Gemcitabine-resistant Pancreatic Cancer Cells Research Articles

Identifying microRNA-mRNA regulatory network in gemcitabine-resistant cells derived from human pancreatic cancer cells.

Pancreatic cancer is unresectable in over 80 % of patients owing to difficulty in early diagnosis. Chemotherapy is the most frequently adopted therapy for advanced pancreatic cancer. The development of drug resistance to gemcitabine (GEM), which is always used in standard chemotherapy, often results in therapeutic failure. However, the molecular mechanisms underlying the gemcitabine resistance remain unclear. Therefore, we sought to explore the microRNA-mRNA network that is associated with the development of gemcitabine resistance and to identify molecular targets for overcoming the gemcitabine resistance. By exposing SW1990 pancreatic cancer cells to long-term gemcitabine with increasing concentrations, we established a gemcitabine-resistant cell line (SW1990/GEM) with a high IC50 (the concentration needed for 50 % growth inhibition, 847.23 μM). The mRNA and microRNA expression profiles of SW1990 cells and SW1990/GEM cells were determined using RNA-seq analysis. By comparing the results in control SW1990 cells, 507 upregulated genes and 550 downregulated genes in SW1990/GEM cells were identified as differentially expressed genes correlated with gemcitabine sensitivity. Gene ontology (GO) analysis showed that the differentially expressed genes were related to diverse biological processes. The upregulated genes were mainly associated with drug response and apoptosis, and the downregulated genes were correlated with cell cycle progression and RNA splicing. Concurrently, the differentially expressed microRNAs, which are the important player in drug resistance development, were also examined in SW1990/GEM cells, and 56 differential microRNAs were identified. Additionally, the expression profiles of selected genes and microRNAs were confirmed by using Q-PCR assays. Furthermore, combining the differentially expressed microRNAs and mRNAs as well as the predicted targets for these microRNAs, a core microRNA-mRNA regulatory network was constructed, which included hub microRNAs, such as hsa-miR-643, hsa-miR-4644, hsa-miR-4650-5p, hsa-miR-4455, hsa-miR-1261, and hsa-miR-3676. The predicted targets of these hub microRNAs in the microRNA-mRNA network were also observed in the identified differential genes. As a result, a differential gene and microRNA expression pattern was constructed in gemcitabine-resistant pancreatic cancer cells. Therefore, these data may be useful for the detection and treatment of drug resistance in pancreatic cancer patients, and the microRNA-mRNA network-based analysis is expected to be more effective and provides deep insights into the molecular mechanism of drug resistance.

Ribonucleotide reductase is an effective target to overcome gemcitabine resistance in gemcitabine-resistant pancreatic cancer cells with dual resistant factors

Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.

Down-regulation of miR-223 reverses epithelial-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells.

Recent studies have demonstrated that acquisition of epithelial-to-mesenchymal transition (EMT) is associated with drug resistance in pancreatic cancer cells; however, the underlying mechanisms are not fully elucidated. Emerging evidence suggests that microRNAs play a crucial role in controlling EMT. The aims of this study were to explore the potential role of miR-223 in governing EMT in gemcitabine-resistant (GR) pancreatic cancer cells. To achieve this goal, real-time reverse transcription-PCR and western blot analysis were used to validate whether GR cells acquired EMT in AsPC-1 and PANC-1 cells. Invasion, migration, and detachment assays were performed to further identify the EMT characteristics in GR cells. The miR-223 inhibitor was used to determine its role in GR-induced EMT. We found that GR cells acquired EMT features, which obtained elongated fibroblastoid morphology, decreased expression of epithelial marker E-cadherin, and up-regulation of mesenchymal markers. Furthermore, we observed that GR cells are associated with high expression of miR-223. Notably, inhibition of miR-223 led to the reversal of EMT phenotype. More importantly, miR-223 governs GR-induced EMT in part due to down-regulation of its target Fbw7 and subsequent upregulation of Notch-1 in pancreatic cancer. Our study implied that down-regulation of miR-223 could be a novel therapy for pancreatic cancer.

Ratio of phosphorylated HSP27 to nonphosphorylated HSP27 biphasically acts as a determinant of cellular fate in gemcitabine-resistant pancreatic cancer cells

Gemcitabine has been used most commonly as an anticancer drug to treat advanced pancreatic cancer patients. However, intrinsic or acquired resistance of pancreatic cancer to gemcitabine was also developed, which leads to very low five-year survival rates. Here, we investigated whether cellular levels of HSP27 phosphorylation act as a determinant of cellular fate with gemcitabine. In addition we have demonstrated whether HSP27 downregulation effectively could overcome the acquisition of gemcitabine resistance by using transcriptomic analysis. We observed that gemcitabine induced p38/HSP27 phosphorylation and caused acquired resistance. After acquisition of gemcitabine resistance, cancer cells showed higher activity of NF-κB. NF-κB activity, as well as colony formation in gemcitabine-resistant pancreatic cancer cells, was significantly decreased by HSP27 downregulation and subsequent TRAIL treatment, showing that HSP27 was a common network mediator of gemcitabine/TRAIL-induced cell death. After transcriptomic analysis, gene fluctuation after HSP27 downregulation was very similar to that of pancreatic cancer cells susceptible to gemcitabine, and then in opposite position to that of acquired gemcitabine resistance, which makes it possible to downregulate HSP27 to overcome the acquired gemcitabine resistance to function as an overall survival network inhibitor. Most importantly, we demonstrated that the ratio of phosphorylated HSP27 to nonphosphorylated HSP27 rather than the cellular level of HSP27 itself acts biphasically as a determinant of cellular fate in gemcitabine-resistant pancreatic cancer cells.

Gemcitabine resistant pancreatic cancer cell lines acquire an invasive phenotype with collateral hypersensitivity to histone deacetylase inhibitors

Gemcitabine based treatment is currently a standard first line treatment for patients with advanced pancreatic cancer, however overall survival remains poor, and few options are available for patients that fail gemcitabine based therapy. To identify potential molecular targets in gemcitabine refractory pancreatic cancer, we developed a series of gemcitabine resistant (GR) cell lines. Initial drug exposure selected for an early resistant phenotype that was independent of drug metabolic pathways. Prolonged drug selection pressure after 16 weeks, led to an induction of cytidine deaminase (CDA) and enhanced drug detoxification. Cross resistance profiles demonstrate approximately 100-fold cross resistance to the pyrimidine nucleoside cytarabine, but no resistance to the same in class agents, azacytidine and decitabine. GR cell lines demonstrated a dose dependent collateral hypersensitivity to class I and II histone deacetylase (HDAC) inhibitors and decreased expression of 3 different global heterochromatin marks, as detected by H4K20me3, H3K9me3 and H3K27me3. Cell morphology of the drug resistant cell lines demonstrated a fibroblastic type appearance with loss of cell-cell junctions and an altered microarray expression pattern, using Gene Ontology (GO) annotation, consistent with progression to an invasive phenotype. Of particular note, the gemcitabine resistant cell lines displayed up to a 15 fold increase in invasive potential that directly correlates with the level of gemcitabine resistance. These findings suggest a mechanistic relationship between chemoresistance and metastatic potential in pancreatic carcinoma and provide evidence for molecular pathways that may be exploited to develop therapeutic strategies for refractory pancreatic cancer.

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 <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 694: Different pancreatic cancer cells have their own core signaling pathway

Abstract Gemcitabine as the standard chemotherapy agent to pancreatic cancer has proved effective; however, the response rate remains at 5.4% and the 5-year survival rate is extremely poor. Due to the shortcomings of gemcitabine and the presence of gemcitabine-resistant pancreatic cancer, the development of targeted agents into an anti-neoplastic agent remains a viable option. We have investigated the transforming growth factor-beta (TGF-β), which is known to play a central role in pancreas cell function. We have already observed that the suppression of TGF-β2 induced an unexpected down-regulation of both TGF-β1 and TGF-β3 with concomitant decreases of survival-related signals, such as N-caderin, β-catenin, pp65 in melanoma cell line. Here, two different pancreatic cancer cell lines (HPAC, BxPC3), which have opposite genetic backgrounds (K-ras, p53, Smad4, EGFR), have shown differing sensitivities to the down-regulation of TGF-β2. Through transcriptome analysis, HPAC showed a high expression of TGF-β2, MUC4, HSP27, BMP4 which are deeply related to tumor progression and metastasis and are composed to core signaling complex initiating from TGF-β2. This was confirmed by TGF-β2 down-regulation with gemcitabine to show a greater decrease in survival potential compared to BxPC3. On the contrary, BxPC3, which has more of the mutant type p53, exhibited greater down-regulation of MUC4 by downregulation of p53 compared to that of TGF-β2. Such observations suggest that BxPC3 is composed of a different core signaling pathway, p53-MUC4 rather than TGF-β2-related signalings.In conclusion, it is necessary that the core signaling pathways which are implicated in individual pancreatic carcinogenesis are more specifically considered so that the various gemcitabine-resistant pancreatic cancer cells on the basis of customized treatment can be controlled. Citation Format: Eunkyung Kim, Sujin Kang, So Y. Kim, Dongxu Kang, Seungha Lee, Hye J. Choi, Joo-Hang Kim, Jae Jin Song. Different pancreatic cancer cells have their own core signaling pathway. [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 694. doi:10.1158/1538-7445.AM2014-694

Reversible epigenetic regulation of 14-3-3σ expression in acquired gemcitabine resistance by uhrf1 and DNA methyltransferase 1.

Although gemcitabine is the most commonly used drug for treating pancreatic cancers, acquired gemcitabine resistance in a substantial number of patients appears to hinder its effectiveness in successful treatment of this dreadful disease. To understand acquired gemcitabine resistance, we generated a gemcitabine-resistant pancreatic cancer cell line using stepwise selection and found that, in addition to the known mechanisms of upregulated expression of ribonucleotide reductase, 14-3-3σ expression is dramatically upregulated, and that 14-3-3σ overexpression contributes to the acquired resistance to gemcitabine and cross-resistance to cytarabine. We also found that the increased 14-3-3σ expression in the gemcitabine-resistant cells is due to demethylation of the 14-3-3σ gene during gemcitabine selection, which could be partially reversed with removal of the gemcitabine selection pressure. Most importantly, the reversible methylation/demethylation of the 14-3-3σ gene appears to be carried out by DNA methyltransferase 1 under regulation by Uhrf1. These findings suggest that the epigenetic regulation of gene expression may play an important role in gemcitabine resistance, and that epigenetic modification is reversible in response to gemcitabine treatment.

Gemcitabine Resistance is Associated with Epithelial-Mesenchymal Transition and Induction of HIF-1α in Pancreatic Cancer Cells

Pancreatic cancer is one of the highly aggressive malignant diseases worldwide. To achieve better treatment outcome of pancreatic cancer, in the current study we explore the underlying molecular mechanism of drug resistance in pancreatic cancer cells. We found that resistance to gemcitabine is associated with epithelial-mesenchymal transition (EMT) phenotype in a panel of pancreatic cancer cell lines. Notably, gemcitabine-resistant pancreatic cancer cells acquire EMT phenotype. Moreover, gemcitabine-resistant cells have increased migration and invasion activities. Furthermore, we observed the high expression of HIF-1α in gemcitabine-resistant cells. More importantly, inhibition of HIF-1α in gemcitabine-resistant cells caused partial reversal of EMT phenotype, suggesting that HIF-1α was critically involved in gemcitabine-resistant-mediated EMT. Therefore, targeting HIF-1α could be an effective strategy for the treatment of pancreatic cancer.

Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells.

Gemcitabine resistance remains a significant clinical challenge. Here, we used a novel glucose transporter (Glut) inhibitor, CG-5, as a proof-of-concept compound to investigate the therapeutic utility of targeting the Warburg effect to overcome gemcitabine resistance in pancreatic cancer. The effects of gemcitabine and/or CG-5 on viability, survival, glucose uptake and DNA damage were evaluated in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cell lines. Mechanistic studies were conducted to determine the molecular basis of gemcitabine resistance and the mechanism of CG-5-induced sensitization to gemcitabine. The effects of CG-5 on gemcitabine sensitivity were investigated in a xenograft tumor model of gemcitabine-resistant pancreatic cancer. In contrast to gemcitabine-sensitive pancreatic cancer cells, the resistant Panc-1 and Panc-1(GemR) cells responded to gemcitabine by increasing the expression of ribonucleotide reductase M2 catalytic subunit (RRM2) through E2F1-mediated transcriptional activation. Acting as a pan-Glut inhibitor, CG-5 abrogated this gemcitabine-induced upregulation of RRM2 through decreased E2F1 expression, thereby enhancing gemcitabine-induced DNA damage and inhibition of cell survival. This CG-5-induced inhibition of E2F1 expression was mediated by the induction of a previously unreported E2F1-targeted microRNA, miR-520f. The addition of oral CG-5 to gemcitabine therapy caused greater suppression of Panc-1(GemR) xenograft tumor growth in vivo than either drug alone. Glut inhibition may be an effective strategy to enhance gemcitabine activity for the treatment of pancreatic cancer.

Gemcitabine-induced pancreatic cancer cell death is associated with MST1/cyclophilin D mitochondrial complexation.

The pancreatic adenocarcinoma remains the most aggressive human malignancy with an extremely low 5-year overall survival. Postoperative gemcitabine could significantly delay recurrence after complete resection of pancreatic cancer. However, the underlying mechanisms are not fully understood. The chemo-resistance factors against gemcitabine still need further characterizations. Here we studied the mechanism of gemcitabine-induced pancreatic cancer cell death by focusing on mammalian sterile 20-like kinase 1 (MST1) and cyclophilin D (Cyp-D). We found that MST1 and Cyp-D expressions were significantly lower in gemcitabine-resistant pancreatic cancer tissues and cell lines. In vitro, gemcitabine activated MST1 through reactive oxygen species (ROS) production, which was prevented by antioxidant n-acetyl-cysteine (NAC). We found that gemcitabine-activated MST1 translocated to mitochondria and formed a complex with the local protein Cyp-D. Gemcitabine-induced cell death was alleviated by MST1 or Cyp-D shRNA silencing, but was aggravated by MST1 or Cyp-D over-expression. Further, cyclosporin A (CsA), the Cyp-D inhibitor, prevented gemcitabine-induced MST1/Cyp-D mitochondrial complexation and cancer cell death. We suggest that gemcitabine-induced death of pancreatic cancer cells requires MST1/Cyp-D mitochondrial complexation.

Development of High-Content Gemcitabine PEGylated Liposomes and Their Cytotoxicity on Drug-Resistant Pancreatic Tumour Cells

The objective of this study was to develop high-content gemcitabine PEGylated liposomes to reverse gemcitabine resistance in pancreatic tumour cells. The mechanism of drug loading into liposomes was also investigated. To increase the drug entrapment efficiency (EE) and drug loading (DL), a novel passive loading approach named Small Volume Incubation method (SVI) was developed and compared to the reverse phase evaporation (REV) and remote loading methods. The in vitro cytotoxicity was evaluated using MIA PaCa-2 and Panc-1 cell lines. The EE for remote loading was 12.3 ± 0.3%, much lower than expected and a burst release was observed with the resultant liposomes. Using the optimized SVI method, increased EE (37 ± 1%) and DL (4%, w/w) were obtained. The liposomes (200 ± 5nm) showed minimal drug leakage, good stability, and significant improvement in cytotoxicity to the gemcitabine-resistant pancreatic cancer cell lines. Remote loading was not suitable for loading gemcitabine into liposomes. pKa > 4.6 for basic drugs and intra-liposomal precipitation of loaded compounds were suggested as an additional requirement to the current criteria for remote loading using ammonium sulphate gradient (pKa < 11). High DL is essential for liposomes to reverse gemcitabine resistance in pancreatic cell lines.

Mass Spectrometry-Based Metabolic Profiling of Gemcitabine-Sensitive and Gemcitabine-Resistant Pancreatic Cancer Cells

Gemcitabine resistance (GR) is one of the critical issues for therapy for pancreatic cancer, but the mechanism still remains unclear. Our aim was to increase the understanding of GR by metabolic profiling approach. To establish GR cells, 2 human pancreatic cancer cell lines, SUIT-2 and CAPAN-1, were exposed to increasing concentration of gemcitabine. Both parental and chemoresistant cells obtained by this treatment were subjected to metabolic profiling based on liquid chromatography-mass spectrometry. Multivariate statistical analyses, both principal component analysis and orthogonal partial least squares discriminant analysis, distinguished metabolic signature of responsiveness and resistance to gemcitabine in both SUIT-2 and CAPAN-1 cells. Among significantly different (P < 0.005) metabolite peaks between parental and GR cells, we identified metabolites related to several metabolic pathways such as amino acid, nucleotide, energy, cofactor, and vitamin pathways. Decreases in glutamine and proline levels as well as increases in aspartate, hydroxyproline, creatine, and creatinine levels were observed in chemoresistant cells from both cell lines. These results suggest that metabolic profiling can isolate distinct features of pancreatic cancer in the metabolome of gemcitabine-sensitive and GR cells. These findings may contribute to the biomarker discovery and an enhanced understanding of GR in pancreatic cancer.

Abstract C237: Overcoming gemcitabine resistance with TRAIL plus enhancer of anti-survival potential in pancreatic cancer.

Abstract Although the combination of gemcitabine with a variety of cytotoxic and targeted agents has generally shown no significant survival advantage as compared with gemcitabine alone, still, many targeted agents against various pathways remain to be tested in pancreatic cancer. Previously, we have observed that the combined treatment with TRAIL plus shRNA of HSP27 induced complete cell death in MiaPaCa-2 pancreatic cell. Here three different targets were invited with TRAIL for chemotherapy combination with gemcitabine using three pancreatic cancer cell lines. We observed that TRAIL treatment to all three pancreatic cells have relieved gemcitabine-induced resistance. More importantly, three different pancreatic cancer cell lines showed their own unique differential sensitivities to targeted agents examined. For example, in case of MiaPaCa-2, gemcitabine/TRAIL/shRNA of survivin was more effective to cell death than HPAC, whereas TRAIL plus downregulation of survival-related molecules (survivin, XIAP, Bcl-xL) effectively enhanced cell death of Panc-1 even without gemcitabine. Then, we extended the survival-related molecules to upstreams such as Src, PI3K-Akt, or NF-κB and inhibited their downstream signals using PP2, wortmannin, SN50, respectively. From their results, TRAIL plus SN50 decreased NF-κB activation and concomitant significant cell death in MiaPaCa-2 which has an acquired resistance of NF-κB after gemcitabine. Based on this result, it was found that combination of gemcitabine/TRAIL/SN50 dramatically increased cell death of MiaPaCa-2, whereas in case of Panc-1 or HPAC, gemcitabine/TRAIL/PP2 dramatically increased cell death. Taken all together, a better knowledge of the multiple molecular pathways implicated in pancreatic carcinogenesis should be much more specifically considered to control the various gemcitabine-resistant pancreatic cancer cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C237. Citation Format: Seungha Lee, So Y. Kim, Dongxu Kang, Hye J. Choi, Joo-Hang Kim, Jae Jin Song. Overcoming gemcitabine resistance with TRAIL plus enhancer of anti-survival potential in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C237.

Pharmacological Reversal of Histone Methylation Presensitizes Pancreatic Cancer Cells to Nucleoside Drugs: In Vitro Optimization and Novel Nanoparticle Delivery Studies

We evaluated the potential of an investigational histone methylation reversal agent, 3-deazaneplanocin A (DZNep), in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines by increased apoptosis. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents to produce a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs through the reversal of histone methylation, emphasizing the promising clinical utilities of epigenetic reversal agents in future pancreatic cancer combination therapies.

Abstract A30: Inhibiting histone methylation as an epigenetic therapeutic approach for pancreatic cancer

Abstract We evaluated the potential of an investigational histone methylation reversal agent, 3-deazaneplanocin A (DZNep), in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents to produce a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs through the reversal of histone methylation, emphasizing the promising clinical utilities of epigenetic reversal agents in future pancreatic cancer combination therapies. Citation Format: Rajgopal Govindarajan. Inhibiting histone methylation as an epigenetic therapeutic approach for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A30.

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

Abstract 2833: Significant protection against gemcitabine-resistant pancreatic cancer cells with tumor lysate vaccine, engineered to express α-gal epitopes.

Abstract Gemcitabine(Gem)-based chemotherapy is typically offered as a standard care of pancreatic cancer (PC) patients. However most of patients do not survive longer than 6 months, as the PC cells are naturally resistant to current chemotherapy. New therapeutic approaches need to be encouraged. Human natural anti-Gal is an IgG known to be presented in large amounts (∼1% of circulating IgG) in normal subjects and cancer patients. Anti-Gal specifically interacts with α-gal epitopes (Galα1, 3Galβ1, 4GlcNAc-R), synthesized by α1, 3 galactosyltransferase (α1, 3GT) on cell surface glycolipids and glycoproteins. We previously showed the in vitro and in vivo effectiveness of new immunotherapy by vaccination with whole PC cells, expressing α-gal epitopes (Cancer Res; 70(13); 5259-69, 2010). For the clinical development, we proposed that tumor lysate is a suitable source of tumor-associated antigens (TAAs), because it contains several known as well as unknown TAAs that could elicit an anti-tumor immune response against heterogeneous PC cell populations, including Gem-resistant PC cells (Gem-resi cells). In this study, we investigate whether vaccination by PC tumor lysate, expressingα-gal epitopes can efficiently induce anti-tumor response against both PC cells and gem-resi cells. A human PC cell line, PANC1 was transfected by α1,3GT gene (α-gal PANC1), 2×106 of either parental or α-gal PANC1 were injected s.c. into NOD/SCID mice. The grown PANC1 tumors were enucleated and homogenized. α1,3GT KO mice were pre-immunized with pig tissues to produce anti-Gal in their sera like human. These mice were vaccinated intraperitoneally by 10 mg of either parental (control group) or α-gal PANC1 tumor lysate (α-gal group). Tumor lysate of parental PANC1 lacked of α-gal epitopes and it of α-gal PANC1 expressed ∼40 × 106 of these epitopes per 1mg of lysate. To demonstrate in vivo tumor destruction, an animal model was performed. Splenocytes from vaccinated KO mice were prepared, and these cells were transferred ip (30×106 cells/ip ×3 times) into NOD/SCID mice. Transferred mice were challenged with S.C. injection by either 1×107 of live PANC1 or gem-resi PANC1. PANC1 tumors in mice transferred from parental group reached the size of 100 mm2 at 40.3 days after injection. For tumors in mice from α-gal group, regrowth of tumor was completely prevented. Survival of α-gal transferred mice was prolonged [α-gal vs. parental: 82.5±21.9 vs. 48.0±6.7 days, p&amp;lt;0.0001]. For gem-resi tumors in mice from α-gal group, regrowth of them was markedly protected [size of 100 mm2; α-gal vs. parental: 74.3 vs. 37.4 days]. Survival was prolonged [α-gal vs. parental: 93.6±17.7 vs.46.2±7.3 days, p=0.0018]. In conclusion, immunotherapy with tumor lysate vaccine, expressing α-gal epitopes effectively targets both PC cells and gem-resi cells and may provide PC cure by the elimination of the replenishing pool of all PC cells. Citation Format: Masahiro Tanemura, Eiji Miyoshi, Tsukasa Tanida, Hiroaki Nagano, Hidetoshi Eguchi, Kenta Furukawa, Yuji Nonaka, Hirofumi Akita, Naoki Hama, Hiroshi Wada, Koichi Kawamoto, Shyogo Kobayashi, Kiyomi Taniyama, Wataru Kamiike, Masaki Mori, Yuichiro Doki. Significant protection against gemcitabine-resistant pancreatic cancer cells with tumor lysate vaccine, engineered to express α-gal epitopes. [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 2833. doi:10.1158/1538-7445.AM2013-2833

Abstract 1025: Optimized DZNep exposure presensitizes pancreatic cancer cells to anticancer nucleoside analogues: potential clinical implications.

Abstract We evaluated the potential of a histone methylation reversal agent 3-deazaneplanocin A (DZNep) in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents produced a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs and emphasize the promising clinical utilities of histone methylation reversal agents in future pancreatic cancer combination therapies. Citation Format: Sau Wai Hung, Hardik Mody, Sean Marrache, Yangzom D. Bhutia, Franklin Davis, Jong Hyun Cho, Shanta Dhar, Chung K. Chu, Rajgopal Govindarajan. Optimized DZNep exposure presensitizes pancreatic cancer cells to anticancer nucleoside analogues: potential clinical implications. [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 1025. doi:10.1158/1538-7445.AM2013-1025

Differential Processing of let-7a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

Overexpression of ribonucleotide reductase subunit M2 (RRM2), involved in deoxyribonucleotide synthesis, drives the chemoresistance of pancreatic cancer to nucleoside analogs (e.g., gemcitabine). While silencing RRM2 by synthetic means has shown promise in reducing chemoresistance, targeting endogenous molecules, especially microRNAs (miRNAs), to advance chemotherapeutic outcomes has been poorly explored. Based on computational predictions, we hypothesized that the let-7 tumor suppressor miRNAs will inhibit RRM2-mediated gemcitabine chemoresistance in pancreatic cancer. Reduced expression of the majority of let-7 miRNAs with an inverse relationship to RRM2 expression was identified in innately gemcitabine-resistant pancreatic cancer cell lines. Direct binding of let-7 miRNAs to the 3′ UTR of RRM2 transcripts identified post-transcriptional regulation of RRM2 influencing gemcitabine chemosensitivity. Intriguingly, overexpression of human precursor-let-7 miRNAs led to differential RRM2 expression and chemosensitivity responses in a poorly differentiated pancreatic cancer cell line, MIA PaCa-2. Defective processing of let-7a precursors to mature forms, in part, explained the discrepancies observed with let-7a expressional outcomes. Consistently, the ratios of mature to precursor let-7a were progressively reduced in gemcitabine-sensitive L3.6pl and Capan-1 cell lines induced to acquire gemcitabine resistance. Besides known regulators of let-7 biogenesis (e.g., LIN-28), short hairpin RNA library screening identified several novel RNA binding proteins, including the SET oncoprotein, to differentially impact let-7 biogenesis and chemosensitivity in gemcitabine-sensitive versus -resistant pancreatic cancer cells. Further, LIN-28 and SET knockdown in the cells led to profound reductions in cellular proliferation and colony-formation capacities. Finally, defective processing of let-7a precursors with a positive correlation to RRM2 overexpression was identified in patient-derived pancreatic ductal adenocarcinoma (PDAC) tissues. These data demonstrate an intricate post-transcriptional regulation of RRM2 and chemosensitivity by let-7a and that the manipulation of regulatory proteins involved in let-7a transcription/processing may provide a mechanism for improving chemotherapeutic and/or tumor growth control responses in pancreatic cancer.