The Hallmarks of Cancer

Authors: Douglas Hanahan and Robert A. Weinberg

Published: Cell Vol 100 (2000), pages 57-70

Introduction

Cancer genes can be classified into two: the oncogenes with dominant gain of function; and tumor suppressor genes with recessive loss of function. In this paper, the authors described the rules that govern the transformation of cells into malignant states - acquired capabilities - shared across different types of cancers. Virtually, all mammalian cells carry similar molecular machinery that dictates cellular behaviors.

Tumorigenesis in humans is a multistep process, which reflects genetic alterations. Many cancers are diagnosed with age-dependent incidence that implicates 4-7 rate-limiting stochastic events. Lesions represents intermediate steps in which cells evolve toward malignancy. Genomes of tumor cells are invariably altered at multiple sites (i.e. lesions, point mutations, changes in chromosome complement). The transformation process is formally analogous to Darwinian evolution.

An Enumeration of the Traits

The main problem in cancer is that cancer cells have defects in regulatory circuits that govern normal cell proliferation and homeostasis. The large variety of types of cancers raises a number of questions:

• How many distinct regulatory circuits within each type of target cell must be disrupted for the cell to become cancerous?
• Does the same set of circuits suffer disruption in other types of cancer cells in the body?
• Which of the circuits operate cell-autonomously? Which are coupled to signals from environment?
• Can cancer-associated genes be related to these regulatory circuits?

The authors hypothesized that the cancer cell genotypes are the manifestation of 6 essential alterations shared among tumors

• Self-sufficiency in growth signals
• Insensitivity to growth-inhibitory signals
• Evasion of apoptosis
• Limitless replicative potential
• Sustained angiogenesis
• Tissue invasion and metastasis

Each represents successful breaching of the defense mechanism. The multiple mechanisms may be responsible for the relatively rare occurrence of cancer during an average human lifetime.

Self-sufficiency in growth signals

Normal cells require mitogenic growth signals (GS), transmitted by transmembrane receptors. The signaling molecules can be:

• Diffusible growth factors
• ECM components
• Cell-to-cell adhesion/interaction molecules (i.e. integrins)

Many oncogenes mimic normal growth signaling. The tumor cells have greatly reduced dependence on exogenous growth stimulation - generate many of their own growth signals - hence disrupting a critically important homeostatic mechanism. Many dominant oncogenes are found to modulate this.

3 common strategies:

1. Alteration of extracellular growth signals

• While most soluble mitogenic growth factors are made by one cell type to stimulate another (heterotypic signaling), cancer cells can synthesize the growth factors to which they are responsive - positive feedback loop or autocrine stimulation - and obviates dependence on GFs from other cells within the tissue.

2. Alteration of transcellular transducers/receptors

• GF receptors often carry tyrosine kinase activities in the cytoplasmic domains
• In tumor cells, the receptors are overexpressed - hyperresponsive to ambient levels of GFs (normally would not trigger proliferation)
  • epidermal GF receptor (EGF-R/erbB) upregulated in stomach, brain, and breast tumors
  • HER2/neu receptor overexpressed in stomach and mammary carcinomas
• Overexpression >> ligand-independent signaling (can also be achieved by structural alteration of receptors)
  • truncated EGF-R lacking cytoplasmic domain is activated constitutively
• Tumor cells can switch types of extracellular matrix receptors (integrins) they express - favors those that transmit progrowth signals
  • These cell surface receptors physically link to ECM
  • Successful binding to specific moieties of ECM >> integrins transduce signals into the cell
  • Failure to do so can impair cell motility, induce apoptosis, or cause cell cycle arrest (in normal cells)
• Both ligand-activated GFRs and progrowth integrins can activate SOS-Ras-Raf-MAPK pathway

3. Alteration of intracellular circuits translating those signals

• receive that process signals transduced by the cell surface receptors
• Structurally altered forms of Ras proteins release flux of mitogenic signals into cells without stimulation by normal upstream regulators

• The cascade is linked via cross-talking connections with other pathways - enabling signals to cause multiple cellular effects
  • Direct interaction of Ras protein with survival-promoting PI3 kinase >> growth signals >> survival signals within the cell

The growth signaling autonomy seems conceptually fine, but also too simplistic. The contributions of the ancillary cells or neighboring, normal cells need to be taken into account. Within normal tissue, cells are mostly instructed by their neighbors (paracrine signals) or via systemic regualtions (endocrine signals). The heterotypic signaling between diverse cell types within a tumor may be important in cell proliferation. The authors hypothesized that growth signals may originate from stromal cells. Successful tumor cells have acquired the ability to induce normal neighboring cells to release abundant amount of growth-stimulating signals. These cooperating cells may eventually become abnormal, coevolving to support tumor cells. Inflammatory cells may be attracted to the sites and promote (rather than kill) the tumor cells.

Insensitivity to antigrowth signals

Normally, antiproliferative signals maintain cell quiescence and tissue homeostasis

• Soluble growth inhibitors
• Immobilized inhibitors in ECM/surfaces of nearby cells

Effects:

• Cells may be forced into <math>G_0</math> quiescent state - may reemerge when Extracellular signals permit
• Cells may be induced to permanently lose proliferation potential by entering into postmitotic states - usually associated with acquisition of differentiation-associated traits

Circuitry that allows normal cells to respond to these signals is associated with cell cycle phases - the components dictating the transition of the cell through <math>G_1</math> phase. Cells monitor external environment for signals. Most antiproliferative signals are received via retinoblastoma protein (pRb) and its relatives (p107, p130)

• In hypophosphorylated state: pRb blocks proliferation by sequestering and altering function of E2F transcription factors
  • Disruption of the pathway liberates E2Fs >> allows cell proliferation (cells are insensitive to antigrowth factors)
• TGF<math>\beta</math> prevent the phosphorylation that inactivates pRb (still elusive)
  • It blocks proliferation
  • In some cell types, TGF<math>\beta</math> suppresses expression of c-myc gene (regulates <math>G_1</math> cell cycle machinery) (in unknown ways)
  • It also causes synthesis of the <math>p15^{INK4B}</math> and p21 proteins (blocks cyclin:CDK complexes responsible for pRb phosphorylation)
• Disruption in pRb signaling
  • From the loss of TGF<math>\beta</math> responsiveness via downregulation of the receptors
  • From mutant, dysfunctional receptors
  • Cytoplasmic Smad4 protein (transduces signals from ligand-activated TGF<math>\beta</math> receptors to downstream components) is lost by mutation
  • Deletion of the locus encoding <math>p15^{INK4B}</math>
  • CDK4, immediate downstream target, may become unresponsive to inhibitory activities of <math>p15^{INK4B}</math> by means of mutaitons (amino acid substitutions in INK4A/B-interacting domain) >> not blocking on cyclin:CDK complexes >> hyperphosphorylation of pRb
  • Functional pRb may be lost through mutaiton
  • Certain DNA virus-induced tumors >> pRb function eliminated by sequestration by viral oncoproteins (e.g. E7 oncoprotein of human papillomavirus)
• Turning off expression of integrins and cell adhesion molecules sending antigrowth signals and favoring those with progrowth signaling functions >> likely lead to pRb circuit

The tissue also constrain cell multiplication by instructing cells to irreveribly enter postmitotic, differentiated states. Tumor cells, on the other hand, use various strategies to evade these states.

• c-myc oncogene - encodes a TF
  • Normal development: growth-stimulating action of Myc in association with Max can be supplanted by Max-Mad complexes >> differentiation-inducing signals
  • Overexpression of c-Myc can reverse the process by shifting back the balance to favor Myc-Max >> promote growth
    • (In human colon carcinogenesis) inactivation of APC/<math>\beta</math>-catenin pathway blocks the egress of enterocytes in the colonic crypts into a differentiated state
    • (Avian erythroblastosis) erbA oncogene prevents irreversible erythrocyte differentiation

Evading Apoptosis

Apoptotic program is in the latent form in all the cell types in the body. It is a precise series of steps, once triggered.

• Cellular membranes are disrupted
• Cytoplasmic and nuclear skeletons are borken down
• Cytosol is extruded
• Chromosomes are degraded
• Nucleus is fragmented

All happens in 30-120 minutes. The result is the cell debris being engulfed by nearby cells, typically within 24 hours.

Two classes of components of the process:

1. Sensors

• Monitor extracellualr and intracellular environment for conditions of normality and abnormality that will influence whether a cell shold live or die

Extracellular sensors

• Cell surface receptors that bind survival factors
  • IGF-1/-2 <-> IGF-1R
  • IL-3 <-> IL-3R
• Cell surface receptors that bind death factors
  • FAS <-> FAS receptor
  • TNF<math>\alpha</math> <-> TNF-R1

Intracellular sensors

• Monitor cell's well-being and activate death pathway once detected abnormalities
  • DNA damage
  • Signaling imbalance by oncogene action
  • Survival signal insufficiency
  • Hypoxia

Note: Cell survival is also maintained by cell-matrix and cell-cell adherence-based survival signals

>> Regulatory signals likely reflect the needs of tissues to maintain cells in appropriate architectural configurations

2. Effectors

• Proapoptotic signals >> mitochondria >> release cytochrome C(potent catalyst of apoptosis)
• Members of Bcl-2 family
  • Proapoptotic: Bax, Bak, Bid, Bim
  • Antiapoptotic: Bcl-2, Bcl-XL, Bcl-W
• They govern mitochondrial death signaling thru cytochrome C release
• p53 tumor suppressor protein >> upregulates expression of Bax in response to sensing DNA damage
• Bax in turn stimulates mitochondria to release cytochrome C
• Ultimate effectors: caspases - two gatekeeper caspases
  • caspase-8: activated by death receptors (e.g. FAS)
  • caspase-9: activated by cytochrome C
  • Downstream caspases got activated and execute death program

People have been speculating that apoptosis is a barrier to cancer since 1972 (Kerr, Wyllie, Currie - described that hormone-dependent tumor cells underwent massive apoptosis once the hormone was removed). The oncogene bcl-2 was discovered by the upregulation via chromosomal translocation in follicular lymphoma (Korsmeyer, 1992). It was found that, when coexpresed with myc oncogene, bcl-2 gene promotes function of B cell lymphomas, via enhancement in lymphocyte survival rather than further stimulating myc-induced proliferation. (Note: myc involves in the regulation of G1 cell cycle)

Effects of a myc oncogene on fibroblasts in low serum culture:

• myc-expressing cells with no serum >> apoptosis
• Cells can be rescued by
  • exogenous survival factors: IGF-1
  • forced overexpression of Bcl-2 or related Bcl-XL protein
  • disruption of FAS death signaling pathway
• Apoptotic program can be triggered by overexpressed oncogene: Barrier!

Another case: pRb

• When pRb tumor suppressor is inactivated >> slowly growing tumors with high apoptotic rates
• Additional loss of p53 >> rapidly growing tumors with low apoptotic rates
• (Extracellular survival factors: knockout of IGF-2 gene expression (activated in tumors) >> impaired tumor growth and progression
  • Not affect proliferation rates >> antiapoptotic survival factor

Most commonly loss of a proapoptotic regulator thru mutation: p53 tumor suppressor gene - key component of DNA damage sensor (normally induce apoptotic effector cascade)

• Other signals, such as hypoxia and oncogene hyperexpression also interact in part with p53

PI3 kinase-AKT/PKB pathway

• transmits antiapoptotic survivial signals >> cells survive!
• likely involved in preventing apoptosis
• activated by
  • extracellular factors: IGF-1/2, IL-3
  • intracellular signals from Ras
  • loss of pTEN tumor suppressor (phospholipid phosphatase normally attenuating AKT survival signal)

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