Mahsa Zarei

HuR Contributes to TRAIL Resistance by Restricting Death Receptor 4 Expression in Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancers, in part, due to resistance to both conventional and targeted therapeutics. TRAIL directly induces apoptosis through engagement of cell surface Death Receptors (DR4 and DR5), and has been explored as a molecular target for cancer treatment. Clinical trials with recombinant TRAIL and DR-targeting agents, however, have failed to show overall positive outcomes. Herein, we identify a novel TRAIL resistance mechanism governed by Hu antigen R (HuR, ELAV1), a stress-response protein abundant and functional in PDA cells. Exogenous HuR overexpression in TRAIL-sensitive PDA cell lines increases TRAIL resistance whereas silencing HuR in TRAIL-resistant PDA cells, by siRNA oligo-transfection, decreases TRAIL resistance. PDA cell exposure to soluble TRAIL induces HuR translocation from the nucleus to the cytoplasm. Furthermore, it is demonstrated that HuR interacts with the 3′-untranslated region (UTR) of DR4 mRNA. Pre-treatment of PDA cells with MS-444 (Novartis), an established small molecule inhibitor of HuR, substantially increased DR4 and DR5 cell surface levels and enhanced TRAIL sensitivity, further validating HuRs role in affecting TRAIL apoptotic resistance. NanoString analyses on the transcriptome of TRAIL-exposed PDA cells identified global HuR-mediated increases in antiapoptotic processes. Taken together, these data extend HuRs role as a key regulator of TRAIL-induced apoptosis. Implications: Discovery of an important new HuR-mediated TRAIL resistance mechanism suggests that tumor-targeted HuR inhibition increases sensitivity to TRAIL-based therapeutics and supports their re-evaluation as an effective treatment for PDA patients. Mol Cancer Res; 14(7); 599–611. ©2016 AACR.

WEE1 inhibition in pancreatic cancer cells is dependent on DNA repair status in a context dependent manner

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease, in part, because of the lack of effective targeted therapeutic options. MK-1775 (also known as AZD1775), a mitotic inhibitor, has been demonstrated to enhance the anti-tumor effects of DNA damaging agents such as gemcitabine. We evaluated the efficacy of MK-1775 alone or in combination with DNA damaging agents (MMC or oxaliplatin) in PDA cell lines that are either DNA repair proficient (DDR-P) or deficient (DDR-D). PDA cell lines PL11, Hs 766T and Capan-1 harboring naturally selected mutations in DNA repair genes FANCC, FANCG and BRCA2 respectively, were less sensitive to MK-1775 as compared to two out of four representative DDR-P (MIA PaCa2 and PANC-1) cell lines. Accordingly, DDR-P cells exhibit reduced sensitivity to MK-1775 upon siRNA silencing of DNA repair genes, BRCA2 or FANCD2, compared to control cells. Only DDR-P cells showed increased apoptosis as a result of early mitotic entry and catastrophe compared to DDR-D cells. Taken together with other recently published reports, our results add another level of evidence that the efficacy of WEE1 inhibition is influenced by the DNA repair status of a cell and may also be dependent on the tumor type and model evaluated.

Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells

Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C > 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR-IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer. Cancer Res; 77(16); 4460-71. ©2017 AACR.

CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States, whereas colorectal cancer is the third most common cancer. The RNA-binding protein HuR (ELAVL1) supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and colorectal cancer tumor cohorts as compared with normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and colorectal cancer (HCT116) cell lines. HuR deficiency has a mild phenotype, in vitro, as HuR-deficient MIA PaCa-2 (MIA.HuR-KO(−/−)) cells had increased apoptosis when compared with isogenic wild-type (MIA.HuR-WT(+/+)) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a two-dimensional culture into three dimensional (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors in vivo compared with control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. Although not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO(−/−)) showed significantly reduced in vivo tumor growth compared with controls (HCT.HuR-WT(+/+)). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes. Implications: The work reported here supports the notion that targeting HuR is a promising therapeutic strategy to treat GI malignancies. Mol Cancer Res; 15(6); 696–707. ©2017 AACR.

Identification of a novel metabolic-related mutation (IDH1) in metastatic pancreatic cancer.

ABSTRACT Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme implicated in cancer cell metabolic reprogramming. This is underscored by the detection of functional, somatic IDH1 mutations frequently found in secondary glioblastoma. To our knowledge, there has never been a reported, validated case of an IDH1 mutation in a pancreatic ductal adenocarcinoma (PDA). Herein, we present a case of a patient with metastatic PDA that harbored a potentially actionable, albeit rare, IDH1 mutation. As part of the Know Your Tumor project (Pancreatic Cancer Action Network), a 48-year-old female was diagnosed with metastatic PDA and subsequently started on standard of care chemotherapy, during which her hepatic lesions progressed. Detailed molecular profiling was performed on a biopsy from a liver lesion that demonstrated an IDH1 mutation, R132H. This mutation was confirmed by an independent sequencing reaction from the tumor sample, and by immunohistochemistry using an antibody specific for the IDH1 R132H mutation. The patient subsequently received a mutant IDH1 inhibitor (AG-120, Agios Pharmaceuticals, Cambridge, MA), but with no response. IDH1 mutations are common in certain cancer types, but have not been reported in PDA. We report the first case of an IDH1 mutation in this tumor type, perhaps providing a rare opportunity for a targeted therapy as a treatment option for PDA.

Abstract 100: Pancreatic cancer cells rely on the NADPH producing enzyme, IDH1, for adaptive survival against acute metabolic stress

We recently demonstrated that pancreatic cancer cells adapt to low nutrient conditions and chemotherapeutic stress through an adaptive response where HuR (ELAVL1) protects cells from oxidative damage induced by metabolic stress. RNA sequencing data and a series of protein-RNA interaction assays proved that HuR stabilizes transcript levels of the NADPH producing enzyme, isocitrate dehydrogenase 1 (IDH1). HuR-knockout cells had near-complete loss of IDH1 expression (manuscript under review). In light of the fact that IDH1-null mice are particularly sensitive to oxidative damage, we hypothesize that this enzyme plays a critical role in PDA survival of acute stress. We examined the expression levels of all eight well-characterized NADPH-generating enzymes in pancreatic cancer cells in vitro, and demonstrate that only IDH1 and phosphogluconate dehydrogenase (PGD) are upregulated by >2-fold after incubation in low glucose (5 mM) for 48 hours. IDH1-knockout MiaPaCa2 cells were generated through CRISPR gene editing, such that mRNA expression was detected at Citation Format: Ali Vaziri-Gohar, Mahsa Zarei, Jonathan R. Brody, Jordan M. Winter. Pancreatic cancer cells rely on the NADPH producing enzyme, IDH1, for adaptive survival against acute metabolic stress [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 100. doi:10.1158/1538-7445.AM2017-100

Abstract B41: Post-transcriptional regulation of IDH1 by the RNA-binding protein HuR is important for pancreatic cancer cell survival under nutrient deprivation

Introduction: Isocitrate dehydrogenase 1(IDH1) has been prioritized in the recent cancer biology literature because of mutations occurring in some tumor types. Additionally the wild type isoenzyme is important for metabolic reprogramming under hypoxic stress. Importantly, the role of IDH1 under other forms of metabolic stress, such as nutrient deprivation (hallmark of the PDA microenvironment) has not been explored, and the regulatory mechanism of IDH1 expression remains unknown. We recently showed that the regulatory RNA binding protein, HuR, binds and directly regulates IDH1 expression in multiple PDA cell lines. The regulatory protein becomes biologically engaged under nutrient deprivation by translocating with the IDH1 transcript form the nucleus to the cytoplasm under glucose deprivation, and HuR silencing sensitized cells under these conditions. Here, we hone in on the regulatory HuR binding site to the IDH1 transcript, explore the importance of HuR in the context of additional metabolic stressors and utilize stable isotope metabolomic profiling to gain mechanistic insight into how HuR is protective under nutrient deprivation. Methods: IDH1 and HuR expression were knocked down by siRNA, and cell viability was determined by PicoGreen and Trypan blue exclusion assays. Immunofluorescence was used to image HuR subcellular localization under glutamine deprivation. Based on computational predictions of 5 HuR binding sites in the IDH1 mRNA 39UTR, we subcloned this entire region into a luciferase reporter construct to further study this regulatory interaction. In order to determine the impact of HuR expression in cellular metabolism targeted tracer fate association studies were performed using GC-MS of pellets from cells cultured with 13C-labeled glucose and glutamine in BxPC3 pancreatic cancer cells that differed only in HuR expression. We calculated Pearson correlations of measured metabolites after HuR silencing. Results: Cell viability was impaired by depletion of both IDH1 and HuR (vs. controls) upon glutamine and glucose withdrawal. Moreover, HuR silencing resulted in potent suppression of IDH1 at the mRNA and protein levels. HuR silencing substantially decremented luciferase activity in the IDH1 39UTR construct compared to the control (> 2-fold decrease). HuR translocated to the cytoplasm under glutamine deprivation, as previously published observed with glucose deprivation by our group. Metabolic tracer fate association studies revealed that HuR silencing impaired carbon flux from glutamine into fatty acid end products (myristate and palmitate, > 0.9-Pearson9s Correlation) under low glucose conditions, implicating HuR9s regulatory role in the IDH1-mediated reductive carboxylation step of this metabolic pathway. Additionally, HuR silencing impaired ribose and glycogen synthesis from glucose, and futile carbon exchange fluxes were prevalent. Conclusions: HuR is important for pancreatic cancer cell survival under glutamine deprivation, as previously observed for glucose deprivation. Carbon flux from glutamine to fatty acid end products suggests a role for HuR in reductive carboxylation of glutamine-derived α-ketoglutarate by IDH1, as a way to maintain adequate lipid synthesis under glucose deprivation. Our Studies provide a rationale to pursue pharmacologic strategies that target HuR or its regulation of IDH1 as a novel treatment of PDA. Citation Format: Mahsa Zarei, Fernando F. Blanco, Laszlo G. Boros, Charles J. Yeo, Jonathan R. Brody, Jordan M. Winter. Post-transcriptional regulation of IDH1 by the RNA-binding protein HuR is important for pancreatic cancer cell survival under nutrient deprivation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B41.

Abstract 2854: CRISPR knockout of HuR in pancreatic cancer cells causes a xenograft lethal phenotype

Pancreatic ductal adenocarcinoma (PDA) is the most prevalent type of pancreatic cancer and will soon become the second leading cause of cancer related deaths in the U.S. Studies show that the nuclear localized mRNA-binding protein HuR (ELAVL1) is activated in PDA cells, with cytoplasmic translocation associated with increased tumor size and poor prognosis. Previous in vitro and in vivo studies have established HuR9s role as a PDA cell survival mechanism. Thus, we explored the phenotypic effect of completely eliminating HuR expression from PDA cells through the use of clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas9 technology to target and disrupt the HuR genomic sequence. Since INDELs are induced randomly, we designed 3 gRNAs to target HuR at different loci. Gene disruption was determined via sequencing and validated through protein and mRNA expression, where homozygous knockouts (HuR −/− ) had undetectable HuR expression as compared to wild-type (HuR +/+ ), heterozygotes (HuR +/− ), and CRISPR/Cas9 negative control. Sanger sequencing confirmed homozygous knockouts with a frame shift mutation on both alleles. When HuR knockout cells were exposed to chemotherapeutic stress including mitomycin C, oxaliplatin, and gemcitabine, no HuR expression (nuclear or cytoplasmic) was detected via immunofluorescence. Phenotypically, HuR −/− cells resulted in increased apoptosis and necrosis as measured via trypan blue assay, and accordingly, had increased caspase 3 activity, a marker of a cell death. HuR −/− cells, when treated with mitomycin C, oxaliplatin, gemcitabine, and glucose deprivation exhibited decreased long and short-term cell survival as compared to control cells. HuR −/− cells, pulse-labeled with bromodeoxyurdine (BrdU), had a higher proportion of cells in S phase and fewer cells in G2/M phase. HuR deletion enhanced premature mitotic entry thereby preventing efficient repair of DNA damage, leading to cell death. Importantly, CRISPR knockout of HuR showed marked impairment in tumor growth in mouse xenografts. The differences in median tumor volume with HuR −/- xenografts was significant as compared to xenografts in mice with HuR (+/+) cells (0.0 mm 3 vs 378.0 mm 3 , P Citation Format: Edwin Cheung, Shruti Lal, Mahsa Zarei, Nicole C. Mambelli-Lisboa, Saswati Chand, Carmella Romeo, Kevin O’Hayer, Eric Londin, Joseph A. Cozzitorto, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. CRISPR knockout of HuR in pancreatic cancer cells causes a xenograft lethal phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2854.

Abstract 1191: The RNA binding protein, HuR, regulates pancreatic cancer cell metabolism

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Few proteins have been found to be master-regulators of pancreatic ductal adenocarcinoma (PDA) cell metabolism (e.g., LKB1, KRAS). We recently demonstrated that the regulatory RNA binding protein, HuR, binds to numerous metabolic mRNA transcripts and regulates their expression. Additionally, HuR silencing with small interfering RNAs(siRNAs) inhibited growth of PDA cells in vitro under glucose deprivation. Herein, we manipulated HuR expression levels in PDA cells and performed isotope tracer fate association studies to better understand HuRs role in metabolic reprogramming. Methods: We used 13C-labeled nutrients to map carbon flux in BxPC3 pancreatic cancer cells that were either transiently transfected with siRNA oligos against HuR (siHuR) or scrambled control (siCTRL). Cells were incubated with standard DMEM or mild glucose deprivation (5 mM glucose) for 48 hours, and media were supplemented with [1,2-13C2]-D-glucose or [U-13C5]-L-glutamine tracers for the final 24 hours. A total of 24 samples were snap frozen and prepared for GC-MS isotopomer analysis (3 replicates X 2 different siRNA oligos X 2 isotope tracers X 2 glucose concentrations). Transfection experiments were validated for reduced HuR expression (>70% reduction) and reduced protein expression of at least one metabolic and established HuR target (e.g., IDH1) by immunoblot. Results: HuR silencing directly impaired fatty acid (Table, line 1-3), ribose (line 5) and glycogen synthesis (line 6). Reductive carboxylation of glutamine-derived isocitrate was impaired and futile carbon exchange fluxes were prevalent (not shown). Conclusions: HuR enhances metabolic efficiency in PDA cells by directly regulating multiple metabolic pathways. Ongoing microarray studies will highlight which metabolic transcripts are post-transcriptionally regulated by HuR, resulting in the observed phenotype. | Altered metabolites due to HuR silencing in PDA cells ([1,2-13C2]-D-glucose tracer) | |:----------------------------------------------------------------------------------- | ---------------------------- | | | Pathway | Metabolite | siHuR 25mM glucose | siHuR 5 mM glucose | Correlation | | 1 | Myristate (C:14) Intracell | 13C enrichment | 70.2 | 71.0 | 1.0 | | 2 | Myristate (C:14) intracell | FNS (direct) | 56.0 | 60.4 | 1.0 | | 3 | Oleate (C:18-1) Intracell | Indirect synthes-m1 | 87.6 | 89.5 | 1.0 | | 4 | Glutam extracell [C2-C5] | 13C-m1 (m/z198) | 97.4 | 97.9 | 1.0 | | 5 | RNA-ribose Intracell [C1-C4] | 13C-m3(m/z242) | 99.3 | 99.1 | 0.984 | | 6 | Glucose intracell [C3-C6] | Peak area, Glycog-gluc | 74.1 | 80.1 | 0.983 | Citation Format: Fernando F. Blanco, Mahsa Zarei, Jonathan R. Brody, Laszlo G. Boros, Jordan M. Winter. The RNA binding protein, HuR, regulates pancreatic cancer cell metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1191. doi:10.1158/1538-7445.AM2015-1191