**14.2. Intrinsic apoptotic pathway**

92 Apoptosis and Medicine

The role of autophagy as a key mediator of survival of hypoxic cells is emerging so that the exact mechanisms underlying this phenotype remain unclear. Because chronic hypoxia leads to major metabolic perturbations in tumour tissues, one can postulate that by recycling basic cellular components, autophagy helps stressed cells cope with the increased metabolic

Epithelial cells critically depend on cell adhesion to extracellular matrix (ECM) for proper growth and survival. Upon detachment of cells from the ECM, cells undergo anoikis, a type of apoptotic cell death that serves the homeostatic function of killing cells that have lost contact with the basement membrane [68]. It has been shown that autophagy is induced in oncogene-transformed cells following matrix detachment [169]. Similarly, in three dimensional (3D) epithelial cell culture models, autophagy is significantly increased in the detached luminal cells. Furthermore, when autophagy is inhibited accelerated luminal clearance occurs [158,169]. Altogether, these results suggest that autophagy is fundamental in anoikis resistance, a process exploited by tumour cells to survive after detachment from

Debnath and colleagues have shown that detachment induces autophagy in both nontumourigenic epithelial cell lines and in primary epithelial cells. Autophagy inhibition through siRNA for ATG genes inhibits detachment-induced autophagy and increases apoptosis. Remarkably, even when apoptosis is inhibited matrix-detached cells still exhibit autophagy. Moreover, inhibition of autophagy in MCF-10 acini enhances luminal apoptosis during morphogenesis and fails to elicit long-term luminal filling [171]. Altogether, these results indicate that autophagy promotes epithelial cell survival during anoikis, including in

In addition to inactivation of proliferative and prosurvival oncogenic pathways, current anticancer strategies deal with reactivation of cancer death cell signalling routes in order to induce tumour regression. The most promising cancer therapies that specifically target

*TRAIL*: This member of the TNF family stimulates the extrinsic apoptotic pathway in a wide variety of cancers upon binding to death receptors, while having no effect on normal cells. Therefore, the use of recombinant TRAIL or agonistic TRAIL-receptor antibodies constitutes a novel therapeutic strategy [172]. TRAIL has been found to bind five receptors in human cells: TRAIL-R1, TRAIL-R2, TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and the soluble receptor osteoprogeterin. However, DcR1 and DcR2 are unable to transduce a death signal and are considered "decoy receptors". Ashkenazi and colleagues have proposed that the specificity

apoptosis, necrosis/necroptosis and autophagy are described in the following section.

demand [168]. However, further studies are needed to validate this hypothesis.

the primary site, as well as while migrating to distant metastatic sites [170].

c. Autophagy is induced in metastatic cells

detached cells harbouring antiapoptotic lesions.

**14. Current therapeutic advances** 

**14.1. Extrinsic apoptotic pathway** 

*Bcl-s inhibitors/BH3 mimetics*: Many efforts in cancer therapeutics are directed towards inhibition of antiapoptotic Bcl-2 like proteins. The first class of drugs being developed was antisense nucleotides. Oblimersen has been tested in clinical trials in different types of cancers. Even though some clinical trials shows efficacy of oblimersen combined with chemotherapy in treating melanoma [183], other studies show that this Bcl-2 inhibitor does not confer significant benefit to cancer patients [178]. Small molecule Bcl-2 inhibitors are also called "BH3 mimetics", as they imitate the function of BH3-only proteins: Gossypol (AT-101) binds Bcl-2, Bcl-XL and MCl-1, inhibiting their binding to Bax and Bak. In combination with other chemotherapeutic agents it promotes apoptosis in preclinical studies and has shown efficacy in at least one clinical trial [178]. Obotoclax is a pan Bcl-2 inhibitor that induces apoptosis in leukemia cells. However, recent studies have shown that its activity is partially mediated by autophagy [184,185]. Therefore, caution should be taken when using these kinds of drugs. ABT-737 and its orally available derivative, ABT-263 (navitoclax), have shown promising results in preclinical studies. They bind Bcl-2, Bcl-XL and Bcl-w [186], but mildly inhibit Mcl-1 [187] or A1 [101]. ABT-727 synergizes with standard chemotherapeutic agents, radiation and tyrosine kinase inhibitors in cancer cells and is effective as a single agent promoting tumour regression in mice. ABT-263 is currently in phase I and II clinical trails as a single agent [78,188]. However, its use is limited because it causes trombocytophenia owing to Bcl-XL inhibition [189]. A new BH-3 mimetic agent, GDC-0199 has been developed and entered clinical trails for lymphomas [101].

*IAP inhibitors and Smac mimetics:* Moreover, the role of IAPs in regulating apoptosis and/or necroptosis has led to the development of small molecules that antagonize IAP, or mimic the function of Smac/DIABLO. In fact, IAPs have been found to be overexpressed in many cancers and to be associated with poor prognosis and chemoresistance [102], making them putative targets for cancer therapeutics. Smac mimetics can sensitize chemoresistant cancer cells to cisplatin, doxorubicin or TRAIL [190], either as single agents or in combination with other drugs [109]. Therefore, some of these agents (AT-406, LCL-161, HGS-1029, TL32711, GDC-0917) [73] have recently entered clinical trials [191] for the treatment of solid tumours and lymphomas [192]. However, the potential of Smac mimetics as antitumour agents has been questioned, since the inhibition of cIAP1 and cIAP2 can stabilize IKK and activate the proinflammatory NFkB pathway [102].

Cell Death and Cancer, Novel Therapeutic Strategies 95

DNA double strand breaks. The current hypothesis is that since PARP is involved in repairs in DNA single strands breaks, inhibition of PARP leads to accumulation of single strand unrepaired breaks and becomes synthetically lethal in BRCA-mutated cancers [197]. Several PARP inhibitors are being evaluated in clinical trials: AG014699, AZD2281 (olaparib), ABT888 (veliparib), BSI-201 (iniparib), INO-1001, GP121016, CEP-9722 and MKI4827 [197,198]. PARP inhibitors may be useful in tumours bearing other types of genomic or

*cFLIP*: This antiapoptotic regulator is expressed as long (c-FLIPL) short (c-FLIPS) or c-FLIPR variants in human cells. In the absence of cFLIP, the ripoptosome triggers apoptotic cell death. On the contrary, expression of cFLIPL, which binds caspase-8, neutralizes its ability to engage the apoptotic machinery, but inhibits necroptosis by preventing RIP1-RIP3 association. On the other hand, c-FLIPS, although is able to bind caspase-8, does not form an active heteromer and necroptosis can develop [78,199]. Increased expression of c-FLIP has been found in several human cancers and is associated with poor prognosis [102]. Downregulation of FLIP seems to be a promising therapeutic strategy. Research efforts are being focused on the development of small interference RNA (siRNA) targeted against c-FLIP. In addition, several antitumour agents can downregulate c-FLIP at the transcriptional

A large series of anticancer drugs (both clinically approved and experimental) are able to induce a significant accumulation of autophagosomes in tumour cells both *in vitro* and *in vivo* [201]. For many years, it was thought that these therapies may kill tumours through autophagy and this massive vacuolization of the cytoplasm has been considered a manifestation of **autophagic cell death**. According to this notion, autophagy would represent another mechanism of cell death and the inhibition of autophagy would protect the cell from dying. However, this perspective is nowadays rather discredited, as the majority of the literature has reported that specific inhibition of autophagy (through siRNA) contributes to cell death during cancer treatment [202-204]. Besides, it is now well accepted that acquired resistant to chemotherapeutic drugs is, in part, due to the adaptive prosurvival response conferred by autophagy. Also, several studies have shown that inhibition of autophagy by pharmacological inhibitors such as Bafilomycin A1, cloroquine, or 3 methyladenine can enhance and accelerate cytotoxic cancer therapy in several tumours [205- 207]. In particular, some reports show that chloroquine as a single agent is sufficient to promote tumour regression in transplanted cancer models [208,209]. Moreover, chloroquine is a well-tolerated nontoxic drug that has entered a clinical trail as monotherapy for pancreatic cancer treatment [78]. This clearly indicates that the induction of autophagy represents an

functional defects in DNA damage response pathways.

**14.4. Necroptosis** 

or posttranscriptional level [200].

a. Autophagy as a protective mechanism

**14.5. Autophagy** 

Other IAP-targeted therapies such as XIAP and survivin antisense oligonucleotides have been developed [102]. Antisense oligonucleotides against survivin synergize with etoposide in non small lung cancer cells [117]. LY2181308 is a second generation antisense oligonucleotide being evaluated in clinical trials. In addition, a small molecule inhibitor of survivin, YM155, has shown potency in preclinical models and has entered phase II clinical trails [110].
