The hallmarks of cancer comprise six biological capabilities acquired during the multiste

p development of human tumors. The hallmarks constitute an organizing principle

for rationalizing the
complexities of neoplastic disease. They include sustaining proliferative signaling, evad
ing growth
suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesi
s, and activating invasion and metastasis. Underlying these hallmarks are genome instabi
lity, which generates
the genetic diversity that expedites their acquisition, and inflammation, which fosters m
ultiple hallmark functions. Conceptual progress in the last decade has added two e
merging hallmarks of potential generality to this list-
reprogramming of energy metabolism and evading immune
destruction. In addition to cancer cells, tumors exhibit another dimension of compl
exity: they
contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisit
ion of hallmark traits by creating the ‘‘tumor microenvironment.’’ Recognition of the wid
espread applicability
of these concepts will increasingly affect the development of new means to treat huma
n cancer.

We have proposed that six hallmarks of cancer together constitute an organizing principle that provides a logical fra
mework for understanding the remarkable diversity of neoplastic diseases
(Hanahan and Weinberg, 2000). Implicit in our discussion was
the notion that as normal cells evolve progressively to
a neoplastic state, they acquire a succession of these hallmark
capabilities, and that the multistep process of human tumor
pathogenesis could be rationalized by the need of incipient
cancer cells to acquire the traits that enable them to become tumorigenic and ultimately malignant.

We noted as an ancillary proposition that tumors are more than

insular masses of proliferating cancer cells. Instead, they are
complex tissues composed of multiple distinct cell types that
participate in heterotypic interactions with one another. We depicted the recruited normal cells, which form tumor-
associated stroma, as active participants in tumorigenesis rather than
passive bystanders; as such, these stromal cells contribute to
the development and expression of certain hallmark capabilities.
During the ensuing decade this notion has been solidified and
extended, revealing that the biology of tumors can no longer
be understood simply by enumerating the traits of the cancer
cells but instead must encompass the contributions of the ‘‘tumor microenvironment’’ to tumorigenesis.

In the course of remarkable progress in cancer research

subsequent to this publication, new observations have served
both to clarify and to modify the original formulation of the hallmark capabilities. In addition, yet other observations h
ave raised questions and highlighted mechanistic concepts that were not
integral to our original elaboration of the hallmark traits. Motivated by these developments, we now revisit the o
riginal hallmarks, consider new ones that might be included in this roster,
and expand upon the functional roles and contributions made by recruited stromal cells to tumor biology.

The six hallmarks of cancer—distinctive and complementary

capabilities that enable tumor growth and metastatic dissemination-
continue to provide a solid foundation for understanding
the biology of cancer (Figure 1; see the Supplemental Information for downloadable versions of the figures for pres
entations). In the first section of this Review, we summarize the essence
of each hallmark as described in the original presentation in
2000, followed by selected illustrations (demarcated by subheadings in italics) of the conceptual progress mad
e over the past decade in understanding their mechanistic underpinnings.
In subsequent sections we address new developments that
broaden the scope of the conceptualization, describing in turn
two enabling characteristics crucial to the acquisition of the six
hallmark capabilities, two new emerging hallmark capabilities,
the constitution and signaling interactions of the tumor microenvironment crucial to cancer phenotypes, and we fina
lly discuss the new frontier of therapeutic application of these concepts.

Arguably the most fundamental trait of cancer cells involves their

ability to sustain chronic proliferation. Normal tissues carefully control the production and release of growth-
promoting signals that instruct entry into and progression through the cell growth-and-
division cycle, thereby ensuring a homeostasis of cell
number and thus maintenance of normal tissue architecture and
function. Cancer cells, by deregulating these signals, become
masters of their own destinies. The enabling signals are
conveyed in large part by growth factors that bind cell-surface
receptors, typically containing intracellular tyrosine kinase
domains. The latter proceed to emit signals via branched intra-
cellular signaling pathways that regulate progression through
the cell cycle as well as cell growth (that is, increases in cell size); often these signals influence yet other cell-
biological properties, such as cell survival and energy metabolism.

Remarkably, the precise identities and sources of the proliferative signals operating within normal tissues were po
orly understood a decade ago and in general remain so. Moreover, we still
know relatively little about the mechanisms controlling the
release of these mitogenic signals. In part, the understanding
of these mechanisms is complicated by the fact that the growth
factor signals controlling cell number and position within tissues
are thought to be transmitted in a temporally and spatially regulated fashion from one cell to its neighbors; such
paracrine signaling is difficult to access experimentally. In addition, the
bioavailability of growth factors is regulated by sequestration in
the pericellular space and extracellular matrix, and by the actions
of a complex network of proteases, sulfatases, and possibly
other enzymes that liberate and activate them, apparently in a highly specific and localized fashion.

The mitogenic signaling in cancer cells is, in contrast, better

understood (Lemmon and Schlessinger, 2010; Witsch et al.,
2010; Hynes and MacDonald, 2009; Perona, 2006). Cancer cells
can acquire the capability to sustain proliferative signaling in
a number of alternative ways: They may produce growth factor
ligands themselves, to which they can respond via the expression of cognate receptors, resulting in autocrine p
roliferative
stimulation. Alternatively, cancer cells may send signals to stimulate normal cells within the supporting tumor-
associated stroma, which reciprocate by supplying the cancer cells with
various growth factors (Cheng et al., 2008; Bhowmick et al.,
2004). Receptor signaling can also be deregulated by elevating
the levels of receptor proteins displayed at the cancer cell
surface, rendering such cells hyperresponsive to otherwise-limiting amounts
of growth factor ligand; the same
outcome can result from structural alterations in the receptor molecules that facilitate ligand-
independent firing.
Growth factor independence may also derive from the constitutive activation of
components of signaling pathways operating downstream of these receptors,
obviating the need to stimulate these pathways by ligand-mediated receptor
activation. Given that a number of distinct downstream signaling pathways radiate from a ligand-
stimulated receptor, the activation of one or another of these downstream pathways, for
example, the one responding to the Ras signal transducer,
may only recapitulate a subset of the regulatory instructions transmitted by an activated receptor.

High-throughput DNA sequencing analyses of cancer cell

genomes have revealed somatic mutations in certain human
tumors that predict constitutive activation of signaling circuits
usually triggered by activated growth factor receptors. Thus,
we now know that 40% of human melanomas contain
activating mutations affecting the structure of the B-Raf protein,
resulting in constitutive signaling through the Raf to mitogen-activated protein (MAP)-
kinase pathway (Davies and Samuels 2010). Similarly, mutations in the catalytic subunit of phosphoinositide 3-
kinase (PI3-kinase) isoforms are being detected in
an array of tumor types, which serve to hyperactivate the PI3-
kinase signaling circuitry, including its key Akt/PKB signal
transducer (Jiang and Liu, 2009; Yuan and Cantley, 2008). The
advantages to tumor cells of activating upstream (receptor)
versus downstream (transducer) signaling remain obscure, as
does the functional impact of crosstalk between the multiple pathways radiating from growth factor receptors.

Recent results have highlighted the importance of negative-

feedback loops that normally operate to dampen various types
of signaling and thereby ensure homeostatic regulation of the
flux of signals coursing through the intracellular circuitry (Wertz
and Dixit, 2010; Cabrita and Christofori, 2008; Amit et al.,
2007; Mosesson et al., 2008). Defects in these feedback mechanisms are capable of enhancing proliferative sign
aling. The prototype of this type of regulation involves the Ras oncoprotein:
the oncogenic effects of Ras do not result from a hyperactivation
of its signaling powers; instead, the oncogenic mutations
affecting ras genes compromise Ras GTPase activity, which operates as an intrinsic negative
feedback mechanism that normally ensures that active signal transmission is transitory.

Analogous negative-feedback mechanisms operate at

multiple nodes within the proliferative signaling circuitry. A prominent example involves the PTEN phosphatase, whic
h counter-acts PI3-kinase by degrading its product, phosphatidylinositol (3,4,5) trisphosphate (PIP3 ). Loss-of-
function mutations in PTEN amplify PI3K signaling and promote tumorigenesis in a variety
of experimental models of cancer; in human tumors, PTEN
expression is often lost by promoter methylation (Jiang and Liu, 2009; Yuan and Cantley, 2008).

Yet another example involves the mTOR kinase, a coordinator

of cell growth and metabolism that lies both upstream and down-
stream of the PI3K pathway. In the circuitry of some cancer cells,
mTOR activation results, via negative feedback, in the inhibition
of PI3K signaling. Thus, when mTOR is pharmacologically
inhibited in such cancer cells (such as by the drug rapamycin),
the associated loss of negative feedback results in increased
activity of PI3K and its effector Akt/PKB, thereby blunting the
antiproliferative effects of mTOR inhibition (Sudarsanam and
Johnson, 2010; O’Reilly et al., 2006). It is likely that compromised negative-
feedback loops in this and other signaling pathways
will prove to be widespread among human cancer cells and
serve as an important means by which these cells can achieve
proliferative independence. Moreover, disruption of such self-
attenuating signaling may contribute to the development of
adaptive resistance toward drugs targeting mitogenic signaling.

Early studies of oncogene action encouraged the notion that ever-

increasing expression of such genes and the signals manifested in their protein products would result in correspond
ingly ncreased cancer cell proliferation and thus tumor growth. More
recent research has undermined this notion, in that excessively
elevated signaling by oncoproteins such as RAS, MYC, and
RAF can provoke counteracting responses from cells, specifically induction of cell senescence and/or apoptosi
s (Collado and Serrano, 2010; Evan and d’Adda di Fagagna, 2009; Lowe
et al., 2004). For example, cultured cells expressing high levels
of the Ras oncoprotein may enter into the nonproliferative but
viable state called senescence; in contrast, cells expressing
lower levels of this protein may avoid senescence and proliferate.

Cells with morphological features of senescence, including

enlarged cytoplasm, the absence of proliferation markers, and expression of the senescence-
induced b-galactosidase enzyme, are abundant in the tissues of mice engineered to over-
express certain oncogenes (Collado and Serrano, 2010; Evan
and d’Adda di Fagagna, 2009) and are prevalent in some cases
of human melanoma (Mooi and Peeper, 2006). These ostensibly
paradoxical responses seem to reflect intrinsic cellular defense
mechanisms designed to eliminate cells experiencing excessive
levels of certain types of signaling. Accordingly, the relative
intensity of oncogenic signaling in cancer cells may represent
compromises between maximal mitogenic stimulation and
avoidance of these antiproliferative defenses. Alternatively,
some cancer cells may adapt to high levels of oncogenic signaling by disabling their senescence-
or apoptosis-inducing circuitry.

In addition to the hallmark capability of inducing and sustaining positively acting growth-
stimulatory signals, cancer cells must also circumvent powerful programs that negatively regulate
cell proliferation; many of these programs depend on the actions
of tumor suppressor genes. Dozens of tumor suppressors that
operate in various ways to limit cell growth and proliferation
have been discovered through their characteristic inactivation
in one or another form of animal or human cancer; many of these
genes have been validated as bona fide tumor suppressors through gain- or loss-of-
function experiments in mice. The two prototypical tumor suppressors encode the RB (retinoblastoma-
associated) and TP53 proteins; they operate as central
control nodes within two key complementary cellular regulatory
circuits that govern the decisions of cells to proliferate or, alternatively, activate senescence and apoptotic program
s.

The RB protein integrates signals from diverse extracellular

and intracellular sources and, in response, decides whether or not a cell should proceed through its growth-and-
division cycle (Burkhart and Sage, 2008; Deshpande et al., 2005; Sherr and
McCormick, 2002). Cancer cells with defects in RB pathway
function are thus missing the services of a critical gatekeeper of cell-
cycle progression whose absence permits persistent cell proliferation. Whereas RB transduces growth-
inhibitory signals that originate largely outside of the cell, TP53 receives
inputs from stress and abnormality sensors that function within
the cell’s intracellular operating systems: if the degree of
damage to the genome is excessive, or if the levels of nucleotide pools, growth-
promoting signals, glucose, or oxygenation are suboptimal, TP53 can call a halt to further cell-cycle progression
until these conditions have been normalized. Alternatively, in the
face of alarm signals indicating overwhelming or irreparable
damage to such cellular subsystems, TP53 can trigger
apoptosis. Notably, the various effects of activated TP53 are
complex and highly context dependent, varying by cell type as
well as by the severity and persistence of conditions of cell stress and genomic damage.

Although the two canonical suppressors of proliferation-TP53 and RB—

have preeminent importance in regulating cell
proliferation, various lines of evidence indicate that each operates as part of a larger network that is wired for functio
nal redundancy. For example, chimeric mice populated throughout their
bodies with individual cells lacking a functional Rb gene are
surprisingly free of proliferative abnormalities, despite the expectation that loss of RB function would allow continuous
firing of the cell division cycle in these cells and their lineal descendants;
some of the resulting clusters of Rb null cells should, by all rights,
progress to neoplasia. Instead, the Rb null cells in such chimeric
mice have been found to participate in relatively normal tissue
morphogenesis throughout the body; the only neoplasia
observed was in the development of pituitary tumors late in life
(Lipinski and Jacks, 1999). Similarly, TP53 null mice develop normally, show largely proper cell and tissue homeos
tasis, and again develop abnormalities later in life, in the form of leukemias
and sarcomas (Ghebranious and Donehower, 1998). Both examples must reflect the operations of redundantly acti
ng mechanisms that serve to constrain inappropriate replication of cells
lacking these key proliferation suppressors.

Four decades of research have demonstrated that the cell-to-

cell contacts formed by dense populations of normal cells propagated in two-
dimensional culture operate to suppress further cell proliferation, yielding confluent cell monolayers. Importantly,
such ‘‘contact inhibition’’ is abolished in various types of cancer
cells in culture, suggesting that contact inhibition is an in vitro
surrogate of a mechanism that operates in vivo to ensure normal
tissue homeostasis, one that is abrogated during the course of
tumorigenesis. Until recently, the mechanistic basis for this
mode of growth control remained obscure. Now, however,
mechanisms of contact inhibition are beginning to emerge.

One mechanism involves the product of the NF2 gene, long

implicated as a tumor suppressor because its loss triggers
a form of human neurofibromatosis. Merlin, the cytoplasmic
NF2 gene product, orchestrates contact inhibition via coupling cell-surface adhesion molecules (e.g., E-
cadherin) to transmembrane receptor tyrosine kinases (e.g., the EGF receptor). In so
doing, Merlin strengthens the adhesivity of cadherin-mediated cell-to-
cell attachments. Additionally, by sequestering growth
factor receptors, Merlin limits their ability to efficiently emit mitogenic signals (Curto et al., 2007; Okada et al., 2005).

A second mechanism of contact inhibition involves the LKB1

epithelial polarity protein, which organizes epithelial structure
and helps maintain tissue integrity. LKB1 can, for example,
overrule the mitogenic effects of the powerful Myc oncogene
when the latter is upregulated in organized, quiescent epithelial
structures; in contrast, when LKB1 expression is suppressed,
epithelial integrity is destabilized, and epithelial cells become susceptible to Myc-
induced transformation (Partanen et al., 2009; Hezel and Bardeesy, 2008). LKB1 has also been identified
as a tumor suppressor gene that is lost in certain human malignancies (Shaw, 2009), possibly reflecting its normal f
unction as a suppressor of inappropriate proliferation. It remains to be
seen how frequently these two mechanisms of contact-
mediated growth suppression are compromised in human cancers; no doubt yet other contact-
induced proliferative barriers are yet to be discovered. Clearly mechanisms like these that enable
cells to construct and maintain architecturally complex tissues
represent important means of suppressing and counterbalancing inappropriate proliferative signals.