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Pluripotency is an important feature of cancer stem cells (CSCs) that contributes to self-renewal and chemoresistance. The maintenance of pluripotency of CSCs under various pathophysiological conditions requires a complex interaction between various cellular pathways including those involved in homeostasis and energy metabolism. However, the exact mechanisms that maintain the CSC pluripotency remain poorly understood. In this report, using both human and murine models of CSCs, we demonstrate that basal levels of autophagy are required to maintain the pluripotency of CSCs, and that this process is differentially regulated by the rate-limiting enzyme in the NAD C synthesis pathway NAMPT (nicotinamide phosphoribosyltransferase) and the transcription factor POU5F1/OCT4 (POU class 5 homeobox 1). First, our data show that the pharmacological inhibition and knockdown (K D ) of NAMPT or the K D of POU5F1 in human CSCs significantly decreased the expression of pluripotency markers POU5F1, NANOG (Nanog homeobox) and SOX2 (SRY-box 2), and upregulated the differentiation markers TUBB3 (tubulin b 3 class III), CSN2 (casein b), SPP1 (secreted phosphoprotein 1), GATA6 (GATA binding protein 6), T (T brachyury transcription factor) and CDX2 (caudal type homeobox 2). Interestingly, these pluripotency-regulating effects of NAMPT and POU5F1 were accompanied by contrasting levels of autophagy, wherein NAMPT K D promoted while POU5F1 K D inhibited the autophagy machinery. Most importantly, any deviation from the basal level of autophagy, either increase (via rapamycin, serum starvation or Tat-beclin 1 [Tat-BECN1] peptide) or decrease (via ATG7 or ATG12 K D ), strongly decreased the pluripotency and promoted the differentiation and/or senescence of CSCs. Collectively, these results uncover the link between the NAD C biosynthesis pathway, CSC transcription factor POU5F1 and pluripotency, and further identify autophagy as a novel regulator of pluripotency of CSCs.

A lack of effective treatment is one of the main factors contributing to gastric cancerrelated death. Discovering effective targets and understanding their underlying anticancer mechanism is key to achieving the best response to treatment and to limiting side effects. Although recent studies have shown that the cation channel transient receptor potential melastatin-2 (TRPM2) is crucial for cancer cell survival, the exact mechanism remains unclear, limiting its therapeutic potential. Here, using molecular and functional assays, we investigated the role of TRPM2 in survival of gastric cancer cells. Our results indicated that TRPM2 knockdown in AGS and MKN-45 cells decreases cell proliferation and enhances apoptosis. We also observed that the TRPM2 knockdown impairs mitochondrial metabolism, indicated by a decrease in basal and maximal mitochondrial oxygen consumption rates (OCRs) and ATP production. These mitochondrial defects coincided with a decrease in autophagy and mitophagy, indicated by reduced levels of autophagyand mitophagy-associated proteins (i.e. ATGs, LC3A/B II, and BNIP3). Moreover, we found that TRPM2 modulates autophagy through a JNK-dependent and mechanistic target of rapamycin (mTOR)-independent pathway. We conclude that in the absence of TRPM2, down-regulation of the JNK signaling pathway impairs autophagy, ultimately causing the accumulation of damaged mitochondria and death of gastric cancer cells. Of note, by inhibiting cell proliferation and promoting apoptosis, the TRPM2 down-regulation enhanced the efficacy of paclitaxel and doxorubicin in gastric cancer cells. Collectively, we provide compelling evidence that TRPM2 inhibition may benefit therapeutic approaches for managing gastric cancer.Gastric cancer is the fifth most common type of cancer worldwide, affecting millions each year (1-4). The five-year survival rate is estimated at approximately 30% (5) making it one of the deadliest malignancies in the world and the second leading cause of cancer-related mortality in Eastern Asia (6, 7). Currently, surgery is the most effective available therapy against gastric cancer; however, its efficacy is limited to the early-stage gastric cancer patients (8, 9). For patients with late-stage tumors, surgery is not an option and despite systemic chemotherapy, the disease is deemed incurable (10)(11)(12). Considering the poor efficacy of current anticancer agents, the increasing resistance to chemotherapy drugs and the lack of treatment options for late stage patients, the development of novel and effective therapeutic approaches is of critical importance.Over the last decade, Transient Receptor Potential (TRP) channels have gained considerable attention in the field of cancer-targeted therapy (13-16). TRP channels are often altered in cancer cells and disruption in their normal function can affect various signalling pathways, ultimately leading to cancer progression and growth (17, 18). The TRP family is divided into seven subfamilies consisting of a total of 28 members. Some of members inclu...

Mitochondria require cholesterol for biogenesis and membrane maintenance, and for the synthesis of steroids, oxysterols and hepatic bile acids. Multiple pathways mediate the transport of cholesterol from different subcellular pools to mitochondria. In steroidogenic cells, the steroidogenic acute regulatory protein (StAR) interacts with a mitochondrial protein complex to mediate cholesterol delivery to the inner mitochondrial membrane for conversion to pregnenolone. In non-steroidogenic cells, several members of a protein family defined by the presence of a StAR-related lipid transfer (START) domain play key roles in the delivery of cholesterol to mitochondrial membranes. Subdomains of the endoplasmic reticulum (ER), termed mitochondria-associated ER membranes (MAM), form membrane contact sites with mitochondria and may contribute to the transport of ER cholesterol to mitochondria, either independently or in conjunction with lipid-transfer proteins. Model systems of mitochondria enriched with cholesterol in vitro and mitochondria isolated from cells with (patho)physiological mitochondrial cholesterol accumulation clearly demonstrate that mitochondrial cholesterol levels affect mitochondrial function. Increased mitochondrial cholesterol levels have been observed in several diseases, including cancer, ischemia, steatohepatitis and neurodegenerative diseases, and influence disease pathology. Hence, a deeper understanding of the mechanisms maintaining mitochondrial cholesterol homeostasis may reveal additional targets for therapeutic intervention. Here we give a brief overview of mitochondrial cholesterol import in steroidogenic cells, and then focus on cholesterol trafficking pathways that deliver cholesterol to mitochondrial membranes in non-steroidogenic cells. We also briefly discuss the consequences of increased mitochondrial cholesterol levels on mitochondrial function and their potential role in disease pathology.