Section 4 -

Endometrial Carcinoma. Pathology and Genetics: Role of Apoptosis Xavier Matias-Guiu Hospital Universitari Arnau de Vilanova Universitat de Lleida, Spain

Apoptosis is a key process in the regulation of cellular homeostasis. Deregulation of apoptosis plays an important role in development and progression of cancer. The lack of response to such stimuli can originate a survival advantage, and the expansion of a population of neoplastic cells. Moreover, cells resistant to apoptosis are likely to escape the immune surveillance, but they may be also resistant to therapy. Apoptosis-resistant cells may expand while the patients receive anticancer treatment, and be responsible for relapse. Apoptosis can be initiated by two main mechanisms: the intrinsic pathway, which has its origin in the mitochondria, and the extrinsic apoptotic pathway, triggered by the activation of death receptors situated in the cell surface. A final common feature for execution of the apoptotic programme is the activation of a cascade of caspases, which are proteases that have a cysteine containing active site that cleaves protein substrates at specific amino acid motifs containing an aspartic acid residue. Diverse stress injuries may cause the release of mitochondrial proteins to activate the 'intrinsic pathway'. Mitochondrial protein release often occurs after BH-3-only members of the Bcl-2 family bind to and neutralize anti-apoptotic members of the Bcl-2 family. This promotes release of some mitochondrial proteins, including cytochrome c, through an as yet incompletely characterized mechanism. Released cytochrome c interacts with Apaf-1, pro-caspase 9 and dATP to form a complex called the apoptosome. This complex dimerizes and activates caspase 9, which then activates effector caspases to induce apoptosis. The 'extrinsic pathway', is activated by ligand-bound death receptors such as tumor necrosis factor (TNF), Fas or TRAIL receptors . Although death receptors can promote cell growth under at least some situations, the ability of these receptors to induce apoptosis is critical in several diseases. The six known death receptors contain an intracellular interaction domain called a death domain (DD). After ligand binding, the activated death receptors recruit an adaptor protein named Fas Associated Death Domain (FADD). FADD consists of two protein interaction domains: a death domain and a death effector domain (DED). FADD binds (directly or through another adaptor named TRADD, which binds to TNFR1) to the receptor through interactions between DDs and to pro-caspase-8 through DED interactions to form a complex at the receptor called the Death Inducing Signalling Complex (DISC). Recruitment of caspase- 8 through FADD leads to its auto-cleavage and activation. Active caspase-8 in turn activates effector caspases such as caspase-3 causing the cell to undergo apoptosis by digesting upwards of a hundred or so proteins. One of the key regulators of this signalling is c-FLIP, which shares a high degree of homology with caspase- 8 but lacks protease activity. Thus it functions by competing with caspase-8 for binding to the DISC. In some type of cells this relatively simple pathway is enough to trigger apoptosis, but in other types of cells, the death receptor apoptotic pathway requires mitochondrial amplification. The BH3-only protein Bid is cleaved by caspase-8 and is then translocated to the mitochondria to activate the intrinsic pathway, thus connecting the two caspase activation pathways and amplifying the death receptor apoptotic signal. Thus, alterations in the mitochondrial pathway may affect the ability to induce apoptosis by death receptors. Different steps of apoptosis process have been found to be deregulated in many tumors. Down-regulation of death receptors, like Fas, has been detected in colorectal and hepatocellular carcinomas; and loss-of-function mutations and deletions on receptors DR4 and DR5 occur in metastasic breast carcinomas and lung carcinomas. In other tumors there is up-regulation of decoy receptors (DcR1 and DcR2) that compete with the functional receptors for the ligand binding, and may result in a diminished binding of TRAIL to their functional receptors and decreased apoptosis. Signalling intermediates of death receptor signalling can also be alterated. Increased levels of FLIP can inhibit caspase-8 recruitment and apoptosis. Recent evidences indicate that FLIP is constitutively or highly expressed in prostate cancer, Hodgkin lymphoma, gastric cancer, bladder carcinoma and malignant mesothelial cell lines. Moreover, the genes of the Bcl-2 family have been found to be involved in development and progression of a high number of tumors; and they may be responsible for alterations in both the "intrinsic" and the "extrinsic" pathways. Since in some type of cells death receptors require of mitochondrial amplification, there is increasing evidence that alterations in Bcl-2 family members may decrease the sensitivity to death ligands. Finally, other alterations that may play a key role in a diminished sensitivity to apoptosis are those present in proteins of signal transduction pathways involved in survival and cell growth promotion, such as the PI3kinase and the NF-κB signaling pathways. These pathways can modulate the sensibility to death receptor-triggered apoptosis, and have a well-known role in the development and progression of many tumours There are many evidences suggesting that alteration of apoptosis is important in development and progression of endometrioid adenocarcinomas of the endometrium. Several of the molecular abnormalities that have been detected in endometrioid adenocarcinomas may be associated with apoptosis deregulation. Endometrioid carcinomas of the endometrium show a high frequency of mutations in PTEN, which lead to constitutively active Akt, which in turn suppresses apoptosis triggered by various stimuli. Moreover, activation of the PI3 kinase pathway by loss of function of PTEN may lead to enhanced survival, by different mechanisms that ultimately cause phosphorylation of BAD, inhibition of caspase 9 or downregulation of Fas ligand. Moreover, the recent evidence that NF-κB activation is frequent in endometrial carcinoma may explain the presence of apoptosis resistance by activation of target genes such as FLIP and Bcl-XL. p53 alterations, which are characteristic of non-endometrioid carcinomas, may also occur in endometrioid tumors, particularly in those neoplasms showing overlapping features between types I and II tumors; and they may have an impact in apoptosis at several different levels. Also, members of the Bcl-2 family of genes are abnormal in endometrial carcinoma. For example, BAX is a target gene for mutations in endometrioid carcinomas with microsatellite instability, and may have a role in resistance to apoptosis in these tumors. Finally, several other proteins involved in apoptotic control (survivin) have also been shown to be abnormal in endometrial carcinoma. An important protein responsible for apoptosis resistance in endometrial carcinoma is FLIP. FLIP expression is frequent in endometrial carcinomas. A direct evidence of the role of FLIP in TRAIL apoptosis resistance on endometrial carcinoma cells is provided by treatment with specific siRNA targeting FLIP. Transfection of endometrial carcinoma cell lines with FLIP siRNA results in a marked decrease in cell viability after TRAIL exposition. This is accompanied by activation of both caspase-8 and caspase-3 suggesting activation of the extrinsic pathway. Finally, it is worth mentioning that FLIP is an important target of the Nuclear factor-Kappa B (NF-κB) signaling pathway. NF-κB encompasses different members of a family of transcription factors, involved in the regulation of genes encoding cytokines, cytokine receptors, and cell adhesion molecules, that drive immune and inflammatory responses. However, NF-κB has been recently found related to carcinogenesis, by regulating genes involved in apoptosis, the cell cycle, differentiation, and cell migration. NF-κB family members are frequently expressed in endometrial carcinomas. References Pallares J, Martinez-Guitarte JL, Dolcet X, Llobet D, Rue M, Palacios J, Prat J, Matias-Guiu X: Abnormalities in NF-κB family and related proteins in endometrial carcinoma. A tissue microarray study. J Pathol 2004 13:569-577 Pallares J, Martinez-Guitarte JL, Dolcet X, Llobet D, Rue M, Palacios J, Prat J, Matias-Guiu X. Survivin expression in endometrial carcinoma. A tissue microarray study with correlation with PTEN and STAT-3. Int J Gynecol Pathol (in press) Dolcet X, Llobet D, Pallares J, Rue M, Matias-Guiu X. Flip is frequently expressed in endometrial carcinoma and has a role in resistance to TRAIL-induced apoptosis. Lab Invest (in press) Matias-Guiu X, Catasus L, Bussaglia E, Lagarda H, Garcia A, Pons C, Muñoz J, Garcia A, Pons C, Muñoz J, Argüelles R, Machin P, Prat J: Molecular Pathology of endometrial hyperplasia and carcinoma. Hum Pathol 2001 32:569-77