**11. Apoptosis, chemoresistance and cancer**

So far, we have reviewed generalities of different modalities of cell death that can take place after various pathologies: inflammation, stroke, ischemic injury, neurodegenerative disorders, viral infection, neoplasia, etc. The implication of apoptosis in cancer was initially observed as the type of cell death occurring in untreated tumours and in tumour regression after radiotherapy [4]. The oncogenic process requires accumulation of diverse alterations within a cell that disrupt its normal homeostasis of cell death and growth. It is well established that excessive proliferation is not only due to oncogene activation but also to failure of the pathways controlling programmed cell death mechanisms [94]. A malignant cell can be protected from cell demise though expression and/or activation of antiapoptotic factors (acting as oncogenes) or through inactivation of antiapoptotic factors (acting as tumour suppressor genes). Evasion of apoptosis is a hallmark of cancer [95]. Dysregulation of apoptotic pathways renders cells resistant to antitumour strategies since the final outcome of radio and chemotherapy is frequently apoptosis of cancer cells. Therefore, resistance to cell death- in particular apoptotic cell death- is an important aspect of carcinogenesis, as it confers resistance to anticancer agents [96]. In many tumours, chemoresistance acquisition is due to upregulation or modification of key elements of apoptosis control, such as Bcl-2, Bcl-XL and IAP family members [36]. Other mechanisms are characterized by inactivating mutations in proapoptotic proteins, such as p53.

82 Apoptosis and Medicine

vesicles [92,93].

and RIP1 to form *complex I*. RIP1 is polyubiquinated by cIPA1 and cIAP2 and activates the I Kappa B kinase (IKK) complex, triggering NFkB activation. RIP1 can be deubiquitinated by cylindromatosis D (CYLD) and together with RIP3 forms the *complex II* which also contains TRADD, FADD, and caspase-8. Subsequently, caspase-8 cleavage will induce apoptosis, whereas caspase-8 inhibition by caspase inhibitors (zVAD-fmk) for example, will favour necroptosis [89]. A novel death regulation platform named ripoptosome has recently been described. Unlike complex II, the ripoptosome forms independently of death receptors, is activated by genotoxic stress or IAP antagonists and is tightly regulated by IAPs (ciAP1, cIAP2, xIAP) or cellular FLICE-like inhibitory protein (FLIP), a catalytically inactive homologue of caspase-8 [78,90,91]. The executioner mechanisms of necroptosis are unclear. However, in some cases the necroptotic process involves ROS generation, lysosomal membrane permeabilization, AIF release from the mitochondria and PARP activation. When caspase activation is not involved, necroptosis is associated with formation of autophagic

**Figure 8.** TNFR1-mediated apoptosis and necroptosis (taken from Long and Ryan 2012).

So far, we have reviewed generalities of different modalities of cell death that can take place after various pathologies: inflammation, stroke, ischemic injury, neurodegenerative disorders, viral infection, neoplasia, etc. The implication of apoptosis in cancer was initially

**11. Apoptosis, chemoresistance and cancer** 

**Bcl-2.** The first apoptotic gene related to neoplasia was the apoptotic inhibitor *bcl-2*. Therefore, Bcl-2 overexpression can contribute to tumour cell survival. Indeed, it was discovered at the breakpoint of the t(14;18) chromosomal translocation occurring in follicular lymphomas and leukemias [97,98]. Bcl-2 is the first oncogene that acts by inhibiting cell death instead of stimulating cell proliferation. In this respect, it was shown to cooperate with *myc* in immortalization of lymphoid cells [99] and in lymphomagenesis in transgenic mice [100]. Furthermore, it has been shown that Bcl-2 overexpression can confer multidrug resistance (MDR) phenotype and evasion of apoptosis to tumour cells exposed to serum deprivation, certain toxins or chemotherapeutic agents [101]. Mcl-1 and Bcl-XL have been shown to play important roles in tumourigenesis as well. Bcl-2 overexpression can also be the result of gene amplification or reduced expression of micro RNAs (miRNAs) in cancer cells [102].

**BH3-only proteins.** Loss or suppression of proaptotic proteins is frequent in cancer. Bax frameshift mutations appear in 50 % of colon carcinomas with DNA mismatch repair defects [103]. In addition, 17% of mantel cell lymphoma has homozygous Bim deletions [104], Bok and Puma suffer allelic deletions [105], and Bim and Puma are silenced by hypermethylationin Burkitt lymphoma [106,107]. Bim expression can also by silenced by miRNAs in several types of cancers [102]. Some BH-3 only proteins (Bim, Puma and Bmf) are necessary to initiate apoptosis in response to certain antineoplasic drugs [101].

**XIAP.** The X-linked inhibitor factor of apoptosis (XIAP) belongs to the IAP protein family. XIAP can render chemoresistance to cancer cells by inactivating caspases. XIAP overexpression is frequent in several tumour types [108]. Since XIAP is the only protein capable of inactivating both initiator and executioner caspases, it has become a putative biomarker of chemoresistance. It is the only IAP member capable of blocking active caspases [109]. However, XIAP has other roles in malignant transformation apart from preventing apoptosis. XIAP is involved in NFkB, MAPK and ubiquitin-proteasome pathways [108].

**Survivin.** This protein is a member of the IAP family that inhibits apoptosis by inactivating caspases [110] and stimulates DNA repair upon binding to DNA-PK in glioblastoma cell lines [111]. In addition, survivin plays a role in regulation of the mitotic checkpoint. Survivin expression is deregulated in cancer through several mechanisms: amplification of the locus on chromosome 17q25 [112], exon demethylation [113], or increased promoter activity [114]. Overall, survivin overexpression is an unfavourable prognostic marker and correlates with poor prognosis [110]. It is also involved in angiogenesis [115], tumour progression and chemoresistance. It has been shown that survivin inhibition sensitizes tumour cells to paclitaxel, cisplatin, etoposide, gamma radiation and immunotherapy [116].

Cell Death and Cancer, Novel Therapeutic Strategies 85

balance between cell cycle arrest and apoptosis. The frequent loss of p53 in cancer enhances its ability to survive after DNA damage and evade apoptosis. When p53 is mutated, the response to DNA damage depends on mechanisms regulated by Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3 related (ATR) independent of p53, which regulate Chk1 or Chk2 [119]. These kinases in turn activate

*Cell cycle checkpoints*. Checkpoints are mechanisms that respond to internal or external stress by activating machineries that arrest the cell cycle at particular points (G1/S, intra-S-phase, G2/M, or mitotic spindle). Many tumour suppressor genes are components of DNA damage or cell cycle checkpoints: p53, Retinoblastoma (Rb), ATM, p16, BRCA1/2, etc. The frequency of tumour suppressor loss in cancer cells provides an advantage to their growth. The G1 checkpoint is controlled by Rb, which in turn, is phosphorylated by cdk's. One cdk inhibitor is p16. Both Rb and p16 are frequently mutated in diverse types of cancer. The mitotic checkpoint utilizes genes (Mad, Bub, Aurora kinases) to detect spindle defects. When the cell is not able to surpass the damage, it undergoes mitotic catastrophe. In addition, loss of checkpoint controls increases genomic stability, providing cancer cells with adaptive or

NFkB, Akt and survivin [120].

evolutionary advantages [121].

**Figure 10.** DNA damage and cell cycle checkpoints

**p53.** Inactivating mutations in the tumour suppressor p53 account for about 50% of human tumours and are associated with poor prognosis. One role of p53 is the regulation of cell cycle through the DNA damage response. Many chemotherapeutic agents cause DNA damage and activate p53. As a result, the cdk inhibitor p21 can be transcriptionally activated causing cell cycle arrest or Bax can be activated by translocation to the mitochondria inducing apoptosis [56]. When p53 is not mutated, cells showing DNA damage induced by genotoxic stress that cannot be repaired during cell cycle arrest, are induced to apoptosis through p53 activation of the mitochondrial pathway, mostly increasing transcription of the BH-3-only proteins, Bid, Noxa and Puma [52]. In addition, p53 can activate genes of the extrinsic pathway: TRAIL-R2 (DR5) and Fas (CD95/Apo-1) [117].

**Figure 9.** Transcription-dependent and independent effects of p53 activation
