The XAF1 tumor suppressor induces autophagic cell death via upregulation of Beclin-1 and inhibition of Akt pathway

Abstract

Autophagy is designated as type II programmed cell death and may confer a tumor-suppressive function. Our previous studies have shown that XIAP-associated factor 1 (XAF1) induced apoptosis and inhibited tumor growth in gastric cancer cells. In this study, we investigated the effect of XAF1 on the induction of autophagy in gastric cancer cells. We found that adenovirus vector-mediated XAF1 (adeno-XAF1) expression markedly induced autophagy, upregulated the level of Beclin-1 and inhibited phospho-Akt and phospho-p70S6K in gastric cancer cells. The downregulation of Beclin 1 or 3-methyladenine treatment suppressed adeno-XAF1-induced autophagy, but significantly enhanced adeno-XAF1-induced apoptosis. A pan-caspase inhibitor prevented adeno-XAF1-induced apoptosis, but significantly increased adeno-XAF1-induced autophagy. Furthermore, adeno-XAF1 induced autophagy in xenograft tumor and inhibited tumor growth. Our results document that adeno-XAF1 induces autophagy through upregulation of Beclin 1 expression and inhibition of Akt/p70S6K pathway, and reveal a new mechanism of XAF1 tumor suppression.

Introduction

XAF1 is previously identified as a XIAP binding partner that directly interacts with endogenous XIAP [1]. XAF1 is reported to antagonize anti-caspase activity of recombinant XIAP and reverse the protective effect of XIAP overexpression in cell lines [1]. Studies have shown that expression levels of XAF1 are extremely low or undetectable in the NCI 60 cell line panel of cancer cells [1], as well as in many types of human cancer tissues [2], [3], [4], [5]. Low expression of XAF1 is correlated with promoter hypermethylation and strongly associated with tumor staging [2], [3], [4], [5]. Our previous studies and others have shown that the restoration of XAF1 expression induces cancer cell apoptosis and inhibits tumor growth in multiple types of cancers including gastric, colon, liver, pancreatic and prostate cancers [3], [4], [5], [6], [7], [8]. In addition, we also found that XAF1 induced cell cycle arrest in G2/M phase and mitotic catastrophe [7], [9], [10]. XAF1 interacts with and attenuates the trans-activity of Four and a Half LIM protein 2 (FHL2), and inhibits cell invasion [9], [11]. However, the functions of XAF1 tumor suppressor are not fully elucidated.

Autophagy is the major cellular route for degrading long-lived proteins and cytoplasmic organelles under physiology condition. It is characterized by the appearance of double- and multi-membrane cytoplasmic vesicles that engulf cytoplasm and organelles, forming autophagosomes marked by microtubule-associated protein light chain 3 (LC3) [12]. Autophagy is induced in response to various stresses, such as nutrient starvation, endoplasmic reticulum stress, oxidative stress, or hypoxia [13]. Accordingly, the inhibition of autophagy will reduce the capacity to clear protein aggregates or damaged organelles and has been implicated in multiple pathological conditions, including cancer [14].

Autophagy is reported to be involved in malignant transformation [13], [14]. Several genetic links have emerged between defects of autophagy and development of cancer. Beclin 1, a human homolog of the autophagy-related gene Atg6, is monoallelically deleted in 40–70% of human prostate, ovarian and breast cancers and thus considered as a tumor suppressor [13], [15], [16], [17]. Heterozygous deletion of beclin 1 in mice resulted in higher incidence of spontaneous tumor [15], [16], [17]. Beclin 1 has also been shown to form a complex with phosphatidylinositol 3-kinase (PI3K) class III, and is a positive regulator of autophagosome formation [15], [16], [17]. Loss of Beclin 1 promotes genomic instability, which may ultimately lead to tumor progression [18]. Several other tumor suppressor proteins (i.e Atg4c; Bif-1; BH3-only proteins; LKB1/STK11; PTEN and UVRAG) inhibit tumor growth by promoting the autophagic pathway. Conversely, numerous oncoproteins, including PI3K, Akt1 and anti-apoptotic members of the Bcl-2 family suppress autophagy. Akt is an upstream regulator of the mTOR pathway and functions to inhibit autophagy [19]. Negative regulation of autophagy by mTOR is through activation of p70S6K [20]. Moreover, autophagy and apoptosis might be linked to each other and occur simultaneously or sequentially in cell type-, death stimulus-, or context-dependent manners [13]. In this context, several studies have already described a role for autophagy in antagonizing cell survival and promoting cell death.

Tumor suppression can be achieved by inhibition of cell proliferation or by induction of cell death or differentiation. Our previous studies have found that a pan-caspase inhibitor can prevent XAF1-induced apoptosis but can not inhibit cell death completely [6], [7], indicating that XAF1 may induce cell death in a caspase-independent manner. Autophagy is considered as type II cell death, a caspase-independent form of programmed cell death [21]. Thus, in this study, we investigated the effect of XAF1 on autophagy in gastric cancer cells. We showed that adeno-XAF1 induced autophagy in gastric cancer cells through upregulation of Beclin 1 and inhibition of Akt pathway. Our results reveal a new mechanism of XAF1 tumor suppression and provide a molecular link between autophagy and apoptosis.