Pathology_Robbins

Pathology_Robbins. It could be argued that the proliferation of literature on the molecular basis of cancer has outpaced the growth of even the most malignant of tumors. Researchers and students alike can easily get lost in the growing forest of information. But it has become eminently clear that cancer is a disease caused by mutations that alter the function of of a finite subset of the 20,000 or so human genes. For simplicity, we will refer to these genes as cancer genes. Cancer genes can be defined as genes that are recurrently affected by genetic aberrations in cancers, presumably because they contribute directly to the malignant behavior of cancer cells. Causative mutations that give rise to cancer genes may be acquired by the action of environmental agents, such as chemicals, radiation, or viruses, may occur spontaneously, or may be inherited in the germ line. If such mutations drive carcinogenesis, a key prediction is that each cell in an individual tumor should share mutations that were present


Pathology_Robbins

Pathology_Robbins. occur spontaneously, or may be inherited in the germ line. If such mutations drive carcinogenesis, a key prediction is that each cell in an individual tumor should share mutations that were present in the founding cell at the time of transformation. This expectation has been realized in all tumors that have been systematically analyzed by genomic sequencing, providing strong support for the hypothesis that cancer is at its root a genetic disease.


Pathology_Robbins

Pathology_Robbins. Cancer genes number in the hundreds and new ones are still being discovered. Not only are these genes numerous, but many have unpronounceable acronyms for names that are difficult to remember, even for the aficionado. One way to try to simplify this complexity is to consider that cancer genes fall into one of four major functional classes: Oncogenes are genes that induce a transformed phenotype when expressed in cells by promoting increased cell growth. A major discovery in cancer was the realization that oncogenes are mutated or overexpressed versions of normal cellular genes, which are called proto-oncogenes. Most oncogenes encode transcription factors, factors that participate in pro-growth signaling pathways, or factors that enhance cell survival. They are considered dominant genes because a mutation involving a single allele is sufficient to produce a pro-oncogenic effect.


Pathology_Robbins

Pathology_Robbins. Tumor suppressor genes are genes that normally prevent uncontrolled growth and, when mutated or lost from a cell, allow the transformed phenotype to develop. Often both normal alleles of tumor suppressor genes must be damaged for transformation to occur. Tumor suppressor genes can be placed into two general groups, “governors” that act as important brakes on cellular proliferation, and “guardians” that are responsible for sensing genomic damage. Some guardian genes initiate and choreograph a complex “damage control response” that leads to the cessation of proliferation or, if the damage is too great to be repaired, or induce apoptosis. Genes that regulate apoptosis primarily act by enhancing cell survival, rather than stimulating proliferation per se. Understandably, genes of this class that protect against apoptosis are often overexpressed in cancer cells, whereas those that promote apoptosis tend to be under-expressed or functionally inactivated by mutations.


Pathology_Robbins

Pathology_Robbins. To this list may now be added genes that regulate interactions between tumor cells and host cells, as these genes are also recurrently mutated or functionally altered in certain cancers. Particularly important are genes that enhance or inhibit recognition of tumors cells by the host immune system. In most instances, the mutations that give rise to cancer genes are acquired during life and are confined to the cancer cells. However, causative mutations sometimes are inherited in the germ line and are therefore present in http://ebooksmedicine.net Neurofibromatosis1and2NF1, NF2 BreastandovariantumorsBRCA1, BRCA2 Multipleendocrineneoplasia1and2MEN1, RET HereditarynonpolyposiscoloncancerMSH2, MLH1, MSH6 every cell in the body, placing the affected individual at high risk for developing cancer. Understandably, in families in which these germ line mutations are passed from generation to generation, cancer behaves like an inherited trait (


Pathology_Robbins

Pathology_Robbins. Table 6.4 ). We will touch on important familial cancer syndromes and associated genes and cancers later in this chapter. Presented next is a discussion of the varied genetic lesions that underlie altered cancer gene expression and function. The genetic changes found in cancers vary from point mutations involving single nucleotides to abnormalities large enough to produce gross changes in chromosome structure. In certain neoplasms, genetic abnormalities are nonrandom and highly characteristic. Specific chromosomal abnormalities have been identified in most leukemias and lymphomas and in an increasing number of nonhematopoietic tumors, while other tumors are characterized by particular point mutations. It is believed that all recurrent genetic changes alter the activity of one or more cancer genes in a fashion that gives the affected cells a selective advantage, presumably by contributing to one or more of the hallmarks of cancer.


Pathology_Robbins

Pathology_Robbins. In the following sections, we briefly review the types of mutations that are commonly found in cancers. Before doing so, however, we must first touch on the important concept of driver mutations and passenger mutations. Driver mutations are mutations that alter the function of cancer genes and thereby directly contribute to the development or progression of a given cancer. They are usually acquired, but as mentioned earlier, occasionally inherited. By contrast, passenger mutations are acquired mutations that are neutral in terms of fitness and do not affect cellular behavior; they just come along for the proverbial ride. Because they occur at random, passenger mutations are sprinkled throughout the genome, whereas driver mutations tend to be tightly clustered within cancer genes. It is now appreciated that particularly in cancers caused by carcinogen exposure, such as melanoma and smoking-related lung cancer, passenger mutations greatly outnumber driver mutations.


Pathology_Robbins

Pathology_Robbins. Despite their apparently innocuous nature, passenger mutations have nevertheless proven to be important in several ways: In carcinogen-associated cancers, mutational analysis has provided definitive evidence that most genomic damage is directly caused by the carcinogen in question. For example, before sequencing of melanoma genomes, the causative role of sun exposure in this cancer was debated. This is no longer so, as most melanomas have thousands of mutations of a type that is specifically linked to damage caused by ultraviolet light.


Pathology_Robbins

Pathology_Robbins. A second, more nefarious effect of passenger mutations is that they create genetic variants that, while initially neutral, may provide tumor cells with a selective advantage in the setting of therapy. The evidence for this comes from DNA sequence analyses of tumors at the time of recurrence after drug therapy; in many instances, mutations that lead directly to drug resistance are found in most tumor cells. Generally, the same resistance mutations can also be found before therapy, but only in a very small fraction of cells. In such instances, it appears that the selective pressure of therapy “converts” a neutral passenger mutation into a driver mutation, to the benefit of the tumor and the detriment of the patient.


Pathology_Robbins

Pathology_Robbins. Point mutations can either activate or inactivate the protein products of the affected genes depending on their precise position and consequence. Point mutations that convert proto-oncogenes into oncogenes generally produce a gain-of-function by altering amino acid residues in a domain that normally holds the protein’s activity in check. A cardinal example is point mutations that convert the RAS gene into a cancer gene, one of the most comment events in human cancers. By contrast, point mutations (as well as larger aberrations, such as insertions and deletions) in tumor suppressor genes reduce or disable the function of the encoded protein. The tumor suppressor gene that is most commonly affected by point mutations in cancer is TP53, a prototypical “guardian” type tumor suppressor gene (discussed later).


Pathology_Robbins

Pathology_Robbins. Gene rearrangements may be produced by chromosomal translocations or inversions. Specific chromosomal trans-locations and inversions are highly associated with certain malignancies, particularly neoplasms derived from hematopoietic cells and other kinds of mesenchymal cells. http://ebooksmedicine.net These rearrangements can activate proto-oncogenes in two ways: Some gene rearrangements result in overexpression of proto-oncogenes by removing them from their normal regulatory elements and placing them under control of an inappropriate, highly active promoter or enhancer. Two different kinds of B cell lymphoma provide illustrative examples of this mechanism. In more than 90% of cases of Burkitt lymphoma, the cells have a translocation, usually between chromosomes 8 and 14, that leads to overexpression of the MYC gene on chromosome 8 by juxtaposition with immunoglobulin heavy chain gene regulatory elements on chromosome 14 (


Pathology_Robbins

Pathology_Robbins. Fig. 6.14 ). In follicular lymphoma, a reciprocal translocation between chromosomes 14 and 18 leads to overexpression of the anti-apoptotic gene, BCL2, on chromosome 18, also driven by immunoglobulin gene regulatory elements. Other oncogenic gene rearrangements create fusion genes encoding novel chimeric proteins. Most notable Activation of growth factor signaling pathways oncogene Increased expression of pro-growth genes Fig. 6.14 ThechromosomaltranslocationsandassociatedoncogenesinchronicmyelogenousleukemiaandBurkittlymphoma. is the Philadelphia (Ph) chromosome in chronic myeloid leukemia, consisting of a balanced reciprocal translocation between chromosomes 9 and 22 (see


Pathology_Robbins

Pathology_Robbins. is the Philadelphia (Ph) chromosome in chronic myeloid leukemia, consisting of a balanced reciprocal translocation between chromosomes 9 and 22 (see Fig. 6.14 ). As a consequence, the derivative chromosome 22 (the Philadelphia chromosome) appears smaller than normal. This cytogenetic change is seen in more than 90% of cases of chronic myeloid leukemia and results in the fusion of portions of the BCR gene on chromosome 22 and the ABL gene on chromosome 9. The few Philadelphia chromosome–negative cases harbor a cryptic (cytogenetically silent) BCR-ABL fusion gene, the presence of which is the sine qua non of chronic myeloid leukemia. As discussed later, the BCR-ABL fusion gene encodes a novel tyrosine kinase with potent transforming activity.


Pathology_Robbins

Pathology_Robbins. Lymphoid tumors are most commonly associated with recurrent gene rearrangements. This relationship exists because normal lymphocytes express special enzymes that purposefully introduce DNA breaks during the processes of immunoglobulin or T cell receptor gene recombination. Repair of these DNA breaks is error-prone, and the resulting mistakes sometimes result in gene rearrangements that activate proto-oncogenes. Two other types of mesenchymal tumors, myeloid neoplasms (acute myeloid leukemias and myeloproliferative disorders) and sarcomas, also frequently possess gene rearrangements. Unlike lymphoid neoplasms, the cause of the DNA breaks that lead to gene rearrangements in myeloid neoplasms and sarcomas is unknown. In general, the rearrangements that are seen in myeloid neoplasms and sarcomas create fusion genes that encode either hyperactive tyrosine kinases (akin to BCRABL) or novel oncogenic transcription factors. A well-characterized example of the latter is the (11;22)(q24;q12)


Pathology_Robbins

Pathology_Robbins. create fusion genes that encode either hyperactive tyrosine kinases (akin to BCRABL) or novel oncogenic transcription factors. A well-characterized example of the latter is the (11;22)(q24;q12) translocation in Ewing sarcoma. This rearrangement creates a fusion gene encoding a chimeric oncoprotein composed of portions of two different transcription factors called EWS and FLI1.


Pathology_Robbins

Pathology_Robbins. Identification of pathogenic gene rearrangements in carcinomas has lagged because karyotypically evident trans-locations and inversions (which point to the location of important oncogenes) are rare in carcinomas. However, advances in DNA sequencing have revealed recurrent cryptic pathogenic gene rearrangements in carcinomas as well. As with hematologic malignancies and sarcomas, gene rearrangements in solid tumors can contribute to carcinogenesis either by increasing expression of an oncogene or by generation of a novel fusion gene. Examples will be discussed along with specific cancers in other chapters. As with a fusion gene such as BCR-ABL, some of the fusion genes in solid tumors also provide drug targets (e.g., EML-ALK in lung cancer; Chapter 13). Deletions are another prevalent abnormality in tumor cells. Deletion of specific regions of chromosomes may result in the loss of particular tumor suppressor genes.


Pathology_Robbins

Pathology_Robbins. Deletions are another prevalent abnormality in tumor cells. Deletion of specific regions of chromosomes may result in the loss of particular tumor suppressor genes. Tumor suppressors generally require inactivation of both alleles in order for them to contribute to carcinogenesis. A common mechanism for this is an inactivating point mutation in one allele, followed by deletion of the other, non-mutated allele. As discussed later, deletions involving 13q14, the site of the RB gene, are associated with retinoblastoma, and deletion of 17p is associated with loss of TP53, arguably the most important tumor suppressor gene. http://ebooksmedicine.net


Pathology_Robbins

Pathology_Robbins. Proto-oncogenes may be converted to oncogenes by gene amplification, with consequent overexpression and hyperactivity of otherwise normal proteins. Such amplification may produce several hundred copies of the gene, a change in copy number that can be readily detected by molecular hybridization with appropriate DNA probes. In some cases, the amplified genes produce chromosomal changes that can be identified microscopically. Two mutually exclusive patterns are seen: multiple small, extrachromosomal structures called double minutes; and homogeneously staining regions. The latter derive from the insertion of the amplified genes into new chromosomal locations, which may be distant from the normal location of the involved genes; because regions containing amplified genes lack a normal banding pattern, they appear homogeneous in a G-banded karyotype. Two clinically important examples of amplification involve the NMYC gene in neuroblastoma and the HER2 gene in breast cancers. NMYC is


Pathology_Robbins

Pathology_Robbins. pattern, they appear homogeneous in a G-banded karyotype. Two clinically important examples of amplification involve the NMYC gene in neuroblastoma and the HER2 gene in breast cancers. NMYC is amplified in 25% to 30% of neuroblastomas, and the amplification is associated with poor prognosis (


Pathology_Robbins

Pathology_Robbins. Fig. 6.15 ). HER2 (also known as ERBB2) amplification occurs in about 20% of breast cancers, and antibody therapy directed against the receptor encoded by the HER2 gene has proved effective in this subset of tumors. Aneuploidy is defined as a number of chromosomes that is not a multiple of the haploid state; for humans, that is a chromosome number that is not a multiple of 23. Aneuploidy is remarkably common in cancers, particularly carcinomas, and was proposed as a cause of carcinogenesis over 100 years ago. Aneuploidy frequently results from errors of the mitotic checkpoint, the major cell cycle control mechanism that acts to prevent mistakes in chromosome segregation. The mitotic checkpoint prevents aneuploidy by inhibiting the irreversible transition to anaphase until all of the replicated chromosomes have made productive attachments to spindle microtubules. Complete absence of the mitotic checkpoint leads to rapid cell death as a consequence of abnormal chromosome segregation.


Pathology_Robbins

Pathology_Robbins. Mechanistic data establishing aneuploidy as a cause of carcinogenesis, rather than a consequence, have been difficult to generate. However, statistical approaches made possible by detailed analysis of cancer cells suggest (as might be expected) that aneuploidy tends to increase the copy number of key oncogenes and decrease the copy number of potent tumor suppressors. For example, chromosome 8, which almost never is lost and often is present in increased copies in tumor cells, is where the MYC oncogene is located. By contrast, portions of chromosome 17, where the TP53 gene is located, are often lost and are infrequently gained. Thus, tumor development and progression may be molded by changes in chromosome numbers that enhance the dosage of oncogenes while restricting the activity of tumor suppressor genes.


Pathology_Robbins

Pathology_Robbins. Fig. 6.15 AmplificationoftheNMYC geneinhumanneuroblastoma.TheNMYC gene,presentnormallyonchromosome2p,becomesamplifiedandisseeneitherasextrachromosomaldoubleminutesorasachromosomallyintegratedhomogeneous-stainingregion(HSR).Theintegrationinvolvesotherautosomes,suchas4,9,or13.(Modified from Brodeur GM, Seeger RC, Sather H, et al: Clinical implications of oncogene activation in human neuroblastomas. Cancer58:541,1986.Reprinted by permission of Wiley-Liss,Inc,a subsidiary of John Wiley & Sons, Inc.)


Pathology_Robbins

Pathology_Robbins. As discussed in Chapter 1, microRNAs (miRNAs) are non-coding, single-stranded RNAs, approximately 22 nucleotides in length, that function as negative regulators of genes. They inhibit gene expression posttranscriptionally by repressing translation or, in some cases, by messenger RNA (mRNA) cleavage. In view of their important functions in control of cell growth, differentiation, and survival, it is not surprising that accumulating evidence indicates that miRNAs also can contribute to carcinogenesis. Specifically, if the target of a miRNA is a tumor suppressor gene, then overactivity of the miRNA can reduce the tumor suppressor protein. Such miRNAs are sometimes referred to as oncomIRs. Conversely, if an miRNA inhibits the translation of an oncogene, a reduction in the quantity or function of that miRNA will lead to overproduction of the oncogene product. Such relationships have already been established by miRNA profiling of several human tumors. For example, downregulation or deletion


Pathology_Robbins

Pathology_Robbins. of that miRNA will lead to overproduction of the oncogene product. Such relationships have already been established by miRNA profiling of several human tumors. For example, downregulation or deletion of certain miRNAs in some leukemias and lymphomas results in increased expression of BCL2, an anti-apoptotic gene. Thus, by negatively regulating BCL2, such miRNAs behave as tumor suppressor genes. Dysregulation of other miRNAs that control the expression of the RAS and MYC oncogenes also has been detected in lung tumors and in certain B-cell leukemias, respectively.


Pathology_Robbins

Pathology_Robbins. You will recall from Chapter 1 that epigenetics refers to reversible, heritable changes in gene expression that occur without mutation. Such changes involve posttranslational modifications of histones and DNA methylation, both of which affect gene expression. In normal, differentiated cells, the major portion of the genome is not expressed. These regions of the genome are silenced by DNA methylation and histone modifications. On the other hand, cancer cells are characterized by a global DNA hypomethylation and selective promoter-localized hypermethylation. Indeed, it has become evident during the past several years that tumor suppressor genes are sometimes silenced by hypermethylation of promoter sequences, rather than by mutation. In addition, genome-wide hypomethylation has been shown to cause chromosomal instability and can induce tumors in mice. Thus, epigenetic changes may influence carcinogenesis in many ways. As an added wrinkle, deep sequencing of cancer genomes has identified


Pathology_Robbins

Pathology_Robbins. cause chromosomal instability and can induce tumors in mice. Thus, epigenetic changes may influence carcinogenesis in many ways. As an added wrinkle, deep sequencing of cancer genomes has identified mutations in genes that regulate epigenetic modifications in many cancers. Thus, certain genetic changes in cancers may be selected because they lead to alterations of the “epigenome” that favor cancer growth and survival.


Pathology_Robbins

Pathology_Robbins. http://ebooksmedicine.net The epigenetic state of particular cell types—a feature described as the epigenetic context—also dictates their response to signals that control growth and differentiation. As mentioned earlier, epigenetic modifications regulate gene expression, allowing cells with the same genetic makeup (e.g., a neuron and a keratinocyte) to have completely different appearances and functions. In some instances, the epigenetic state of a cell dramatically affects its response to otherwise identical signals. For example, the NOTCH1 gene has an oncogenic role in T-cell leukemia, yet acts as a tumor suppressor in squamous cell carcinomas. As would be expected, this dichotomy exists because activated NOTCH1 turns on pro-growth genes in T-cell progenitors and tumor suppressor genes in keratinocytes. Mutationsincancercellsfallintotwomajorclasses,driver(pathogenic)mutationsandpassenger(neutral)mutations.


Pathology_Robbins


Pathology_Robbins

Pathology_Robbins. OverexpressionofmiRNAscancontributetocarcinogenesisbyreducingtheexpressionoftumorsuppressors,whiledeletionorlossofexpressionofmiRNAscanleadtooverexpressionofproto-oncogenes. TumorsuppressorgenesandDNArepairgenesalsomaybesilencedbyepigeneticchanges,whichinvolvereversible,heritablechangesingeneexpressionthatoccurnotbymutationbutbymethylationofthepromoter. CARCINOGENESIS:AMULTISTEPPROCESS Fortunately, in most if not all instances, no single mutation is sufficient to transform a normal cell into a cancer cell. Carcinogenesis is thus a multistep process resulting from the accumulation of multiple genetic alterations that collectively give rise to the transformed phenotype and all of its associated hallmarks, discussed later. As mentioned earlier, the presence of driver mutations in some non-neoplastic precursor lesions suggest the need for additional mutations for transition to a full blown cancer and thus support this model.


Pathology_Robbins

Pathology_Robbins. Beyond tumor initiation from a single founding cell, it is important to recognize that cancers continue to undergo Darwinian selection and therefore continue to evolve (


Pathology_Robbins

Pathology_Robbins. Fig. 6.16 ). It is well established that during their course cancers generally become more aggressive and acquire greater malignant potential, a phenomenon referred to as tumor progression. At the molecular level, tumor progression most likely results from mutations that accumulate independently in different cells. Some of these mutations may be lethal, but others may affect the function of cancer genes, thereby making the affected cells more adept at growth, survival, invasion, metastasis, or immune evasion. Due to this selective advantage, subclones that acquire these mutations may come to dominate one area of a tumor, either at the primary site or at sites of metastasis. As a result of continuing mutation and Darwinian selection, even though malignant tumors are monoclonal in origin they are typically genetically heterogeneous by the time of their clinical presentation. In advanced tumors exhibiting genetic instability, the extent of genetic heterogeneity may be enormous.


Pathology_Robbins

Pathology_Robbins. Genetic evolution shaped by darwinian selection can explain the two most pernicious properties of cancers: the tendency over time for cancers to become both more aggressive and less responsive to therapy. Thus, genetic heterogeneity has implications not only for cancer progression but also for its response to therapy. Experience has shown that when tumors recur after chemotherapy, the recurrent tumor is almost always resistant to the original drug regimen if it is given again. Experimental data suggest that this acquired resistance stems from the outgrowth of subclones that have, by chance, mutations (or epigenetic alterations) that impart drug resistance.


Pathology_Robbins

Pathology_Robbins. This overview serves as background for a more detailed consideration of the molecular pathogenesis of cancer. As mentioned earlier, bona fide cancer genes number in the hundreds, at a minimum. While it is traditional to describe the function of cancer genes one gene at a time, the blizzard of mutated genes emerging from the sequencing of cancer genomes has blanketed the landscape and revealed the limitations of trying to grasp the fundamental properties of cancer, gene by gene. A much more tractable and conceptually satisfying way to think about the biology of cancer is to consider the common phenotypic and http://ebooksmedicine.net Accumulation of driver and passenger mutations Carcinogen-induced Additional driver Additional mutations, mutation Emergence of subclones Diagnosis Normal Initiated precursor with Founding


Pathology_Robbins

Pathology_Robbins. Accumulation of driver and passenger mutations Carcinogen-induced Additional driver Additional mutations, mutation Emergence of subclones Diagnosis Normal Initiated precursor with Founding Fig. 6.16 Developmentofcancerthroughstepwiseaccumulationofcomplementarydrivermutations.Theorderinwhichvariousdrivermutationsoccurisusuallyunknownandmayvaryfromtumortotumor. biological properties of cancer cells. It appears that all cancers display eight fundamental changes in cell physiology, which are considered the hallmarks of cancer. These changes are illustrated in Fig. 6.17 and consist of the following: Insensitivity to growth-inhibitory signals Evasion of apoptosis Evasion of immune surveillance The acquisition of the genetic and epigenetic alterations that confer these hallmarks may be accelerated by cancer-promoting inflammation and by genomic instability. These are considered enabling characteristics because they promote cellular transformation and subsequent tumor progression.


Pathology_Robbins

Pathology_Robbins. Mutations in genes that regulate some or all of these cellular traits are seen in every cancer; accordingly, these traits form the basis of the following discussion of the molecular origins of cancer. Of note, by convention, gene symbols are italicized but their protein products are not (e.g., RB gene and RB protein, TP53 and p53, MYC and MYC). The self-sufficiency in growth that characterizes cancer cells generally stems from gain-of-function mutations that convert proto-oncogenes to oncogenes. Oncogenes encode proteins called oncoproteins that promote cell growth, even in the absence of normal growth-promoting signals. To appreciate how oncogenes drive inappropriate cell growth, it is helpful to review briefly the sequence of events that characterize normal cell proliferation (introduced in Chapter 1). Under physiologic conditions, cell proliferation can be readily resolved into the following steps: 1. Binding of a growth factor to its specific receptor on the cell membrane 2.


Pathology_Robbins

Pathology_Robbins. Binding of a growth factor to its specific receptor on the cell membrane 2. Transient and limited activation of the growth factor receptor, which in turn activates several signaltransducing proteins on the inner leaflet of the plasma membrane 3. Transmission of the transduced signal across the cytosol to the nucleus by second messengers or a cascade of signal transduction molecules 4. Induction and activation of nuclear regulatory factors that initiate and regulate DNA transcription and the biosynthesis of other cellular components that are needed for cell division, such as organelles, membrane components, and ribosomes 5. Entry and progression of the cell into the cell cycle, resulting ultimately in cell division


Pathology_Robbins

Pathology_Robbins. Entry and progression of the cell into the cell cycle, resulting ultimately in cell division Fig. 6.17 Eightcancerhallmarksandtwoenablingfactors(genomicinstabilityandtumor-promotinginflammation).Mostcancercellsacquirethesepropertiesduringtheirdevelopment,typicallyduetomutationsincriticalgenes.(From Hanahan D,Weinberg RA: Hallmarks of cancer: the next generation. Cell144:646, 2011.) The mechanisms that endow cancer cells with the ability to proliferate can be grouped according to their role in the growth factor–induced signal transduction cascade and cell cycle regulation. Indeed, each one of the listed steps is susceptible to corruption in cancer cells. http://ebooksmedicine.net


Pathology_Robbins

Pathology_Robbins. http://ebooksmedicine.net Cancers may secrete their own growth factors or induce stromal cells to produce growth factors in the tumor microenvironment. Most soluble growth factors are made by one cell type and act on a neighboring cell to stimulate proliferation (paracrine action). Normally, cells that produce the growth factor do not express the cognate receptor, preventing the formation of positive feedback loops within the same cell. This “rule” may be broken by cancer cells in several different ways. Some cancer cells acquire growth self-sufficiency by acquiring the ability to synthesize the same growth factors to which they are responsive. For example, many glioblastomas secrete platelet-derived growth factor (PDGF) and express the PDGF receptor, and many sarcomas make both transforming growth factor-α (TGF-α) and its receptor. Similar autocrine loops are fairly common in many types of cancer.


Pathology_Robbins

Pathology_Robbins. Another mechanism by which cancer cells acquire growth self-sufficiency is by interaction with stroma. In some cases, tumor cells send signals to activate normal cells in the supporting stroma, which in turn produce growth factors that promote tumor growth.


Pathology_Robbins

Pathology_Robbins. The next group in the sequence of signal transduction is growth factor receptors. Some growth factor receptors have an intrinsic tyrosine kinase activity that is activated by growth factor binding, while others signal by stimulating the activity of downstream proteins. Many of the myriad growth factor receptors function as oncoproteins when they are mutated or if they overexpressed. The best-documented examples of overexpression involve the epidermal growth factor (EGF) receptor family. ERBB1, the EGF receptor, is overexpressed in 80% of squamous cell carcinomas of the lung, 50% or more of glioblastomas, and 80% to 100% of epithelial tumors of the head and neck. As mentioned earlier, the gene encoding a related receptor, HER2 (ERBB2), is amplified in approximately 20% of breast cancers and in a smaller fraction of adenocarcinomas of the lung, ovary, stomach, and salivary glands. These tumors are exquisitely sensitive to the mitogenic effects of small amounts of growth factors. The


Pathology_Robbins

Pathology_Robbins. and in a smaller fraction of adenocarcinomas of the lung, ovary, stomach, and salivary glands. These tumors are exquisitely sensitive to the mitogenic effects of small amounts of growth factors. The significance of HER2 in the pathogenesis of breast cancers is illustrated dramatically by the clinical benefit derived from blocking the extracellular domain of this receptor with anti-HER2 antibodies, an elegant example of “bench to bedside” medicine. In other instances, tyrosine kinase activity is stimulated by point mutations or small indels that lead to subtle but functionally important changes in protein structure, or gene rearrangements that create fusion genes encoding chimeric receptors. In each of these cases, the mutated receptors are constitutively active, delivering mitogenic signals to cells even in the absence of growth factors. These types of mutations are most common in leukemias, lymphomas, and certain forms of sarcoma.


Pathology_Robbins

Pathology_Robbins. Cancer cells often acquire growth autonomy as a result of mutations in genes that encode components of signaling pathways downstream of growth factor receptors. The signaling proteins that couple growth factor receptors to their nuclear targets are activated by ligand binding to growth factor receptors. The signals are trasnmitted to the nucleus through various signal transduction molecules. Two important oncoproteins in the category of signaling molecules are RAS and ABL. Each of these is discussed briefly next.


Pathology_Robbins

Pathology_Robbins. RAS is the most commonly mutated oncogene in human tumors. Approximately 30% of all human tumors contain mutated RAS genes, and the frequency is even higher in some specific cancers (e.g., pancreatic adenocarcinoma). RAS is a member of a family of small G proteins that bind guanosine nucleotides (guanosine triphosphate [GTP] and guanosine diphosphate [GDP]). Signaling by RAS involves the following sequential steps: • Normally, RAS flips back and forth between an excited signal-transmitting state and a quiescent state. RAS is inactive when bound to GDP; stimulation of cells by growth factors such as EGF and PDGF leads to exchange of GDP for GTP and subsequent conformational changes Activation of transcription


Pathology_Robbins

Pathology_Robbins. Activation of transcription Fig. 6.18 Model for action of RAS. When a normal cell is stimulated through a growth factor receptor, inactive (GDP-bound) RAS is activated to a GTP-bound state. Activated RAS transduces proliferative signals to the nucleus along two pathways: the so-called “RAF/ERK/MAP kinase pathway” and the PI3 kinase/AKT pathway. GDP, Guanosine diphosphate; GTP, guanosine triphosphate; MAP, mitogen-activated protein; PI3, phosphatidylinositol-3. Fig. 6.18 ). This excited signal-emitting state is short-lived, however, because the intrinsic guanosine triphosphatase (GTPase) activity of RAS hydrolyzes GTP to GDP, releasing a phosphate group and returning the protein to its quiescent GDP-bound state. The GTPase activity of activated RAS is magnified dramatically by a family of GTPase-activating proteins (GAPs), which act as molecular brakes that prevent uncontrolled RAS activation by favoring hydrolysis of GTP to GDP.


Pathology_Robbins

Pathology_Robbins. • Activated RAS stimulates downstream regulators of proliferation by several interconnected pathways that converge on the nucleus and alter the expression of genes that regulate growth, such as MYC. While details of the signaling cascades (some of which are illustrated in Fig. 6.18 ) downstream of RAS are not discussed here, an important point is that mutational activation of these signaling intermediates mimics the growth promoting effects of activated RAS. For example, BRAF, which lies in the so-called “RAF/ERK/MAP kinase pathway” is mutated in more than 60% of melanomas and is associated with unregulated cell proliferation. Mutations of phosphatidly inositol-3 kinase (PI3 kinase) in the PI3K/AKT pathway also occur with high frequency in some tumor types, with similary consequences.


Pathology_Robbins

Pathology_Robbins. RAS most commonly is activated by point mutations in amino acid residues that are either within the GTP-binding pocket or in the enzymatic region that carries out GTP hydrolysis. Both kinds of mutations interfere with breakdown of GTP, which is essential to inactivate RAS. RAS is thus trapped in its activated, GTP-bound form, and the cell is forced into a continuously proliferating state. It follows from this scenario that the consequences of activating mutations in RAS should be mimicked by loss-offunction mutations in GAPs, which would lead to a failure to simulate GTP hydrolysis and thereby restrain RAS. Indeed, the GAP neurofibromin-1 (NF1) is mutated in the cancer-prone familial disorder neurofibromatosis type 1 (Chapter 22) and is a bona fide tumor suppressor. Similarly, another important tumor suppressor called PTEN is a negative inhibitor of PI3 kinase and is frequently mutated in carcinomas, certain leukemias, and other cancers as well.


Pathology_Robbins

Pathology_Robbins. Several non–receptor tyrosine kinases function as signal transduction molecules. In this group, ABL is the best defined with respect to carcinogenesis. The ABL proto-oncoprotein has tyrosine kinase activity that is dampened by internal negative regulatory domains. As discussed earlier (see Fig. 6.14 ), in chronic myeloid leukemia and certain acute leukemias, a part of the ABL gene is translocated from its normal abode on chromosome 9 to chromosome 22, where it fuses with part of the breakpoint cluster region (BCR) gene. This fusion gene encodes as BCR-ABL hybrid protein that contains the ABL tyrosine kinase domain and a BCR domain that self-associates, an event that unleashes a constitutive tyrosine kinase activity. Of interest, the BCR-ABL protein activates all of the signals that are downstream of RAS, making it a potent stimulator of cell growth.


Pathology_Robbins

Pathology_Robbins. The crucial role of BCR-ABL in cancer has been confirmed by the dramatic clinical response of patients with chronic myeloid leukemia to BCR-ABL kinase inhibitors. The prototype of this kind of drug, imatinib mesylate (Gleevec), galvanized interest in design of drugs that target specific molecular lesions found in various cancers (so-called “targeted therapy”). BCR-ABL also is an example of the concept of oncogene addiction, wherein a tumor is profoundly dependent on a single signaling molecule. BCR-ABL fusion gene formation is an early, perhaps initiating, event that drives leukemogenesis. Development of leukemia probably requires other collaborating mutations, but the transformed cell continues to depend on BCR-ABL for signals that mediate growth and survival. BCR-ABL signaling can be seen as the central lodgepole around which the transformed state is “built”. If the lodgepole is removed by inhibition of the BCR-ABL kinase, the structure collapses. In view of this level of


Pathology_Robbins

Pathology_Robbins. can be seen as the central lodgepole around which the transformed state is “built”. If the lodgepole is removed by inhibition of the BCR-ABL kinase, the structure collapses. In view of this level of dependency, it is not surprising that acquired resistance of tumors to BCR-ABL inhibitors often is due to the outgrowth of a subclone with a mutation in BCR-ABL that prevents binding of the drug to the BCR-ABL protein.


Pathology_Robbins

Pathology_Robbins. The ultimate consequence of signaling through oncoproteins such as RAS or ABL is inappropriate and continuous stimulation of nuclear transcription factors that drive the expression of growth-promoting genes. Growth autonomy may thus be a consequence of mutations affecting genes that regulate DNA transcription. A host of oncoproteins, including products of the MYC, MYB, JUN, FOS, and REL oncogenes, function as transcription factors that regulate the expression of growth-promoting genes, such as cyclins. Of these, MYC is involved most commonly in human tumors.


Pathology_Robbins

Pathology_Robbins. Dysregulation of MYC promotes tumorigenesis by simultaneously promoting the progression of cells through the cell cycle and enhancing alterations in metabolism that support cell growth. MYC primarily functions by activating the transcription of other genes. Genes activated by MYC include several growth-promoting genes, including cyclin-dependent kinases (CDKs), whose products drive cells into the cell cycle (discussed next), and genes that control pathways that produce the building blocks (e.g., amino acids, lipids, nucleotides) that are needed for cell growth and division. As mentioned earlier (see Fig. 6.14 ), dysregulation of MYC results from a (8;14) translocation in Burkitt lymphoma, a highly aggressive B-cell tumor. MYC also is amplified in breast, colon, lung, and many other cancers, while the related NMYC and LMYC genes are amplified in neuroblastomas and small cell cancers of lung, respectively.


Pathology_Robbins

Pathology_Robbins. As mentioned in Chapter 1, growth factors transduce signals that stimulate the orderly progression of cells through the various phases of the cell cycle, the process by which cells replicate their DNA in preparation for cell division. You will recall that progression of cells through the cell cycle is orchestrated by cyclin-dependent kinases (CDKs), which are activated by binding to cyclins, so called because of the cyclic nature of their production and degradation. http://ebooksmedicine.net The CDK-cyclin complexes phosphorylate crucial target proteins that drive cells forward through the cell cycle. While cyclins arouse the CDKs, CDK inhibitors (CDKIs), of which there are many, silence the CDKs and exert negative control over the cell cycle. Expression of these inhibitors is downregulated by mitogenic signaling pathways, thus promoting the progression of the cell cycle.


Pathology_Robbins

Pathology_Robbins. There are two main cell cycle checkpoints, one at the G1/S transition and the other at the G2/M transition, each of which is tightly regulated by a balance of growth-promoting and growth-suppressing factors, as well as by sensors of DNA damage (Chapter 1). If activated, these DNA-damage sensors transmit signals that arrest cell cycle progression and, if cell damage cannot be repaired, initiate apoptosis. Once cells pass through the G1/S checkpoint, they are committed to undergo cell division. Understandably, then, defects in the G1/S checkpoint are particularly important in cancer, since these lead directly to increased cell division. Indeed, all cancers appear to have genetic lesions that disable the G1/S checkpoint, causing cells to continually reenter the S phase. For unclear reasons, particular lesions vary widely in frequency across tumor types, but they fall into two major categories.


Pathology_Robbins

Pathology_Robbins. Gain-of-function mutations involving CDK4 or D cyclins. Mishaps increasing the expression of cyclin D or CDK4 are common events in neoplastic transformation. The cyclin D genes are overexpressed in many cancers, including those affecting the breast, esophagus, liver, and a subset of lymphomas and plasma cell tumors. Amplification of the CDK4 gene occurs in melanomas, sarcomas, and glioblastomas. Mutations affecting cyclins B and E and other CDKs also occur, but they are much less frequent than those affecting cyclin D and CDK4.


Pathology_Robbins

Pathology_Robbins. Loss-of-function mutations involving CDKIs. CDKIs frequently are disabled by mutation or gene silencing in many human malignancies. For example, germline mutations of CDKN2A, a gene that encodes the CDK inhibitor p16, are present in 25% of melanoma-prone kindreds, and acquired deletion or inactivation of CDKN2A is seen in 75% of pancreatic carcinomas, 40% to 70% of glioblastomas, 50% of esophageal cancers and certain leukemias, and 20% of non–small cell lung carcinomas, soft-tissue sarcomas, and bladder cancers. A final consideration of importance in a discussion of growth-promoting signals is that the increased production of oncoproteins does not by itself lead to sustained proliferation of cancer cells. There are two built-in mechanisms, cell senescence and apoptosis, that oppose oncogene-mediated cell growth. As discussed later, genes that regulate these two braking mechanisms must be disabled to allow the action of oncogenes to proceed unopposed.


Pathology_Robbins


Pathology_Robbins

Pathology_Robbins. RAScommonlyismutatedinhumancancersandnormallyflipsbetweenrestingGDP-boundstateandactiveGTP-boundstate;mutationsblockhydrolysisofGTPtoGDP,leadingtouncheckedsignaling ComplexesofcyclinswithCDKsdrivethecellcyclebyphosphorylatingvarioussubstratesandnormallyarecontrolledbyCDKinhibitors.Mutationsingenesencodingcyclins,CDKs,andCDKinhibitorsresultinuncontrolledcellcycleprogressionandarefoundinawidevarietyofcancersincludingmelanomasandbrain,lung,andpancreaticcancers. InsensitivitytoGrowthInhibitorySignals:TumorSuppressorGenes Isaac Newton theorized that every action has an equal and opposite reaction. Although Newton was not a cancer biologist, his formulation holds true for cell growth.


Pathology_Robbins

Pathology_Robbins. Isaac Newton theorized that every action has an equal and opposite reaction. Although Newton was not a cancer biologist, his formulation holds true for cell growth. Whereas oncogenes encode proteins that promote cell growth, the products of tumor suppressor genes apply brakes to cell proliferation. Disruption of such genes renders cells refractory to growth inhibition and mimics the growth-promoting effects of oncogenes. The following discussion describes tumor suppressor genes, their products, and possible mechanisms by which loss of their function contributes to unregulated cell growth.


Pathology_Robbins

Pathology_Robbins. In principle, anti-growth signals can prevent cell proliferation by several complementary mechanisms. The signal may cause dividing cells to enter G0 (quiescence), where they remain until external cues prod their reentry into the proliferative pool. Alternatively, the cells may enter a post-mitotic, differentiated pool and lose replicative potential. Nonreplicative senescence, alluded to earlier, is another mechanism of escape from sustained cell growth. And, as a last-ditch effort, the cells may be programmed for death by apoptosis. As we will see, tumor suppressor genes have all these “tricks” in their toolbox designed to halt wayward cells from becoming malignant. RB: Governor of the Cell Cycle


Pathology_Robbins

Pathology_Robbins. RB: Governor of the Cell Cycle RB, a key negative regulator of the cell cycle, is directly or indirectly inactivated in most human cancers. The http://ebooksmedicine.net retinoblastoma gene (RB) was the first tumor suppressor gene to be discovered and is now considered the prototype of this family of cancer genes. As with many advances in medicine, the discovery of tumor suppressor genes was accomplished by the study of a rare disease—in this case, retinoblastoma, an uncommon childhood tumor. Approximately 60% of retinoblastomas are sporadic, while the remaining ones are familial, the predisposition to develop the tumor being transmitted as an autosomal dominant trait. To account for the sporadic and familial occurrence of an identical tumor, Knudson, in 1974, proposed his now famous two-hit hypothesis, which in molecular terms can be stated as follows:


Pathology_Robbins

Pathology_Robbins. Two mutations (hits) are required to produce retinoblastoma. These involve the RB gene, which has been mapped to chromosomal locus 13q14. Both of the normal alleles of the RB locus must be inactivated (hence the two hits) for the development of retinoblastoma ( Fig. 6.19 In familial cases, children inherit one defective copy of the RB gene in the germ line; the other copy is normal. Retinoblastoma develops when the normal RB gene is lost in retinoblasts as a result of somatic mutation. Because in retinoblastoma families a single germ line mutation is sufficient to transmit disease risk, the trait has an autosomal dominant inheritance pattern. • In sporadic cases, both normal RB alleles are lost by somatic mutation in one of the retinoblasts. The end result is the same: a retinal cell that has lost both of the normal copies of the RB gene becomes cancerous.


Pathology_Robbins

Pathology_Robbins. From the above, it is evident that although the risk for developing retinoblastoma in retinoblastoma families is inherited as a dominant trait, at the level of the cell, one intact RB gene is all that is needed for normal function. Although the loss of normal RB genes initially was discovered in retinoblastomas, it is now evident that biallelic loss of this gene is a fairly common feature of several tumors, including breast cancer, small cell cancer of the lung, and bladder cancer. Patients with familial retinoblastoma also are at greatly increased risk for developing osteosarcomas and some soft-tissue sarcomas. Germ cells Zygote Somatic cells of child Retinal cells Retinoblastoma


Pathology_Robbins

Pathology_Robbins. Germ cells Zygote Somatic cells of child Retinal cells Retinoblastoma Fig. 6.19 Pathogenesisofretinoblastoma.TwomutationsoftheRB chromosomallocus,on13q14,leadtoneoplasticproliferationoftheretinalcells.Inthesporadicform,bothRB mutationsinthetumor-foundingretinalcellareacquired.Inthefamilialform,allsomaticcellsinheritonemutantRB genefromacarrierparent,andasaresultonlyoneadditionalRB mutationinaretinalcellisrequiredforcompletelossofRB function. http://ebooksmedicine.net


Pathology_Robbins

Pathology_Robbins. http://ebooksmedicine.net The function of the RB protein is to regulate the G1/S checkpoint, the portal through which cells must pass before DNA replication commences. Although each phase of the cell cycle circuitry is monitored carefully, the transition from G1 to S is an extremely important checkpoint in the cell cycle “clock.” In the G1 phase, diverse signals are integrated to determine whether the cell should progress through the cell cycle, or exit the cell cycle and differentiate. The RB gene product, RB, is a DNA-binding protein that serves as a point of integration for these diverse signals, which ultimately act by altering the phosphorylation state of RB. Specifically, signals that promote cell cycle progression lead to the phosphorylation and inactivation of RB, while those that block cell cycle progression act by maintaining RB in an active hypophosphorylated state.


Pathology_Robbins

Pathology_Robbins. To appreciate this crucial role of RB in the cell cycle, it is helpful to review the mechanisms that enforce the G1/S transition. The initiation of DNA replication (S phase) requires the activity of cyclin E/CDK2 complexes, and expression of cyclin E is dependent on the E2F family of transcription factors. Early in G1, RB is in its hypophosphorylated active form, and it binds to and inhibits the E2F family of transcription factors, preventing transcription of cyclin E. Hypophosphorylated RB blocks E2F-mediated transcription in at least two ways ( Fig. 6.20 ). First, it sequesters E2F, preventing it from interacting with other transcriptional activators. Second, RB recruits chromatin remodeling proteins, such as histone deacetylases and histone methyltransferases, which bind to the promoters of E2F-responsive genes such as cyclin E. These enzymes modify chromatin at the promoters to make DNA insensitive to transcription factors.


Pathology_Robbins

Pathology_Robbins. This situation is changed on mitogenic signaling. Growth factor signaling leads to cyclin D expression and activation of cyclin D–CDK4/6 complexes. The level of cyclin D-CDK4/6 activity is tempered by antagonists such as p16, which is itself subject to regulation by growth inhibitors such as TGFβ that serve to set a threshold for mitogenic responses. If the stimulus is sufficently strong, cyclin D-CDK4/6 complexes phosphorylate RB, inactivating the protein and releasing E2F to induce target genes such as cyclin E. Cyclin E/CDK complexes then stimulate DNA replication and progression through the cell cycle. When the cells enter S phase, they are committed to divide without additional growth factor stimulation. During the ensuing M phase, the phosphate groups are removed from RB by cellular phosphatases, regenerating the hypophosphorylated form of RB.


Pathology_Robbins

Pathology_Robbins. E2F is not the sole target of RB. The versatile RB protein binds to a variety of other transcription factors that regulate cell differentiation. For example, RB stimulates myocyte-, adipocyte-, melanocyte-, and macrophage-specific transcription factors. Thus, the RB pathway couples control of cell cycle progression at G1 with differentiation, which may explain how differentiation is associated with exit from the cell cycle. In view of the centrality of RB to the control of the cell cycle, an interesting question is why RB is not mutated in every cancer. In fact, mutations in other genes that control PPPPPPPPPPE2F site S phase genes Stimulate Inactivate ActivateCDK Inhibitors p16 Cyclins D/CDK4,6 Cyclin E/CDK2 Cyclins D/CDK4,6 Cyclin E/CDK2 E2F Hyperphosphorylated RB Hypophosphorylated RB GROWTH FACTORS (EGF, PDGF) GROWTH INHIBITORS (TGF-˜, p53, others) E2F E2F site Histone deacetylase Histone methyl-transferase S phase genes


Pathology_Robbins

Pathology_Robbins. RB phosphorylation can mimic the effect of RB loss and are commonly found in many cancers that have normal RB genes. For example, mutational activation of CDK4 and overexpression of cyclin D favor cell proliferation by facilitating RB phosphorylation and inactivation. Indeed, cyclin D is overexpressed in many tumors because of amplification or translocation of the cyclin D1 gene. Mutational inactivation of genes encoding CDKIs also can drive the cell cycle by removing important brakes on cyclin/ CDK activity. As mentioned earlier, the CDKN2A gene, which encodes the CDK inhibitor p16, is an extremely common target of deletion or mutational inactivation in human tumors.


Pathology_Robbins

Pathology_Robbins. It is now accepted that loss of normal cell cycle control is central to malignant transformation and that at least http://ebooksmedicine.net one of the four key regulators of the cell cycle (p16, cyclin D, CDK4, RB) is mutated in most human cancers. Notably, in cancers caused by certain oncogenic viruses (discussed later), this is achieved through direct targeting of RB by viral proteins. For example, the human papillomavirus (HPV) E7 protein binds to the hypophosphorylated form of RB, preventing it from inhibiting the E2F transcription factors. Thus, RB is functionally deleted, leading to uncontrolled growth. Likeothertumorsuppressorgenes,bothcopiesofRB mustbedysfunctionalfortumordevelopmenttooccur. Incasesoffamilialretinoblastoma,onedefectivecopyoftheRB geneispresentinthegermline,sothatonlyoneadditionalsomaticmutationisneededtocompletelyeliminateRBfunction.


Pathology_Robbins

Pathology_Robbins. ManyoncogenicDNAviruses,likeHPV,encodeproteins(e.g.,E7)thatbindRBandrenderitnonfunctional. TP53: Guardian of the Genome The p53-encoding tumor suppressor gene, TP53, is the most commonly mutated gene in human cancer. The p53 protein is a transcription factor that thwarts neoplastic transformation by three interlocking mechanisms: activation of temporary cell cycle arrest (termed quiescence), induction of permanent cell cycle arrest (termed senescence), or triggering of programmed cell death (termed apoptosis). If RB is a “sensor” of external signals, p53 can be viewed as a central monitor of internal stress, directing the stressed cells toward one of these pathways. A variety of stresses trigger the p53 response pathways, including anoxia, inappropriate pro-growth stimuli (e.g., unbridled MYC or RAS activity), and DNA damage. By managing the DNA damage response, p53 plays a central role in maintaining the integrity of the genome, as discussed next.


Pathology_Robbins

Pathology_Robbins. In nonstressed, healthy cells, p53 has a short half-life (20 minutes) because of its association with MDM2, a protein that targets p53 for destruction. When the cell is stressed, for example, by an assault on its DNA, “sensors” that include protein kinases such as ATM (ataxia telangiectasia mutated) are activated. These activated sensors catalyze posttranslational modifications in p53 that release it from MDM2, increasing its half-life and enhancing its ability to drive the transcription of target genes. Hundreds of genes whose transcription is triggered by p53 have been found. These genes suppress neoplastic transformation by three mechanisms: p53-mediated cell cycle arrest may be considered the primordial response to DNA damage (


Pathology_Robbins

Pathology_Robbins. Fig. 6.21 ). It occurs late in the G1 phase and is caused mainly by p53-dependent transcription of the CDKI gene CDKN1A (p21). The p21 protein inhibits cyclin–CDK complexes and prevents phosphorylation of RB, thereby arresting cells in the G1 phase. Such a pause in cell cycling is welcome, because it gives the cells “breathing time” to repair DNA damage. The p53 protein also induces expression of DNA damage repair genes. If DNA damage is repaired successfully, p53 upregulates transcription of MDM2, leading to its own destruction and relief of the cell cycle block. If the damage cannot be repaired, the cell may enter p53induced senescence or undergo p53-directed apoptosis.


Pathology_Robbins

Pathology_Robbins. p53-induced senescence is a form of permanent cell cycle arrest characterized by specific changes in morphology and gene expression that differentiate it from quiescence or reversible cell cycle arrest. Senescence requires activation of p53 and/or Rb and expression of their mediators, such as the CDKIs. The mechanisms of senescence are unclear but seem to involve global chromatin changes, which drastically and permanently alter gene expression. p53-induced apoptosis of cells with irreversible DNA damage is the ultimate protective mechanism against neoplastic transformation. It is mediated by upregulation of several proapoptotic genes, including BAX and PUMA (discussed later). To summarize, p53 is activated by stresses such as DNA damage and assists in DNA repair by causing G1 arrest and inducing the expression of DNA repair genes. A cell with damaged DNA that cannot be repaired is directed by p53 to either enter senescence or undergo apoptosis (see


Pathology_Robbins

Pathology_Robbins. Fig. 6.21 ). In view of these activities, p53 has been rightfully called the “guardian of the genome.” With loss of normal p53 function, DNA damage goes unrepaired, mutations become fixed in dividing cells, and the cell turns onto a one-way street leading to malignant transformation.


Pathology_Robbins

Pathology_Robbins. Confirming the importance of TP53 in controlling carcinogenesis, more than 70% of human cancers have a defect in this gene, and the remaining malignant neoplasms often have defects in genes upstream or downstream of TP53. Biallelic abnormalities of the TP53 gene are found in virtually every type of cancer, including carcinomas of the lung, colon, and breast—the three leading causes of cancer deaths. In most cases, mutations affecting both TP53 alleles are acquired in somatic cells. In other tumors, such as certain sarcomas, the TP53 gene is intact but p53 function is lost because of amplification and over-expression of the MDM2 gene, which encodes a potent inhibitor of p53. Less commonly, patients inherit a mutant TP53 allele; the resulting disorder is called the Li-Fraumeni syndrome. As in the case with familial retinoblastoma, inheritance of one mutant TP53 allele predisposes affected http://ebooksmedicine.net


Pathology_Robbins

Pathology_Robbins. Normal cell Cell with (p53 normal) mutations or loss of p53 p53-dependent genesbinds to DNA No DNANo cell repair, noTranscription dependent and cycle arrest senescenceindependent effects on targets Fig. 6.21 The role of p53 in maintaining the integrity of the genome. Activation of normal p53 by DNA-damaging agents or by hypoxia leads to cell cycle arrest in G1 and induction of DNA repair, by transcriptional upregulation of the cyclin-dependent kinase inhibitor CDKN1A (p21) and the GADD45 genes. Successful repair of DNA allows cells to proceed with the cell cycle; if DNA repair fails, p53 triggers either apoptosis or senescence. In cells with loss or mutations of TP53, DNA damage does not induce cell cycle arrest or DNA repair, and genetically damaged cells proliferate, giving rise eventually to malignant neoplasms.


Pathology_Robbins

Pathology_Robbins. individuals to develop malignant tumors because only one additional hit is needed to inactivate the second, normal allele. Patients with the Li-Fraumeni syndrome have a 25-fold greater chance of developing a malignant tumor by 50 years of age compared with the general population. In contrast to tumors developing in patients who inherit a mutant RB allele, the spectrum of tumors that develop in patients with the Li-Fraumeni syndrome is much more varied; the most common types are sarcomas, breast cancer, leukemia, brain tumors, and carcinomas of the adrenal cortex. Compared with individuals diagnosed with sporadic tumors, patients with Li-Fraumeni syndrome develop tumors at a younger age and may develop multiple primary tumors.


Pathology_Robbins

Pathology_Robbins. As with RB, normal p53 also can be rendered nonfunctional by certain oncogenic DNA viruses. Proteins encoded by oncogenic HPVs, certain polyoma viruses, and hepatitis B virus bind to p53 and nullify its protective function. Thus, transforming DNA viruses subvert two of the best-understood tumor suppressors, RB and p53. TP53: GUARDIAN OF THE GENOME TP53 encodes p53, the central monitor of stress in the cell, which can be activated by anoxia, inappropriate oncogenesignaling, or DNA damage. Activated p53 controls the expression and activity of genes involved in cell cycle arrest, DNArepair, cellular senescence, and apoptosis. DNA damage leads to activation of p53 by phosphorylation. Activated p53 drives transcription of CDKN1A (p21), whichprevents RB phosphorylation, thereby causing a G1-S block in thecellcycle.ThispauseallowsthecellstorepairDNAdamage. IfDNAdamagecannotberepaired,p53inducescellularsenescenceorapoptosis.


Pathology_Robbins


Pathology_Robbins

Pathology_Robbins. AswithRB,p53canbeincapacitatedwhenboundbyproteinsencodedbyoncogenicDNAvirusessuchasHPV. Although much is known about the circuitry that applies brakes to the cell cycle, the molecules that transmit anti-proliferative signals to cells are less well characterized. Best-known is TGF-β, a member of a family of dimeric growth factors that includes bone morphogenetic proteins and activins. In most normal epithelial, endothelial, and hematopoietic cells, TGF-β is a potent inhibitor of proliferation. It regulates cellular processes by binding to a complex composed of TGF-β receptors I and II. Dimerization of the receptor upon ligand binding leads to a cascade of events that result in the transcriptional activation of CDKIs with growth-suppressing activity, as well as repression of growth-promoting genes such as MYC and CDK4.


Pathology_Robbins

Pathology_Robbins. In many forms of cancer, the growth-inhibiting effects of the TGF-β pathways are impaired by mutations affecting TGF-β signaling. These mutations may alter the type II TGF-β receptor or SMAD molecules that serve to transduce anti-proliferative signals from the receptor to the nucleus. Mutations affecting the type II receptor are seen in cancers of the colon, stomach, and endometrium. Mutational inactivation of SMAD4, 1 of the 10 proteins known to be involved in TGF-β signaling, is common in pancreatic cancers. In other cancers, by contrast, loss of TGF-β–mediated growth control occurs at a level downstream of the core TGF-β signaling pathway; for example, there may be loss of p21 expression and/or overexpression of MYC. These tumor cells can then use other elements of the TGF-β–induced program, including immune system suppression or promotion of angiogenesis, to facilitate tumor progression. Thus, TGF-β can function to prevent or promote tumor growth, depending on the state of other


Pathology_Robbins

Pathology_Robbins. including immune system suppression or promotion of angiogenesis, to facilitate tumor progression. Thus, TGF-β can function to prevent or promote tumor growth, depending on the state of other genes in the cell. Indeed, in many late-stage tumors, TGF-β signaling activates epithelial-to-mesenchymal transition (EMT), a process that promotes migration, invasion, and metastasis, as discussed later.


Pathology_Robbins

Pathology_Robbins. Contact Inhibition, NF2, and APC When cancer cells are grown in the laboratory, their proliferation fails to be inhibited when they come in contact with each other. This is in sharp contrast to nontransformed cells, which stop proliferating once they form confluent monolayers. The mechanisms that govern contact inhibition are only now being discovered.


Pathology_Robbins

Pathology_Robbins. Cell–cell contacts in many tissues are mediated by homodimeric interactions between transmembrane proteins called cadherins. E-cadherin (E for epithelial) mediates cell–cell contact in epithelial layers. Two mechanisms have been proposed to explain how E-cadherin maintains contact inhibition: • One mechanism is mediated by the tumor suppressor gene NF2. Its product, neurofibromin-2, more commonly called merlin, acts downstream of E-cadherin in a signling pathway that helps fo maintain contact inhibition. Homozygous loss of NF2 is known to cause certain neural tumors, and germ line mutations in NF2 are associated with a tumor-prone hereditary condition called neurofibromatosis type 2.


Pathology_Robbins

Pathology_Robbins. • A second mechanism by which E-cadherin may regulate contact inhibition involves its ability to bind β-catenin, another signaling protein. β-catenin is a key component of the WNT signaling pathway (described below), which has broad but as of yet incompletely understood roles in regulating the morphology and organization of epithelial cells lining structures such as the gut.


Pathology_Robbins

Pathology_Robbins. A further clue to the important of E-cadherin and β-catenin in epithelial cancers is illustrated by the rare hereditary disease adenomatous polyposis coli (APC). This disorder is characterized by the development of numerous adenomatous polyps in the colon that have a very high incidence of transformation into colonic cancers. The polyps consistently show loss of a tumor suppressor gene called APC (named for the disease), which exerts anti-proliferative effects in an unusual manner. APC encodes a cytoplasmic protein whose dominant function is to promote the degradation of β-catenin, which has several functions. In addition to binding E-cadherin, β-catenin also is a key component of the WNT signaling pathway (illustrated in Fig. 6.22).


Pathology_Robbins

Pathology_Robbins. Fig. 6.22). WNTs are soluble factors that bind WNT receptors, which in turn transmit signals that prevent the APC-mediated degradation of β-catenin, allowing it to translocate to the nucleus, where it acts as a transcriptional activator. In quiescent cells that have not been exposed to WNT, cytoplasmic β-catenin is degraded by a destruction complex, of which APC is an integral part. With loss of APC (e.g., in colon cancers), β-catenin degradation is prevented, and the WNT signaling response is inappropriately activated even in the absence of WNT factors. In colonic epithelium this leads to increased transcription of growth-promoting genes, such as cyclin D1 and MYC, as well as transcriptional regulators, such as TWIST and SLUG, which repress Ecadherin expression and thus reduce contact inhibition.


Pathology_Robbins

Pathology_Robbins. APC behaves as a typical tumor suppressor gene. Individuals born with one mutated allele typically are found to have hundreds to thousands of adenomatous polyps in the colon by their teens or twenties; these polyps show loss of the other APC allele. Almost invariably, one or more polyps undergo malignant transformation, as discussed later. APC mutations are seen in 70% to 80% of sporadic colon cancers. Colonic cancers with normal APC genes sometimes show activating mutations of β-catenin that render them refractory to the degrading action of APC. http://ebooksmedicine.net


Pathology_Robbins

Pathology_Robbins. http://ebooksmedicine.net Fig. 6.22 TheroleofAPCinregulatingthestabilityandfunctionofβ-catenin.APCandβ-cateninarecomponentsoftheWNTsignalingpathway.(A)Inrestingcells(notexposedtoWNT),β-cateninformsamacromolecularcomplexcontainingtheAPCprotein.Thiscomplexleadstothedestructionofβ-catenin,andintracellularlevelsofβ-cateninarelow.(B)WhencellsarestimulatedbysecretedWNTmolecules,thedestructioncomplexisdeactivated,β-catenindegradationdoesnotoccur,andcytoplasmiclevelsincrease.β-Catenintranslocatestothenucleus,whereitbindstoTCF,atranscriptionfactorthatactivatesseveralgenesinvolvedinthecellcycle.(C)WhenAPCismutatedorabsent,thedestructionofβ-catenincannotoccur.β-Catenintranslocatestothenucleusandcoactivatesgenesthatpromotethecellcycle,andcellsbehaveasiftheyareunderconstantstimulationbytheWNTpathway. TGF-β,CONTACTINHIBITION,ANDAPC-βCATENINPATHWAYSRESTING CELL WNT STIMULATION TUMOR CELL WITH RESTING CELL WNT STIMULATION TUMOR CELL WITH


Pathology_Robbins

Pathology_Robbins. TGF-β,CONTACTINHIBITION,ANDAPC-βCATENINPATHWAYSRESTING CELL WNT STIMULATION TUMOR CELL WITH RESTING CELL WNT STIMULATION TUMOR CELL WITH TGF-β inhibitsproliferationofmanycelltypesbyactivationofgrowth-inhibitinggenessuchasCDKIs andsuppressionofgrowth-promotinggenessuchasMYC andthoseencodingcyclins. TGF-β functioniscompromisedinmanytumorsbymutationsinitsreceptors(colon,stomach,endometrium)orbymutationalinactivationofSMAD genesthattransduceTGF-β signaling(pancreas). E-cadherinmaintainscontactinhibition,whichislostinmalignantcells. TheAPC geneexertsanti-proliferativeactionsbyregulatingthedestructionofthecytoplasmicproteinβ-catenin.WithalossofAPC,β-cateninisnotdestroyed,andittranslocatestothenucleus,whereitactsasagrowth-promotingtranscriptionfactor.


Pathology_Robbins

Pathology_Robbins. Even in the presence of ample oxygen, cancer cells demonstrate a distinctive form of cellular metabolism characterized by high levels of glucose uptake and increased conversion of glucose to lactose (fermentation) via the glycolytic pathway. This phenomenon, called the Warburg effect and also known as aerobic glycolysis, has been recognized for many years (indeed, Otto Warburg received the Nobel Prize in 1931 for its discovery). Clinically, the “glucose-hunger” of tumors is used to visualize tumors via positron emission tomography (PET) scanning, in which patients are injected with 18F-fluorodeoxyglucose, a glucose derivative that is preferentially taken up into tumor cells (as well as normal, actively dividing tissues such as the bone marrow). Most tumors are PET-positive, and rapidly growing ones are markedly so.


Pathology_Robbins

Pathology_Robbins. Warburg’s discovery was largely neglected for many years, but over the past decade, metabolism has become one of the most active areas of cancer research. Metabolic pathways (like signaling pathways) in normal and cancer cells are still being elucidated, and the details are complex, but at the heart of the Warburg effect lies a simple question: why is it advantageous for a cancer cell to rely on seemingly inefficient glycolysis (which generates two molecules of ATP per molecule of glucose) instead of oxidative phosphorylation (which generates up to 36 molecules of ATP per molecule of glucose)? While pondering this question, it is important to recognize that rapidly proliferating normal cells, such as in embryonic tissues and lymphocytes during immune responses, also rely on aerobic fermentation. Thus, “Warburg metabolism” is not cancer specific, but instead is a general property of growing cells that becomes “fixed” in cancer cells. http://ebooksmedicine.net


Pathology_Robbins

Pathology_Robbins. http://ebooksmedicine.net The answer to this riddle is simple: Aerobic glycolysis provides rapidly dividing tumor cells with metabolic intermediates that are needed for the synthesis of cellular components, whereas mitochondrial oxidative phosphorylation does not. The reason growing cells rely on aerobic glycolysis becomes readily apparent when one considers that a growing cell has a strict biosynthetic requirement; it must duplicate all of its cellular components—DNA, RNA, proteins, lipid, and organelles—before it can divide and produce two daughter cells. While oxidative phosphorylation yields abundant ATP, it fails to produce any carbon moieties that can be used to build the cellular components needed for growth (proteins, lipids, and nucleic acids). Even cells that are not actively growing must shunt some metabolic intermediates away from oxidative phosphorylation in order to synthesize macromolecules that are needed for cellular maintenance.


Pathology_Robbins

Pathology_Robbins. By contrast, in actively growing cells only a small fraction of the cellular glucose is shunted through the oxidative phosphorylation pathway, such that on average each molecule of glucose metabolized produces approximately four molecules of ATP. Presumably, this balance (heavily biased toward aerobic fermentation, with a bit of oxidative phosphorylation) hits a metabolic “sweet spot” that is optimal for growth. It follows that growing cells do rely on mitochondrial metabolism. However, a major function of mitochondria in growing cells is not to generate ATP, but rather to carry out reactions that generate metabolic intermediates that can be shunted off and used as precursors in the synthesis of cellular building blocks. For example, lipid biosynthesis requires acetyl-CoA, and acetyl-CoA is largely synthesized in growing cells from intermediates such as citrate that are generated in mitochondria.


Pathology_Robbins

Pathology_Robbins. So how is this profound reprogramming of metabolism, the Warburg effect, triggered in growing normal and malignant cells? As might be guessed, metabolic reprogramming is produced by signaling cascades downstream of growth factor receptors, the very same pathways that are deregulated by mutations in oncogenes and tumors suppressor genes in cancers. Thus, whereas in rapidly dividing normal cells aerobic glycolysis ceases when the tissue is no longer growing, in cancer cells this reprogramming persists due to the action of oncogenes and the loss of tumor suppressor gene function. Some of the important points of cross-talk between pro–growth signaling factors and cellular metabolism are shown in Fig. 6.23 and include the following: • Growth factor receptor signaling. In addition to transmitting growth signals to the nucleus, signals from growth factor receptors also influence metabolism by


Pathology_Robbins

Pathology_Robbins. Fig. 6.23 Metabolismandcellgrowth.QuiescentcellsrelymainlyontheKrebscycleforATPproduction;ifstarved,autophagy(self-eating)isinducedtoprovideasourceoffuel.Whenstimulatedbygrowthfactors,normalcellsmarkedlyupregulateglucoseandglutamineuptake,whichprovidecarbonsourcesforsynthesisofnucleotides,proteins,andlipids.Incancers,oncogenicmutationsinvolvinggrowthfactorsignalingpathwaysandotherkeyfactorssuchasMYCderegulatethesemetabolicpathways,analterationknownastheWarburg effect. Krebs cycle ATP CO2 GROWTHtransporter Lipids RTK LysosomeLipid and amino acid catabolism Autophagy transporter Increased glycolytic intermediates Increased glutamine utilization Lactate RTK Growth factor RTK Glutamine RTK Mitochondrial metabolites PI3K Protein synthesis Lipogenesis Nucleotide, amino acid synthesis Akt MYC http://ebooksmedicine.net upregulating glucose uptake and inhibiting the activity of pyruvate kinase, which catalyzes the last step in the glycolytic pathway, the conversion of phosphoenolpyruvate to


Pathology_Robbins

Pathology_Robbins. http://ebooksmedicine.net upregulating glucose uptake and inhibiting the activity of pyruvate kinase, which catalyzes the last step in the glycolytic pathway, the conversion of phosphoenolpyruvate to pyruvate. This creates a damming effect that leads to the buildup of upstream glycolytic intermediates, which are siphoned off for synthesis of DNA, RNA, and protein.


Pathology_Robbins

Pathology_Robbins. RAS signaling. Signals downstream of RAS upregulate the activity of glucose transporters and multiple glycolytic enzymes, thus increasing glycolysis; promote shunting of mitochondrial intermediates to pathways leading to lipid biosynthesis; and stimulate factors that are required for protein synthesis. MYC. As mentioned earlier, pro-growth pathways upregulate expression of the transcription factor MYC, which drives changes in gene expression that support anabolic metabolism and cell growth. Among the MYC-regulated genes are those for several glycolytic enzymes and glutaminase, which is required for mitochondrial utilization of glutamine, a key source of carbon moieties needed for biosynthesis of cellular building blocks.


Pathology_Robbins

Pathology_Robbins. The flipside of the coin is that tumor suppressors often inhibit metabolic pathways that support growth. We have already discussed the “braking” effect of the tumor suppressors NF1 and PTEN on signals downstream of growth factor receptors and RAS, allowing them to oppose the Warburg effect. Indeed, it may be that many (and perhaps all) tumor suppressors that induce growth arrest suppress the Warburg effect. For example, p53, arguably the most important tumor suppressor, upregulates target genes that collectively inhibit glucose uptake, glycolysis, lipogenesis, and the generation of NADPH (a key cofactor needed for the biosynthesis of macromolecules). Thus, it is increasingly clear that the functions of many oncoproteins and tumor suppressors are inextricably intertwined with cellular metabolism.


Pathology_Robbins

Pathology_Robbins. Beyond the Warburg effect, there are two other links between metabolism and cancer that are of sufficient importance to merit brief mention, autophagy and an unusual set of oncogenic mutations that lead to the creation of oncometabolites, small molecules that appear to directly contribute to the transformed state.


Pathology_Robbins

Pathology_Robbins. Autophagy is a state of severe nutrient deficiency in which cells not only arrest their growth, but also cannibalize their own organelles, proteins, and membranes as carbon sources for energy production (Chapter 2). If this adaptation fails, the cells die. Tumor cells often seem to be able to grow under marginal environmental conditions without triggering autophagy, suggesting that the pathways that induce autophagy are deranged. In keeping with this, several genes that promote autophagy are tumor suppressors. Whether autophagy is always bad from the vantage point of the tumor, however, remains a matter of active investigation and debate. For example, under conditions of severe nutrient deprivation, tumor cells may use autophagy to become “dormant,” a state of metabolic hibernation that allows cells to survive hard times for long periods. Such cells are believed to be resistant to therapies that kill actively dividing cells, and could therefore be responsible for therapeutic failures.


Pathology_Robbins

Pathology_Robbins. cells to survive hard times for long periods. Such cells are believed to be resistant to therapies that kill actively dividing cells, and could therefore be responsible for therapeutic failures. Thus, autophagy may be a tumor’s friend or foe depending on how the signaling pathways that regulate it are “wired” in a given tumor.


Pathology_Robbins

Pathology_Robbins. Another surprising group of genetic alterations discovered through tumor genome sequencing studies are mutations in enzymes that participate in the Krebs cycle. Of these, mutations in isocitrate dehydrogenase (IDH) have garnered the most interest, as they have revealed a new mechanism of oncogenesis termed oncometabolism ( Fig. 6.24). The proposed steps in the oncogenic pathway involving IDH are as follows: IDH acquires a mutation that leads to a specific amino acid substitution involving residues in the active site of the enzyme. As a result, the mutated protein loses it ability to function as an isocitrate dehydrogenase and instead acquires a new enzymatic activity that catalyzes the production of 2-hydroxglutarate (2-HG). 2-HG in turn acts as an inhibitor of several other enzymes that are members of the TET family, including TET2.


Pathology_Robbins

Pathology_Robbins. 2-HG in turn acts as an inhibitor of several other enzymes that are members of the TET family, including TET2. TET2 is one of several factors that regulate DNA methylation, which you will recall is an epigenetic modification that controls normal gene expression and often goes awry in cancer. According to the model, loss of TET2 activity leads to abnormal patterns of DNA methylation. Abnormal DNA methylation in turn leads to misexpression of currently unknown cancer genes, which drive cellular transformation and oncogenesis. Thus, according to this scenario, mutated IDH acts as an oncoprotein by producing 2-HG, which is considered a prototypical oncometabolite. Oncogenic IDH mutations Epigenetic changes (DNA methylation, histone modification) Fig. 6.24 Proposedactionoftheoncometabolite2-hydroxyglutarate(2-HG)incancercellswithmutatedisocitratedehydrogenase(mIDH).


Pathology_Robbins

Pathology_Robbins. Epigenetic changes (DNA methylation, histone modification) Fig. 6.24 Proposedactionoftheoncometabolite2-hydroxyglutarate(2-HG)incancercellswithmutatedisocitratedehydrogenase(mIDH). http://ebooksmedicine.net have now been described in a diverse collection of cancers, including a sizable fraction of cholangiocarcinomas, gliomas, acute myeloid leukemias, and sarcomas. Of clinical significance, because the mutated IDH proteins have an altered structure, it has been possible to develop drugs that inhibit mutated IDH and not the normal IDH enzyme. These drugs are now being tested in cancer patients and have produced encouraging therapeutic responses. This developing story is a remarkable example of how detailed understanding of oncogenic mechanisms can yield entirely new kinds of anti-cancer drugs.


Pathology_Robbins

Pathology_Robbins. Warburg metabolism is a form of pro-growth metabolismfavoring glycolysis over oxidative phosphorylation. It is inducedin normal cells by exposure to growth factors and becomesfixed in cancer cells due to the action of certain drivermutations. Many oncoproteins (RAS, MYC, mutated growth factor receptors) induce or contribute to Warburg metabolism, and manytumor suppressors (PTEN, NF1, p53) oppose it. Stress may induce cells to consume their components in aprocess called autophagy. Cancer cells may accumulate mutations to avoid autophagy, or may corrupt the process toprovide nutrients for continued growth and survival. Some oncoproteins such as mutated IDH act by causing theformation of high levels of “oncometabolites” that alter theepigenome, thereby leading to changes in gene expression thatare oncogenic.


Pathology_Robbins

Pathology_Robbins. Tumor cells frequently contain mutations in genes that regulate apoptosis, making the cells resistant to cell death. As discussed in Chapter 2, apoptosis, or regulated cell death, refers to an orderly dismantling of cells into component pieces, which are then efficiently consumed by neighboring cells and professional phagocytes without stimulating inflammation. You will recall that there are two pathways that lead to apoptosis: the extrinsic pathway, triggered by the death receptors FAS and FAS-ligand; and the intrinsic pathway (also known as the mitochondrial pathway), initiated by perturbations such as loss of growth factors and DNA damage. Cancer cells are subject to a number of intrinsic stresses that can initiate apoptosis, particularly DNA damage, but also metabolic disturbances stemming from dysregulated growth as well as hypoxia caused by insufficient blood supply. These stresses are enhanced manyfold when tumors are treated with chemotherapy or radiation therapy, which kill


Pathology_Robbins

Pathology_Robbins. from dysregulated growth as well as hypoxia caused by insufficient blood supply. These stresses are enhanced manyfold when tumors are treated with chemotherapy or radiation therapy, which kill tumor cells by activating the intrinsic pathway of apoptosis. Thus, there is strong selective pressure, both before and during therapy, for cancer cells to develop resistance to intrinsic stresses that may induce apoptosis. Accordingly, evasion of apoptosis by cancer cells occurs mainly by way of acquired mutations and changes in gene expression that disable key components of the intrinsic pathway, or that reset the balance of regulatory factors so as to favor cell survival in the face of intrinsic stresses (


Pathology_Robbins

Figure 6.25

Pathology_Robbins. Figure 6.25


Pathology_Robbins

Pathology_Robbins. Before delving into modes of resistance to apoptosis, a brief review of the intrinsic pathway is in order. Activation of this pathway leads to permeabilization of the mitochondrial outer membrane and release of molecules, such as cytochrome c, that initiate apoptosis. The integrity of the mitochondrial outer membrane is determined by a delicate balancing act between pro-apoptotic and anti-apoptotic members of the BCL2 protein family. The pro-apoptotic proteins BAX and BAK are required for apoptosis and directly promote mitochondrial permeabilization. Their action is inhibited by the anti-apoptotic members of this family, which are exemplified by BCL2, BCL-XL, and MCL1. A third set of proteins, the so-called “BH3-only proteins,” which include BAD, BID, and PUMA, shift the balance between the pro-apoptotic and anti-apoptotic family members by neutralizing the actions of anti-apoptotic proteins like BCL2 and BCL-XL, thereby promoting apoptosis. When the sum total of all the BH3-only


Pathology_Robbins

Pathology_Robbins. the pro-apoptotic and anti-apoptotic family members by neutralizing the actions of anti-apoptotic proteins like BCL2 and BCL-XL, thereby promoting apoptosis. When the sum total of all the BH3-only proteins “overwhelms” the protective BCL2/BCL-XL factors, BAX and BAK are activated and form pores in the mitochondrial membrane. These allow mitochondrial cytochrome c to


Pathology_Robbins

Pathology_Robbins. Deficiency of ROS growth factors Radiation and survival Chemicals signals Fig. 6.25 Intrinsic pathway of apoptosis and major mechanisms used by tumor cells to evade cell death. (1) Loss of p53, either through mutation or through antagonism by MDM2. (2) Reduced egress of cytochrome c from mitochondria as a result of upregulation of anti-apoptotic factors such as BCL2, BCL-XL, and MCL-1. IAP, Inhibitor of apoptosis. leak out into the cytosol, where it binds to APAF-1 and activates caspase-9, which in turn cleaves and activates executioner caspases such as caspase-3. Another group of factors that function as negative regulators of the intrinsic pathway is known as inhibitor of apoptosis proteins (IAPs), which bind caspase-9 and prevent apoptosis. Within this framework, it is possible to illustrate the major mechanisms by which apoptosis is evaded by cancer cells (


Pathology_Robbins

Pathology_Robbins. Within this framework, it is possible to illustrate the major mechanisms by which apoptosis is evaded by cancer cells ( Figure 6.25 ). These mainly involve loss of p53, the key component of early steps in the intrinsic pathway, and increased expression of anti-apoptotic members of the BCL2 family. Loss of TP53 function. As already discussed, TP53 is commonly mutated in cancers at diagnosis, and the frequency of TP53 mutations is even higher in tumors that relapse after therapy. In addition to mutation of TP53, other lesions in cancers impair p53 function indirectly, most notably amplification of MDM2, which encodes an inhibitor of p53. Loss of p53 function prevents the upregulation of PUMA, a pro-apoptotic BH3-only member of the BCL2 family that is a direct target of p53. As a result, cells survive levels of DNA damage and cell stress that otherwise would result in their death.


Pathology_Robbins

Pathology_Robbins. Overexpression of anti-apoptotic members of the BCL2 family. Overexpression of BCL2 is a common event leading to the protection of tumor cells from apoptosis and occurs through several mechanisms. One of the best understood examples is found follicular lymphoma (Chapter 12), a B-cell tumor carrying a characteristic (14;18) (q32;q21) translocation that fuses the BCL2 (located at 18q21) to the transcriptionally active immunoglobulin heavy chain gene (located at 14q32). The resulting overabundance of BCL2 protects lymphocytes from apoptosis and allows them to survive for abnormally long periods, producing a steady accumulation of B lymphocytes that results in lymphadenopathy. Because BCL2overexpressing follicular lymphomas arise in large part through reduced cell death rather than explosive cell proliferation, they tend to be indolent (slow-growing). In other tumors such as chronic lymphocytic leukemia (Chapter 12), it appears that BCL2 is upregulated because of loss of expression of


Pathology_Robbins

Pathology_Robbins. cell proliferation, they tend to be indolent (slow-growing). In other tumors such as chronic lymphocytic leukemia (Chapter 12), it appears that BCL2 is upregulated because of loss of expression of specific micro-RNAs that normally dampen BCL2 expression. Many other mechanisms leading to overexpression of anti-apoptotic members of the BCL2 family have been described, particularly in the setting of resistance to chemotherapy. For example, amplification of the MCL1 gene is seen in a subset of lung and breast cancers.


Pathology_Robbins

Pathology_Robbins. Recognition of the mechanisms by which cancers evade cell death has stimulated several lines of targeted drug development. Restoration of p53 function in TP53-mutated tumors is a daunting problem (because of the inherent difficulty of “fixing” defective genes), but is possible in tumors in which p53 is inactive because of overexpression of its inhibitor, MDM2. Indeed, inhibitors of MDM2 that reactivate p53 and induce apoptosis in tumors with MDM2 gene amplification, such as certain types of sarcoma, are being tested in clinical trials. Even more promising results are being generated with drugs that mimic the activities of BH3-only proteins and inhibit the function of antiapoptotic members of the BCL2 family, particularly BCL2 itself. These drugs have potent activity against tumors characterized by BCL2 overexpression (such as chronic lymphocytic leukemia) and are likely to become a standard part of cancer treatment over the course of the next few years.


Pathology_Robbins


Pathology_Robbins

Pathology_Robbins. InhibitorsofMDM2(whichactivatep53)andinhibitorsofBCL2familymembersinducethedeathofcancercellsbystimulatingtheintrinsicpathwayofapoptosisandarebeingdevelopedastherapeuticagents. Tumor cells, unlike normal cells, are capable of limitless replication. As discussed previously in the context of cellular aging (Chapter 2), most normal human cells have a capacity of at most 70 doublings. Thereafter, the cells lose the ability to divide and enter replicative senescence. This phenomenon has been ascribed to progressive shortening of telomeres at the ends of chromosomes. Markedly eroded telomeres are recognized by the DNA repair machinery as double-stranded DNA breaks, leading to cell cycle arrest and senescence, mediated by TP53 and RB. In cells in which TP53 or RB mutations are disabled by mutations, the nonhomologous end-joining pathway is activated in a last-ditch effort to save the cell, joining the shortened ends of two chromosomes.


Pathology_Robbins

Pathology_Robbins. Such an inappropriately activated repair system results in dicentric chromosomes that are pulled apart at anaphase, resulting in new double-stranded DNA breaks. The resulting genomic instability from the repeated bridge– fusion–breakage cycles eventually produces mitotic catastrophe, characterized by massive apoptosis. It follows that for tumors to acquire the ability to grow indefinitely, loss of growth restraints is not enough; both cellular senescence and mitotic catastrophe must also be avoided (


Pathology_Robbins

Pathology_Robbins. It follows that for tumors to acquire the ability to grow indefinitely, loss of growth restraints is not enough; both cellular senescence and mitotic catastrophe must also be avoided ( Fig. 6.26 ). If during crisis a cell manages to reactivate telomerase, the bridge–fusion–breakage cycles cease, and the cell is able to avoid death. However, during this period of genomic instability that precedes telomerase activation, numerous mutations could accumulate, helping the cell march toward malignancy. Telomerase, active in normal stem cells, is absent from or present at very low levels in most somatic cells. By contrast, telomere maintenance is seen in virtually all types of cancers. In 85% to 95% of cancers, this is due to upregulation of the enzyme telomerase. A few tumors use other mechanisms, termed alternative lengthening of telomeres, which depend on DNA recombination. http://ebooksmedicine.net


Pathology_Robbins

Pathology_Robbins. http://ebooksmedicine.net Fig. 6.26 Escapeofcellsfromreplicativesenescenceandmitoticcatastrophecausedbytelomereshortening. Of interest, in a study of the progression from colonic adenoma to colonic adenocarcinoma, early lesions had a high degree of genomic instability with low telomerase expression, whereas malignant lesions had complex karyotypes with high levels of telomerase activity, consistent with a model of telomere-driven tumorigenesis in human cancer. Thus, it appears that unregulated proliferation in incipient tumors leads to telomere shortening, followed by chromosomal instability and the accumulation of mutations. If telomerase is then reactivated in these cells, telomeres are extended and these mutations become fixed, contributing to tumor growth. •Innormalcells,whichlackexpressionoftelomerase,theshortenedtelomeresgeneratedbycelldivisioneventuallyactivatecellcyclecheckpoints,leadingtosenescenceandplacingalimitonthenumberofdivisionsacellmayundergo.


Pathology_Robbins

Pathology_Robbins. Even if a solid tumor possesses all of the genetic aberrations that are required for malignant transformation, it cannot enlarge beyond 1 to 2 mm in diameter unless it has the capacity to induce angiogenesis. Like normal tissues, tumors require delivery of oxygen and nutrients and removal of waste products; presumably the 1to 2-mm zone represents the maximal distance across which oxygen, nutrients, and waste can diffuse to and from blood vessels. Growing cancers stimulate neoangiogenesis, during which vessels sprout from previously existing capillaries. Neovascularization has a dual effect on tumor growth: perfusion supplies needed nutrients and oxygen, and newly formed endothelial cells stimulate the growth of adjacent tumor cells by secreting growth factors, such as insulin-like growth factors (IGFs) and PDGF. While the resulting tumor vasculature is effective at delivering nutrients and removing wastes, it is not entirely normal; the vessels are leaky and dilated, and have a


Pathology_Robbins

Pathology_Robbins. factors (IGFs) and PDGF. While the resulting tumor vasculature is effective at delivering nutrients and removing wastes, it is not entirely normal; the vessels are leaky and dilated, and have a haphazard pattern of connection, features that can be appreciated on angiograms. By permitting tumor cells access to these abnormal vessels, angiogenesis also contributes to metastasis. Angiogenesis is thus an essential facet of malignancy.


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Pathology_Robbins. http://ebooksmedicine.net


Pathology_Robbins

Pathology_Robbins. How do growing tumors develop a blood supply? The current paradigm is that angiogenesis is controlled by a balance between angiogenesis promoters and inhibitors; in angiogenic tumors this balance is skewed in favor of promoters. Early in their development, most human tumors do not induce angiogenesis. Starved of nutrients, these tumors remain small or in situ, possibly for years, until an angiogenic switch terminates this stage of vascular quiescence. The molecular basis of the angiogenic switch involves increased production of angiogenic factors and/ or loss of angiogenic inhibitors. These factors may be produced by the tumor cells themselves or by inflammatory cells (e.g., macrophages) or resident stromal cells (e.g., tumor-associated fibroblasts). Proteases, either elaborated by the tumor cells or by stromal cells in response to the tumor, are also involved in regulating the balance between angiogenic and anti-angiogenic factors. Many proteases can release proangiogenic basic


Pathology_Robbins

Pathology_Robbins. tumor cells or by stromal cells in response to the tumor, are also involved in regulating the balance between angiogenic and anti-angiogenic factors. Many proteases can release proangiogenic basic fibroblast growth factors (bFGF) that are stored in the ECM; conversely, the angiogenesis inhibitors angiostatin and endostatin are produced by proteolytic cleavage of plasminogen and collagen, respectively.


Pathology_Robbins

Pathology_Robbins. The local balance of angiogenic and anti-angiogenic factors is influenced by several factors: Relative lack of oxygen due to hypoxia stabilizes HIF1α, an oxygen-sensitive transcription factor mentioned earlier, which then activates the transcription of proangiogenic cytokines such as VEGF. These factors create an angiogenic gradient that stimulates the proliferation of endothelial cells and guides the growth of new vessels toward the tumor. Mutations involving tumor suppressors and oncogenes in cancers also tilt the balance in favor of angiogenesis. For example, p53 stimulates expression of antiangiogenic molecules, such as thrombospondin-1, and represses expression of proangiogenic molecules, such as VEGF. Thus, loss of p53 in tumor cells provides a more permissive environment for angiogenesis.


Pathology_Robbins

Pathology_Robbins. The transcription of VEGF also is influenced by signals from the RAS-MAP kinase pathway, and gain-offunction mutations in RAS or MYC upregulate the production of VEGF. Notably, elevated levels of VEGF can be detected in the serum and urine of a significant fraction of cancer patients.


Pathology_Robbins

Pathology_Robbins. The idea that angiogenesis is essential if solid tumors are to grow to clinically significant sizes has provided a powerful impetus for the development of therapeutic agents that block angiogenesis. These agents are now a part of the armamentarium that oncologists use against cancers; a cardinal example is bevacizumab, a monoclonal antibody that neutralizes VEGF activity and is approved for use in the treatment of multiple cancers. However, angiogenesis inhibitors have not been nearly as effective as was originally hoped; they can prolong life, but usually for only a few months and at high financial cost. The mechanisms that underlie the persistence and ultimate progression of cancers in the face of therapy with angiogenesis inhibitors are not yet clear. The modest benefit of anti-angiogenic therapy highlights the pernicious nature of advanced cancers, which can even elude therapies directed at stromal support cells such as endothelium.