Apoptosis and Cancer Induction

With the evolving role of apoptosis in tissue homeostasis and removal of genetically damaged cells, it has been postulated that the loss of its regulation can be an important factor in the induction and progression of cancers. Mutations or dysregulation of the apoptotic machinery has been implicated in GI cancers and includes the tumor suppressor gene p53, the anti-apoptotic protein Bcl-2, and the pro-apoptotic protein Bax. The loss of the apoptotic pathway results in increased survival of cells with genetic mutations resulting in the increased probability of further mutations or oncogenic progression with subsequent tumor formation (Fig. 4.5). In this section we will discuss the current theories and evidence involved in the implication of these factors in cancers of the GI tract.

The tumor suppressor gene p53 is involved in surveying the genome for DNA damage, and functions as a regulator of the cell cycle and an inducer of apoptosis.78 Once DNA damage is detected, p53 expression is upregulated at the translational level and is translocated to the nucleus where it binds to cis-activating DNA sequences that regulate transcription of various DNA damage response genes.78,91 These DNA damage response genes encode the following proteins: (1) p21Waf1, a CKI which regulates G1 cell cycle arrest; (2) Bax, a pro-apoptotic regulator protein; (3) GADD45, a DNA damage-induced protein of unknown function; and (4) MDM2, a cellular protein that binds to and regulates p53. The p53 induction of p21 expression results in G1 cell cycle arrest (Fig. 4.3) which allows the cell to either repair the damaged DNA, or commit to cell death by apoptosis. The significance of this event is that it protects the cell with DNA damage from entry into S-phase, which can result in the daughter cells having unstable chromosomal structure allowing possible recombination events, or stable mutations with resulting malignant transformations. The complex regulatory role of p53 in the differential response to cell cycle arrest and DNA repair versus the induction of apoptosis has been thought to be cell type specific. However, others have shown that p53 response levels to DNA damage may be implicated in this differential function.79,92 Low to moderate p53 expression results in cell cycle arrest with subsequent DNA repair, while a high level of p53 response results in cell progression to apoptosis. Collectively, the role of p53 is an essential process functioning to prevent mutant cell propagation through regulation of DNA repair and repair enzymes, cellular growth arrest, and inducing apoptosis following genotoxic stress.

Our current knowledge of p53 function suggests that the loss of p53 activity or expression through mutation would result in decreased apoptosis with a subsequent increased population of cells bearing DNA damage (Fig. 4.5). Although it has been shown that p53 mutations have no bearing on subsequent mutation, it does promote the propagation of mutant cells by increasing cell survival.93 The p53 gene is the most commonly mutated gene in human malignancy. Despite traditional thinking that p53 mutations occur late in tumorigenesis, there is increasing evidence that mutations of p53 in the neoplastic process

Fig. 4.5. The role of the apoptotic pathway in the induction and progression of cancer. The normal function of wild type p53 tumor suppressor gene after cell exposure to apoptotic stimuli or DNA damage is the induction of apoptotic cell death. One of the mechanisms involved in p53-mediated apoptosis is increased expression of the pro-apoptotic protein, Bax. Mutations in p53, or increased expression of negative regulators of apoptosis (i.e., Bcl-2, Bcl-xL or Bfl-1) leads to prolonged cell survival. This prolonged longevity increases cell exposure to mutagens with increased risk for further oncogenic lesions. The culmination of these events leads to cancer cell transformation and malignant progression. Reproduced with permission Pan H et al. Apoptosis and cancer mechanisms. In: Kastan MB (ed). Cancer Surveys. Checkpoint Controls and Cancer 1997; 29:314.©Cold Spring Harbor Laboratory Press; Plainview, NY.

Fig. 4.5. The role of the apoptotic pathway in the induction and progression of cancer. The normal function of wild type p53 tumor suppressor gene after cell exposure to apoptotic stimuli or DNA damage is the induction of apoptotic cell death. One of the mechanisms involved in p53-mediated apoptosis is increased expression of the pro-apoptotic protein, Bax. Mutations in p53, or increased expression of negative regulators of apoptosis (i.e., Bcl-2, Bcl-xL or Bfl-1) leads to prolonged cell survival. This prolonged longevity increases cell exposure to mutagens with increased risk for further oncogenic lesions. The culmination of these events leads to cancer cell transformation and malignant progression. Reproduced with permission Pan H et al. Apoptosis and cancer mechanisms. In: Kastan MB (ed). Cancer Surveys. Checkpoint Controls and Cancer 1997; 29:314.©Cold Spring Harbor Laboratory Press; Plainview, NY.

of colon cancer may be an early event.94,95 On the basis of crystallographic data and biochemical studies, the p53 amino acid residues most frequently mutated in human cancers alter or destroy p53 sequence-specific DNA binding function.96,97 Therefore, the transcriptional regulation of DNA repair genes, cell cycle regulators, and pro-apoptotic regulators may be the mechanism by which p53 mutations influence tumorigenesis.

One of the theories that explain the prevalence of p53 mutation in cancers lies in its role in the induction of apoptosis following hypoxia. In tissue regions exhibiting low oxygen tension, such as the center of poorly vascularized solid tumors, cells bearing wild type p53 are more readily removed from the malignant tissue so that the majority of cells would be those with the mutated gene.98 This process may be one of the selective advantages of p53 mutations in the progression of malignant disease.

Other regulators of the apoptotic cell death pathway implicated in tumor induction are the Bcl-2 family of genes. Bcl-2 is a negative regulator of apoptosis. The bcl-2 gene was first isolated from human follicular B cell lymphoma, where chromosomal t(14:18) translocation moved the gene into juxtaposition with the immunoglobulin heavy chain transcription enhancer resulting in increased Bcl-2 mRNA synthesis and protein expression. The cancer resulting from this mutation was a consequence of extended B cell lifespan due to the Bcl-2-mediated suppression of apoptosis which resulted in predisposition to B cell lymphoma.87,88 Increased expression of other anti-apoptotic regulators such as Bcl-xL and Bfl-1, and decreased expression or mutation of pro-apoptotic genes (e.g., Bax and Bak), would lead to decreased apoptosis with increased cell survival. Therefore, alterations in apoptotic regulators can increase cell survival and cell number resulting in cancer formation.

Cancers of the Small Bowel and Colon

The incidence of cancer in the colon is about 100 times more prevalent than cancer of the small bowel. Traditionally it has been thought that this was secondary to the rapid transit time of the small bowel, decreasing exposure to carcinogens and intraluminal toxins. As described above, the tissue homeostasis of both the small bowel and colonic epithelium are very dynamic where the proliferating stem cell compartment located in the crypts rapidly replaces cells loosed from the mucosal surface or villus. Recent evidence has shown that apoptosis plays a significant role in regulating the crypt stem cell units. Furthermore, the differences in expression of apoptotic mediators and location of apoptosis in the proliferative unit of the small bowel and colon may provide new insight into the differences between cancer induction in these organs.

In the normal small intestine, there is a persistent low frequency (<1% of crypt cell number) of apoptosis found in the stem cell zone, and has been termed spontaneous apoptosis.69,99 It is believed that apoptosis in this region functions to regulate stem cell number. Tight control of the stem cell population within the crypts helps maintain villus size through steady migration of cells from the crypt to villus axis. In the colon, spontaneous apoptosis in the proliferating zone is a rare event.69 The location of these apoptotic events are scattered throughout the crypt and are rarely located in the stem cell unit. Therefore, spontaneous apoptosis in the colon is unlikely to regulate stem cell number. Potten et al69 proposed that the positional differences in apoptotic incidence between the small bowel and colon has direct implications on cancer incidence. They argue that the small bowel, capable of removing excess cells from the proliferating crypt unit, can regulate the output of progenitor cells to maintain constant cell number in the villus. Without apoptosis as a regulating mechanism, the colon is unable to remove excess stem cells, which can result in an increased cell number in the proliferating unit resulting in a hyperplastic crypt. These hyperplastic crypts result in an increased cell number exposed to, and therefore susceptible to, carcinogens and mutagens.

In addition to the positional differences of apoptosis between the small and large bowel, Merrit et al100,101 found important regional differences in the expression of p53 and Bcl-2 family proteins which directly relate to tumor incidence in these regions of the GI tract. In the small bowel, the anti-apoptotic protein Bcl-2 was not expressed in the small intestinal epithelial cells, whereas the pro-apoptotic protein Bax was present.85,101 This natural organ-specific distribution of apoptotic protein regulators would localize apoptosis to specific small bowel enterocytes, which would explain the relatively low incidence of small bowel adenocarcinomas despite their rapid proliferative rate. In contrast, Bcl-2 is expressed in the base of the colonic crypts,101 suggesting that Bcl-2 expression predispose the colonic epithelium to the formation of adenomas by prolonging the survival of stem cells and increasing the time of exposure to intraluminal carcinogens.

It has become increasingly evident that genes that regulate apoptosis are mutant in colon cancers, and that the transformation of the colorectal epithelium to carcinoma is associated with a progressive inhibition of apoptosis.102 Fearon and Vogelstein103 reported that colorectal tumorigenesis was a result of mutational activation of oncogenes coupled to the mutational inactivation of tumor suppressor genes. Furthermore, formation of malignant tumors required the accumulation of multiple mutational events.104 In adenocarcinomas of the colon, p53 mutations and decreased Bcl-2 expression are frequent, and result in the loss of cell ability to regulate apoptosis (Fig. 4.5).105,106 The importance of p53-mediated apoptotic cell death in the induction and progression of colon cancer has been shown by the introduction of wild type p53 to colon cancer cell lines with known p53 mutations. These wild type p53 transfection experiments resulted in inhibition of cell growth and the induction of apoptosis.107,108 These data suggest that functional loss of p53 activity increases cancer cell survival and propagation of mutational events through the loss of the apoptotic pathway. The tumor suppressor gene p53 is located on chromosome 17p, which regulates cell cycle progression and the induction of apoptosis as a result of DNA damage. In colon cancer, loss of heterozygosity on chromosome 17p is associated with a worse prognosis and advanced metastatic disease.109 The bcl-2 gene is located on chromosome 18q, and is a negative regulator of apoptosis. It has been shown that the progression from normal mucosa to carcinoma is associated with a dramatic reduction in apoptosis102 through differential expression of the pro-apoptotic gene bcl-2 and mutation of p53.105 Immunohistochemical analysis of tissues for Bcl-2 protein revealed an increased expression in adenomas, and a most striking expression in colorectal carcinomas.105,110 Furthermore, there is an inverse relationship of Bcl-2 to p53 expression in adenomas, with coexpression in adenocarcinomas.105 The recent discovery of Bfl-1, an anti-apoptotic protein, has led to the observation that there is an increased expression through the development of colonic adenomas and carcinomas.111 These data provide evidence that increased cell survival through increased expression of anti-apoptotic regulators (Bcl-2 and Bfl-1) is an early event, and p53 mutation is a late event in the induction and progression of adenocarcinoma of the colon. Others have shown a loss in the expression of Bcl-2 once the tumor has progressed to a carcinoma.112 This is partly because 69% of colorectal cancers are associated with a loss of chromosome 18q, which is the locus of the bcl-2 gene.113 The loss of Bcl-2 expression may be from the increased expression of p53, since both wild type and mutant p53 downregulate bcl-2 by binding to a transcriptional silencer element within the bcl-2 promoter.114 Clinically, the relevance of Bcl-2 expression in certain subsets of colon cancer is a favorable clinical outcome.105 This seems counter-intuitive because of the association of Bcl-2 with cell survival, but whether or not this is a mutated protein is not known. Furthermore, it has been postulated that the loss of Bcl-2 expression may be just a marker of acquisition of a more potent survival factor.115

Finally, immunohistochemistry staining of FasR and Fas ligand (FasL) expression has been studied in colorectal adenomas and carcinomas.116,117 FasL is a triggering agent of FasR-mediated apoptosis within the immune system.68 Expression of FasR is also seen in the normal colonic epithelium,118 and is also responsible for the induction of apoptosis in these cells. Moller et al118 showed that FasR expression is decreased or lost in 40 to 50% of colorectal cancers examined, and 100% of colon cancers with established metastasis. Recent evidence suggests that the decreased expression of FasR in colon cancer is not a result of Fas gene disruption, but a loss of Fas gene transcription through epigenetic gene silencing.116 These data support the findings of decreased apoptosis in the induction and progression of colorectal tumors through the loss of an apoptosis-inducing receptor. Furthermore, others have shown that there is an increased FasL expression in colon cancer, which induces apoptosis in activated T cells in culture.117,119 Collectively, these data suggest a FasL counterattack model for immune escape and metastatic establishment of colorectal cancers (Fig. 4.6).

Cancers of the Esophagus and Stomach

Gastric carcinomas have also been associated with dysregulation of apoptosis. Increased Bcl-2 expression is a frequent occurrence in malignant and premalignant conditions of the stomach such as chronic atrophic gastritis, gastric epithelial dysplasia and gastric cancer.120 There is accumulating evidence to suggest different genetic pathways for well differentiated and poorly differentiated gastric cancers.57,58 Kasagi et al121 measured apoptosis in various gastric cancer tissues using the TUNEL assay. Their results showed a decrease in apoptotic index between well differentiated and poorly differentiated tumors (10.9% vs 4.0%). Furthermore, the inhibition of apoptosis through Bcl-2 expression appears to be specifically associated with the promotion of poorly differentiated gastric adenocarcinomas. Studies have shown that the loss of heterozygosity of the bcl-2 gene is a common event in well differentiated adenocarcinoma, whereas overexpression of bcl-2 gene is observed in poorly differentiated adenocarcinoma.122 Bcl-2 expression is also increased in gastric adenocarcinomas of the intestinal morphotype, but have no correlation to patient survival.120 Finally,

Fig. 4.6. Hypothetical model of FasL-expressing human colonic adenocarcinomas in tumor escape from immune cell surveillance and liver colonization. FasR induces FasL-mediated apoptosis. FasR is normally expressed in immunocytes, hepatocytes, and epithelial cells of the gut. Colon cancer formation is associated with decreased expression or functional loss of FasR with increase in FasL. Increased FasL expression results in immune cell apoptosis and loss of normal tumor cell surveillance and elimination. FasL expression is also thought to play a role in liver metastatic establishment through the induction of hepatocyte apoptosis. Reproduced with permission, Shiraki K et al. Proc Natl Acad Sci USA 1997; 94:6424. ©The National Academy of Sciences.

Fig. 4.6. Hypothetical model of FasL-expressing human colonic adenocarcinomas in tumor escape from immune cell surveillance and liver colonization. FasR induces FasL-mediated apoptosis. FasR is normally expressed in immunocytes, hepatocytes, and epithelial cells of the gut. Colon cancer formation is associated with decreased expression or functional loss of FasR with increase in FasL. Increased FasL expression results in immune cell apoptosis and loss of normal tumor cell surveillance and elimination. FasL expression is also thought to play a role in liver metastatic establishment through the induction of hepatocyte apoptosis. Reproduced with permission, Shiraki K et al. Proc Natl Acad Sci USA 1997; 94:6424. ©The National Academy of Sciences.

Bcl-2 expression has been shown to improve cancer cell survival and enhance peritoneal dissemination with a 4-fold tumor weight increase in an in vivo model.123 Therefore, increased Bcl-2 expression appears to be an early event in gastric carcinogenesis, and a factor for tumor aggressiveness and peritoneal dissemination.

Transforming growth factor-beta 1 (TGF-pi) can induce apoptosis through a receptor-mediated event. Ito et al124 and co-workers demonstrated reduced levels of the TGF-P receptor in poorly differentiated tumors. These data provide further mechanistic implications for the differences in the apoptotic index between well and poorly differentiated gastric adenocarcinomas.

The tumor suppressor gene p53 has also been implicated in the induction and progression of gastric cancers. Allele loss and mutations of the p53 gene are detected in >60% of gastric cancers regardless of histological type.58 However, p53 abnormalities are found in 30% of gastric adenomas, and are a good correlation to histological atypia.125 It is known that activation of wild type p53 induces bax expression to accelerate apoptosis.78,91 The Bcl-2 and Bax proteins can form heterodimers which regulate the ability of Bcl-2 protein to block apoptosis. Decreased pro-apoptotic regulatory proteins (Bax and Bak) have been found in 25 to 70% of gastric cancer cases when compared to normal tissues.126 However, the expression of Bax does not correlate to gastric tumor histology.127 These data suggest that p53 mutations alter pro-apoptotic protein expression, which may be involved in the increased survival and chemoresistance of gastric cancer cells in vitro and in vivo.

The induction of esophageal cancers has also been associated with the activation of proto-oncogenes and mutations in tumor suppressor genes. Among them, p53 was the first target tumor suppressor gene shown to undergo frequent point mutation in esophageal cancer tissues and cell lines.128 Similar findings were found in Barrett's esophagus.129 These data were supported by other studies showing the high prevalence of p53 mutations in squamous cell and adenocarcinoma of the esophagus.130 In all, p53 mutations are found in approximately 50-80% of esophageal cancers.131,132 The mutation of p53 and accumulation of nuclear p53 protein occur during Barrett's metaplasia progression both before and after cancer diagnosis.133,134 The accumulation of p53 in Barrett's esophagus has been associated with an increased potential (56%) for malignant progression.135 These data suggest that p53 mutation is an early event in the development of squamous and adenocarcinoma of the esophagus,129,130 and may be of diagnostic significance in identifying the early progression of Barrett's esophagus to adenocarcinoma.

As in other cancers of the GI tract, the Bcl-2 gene family of apoptotic regulators is altered in a subset of esophageal cancers. The bcl-2 proto-oncogene is a known inhibitor of apoptosis. Immunohistochemical staining studies have examined Bcl-2 protein expression in both normal and cancer tissues of the esophagus. Immunoreactivity to Bcl-2 was found in 27% of invasive squamous cell carcinomas, and was more frequently expressed in poorly differentiated cell types.136 However, Bcl-2 expression showed no correlation to tumor size, depth of invasion, nodal status, or overall survival. The expression of Bcl-xL, another negative regulator of apoptosis, has been examined in the progression of normal to squamous cell carcinoma in the esophageal epithelium. In this study, Bcl-xL protein expression was decreased in malignant lesions of the esophagus, and correlated with decreased tumor differentiation and decreased patient survival.137 However, there was an inverse correlation of Bcl-xL to Bcl-2 expression. Bax expression, a pro-apoptotic regulatory protein, was also examined in normal and cancer tissues of the esophagus. Cytoplasmic staining for Bax protein was found uniformly in all layers of the normal esophageal squamous epithelium, with a gradual loss of expression in a fraction of preneoplastic and neoplastic lesions.136 This may be a result of loss of transcriptional regulation of the Bax gene through p53 mutations. However, as with Bcl-2, Bax expression showed no correlation to tumor size, depth of invasion, nodal status, or overall survival. These data indicate that the collective alterations in the apoptotic regulatory machinery may play a role in the development of esophageal cancers, but other factors determine tumor aggressiveness and metastatic spread.

As described above, FasR is a cell surface receptor that mediates the induction of apoptosis by binding with FasL. FasR and FasL have been shown to be expressed in the normal epithelium of the esophagus in a positional-dependent manner.138 Squamous cell esophageal cancer cell lines were shown to possess FasR and FasL through reverse tran-scriptase-PCR analysis. Functional FasL was determined through the induction of apoptosis in Jurkat T leukemia cells, which are sensitive to FasL. Furthermore, through immunohistochemical staining, this study showed that FasL expression was found in >50% of tumor cells in 95% of invasive esophageal squamous cell carcinomas. In contrast, 80% of tumors showed no FasR expression. Therefore, similar to that seen in cancers of the colon, upregulation of FasL and downregulation of FasR expression are early events frequently associated with the evolution of esophageal squamous cell carcinomas.

Pancreatic Cancer

Pancreatic adenocarcinoma is a disease with extremely poor prognosis, and is the fifth leading cause of cancer death. Insights into the molecular aberrancies that result in pancreatic cancer formation have evolved through the study of apoptosis-related genes, specifically p53 and bcl-2. Mutations in the p53 gene are frequent in pancreatic adenocarcinoma, occurring in 50 to 70% of the tumors.139-141 Investigation of premalignant hyperplastic lesions and carcinoma in situ of the pancreas revealed the presence of p53 mutations in 35% of cases.142 These data suggest that mutations of the p53 gene may represent early genetic transformation in the induction of pancreatic cancer, and may help identify precursor lesions with malignant predisposition. The prognostic significance of p53 mutations in this disease is somewhat unclear. One study reports that p53 mutations are an independent prognostic factor and conferred no significant differences in overall survival rates in pancreatic cancers.143 However, when evaluated in surgical patients undergoing resection or palliation, p53 mutations were associated with decreased survival (6.2 months vs. 15.0 months). Furthermore, in patients undergoing curative resection, the median survival of those with p53 mutations was 12.8 months vs. 38.6 months in those patients without p53 mutations.144 Therefore, it does appear that p53 mutations have a prognostic significance in pancreatic cancers, but further studies are required to confirm these findings.

A prevalent mechanism in the increased survival of cancer cells and the induction of malignant disease is through the increased expression of Bcl-2, a negative regulator of apoptosis. Normal pancreatic ductal cells exhibit no Bcl-2 staining.145 Recent immunohis-tochemical staining experiments have revealed increased Bcl-2 expression in 53% of pancreatic adenocarcinomas studied. Bcl-2 expression was not related to tumor grade, DNA ploidy or S-phase fraction, but did predict a favorable outcome when compared to Bcl-2 negative tumors.146 These data indicate that pancreatic cancers with increased Bcl-2 expression behave less aggressively; however, the mechanism remains unknown. Overall, the prognostic value of Bcl-2 expression is unclear, since other tumors show no prognostic significance with Bcl-2 expression.147,148

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