**13.4. Role of autophagy in tumour survival**

90 Apoptosis and Medicine

have been described:

such as ROS.

is speculated to be essential in autophagy.

**13.3. How does autophagy suppress tumourigenesis?** 

a. Autophagy maintains the integrity of the genome

and gene amplification, especially after ischemic stress.

b. Autophagy limits necrosis-mediated inflammation

promote cell growth to replace the damaged tissue [161,162].

*Bcl-2* forms part of the Bcl-2 family proteins. For almost two decades, Bcl-2 has been regarded to function as an antiapoptotic protein that contributes to tumourigenesis. The role of Bcl-2 in autophagy was realized in 1998 when Liang identified Beclin 1 as Bcl-2 interacting protein [140]. Bcl-2 acts as a negative regulator of autophagy by inhibiting Beclin 1. It binds constitutively to Beclin 1, blocking the interaction between Beclin 1 and PI3KC3 [154-156]. As a result, PI3KC3 activity is decreased and autophagy is downregulated [155]. The binding of Bcl-2 with Beclin 1 seems to be constitutive, and its detachment from Beclin 1

The combined data presented above strongly support the idea that autophagy functions as a tumour suppressor process. Consistent with this, oncogenes that have a role in autophagy are potent inhibitors of this process. Although the molecular mechanisms by which autophagy functions in tumour suppression are poorly defined, at least two mechanisms

The first hypothesis is that autophagy may function as a housekeeping pathway to exert the quality control of organelles, proteins and DNA. Mathews and colleague observed that in autophagy deficient tumour cells, metabolic stress promotes the accumulation of p62, damaged mitochondria and ROS generation, promoting genomic instability and leading to oncogene activation and tumour progression [157,158]. Furthermore, immortalized mouse epithelial cells with a defect in ATG genes (loss of Beclin 1 or Atg 5) display increased DNA damage, centrosome abnormalities, numerical and structural chromosomal abnormalities

However, the mechanisms through which autophagy preserves the integrity of the genome remains elusive. One possibility could be that autophagy may contribute to cell cycle regulation, for example by degrading organelles and/or proteins involved in the cell cycle checkpoints [159]. Another possibility is that autophagy might simply function at a more general level to ensure the minimal amount of ATP and other metabolites required for DNA repair. Finally, autophagy may act by removing old and/or damaged organelles (for example, uncoupled mitochondria) which may act as a source of genotoxic chemical species

Necrosis normally results from physical injury in which the cell lyses and releases its intracellular contents, which activate the innate immune system and a wound-healing response [160]. As a result, inflammatory cells are recruited and cytokines are released to

In contrast, apoptosis may be the preferred means of cell demise for cells upon metabolic stress, as cells are eliminated without inflammation. However, in cancer cells with a defect The data presented above strongly supports the idea that autophagy functions as a tumour suppressor process and that inhibition of autophagy leads to carcinogenesis. However, there are some circumstances where autophagy contributes to tumour survival promoting carcinogenesis:

a. Autophagy is induced by nutrient starvation

The survival role of autophagy during nutrient limitation is well established. When cells encounter environmental stresses such as nutrient starvation, autophagy can be activated and protects cells by preventing them from undergoing apoptosis. Through autophagy, starving cells degrade cytoplasmic material to generate both nutrients and energy [85]. Consistent with this, during nutrient starvation, inhibition of autophagy promotes apoptosis [164].

b. Autophagy is induced by hypoxia

Hypoxia in tumours results from inadequate tumour vasculature and is associated with a more malignant phenotype, higher predisposition for metastasis, and poor prognosis. Hypoxic stress selects for cells that are resistant to apoptosis as well as poses a major barrier to chemotherapy and radiotherapy.

White and colleagues first showed that autophagy is induced specifically in the hypoxic core of tumours, where it promotes survival [163]. Further studies have unveiled the molecular connections between hypoxia and the activation of autophagy. For example, it has been reported that when oxygen concentration falls below 5% hypoxia inducible factor 1 (HIF1) is activated and this transcription factor activates key autophagy inducers (BNIP3), which in turn activate the key autophagy complex formed by PI3K III [165,166]. Further mechanistic studies have revealed that induction of BNIP3 and BNIP3L in hypoxic cells disrupts the Becn1-Bcl-2 complex, thereby releasing Becn1 to induce autophagy [167].

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

Cell Death and Cancer, Novel Therapeutic Strategies 93

of TRAIL for cancer cells is due to the fact that normal cells express more frequently these decoy receptors [173]. Moreover, TRAIL only weakly induces the prosurvival NFkB pathway [78]. Unfortunately, many primary tumour cell lines have shown resistance to TRAIL [174]. It has been shown that in preclinical studies, the combination of recombinant human TRAIL (rhTRAIL, AMG951, Dulanermin) or agonistic antibodies with irradiation, several chemotherapeutic agents, Akt or proteosome inhibitors induced cell death in tumours resistant to TRAIL [175-177]. Therefore, the efficacy of rhTRAIL or anti-TRAIL-R1/2 antibodies is being examined in different phase I and phase II clinical trails alone or in

*Fas activating compounds:* Fas, also called CD95 or APO-1 is a member of the TNF family of death receptors involved in the extrinsic apoptotic pathway. Fas plays an important role in regulation of the immune system. This receptor belongs to a subgroup within the TNFR family which includes TNFR1, DR3, TRAIL-R4 and TRAIL-R5 [180]. Fas has been recently implicated in the activation of RIP1, resulting in necroptosis when caspase-8 is inactive [181,182]. In addition, Tschopp and colleagues have demonstrated that Fas can activate other signalling pathways involved in proliferation or differentiation [109]. Fas agonists, such as highly aggregated FasL or anti-Fas antibodies are not suitable for cancer therapy owing to their profound liver toxicity. However, weaker Fas agonists may be useful. In this

*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

combination with other chemotherapeutic agents [172,178,179].

respect, APO010 is undergoing clinical trials for solid tumours [109].

has been developed and entered clinical trails for lymphomas [101].

**14.2. Intrinsic apoptotic pathway** 

c. Autophagy is induced in metastatic cells

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

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 detached cells harbouring antiapoptotic lesions.
