:e281.
	 58.	 Thienpont B, Van Dyck L, Lambrechts D. Tumors smother their
epigenome. Mol Cell Oncol 2016;3:e1240549.
	 59.	 Gameiro PA, Struhl K. Nutrient deprivation elicits a transcriptional
and translational inflammatory response coupled to decreased protein
synthesis. Cell Rep 2018;24:1415–24.
	 60.	 Lin GL, Monje M. Understanding the deadly silence of posterior
fossa A ependymoma. Mol Cell 2020;78:999–1001.
	61.	Michealraj KA, Kumar SA, Kim LJY, Cavalli FMG, Przelicki D,
Wojcik JB, et al. Metabolic regulation of the epigenome drives lethal
infantile ependymoma. Cell 2020;181:1329–45.
	 62.	 Bakir B, Chiarella AM, Pitarresi JR, Rustgi AK. EMT, MET, plasticity,
and tumor metastasis. Trends Cell Biol 2020;30:764–76.
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
	 JANUARY 2022 CANCER DISCOVERY | 45
Hallmarks of Cancer: New Dimensions REVIEW
	 63.	 Gupta PB, Pastushenko I, Skibinski A, Blanpain C, Kuperwasser C.
Phenotypic plasticity: driver of cancer initiation, progression, and
therapy resistance. Cell Stem Cell 2019;24:65–78.
	 64.	 Lambert AW, Weinberg RA. Linking EMT programmes to normal
and neoplastic epithelial stem cells. Nat Rev Cancer 2021;21:325–38.
	 65.	 Lindner P, Paul S, Eckstein M, Hampel C, Muenzner JK, ErlenbachWuensch
K, et al. EMT transcription factor ZEB1 alters the epigenetic
landscape of colorectal cancer cells. Cell Death Dis 2020;11:147.
	66.	 Javaid S, Zhang J, Anderssen E, Black JC, Wittner BS, Tajima K,
et al. Dynamic chromatin modification sustains epithelial-mesenchymal
transition following inducible expression of Snail-1. Cell
Rep 2013;5:1679–89.
	 67.	 Serrano-Gomez SJ, Maziveyi M, Alahari SK. Regulation of epithelialmesenchymal
transition through epigenetic and post-translational
modifications. Mol Cancer 2016;15:18.
	68.	Skrypek N, Goossens S, De Smedt E, Vandamme N, Berx G.
Epithelial-to-mesenchymal transition: epigenetic reprogramming
driving cellular plasticity. Trends Genet TIG 2017;33:943–59.
	 69.	 Li L, Hanahan D. Hijacking the neuronal NMDAR signaling circuit
to promote tumor growth and invasion. Cell 2013;153:86–100.
	 70.	 LiL,ZengQ,BhutkarA,GalvánJA,KaramitopoulouE,Noordermeer
D, et al. GKAP acts as a genetic modulator of NMDAR signaling to
govern invasive tumor growth. Cancer Cell 2018;33:736–51.
	 71.	 Mohammadi H, Sahai E. Mechanisms and impact of altered tumour
mechanics. Nat Cell Biol 2018;20:766–74.
	72.	Odenthal J, Takes R, Friedl P. Plasticity of tumor cell invasion:
governance by growth factors and cytokines. Carcinogenesis 2016;
37:1117–28.
	73.	Torres CM, Biran A, Burney MJ, Patel H, Henser-Brownhill T,
Cohen A-HS, et al. The linker histone H1.0 generates epigenetic and
functional intratumor heterogeneity. Science 2016;353:aaf1644.
	 74.	 Puram SV, Tirosh I, Parikh AS, Patel AP, Yizhak K, Gillespie S, et al.
Single-cell transcriptomic analysis of primary and metastatic tumor
ecosystems in head and neck cancer. Cell 2017;171:1611–24.
	 75.	 Kinker GS, Greenwald AC, Tal R, Orlova Z, Cuoco MS, McFarland
JM, et  al. Pan-cancer single-cell RNA-seq identifies recurring programs
of cellular heterogeneity. Nat Genet 2020;52:1208–18.
	76.	Murtha M, Esteller M. Extraordinary cancer epigenomics: thinking
outside the classical coding and promoter box. Trends Cancer
2016;2:572–84.
	 77.	 Nebbioso A, Tambaro FP, Dell’Aversana C, Altucci L. Cancer epigenetics:
moving forward. PLoS Genet 2018;14:e1007362.
	78.	Tavernari D, Battistello E, Dheilly E, Petruzzella AS, Mina M,
Sordet-Dessimoz J, et al. Non-genetic evolution drives lung adenocarcinoma
spatial heterogeneity and progression. Cancer Discov
2021;11:1490–507.
	 79.	 Heyn H, Vidal E, Ferreira HJ, Vizoso M, Sayols S, Gomez A, et al.
Epigenomic analysis detects aberrant super-enhancer DNA methylation
in human cancer. Genome Biol 2016;17:11.
	 80.	 Saghafinia S, Mina M, Riggi N, Hanahan D, Ciriello G. Pan-cancer
landscape of aberrant DNA methylation across human tumors. Cell
Rep 2018;25:1066–80.
	 81.	 Audia JE, Campbell RM. Histone modifications and cancer. Cold
Spring Harb Perspect Biol 2016;8:a019521.
	 82.	 Corces MR, Granja JM, Shams S, Louie BH, Seoane JA, Zhou W,
et al. The chromatin accessibility landscape of primary human cancers.
Science 2018;362:eaav1898.
	83.	Esteve-Puig R, Bueno-Costa A, Esteller M. Writers, readers and
erasers of RNA modifications in cancer. Cancer Lett 2020;474:
127–37.
	84.	Janin M, Coll-SanMartin L, Esteller M. Disruption of the RNA
modifications that target the ribosome translation machinery in
human cancer. Mol Cancer 2020;19:70.
	85.	Hanahan D, Coussens LM. Accessories to the crime: functions
of cells recruited to the tumor microenvironment. Cancer Cell
2012;21:309–22.
	 86.	 Lu Z, Zou J, Li S, Topper MJ, Tao Y, Zhang H, et al. Epigenetic therapy
inhibits metastases by disrupting premetastatic niches. Nature
2020;579:284–90.
	 87.	 Thomas S, Izard J, Walsh E, Batich K, Chongsathidkiet P, Clarke G,
et al. The host microbiome regulates and maintains human health:
a primer and perspective for non-microbiologists. Cancer Res
2017;77:1783–812.
	 88.	 Dzutsev A, Badger JH, Perez-Chanona E, Roy S, Salcedo R, Smith CK,
et al. Microbes and cancer. Annu Rev Immunol 2017;35:199–228.
	 89.	 Helmink BA, Khan MAW, Hermann A, Gopalakrishnan V, Wargo JA.
The microbiome, cancer, and cancer therapy. Nat Med 2019;25:
377–88.
	 90.	 Sears CL, Garrett WS. Microbes, microbiota, and colon cancer. Cell
Host Microbe 2014;15:317–28.
	91.	Pleguezuelos-Manzano C, Puschhof J, Rosendahl Huber A, van
Hoeck A, Wood HM, Nomburg J, et  al. Mutational signature in
colorectal cancer caused by genotoxic pks+ E. coli. Nature 2020;580:
269–73.
	92.	Okumura S, Konishi Y, Narukawa M, Sugiura Y, Yoshimoto S,
Arai  Y, et  al. Gut bacteria identified in colorectal cancer patients
promote tumourigenesis via butyrate secretion. Nat Commun 2021;
12:5674.
	 93.	 Salvi PS, Cowles RA. Butyrate and the intestinal epithelium: modulation
of proliferation and inflammation in homeostasis and disease.
Cells 2021;10:1775.
	 94.	 Fessler J, Matson V, Gajewski TF. Exploring the emerging role of
the microbiome in cancer immunotherapy. J Immunother Cancer
2019;7:108.
	 95.	 Gopalakrishnan V, Helmink BA, Spencer CN, Reuben A, Wargo JA.
The influence of the gut microbiome on cancer, immunity, and
cancer immunotherapy. Cancer Cell 2018;33:570–80.
	 96.	 Zitvogel L, Ma Y, Raoult D, Kroemer G, Gajewski TF. The microbiome
in cancer immunotherapy: diagnostic tools and therapeutic
strategies. Science 2018;359:1366–70.
	 97.	 BaruchEN,YoungsterI,Ben-BetzalelG,OrtenbergR,LahatA,Katz L,
et  al. Fecal microbiota transplant promotes response in immunotherapy-refractory
melanoma patients. Science 2021;371:602–9.
	 98.	 Davar D, Dzutsev AK, McCulloch JA, Rodrigues RR, Chauvin J-M,
Morrison RM, et al. Fecal microbiota transplant overcomes resistance
to anti–PD-1 therapy in melanoma patients. Science 2021;
371:595–602.
	99.	Griffin ME, Espinosa J, Becker JL, Luo J-D, Carroll TS, Jha JK,
et al. Enterococcus peptidoglycan remodeling promotes checkpoint
inhibitor cancer immunotherapy. Science 2021;373:1040–6.
	100.	 Mager LF, Burkhard R, Pett N, Cooke NCA, Brown K, Ramay H,
et  al. Microbiome-derived inosine modulates response to checkpoint
inhibitor immunotherapy. Science 2020;369:1481–9.
	101.	 Ansaldo E, Belkaid Y. How microbiota improve immunotherapy.
Science 2021;373:966–7.
	102.	 Ansaldo E, Farley TK, Belkaid Y. Control of immunity by the microbiota.
Annu Rev Immunol 2021;39:449–79.
	103.	 Zhang Q, Ma C, Duan Y, Heinrich B, Rosato U, Diggs LP, et al. Gut
microbiome directs hepatocytes to recruit MDSCs and promote
cholangiocarcinoma. Cancer Discov 2021;11:1248–67.
	104.	 Ding T, Schloss PD. Dynamics and associations of microbial community
types across the human body. Nature 2014;509:357–60.
	105.	 Byrd AL, Liu M, Fujimura KE, Lyalina S, Nagarkar DR, Charbit B,
et  al. Gut microbiome stability and dynamics in healthy donors
and patients with non-gastrointestinal cancers. J Exp Med 2021;
218:e20200606.
	106.	 Healy CM, Moran GP. The microbiome and oral cancer: more questions
than answers. Oral Oncol 2019;89:30–3.
	107.	 Swaney MH, Kalan LR. Living in your skin: microbes, molecules and
mechanisms. Infect Immun 2021;89:e00695–20.
	108.	Willis JR, Gabaldón T. The human oral microbiome in health
and disease: from sequences to ecosystems. Microorganisms 2020;
8:308.
	109.	 Xu J, Peng J-J, Yang W, Fu K, Zhang Y. Vaginal microbiomes and
ovarian cancer: a review. Am J Cancer Res 2020;10:743–56.
	110.	 Nejman D, Livyatan I, Fuks G, Gavert N, Zwang Y, Geller LT, et al.
The human tumor microbiome is composed of tumor type-specific
intracellular bacteria. Science 2020;368:973–80.
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022
46 | CANCER DISCOVERY JANUARY 2022	AACRJournals.org
HanahanREVIEW
	111.	 Jin C, Lagoudas GK, Zhao C, Bullman S, Bhutkar A, Hu B, et al.
Commensal microbiota promote lung cancer development via γδ T
cells. Cell 2019;176:998–1013.
	112.	Pushalkar S, Hundeyin M, Daley D, Zambirinis CP, Kurz E,
Mishra A, et al. The pancreatic cancer microbiome promotes oncogenesis
by induction of innate and adaptive immune suppression.
Cancer Discov 2018;8:403–16.
	113.	 McAllister F, Khan MAW, Helmink B, Wargo JA. The tumor microbiome
in pancreatic cancer: bacteria and beyond. Cancer Cell
2019;36:577–9.
	114.	 Kadosh E, Snir-Alkalay I, Venkatachalam A, May S, Lasry A, Elyada
E, et al. The gut microbiome switches mutant p53 from tumoursuppressive
to oncogenic. Nature 2020;586:133–8.
	115.	 Birch J, Gil J. Senescence and the SASP: many therapeutic avenues.
Genes Dev 2020;34:1565–76.
	116.	Faget DV, Ren Q, Stewart SA. Unmasking senescence: contextdependent
effects of SASP in cancer. Nat Rev Cancer 2019;19:
439–53.
	117.	Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O,
Bishop C, et al. Cellular senescence: defining a path forward. Cell
2019;179:813–27.
	118.	 He S, Sharpless NE. Senescence in health and disease. Cell 2017;
169:1000–11.
	119.	 Kowald A, Passos JF, Kirkwood TBL. On the evolution of cellular
senescence. Aging Cell 2020;19:e13270.
	120.	 Lee S, Schmitt CA. The dynamic nature of senescence in cancer. Nat
Cell Biol 2019;21:94–101.
	121.	Wang B, Kohli J, Demaria M. Senescent cells in cancer therapy:
friends or foes? Trends Cancer 2020;6:838–57.
	122.	 Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, et al.
Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan.
Nature 2016;530:184–9.
	123.	Ruhland MK, Loza AJ, Capietto A-H, Luo X, Knolhoff BL,
Flanagan KC, et al. Stromal senescence establishes an immunosuppressive
microenvironment that drives tumorigenesis. Nat Commun
2016;7:11762.
	124.	Hwang HJ, Lee Y-R, Kang D, Lee HC, Seo HR, Ryu J-K, et  al.
Endothelial cells under therapy-induced senescence secrete CXCL11,
which increases aggressiveness of breast cancer cells. Cancer Lett
2020;490:100–10.
	125.	 Wang D, Xiao F, Feng Z, Li M, Kong L, Huang L, et al. Sunitinib
facilitates metastatic breast cancer spreading by inducing endothelial
cell senescence. Breast Cancer Res 2020;22:103.
	126.	 Amor C, Feucht J, Leibold J, Ho Y-J, Zhu C, Alonso-Curbelo D, et al.
Senolytic CAR T cells reverse senescence-associated pathologies.
Nature 2020;583:127–32.
	127.	 Aster JC, Pear WS, Blacklow SC. The varied roles of notch in cancer.
Annu Rev Pathol 2017;12:245–75.
	128.	 Kuczynski EA, Vermeulen PB, Pezzella F, Kerbel RS, Reynolds AR.
Vessel co-option in cancer. Nat Rev Clin Oncol 2019;16:469–93.
Downloadedfromhttp://aacrjournals.org/cancerdiscovery/article-pdf/12/1/31/3052722/31.pdfbyMasarykUniversityuseron10September2022