Mechanisms Of Gemcitabine Resistance Research Articles

Epigenetic Activation of the CMTM6-IGF2BP1-EP300 Positive Feedback Loop Drives Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a dismal prognosis. Gemcitabine-based chemotherapy has emerged as a first-line treatment for PDAC. However, the development of gemcitabine resistance often results in therapeutic failure. In order to uncover the underlying mechanisms of gemcitabine resistance, gemcitabine-resistant PDAC cell lines and patient-derived xenograft (PDX) models are established and subjected to RNA sequencing. It is found that CMTM6 is closely related to gemcitabine resistance in PDAC. Multi-omics analysis revealed that EP300-mediated H3K27ac modification is involved in the transcriptional activation of CMTM6, which maintains IGF2BP1 expression by preventing its ubiquitination. The m6A reader IGF2BP1 stabilizes the EP300 and MYC mRNAs by recognizing m6A modifications, forming a positive feedback loop that enhances tumor stemness and ultimately contributes to PDAC resistance. The combined application of the EP300 inhibitor inobrodib and gemcitabine exerts a synergistic effect on PDAC. Overall, these findings reveal that the EP300-CMTM6-IGF2BP1 positive feedback loop facilitates gemcitabine resistance via epigenetic reprogramming and the combined use of inobrodib and gemcitabine represents a promising strategy for overcoming chemoresistance in PDAC, warranting further investigation in clinical trials.

The interaction between UBR7 and PRMT5 drives PDAC resistance to gemcitabine by regulating glycolysis and immune microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is a common malignant tumor of the digestive tract. Although gemcitabine and other therapeutic agents are effective in patients with advanced and metastatic pancreatic cancer, drug resistance has severely limited their use. However, the mechanisms of gemcitabine resistance in pancreatic cancer are poorly understood. In this study, ATAC-seq, ChIP-seq, and RNA-seq were performed to compare chromatin accessibility and gene expression in a patient-derived tumor xenograft (PDX) model of pancreatic cancer with or without gemcitabine resistance. Analyzing these sequencing data, we found a dramatic change in chromatin accessibility in the PDX model of gemcitabine-resistant tissues and identified a key gene, UBR7, which plays an important role in mediating gemcitabine resistance. Further research found that depletion of UBR7 significantly increased pancreatic carcinogenesis and the immunosuppressive microenvironment. Mechanistically, depleted UBR7 increased the stability of PRMT5, thereby promoting glycolysis in pancreatic cancer cells. Finally, an inhibitor that blocks PRMT5 (DS-437) significantly reduced gemcitabine resistance in pancreatic cancer caused by UBR7 depletion. In conclusion, our results illustrate that the UBR7-PRMT5 axis is a key metabolic regulator of PDAC and a promising target for the clinical treatment of gemcitabine resistance in PDAC.

Effect of UBR7 deficiency on glycolysis and PDAC resistance to gemcitabine by promoting PRMT5 stability.

e16265 Background: Pancreatic ductal adenocarcinoma (PDAC) is a common malignant tumor of the digestive tract. Although gemcitabine and other therapeutic agents are effective in patients with advanced and metastatic pancreatic cancer, drug resistance has severely limited their use. However, the mechanisms of gemcitabine resistance in pancreatic cancer are poorly understood. Methods: In this study, ATAC-seq, ChIP-seq, and RNA-seq were performed to compare chromatin accessibility and gene expression in a patient-derived tumor xenograft (PDX) model of pancreatic cancer with or without gemcitabine resistance. Results: Analyzing these sequencing data, we found a dramatic change in chromatin accessibility in the PDX model of gemcitabine-resistant tissues and identified a key gene, UBR7, which plays an important role in mediating gemcitabine resistance. Further research found that depletion of UBR7 significantly increased pancreatic carcinogenesis and the immunosuppressive microenvironment. Mechanistically, depleted UBR7 increased the stability of PRMT5, thereby promoting glycolysis in pancreatic cancer cells. Finally, an inhibitor that blocks PRMT5 (DS-437) significantly reduced gemcitabine resistance in pancreatic cancer caused by UBR7 depletion. Conclusions: In conclusion, our results indicated that the UBR7-PRMT5 axis is a key metabolic regulator of PDAC and a promising target for the clinical treatment of gemcitabine resistance in PDAC.

ELF3 promotes gemcitabine resistance through PKMYT1/CDK1 signaling pathway in gallbladder cancer.

Gemcitabine is the standard treatment for gallbladder cancer (GBC) patients, and the development of resistance frequently limits its efficacy. However, the molecular features and mechanisms of gemcitabine resistance (Gem-R) in GBC cells remain unknown. Herein, we aimed to explore the role of ELF3 in Gem-R of GBC, including the underlying mechanisms. RNA sequencing was used to screen the essential genes related to the generation of Gem-R in GBC tissues. The correlation between Gem-R and ELF3 expression was identified in GDSC, GEO database, GBC tissues, and 3 GBC cell lines. Immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blot were used to examine the expression of ELF3, PKMYT1, and CDK1. Luciferase reporter assays were used to identify the binding site of ELF3 in the PKMYT1 promoter region. CCK-8 assay and clonogenic survival assays were used to evaluate the sensitivity of gemcitabine in GBC cells. A GBC xenograft model was used to evaluate the influence of ELF3 on the therapeutic effect of gemcitabine. A consistently positive correlation between ELF3 expression and Gem-R, both in newly generated GBC RNA-seq data and in the datasets from GDSC and GEO. Gem-R in GBC cells was facilitated by ELF3 overexpression, whereas ELF3 knockdown had the opposite effect. In vivo experiments further proved that reducing ELF3 expression promoted the gemcitabine sensitivity of GBC cells and extended the survival time of mice that received orthotopic xenografted tumors. Mechanistically, ELF3 upregulated PKMYT1 expression by interacting with the DNA binding region of PKMYT1 in GBC cells, thereby promoting the phosphorylation of CDK1 and inducing Gem-R. Treatment with a combination of the PKMYT1 shRNA and gemcitabine significantly reduced the growth of GBC cells induced by overexpression of ELF3 in vitro and in vivo. ELF3/PKMYT1/CDK1 axis significantly regulates Gem-R to GBC cells and may represent a promising drug target for treating GBC patients.

MiR-520a-5p/PPP5C regulation pattern is identified as the key to gemcitabine resistance in pancreatic cancer.

ObjectiveTo explore the effects of the expression level of miR-520-5p/PPP5C in pancreatic cancer cells and exosomes on cell viability, angiogenesis, autophagy, which involved in the mechanism of gemcitabine resistance in pancreatic cancer.MethodsAPSC-1 cell line was treated with gemcitabine, after which its exosomes were extracted for NTA assay. Subsequently, the drug resistance of APSC-1 cells was assayed using CCK8, as well as the activity of HUVEC cells treated with exosomes from each group of APSC-1 cells after drug resistance treatment as well as overexpression treatment. Five groups of HUVEC cells treated with exosomes were subjected to in vitro tubule formation assay. levels of PPP5C in each group of ASPC-1 cells and their exosomes, levels of overexpressed PPP5C, and related exosomal proteins were examined by WB. mRNA expression levels of PPP5C and levels of miR-520a were examined by qPCR The relationship between miR-520a-5p and PPP5C was investigated. After that, the autophagy of PPP5C was detected. Finally, it was analyzed by TCGA database for survival prognosis analysis.ResultsAPSC-1 cells had an IC50 value of 227.1 μM for gemcitabine, elevated PPP5C expression, drug resistance, and enhanced HUVEC cell activity; exosomes CD9, CD63, and CD81 were significantly expressed in all groups; meanwhile, enhanced PPP5C expression not only promoted in vitro tubule formation but also increased autophagy levels; meanwhile, its relationship with miR-520-5p and There was a targeted inhibitory relationship between its level and miR-520-5p and PPP5C, and its elevated level also led to a decrease in the survival level of patients over 3-5 years.ConclusionPPP5C has a prognostic role in pancreatic cancer by promoting the value-added and invasion of pancreatic cancer cells, and a targeted inhibitory relationship between miR-520-5p and PPP5C was found.

Tumor microenvironment and metabolic remodeling in gemcitabine-based chemoresistance of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a solid malignant tumor with a very low operative rate and a poor patient prognosis. Therefore, gemcitabine (GEM)-based chemotherapy remains one of the most important treatment choices for PDAC. However, the efficacy of GEM monotherapy or GEM combination chemotherapy in improving the survival of patients with advanced PDAC is very limited, primarily due to GEM resistance. The mechanism of GEM resistance is complex and unclear. An extensive and dense fibrous matrix in the tumor microenvironment (TME) is an important feature of PDAC. Increasing evidence indicates that this fibrotic TME not only actively participates in the growth and spread of PDAC but also contributes to the induction of GEM resistance. Metabolic remodeling reduces GEM transport and synthesis in PDAC. This review focuses on the main cellular and molecular mechanisms underlying the involvement of the extracellular matrix (ECM), immune cells, and metabolic remodeling in the induction of GEM resistance; highlights the prospect of targeting the TME as an essential strategy to overcome GEM resistance; and provides new precise interventions for chemotherapy sensitization and improving the overall prognosis of patients with PDAC.

Possible Role of Cytochrome P450 1B1 in the Mechanism of Gemcitabine Resistance in Pancreatic Cancer.

Patient-derived xenograft models reportedly represent original tumor morphology and gene mutation profiles. In addition, patient-derived xenografts are expected to recapitulate the parental tumor drug responses. In this study, we analyzed the pathways involved in gemcitabine resistance using patient-derived xenograft models of pancreatic cancer. The patient-derived xenograft models were established using samples from patients with pancreatic cancer. The models were treated with gemcitabine to better understand the mechanism of resistance to this anti-cancer drug. We performed comparative gene analysis through the next-generation sequencing of tumor tissues from gemcitabine-treated or non-treated patient-derived xenograft mice and gene set enrichment analysis to analyze mRNA profiling data. Pathway analysis of gemcitabine-treated patient-derived xenografts disclosed the upregulation of multiple gene sets and identified several specific gene pathways that could potentially be related to gemcitabine resistance in pancreatic cancer. Further, we conducted an in vitro analysis to validate these results. The mRNA expression of cytochrome P450 1B1 and cytochrome P450 2A6 was upregulated in a concentration-dependent manner following gemcitabine treatment. Moreover, the sensitivity to gemcitabine increased, and viable cells were decreased by the cytochrome P450 1B1 inhibitor, indicating that the cytochrome P450 1B1 pathway may be related to gemcitabine resistance in pancreatic cancer.

A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient.

Simple SummaryIntrahepatic cholangiocarcinoma (ICC) has limited prognosis and therapies. The first-line gemcitabine-based therapy provided poor benefits in terms of survival due to the development of resistance. Gemcitabine-resistance mechanisms were studied on pancreatic cancer models or cell lines derived from ICC patients of Eastern countries. Since ICC has different etiology and genetic/molecular characteristics depending on the ethnicity, appropriate preclinical models that recapitulate their biology are required. Thus, we aimed to establish and characterize an Italian ICC cell line, named 82.3. Cells were isolated from a patient-derived xenograft. After one year, immunophenotypical, biological, genetic, molecular features, and in vivo tumorigenicity in NOD/SCID mice were investigated. Furthermore, 82.3 cells displayed resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin. This model could be exploited either to investigate drug resistance mechanisms or to test alternative drugs through the identification of suitable targets to overcome drug resistance.Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α–fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.

Establishment and characterization of the gemcitabine-resistant human pancreatic cancer cell line SW1990/gemcitabine.

Due to its rapid progression, metastasis and resistance to chemotherapy, pancreatic cancer is one of the most malignant tumor types to affect the digestive system. Gemcitabine chemotherapy is typically the first choice of treatment for advanced pancreatic cancer; however, chemoresistance is a major obstacle to successful treatment. In order to elucidate the underlying mechanisms of gemcitabine resistance in pancreatic cancer, the drug-resistant cell line SW1990-gemcitabine (SW1990-GZ) was established using the human pancreatic cancer cell line SW1990. The IC50, resistance index and growth of SW1990 and SW1990-GZ cells were also assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays. The cellular uptake of gemcitabine in SW1990 and SW1990-GZ was measured using high performance liquid chromatography (HPLC). The protein expression of p53 was also assessed by western blot analysis. The results demonstrated that the IC50 of SW1990 and SW1990-Gz was 0.07±0.0021 and 87.5±3.24 µg/ml, respectively, and that the resistance index ratio of SW1990-Gz was 1,250. The growth rate of SW1990-GZ cells was low compared with that of SW1990 cells. The HPLC results indicated that gemcitabine uptake was markedly reduced in SW1990-GZ cells compared with in SW1990 cells at different time points. The protein expression of p53 was significantly higher in GEM-resistant SW1990-GZ cells compared with that in SW1990 cells (P<0.01). These results suggest that a human gemcitabine-resistant pancreatic cancer cell line was successfully established, with stable and significant drug resistance. The results of the present study suggest that the decreased cellular uptake of gemcitabine may serve an important role in gemcitabine chemoresistance in SW1990-GZ cells; thus, this cell line may be used as an effective in vitro model to improve our understanding of gemcitabine-resistance in pancreatic cancer.

Abstract 1283: Targeting c-MYC and MAPK pathway to overcome pancreatic cancer drug resistance

Abstract Background: Acquired resistance to systemic chemotherapy is the main complication in pancreatic ductal adenocarcinoma (PDAC) treatment. Although there are studies focused on gemcitabine resistance mechanisms, our understanding of the mechanisms of nab-paclitaxel (n-PTX) treatment failure remains extremely limited. To enhance the use of properly powered patient-derived platforms, we adopted the conditionally reprogrammed (CR) cell culture technique in order to develop both parental and nab-PTX-resistant cells. The CR approach allowed us to identify the critical role of c-MYC and ERK in the PDAC drug response. Small molecule activators of PP2A (SMAPS) have showed activity in inhibiting lung KRAS-mutant tumor growth. We used SMAPS as new therapeutic agents in PDAC, for its ability to alter c-MYC activity through PP2A dysregulation and enhance PDAC sensitivity to n-PTX. Methods: Long-term cultures of PDAC CRs were established from treatment-naive PDAC patients’ biopsies, and used to generate drug-resistant cells. Zebrafish and mouse model were used to test the cells’ ability to form tumors and to verify the drug resistance in vivo. Molecular analyses were used to characterize the drug-resistant cells and to identify key pathways involved in the drug resistance evolution. Genomic and chemical alterations of the key proteins were used to confirm the involvement in the drug resistance mechanism. We regulated the expression of c-MYC and ERK using SMAPS as a new targeting agent and trametenib to verify the direct correlation between c-MYC and ERK and the drug resistance mechanism. Results: Using the credentialed KRAS-mutant CR cultures, we generated n-PTX-resistant cell lines. The parental and nab-PTX resistant cells were subjected to subcutaneous injections in nude mice, and formed tumors in 2-3 weeks. Histological evaluation showed that the CRs self-assembled into ductal structures, surrounded by a desmoplastic stromal microenvironment that faithfully recapitulates human PDAC. Resistant profiles were verified both in mouse and Zebrafish model. RNA microarrays identified a sustained induction of a pro-inflammatory pathway leading to c-MYC overexpression. c-MYC silencing and overexpression confirmed the role of c-MYCin the evolution of nab-PTX resistance. Treatment of the resistant CRs with either trametenib or with SMAPS resulted in enhanced sensitivity to nab-PTX. We furtherverified that the enhanced sensitivity was commensurate with a reduction in p-Erk and c-Myc. Conclusion: The CR methodology addresses the need for a reliable method for generating primary cell lines on a single patient basis. The ability to rapidly model in vitro, and verify in vivo, that the overexpression of c-MYC contributes to the development of n-PTX resistance is a significant advancement in the field. Our data showed that SMAPs or trametanib overcome a significant component of the n-PTX resistance providing new hope for refractory PDAC. Citation Format: Erika Maria Parasido, George S. Avetian, Jonathan Brody, Jordan Winter, Eric Londin, Michael Pishvaian, Eric Glasgow, Stephen Byers, Goutham Narla, Christopher Albanese. Targeting c-MYC and MAPK pathway to overcome pancreatic cancer drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1283.

Harmine suppresses the proliferation of pancreatic cancer cells and sensitizes pancreatic cancer to gemcitabine treatment.

PurposePancreatic carcinoma is one of the most deadliest types of cancer, and relatively insensitive to the currently available chemotherapy. Thus, the discovery of novel therapeutic agents to prolong the survival times of patients with pancreatic cancer is urgently required.MethodsCell proliferation was assessed using the sulforhodamine B and cell clone formation assay, apoptosis was analyzed through Annexin V/PI staining, analysis of cell cycle distribution was determined by PI staining, and the expression of proteins was detected via Western blotting.ResultsOur data showed that harmine exerted an anti-proliferative effect and cell cycle arrest at G2/M in pancreatic cancer cells. Meanwhile, harmine plus gemcitabine showed strong synergy in inhibiting the proliferation of pancreatic cancer cells. Furthermore, harmine induced apoptosis and enhanced the gemcitabine-induced apoptosis in pancreatic cancer cells. The AKT/mTOR pathway is involved in mechanisms of gemcitabine resistance in pancreatic cancer cells, our data demonstrated that harmine plus gemcitabine significantly suppressed the AKT/mTOR signaling pathway.ConclusionHarmine may be a potential candidate for the treatment of pancreatic cancer. Morever, the combination of harmine with gemcitabine appears to be an attractive option for the treatment of patients with pancreatic cancer.

Circular RNA Signature Predicts Gemcitabine Resistance of Pancreatic Ductal Adenocarcinoma.

Gemcitabine resistance is currently the main problem of chemotherapy for advanced pancreatic cancer patients. The resistance is thought to be caused by altered drug metabolism or reduced apoptosis of cancer cells. However, the underlying mechanism of Gemcitabine resistance in pancreatic cancer remains unclear. In this study, we established Gemcitabine resistant PANC-1 (PANC-1-GR) cell lines and compared the circular RNAs (circRNAs) profiles between PANC-1 cells and PANC-1-GR cells by RNA sequencing. Differentially expressed circRNAs were demonstrated using scatter plot and cluster heatmap analysis. Gene ontology and pathway analysis were performed to systemically map the genes which are functionally associated to those differentially expressed circRNAs identified from our data. The expression of the differentially expressed circRNAs picked up by RNAseq in PANC-1-GR cells was further validated by qRT-PCR and two circRNAs were eventually identified as the most distinct targets. Consistently, by analyzing plasma samples form pancreatic ductal adenocarcinoma (PDAC) patients, the two circRNAs showed more significant expression in the Gemcitabine non-responsive patients than the responsive ones. In addition, we found that silencing of the two circRNAs could restore the sensitivity of PANC-1-GR cells to Gemcitabine treatment, while over-expression of them could increase the resistance of normal PANC-1 and MIA PACA-2 cells, suggesting that they might serve as drug targets for Gemcitabine resistance. Furthermore, the miRNA interaction networks were also explored based on the correlation analysis of the target microRNAs of these two circRNAs. In conclusion, we successfully established new PANC-1-GR cells, systemically characterized the circRNA and miRNA profiles, and identified two circRNAs as novel biomarkers and potential therapeutic targets for Gemcitabine non-responsive PDAC patients.

Different mechanisms of gemcitabine resistance in Suit-2 cell lines and double stranded DNA Breaks

Different mechanisms of gemcitabine resistance in Suit-2 cell lines and double stranded DNA Breaks

Two contrasting gemcitabine resistance mechanisms involving ribonucleotide reductase and cyclin D1b

Two contrasting gemcitabine resistance mechanisms involving ribonucleotide reductase and cyclin D1b

Leveraging transcriptomic data to pinpoint mechanisms of gemcitabine resistance and potential combination therapies for pancreatic cancer.

e15730Background: Pancreatic cancer has an estimated 5-year survival rate of only 5-6% and is often diagnosed after metastasis has occurred. Gemcitabine or gemcitabine combination therapies are fre...

Abstract A49: Carcinoma-associated fibroblasts in the tumor microenvironment affect growth, invasiveness, and drug response of human pancreatic cancer cells

Abstract Pancreatic cancer is one of the most aggressive malignancies, with a 5-year overall survival of less than 5%. Tumor drug resistance to conventional chemotherapy, such as gemcitabine, is often a significant contributor to poor overall survival. One of the common mechanisms of gemcitabine resistance is activation of cell signaling via increased phosphorylation of Mitogen-Activated kinase (MAP) kinases, leading to increased tumor survival and reduced sensitivity to chemotherapeutic agents. A growing body of evidence suggests that the CXCL12/CXCR4 signal transduction axis in the tumor microenvironment is an important mediator of tumor migration, growth, and drug resistance. We hypothesized that carcinoma-associated fibroblasts (CAFs), an important cellular component of the TME, play a contributory role in the growth, invasiveness, and drug response of pancreatic cancer cells (PCCs) by activating CXCL12/CXCR4-mediated signal transduction. To test this, we used an in vitro model system to study the growth, invasion, and drug response of human PCCs in the presence or absence of in vitro-generated CAFs. Functional studies demonstrated that co-culture of PCCs with CAFs led to significant increase in tumor cell invasion, which was abrogated by blockade of CXCR4 by AMD3100-plerixafor (AMD3100), a CXCR4 antagonist, and by siRNA-mediated knockdown of CXCR4 in CAFs. Increased invasion of PCCs after co-culture with orthotopically derived CAFs was also abrogated by CXCR4 blockade, demonstrating a critical role for this receptor in regulating the tumor-promoting abilities of CAFs. Additionally, we investigated the effect of CAFs on the MAPK Signaling pathway (MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, Akt, and p-Akt) via Western blot assays. Further, we examined the effect of AMD3100 on the chemo-resistance of PCCs to gemcitabine. Our results indicated that AMD3100 reversed gemcitabine-mediated chemo-resistance of PCCs cells in the presence of CAFs or CAF-conditioned media. In co-culture with pancreatic cancer cells, CAFs induced activation of MAPK signaling pathways and enhanced transcription of Mn-SOD, G6PD, and catalase, genes involved in reactive oxygen species (ROS) pathways. Using phloretin, a ROS inhibitor, we observed attenuation of MAPK signaling, SOD activity, and reversal of drug resistance in CAF-exposed pancreatic cancer cells. These findings implicate CXCL12/CXCR4-dependent MAPK signaling and ROS pathways in CAF-mediated modulation of the growth, migration, and drug resistance of PCCs. Citation Format: Ahlim Al. Sanani, Brijsh Patel, Alix Duarte, Nitu Bansal, Tushar Bhagat, Yanique Rattigan, Anirban Maitra, Amit Verma, Debabrata Banerjee. Carcinoma-associated fibroblasts in the tumor microenvironment affect growth, invasiveness, and drug response of human pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A49.

Bitter melon juice targets molecular mechanisms underlying gemcitabine resistance in pancreatic cancer cells.

Pancreatic cancer (PanC) is one of the most lethal malignancies, and resistance towards gemcitabine, the front-line chemotherapy, is the main cause for dismal rate of survival in PanC patients; overcoming this resistance remains a major challenge to treat this deadly malignancy. Whereas several molecular mechanisms are known for gemcitabine resistance in PanC cells, altered metabolism and bioenergetics are not yet studied. Here, we compared metabolic and bioenergetic functions between gemcitabine-resistant (GR) and gemcitabine-sensitive (GS) PanC cells and underlying molecular mechanisms, together with efficacy of a natural agent bitter melon juice (BMJ). GR PanC cells showed distinct morphological features including spindle-shaped morphology and a decrease in E-cadherin expression. GR cells also showed higher ATP production with an increase in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Molecular studies showed higher expression of glucose transporters (GLUT1 and 4) suggesting an increase in glucose uptake by GR cells. Importantly, GR cells showed a significant increase in Akt and ERK1/2 phosphorylation and their inhibition decreased cell viability, suggesting their role in survival and drug resistance of these cells. Recently, we reported strong efficacy of BMJ against a panel of GS cells in culture and nude mice, which we expanded here and found that BMJ was also effective in decreasing both Akt and ERK1/2 phosphorylation and viability of GR PanC cells. Overall, we have identified novel mechanisms of gemcitabine resistance in PanC cells which are targeted by BMJ. Considering the short survival in PanC patients, our findings could have high translational potential in controlling this deadly malignancy.

Abstract 698A: Bitter melon juice targets molecular mechanisms underlying gemcitabine resistance in pancreatic cancer cells

Abstract Pancreatic cancer is an aggressive disease and is usually advanced at the time of diagnosis. Median life of pancreatic cancer patients post-diagnosis is &amp;lt;6 months and an overall 5-year survival rate is 3-5%. In 2013, ∼44,980 new cases of pancreatic cancer are expected in United States with ∼38,460 deaths. In most cases, disease relapse occurs due to development of resistance towards gemcitabine (the frontline therapy for pancreatic cancer) and is the main cause for dismal rate of survival in pancreatic cancer patients. Therefore, overcoming gemcitabine resistance remains a major challenge towards effectively treating this deadly malignancy. Several molecular mechanisms are known to contribute to gemcitabine-resistance in pancreatic cancer; however, metabolism and bioenergetic alterations in gemcitabine-resistant pancreatic cancer cells have not yet been characterized. Here, we analyzed and compared the metabolic and bioenergetic functions between gemcitabine-resistance and sensitive pancreatic cancer cells along with underlying molecular mechanisms. Furthermore, we also assessed the anticancer efficacy of a natural agent bitter melon (Momordica charantia, Family: Cucurbitaceae) juice (BMJ) against gemcitabine-resistant pancreatic cancer cells. Results showed that gemcitabine-resistant human pancreatic adenocarcinoma AsPC-1 and MiaPaCa-2 cells have distinct morphological features including spindle-shaped morphology and a decrease in E-cadherin expression. Interestingly, gemcitabine-resistant cells showed higher ATP production along with an increase in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) confirming an increase in both cellular oxidative phosphorylation and glycolysis. Molecular studies showed a higher expression of glucose transporters GLUT1 and GLUT4 suggesting an increase in glucose uptake by gemcitabine-resistant cells. Importantly, gemcitabine-resistant cells exhibited a significant increase in Akt and ERK1/2 phosphorylation, and Akt and/or ERK1/2 inhibition by specific chemical inhibitors significantly decreased the gemcitabine-resistant cells viability, suggesting the critical role of these molecules in the survival and drug resistance of these cells. Recently, BMJ has shown strong efficacy in a panel of gemcitabine-sensitive cells in culture and nude mice, which we expanded here and found that BMJ was also effective in strongly decreasing both Akt and ERK1/2 phosphorylation and viability of gemcitabine-resistant pancreatic cancer cells. Overall, we have identified novel mechanisms of gemcitabine-resistance in pancreatic cancer cells that are targeted by BMJ. Considering a short survival-rate in pancreatic cancer patient, our findings could have high translational potential in controlling this deadly malignancy. Citation Format: Ranganatha Somasagara, Gagan Deep, Sangeeta Shrotriya, Manisha Patel, Chapla Agarwal, Rajesh Agarwal. Bitter melon juice targets molecular mechanisms underlying gemcitabine resistance in pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 698A. doi:10.1158/1538-7445.AM2014-698A

Abstract 896: miR-198 silencing is a mechanism of gemcitabine resistance in human pancreatic adenocarcinoma.

Abstract Pancreatic adenocarcinoma (PAC) is a very aggressive disease with extremely high mortality rate. Chemotherapy with gemcitabine (G) is a standard of care for advanced disease, but chemotherapy resistance is commonly noted. Human PAC is characterized by overexpression of the proto-oncogene c-met. Increased activation of c-met has been reported in G-resistant PAC cells. Also, aberrant expression of microRNAs (miRNAs) has been linked to cancer progression. Furthermore, a recent study showed that miR-198 regulates c-met expression by binding to the 3′-untranslated region of c-met mRNA. However, the role of miR-198 in chemotherapy resistance in PAC has not previously been studied. Consequently, in this study we used the G-sensitive and G-resistant subclones of the L3.6pl human PAC cell line to examine the role of miR-198 in G-resistant PAC. We observed that the G-resistant subclone had increased expression of c-met (using qPCR and western blotting) and phosphorylated c-met (using western blotting), but not hepatocyte growth factor (HGF). Furthermore, using qPCR we observed significant silencing of miR-198 expression in the G-resistant subclone in comparison to the G-sensitive subclone (P &amp;lt; 0.01). To determine the functional significance of miR-198 silencing in G resistance, we silenced miR-198 expression in the G-sensitive subclone by transfection of a specific oligonucleotide inhibitor of miR-198. We observed that miR-198 silencing resulted in increased expression of c-met and phosphorylated c-met. Further, miR-198 silencing on its own increased total RNA concentration significantly by 146% (P &amp;lt; 0.05) and cell viability by 14.3% (P &amp;lt; 0.05; using MTT assay), both suggestive of a proliferative effect. However, significant effects on apoptosis were undetectable (using Hoechst nuclear staining). Furthermore, miR-198 silencing significantly abrogated G-induced apoptosis (P &amp;lt; 0.05) and G-induced reduction of cell viability (P &amp;lt; 0.05). We conclude that G-resistant human PAC cells have silencing of miR-198. Moreover, miR-198 silencing in PAC cells induces increased expression and phosphorylation of c-met, increased cell proliferation and abrogation of G-induced apoptosis and induction of proliferation. Therefore, miR-198 silencing is a mechanism of G-resistance in PAC. Furthermore, therapeutic strategies to enhance miR-198 expression may be beneficial in PAC. Citation Format: Olorunseun O. Ogunwobi, Jian Li, Xiangxuan Zhao, Jose Trevino, Thomas George, Chen Liu. miR-198 silencing is a mechanism of gemcitabine resistance in human pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 896. doi:10.1158/1538-7445.AM2013-896

Abstract 3426: Curcumin is effective in killing gemcitabine resistant pancreatic cancer cells: Studies of the cell cycle, and apoptosis.

Abstract Background Gemcitabine resistance is a major challenge in pancreatic cancer where the drug is considered a “gold standard” treatment. Although many studies have attempted to address mechanisms of gemcitabine resistance, these are not yet clear. The complete elimination of these resistant cells will be a major breakthrough in treatment. In many cancers, curcumin is of interest due to its inherently low toxicity to patients even at high doses: The drug can inhibit pancreatic cancer cell growth, and also acts in synergy with gemcitabine reducing viability of several established cancer cell lines. Identifying curcumins efficacy after transition from gemcitabine sensitive to resistant disease is therefore a key question for progress toward treatment. We have created a novel cell line from a parental MIA-PaCa-2 pancreatic adenocarcinoma which is 286 times more resistant to gemcitabine exposure than the parent line. Here we investigated the cell cycling properties of this cell line and its ability to be killed by curcumin. Methods The gemcitabine resistant MIA-PaCa-2 cell line was created by continuous gemcitabine dosing which increased its EC50 for the drug to 286-fold greater than that of the parent line. The extent of resistance was confirmed by MTT for viability and BrdU ELISA for proliferation. The sensitive and resistant Mia-PaCa-2 cells were then incubated in the absence, or presence of varying doses of curcumin (0.01-150μM). Cells were analysed with respect to their cell cycling profiles by staining initially with EdU over a time course, followed by 7-AAD DNA staining of permeabilised cells with subsequent flow cytometric analysis. Early apoptotic, late apoptotic and necrotic fractions of these cells were determined by staining with annexin-V and propidium iodide. Results: Curcumin inhibited growth of both gemcitabine sensitive and resistant cell lines to a similar extent (IC50s =13.7 μM and 16.6 μM for sensitive and resistant lines respectively). Whereas gemcitabine was incapable of causing apoptosis in the resistant strain, curcumin induced similar levels of apoptosis in both lines at doses between 1 and 56μM. With respect to the cell cycle, gemcitabine caused complete S-phase arrest in the sensitive strain and had no effect on the resistant strain, whereas curcumin caused G2/M phase arrest (no increase in G1 and 100% loss of second cycle S-phase) in both cell lines. Conclusions Curcumin is effective as a chemotherapeutic drug against gemcitabine resistant cells due to its ability to inhibit the cell cycle at the G2/M boundary after gemcitabine fails to activate the restriction point. The ability of curcumin to avoid cross resistance after gemcitabine is rendered ineffective should be studied as this could provide mechanisms for the creation of new drugs or highlight the possibilities of curcumin analogues as a second-line therapeutic for the placation of pancreatic adenocarcinoma. Citation Format: Alan Levett, Rachel Allen, Christine Galustian, Angus George Dalgleish. Curcumin is effective in killing gemcitabine resistant pancreatic cancer cells: Studies of the cell cycle, and apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3426. doi:10.1158/1538-7445.AM2013-3426