Diindolylmethane and its halogenated derivatives induce protective autophagy in human prostate cancer cells via induction of the oncogenic protein AEG-1 and activation of AMP-activated protein kinase (AMPK)

Highlights

• DIM and ring-DIMs induce the formation of autophagic vacuoles in prostate cancer cells.

• AEG-1/AMPK downregulation sensitizes prostate cancer cells to death in presence of subtoxic concentrations of (ring-)DIM.

• DIM- and ring-DIM-induced protective autophagy is mediated by the induction of AEG-1 and subsequent activation of AMPK.

• Downregulation of AEG-1 induces senescence in prostate cancer cells.

Abstract

3,3′-Diindolylmethane (DIM) and its synthetic halogenated derivatives 4,4′-Br₂- and 7,7′-Cl₂DIM (ring-DIMs) have recently been shown to induce protective autophagy in human prostate cancer cells. The mechanisms by which DIM and ring-DIMs induce autophagy have not been elucidated. As DIM is a mitochondrial ATP-synthase inhibitor, we hypothesized that DIM and ring-DIMs induce autophagy via alteration of intracellular AMP/ATP ratios and activation of AMP-activated protein kinase (AMPK) signaling in prostate cancer cells. We found that DIM and ring-DIMs induced autophagy was accompanied by increased autophagic vacuole formation and conversion of LC3BI to LC3BII in LNCaP and C42B human prostate cancer cells. DIM and ring-DIMs also induced AMPK, ULK-1 (unc-51-like autophagy activating kinase 1; Atg1) and acetyl-CoA carboxylase (ACC) phosphorylation in a time-dependent manner. DIM and the ring-DIMs time-dependently induced the oncogenic protein astrocyte-elevated gene 1 (AEG-1) in LNCaP and C42B cells. Downregulation of AEG-1 or AMPK inhibited DIM- and ring-DIM-induced autophagy. Pretreatment with ULK1 inhibitor MRT 67307 or siRNAs targeting either AEG-1 or AMPK potentiated the cytotoxicity of DIM and ring-DIMs. Interestingly, downregulation of AEG-1 induced senescence in cells treated with overtly cytotoxic concentrations of DIM or ring-DIMs and inhibited the onset of apoptosis in response to these compounds. In summary, we have identified a novel mechanism for DIM- and ring-DIM-induced protective autophagy, via induction of AEG-1 and subsequent activation of AMPK. Our findings could facilitate the development of novel drug therapies for prostate cancer that include selective autophagy inhibitors as adjuvants.

Introduction

Prostate cancer is a major health problem worldwide, ranking as the second most common cancer in males [1] and the third leading cause of cancer-related deaths among American and Canadian men [2]. Treatment with drugs targeting androgen receptor (AR) signaling is the main therapy for early-stage androgen-dependent (AD) prostate cancer [3]. Unfortunately, many patients with AD prostate cancer will progress to an androgen-independent (AI) phenotype, which is harder to treat and often fatal [4], [5]. Diindolylmethane (DIM) is a promising anticancer agent derived from the ingestion of Brassica plants (cabbage, broccoli, etc.) [6]. We have shown that several di-halogenated analogs of DIM (ring-DIMs) have anti-androgenic effects in human AD LNCaP prostate cancer cells [7] and induce apoptosis and necrosis in LNCaP and human AI PC-3 prostate cancer cells with greater potencies than DIM [8]. More recently, we have shown that DIM and ring-DIMs induce ER stress, mitochondrial dysfunction, and autophagy in prostate cancer cells [9]. Autophagy is a self-digestion process activated by cellular stress, in which dysfunctional organelles and protein aggregates are sequestered in double-membraned vesicles [10], and then transported to lysosomes for proteolytic degradation and recycling to maintain cellular homeostasis [11].

Autophagy plays an important role in cancer cell progression; its induction in response to stresses following chemotherapy may promote cancer cell survival. However, excessive autophagy could activate a cell death mechanism known as cytotoxic autophagy, which is different from programmed cell death (apoptosis) [12], [13]. Uncontrolled growth of cancer cells is facilitated by the inactivation of cell death pathways, such as apoptosis, and stimulation of cell survival pathways [14]. Thus, the dysregulation of autophagic machinery in cancer cells, leading to imbalances in the activation of cell death- or survival-related pathways, may have a critical influence on either tumor progression or regression. Various Atg (autophagy-related) proteins are involved in the initiation and regulation of autophagy [15]. The conversion of LC3B (Atg3) from its diffuse LC3BI form to the punctuated LC3BII, which associates with the autophagosome [16], is used as a classic marker of autophagy. AMP-activated protein kinase (AMPK) regulates autophagy via phosphorylation of the autophagy-initiating protein ULK1 (Atg1) [17]. The oncogenic protein, astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) or protein LYRIC, is overexpressed in various cancer including that of the prostate [18], [19], where it acts as a mediator of AMPK activity and autophagy in response to cellular metabolic stress [20], [21]. AEG-1 contributes to chemoresistance in hepatocellular carcinoma (HCC) cells [22] and promotes hepato-carcinogenisis through inhibition of senescence in transgenic mice that overexpress hepatocyte-specific AEG-1 [23].

Although DIM is known to be a mitochondrial ATP synthase inhibitor [24] that alters AMP/ATP ratios leading to activation of AMPK [25], the exact mechanism(s) of DIM-induced protective autophagy has not been elucidated. Previous studies have shown that AEG-1 induces protective autophagy via activation of AMPK [21]. Therefore, we wished to determine the possible involvement of AEG-1 in AMPK activation as well as DIM- and ring-DIM-mediated induction of autophagy.