**11.1. Defective checkpoints**

*p53 and the DNA damage response*. DNA damaging agents can induce different types of lesions that culminate in cell death. DNA damage is detected by sensors within the cell that relay a signal causing cell cycle arrest and DNA repair or apoptosis. These networks of genome surveillance are known as replication checkpoints. When DNA damage is low the lesions can be repaired by several mechanisms. On the other hand, when the DNA damage is high or persistent, the cell undergoes apoptosis [118]. For example, as a response to genotoxic insults, p53 is phosphorylated by Checkpoint kinase 1 (Chk1) and Checkpoint kinase 2 (Chk2) and cannot be ubiquinated by mdm2 and becomes stabilized. Consequently, p21 is activated and cells are arrested either in the G1 or the G2 phase of the cell cycle. Conversely, if p21 is absent, apoptosis prevails. Therefore, p21 is critical in maintaining the 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 NFkB, Akt and survivin [120].

84 Apoptosis and Medicine

**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

**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

**Transactivation**

**Transactivation (nucleus)**

**Translocation (mitochondrial)**

**(nucleus) p21 Cell cycle**

**arrest**

**Bax Apoptosis**

**Apoptosis**

**BH3-only proteins**

**TRAIL-R2, Fas**

paclitaxel, cisplatin, etoposide, gamma radiation and immunotherapy [116].

extrinsic pathway: TRAIL-R2 (DR5) and Fas (CD95/Apo-1) [117].

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

*p53 and the DNA damage response*. DNA damaging agents can induce different types of lesions that culminate in cell death. DNA damage is detected by sensors within the cell that relay a signal causing cell cycle arrest and DNA repair or apoptosis. These networks of genome surveillance are known as replication checkpoints. When DNA damage is low the lesions can be repaired by several mechanisms. On the other hand, when the DNA damage is high or persistent, the cell undergoes apoptosis [118]. For example, as a response to genotoxic insults, p53 is phosphorylated by Checkpoint kinase 1 (Chk1) and Checkpoint kinase 2 (Chk2) and cannot be ubiquinated by mdm2 and becomes stabilized. Consequently, p21 is activated and cells are arrested either in the G1 or the G2 phase of the cell cycle. Conversely, if p21 is absent, apoptosis prevails. Therefore, p21 is critical in maintaining the

**11.1. Defective checkpoints** 

**p53**

**DNA damaging agents**

*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 evolutionary advantages [121].

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

Cell Death and Cancer, Novel Therapeutic Strategies 87

prevents the nuclear localization of p53 upon binding and phosphorylation of mdm2 [120]. Therefore, deregulation of the PI3K-Akt pathway can be considered as a cause of

It is becoming more evident that the immune response facilitates the effects of chemotherapy. Physiological death avoids autoimmunity. However cancer cell death triggered by radiotherapy or some chemotherapeutic agents such as anthracyclines can be immunogenic [128]. Immunogenic death involves changes in the composition of the cell membrane and the release of molecules called Damage Associated Molecular Patterns or alarmins. In particular, calreticulin has been shown to be crucial for immunogenic cancer cell death [129]. The immune system determines the long-term success of antitumor therapies. It seems that mitochondrial events as well as the ER response in conjunction with autophagy can establish whether cancer cells die in response to chemotherapy [130]. It has been found that calreticulin is the dominant pro-phagocytic signal on several cancers including neuroblastoma, non Hodgkin's lymphoma and bladder cancer. However, calreticulin is counterbalanced by the "don´t eat me" signal CD47, which prevents cancer cell phagocytosis and is also highly expressed in these tumours [131]. Moreover, since CD47 is expressed on the surface of all human cancer cells but not in normal cells, blocking CD47

function with antibodies is emerging as a novel potential cancer strategy [132].

potential for cancer treatment, but requires further investigation [135].

We have already mentioned that strong p53 activation induces apoptosis. However, Leontieva and colleagues have shown that a weak and sustained p53 activation during cell cycle arrest can promote a different type of cell demise known as **senescence** [133]. Cellular senescence, induced after an irreversible cell cycle arrest, is a protective mechanism that limits proliferation of cells exposed to endogenous or exogenous insults. This cell death type can take part in processes of tumour suppression, tumour promotion, aging, and tissue repair [134]. Senescent cells become flattened and enriched with vacuoles. Some of the biochemical changes characteristic of senescence include elevation in β-galactosidase activity at an acidic pH, increase in senescence markers (p16, p15, p21, p53, ARF, etc.), heterochromatinization and, similarly to autophagy, formation of autolysosomes. It has been recently shown that chemotherapeutic agents may cause a form of "premature senescence" that can be considered as a tumour suppressor mechanism. Therefore, the induction of premature senescence as a drug-inducible arrest program has therapeutic

Regarding the role played by autophagy in cancer, mounting evidences suggest that autophagic cell death functions as a tumour suppressor mechanism. Several tumour

chemoresistance [127].

**12. Senescence and cancer** 

**13. Autophagy and cancer** 

**11.2. Immunogenecity of cancer cell death** 

**Figure 11.** Akt signalling pathway

**Akt.** Classically, the phosphoinositide-3-kinase (PI3K)-Akt-mTOR pathway is described as a key signal transduction cascade that integrates signals from growth factors and nutrients to regulate cell growth and proliferation [122]. Following growth factor binding to cell surface receptors, PI3K is activated and phosphorylates phosphatidylinositol-4,5-biphosphate (PIP2) to generate the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3). This process can be inhibited by the tumour suppressor protein phosphatase and tensin homolog (PTEN), which dephosphorylates PIP3 and terminates PI3K signalling. Then, the accumulated PIP3 recruits PDK1 and Akt through their Pleckstrin homology domains, and Akt is activated [123]. Akt recognizes and phosphorylates a consensus sequence that is present in many proteins. These substrates control key cellular processes such as apoptosis, cell cycle progression, transcription, and translation, all of which are critical events in cancer [124]. The Akt signal transduction pathway is probably the best survival pathway characterized. Moreover, Akt is constitutively activated in several malignant tumours, such as prostate, breast, ovary, lung and liver carcinomas [125]. Akt suppresses apoptosis through different mechanisms, including phosphorylation of forkhead transcription factors, which regulate proapoptotic proteins such as Bim and Fas ligand. The phosphorylated forkhead proteins are trapped in the cytosol and cannot enter the nucleus. Akt also phosphorylates and inactivates several proapoptotic proteins like Bad and caspase-9. Importantly, it activates IKK inducing the transcription factor NF-κB, leading to transcription of several antiapoptotic proteins such as Bcl-xL, and XIAP [96,126]. Akt also prevents the nuclear localization of p53 upon binding and phosphorylation of mdm2 [120]. Therefore, deregulation of the PI3K-Akt pathway can be considered as a cause of chemoresistance [127].
