Autophagy In Pancreatic Ductal Adenocarcinoma Research Articles

Abstract IA-01: Targeting autophagy in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most vexing problems in cancer with 5-year overall survival rates of 13%, thus there is a need for new therapeutic targets. PDAC have a distinct dependence on autophagy, a conserved eukaryotic catabolic pathway that serves to degrade proteins, other macromolecules, and organelles via the lysosome as part of a recycling process to maintain cellular homeostasis during basal and stress conditions. The unique importance of autophagy in PDAC suggests that there may be a subset of proteins that are specifically targeted by autophagy for degradation, which would promote proliferation and survival, and that these may represent new targets for therapy. Using a quantitative PDAC cellular autophagosomal proteomics approach, we identified NCOA4 as the autophagy receptor for ferritin, the cellular iron storage complex, and demonstrated that ferritin autophagy (ferritinophagy) is a highly regulated process and plays a central role in the larger context of cellular and organismal iron homeostasis. Given the reliance of PDAC, on both high levels of autophagy as well as iron, we studied the role of NCOA4 and ferritinophagy in PDAC thereby identifying NCOA4-mediated ferritinophagy and more broadly lysosomal iron metabolism as a PDAC dependency and therapeutic target. We recently determined the cryo-EM structure of the NCOA4-Ferritin complex for future drug design. To identify the role of autophagy and the lysosome in PDAC metastatic progression, we co-developed an in vivo capable lysosomal enrichment that enables capture and proteomic profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. Employing this strategy, we successfully captured lysosomal proteins from primary tumors (pancreas), as well as lung and liver metastases. Our preliminary analysis revealed an enrichment of resident lysosomal membrane and luminal proteins in the datasets, confirming successful isolation of intact lysosomes from these complex tissues. Additionally, we identified non-resident lysosomal proteins (termed cargo) that are enriched and co-evolve with advancing tumor stages. Notably, our results reveal distinct differences in the lysosomal proteome of tumor cells across different tissues. These findings emphasize the critical role of lysosomal function in tumor progression and suggest that targeting lysosomal components could be a promising therapeutic strategy for treating metastatic PDAC. Finally, given the challenging pharmacokinetic and pharmacodynamic properties and limited clinical efficacy of hydroxychloroquine as an autophagy/lysosomal inhibition strategy, we are now exploring novel autophagy inhibition strategies in PDAC to effectively target this pathway for clinical benefit. Citation Format: Joseph D. Mancias. Targeting autophagy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-01.

Abstract 1634: Novel autophagy inhibitor ESK981 potentiates MEK inhibition cytotoxicity in pancreatic cancer

Abstract Background Pancreatic Ductal Adenocarcinoma (PDAC) is extremely deadly with a dismal 5-year survival rate of only 11%. In PDAC, the overactive KRAS-MEK-ERK pathway is critical to maintaining metabolic homeostasis. Important recent work has shown that when MEK or ERK are inhibited, PDAC upregulates and becomes increasingly dependent on autophagy. Autophagy, the recycling of cellular material through lysosomal degradation, has already been previously established to be upregulated in and crucial to PDAC in order survive in its harsh microenvironment and allows PDAC to evade the immune system. Unfortunately, chloroquine (CQ), the only clinical-grade autophagy inhibitor tested in PDAC failed to improve patient survival in combination with chemotherapies in phase II trials. Recently, our lab reported a novel phase I-cleared multi-tyrosine kinase inhibitor ESK981 which disrupted autophagy and had substantial single agent efficacy in prostate cancer models. Given the importance of autophagy in PDAC and the ongoing clinical trials testing autophagy inhibitor CQ in combination with MEK/ERK inhibitors, this study aims to evaluate ESK981 as a therapeutic agent for PDAC, particularly its ability to potentiate MEK inhibitors’ antiproliferative effects in PDAC. Methods and Results In mouse and human PDAC cell lines, we observed an accumulation of LC3A/B as well as P62 when treated with ESK981, suggesting a disruption of autophagic flux. To confirm an inhibition of autophagy, we used the GFP-LC3-RFP-LC3ΔG reporter system in Mia-Paca2 cells and saw that the GFP:RFP ratio was maintained near 1:1. To assess the effects of ESK981 on cell growth, we used Cell-Titer Glo assays which revealed that ESK981 suppresses proliferation of a panel of human and mouse PDAC cell lines at nanomolar concentrations. To further evaluate ESK981’s potential clinical applications, we evaluated ESK981’s ability to synergize with MEK inhibitor Trametinib. Using the GFP-LC3-RFP-LC3ΔG reporter system we saw that Trametinib decreased the GFP:RFP ratio as expected. When cells were pre-treated with ESK981, the ratio was increased, showing that ESK981 was able to block Trametinib-induced autophagic flux. Finally, using Cell-Titer Glo, we determined that ESK981 and Trametinib had a strong synergistic effect on suppressing cell proliferation (BLISS Score >20) on mouse KPC line 7940B. Taken together, ESK981 is a promising autophagy inhibitor that shows strong anti-proliferative effects on both human and mouse PDAC cell lines as a single agent as well as in combination with Trametinib. Citation Format: Caleb Cheng, Jasmine Wisniewski, Bailey Jackson, Yi Bao, Yuanyuan Qiao, Costas A. Lyssiotis, Arul M. Chinnaiyan. Novel autophagy inhibitor ESK981 potentiates MEK inhibition cytotoxicity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1634.

Perturbation of Autophagy by a Beclin 1-Targeting Stapled Peptide Induces Mitochondria Stress and Inhibits Proliferation of Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, with a dismal five-year survival rate of less than 10%. PDAC possesses prominent genetic alterations in the oncogene KRAS and tumor suppressors p53, SMAD4 and CDKN2A. However, efforts to develop targeted drugs against these molecules have not been successful, and novel therapeutic modalities for PDAC treatment are urgently needed. Autophagy is an evolutionarily conserved self-degradative process that turns over intracellular components in a lysosome-dependent manner. The role of autophagy in PDAC is complicated and context-dependent. Elevated basal autophagy activity has been detected in multiple human PDAC cell lines and primary tumors resected from patients. However, clinical trials using chloroquine (CQ) to inhibit autophagy failed to show therapeutic efficacy. Here we show that a Beclin 1-targeting stapled peptide (Tat-SP4) developed in our lab further enhanced autophagy in multiple PDAC cell lines possessing high basal autophagy activity. Tat-SP4 also triggered faster endolysosomal degradation of EGFR and induced significant mitochondria stress as evidenced by partial loss of Δψ, increased level of ROS and reduced OXPHOS activity. Tat-SP4 exerted a potent anti-proliferative effect in PDAC cell lines in vitro and prohibited xenograft tumor growth in vivo. Intriguingly, excessive autophagy has been reported to trigger a unique form of cell death termed autosis. Tat-SP4 does induce autosis-like features in PDAC cells, including mitochondria stress and non-apoptotic cell death. Overall, our study suggests that autophagy perturbation by a Beclin 1-targeting peptide and the resulting autosis may offer a new strategy for PDAC drug discovery.

Matrix stiffness triggers chemoresistance through elevated autophagy in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a signature of extremely high matrix stiffness caused by a special desmoplastic reaction, which dynamically stiffens along with the pathological process. The poor prognosis and low five-year survival rate of PDAC are partly owing to chemoresistance triggered by substrate stiffness. Understanding the potential mechanisms of matrix stiffness causing PDAC chemoresistance is of great significance. In this study, methacrylated gelatin hydrogel was used as platform for PANC-1 and MIA-PaCa2 cell culture. The results indicated that compared to soft substrate, stiff substrate distinctively reduced the gemcitabine sensitivity of pancreatic cancer. Intriguingly, transmission electron microscopy, immunofluorescence staining, western blot and qRT-PCR assay showcased that the number of autophagosomes and the expression of LC3 were elevated. The observations indicate that matrix stiffness may regulate the autophagy level, which plays a vital role during chemoresistance. In brief, soft substrate exhibited low autophagy level, while the counterpart displayed elevated autophagy level. In order to elucidate the underlying interaction between matrix stiffness-mediated cell autophagy and chemoresistance, rescue experiments with rapamycin and chloroquine were conducted. We found that inhibiting cell autophagy dramatically increased the sensitivity of pancreatic cancer cells to gemcitabine in the stiff group, while promoting autophagy-driven chemoresistance in the soft group, demonstrating that matrix stiffness modulated chemoresistance via autophagy. Furthermore, RNA-seq results showed that miR-1972 may regulate autophagy level in response to matrix stiffness. Overall, our research shed light on the synergistic therapy of PDAC combined with gemcitabine and chloroquine, which is conducive to promoting a therapeutic effect.

Sirtuin 4 activates autophagy and inhibits tumorigenesis by upregulating the p53 signaling pathway

The role of autophagy in cancer is context-dependent. In the present study, we aimed to investigate the regulator and underlying mechanism of autophagy. We found that a sirtuin (SIRT) family member, SIRT4, was significantly associated autophagy pathway in pancreatic ductal adenocarcinoma (PDAC). Specifically, in vitro cell culture experiments and in vivo transgenic and xenografted animal models revealed that SIRT4 could inhibit tumor growth and promote autophagy in PDAC. In terms of the mechanism, we demonstrated that SIRT4 activated the phosphorylation of p53 protein by suppressing glutamine metabolism, which was crucial in SIRT4-induced autophagy. AMPKα was implicated in the regulation of autophagy and phosphorylation of p53 mediated by SIRT4, contributing to the suppression of pancreatic tumorigenesis. Notably, the clinical significance of the SIRT4/AMPKα/p53/autophagy axis was demonstrated in human PDAC specimens. Collectively, these findings suggested that SIRT4-induced autophagy further inhibited tumorigenesis and progression of PDAC, highlighting the potential of SIRT4 as a therapeutic target for cancer.

In This Issue

In This Issue

Therapeutic Targeting of Autophagy in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by early metastasis, late detection, and poor prognosis. Progress towards effective therapy has been slow despite significant efforts. Novel treatment approaches are desperately needed and autophagy, an evolutionary conserved process through which proteins and organelles are recycled for use as alternative energy sources, may represent one such target. Although incompletely understood, there is growing evidence suggesting that autophagy may play a role in PDAC carcinogenesis, metastasis, and survival. Early clinical trials involving autophagy inhibiting agents, either alone or in combination with chemotherapy, have been disappointing. Recently, evidence has demonstrated synergy between the MAPK pathway and autophagy inhibitors in PDAC, suggesting a promising therapeutic intervention. In addition, novel agents, such as ONC212, have preclinical activity in pancreatic cancer, in part through autophagy inhibition. We discuss autophagy in PDAC tumorigenesis, metabolism, modulation of the immune response, and preclinical and clinical data with selected autophagy modulators as therapeutics.

Abstract LB184: Induction of ulk1 regulated autophagy by a novel antipsychotic drug leads to apoptosis in pancreatic cancer cells

Abstract Pancreatic cancer patients have limited treatment options in spite of several advanced treatment strategies. Pancreatic tumors exhibit high basal autophagy compared to other cancers. Several studies including from our lab reported that enhanced autophagy leads to apoptosis in cancer cells. In this study, we have demonstrated that pimavanserin (PVT) suppresses pancreatic tumor growth by inducing autophagy-mediated apoptosis. Our results indicated that PVT induced 1.8 - 5 fold autophagy in pancreatic ductal adenocarcinoma (PDAC) cells, as evaluated by increased acridine orange staining. Furthermore, accumulation of double-membrane vesicles (autophagosomes) and formation of autophagolysosomes were observed with PVT treatment. Genomic analysis by R2 &GEPIA database revealed that PDAC patients exhibited reduced levels of autophagy initiator gene, ULK1. PVT induced the expression of autophagy markers ULK1, FIP200, Atg101, Beclin-1, Atg5, and LC3A/B in several pancreatic cancer cells. In addition, phosphorylation of ULK1 atSer757 was inhibited with PVT treatment. Apoptotic effects of PVT in pancreatic cancer cells were validated by increase in cleavage of caspase3. ULK1 agonist LYN-1604 enhanced the autophagic and apoptotic effects of PVT. On the other hand, autophagy inhibitors chloroquine and bafilomycin blocked the autophagic and apoptotic effects of PVT in pancreatic cancer cells. Our in vivo findings demonstrated that chloroquine abrogated the growth-suppressive effects of PVT by 21% in sub-cutaneously implanted BxPC3 tumor xenografts. Moreover, chloroquine reduced the effects of PVT in inducing the expression of ULK1 and the cleavage of caspase 3 as evaluated by western blotting. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopically implanted PANC1 tumors by 77%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Moreover, long-term administration of PVTdid not altered the clinical chemistry parameters like ALT, AST, total serum protein, calcium, creatinine, BUN, and albumin. Collectively, our results indicate that PVT-mediated pancreatic tumor growth suppression was associated with the induction of autophagy-mediated apoptosis. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for pancreatic cancer therapy. Citation Format: Sharavan Ramachandran, Sanjay K. Srivastava. Induction of ulk1 regulated autophagy by a novel antipsychotic drug leads to apoptosis in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB184.

Pimavanserin: A novel autophagic regulator for pancreatic cancer treatment

Pancreatic cancer patients have limited treatment options in spite of several advanced treatment strategies. Pancreatic tumors exhibit high basal autophagy compared to other cancers. Several studies including from our lab reported that enhanced autophagy leads to apoptosis in cancer cells. In this study, we have demonstrated that pimavanserin (PVT) suppresses pancreatic tumor growth by inducing autophagy-mediated apoptosis. Our results indicated that PVT induced 1.8 – 5 fold autophagy in pancreatic ductal adenocarcinoma (PDAC) cells, as evaluated by increased acridine orange staining. Furthermore, accumulation of double membrane vesicles (autophagosomes) and formation autophagolysosomes were observed with PVT treatment. Genomic analysis by R2 & GEPIA database revealed that PDAC patients exhibited reduced levels of autophagy initiator gene, ULK1. PVT induced the expression of autophagy markers ULK1, FIP200, Atg101, Beclin-1, Atg5, and LC3A/B in several pancreatic cancer cells. In addition, phosphorylation of ULK1 at Ser757 was inhibited with PVT treatment. Apoptotic effects of PVT in pancreatic cancer cells was validated by increase in cleavage of caspase3. ULK1 agonist LYN-1604 enhanced the autophagic and apoptotic effects of PVT. On the other hand, autophagy inhibitors chloroquine and bafilomycin blocked the autophagic and apoptotic effects of PVT in pancreatic cancer cells. Our in vivo findings demonstrated that chloroquine abrogated the growth suppressive effects of PVT by 21% in sub-cutaneously implanted BxPC3 tumor xenografts. Moreover, chloroquine reduced the effects of PVT in inducing the expression of ULK1 and the cleavage of caspase 3 as evaluated by western blotting. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopicaly implanted PANC1 tumors by 77%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Moreover, long-term administration of PVT did not altered the clinical chemistry parameters like ALT, AST, total serum protein, calcium, creatinine, BUN and albumin. Collectively, our results indicate that PVT mediated pancreatic tumor growth suppression was associated with induction of autophagy mediated apoptosis. Since, PVT is already available in clinic with an established safety profile, our results will accelerate its clinical development for pancreatic cancer therapy.

Autophagy is required for proper cysteine homeostasis in pancreatic cancer through regulation of SLC7A11

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.

Autophagy-mediated negative feedback attenuates the oncogenic activity of YAP in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy in humans, and new therapeutic targets are urgently needed. Yes-associated protein (YAP) plays a significant role in cancer progression. Autophagy is also closely associated with various human cancers. However, the interplay between YAP and autophagy in PDAC remains poorly understood. In this study, we found that YAP was upregulated and activated in PDAC. Further analysis revealed that there is a YAP-autophagy feedback loop in pancreatic cancer. Mechanistically, YAP activates autophagy by promoting Atg5 transcription via TEAD1-mediated binding, while autophagy negatively regulates YAP through autophagic degradation. The hyperactivation of YAP in PDAC unbalances the YAP-autophagy circuit and promotes cancer progression. Inhibition of autophagy enhances the oncogenic activity of YAP in PDAC. The autophagy activator rapamycin promotes the antitumor effect of verteporfin, a YAP inhibitor. Therefore, our study elucidated the interaction between YAP and autophagy in PDAC and our results suggest that targeting the YAP-autophagy circuit may be a new therapeutic strategy for pancreatic cancer.

Autophagy Regulatory Genes MET and RIPK2 Play a Prognostic Role in Pancreatic Ductal Adenocarcinoma: A Bioinformatic Analysis Based on GEO and TCGA.

Pancreatic ductal adenocarcinoma is a common malignant tumor with a poor prognosis. Autophagy activity changes in both cancer cells and microenvironment and affects the progression of pancreatic ductal adenocarcinoma. The purpose of this study was to predict the prognostic autophagy regulatory genes and their role in the regulation of autophagy in pancreatic ductal adenocarcinoma. We draw conclusions based on gene expression data from different platforms: GSE62165 and GSE85916 from the array platform, TCGA from the bulk RNA-seq platform, and GSE111672 from the single-cell RNA-seq platform. At first, we detected differentially expressed genes in pancreatic ductal adenocarcinoma compared with normal pancreatic tissue based on GSE62165. Then, we screened prognostic genes based on GSE85916 and TCGA. Furthermore, we constructed a risk signature composed of the prognostic differentially expressed genes. Finally, we predicted the probable role of these genes in regulating autophagy and the types of cell expressing these genes. According to our screening criteria, there were only two genes: MET and RIPK2, selected into the development of the risk signature. However, evaluated by log-rank tests, receiver operating characteristic curves, and calibration curves, the risk signature was worth considering its clinical application because of good sensitivity, specificity, and stability. Besides, we predicted that both MET and RIPK2 promote autophagy in pancreatic ductal adenocarcinoma by gene set enrichment analysis. Analysis of single-cell RNA-seq data from GSE111672 revealed that both MET and RIPK2 were expressed in cancer cells while RIPK2 was also expressed in monocytes and neutrophils. After comprehensive analysis, we found that both MET and RIPK2 are related to the prognosis of pancreatic ductal adenocarcinoma and provided some associated clues for clinical application and basic experiment research.

The Anti-Tumor Activity of Afatinib in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic Ductal Adenocarcinoma (PDAC) is the most common form of pancreatic cancer and leading causes of pancreatic cancer death because of most PDAC patients with advanced unresectable disease at that time, which is remarkably resistant to all forms of chemotherapy and radiotherapy. PDAC increases the social and patient's family burden. However, the PDAC pathogenesis is not identified. We are trying to uncover the underlying mechanism in the future. In our research, the drug-resistant cell line was successfully induced in the vitro by progressive concentrations of Afatinib, which we named it as BxPC3-AR. It has been observed that the effect of autophagy was on the resistance of BxPC3-AR to Afatinib. It has been confirmed that autophagy plays a certain role in BxPC3-AR resistance to Afatinib. Our findings provide a new perspective on the role of autophagy in pancreatic ductal adenocarcinoma.

The Role of Autophagy in Pancreatic Cancer-Recent Advances.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers with a 5-year survival rate of only 9%, despite ongoing efforts to improve treatment. This dismal prognosis is due to the difficulty of early stage diagnosis, drug resistance, and likelihood of metastasis development. It is therefore of great importance to identify appropriate therapeutic targets and gain a greater understanding of PDAC biology. Autophagy is a membrane-mediated degradation and recycling mechanism, which is crucial for cell homeostasis. There is evidence for both a tumor-suppressive and a tumor-promoting role of autophagy in cancer, and this is likely context dependent. Within PDAC, a large body of evidence points towards autophagy being required for tumor survival and metabolism. In this review, we describe the recent advances in the understanding of the role and regulation of autophagy in PDAC.

Levels of the Autophagy-Related 5 Protein Affect Progression and Metastasis of Pancreatic Tumors in Mice

Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5(Atg5) protein on pancreatic tumor formation and progression. We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration andinvasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin Land D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.

RETRACTED ARTICLE: LncRNA PVT1 triggers Cyto-protective autophagy and promotes pancreatic ductal adenocarcinoma development via the miR-20a-5p/ULK1 Axis

BackgroundDefective autophagy is thought to contribute to the pathogenesis of many diseases, including cancer. Human plasmacytoma variant translocation 1 (PVT1) is an oncogenic long non-coding RNA that has been identified as a prognostic biomarker in pancreatic ductal adenocarcinoma, but how PVT1 operates in the regulation of autophagy in pancreatic ductal adenocarcinoma (PDA) is unclear.MethodsPVT1 expression level was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and hybridization in situ (ISH). Western blot or qRT-PCR was performed to assess the ULK1 protein or mRNA level. Autophagy was explored via autophagic flux detection under a confocal microscope and autophagic vacuoles investigation under a transmission electron microscopy (TEM). The biological role of PVT1 in autophagy and PDA development was determined by gain-of-function and loss-of-function assays.ResultsWe found that PVT1 levels paralleled those of ULK1 protein in PDA cancer tissues. PVT1 promoted cyto-protective autophagy and cell growth by targeting ULK1 both in vitro and in vivo. Moreover, high PVT1 expression was associated with poor prognosis. Furthermore, we found that PVT1 acted as sponge to regulate miR-20a-5p and thus affected ULK1 expression and the development of pancreatic ductal adenocarcinoma.ConclusionsThe present study demonstrates that the “PVT1/miR-20a-5p/ULK1/autophagy” pathway modulates the development of pancreatic ductal adenocarcinoma and may be a novel target for developing therapeutic strategies for pancreatic ductal adenocarcinoma.

Abstract 1198: Neuropilin-1 blockade chemosensitizes pancreatic cancer cells via dual inhibition of epithelial-mesenchymal transition and autophagy

Abstract Introduction: Fibrotic and malignant pancreatic ductal adenocarcinoma (PDAC) tumors are often resistant to chemotherapy, and remain a leading cause of morbidity and mortality. Epithelial–mesenchymal transition (EMT), a process by which epithelial cells lose their cell polarity and adhesion to become migratory and invasive mesenchymal stem cells, contributes to chemoresistance in PDAC. Likewise, PDAC tumors benefit from autophagy, a self-degradation pathway that confers a survival benefit against metabolic stress. Over-activation of EMT and autophagy has been linked to increased drug resistance in PDAC. Both these processes are largely mediated by TGFβ1. Recently, we demonstrated the role of neuropilin-1 (NRP-1) in TGFβ1-dependent endothelial-mesenchymal transition and fibrosis in PDAC. However, the potential mechanisms linking NRP-1 with EMT and autophagy-mediated chemoresistance remain unexplored. We hypothesized that NRP-1 blockade will enhance chemotherapeutic effect, by suppressing EMT and autophagy in PDAC. Methods: Human PDAC cells (BxPC-3) or serially gemcitabine exposed chemoresistant (CR-BxPC-3) cells were transfected with siNRP-1 or scramble using DharmaFECT-2. For EMT and chemotherapy studies, transfected cells were treated with TGFβ1 (5ng/mL, 24h), followed by gemcitabine (1μM) for 48h. RNA (24h) and protein (48h) was isolated using TRIzolTM and RIPA, respectively. EMT markers were evaluated by RT-PCR and immunoblotting/immunostaining. For autophagy studies, cells were serum starved post NRP-1 silencing and protein was isolated. For autophagy inhibition, cells were treated with bafilomycin-A1 (100nM) for 24h post NRP-1 silencing and protein was isolated. Autophagy flux (LC3-II/I) and P62 were measured by immunoblotting/immunostaining. Cell viability was evaluated by MTT assay. Results: SiNRP-1 treatment in BxPC-3 and CR-BxPC-3 cells resulted in NRP-1 knockdown and promoted cell-death after chemotherapy. TGFβ1-stimulated BxPC-3 cells demonstrated a morphological change consistent with EMT that was revoked by siNRP-1. Also, siNRP-1 maintained epithelial cellular morphology via gain of epithelial and loss of mesenchymal markers. Remarkably, CR-BxPC-3 cells exhibited enhanced EMT features. SiNRP-1 inhibited EMT and promoted cell-death by overcoming gemcitabine resistance in CR-BxPC-3 cells, possibly through reduced ERK signaling. Moreover, siNRP-1 inhibited autophagy (decreased LC3-II/I ratio and increased P62 levels) following starvation or chemotherapy stress, and enhanced cell-death after chemotherapy and autophagy inhibition in BxPC-3 cells. Conclusions: Our findings define a previously undetermined role of NRP-1 in regulating autophagy and TGFβ1-induced EMT in PDAC, suggesting that NRP-1 may represent a novel therapeutic target to overcome chemoresistance through reduced EMT and autophagy. Citation Format: Pratiek N. Matkar, Krishna K. Singh, Gerald Prud’homme, David Hedley, Howard Leong-Poi. Neuropilin-1 blockade chemosensitizes pancreatic cancer cells via dual inhibition of epithelial-mesenchymal transition and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1198. doi:10.1158/1538-7445.AM2017-1198

Transforming Growth Factor TGFβ Increases Levels of Microtubule-Associated Protein MAP1S and Autophagy Flux in Pancreatic Ductal Adenocarcinomas.

Background and AimAutophagy is a cellular process to regulate the turnover of misfolded/aggregated proteins or dysfunctional organelles such as damaged mitochondria. Microtubule-associated protein MAP1S (originally named C19ORF5) is a widely-distributed homologue of neuronal-specific MAP1A and MAP1B with which autophagy marker light chain 3 (LC3) was originally co-purified. MAP1S bridges autophagic components with microtubules and mitochondria through LC3 and positively regulates autophagy flux from autophagosomal biogenesis to degradation. The MAP1S-mediated autophagy suppresses tumorigenesis as suggested in a mouse liver cancer model and in prostate cancer patients. The TGFβ signaling pathway plays a central role in pancreatic tumorigenesis, and high levels of TGFβ suggest a tumor suppressive function and predict a better survival for some patients with resectable pancreatic ductal adenocarcinoma. In this study, we try to understand the relationship between TGFβ and MAP1S-mediated autophagy in pancreatic ductal adenocarcinoma.MethodsWe collected the tumor and its adjacent normal tissues from 33 randomly selected patients of pancreatic ductal adenocarcinomas to test the association between TGFβ and autophagy markers MAP1S and LC3. Then we tested the cause and effect relation between TGFβ and autophagy markers in cultured pancreatic cancer cell lines.ResultsHere we show that levels of TGFβ and autophagy markers MAP1S and LC3 are dramatically elevated in tumor tissues from patients with pancreatic ductal adenocarcinomas. TGFβ increases levels of MAP1S protein and enhances autophagy flux.ConclusionTGFβ may suppress the development of pancreatic ductal adenocarcinomas by enhancing MAP1S-mediated autophagy.

Autophagy: Surviving stress in pancreatic cancer.

Pereraet al. investigated the high levels of autophagy in pancreatic ductal adenocarcinoma (PDAC).

Identification of Critical Biomarkers Responsive to Anti-Autophagy Therapies for Pancreatic Ductal Adenocarcinoma through a Performance Analysis of miRNA Platforms

Background: Autophagy is a fundamental catabolic pathway that involves the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. The role of autophagy in cancer is complex, exhibiting tumor suppressive or tumor proliferative effects depending on the cancer type. Pancreatic ductal adenocarcinoma has constitutively activated autophagy that results in the selective proliferation of its tumor cells. Understanding the mechanisms by which the inhibition of autophagy occurs could shape our current therapeutic strategies for pancreatic cancer. Recent research has implicated microRNAs as important regulators of autophagy, but how miRNAs influence this inhibition is largely unexplored. Results: It is known that there exists inter-platform variability across miRNA platforms and therefore in order to identify bona fide miRNAs that are involved with the inhibition of autophagy we carried out a performance analysis of miRNA arrays from three vendors. In this paper we report our findings from this cross-platform analysis of miRNA arrays and the identity of several critical biomarkers of response to anti-autophagy therapies in the immortalized human pancreatic ductal adenocarcinoma cells 8988T as compared to normal human pancreatic ductal epithelial cells. Our cross-platform analysis of three different miRNA arrays revealed that RT-PCR-based technologies provide higher inter-platform reproducibility than hybridization-based technologies, a greater sensitivity indicated by larger dynamic ranges and reduced processing time. We also discovered two miRNAs, miRNA 720 and miRNA 29b to play a role in the inhibition of autophagy in 8988T cells and found miRNA 30a to be involved with the inhibition of autophagy in normal human pancreatic ductal epithelial cells. Conclusion: This cross-platform analysis of miRNA arrays contributed significant information about miRNA expression systems and led to the first report of miRNAs implicated in the inhibition of autophagy in pancreatic ductal adenocarcinoma. Our findings should provide new insights for developing therapeutic solutions for this extremely intractable disease.