Hallmarks of Cancer The Next Generation

Leading Edge oo o, r M a u s t h io t g a e e cells but instead must encompass the contributions of the the new frontier of therapeutic application of these concepts. subsequent to this publication, new observations have served both to clarify and to modify the original formulation of the hall- mark capabilities. In addition, yet other observations have raised 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 questions and highlighted mechanistic concepts that were not integral to our original elaboration of the hallmark traits. Moti- that instruct entry into and progression through the cell growth- and-division cycle, thereby ensuring a homeostasis of cell ‘‘tumor microenvironment’’ to tumorigenesis. In the course of remarkable progress in cancer research Sustaining Proliferative Signaling INTRODUCTION We have proposed that six hallmarks of cancer together consti- tute an organizing principle that provides a logical framework 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 aremore 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 de- picted 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 vated by these developments, we now revisit the original hall- marks, 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. HALLMARK CAPABILITIES—CONCEPTUAL PROGRESS The six hallmarks of cancer—distinctive and complementary capabilities that enable tumor growth and metastatic dissemina- tion—continue to provide a solid foundation for understanding the biology of cancer (Figure 1; see the Supplemental Informa- tion for downloadable versions of the figures for presentations). 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 sub- headings in italics) of the conceptual progress made 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 microen- vironment crucial to cancer phenotypes, and we finally discuss Hallmarks of Cancer: Th Douglas Hanahan1,2,* and Robert A. Weinberg3,* 1The Swiss Institute for Experimental Cancer Research (ISREC), Sch 2The Department of Biochemistry & Biophysics, UCSF, San Francisc 3Whitehead Institute for Biomedical Research, Ludwig/MIT Center fo MA 02142, USA *Correspondence: dh@epfl.ch (D.H.), weinberg@wi.mit.edu (R.A.W.) DOI 10.1016/j.cell.2011.02.013 The hallmarks of cancer comprise six biological c ment of human tumors. The hallmarks constit complexities of neoplastic disease. They include suppressors, resisting cell death, enabling replica vating invasion andmetastasis. Underlying these the genetic diversity that expedites their acquisit mark functions. Conceptual progress in the las potential generality to this list—reprogrammin destruction. In addition to cancer cells, tumors contain a repertoire of recruited, ostensibly norm mark traits by creating the ‘‘tumor microenvironm of these concepts will increasingly affect the dev 646 Cell 144, March 4, 2011 ª2011 Elsevier Inc. Review e Next Generation l of Life Sciences, EPFL, Lausanne CH-1015, Switzerland CA 94158, USA olecular Oncology, and MIT Department of Biology, Cambridge, pabilities acquired during themultistep develop- te an organizing principle for rationalizing the ustaining proliferative signaling, evading growth ive immortality, inducing angiogenesis, and acti- allmarks are genome instability, which generates n, and inflammation, which fosters multiple hall- decade has added two emerging hallmarks of of energy metabolism and evading immune exhibit another dimension of complexity: they l cells that contribute to the acquisition of hall- nt.’’ Recognition of the widespread applicability lopment of new means to treat human cancer. 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 prop- erties, such as cell survival and energy metabolism. Remarkably, the precise identities and sources of the prolifer- ative signals operating within normal tissues were poorly under- stood 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 regu- lated 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 expres- sion of cognate receptors, resulting in autocrine proliferative stimulation. Alternatively, cancer cells may send signals to stim- ulate 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 resulting in constitutiv activated protein (MA 2010). Similarly, mutat nositide 3-kinase (PI3 an array of tumor type kinase signaling circ transducer (Jiang and advantages to tumor versus downstream (t does the functional im pathways radiating fro Disruptions of Nega Attenuate Proliferat Recent results have feedback loops that n of signaling and there flux of signals coursin and Dixit, 2010; Cab 2007; Mosesson et al. anisms are capable o prototype of this type o the oncogenic effects of its signaling pow affecting ras genes c Cell 1 ffecting the structure of the B-Raf protein, e signaling through the Raf to mitogen- P)-kinase pathway (Davies and Samuels ions in the catalytic subunit of phosphoi- -kinase) isoforms are being detected in s, which serve to hyperactivate the PI3- uitry, including its key Akt/PKB signal Liu, 2009; Yuan and Cantley, 2008). The cells of activating upstream (receptor) ransducer) signaling remain obscure, as pact of crosstalk between the multiple m growth factor receptors. tive-Feedback Mechanisms that Figure 1. The Hallmarks of Cancer This illustration encompasses the six hallmark capabilities originally proposed in our 2000 per- spective. The past decade has witnessed remarkable progress toward understanding the mechanistic underpinnings of each hallmark. surface, rendering such cells hyperre- sponsive to otherwise-limiting amounts of growth factor ligand; the same outcome can result from structural alter- ations in the receptor molecules that facilitate ligand-independent firing. Growth factor independence may also derive from the constitutive activation of components of signaling pathways oper- ating 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 activa- tion 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. Somatic Mutations Activate Additional Downstream Pathways 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 a ive Signaling highlighted the importance of negative- ormally operate to dampen various types by ensure homeostatic regulation of the g through the intracellular circuitry (Wertz rita and Christofori, 2008; Amit et al., , 2008). Defects in these feedback mech- f enhancing proliferative signaling. The f regulation involves the Ras oncoprotein: of Ras do not result from a hyperactivation ers; instead, the oncogenic mutations ompromise Ras GTPase activity, which 44, March 4, 2011 ª2011 Elsevier Inc. 647 operates as an intrinsic negative-feedback mechanism that nor- mally ensures that active signal transmission is transitory. Analogous negative-feedback mechanisms operate at multiple nodes within the proliferative signaling circuitry. A prom- inent example involves the PTEN phosphatase, which 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 andmetabolism 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. Excessive Proliferative Signaling Can Trigger Cell Senescence Early studies of oncogene action encouraged the notion that ever-increasing expression of such genes and the signals mani- fested in their protein products would result in correspondingly increased 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, specifi- cally induction of cell senescence and/or apoptosis (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 proteinmay 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. 648 Cell 144, March 4, 2011 ª2011 Elsevier Inc. Evading Growth Suppressors 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 (retinoblas- toma-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, alter- natively, activate senescence and apoptotic programs. 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 oper- ates as part of a larger network that is wired for functional redun- dancy. For example, chimeric mice populated throughout their bodies with individual cells lacking a functional Rb gene are surprisingly free of proliferative abnormalities, despite the expec- tation that loss of RB functionwould allow continuous firing of the cell division cycle in these cells and their lineal descendants; some of the resulting clusters ofRb 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 nor- mally, show largely proper cell and tissue homeostasis, and again develop abnormalities later in life, in the form of leukemias and sarcomas (Ghebranious and Donehower, 1998). Both exam- ples must reflect the operations of redundantly acting mecha- nisms that serve to constrain inappropriate replication of cells lacking these key proliferation suppressors. Mechanisms of Contact Inhibition and Its Evasion Four decades of research have demonstrated that the cell-to- cell contacts formed by dense populations of normal cells prop- agated 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 transmem- brane 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 mito- genic 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 malig- nancies (Shaw, 2009), possibly reflecting its no