Abeloff's Clinical Oncology, 4th Edition

Part I – Science of Clinical Oncology

Section B – Genesis of Cancer

Chapter 14 – Progressing from Gene Mutations to Cancer

Eric R. Fearon,Guido T. Bommer

SUMMARY OF KEY POINTS

  

   

A root cause of cancer is the accumulation of defects in genes that play critical roles in regulating cell proliferation, cellular differentiation, and cell death. The mutations in cancer cells are of two types: gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes.

  

   

Epigenetic mechanisms can substantially alter expression of proto-oncogenes and tumor suppressor genes, leading to essentially the same consequences as if the structure and/or sequence of the genes were affected by mutation.

  

   

Clinical and pathologic studies indicate that most cancers arise from pre-existing benign lesions, and it is estimated that six to seven mutations are needed for development of a clinically recognizable cancer. On the basis of molecular analyses of cancers, multiple gene defects may often accumulate in a cancer cell during its development, and benign lesions generally have fewer defects than do their malignant counterparts.

  

   

A process termed clonal selection has a key role in determining the particular constellation of genetic and epigenetic defects that are present in a cancer cell. Clonal selection is essentially a punctuated evolutionary process that promotes outgrowth of precancerous and cancerous cells carrying those mutations and gene expression changes that confer the most potent proliferative and survival properties upon the cancer cells, in a given context.

  

   

Although a diverse array of mutations and gene expression changes have been implicated in cancer pathogenesis, the defects appear to affect a more limited number of conserved signaling pathways or networks. The proto-oncogenes and tumor suppressor genes that are most frequently mutated in cancer cells most likely represent particularly critical hubs in the cell's regulatory circuitry.

  

   

Although cancer represents a very heterogeneous collection of diseases, the development of all cancers, regardless of type, appears to be critically dependent on the acquisition of certain traits that allow the cancer cells to grow in an unchecked fashion in their tissue of origin and to grow as metastatic lesions in distant sites in the body. Signature traits that are likely to be inherent in the majority, if not all, of cancer cells include the following: (1) an increased tendency to manifest a stem cell or progenitor-like phenotype, (2) an enhanced response to growth-promoting signals, (3) a relative resistance to growth inhibitory cues, (4) an increased mutation rate to allow for the rapid generation of new variant daughter cells, (5) the ability to attract and support a new blood supply (angiogenesis), (6) the capacity to minimize an immune response and/or evade destruction by immune effector cells, (7) the capacity for essentially limitless cell division, (8) a failure to respect tissue boundaries, allowing for invasion into adjacent tissues and organs as well as blood vessels and lymphatics, and (9) the ability to grow in organ sites with microenvironments that are markedly different from the one where the cancer cells arose.

  

   

Certain gene defects in cancer cells may contribute to a few or perhaps even only one of the signature traits of cancer cells. However, many of the gene defects and expression changes might have been selected for in large part because they exert pleiotropic effects on the cancer cell phenotype.

  

   

Despite the fact that some gene defects may arise early in the development of certain cancer types, advanced cancer cells might still be critically dependent on the “early gene defects” for continued growth and survival. Such findings imply that agents that specifically target key signaling pathways and proteins could have utility in advanced cancers even if the signaling pathway defect arose very early in cancer development.

  

   

Future studies will further clarify the role of gene defects in cancer phenotype, allowing more definitive and more specific strategies for inhibiting cancer cells.

INTRODUCTION

A genetic basis for human cancer has been recognized for perhaps more than a century and has been supported by data from familial and epidemiologic studies and animal studies. However, only in the past 25 years has convincing molecular evidence been obtained to support the view that cancer is a genetic disease. Studies from many different fields, including tumor virology, chemical carcinogenesis, molecular biology, somatic cell genetics, and genetic epidemiology, have provided fundamental insights into mechanisms underlying cancer development. While environmental and dietary factors as well as other genes undoubtedly have substantial roles in cancer development, it is now well established that the accumulation of multiple mutations in a single cell plays a fundamental role in the pathogenesis of cancer. The mutations occur in two distinct classes of cellular genes: oncogenes and tumor suppressor genes. As was noted in earlier chapters, some mutations may be present in individuals’ germline and may predispose to particular cancers. Such mutations can also be passed on to future generations. The nature and role of germline mutations in cancer development are of great interest to cancer biologists because the mutations provide powerful clues about the identity of genes and pathways that play particularly critical roles in the malignant conversion of cells. Nevertheless, germline mutations in oncogenes or tumor suppressor genes likely have a major contributing role in only a small fraction of cancers, and the vast majority of mutations in cancer are somatic (i.e., present only in the tumor cells).

A subset of the cellular genes that are affected by inherited and somatic mutations in human cancer will be discussed in more detail later. Brief mention will be made here of some general properties of the genes. Oncogenes, when mutated, act in a positive fashion to promote tumorigenesis. Their normally functioning cellular counterparts, termed proto-oncogenes, have been found to be important regulators of many aspects of cell growth. The proteins that are encoded by various proto-oncogenes can be found in virtually all subcellular compartments. The term proto-oncogene does not imply that genes of this class lie dormant in the cell with the purpose of promoting tumorigenesis. Rather, the terminology reflects the fact that mutations in cancer cells alter the normal structure and/or expression pattern of the proto-oncogene, generating oncogenic variant forms with altered function. In genetic terms, oncogenic alleles have gain-of-function mutations that confer enhanced or novel functions.

In contrast to the activating mutations in oncogenes, loss-of-function defects in tumor suppressor genes are found in cancer cells. The term antioncogene has sometimes been used with respect to the tumor suppressor class of genes. The term suggests that the primary function of the genes might be to act in direct opposition to activated oncogenes. While some of the proteins that are encoded by tumor suppressor genes do in fact bind to and regulate the function of proto-oncogenes or function in pathways that directly regulate proto-oncogene activity, it is not by necessity a general principle.[1] Hence, genes that contribute to cancer by virtue of inactivating or loss-of-function mutations in human cancers will be referred to here as tumor suppressor genes. Similarly to the proto-oncogenes, the normal functions of tumor suppressor genes are diverse, and the proteins that are encoded by these genes are found in essentially all compartments of the cell. Compared to the vast array of oncogenic alleles that are seen in human cancer, a somewhat more limited number of tumor suppressor genes have been identified at the molecular level thus far. This finding might not be an accurate representation of the prevalence of oncogene versus tumor suppressor gene mutations in cancer. Rather, it might reflect the practical difficulties that are associated with experimental strategies to identify genes that negatively regulate the growth of cells.

Much evidence indicates that mutations in genes that regulate the recognition and repair of DNA damage play critical roles in tumorigenesis. The DNA damage recognition and repair genes could be considered to constitute a distinct class of cancer genes. However, because DNA repair genes appear nearly invariably to be affected by loss-of-function mutations in cancer, they will be classified here as tumor suppressor genes. Nevertheless, on the basis of certain features, DNA damage recognition and repair genes might constitute a potentially unique subset of tumor suppressor genes. Specifically, in comparison to the presumed direct role of many tumor suppressor genes in regulation of cell growth and programmed cell death, at least some of the DNA repair proteins might have a more passive role in growth, differentiation, and cell survival. Their inactivation in tumor cells might lead predominantly to the acquisition of a “mutator phenotype,” with a resultant increased rate of mutations in other cellular genes with rate-determining roles in the cancer process, that is, oncogenes and tumor suppressor genes.

In addition to the well-established role of oncogene and tumor suppressor gene mutations in cancer, there is a large and growing body of data indicating that epigenetic mechanisms might play critical roles in altering the patterns and levels of expression of certain proto-oncogenes and tumor suppressor genes in cancer. For instance, in some cancers, defects in transcriptional regulatory mechanisms can lead to markedly increased levels of proto-oncogene expression, akin to the level that is seen in cancer cells with mutational defects that alter the structure or copy number of the proto-oncogene. Conversely, gene-silencing mechanisms can exert dramatic effects on the expression of certain tumor suppressor genes in cancer cells, essentially rendering the genes functionally inactive in the absence of any mutations.

Given the enormous advances over the past two decades in defining oncogene and tumor suppressor gene mutations and gene expression defects in cancer, it will not be possible in this chapter to review in a comprehensive fashion the vast collection of gene defects that have been identified in human cancers. Nor will it be possible to discuss in great detail the possible contributions of the many different gene defects to alterations in cell signaling and cell physiology. Rather, the primary aim of this chapter will be to offer a framework for understanding the relationship between gene defects in cancer cells and the impact of the accumulated defects on the cancer cell phenotype. Although some details on the identity and nature of gene defects in cancer will be offered here, the emphasis will be on concepts that are likely to have biologic and clinical significance.

CANCERS ARISE FROM THE ACCUMULATION OF MULTIPLE GENE DEFECTS

On the basis of a simple consideration of the likely large number of mutations that arise in normal cells during the many years of life, it would seem quite unlikely that cancers arise as the result of any single gene defect. Even in individuals who are strongly predisposed to cancer as a result of a germline mutation in a specific oncogene or tumor suppressor gene, the vast majority of cells in the individual never develop into cancer or even display definitive morphologic changes akin to those that are seen in benign tumors. In fact, depending on the inherited cancer syndrome, a significant fraction of those that carry specific germline mutations in oncogenes or tumor suppressor genes never develop cancer. Therefore, any model for cancer must incorporate these data, suggesting that cancers likely arise as the result of the accumulation of multiple gene defects in an affected cell. Another issue to consider before formulating genetic models for cancer development is that clinical and histopathologic data indicate that the development of nearly all cancers, regardless of the organ site, is often, if not invariably, preceded by precancerous phases or stages in which the neoplastic cells manifest increasing disordered patterns of differentiation and morphology.

Given this background, it would appear that there is compelling evidence that cancers arise from accumulated defects in several genes and precancerous (benign) precursor lesions contain by necessity fewer of the key gene defects. One question, therefore, is how many rate-limiting defects or “hits” are required for cancer development? While a definitive answer to this question cannot be given at this point, some estimates can be offered. Most common cancers show dramatically increased incidence with increasing age. On the basis of analysis of the age-specific incidence of a number of common cancers and some straightforward assumptions about the rate of mutations and the size of the target cell population, it was argued as early as the mid-1950s that most common epithelial cancers arise as the result of four to seven rate-limiting events. [2] [3] It was inferred that these rate-limiting events represented mutational events. Moreover, benign lesions were inferred to arise as the result of fewer gene defects, consistent with the fact that recognizable benign lesions that are often found show an age-incidence distribution that was shifted roughly one to two decades earlier in life than cancers arising in the corresponding organ or tissue sites. Nevertheless, confounding the use of age-incidence data to model the number of rate-limiting mutations were questions about certain key biologic assumptions underlying the multihit models. Given the attendant uncertainties with estimates of rate-limiting mutation numbers derived largely or solely from age-incidence data and the practical difficulties in defining the nature and significance of all inherited and somatic gene defects in cancer, a definitive answer to the number of rate-determining mutations for a particular cancer type has not yet been obtained. However, at the very least, it is encouraging to note that molecular analyses of a number of common cancers, such as those of the colon, [4] [5] breast,[5] lung,[6] and pancreas,[7] indicate that five or more gene defects are not infrequently seen in cancers, and fewer of the gene defects are seen in certain of the precancerous precursor lesions that are associated with these common cancers.

CLONAL SELECTION AND EVOLUTION IN CANCER

As was noted previously, most, if not all, cancers are thought to arise from pre-existing precancerous populations of cells, and multiple rate-limiting mutations (events) are likely needed for conversion of a normal cell to a cancerous cell. Molecular studies of cancers of various types and their corresponding associated precancerous lesions have yielded some fundamental insights into the processes likely to be critical in emergence of the cancer. First, while normal tissues and tissues from noncancerous disease states display polyclonal (balanced) cell populations, the neoplastic component that is present in benign lesions and cancers invariably displays a clonally related cell population, consistent with the notion that neoplastic transformation of one or at most a few cells within a tissue gave rise to all daughter cells that are present in the tumor. Second, in tumors where it has been possible to analyze both cancer cells and associated precancerous cell populations, a subset of the somatic gene defects that are present in the cancer are clonally represented in the precancerous cell population. Other somatic gene defects appear to be acquired during progression from the precancer subclones to the dominant subclone in the cancer.

These molecular findings in benign and malignant tumors are essentially consistent with a model that was initially proposed by Foulds[8] and subsequently advanced by Nowell[9] ( Fig. 14-1 ). In brief, the clonal evolution model predicts that cancers arise as the result of successive expansions of clonally related cell populations. The successive expansions are driven by the punctuated acquisition of mutations and gene expression changes that endow a particular cell and its progeny with a selective growth and survival advantage over cells that do not harbor the gene defects. In essence, clonal selection is an evolutionary process that allows the outgrowth of precancerous and cancerous cells that carry mutations and gene expression changes that confer the most potent proliferative and survival properties on the cancer cells. It is important to note the specific constellation of genetic and epigenetic changes that are present in precancerous and cancerous cells is context-dependent and likely varies considerably from one cancer type to another and perhaps even to a significant degree among cancers that display quite similar clinical and histopathologic features. The basis for the context-dependent relationship of the changes that confer a selective growth advantage in a particular cancer may reflect physiologic differences in organ site and the tissue microenvironment within the organ site, the identities of the preceding somatic gene defects in the precancerous or cancerous clone, and even the constitutional sequence variations and particular gene expression patterns that are present in nonneoplastic tissues of a given patient. This issue of context-dependent effects of gene defects that promote clonal selection in neoplastic cells will be addressed further in the following sections.

 
 

Figure 14-1  Role of clonal selection in cancer development and progression. Clonal selection is essentially a punctuated evolutionary process that promotes outgrowth of precancerous and cancerous cells that carry the mutations and gene expression changes that confer the most potent proliferative and survival properties on the cancer cells in a given context. The schematic diagram indicates that the stepwise emergence of benign and malignant cells over time is critically influenced by mutations and epigenetic defects. Neoplasms most likely arise from a stem cell or progenitor cell population that is capable of additional cell divisions and acquisition of certain differentiated characteristics. Following the accumulation of a particular constellation of mutations and epigenetic defects in oncogenes and tumor suppressor genes, a successful malignant subclone will outgrow the various competing neoplastic subclones. Further genetic heterogeneity within the malignant subclone (not depicted) is possible, and the genetic heterogeneity in the malignant subclone might give rise to new subclones that display increased invasive and metastatic potential.  (Modified from Kern SE: Progressive genetic abnormalities in human neoplasia. In Mendelsohn J, Howley PM, Israel MA, Liotta LA [eds]: The Molecular Basis of Cancer, 2nd ed. Philadelphia, WB Saunders, 2001, pp 41–69.)

 



The clonal evolution model has some important biologic and clinical ramifications, just a few of which will be mentioned here. First, the clonal gene defects that are present in a cancer can be traced in precancerous lesions from the same organ site with a goal of attempting to clarify the preferred order in which gene defects arise in the natural history of a particular cancer type. The particular order in which defects accumulate during the initiation and subsequent progression of one cancer type often differs from that in another cancer type. As a result, a genetic or epigenetic change that is critical in tumor initiation in one cancer type might contribute to tumor progression in another tumor type and vice versa. Second, defects that arise at “early” stages of tumorigenesis might play a vital role not only in tumor initiation but also in the aggressive behavior of advanced stage cancers. Third, the model predicts that the acquisition of further genetic heterogeneity will be a common and important factor in primary cancer lesions and metastases. Genetic heterogeneity likely plays a significant role in resistance to chemotherapy and the emergence of aggressive cell populations in patients with advanced cancer.[10]

It is important to note that clonal somatic mutations are often presumed to have a causal role in promoting further tumor outgrowth or progression because somatic mutations can become clonal (i.e., present in all neoplastic cells) by only a limited number of mechanisms. For instance, the genetic alteration itself could have been selected for because it provided the neoplastic cell with a growth advantage, allowing it to become the predominant cell type in the tumor (clonal expansion). Genes with critical roles in promoting clonal outgrowth in a given cancer have been termed drivers.[11] Alternatively, a somatic mutation, when detected, might have arisen essentially coincident with another, perhaps undetected, alteration that was the crucial change underlying clonal outgrowth. Somatic mutations of this latter type have been termed passengers.[11] Genes that are mutant in a significant fraction of cancers and for which other lines of evidence link them to the cancer process can more readily be classified as drivers. However, on the basis of early data from some large-scale sequencing analyses that reveal large numbers of distinct genes that are each mutated in only a minority of cancers of a given type, [5] [12] [13] sorting out drivers and passengers might not be entirely straightforward, based solely on sequencing data, but will likely require a significant body of functional studies and data.[11]

Given this discussion about the potential uncertainties associated with linking specific somatic mutations to cancer development, it is perhaps apparent why it is even more problematic to assign causal significance to gene expression changes in precancerous and cancerous cells. In particular, the ambiguities in assigning a causal role to gene expression and epigenetic changes in the absence of mutations are in large part due to difficulties in determining whether these changes merely reflect or are causally involved in the cancer process. Nonetheless, if a specific gene alteration can be shown to promote tumorigenesis or neoplastic transformation in in vitro models or animal model experiments or if the same gene or chromosomal region is recurrently altered in tumors, then it might be reasonable to infer that the particular defect might indeed have a causal (driver) role in tumorigenesis.

ONCOGENE AND TUMOR SUPPRESSOR GENE DEFECTS IN CANCER TARGET CONSERVED SIGNALING PATHWAYS

Recurrent Mutational Targets in Cancer

As was noted previously, in genetic terms, oncogenic alleles have gain-of-function mutations. Oncogenic variant alleles that are present in cancer are generated from the normal counterpart proto-oncogenes by various mutational mechanisms, including point or localized mutations, gross chromosomal rearrangements, or gene amplification. Some representative oncogene mutations in human cancer are summarized in Table 14-1 . From a brief review of the data in Table 14-1 , several generalizations are apparent. First, the mutations affect proteins functioning in various compartments of the cell, including growth factor receptors, cytoplasmic signal tranducers, and nuclear proteins, such as transcription factors. Second, although some oncogene mutations may be unique to cancers of a particular type, such as the specific chromosomal translocations and resultant fusion proteins that are seen in cancers of hematopoietic origin (e.g., the BCR-ABL translocation that is seen in chronic myelogenous leukemia and a subset of acute lymphoid leukemias and the PML-RARa translocation that is seen in acute promyelocytic leukemia), other mutations, such as those affecting the K-RAS, β-catenin, and c-MYC genes, are found in a broad spectrum of different cancer types. Third, oncogene mutations in cancer are nearly always somatic, as only a very limited number of germline mutations in proto-oncogenes have been linked to cancer predisposition thus far.[14] Fourth, some proto-oncogenes, such as K-RAS or BCL2, are somatically altered in cancer by a single mutational mechanism, namely, point mutations in the K-RAS gene and chromosomal translocations affecting the BCL2 gene. In contrast, other proto-oncogenes, such as c-MYC, may be activated by more than one mechanism in cancer, including chromosomal translocation and gene amplification. Both mutational mechanisms lead to increased levels of c-MYC transcripts and protein. In fact, specific missense mutations at threonine58 of the c-MYC gene in some lymphomas may further enhance c-MYC protein levels by abrogating a phosphorylation-ubquitination mechanism targeting c-MYC for proteasomal degradation. [15] [16]


Table 14-1   -- Representative Oncogene Mutations in Cancer

Gene

Activation Mechanism

Protein Properties

Tumor Types

K-RAS

Point mutation

Signal transducer

Pancreatic, colorectal, lung (adeno), endometrial, other carcinomas

N-RAS

Point mutation

Signal transducer

Myeloid leukemia, colorectal cancer

H-RAS

Point mutation

Signal transducer

Bladder carcinoma

EGFR (ERBB)

Amplification, mutation

Growth factor (EGF) receptor

Gliomas, lung (non-small cell) carcinoma

NEU (HER2/ERBB2)

Amplification

Growth factor receptor

Breast, ovarian, gastric, other carcinomas

c-MYC

Chromosome translocation

Transcription factor

Burkitt's lymphomas

 

Amplification

 

Small cell lung carcinoma (SCLC); other carcinomas; glioblastoma

N-MYC

Amplification

Transcription factor

Neuroblastoma, SCLC; glioblastoma

L-MYC

Amplification

Transcription factor

SCLC, ovarian carcinoma

BCL-2

Chromosome translocation

Antiapoptosis protein

B-cell lymphoma (follicular type)

CYCD1

Amplification

Cyclin D, cell cycle control

Breast and other carcinomas

 

Chromosome translocation

 

B-cell lymphoma, parathyroid adenoma

BCR-ABL

Chromosome translocation

Chimeric nonreceptor tyrosine kinase

CML, ALL (T cell)

RET

Chromosome translocation

GDNF receptor tyrosine kinase

Thyroid cancer (papillary type)

 

Point mutation

 

Thyroid cancer (medullary type: germline mutations)

CDK4

Amplification

Cyclin-dependent kinase

Sarcoma, glioblastoma

 

Point mutation

 

 

MET

Point mutation

Hepatocyte growth factor (HGF) receptor

Renal carcinoma (papillary type: germline mutations)

SMO

Point mutations

Transmembrane signaling molecule in sonic hedgehog pathway

Basal cell skin cancer

β-CAT (CTNNB1)

Point mutation, in-frame deletion

Transcriptional coactivator, links

Melanoma; colorectal, endometrial, ovarian, hepatocellular, and other carcinomas, hepatoblastoma, Wilms' tumor

 

 

E-cadherin to cytoskeleton

 

HST

Amplification

Growth factor (FGF-like)

Gastric carcinoma

PML-RARα

Chromosome translocation

Chimeric transcription factor

APL

E2A-PBX1

Chromosome translocation

Chimeric transcription factor

Pre-β ALL

MDM-2

Amplification

p53 binding protein

Sarcoma

GLI

Amplification

Transcription factor

Sarcoma, glioma

TTG

Chromosome translocation

Transcription factor

T-cell ALL

AKT2

Amplification

Signal transducer (serine/threonine kinase; downstream effector of PI3K)

Pancreatic and ovarian carcinoma

PIK3CA

Amplification

Catalytic subunit of PI3K

Ovarian carcinoma

STK15

Amplification

Centrosome-associated kinase

Breast, colon, ovarian, and prostate carcinomas gliomas

TMPRSS2-ERG

Chromosome translocation

Transcription factor (ETS family)

Prostate cancer

TMPRSS2-ETV1

 

 

 

TMPRSS2-ETV4

 

 

 

ALL, acute lymphocytic leukemia; APL, acute promyelocytic leukemia; CML, chronic myelogenous leukemia; EGF, epidermal growth factor; FGF, fibroblast growth factor; GDNF, glial-derived neurotrophic factor; GTPase, guanine trinucleotide phosphatase; HGF, hepatocyte growth factor; PI3K, phosphatidylinositol 3-kinase; SCLC, small cell carcinoma of the lung.

 

 

 

In contrast to the activating mutations that generate oncogenic alleles from proto-oncogenes, inactivation of the normal function of tumor suppressor genes is critical in tumorigenesis. Akin to the proto-oncogenes, the functions of tumor suppressor genes are diverse, and proteins that are encoded by these genes reside in practically all subcellular compartments ( Table 14-2 ). Many tumor suppressor genes were identified by virtue of the fact that they are mutated in the germline of individuals who are affected by a known Mendelian cancer syndrome or who at the very least display a markedly elevated risk of cancer. The link between a germline-inactivating mutation in a purported tumor suppressor gene and increased cancer predisposition provides very persuasive evidence of the functional significance of the gene in the cancer process. Nevertheless, for the vast majority of tumor suppressor genes, in terms of their magnitude, somatic inactivating mutations play a far more significant role in cancer development than do germline mutations.


Table 14-2   -- Representative Tumor Suppressor Gene Mutations in Cancer

Gene

Associated Inherited Cancer Syndrome

Cancers with Somatic Mutations

Presumed Function of Protein

RB1

Familial retinoblastoma

Retinoblastoma, osteosarcoma, SCLC, breast, prostate, bladder, pancreas, esophageal, others

Transcriptional regulator; E2F binding

TP53

Li-Fraumeni syndrome

Approximately 50% of all cancers (rare in some types, such as prostate carcinoma and neuroblastoma)

Transcription factor; regulates cell cycle and apoptosis

p16INK4A

Familial melanoma, familial pancreatic carcinoma

Approximately 25% to 30% of many different cancer types (e.g., breast, lung, pancreatic, bladder)

Cyclin-dependent kinase inhibitor (i.e., CDK4 and CDK6)

p14Arf (p19Arf)

Familial melanoma(?)

Approximately 15% of many different cancer types

Regulates Mdm-2 protein stability and hence p53 stability; alternative reading frame of p16INK4A gene

APC

Familial adenomatous polyposis coli (FAP), Gardner syndrome, Turcot's syndrome

Colorectal carcinomas, desmoid tumors hepatocellular carcinoma, breast (rare)

Regulates levels of β-catenin protein in the cytosol; binding to EB1 and microtubules

WT-1

WAGR, Denys-Drash syndrome

Wilms' tumor

Transcription factor

NF-1

Neurofibromatosis type 1

Melanoma, neuroblastoma

p21ras-GTPase

NF-2

Neurofibromatosis type 2

Schwannoma, meningioma, ependymoma

Juxtamembrane link to cytoskeleton at adherens junction

VHL

Von Hippel Lindau syndrome

Renal (clear cell type), hemangioblastoma

Regulator of protein stability

BRCA1

Inherited breast and ovarian cancer

Ovarian (∼10%), rare in breast cancer

DNA repair; complexes with Rad 51 and BRCA2; transcriptional regulation

BRCA2

Inherited breast (both female and male), pancreatic cancer

Rare mutations in pancreatic, others (?)

DNA repair; complexes with Rad 51 and BRCA1

MEN-1

Multiple endocrine neoplasia type 1

Parathyroid adenoma, pituitary adenoma

Nuclear protein; unknown function

 

 

Endocrine tumors of the pancreas

 

PTCH

Gorlin syndrome, hereditary basal cell carcinoma syndrome

Basal cell skin carcinoma, medulloblastoma

Transmembrane receptor for sonic hedgehog factor; negative regulator of smoothened protein

PTEN/MMAC1

Cowden's syndrome; sporadic cases of juvenile polyposis syndrome

Glioma, breast, prostate, follicular thyroid carcinoma, head and neck squamous carcinoma

Phosphoinositide 3-phosphatase; protein tyrosine phosphatase

SMAD4

Familial juvenile polyposis syndrome

Pancreatic (∼50%), approximately 10% to 15% of colorectal cancers, rare in others

Transcriptional factor in TGF-β-signaling pathway

BMPR1A

Familial juvenile polyposis syndrome

Not known

Receptor for bone morphogenetic protein

MSH2, MLH1 PMS1, PMS2, MSH6

Hereditary nonpolyposis colorectal cancer

Colorectal, gastric, endometrial, ovarian

DNA mismatch repair

CDH1

Familial diffuse-type gastric cancer

Gastric (diffuse type), lobular breast carcinoma, rare in other types (e.g., ovarian)

E-cadherin cell-cell adhesion molecule

LKB1/STK11

Peutz-Jeghers syndrome

Lung adenocarcinoma (∼30%); rare pancreas cancers; absent in most other cancers

Serine/threonine protein kinase

EXT1

Hereditary multiple exostoses

Not known

Glycosyltransferase; heparan sulfate chain elongation

EXT2

Hereditary multiple exostoses

Not known

Glycosyltransferase; heparan sulfate chain elongation

TSC1

Tuberous sclerosis

Not known

Hamartin; binds tuberin (TSC2); regulates cell size by inhibiting target of rapamycin (TOR) function and protein synthesis

TSC2

Tuberous sclerosis

Not known

Tuberin (see above regarding TSC1)

 

 

Another important point to consider is that much of the attention for tumor suppressor genes has been focused on demonstrating that cancer cells carry biallelic inactivating mutations. Clearly, a diverse array of mechanisms can inactivate gene function, including nonsense, frameshift, and nonconservative missense mutations, as well as gross deletions of the gene or even the chromosome region that contains the gene. In a number of cases, studies of the chromosomal mechanisms associated with tumor suppressor gene inactivation in cancer tissues, such as loss of the parental heterozygosity (i.e., LOH) that is present in normal tissues, have even been used to infer the existence of tumor suppressor genes in particular chromosomal regions prior to the actual identification of the tumor suppressor gene of interest. The emphasis on defining biallelic inactivating mutations in tumor suppressor genes has been stimulated in large part by the Knudson hypothesis, [2] [17] which predicted that recessive genetic determinants played a critical role in retinoblastoma and many other cancers and that inactivation of both alleles of a tumor suppressor gene was needed to abrogate tumor suppressor gene activity. Nevertheless, as will be discussed in a bit more detail in the following sections, a variety of observations indicate that epigenetic (nonmutational) mechanisms might play a prominent role in inactivating tumor suppressor gene function in sporadic tumors. Furthermore, for certain tumor suppressor genes, inactivation of only one of the two alleles of a tumor suppressor gene might significantly impair cell growth regulation or programmed cell death. For example, p53 proteins that carry missense mutations in the central (DNA-binding region) of the protein likely potently interfere via dominant negative mechanisms with the wild-type p53 protein in the cell because p53 functions as a homotetrameric protein and all subunits must be wild type for intact p53 function in transcriptional regulation. [18] [19] In addition to the likely dominant negative role of mutant p53 in inhibiting the function of wild-type p53, several lines of evidence suggest that mutant p53 protein might have some gain-of-function properties that contribute to the tumor phenotype. [18] [19] For other tumor suppressor proteins, such as the cyclin-dependent kinase inhibitory protein p27, reduction of protein levels to 50% of the levels present in normal cells might result in significant detrimental effects on the ability of the cell to appropriately regulate growth. [20] [21]

Epigenetic Mechanisms of Proto-oncogene Activation and Tumor Suppressor Inactivation

The preceding discussion largely emphasized the role and significance of somatic and germline mutations in proto-oncogenes and tumor suppressor genes in cancer. The rationale for focusing on the role ofDNA sequence alterations in cancer initiation and progression is based chiefly on the view that it is arguably more straightforward to ascribe a causal role in cancer to specific mutations in the tumor cell genome than it is to attribute a causal role in cancer to apparent changes in the levels and/or patterns of gene expression in cancer cells. Potential difficulties in assigning a causal role in cancer to changes in the expression of various proto-oncogenes or tumor suppressor genes include uncertainties about whether the apparent differences in gene expression reflect authentic changes in gene expression or simply the possibility that the cancer might have arisen from neoplastic transformation of a cell type that had differences in gene expression than the majority of normal cells in the tissue from which the cancer originated.

In spite of the fact that changes solely in the expression, but not the structure or sequence, of proto-oncogenes and tumor suppressor genes have been more difficult to implicate definitively in the cancer process, genuine progress in the cancer epigenetics area has been made. Arguably the most compelling data for assigning a critical and likely causal role to changes in gene expression in the cancer process are for those genes that have already been well established in prior studies to function as tumor suppressor genes. Hence, the emphasis here will be placed on illustrating how epigenetic mechanisms have been assigned a causal role in silencing tumor suppressor genes in cancer. This emphasis is largely due to space limitations and not simply because of the absence of data implicating epigenetic mechanisms in proto-oncogene activation in cancer. Indeed, a priori, there is no reason why epigenetic mechanisms cannot lead to substantial increases in the expression of proto-oncogenes, with expression changes in some cancers perhaps on the order of those that are seen in cancers with high copy amplification of the respective proto-oncogene. In some cases, epigenetic mechanisms likely do lead to overexpression of certain proto-oncogenes in a variety of cancer types, such as for c-MYC,[22] the epidermal growth factor receptor,[23] and the aurora-2 kinase and closely related kinases.[24]

As is summarized in Table 14-2 , somatic inactivation of tumor suppressor genes resulting from well-established genetic mechanisms has been seen in many cancers. However, there is also a robust and growing body of data to support the view that epigenetic mechanisms somatically inactivate selected tumor suppressor genes in certain cancer types[25] ( Fig. 14-2 ). While data are only now emerging on the specific transcriptional and chromatin remodeling mechanisms that are responsible for epigenetic silencing of tumor suppressor genes, many studies have demonstrated that increases in the methylation of CpG-rich sequences (CpG islands) in the regulatory regions (i.e., promoter/enhancer) of tumor suppressor genes are often linked to loss of tumor suppressor gene expression. For instance, while the VHLgene is inactivated by mutational mechanisms in roughly 80% of renal carcinomas of clear cell type, in the majority of the clear cell renal carcinomas in which specific VHL mutations cannot be detected, loss of VHL gene expression appears to be tightly linked to hypermethylation of the VHL promoter.[25] For some other tumor suppressor genes, including the p16INK4aBRCA1, and MLH1 genes, promoter hypermethylation has also been implicated as key mechanism of inactivation.[26] In fact, on the basis of studies of genes that display extensive CpG island methylation and decreased or absent gene expression in cancer cells of one type or another, it has been suggested that aberrant CpG methylation might play a very broad and important role in the cancer process. [25] [26]

 
 

Figure 14-2  Knudson's two-hit hypothesis revised. In brief, Knudson's hypothesis predicted that both alleles of a tumor suppressor gene would need to be inactivated by germline and/or somatic mutations to elicit critical phenotypic alterations associated with cancer development. The revised version of Knudson's two-hit hypothesis considers the possibility that tumor suppressor gene inactivation can result from either genetic (mutation) or epigenetic silencing events. The two functional alleles of a given tumor suppressor gene are indicated by the two purple boxes (top). The first inactivating event affecting one of the two tumor suppressor gene alleles could be either a mutation, such as the localized defect indicated by the yellow box (left), or transcriptional silencing associated with or caused by hypermethylation of CpG-rich sequences in the promoter/regulatory region (right). The inactivating event for the second tumor suppressor gene allele (i.e., “Hit #2”) could be a nondisjunction event resulting in loss of the chromosome containing the wild-type tumor suppressor gene allele (loss of heterozygosity, LOH) or epigenetic silencing.  (Modified from Jones PA, Laird PW: Cancer epigenetics comes of age. Nat Genet 1999;21:163–167.)

 

 

 

Nevertheless, at this point, it might be reasonable to offer a few cautionary comments regarding the linkages between CpG island methylation, gene silencing, and tumor suppressor genes. One issue to reflect on is the uncertainty about what fraction of the genes whose promoters show increased methylation in cancers actually function in vivo as tumor suppressor genes. Promoter hypermethylation and loss of gene expression might be best considered as potentially useful but insufficient criteria for establishing tumor suppressor gene function in the absence of other supporting data. For instance, additional supportive evidence of tumor suppressor gene function might include data indicating that the methylation status of a promoter is tightly linked to its expression in a large panel of primary cancer specimens and data showing that gene expression can be readily and fully restored by treatment of cancer cells with demethylating agents and/or other agents that affect chromatin functional state, such as histone deacetylase inhibitors. In addition, data showing that biallelic inactivation of the methylated gene occurs by mutational mechanisms (e.g., localized mutation and LOH) or a combination of mutational and epigenetic mechanisms in at least some cancers might also represent a potentially critical set of observations. Yet another caveat to be aware of is that because several transcription factors that specifically act to repress tumor suppressor gene expression have been identified, such as the Snail and Slug proteins and their ability to repress E-cadherin in breast cancer[27] and the bmi-1 oncoprotein and itsrepression of p16INK4a and p19ARF,[28] in some cases promoter hypermethylation may be principally a reflection rather than a proximate cause of tumor suppressor gene inactivation in cancer. It should be noted, however, that certain subtypes of colorectal cancers have been identified that preferentially inactivate genes by promoter hypermethylation, suggesting that in at least some of the cases, a so far unidentified alteration in the methylation/demethylation machinery might support the development of cancers.[29]

Alterations in Cancer Target Conserved Signaling Pathways and Networks

As was noted previously and summarized in Tables 14-1 and 14-2 [1] [2], the protein products of proto-oncogenes and tumor suppressor genes have been implicated in diverse cellular processes. In light of the potentially vast complexity that is suggested by the diverse array of gene defects in cancer, it is somewhat reassuring to note that some general concepts have emerged with respect to the means by which genetic and epigenetic alterations contribute to cancer initiation and progression. Perhaps the principal overarching theme is that the protein products of oncogenes and tumor suppressor genes function in highly conserved signaling pathways and regulatory networks.

The network in which the pRb tumor suppressor protein functions is one of the more intensively studied oncogene-tumor suppressor gene networks. [30] [31] The pRb protein regulates cell cycle progression, in large part via its ability to bind to E2F transcription factor proteins. [30] [31] In addition to its role as a cell cycle regulator, the pRb protein has been implicated in regulation of cellular differentiation, survival, and even angiogenesis in certain settings. [32] [33] The binding of pRb to E2F proteins allows pRb to silence expression of E2F-regulated or “target” genes, such as those needed for the DNA synthetic (S) phase of the cell cycle. The ability of the pRb protein to bind to E2F proteins and to function in transcriptional repression appears to be tightly linked to its phosphorylation status, with the hyperphosphorylated forms of pRb incapable of binding to and regulating E2F proteins. Strong evidence indicates that the cyclin D1 protein and its associated protein kinase, cyclin-dependent kinase 4 (CDK4), negatively regulate pRb by phosphorylating it. The p16INK4a tumor suppressor protein is a critical inhibitor of the CDK4/cyclin D1 complex ( Fig. 14-3 ) and can therefore prevent the inactivation of pRb. As is noted in Table 14-2 , a subset of sporadic cancers of various types has inactivating mutations in the RB1 gene.[34] In other cancers, pRb function appears to be critically compromised as a result of mutations in other components of the network or pathway.[34] For example, in many cancers that lack RB1 mutations, inactivating mutations in the p16INK4a gene have been noted. In others, including some breast cancers, gene amplification and overexpression of cyclin D1 is found. In yet others, such as some glioblastomas and sarcomas, amplification and overexpression of the CDK4 gene has been seen. The net effect of mutations in the pRb pathway, whether in RB1 itself or in other genes, such as p16INK4acyclin D1, or CDK4, is to inactivate pRb function and its ability to regulate expression of critical E2F target genes (see Fig. 14-3 ).

 
 

Figure 14-3  Recurrent gene defects in conserved signaling pathways in cancer. Three signaling pathways that are commonly affected by mutations in various cancers are shown. Selected interactions between components of the pRb (left), APC/β-catenin (middle), and p53 pathways (right) are shown. Tumor suppressor proteins are indicated with red symbols, oncogene products are indicated by green, and those proteins that are not known to be affected by mutational or epigenetic defects in human cancer are indicated in yellow. Inhibitory interactions between proteins are indicated by perpendicular lines, and activating effects are indicated by arrows. Presumptive downstream genes whose expression is affected by the pathways are noted. APC, adenomatous polyposis coli; β-CAT, β-catenin; Cdk4, cyclin-dependent kinase 4; CYC D1, cyclin D1; DHFR, dihydrofolate reductase; DNA Pola, DNA polymerase a; GADD45, growth arrest and DNA damage inducible gene 45; GSK3β, glycogen synthase 3β; MMP-7, matrix metalloproteinase 7, p21CIP1, p21 CDK-interacting protein 1; p53AIP1, p53-regulated apoptosis-inducing protein 1; RNR, ribonucleotide reductase; TCF-4, T-cell factor-4; TS, thymidylatesynthase; TSP1, thrombospondin 1.

 

 

Studies of other proto-oncogenes and tumor suppressor genes have also supported the existence of conserved regulatory networks in which multiple different tumor suppressor gene and proto-oncogene protein products function. Although the APC (adenomatous polyposis coli) tumor suppressor protein may have roles in regulating various processes in the cell, [35] [36] [37] a key function of APC is to participate in a multiprotein complex that regulates the levels of the β-catenin protein in the cytoplasm and nucleus (see Fig. 14-3 ). Components of the multiprotein complex that regulates β-catenin include the APC protein, another tumor suppressor protein known as AXIN1, and a kinase known as glycogen synthase kinase 3β (GSK3β). Inactivation of APC or AXIN1 function in cancer cells appears to lead to an inability to phosphorylate β-catenin and hence target it for recognition and subsequent ubiquitination by the bTrCP ubiquitin ligase and ultimately its destruction by the proteasome. As a result, cancer cells with APC or AXIN1 inactivation display increased levels of β-catenin in the cytoplasm and nucleus and essentially constitutive complexing of β-catenin with transcription factors of the T cell factor (TCF) family, such as TCF-4. When bound to TCF-4, β-catenin can function as a transcriptional coactivator, and in cancers with APC inactivation, such as colorectal carcinomas, or cancers with AXIN1 inactivation, such as hepatocellular carcinomas, TCF transcriptional activity is clearly deregulated. In a subset of the colorectal carcinomas that lack APC inactivation and in a variety of other cancer types (see Table 14-1 ), activating (oncogenic) mutations in the β-catenin protein have been found.[38] These missense and in-frame deletion mutations affect key phosphorylation sites in the N-terminus of β-catenin, essentially rendering β-catenin resistant to regulation by the APC/AXIN/GSK3β complex. Hence, the mutant β-catenin protein accumulates in the cell and deregulates TCF transcription. Of some interest, and a point that will be discussed more later, transcription of several proto-oncogenes appears to be activated directly by the β-catenin/TCF complex, including the c-MYC and cyclin D1 genes.[38]

Other tumor suppressor gene regulatory networks have been defined, including the p53/MDM2/p19Arf pathway (see Fig. 14-3 ), the PTCH/SMO/GLI pathway, and the MSH2/MLH1/PMS2 DNA mismatch recognition and repair pathway. Similar to the situation for the pRb, APC/β-catenin, and p53 pathways, mutations in cancer cells not infrequently target the PTCH/SMO/GLI and MSH2/MLH1/PMS2 pathways, either activating an oncogene within the pathway (e.g., SMO or GLI for the PTCH/SMO/GLI pathway) or inactivating one of the key tumor suppressors (e.g., either MLH1 or MSH2 in the mismatch repair pathway).

Although a large collection of genetic and biochemical data support the proposed protein functions and interactions depicted in Figure 14-3 , it seems likely that the situation in vivo is far more complex. For example, on the basis of the regulatory scheme outlined for the pRb pathway in Figure 14-3 , it might appear that the phenotypic consequences of pRb or p16INK4a inactivation are functionally equivalent. However, patients with germline mutations that inactivate pRb are predisposed to retinoblastomas and osteosarcomas, while those with germline defects in p16INK4a are predisposed predominantly to melanoma and pancreatic cancer. [39] [40] Furthermore, while those with germline mutations that affect pRb or p16Ink4a are predisposed to a rather limited spectrum of cancers, somatic defects in the pRb pathway (e.g., including mutations in pRb, p16Ink4a, cyclin D1, and CDK4) are seen in the majority of a broad array of cancer types. [39] [40] Unfortunately, at present, although there is no compelling mechanistic explanation for these observations, perhaps a general explanation can be offered. Specifically, the genetic pathways in which certain oncogenes and tumor suppressor genes function are not simply linear pathways as indicated schematically in Figure 14-3 but more likely represent much more complex networks. The branches of the network may even vary considerably, depending on cell type and developmental context, though it seems reasonable to predict that the genes and the protein products recurrently affected by mutation in human cancer represent particularly critical hubs in the pathways and networks.

CONTRIBUTION OF GENE DEFECTS TO THE SIGNATURE TRAITS OF CANCER CELLS

Defining Signature Traits of Cancer Cells

Cancer represents a highly heterogeneous collection of diseases. Each cancer type has distinct biologic and clinical features and a variable prognosis. Even cancers that arise in a single organ site, such as the ovary, kidney, or lung, represent a hodgepodge of different diseases. Morphologic features often allow the particular cancer types to be distinguished to some degree from one another. Yet even for patients whose cancers have essentially identical gross and microscopic appearances and very similar clinical manifestations, there may be vast differences in outcome. In spite of this complexity, the development of all cancers, regardless of type, is likely to be critically dependent on the acquisition of certain phenotypic features that allow the cancer cells not only to grow in an unchecked fashion in their tissue of origin, but also to gain the ability to disseminate into surrounding tissues and organs, lymphatics, and the bloodstream and ultimately to grow as metastatic lesions in distant sites in the body.[41] As is indicated in Figure 14-4 , among the signature traits that are likely to be inherent in the majority, if not all, of cancer cells are the following: (1) an increased tendency to manifest a stem cell or progenitor-like phenotype, (2) an enhanced response to growth-promoting signals, (3) a relative resistance to growth-inhibiting cues, (4) an increased mutation rate to allow for the rapid generation of new variant daughter cells, (5) the ability to attract and support a new blood supply (angiogenesis), (6) the capacity to minimize an immune response and/or evade destruction by immune effector cells, (7) the capacity for essentially limitless cell division, (8) a failure to respect tissue boundaries, allowing for invasion into adjacent tissues and organs as well as blood vessels and lymphatics, and (9) the ability to grow in organ sites with microenvironments markedly different from the one where the cancer cells arose. The development of some traits is likely to be associated with certain stages of tumorigenesis (see Fig. 14-4), but acquisition of signature traits in cancers is far more likely to show a preferred order than an invariant order.[41] Furthermore, many of the signature traits of cancer cells that were elaborated previously represent complex biologic capabilities (e.g., angiogenic activity, immune evasion/resistance, metastatic competence). Therefore, it is likely that substantial changes in many signaling pathways are needed for the cancer cell to manifest the traits.

 
 

Figure 14-4  Acquisition of signature traits in neoplastic cells during cancer progression. Depicted in the figure are representative stages in the development of a cancer, perhaps a typical epithelial cancer, such as those that typically arise in the lung, colon, breast, or prostate. The schema suggests that most advanced cancers arise via clonal selection from subclones present in earlier stage benign and localized lesions (e.g., carcinoma in situ and locally invasive carcinoma). Some of the properties of advanced cancer cells are depicted by the ability of the cells to enter the bloodstream and to seed and grow in distant organ sites, such as the liver (bottom). Nine signature traits of cancer cells are listed. The relative time at which neoplastic cells acquire some of the traits is uncertain, though it seems likely that some traits, such as the expression of a stem/progenitor cell phenotype or enhanced response to growth-promoting signals, may be acquired earlier in cancer development. Other traits, such as invasive capacity and/or metastatic competence, may be acquired later. Many signature traits of cancer cells are in fact complicated biologic capabilities (e.g., angiogenic activity, immune evasion/resistance, metastatic competence) and likely depend on defects in a number of different factors and signaling pathways.

 

 

An exhaustive cataloging of the observations that link specific gene defects to the altered phenotype of cancer cells will not be offered here, in part because of space limitations and in part because of uncertainties about the significance of some of the linkages between single gene defects and cancer phenotype. Nonetheless, some general concepts regarding the relationships among gene defects and cancer cell phenotype have emerged, and two of these concepts are offered here. First, while some specific mutations and major gene expression defects that are seen in cancer cells may contribute predominantly to a few or perhaps even only one of the signature traits of cancer cells listed in the preceding list, it seems likely that many of the gene defects and expression changes have been selected for in large part because they exert pleiotropic effects on the cancer cell phenotype. Second, for some tumor types, it has been possible to gain insights into the apparent order in which genetic and epigenetic changes might arise and contribute to cancer pathogenesis. The data suggest that defects in certain genes and signaling pathways might be strongly selected for at certain point in cancer development and progression, perhaps in large part because the alterations allow the precancerous or cancerous cells to acquire certain critical phenotypic features. Of some interest, gene defects that might be nearly uniformly present in early-stage lesions of one tumor type might preferentially arise in later-stage tumors in another organ site. These data suggest that cellular and tissue context have critical, albeit poorly understood, modifying effects on the specific genetic defects that give rise to neoplastic transformation and clonal outgrowth. The two concepts—the often pleiotropic effects of the gene defects that are present in cancer cells and the context-dependent effects of the defects—will be expanded on later, with presentation of some concrete examples.

Contribution of APC Inactivation and β-Catenin Deregulation to Cancer Phenotype

As was noted previously, mutational defects that lead to deregulation of the signaling activity of the β-catenin protein are present in a relatively broad array of cancer types, including colorectal tumors, in which upward of 80% to 90% of colorectal adenomas and carcinomas harbor inactivating mutations in APC or AXIN1 or AXIN2 or gain-of-function mutations in β-catenin itself.[36] On the basis of the observation that germline-inactivating mutations in APC markedly increase the rate at which adenomatous lesions arise in the colon and rectum and extensive descriptive molecular studies showing that somatic mutations in APC or β-catenin are present in even microscopic adenomatous lesions in the colon, [42] [43] it appears that dysregulation of β-catenin likely plays a central role in the earliest stages of colon cancer development. In other cancer types in which β-catenin is often deregulated by mutational mechanisms,[36] such as hepatocellular and endometrial cancers, the timing of β-catenin mutations in the natural history of disease is less certain.

Given this background, what might be the contribution of β-catenin defects to the development and biologic behavior of colon and perhaps other cancers? As was highlighted previously, β-catenin, upon its association with TCF transcription factors, has been implicated in activating expression of genes that likely play critical roles in stimulating progression through the G1-to-S transition, including cyclin D1 and c-MYC. Effects of cyclin D1 activation on the pRb pathway and progression through the G1/S phase of the cell cycle were mentioned in the preceding discussion. As is indicated in Figure 14-5 , one of the apparent consequences of c-MYC activation of colon cancer cells is repression of the expression of the p21CIP1 cyclin-dependent kinase inhibitor, [44] [45] and inhibition of p21CIP1 might contribute to defective cell cycle control. [44] [45] [46] In addition to effects on cyclin D1 and c-MYC, deregulation of β-catenin/TCF transcription has been implicated in activation of a number of genes that might play a role in maintaining or inducing a progenitor cell or stem cell-like phenotype in colon cells that should otherwise be destined for differentiation or apoptosis [38] [45] (see Fig. 14-5 ). Genes that might play a role in conferring a progenitor cell phenotype include the cell surface protein CD44 and the EphB2 and EphB3 receptors and their ligand ephrin-B1. [45] [47] Either individually or collectively along with other molecules, the cell surface proteins might exert potent effects on colonic epithelial cell fate, perhaps in part by inhibiting appropriate migration of cells in the crypt and hence favoring response to proliferation-inducing signals over differentiation-inducing cues. [45] [47]

 
 

Figure 14-5  Potential contributions of β-catenin deregulation to cancer cell signature traits. Inactivation of the APC tumor suppressor protein or activating (oncogenic) mutations in β-catenin can lead to marked increases in the levels of free β-catenin protein in the cytoplasm and nucleus, enhanced binding of β-catenin to the TCF-4 (T cell factor-4) transcription factor, and activation of β-catenin/TCF-4 regulated genes. Some of the genes that might be directly regulated by the β-catenin-TCF-4 transcription process include the genes for c-MYC, cyclin D1, MMP-7, survivin, CD44, the EPHB2 and EPHB3 receptors, and their ligand Ephrin-B1. Some of the potential effects resulting from activation of these target genes are indicated. In addition to β-catenin's role in activating expression of TCF-4-related genes, β-catenin appears to inhibit the activity of NF-κB and NF-κB's ability to activate genes with potential roles in apoptosis, such as Fas and TRAF1. The potential contributions of β-catenin to cancer cell traits are discussed in more detail in the text.

 

 

Another presumptive β-catenin/TCF target gene with a potential role in acquisition of several critical cancer phenotypic traits is the matrix metalloproteinase-7 (MMP-7, also known as matrilysin)[48] (seeFig. 14-5 ). MMP-7 itself has a number of potential roles in cancer progression, [49] [50] including the ability to cleave and downregulate the activity of the E-cadherin tumor suppressor protein[51] and the ability to cleave osteopontin and apparently activate osteopontin's cell migration-stimulating activity.[52] Besides the role of dysregulated β-catenin in activating transcription of TCF target genes, there is evidence that elevated levels of nuclear β-catenin in cancer cells can interfere with NF-κB function and NF-κB's ability to activate key downstream effectors of apoptosis, such as Fas and TRAF1[53] (seeFig. 14-5 ). Finally, the carboxyl-terminus region of the APC tumor suppressor protein confers binding to the EB1 protein, a microtubule-binding protein, as well as to microtubules.[35] Given the apparent role of EB1 in regulating microtubule dynamics, cell polarity, and chromosome stability and the evidence that APC inactivation in certain cellular contexts could confer a chromosome instability phenotype, it is possible that APC inactivation also contributes to a chromosomal instability phenotype in colorectal cancer cells. [35] [54] [55]

Contribution of RAS-Signaling Pathway Defects to Cancer Phenotype

RAS gene mutations were the first somatic gene defects to be characterized at the molecular level in cancer cells, and we now know that mutations in the three RAS genes—H-RAS, K-RAS, and N-RAS—are among the most common oncogene defects in cancer, with an estimated 20% of all cancers carrying a point-mutated, activated RAS allele. [56] [57] [58] RAS proteins appear to play key roles in several important signaling pathways ( Fig. 14-6 ), and proteins that function upstream or downstream of the RAS proteins are affected by mutations in certain cancers. For instance, mutational activation and/or overexpression of growth factors upstream of RAS, such as epidermal growth factor receptor and/or ERBB2 (also known as HER2/Neu), can be commonly seen in a number of epithelial cancers, including breast and ovarian carcinomas (see Table 14-1 ). Mutations in effectors downstream of RAS can also be seen in cancers, including defects in the mitogen-activated protein kinase (MAPK) pathway as a result of BRAF gene mutations [56] [59] or defects in phosphatidylinositol 3-kinase (PI3K) signaling, as result of amplification and overexpression of AKT2, activating mutations of AKT1, or inactivating mutations in the PTEN (phosphatase and tensin homolog) tumor suppressor gene. [58] [59] [60] [61] Sometimes mutations in Ras and the downstream effectors are mutually exclusive (e.g., Ras and β-Raf), while in other cases (e.g., Ras and PI3K catalytic subunit), the mutations coexist. [5] [58]

 
 

Figure 14-6  Potential contribution of RAS pathway defects to the cancer cell phenotype. The RAS protein is indicated near the top of the figure, and selected upstream and downstream factors are also indicated relative to their likely location in the cell. RAS pathway-signaling interactions are complex, and only selected functional interactions upstream and downstream of RAS are indicated. As is indicated in the figure and discussed in more detail in the text, RAS pathway activation can likely enhance the response to growth-promoting stimuli via effects on cyclin D1 (CYC D1) expression and pRb phosphorylation and activity. These effects on cyclin D1 expression are likely to be mediated by the RAF/MAPK (mitogen activated protein kinase) signaling cascade and its effects on downstream conscription factors, including JUN and the ETS-related protein ELK1, which activates expression of JUN's dimeric partner, FOS. RAS pathway activation also likely acts to increase resistance to apoptosis in some settings through effects on AKT and its ability to inhibit factors with proapoptotic roles (e.g., Forkhead in human rhabdomyosarcoma [FKHR] or BAD) or AKT's ability to activate NF-κB's survival function. Besides these effects, RAS pathway activation might act to stimulate vascular endothelial growth factor (VEGF) production and angiogenesis. RAS can enhance cell migration and invasion via RAC/CDC42-mediated effects on the cytoskeleton and possibly FOS/JUN-mediated increases in the expression of some matrix metalloproteases, such as MMP-9. Finally, MMP-9 can also promote release of VEGF from extracellular reservoirs, perhaps further enhancing VEGF effects on angiogenesis.

 

 

The consequences of RAS mutations specifically and RAS-signaling pathway defects more generally are varied and undoubtedly depend on cell context, because constitutive activation of RAS signaling in certain contexts can promote apoptosis rather than cell proliferation or neoplastic transformation. [62] [63] In brief, activated mutant RAS alleles have been implicated in enhanced response to proliferative cures, perhaps to a certain extent owing to the ability of RAS-MAPK activation to enhance expression of cyclin D1, and cyclin D1′s ability to inactivate pRb function via phosphorylation [59] [63] (see Fig. 14-6 ). RAS pathway activation can also interfere with apoptosis induction, perhaps in part via activation of the PI3K pathway and its ability to antagonize the proapoptotic factor BAD and perhaps other molecules that are important in promoting programmed cell death [59] [61] (see Fig. 14-6 ). In addition to the potential ability of RAS pathway activation to enhance cell proliferation and inhibit programmed cell death in cancer cells, RAS activation has been implicated in transcriptional activation of vascular endothelial growth factor (VEGF),[64] a potent stimulator of angiogenesis. Furthermore, RAS activation has been linked to increased invasive potential.[59] The role of RAS in promoting invasiveness might be mediated through RAC-dependent effects on the cytoskeleton as well as MAPK pathway-dependent activation of matrix metalloproteinases,[59] such as MMP-9, which can function in degradation of the basement membrane component type IV collagen[50] (see Fig. 14-6 ). Interestingly, MMP-9 has also been implicated in promoting angiogenesis in some settings via its ability to stimulate release of VEGF from poorly defined extracellular reservoirs.[65]

MicroRNAs as Post-transcriptional Regulators of Gene Function

It has been recognized in the last few years that the translation of mRNA transcripts into proteins is highly regulated by a novel class of short noncoding RNAs, the so-called microRNAs (miRNAs). The mature forms of miRNAs are 18 to 24 nucleotides in length and are generated by successive cleavages by the Drosha and Dicer nucleases from longer precursor transcripts that contain characteristic hairpin formations.[66] Recognition of target transcripts occurs by binding of the miRNA to the 3′ untranslated regions (3′UTR). Depending on the degree of homology to their target sequence, miRNAs induce translational repression or cleavage of mRNAs. More than 500 human miRNAs have been described, and each single miRNA can target hundreds to a thousand or more mRNAs.[66] This ability allows for substantial combinatorial complexity and functional redundancy, making the identification of specific functions of miRNAs as well as their involvement in oncogenic or tumor suppressive networks difficult. The fact that miRNAs act via base-pairing of the miRNA with the 3′UTR also implies that alterations of both miRNA sequence and miRNA target sequence might have functional consequences. So far, large-scale sequencing efforts have been focused mainly on the coding sequence of genes, which means that a significant number of functionally relevant alterations might have been missed.

Several lines of evidence make it likely that miRNAs do play a role in the development of cancers. For instance, global inhibition of miRNA production seems to facilitate the acquisition of a neoplastic phenotype in primary mouse cells, suggesting that the net effect of all miRNAs might be a tumor suppressive effect.[67]

The genomic location of many miRNAs maps close to common chromosomal breakpoints in cancer. In most cases, however, it is still unclear whether the miRNAs are actually involved in conferring the selective advantage that is gained by these translocations or whether miRNAs for other reasons are localized in regions of high genomic fragility.[68]

Comprehensive analyses of miRNA expression patterns in human cancers have revealed that different cancer types have distinct miRNA expression patterns. In fact, in many instances, miRNA expression patterns might be more precise in determining the tissue of origin than mRNA expression profiles.[69] Similar to the situation with protein-coding genes, in most cases, it is unclear which expression changes are causative and which are secondary to the development of the tumors.[70]

Several miRNAs seem to play a role as part of classical tumor suppressive or oncogenic signal transduction pathways ( Table 14-3 ). The miRNA17–92 locus has been described as a direct transcriptional target of the c-MYC and E2F oncogenes.[71] This polycistron locus encodes for seven miRNAs and has been shown to be genomically amplified and overexpressed in some human β-cell lymphomas and lung cancers. When overexpressed, it can cooperate with c-MYC to accelerate lymphoma development in a murine model system. In addition, miRNA372 and miRNA373 have been implicated as oncogenes in the development of germ cell tumors at least in part by inactivating the p53 tumor suppressor pathway.[72] Recently miRNA10a has been suggested to be a major driving force behind the metastatic progression of breast cancer cell lines.[73]


Table 14-3   -- Putative Tumor Suppressor or Oncogenic miRNAs

miRNA

Chromosomal Location

Expression

Target Genes/Effect

PUTATIVE TUMOR SUPPRESSOR miRNAs

miRNA15/16

13q14

CLL and pituitary adenoma

BCL2

miRNA34a

1p36

p53 target gene; loss in neuroblastoma

BCL2, MET, Cyclin E2

miRNA34b/c

11q21

p53 target gene; loss in NSCLC

BCL2, MET

let-7a-2

11q24

Loss in lung cancer

K-RAS, HMGA2

PUTATIVE ONCOGENIC miRNAs

miRNA17–92

13q14

c-MYC target gene

Cooperates with c-MYC and regulates E2F1

 

 

Up in β-cell lymphoma, lung cancer

 

miRNA372/373

19q13

Up in germ cell tumors

LATS2 tumor suppressor ➙ p53 inactivation

miRNA10a

19q13

Up in metastatic breast cancer

Metastatic phenotype

miRNA155

21q21

Up in breast cancer, lymphoma

?

miRNA21

17q23

Up in pancreatic, breast and CNS cancer

PTEN tumor suppressor

TRANSLOCATIONS INVOLVING miRNAs OR miRNA TARGET SITES

miRNA142

t(8;17)(q24;q22)

B-cell lymphoma

miRNA promoter drives c-MYC expression

HMGA2

12q15 translocations

Benign salivary gland tumors

Translocation removes let-7 recognition sites from the HMGA2 transcript

Modified from Calin GA, Croce CM: Chromosomal rearrangements and microRNAs: a new cancer link with clinical implications. J Clin Invest 2007;117:2059–2066; and Garzon R, Fabbri M, Cimmino A, et al: 206 MicroRNA expression and function in cancer. Trends Mol Med 2006;12:580–587.

CLL, chronic lymphocytic leukemia; NSCLC, non-small-cell lung cancer.

 

 

 

 

Members of the let-7 family of miRNAs have been proposed as tumor suppressor genes in lung cancer, supposedly in part by their ability to inhibit the translation of the K-Ras and HMGA2 oncogenes.[74]The members of the miRNA34 family have recently been shown to be direct transcriptional targets of the p53 tumor suppressor gene and seem to play a significant role in p53 activity. [75] [76]

Role of Tissue and Context Differences in the Contributions of Gene Defects to Cancer Cell Phenotype

The published literature on the potential contributions of gene defects to the altered phenotype of cancer cells has offered some suggestions about how to consider the role of the gene defects in cancer pathogenesis. For instance, terms such as gatekeeper and caretaker have been used to classify the contributions of genes to cancer development.[77] “Gatekeeper” genes have been suggested to be those genes that play particularly critical roles in regulating cell proliferation and inhibiting cancer development in certain tissues, such as the APC gene in colorectal cancer, and the tumor suppressive function of the genes must be overcome for cancers to arise in a given tissue or organ site.[77] “Caretakers” have been generally defined as those genes that do not play direct roles in growth control but rather likely play important roles in a number of tissues in maintaining the fidelity of the genome via their role in DNA damage recognition and repair processes. The MLH1 and MSH2 mismatch repair genes have been proposed to be representative caretaker genes,[77] and some researchers have suggested that perhaps the BRCA1 and BRCA2 genes also represent caretaker genes.[78] The use of terms such as gatekeeperand caretaker might have some merit. However, as will be illustrated in the following discussion, given the apparently important role of “gatekeeper” gene defects in cancers that arise in various organ sites but the quite variable timing of “gatekeeper” gene defects in the natural history of one cancer type versus another, the term gatekeeper might be more confusing than illuminating. In the case of some presumed “caretaker” genes, the genes might not be playing the passive role in the cancer process that has been assigned to them. This view is based on three lines of argument: (1) the apparent tissue specificity of the tumors that arise in individuals who harbor germline mutations in the “caretaker” genes, such as in individuals who are affected by hereditary nonpolyposis colorectal cancer and germline MLH1 or MSH2 mutations[79]; (2) the likely variable time in cancer development at which “caretaker” mutations may arise from one tumor to the next, with sporadic colon tumors not usually manifesting mismatch repair gene inactivation and microsatellite instability until the carcinoma stage, despite the fact that adenomas in individuals with hereditary nonpolyposis colorectal cancer often show high-frequency microsatellite instability [80] [81]; and (3) the evidence that “caretaker” genes might actually play key roles in regulating cell proliferation and promoting apoptosis in certain contexts.[82]

In general, individuals who harbor a germline mutation in a specific tumor suppressor gene or proto-oncogene are predisposed to a very limited spectrum of cancer types. This observation is puzzling for a couple of reasons. The majority of genes that are affected by germline mutations in specific inherited cancer syndromes are essentially ubiquitously expressed in adult tissues. Furthermore, for a number of the tumor suppressor genes, mutations are often found to inactivate the gene in a much broader collection of sporadic cancer types than the types that commonly arise in germline mutation carriers. Children who carry a germline mutation in the RB1 gene have a very elevated risk of developing retinoblastoma and a more modest risk of developing osteosarcoma but no dramatic increase in the risk of most common adult cancers. Yet somatic defects in pRb have been found and are believed to be critical in the development of many different cancers, such as small cell lung carcinomas, in which the vast majority have pRb defects. [40] [82]

There are potential explanations for these puzzling observations. For instance, while pRb might have an essential role in regulating retinoblast cell proliferation and/or differentiation, in other tissues, such as lung or breast epithelial cells, pRb might have a redundant role in growth control, perhaps because of the contribution of pRb-related proteins, such as p107 and p130. [30] [31] Under this scenario, pRb inactivation in most cell types might not promote neoplastic growth unless other defects, such as those in pRb-related proteins, are also present. An alternative and perhaps more likely possibility is that somatic inactivation of pRb might trigger apoptosis in many cell types unless other somatic gene defects have arisen previously and these other defects interfere with the cell's ability to undergo apoptosis following disruption of RB1 function ( Fig. 14-7 ). Evidence that pRb inactivation can act in a context-dependent fashion to promote apoptosis versus neoplastic transformation has been offered. [83] [84] The tissue specificity of cancers that are seen in individuals who carry germline mutations in inherited cancer genes is not restricted to the case of pRb. Germline p53 mutations predispose primarily to osteosarcoma, soft tissue sarcoma, leukemia, brain tumors, and breast cancer in women; and p16INK4a germline mutations predispose primarily to melanoma and pancreatic cancer. In spite of the relatively limited spectrum of cancer types that are seen in people who carry germline p53 or p16INK4a mutations, the p53 and p16INK4a genes are very commonly altered in human cancer, each of the genes being inactivated in upward of 35% to 50% of many different sporadic cancer types.

 
 

Figure 14-7  Mutations in the retinoblastoma tumor suppressor gene (RB1) contribute to inherited and sporadic cancers. The figure indicates that cell context affects the contribution of RB1 mutations to cancer development. In individuals who carry a germline mutation in oneRB1 allele, somatic inactivation of the remaining RB1 allele is an early and rate-limiting event in retinoblastoma formation. Sporadic forms of retinoblastoma are dependent on inactivation of both RB1 alleles. Because somatic inactivation of both RB1 alleles must occur in a single developing retinoblast before tumor formation can ensue, retinoblastoma is a rare disease in people who do not carry a germline RB1 mutation (i.e., the general population). Those who carry a germline RB1 mutation do not manifest a markedly increased risk to many common cancers, such as lung cancer, despite the fact that RB1 mutations are frequently observed in sporadic forms of lung cancer (e.g., small cell lung carcinoma). These observations imply that RB1 mutations might contribute to tumor progression rather than tumor initiation in most cancer types other than retinoblastoma and perhaps osteosarcoma. Possible explanations for this phenomenon include the possibility that inactivation of both RB1 alleles prior to the acquisition of defects in other oncogenes or tumor suppressor genes is not associated with any growth advantage and RB1 activation in some contexts might even induce apoptosis.  (Modified from Haber DA, Fearon ER: The promise of cancer genetics. Lancet 1998;351[suppl 2]:1–8.)

 



Finally, some genes with prominent roles in the development of a variety of different cancer types are sometimes presumed to have essentially singular functions in the cancer process in spite of data that suggest otherwise. As an example, the E-cadherin protein plays an important role in cell-cell adhesion via the ability of its extracellular domain to form adhesive interactions with E-cadherin molecules on opposing cell surfaces and the ability of the E-cadherin cytoplasmic domain to link to the actin cortical cytoskeleton via interactions with catenin proteins at the plasma membrane.[85] Early functional studies have suggested that restoration of E-cadherin in cancer cells that had endogenous E-cadherin defects interfered with the invasive properties of cancer cells in selected in vitro assays.[86] Perhaps in large part because of these observations, the loss of E-cadherin expression in cancer has nearly invariably been assigned a role in promoting invasive behavior in advanced cancer cells. While loss of E-cadherin function might indeed contribute to invasive behavior in cancers arising in vivo, it is worth bearing in mind that defects in E-cadherin could in fact play a distinct role in altering cell growth very early in the neoplastic transformation process in some tumor types, such as the gastric carcinomas that arise in patients who carry germline E-cadherin mutations.[87]

CLINICAL IMPLICATIONS

On the basis of review of the data on the apparent contributions of gene defects to cancer phenotype, some clinical implications are apparent. The genes and the protein products that are recurrently altered by mutations and/or epigenetic defects in human cancer more than likely represent particularly critical hubs in the pathways and networks that regulate cell growth, differentiation, and programmed cell death. Hence, efforts to target the proteins and pathways with apparently central roles in the pathogenesis of a number of different cancer types would appear to offer potentially the broadest impact. Additionally, because the gene defects that promote clonal selection during cancer development might have particularly pleiotropic effects on the cancer cell phenotype, targeting of the central pathways in cancer might also be expected to have some of the most dramatic effects on cancer cells. For example, in light of the important role of RAS pathway defects in a broad array of cancer types and the view that RAS pathway deregulation exerts pleiotropic effects on the cancer cell phenotype, drugs that inhibit the activity of Raf, MEK, or MAPK might have potent anticancer activity in a subset of the many cancers with RAS pathway defects.[88] Moreover, even though RAS pathway defects may arise at early to intermediate stages of tumor development in some cancer types, such as colorectal cancer, it is encouraging to learn that quite dramatic effects can be seen on the growth of advanced cancer cells when the activity of the mutant RAS protein is antagonized. [89] [90]

In spite of the generally optimistic view that is offered as a result of our rapidly expanding understanding of the nature and contribution of gene defects in cancer pathogenesis, some significant challenges remain if novel and specific new anticancer therapies are to be achieved in the near term. In particular, for a fair number of the specific signaling pathways that are commonly disrupted in cancer, it might prove difficult to define readily tractable targets for therapeutic intervention. For instance, in the case of the p53 pathway, it is unclear how effectively p53 function can be restored in the proteins that carry missense substitutions or when the p53 protein is intact and simply antagonized by upstream defects (e.g., p19ARF or MDM2 defects). In the case of cancers with mutations leading to β-catenin deregulation, a potential goal might be to define agents that specifically interfere with the nuclear function of β-catenin in transcriptional activation of β-catenin/TCF-regulated target genes. While this is not an unreasonable notion, given the rather limited successes to date in defining small molecules that specifically affect transcription factor complexes, it could be very challenging to target β-catenin via conventional pharmacologic approaches. Similar concerns could be perhaps raised regarding the merits of attempting to specifically target other nuclear proteins and transcription factors that are deregulated in cancer cells.

Given these concerns regarding the likelihood for success in defining small molecules that effectively target some of the pathways that are most commonly deregulated in cancer, emphasis for therapeutic targeting in the near term might be placed on potentially more promising molecular targets in cancer, such as CDK4 (e.g., for cancers with an intact pRb protein but defects in p16INK4a, cyclin D1, or CDK4) or various effector molecules in the PI3K-AKT pathway, such as the AKT or mTOR proteins (e.g., for cancers with PTEN defects).[61] While the success of approaches to target cancer cells more selectively remains to be broadly established, it is obvious that the efforts to understand the relationship between gene defects, altered cell signaling and physiology, and cancer phenotype have already shaped and will continue to shape our views of how best to proceed with novel therapeutic interventions.

REFERENCES

  1. Sherr CJ: Principles of tumor suppression.  Cell2004; 116:235-246.
  2. Knudson AG: Two genetic hits (more or less) to cancer.  Nature Rev2001; 1:157-162.
  3. Renan MJ: How many mutations are required for tumorigenesis? Implications from human cancer data.  Molecular Carcinog1993; 7:139-146.
  4. Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis.  Cell1990; 61:759-767.
  5. Wood LD, Parsons DW, Jones S, et al: The genomic landscapes of human breast and colorectal cancers.  Science2007; 318:1108-1113.
  6. Sato M, Shames DS, Gazdar AF, Minna JD: A translational view of the molecular pathogenesis of lung cancer.  J Thorac Oncol2007; 2:327-343.
  7. Maitra A, Kern SE, Hruban RH: Molecular pathogenesis of pancreatic cancer.  Best Pract Res2006; 20:211-226.
  8. Foulds L: The natural history of cancer.  J Chronic Dis1958; 8:2-37.
  9. Nowell PC: The clonal evolution of tumor cell populations.  Science1976; 194:23-28.
  10. Blagosklonny MV: Oncogenic resistance to growth-limiting conditions.  Nat Rev2002; 2:221-225.
  11. Haber DA, Settleman J: Cancer: drivers and passengers.  Nature2007; 446:145-146.
  12. Greenman C, Stephens P, Smith R, et al: Patterns of somatic mutation in human cancer genomes.  Nature2007; 446:153-158.
  13. Thomas RK, Baker AC, Debiasi RM, et al: High-throughput oncogene mutation profiling in human cancer.  Nat Genet2007; 39:347-351.
  14. Schubbert S, Shannon K, Bollag G: Hyperactive Ras in developmental disorders and cancer.  Nat Rev2007; 7:295-308.
  15. Bahram F, von der Lehr N, Cetinkaya C, Larsson LG: c-Myc hot spot mutations in lymphomas result in inefficient ubiquitination and decreased proteasome-mediated turnover.  Blood2000; 95:2104-2110.
  16. Hemann MT, Bric A, Teruya-Feldstein J, et al: Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants.  Nature2005; 436:807-811.
  17. Knudson AG: Mutations and cancer: statistical study of retinoblastoma.  Proc Natl Acad Sci USA1971; 17:317-352.
  18. Lozano G: The oncogenic roles of p53 mutants in mouse models.  Curr Opin Genet Dev2007; 17:66-70.
  19. Soussi T: p53 alterations in human cancer: more questions than answers.  Oncogene2007; 26:2145-2156.
  20. Fero ML, Randel E, Gurley KE, et al: The murine gene p27Kip1 is haplo-insufficient for tumour suppression.  Nature1998; 396:177-180.
  21. Payne SR, Kemp CJ: Tumor suppressor genetics.  Carcinogenesis2005; 26:2031-2045.
  22. Adhikary S, Eilers M: Transcriptional regulation and transformation by Myc proteins.  Nat Rev Mol Cell Biol2005; 6:635-645.
  23. Ritter CA, Arteaga CL: The epidermal growth factor receptor-tyrosine kinase: a promising therapeutic target in solid tumors.  Semin Oncol2003; 30:3-11.
  24. Fu J, Bian M, Jiang Q, Zhang C: Roles of Aurora kinases in mitosis and tumorigenesis.  Mol Cancer Res2007; 5:1-10.
  25. Jones PA, Baylin SB: The epigenomics of cancer.  Cell2007; 128:683-692.
  26. Esteller M: Epigenetic gene silencing in cancer: the DNA hypermethylome.  Hum Mol Genet2007; 16(spec no 1):R50-R59.
  27. Hajra KM, Chen DY, Fearon ER: The SLUG zinc-finger protein represses E-cadherin in breast cancer.  Cancer Res2002; 62:1613-1618.
  28. Jacobs JJ, Kieboom K, Marino S, et al: The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus.  Nature1999; 397:164-168.
  29. Weisenberger DJ, Siegmund KD, Campan M, et al: CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer.  Nat Genet2006; 38:787-793.
  30. Classon M, Harlow E: The retinoblastoma tumour suppressor in development and cancer.  Nat Rev Cancer2002; 2:910-917.
  31. Giacinti C, Giordano A: RB and cell cycle progression.  Oncogene2006; 25:5220-5227.
  32. De Falco G, Comes F, Simone C: pRb: master of differentiation: coupling irreversible cell cycle with-drawal with induction of muscle-specific transcription.  Oncogene2006; 25:5244-5249.
  33. Gabellini C, Del Bufalo D, Zupi G: Involvement of RB gene family in tumor angiogenesis.  Oncogene2006; 25:5326-5332.
  34. Goodrich DW: The retinoblastoma tumor-suppressor gene, the exception that proves the rule.  Oncogene2006; 25:5233-5243.
  35. Nathke I: Cytoskeleton out of the cupboard: colon cancer and cytoskeletal changes induced by loss of APC.  Nature Rev2006; 6:967-974.
  36. Polakis P: The many ways of Wnt in cancer.  Curr Opin Genet Dev2007; 17:45-51.
  37. Willert K, Jones KA: Wnt signaling: is the party in the nucleus?.  Genes Dev2006; 20:1394-1404.
  38. Giles RH, van Es JH, Clevers H: Caught up in a Wnt storm: Wnt signaling in cancer.  Biochim Biophysica Acta2003; 1653:1-24.
  39. Fearon ER: 1997 Human cancer syndromes: clues to the origin and nature of cancer.  Science1997; 278:1043-1050.
  40. Sellers WR, Kaelin Jr WG: Role of the retinoblastoma protein in the pathogenesis of human cancer.  J Clin Oncol1997; 15:3301-3312.
  41. Hanahan D, Weinberg RA: The hallmarks of cancer.  Cell2000; 100:57-70.
  42. Samowitz WS, Powers MD, Spirio LN, et al: Beta-catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas.  Cancer Res1999; 59:1442-1444.
  43. Takayama T, Ohi M, Hayashi T, et al: Analysis of K-ras, APC, and beta-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.  Gastroenterology2001; 121:599-611.
  44. Seoane J, Le HV, Massague J: Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage.  Nature2002; 419:729-734.
  45. van de Wetering M, Sancho E, Verweij C, et al: The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.  Cell2002; 111:241-250.
  46. Vousden KH: Switching from life to death: the Mizing link between Myc and p53.  Cancer Cell2002; 2:351-352.
  47. Batlle E, Henderson JT, Beghtel H, et al: Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB.  Cell2002; 111:251-263.
  48. Crawford HC, Fingleton BM, Rudolph-Owen LA, et al: The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors.  Oncogene1999; 18:2883-2891.
  49. Lynch CC, Hikosaka A, Acuff HB, et al: MMP-7 promotes prostate cancer-induced osteolysis via the solubilization of RANKL.  Cancer Cell2005; 7:485-496.
  50. Lynch CC, Matrisian LM: Matrix metalloproteinases in tumor-host cell communication.  Differentiation2002; 70:561-573.
  51. Noe V, Fingleton B, Jacobs K, et al: Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1.  J Cell Sci2001; 114:111-118.
  52. Agnihotri R, Crawford HC, Haro H, et al: Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin).  J Biol Chem2001; 276:28261-28267.
  53. Deng J, Miller SA, Wang HY, et al: Beta-catenin interacts with and inhibits NF-kappa B in human colon and breast cancer.  Cancer Cell2002; 2:323-334.
  54. Dikovskaya D, Schiffmann D, Newton IP, et al: Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis.  J Cell Biol2007; 176:183-195.
  55. Fodde R, Kuipers J, Rosenberg C, et al: Mutations in the APC tumour suppressor gene cause chromosomal instability.  Nat Cell Biol2001; 3:433-438.
  56. Davies H, Bignell GR, Cox C, et al: Mutations of the BRAF gene in human cancer.  Nature2002; 417:949-954.
  57. Malumbres M, Barbacid M: RAS oncogenes: the first 30 years.  Nat Rev Cancer2003; 3:459-465.
  58. Rodriguez-Viciana P, Tetsu O, Oda K, et al: Cancer targets in the Ras pathway.  Cold Spring Harb Symp Quant Biol2005; 70:461-467.
  59. Downward J: Targeting RAS signalling pathways in cancer therapy.  Nat Rev Cancer2003; 3:11-22.
  60. Carpten JD, Faber AL, Horn C, et al: A trans-forming mutation in the pleckstrin homology domain of AKT1 in cancer.  Nature2007; 448:439-444.
  61. Vivanco I, Sawyers CL: The phosphatidylinositol 3-kinase AKT pathway in human cancer.  Nat Rev Cancer2002; 2:489-501.
  62. Mayo MW, Wang CY, Cogswell PC, et al: Requirement of NF-kappaB activation to suppress p53-independent apoptosis induced by oncogenic Ras.  Science1997; 278:1812-1815.
  63. Pruitt K, Der CJ: 2001 Ras and Rho regulation of the cell cycle and oncogenesis.  Cancer Lett2001; 171:1-10.
  64. Rak J, Kerbel RS: Ras regulation of vascular endothelial growth factor and angiogenesis.  Methods Enzymol2001; 333:267-283.
  65. Bergers G, Brekken R, McMahon G, et al: Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.  Nat Cell Biol2000; 2:737-744.
  66. Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R: Control of translation and mRNA degradation by miRNAs and siRNAs.  Genes Dev2006; 20:515-524.
  67. Kumar MS, Lu J, Mercer KL, et al: Impaired microRNA processing enhances cellular transformation and tumorigenesis.  Nat Genet2007; 39:673-677.
  68. Calin GA, Croce CM: Chromosomal rearrangements and microRNAs: a new cancer link with clinical implications.  J Clin Invest2007; 117:2059-2066.
  69. Lu J, Getz G, Miska EA, et al: MicroRNA expression profiles classify human cancers.  Nature2005; 435:834-838.
  70. Garzon R, Fabbri M, Cimmino A, et al: 206 MicroRNA expression and function in cancer.  Trends Mol Med2006; 12:580-587.
  71. He L, Thomson JM, Hemann MT, et al: A microRNA polycistron as a potential human oncogene.  Nature2005; 435:828-833.
  72. Voorhoeve PM, le Sage C, Schrier M, et al: A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.  Cell2006; 124:1169-1181.
  73. Ma L, Teruya-Feldstein J, Weinberg RA: Tumour invasion and metastasis initiated by microRNA-10b in breast cancer.  Nature2007; 449:682-688.
  74. Johnson SM, Grosshans H, Shingara J, et al: RAS is regulated by the let-7 microRNA family.  Cell2005; 120:635-647.
  75. He L, He X, Lowe SW, Hannon GJ: MicroRNAs join the p53 network: another piece in the tumour-suppression puzzle.  Nature Rev Cancer2007; 7:819-822.
  76. Bommer GT, Gerin I, Feng Y, et al: p53-mediated activation of miRNA34 candidate tumor-suppressor genes.  Curr Biol2007; 17:1298-1307.
  77. Kinzler KW, Vogelstein B: Cancer-susceptibility genes: gatekeepers and caretakers.  Nature1997; 386:761.763
  78. Levitt NC, Hickson ID: Caretaker tumour suppressor genes that defend genome integrity.  Trends Mol Med2002; 8:179-186.
  79. Lynch HT, de la Chapelle A: Genetic susceptibility to nonpolyposis colorectal cancer.  J Med Genet1999; 36:801-818.
  80. Aaltonen LA, Peltomaki P, Mecklin JP, et al: Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients.  Cancer Res1994; 54:1645-1648.
  81. Young J, Leggett B, Gustafson C, et al: Genomic instability occurs in colorectal carcinomas but not in adenomas.  Hum Mutat1993; 2:351-354.
  82. Kaye FJ: RB and cyclin dependent kinase pathways: Defining a distinction between RB and p16 loss in lung cancer.  Oncogene2002; 21:6908-6914.
  83. Khidr L, Chen PL: RB, the conductor that orchestrates life, death and differentiation.  Oncogene2006; 25:5210-5219.
  84. Morgenbesser SD, Williams BO, Jacks T, DePinho RA: p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens.  Nature1994; 371:72-74.
  85. Gumbiner BM: Regulation of cadherin-mediated adhesion in morphogenesis.  Nat Rev Mol Cell Biol2005; 6:622-634.
  86. Vleminckx K, Vakaet Jr L, Mareel M, et al: Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role.  Cell1991; 66:107-119.
  87. Guilford P, Hopkins J, Harraway J, et al: E-cadherin germline mutations in familial gastric cancer.  Nature1998; 392:402-405.
  88. Sebolt-Leopold JS, Herrera R: Targeting the mitogen-activated protein kinase cascade to treat cancer.  Nat Rev Cancer2004; 4:937-947.
  89. Brummelkamp TR, Bernards R, Agami R: Stable suppression of tumorigenicity by virus-mediated RNA interference.  Cancer Cell2002; 2:243-247.
  90. Shirasawa S, Furuse M, Yokoyama N, Sasazuki T: Altered growth of human colon cancer cell lines disrupted at activated Ki-ras.  Science1993; 260:85-88.