**13.2. Oncogenic genes that regulate autophagy**

88 Apoptosis and Medicine

in cancer.

suppressor proteins have been shown to induce autophagy. Supporting this idea, there are also several works showing that certain oncogenic proteins inhibit autophagy. Most of this oncogene products form part of the classic PI3K-Akt-mTOR pathway. In the following section, we summarize the literature on the role played by key tumour suppressors and oncogene products in the regulation of autophagy and the involvement of autophagic genes

To date, several tumour suppressor proteins that regulate autophagy have been described. These include: Beclin 1, UV irradiation-resistance-associated gene (UVRAG), PTEN, Bcl-2, and p53. The majority of them act as autophagy inducers with the exception of p53 that is

*Role of Beclin 1 in autophagy:* Beclin1 is a key regulator of autophagy. It regulates the autophagic process at different levels by combining with the enzyme class III PI3K (PI3KC3) and specific group of proteins forming unique class III PI3K-Beclin complexes. This PI3KC3- Beclin complex functions as a core complex during autophagy [136,137] by phosphorylating phosphatidylinositol to produce phosphatidylinositol 3-monophosphate, which presumably allows the recruitment of essential autophagy related (Atg) proteins to the membrane. In mammals, at least three types of class III PI3-kinase-Beclin complexes contribute to

*Role of Beclin 1 in tumour suppression vs. autophagy:* The first link between autophagy and cancer was established in 1999, when Liang and colleagues discovered that the autophagic protein Beclin 1 was able to inhibit tumourigenesis [140]. Beclin 1 is a tumour suppressor gene [141,142] monoallelically deleted in many types of cancers, including ovary (75%), breast (50%) and prostate (40%). Several studies suggest that the activation of autophagy by Beclin 1 is tightly associated with its tumour suppression function. For example, overexpression of Beclin-1 in the human MCF-7 breast carcinoma cell line is associated with inhibition of cell proliferation and induces autophagy [140]. In contrast, studies *in vivo* demonstrate that the monoallelic disruption of Beclin 1 gene promotes cellular proliferation and reduces autophagy [142]. Furthermore, genetically engineered mouse models with heterozygous disruption of Beclin 1 have decreased autophagy and are more prone to develop tumours, including lung carcinomas, hepatocellular carcinomas, lymphomas and mammary precancerous lesions [141,143]. Taken together, these studies firmly support the notion that autophagy induction and tumour inhibition function of Beclin 1 are closely

*UVRAG* is another tumour suppressor protein that is able to activate autophagy. UVRAG is a positive regulator of Beclin 1, as it promotes its binding to PI3KC3 to form the complex and enhance Beclin 1 activity. Through mediating Beclin 1-PI3KC3 complex, UVRAG can promote autophagy, thus inhibiting carcinogenesis of human colon cancer cells. It has also been proposed that UVRAG is involved in causing membrane curving [144]. Furthermore,

**13.1. Tumour suppressor genes that regulate autophagy** 

able to activate or inhibit autophagy.

autophagy [84,138,139].

interconnected.

Most of the oncogenic genes that regulate autophagy described to date are key proteins of the PI3K-Akt-mTOR pathway. These proteins inhibit autophagy and promote carcinogenesis.

*mTOR:* the best-studied downstream substrate of Akt is the serine/threonine kinase mammalian target of rapamycin (mTOR). In mammals, mTOR exists in two different complexes, known as mTORC1 and mTORC2. In the mTORC1 complex, mTOR is bound to a protein called Raptor, and in the mTORC2 complex, mTOR is bound to Rictor. mTORC1 is best known for its role in regulating protein synthesis through two of its substrates, 4E-BP1 and p70S6K [151]. Akt can directly phosphorylate and activate mTOR, or activate it indirectly by phosphorylation and inactivation of TSC2. When TSC2 is inactivated by phosphorylation, it can no longer associate with TSC1, thus abolishing the inhibitory effect of TSC1-TSC2 complex on mTORC1. As a result, mTOR is activated and signals to its downstream targets p70S6 kinase, ribosomal protein S6 and 4EBP-1/eIF-4E to control protein translation and proliferation [152]. mTORC1 is not only a key regulator of proliferation and survival, but also a master inhibitor of autophagy. Under nutrient-rich conditions, mTORC1 is activated and phosphorylates ULK1, ULK2 and mAtg13, inhibiting the initiation of autophagy. In contrast, in nutrient starvation conditions mTORC1 is inactivated and dissociates, resulting in activation of ULK1 and ULK2, which initiates the autophagy cascade [151]. The PI3K-Akt-mTOR pathway is probably the most commonly activated signalling pathway in human cancers [124]. This route is activated aberrantly in many types of tumours, and hence, inhibited autophagy and increased proliferation and protein synthesis are often observed. For example, many human cancers are characterized by activating mutations in specific components of the signalling pathway that connect Receptor tyrosine kinases to mTOR, including Ras, PI3K and Akt. Somatic mutations in PIK3CA gene encoding the catalytic subunit of class I PI3K frequently occur in cancers of colon, breast, brain and lung [153]. PTEN, another regulator of PI3KI-Akt pathway, is a tumour suppressor that is found to be mutated in many types of cancer.

*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 is speculated to be essential in autophagy.

Cell Death and Cancer, Novel Therapeutic Strategies 91

in apoptosis, autophagy is induced for cell survival. Through autophagy, cells eliminate damaged organelles and may maintain their normal cellular function under adverse conditions of fluctuating oxygen and nutrient supply. However, this beneficial effect of autophagy functions during short term interruptions in nutrient availability, as in the long

A high proportion of tumours have been seen to present a defect both in autophagy and apoptosis. Degenhardt and colleagues have shown that the inhibition of both processes under conditions of metabolic stress generates a necrotic cell-death, suggesting that apoptosis and autophagy function to limit necrosis [163]. In these necrotic tumours a persistent inflammatory infiltration and cytokine production exists, which is thought to

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

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

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

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

term (excess of autophagy) it can possibly lead to cell death.

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

a. Autophagy is induced by nutrient starvation

b. Autophagy is induced by hypoxia

to chemotherapy and radiotherapy.

autophagy [167].

carcinogenesis:

[164].

promote tumour growth and thus, is associated with poor prognosis.
