Novel role of VMP1 as modifier of the pancreatic tumor cell response to chemotherapeutic drugs

We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress‐induced autophagy‐associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over‐expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi‐based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1‐binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon. J. Cell. Physiol. 228: 1834–1843, 2013. © 2013 Wiley Periodicals, Inc.     

Zymophagy, a novel selective autophagy pathway mediated by VMP1-USP9x-p62, prevents pancreatic cell death

Autophagy has recently elicited significant attention as a mechanism that either protects or promotes cell death, although different autophagy pathways, and the cellular context in which they occur, remain to be elucidated. We report a thorough cellular and biochemical characterization of a novel selective autophagy that works as a protective cell response. This new selective autophagy is activated in pancreatic acinar cells during pancreatitis-induced vesicular transport alteration to sequester and degrade potentially deleterious activated zymogen granules. We have coined the term "zymophagy" to refer to this process. The autophagy-related protein VMP1, the ubiquitin-protease USP9x, and the ubiquitin-binding protein p62 mediate zymophagy. Moreover, VMP1 interacts with USP9x, indicating that there is a close cooperation between the autophagy pathway and the ubiquitin recognition machinery required for selective autophagosome formation. Zymophagy is activated by experimental pancreatitis in genetically engineered mice and cultured pancreatic acinar cells and by acute pancreatitis in humans. Furthermore, zymophagy has pathophysiological relevance by controlling pancreatitis-induced intracellular zymogen activation and helping to prevent cell death. Together, these data reveal a novel selective form of autophagy mediated by the VMP1-USP9x-p62 pathway, as a cellular protective response.     

Molecular cross-talk between the NRF2/KEAP1 signaling pathway, autophagy, and apoptosis

Oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. This, in turn, may cause the induction of autophagy or apoptosis. The NRF2/KEAP1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels. The relation between NRF2/KEAP1 signaling and regulation of apoptosis and autophagy is not well understood. In this hypothesis article we discuss how KEAP1 protein and its direct interactants (such as PGAM5, prothymosin α, FAC1 (BPTF), and p62) provide a molecular foundation for a possible cross-talk between NRF2/KEAP1, apoptosis, and autophagy pathways. We present a hypothesis for how NRF2/KEAP1 may interfere with the cellular apoptosis-regulatory machinery through activation of the ASK1 kinase by a KEAP1 binding partner-PGAM5. Based on very recent experimental evidence, new hypotheses for a cross-talk between NF-κB and the NRF2/KEAP1 pathway in the context of autophagy-related "molecular hub" protein p62 are also presented. The roles of KEAP1 molecular binding partners in apoptosis regulation during carcinogenesis and in neurodegenerative diseases are also discussed.     

The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.     

Myostatin induces p300 degradation to silence cyclin D1 expression through the PI3K/PTEN/Akt pathway

Myostatin is a negative regulator of skeletal muscle growth and affects numerous genes expression involved in cell proliferation, differentiation and metabolism. However, the molecular mechanisms underlying myostatin-regulated genes expression remain to be elucidated. In this study, we showed that myostatin blocked the recruitment of p300 to the cyclin D1 promoter, resulting in the silence of cyclin D1 expression. Our data further demonstrated that myostatin decreased the protein level of p300 by inducing p300 degradation via the ubiquitin-proteasome system. In addition, we provided experimental evidence to show that myostatin-induced p300 degradation was mediated by the phosphatidylinositol 3-kinase/PTEN/Akt signaling pathway and this could be antagonized by IGF-1 or insulin. Results presented in this study uncovered an epigenetic control of genes expression in response to myostatin.     

A novel mammalian trans-membrane protein reveals an alternative initiation pathway for autophagy

Autophagy is an early cellular event during acute pancreatitis, a disease defined as pancreas self-digestion. The Vacuole Membrane Protein 1 (VMP1) is a trans-membrane protein highly activated in acinar cells early during pancreatitis-induced autophagy and it remains in the autophagosomal membrane. We have shown that VMP1 expression is able to trigger autophagy in mammalian cells, even under nutrient-replete conditions. VMP1 is induced by autophagy stimuli and its expression is required for autophagosome development. VMP1 interacts with Beclin 1 through its hydrophilic C-terminal region, which we named Atg domain, as it is essential for autophagy. Remarkably, VMP1 pancreas-specific transgenic expression in mice promotes autophagosome formation. Most of the autophagy-related proteins were described in yeast or have a yeast homologue. VMP1 does not have any known homologue in yeast but its expression is required to start the autophagic process in mammalian cells. These findings support the hypothesis that mammalian cells may regulate autophagy in a different way. We propose that VMP1 is a novel autophagy related trans-membrane protein, which may lead the way in the search for alternative mechanisms of autophagosome formation.     

Essential role for autophagy protein VMP1 in maintaining neuronal homeostasis and preventing axonal degeneration

Vacuole membrane protein 1 (VMP1), the endoplasmic reticulum (ER)-localized autophagy protein, plays a key role during the autophagy process in mammalian cells. To study the impact of VMP1-deficiency on midbrain dopaminergic (mDAergic) neurons, we selectively deleted VMP1 in the mDAergic neurons of VMP1^fl/fl/DAT^CreERT2 bigenic mice using a tamoxifen-inducible CreERT2/loxp gene targeting system. The VMP1^fl/fl/DAT^CreERT2 mice developed progressive motor deficits, concomitant with a profound loss of mDAergic neurons in the substantia nigra pars compacta (SNc) and a high presynaptic accumulation of α-synuclein (α-syn) in the enlarged terminals. Mechanistic studies showed that VMP1 deficiency in the mDAergic neurons led to the increased number of microtubule-associated protein 1 light chain 3-labeled (LC3) puncta and the accumulation of sequestosome 1/p62 aggregates in the SNc neurons, suggesting the impairment of autophagic flux in these neurons. Furthermore, VMP1 deficiency resulted in multiple cellular abnormalities, including large vacuolar-like structures (LVSs), damaged mitochondria, swollen ER, and the accumulation of ubiquitin⁺ aggregates. Together, our studies reveal a previously unknown role of VMP1 in modulating neuronal survival and maintaining axonal homeostasis, which suggests that VMP1 deficiency might contribute to mDAergic neurodegeneration via the autophagy pathway.Subject terms: Neuroscience, Pathogenesis     

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.     

Examining Hedgehog pathway genes GLI3, SHH, and PTCH1 and the p53 target GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster using fluorescence in situ hybridization uncovers GLIPR1/GLIPR1L1/GLIPR1L2 deletion in 9% of patients with multiple myeloma

Mutations in genes regulating cell cycle and apoptosis are considered major culprits for the malignant transformation of cancer cells. Aberrant activation of the Hedgehog (HH) signaling pathway which primarily regulates genes involved in cell growth, proliferation, survival and apoptosis has been demonstrated in multiple myeloma. Mutations resulting in defective components of the p53 pathway, which serves a critical role in mediating cellular stress response by triggering DNA repair, cell cycle arrest, senescence and apoptosis, have also been identified. This study focuses on detecting copy number variations for the GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster of the p53 pathway and three elements of the HH pathway, SHH, PTCH1 and GLI3 in multiple myeloma (MM) using fluorescence in situ hybridization (FISH). In eighteen samples, there was no evidence of abnormal copy number for PTCH1, GLI3 or SHH. Thus, it is unlikely that copy number variations of these genes are linked to multiple myeloma. However, a deletion of the GLIPR1/GLIPR1L1/ GLIPR1L2 gene cluster, all p53 targets, was found in three of 32 samples (9.4%) indicating that these deleted genes may have significant implications in MM. Further studies should be performed to determine the role of the GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster in the pathogenesis of multiple myeloma.