Gemcitabine-resistant Pancreatic Cancer Research Articles

Salvage Therapy with Mitomycin and Ifosfamide in Patients with Gemcitabine-Resistant Metastatic Pancreatic Cancer: A Phase II Trial

Background: At the time of upfront treatment failure, over half of the patients with advanced pancreatic cancer are candidates for further treatment. Methods: Patients with metastatic gemcitabine-resistant pancreatic cancer were treated with mitomycin 8 mg/m² on day 1, ifosfamide 2,500 mg/m² and mesna 3,000 mg/m² on days 1–3 every 28 days until progressive disease. A positive responder was defined as a patient who was progression free at 6 months from trial enrolment. According to the Fleming design, a sample size of 34 patients was estimated assuming p0 = 0.05 and p1 = 0.20. Results: Between May 2006 and December 2007, 21 patients (median age 56 years; median Karnofsky performance score 80) were enrolled. One patient died before receiving any treatment. Eighteen patients interrupted chemotherapy due to progressive disease (n = 15), toxicity (n = 2) or refusal (n = 1). Grade >2 toxicity consisted of neutropenia in 80% of patients, thrombocytopenia and fatigue in 20% and anemia in 10%. Only 1 patient was progression free at 6 months (5%). One patient had a partial response (5%) and 2 patients had stable disease (10%). Median survival was 3.7 months. Conclusion: Based on the poor outcome observed and on the high level of grade 3–4 toxicity, the trial was prematurely stopped and the mitomycin and ifosfamide regimen was considered insufficiently active in gemcitabine-pretreated advanced pancreatic cancer.

CNT1 Expression Influences Proliferation and Chemosensitivity in Drug-Resistant Pancreatic Cancer Cells

Overcoming the inherent chemoresistance of pancreatic cancers remains a major goal of therapeutic investigations in this disease. In this study, we discovered a role for the human concentrative nucleoside transporter-1 (hCNT1; SLC28A1), a high-affinity pyrimidine nucleoside transporter, in determining the chemosensitivity of human pancreatic cancer cells to gemcitabine, the drug used presently as a standard of care. Compared with normal pancreas and pancreatic ductal epithelial cells, hCNT1 expression was frequently reduced in pancreatic tumors and tumor cell lines. In addition, hCNT1-mediated (3)H-gemcitabine transport was lower in pancreatic cancer cell lines and correlated with cytotoxic IC(50) estimations of gemcitabine. In contrast to gemcitabine-sensitive pancreatic cancer cell lines, MIA PaCa-2, a gemcitabine-resistant pancreatic cancer cell line, exhibited relatively restrictive, cell cycle-dependent hCNT1 expression and transport. hCNT1 translation was suppressed in the late G1-enriched MIA PaCa-2 cell population possibly in an miRNA-dependent manner, which corresponded with the lowest hCNT1-mediated gemcitabine transport during this phase. Although hCNT1 protein was induced during G1/S transition, increased hCNT1 trafficking resulted in maximal cell surface recruitment and transport-overshoot in the G2/M phase-enriched cell population. hCNT1 protein was directed predominantly to proteasomal or lysosomal degradation in S or G2/M phase MIA PaCa-2 cells, respectively. Pharmacological inhibition of hCNT1 degradation moderately increased cell surface hCNT1 expression and cellular gemcitabine transport in MIA PaCa-2 cells. Constitutive hCNT1 expression reduced clonogenic survival of MIA PaCa-2 cells and steeply augmented gemcitabine transport and chemosensitization. In addition to supporting a putative tumor suppressor role for hCNT1, our findings identify hCNT1 as a potential candidate to render drug-resistant pancreatic cancer cells amenable to chemotherapy.

MicroRNA Expression as a Predictive Marker for Gemcitabine Response after Surgical Resection of Pancreatic Cancer

BackgroundTo improve the prognosis of patients after resection of pancreatic cancer, the most appropriate and efficient treatment should be provided to specific subsets of patients. Our aim was to identify promising microRNAs as markers to predict responses to gemcitabine in patients with resected pancreatic cancer.MethodsTwo gemcitabine-resistant pancreatic cancer cell lines were established, and global microRNA expression analyses was performed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Eleven miRNAs were selected as putative predictive markers and analyzed by means of macrodissected formalin-fixed, paraffin-embedded samples obtained from 90 patients with or without gemcitabine treatment after resection of pancreatic cancer.ResultsWe identified 24 microRNAs whose expression was altered in gemcitabine-resistant cells. qRT-PCR analyses showed that patients with high miR-142-5p and miR-204 expression had significantly longer survival times than those with low miR-142-5p (P = 0.0077) and miR-204 (P = 0.0054) expression in the gemcitabine-treated group. This was not seen in the nontreated group. Multivariate analyses showed that miR-142-5p expression was an independent prognostic marker only in patients treated with gemcitabine (P = 0.034).ConclusionsmiR-142-5p is a promising predictive marker for gemcitabine response in patients with resected pancreatic cancer.

Three-Dimensional Collagen I Promotes Gemcitabine Resistance in Pancreatic Cancer through MT1-MMP–Mediated Expression of HMGA2

One of the hallmarks of human pancreatic ductal adenocarcinoma (PDAC) is its pronounced type I collagen-rich fibrotic reaction. Although recent reports have shown that the fibrotic reaction can limit the efficacy of gemcitabine chemotherapy, the underlying mechanisms remain poorly understood. In this article, we show that the type I collagen allows PDAC cells to override checkpoint arrest induced by gemcitabine. Relative to cells grown on tissue culture plastic, PDAC cells grown in 3-dimensional collagen microenvironment have minimal Chk1 phosphorylation and continue to proliferate in the presence of gemcitabine. Collagen increases membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent ERK1/2 phosphorylation to limit the effect of gemcitabine. Collagen also increases MT1-MMP-dependent high mobility group A2 (HMGA2) expression, a nonhistone DNA-binding nuclear protein involved in chromatin remodeling and gene transcription, to attenuate the effect of gemcitabine. Overexpression of MT1-MMP in the collagen microenvironment increases ERK1/2 phosphorylation and HMGA2 expression, and thereby further attenuates gemcitabine-induced checkpoint arrest. MT1-MMP also allows PDAC cells to continue to proliferate in the presence of gemcitabine in a xenograft mouse model. Clinically, human tumors with increased MT1-MMP show increased HMGA2 expression. Overall, our data show that collagen upregulation of MT1-MMP contributes to gemcitabine resistance in vitro and in a xenograft mouse model, and suggest that targeting MT1-MMP could be a novel approach to sensitize pancreatic tumors to gemcitabine.

Effect of nicotine on chemoresistant phenotype as mediated through Src-dependent Id1 expression in pancreatic adenocarcinoma cells.

216 Background: The signaling pathways contributing to DNA-binding protein inhibitor Id1 expression and chemoresistance in pancreatic cancer remain unknown. Id1 plays a role in pancreatic tumor progression with tumor-promoting effects of nicotine regulating protein tyrosine kinase Src activation and Id1 expression, both associated with chemoresistance in other systems. We hypothesize Id1 expression regulates chemoresistance in pancreatic cancer through a nicotine-promoting Src-dependent pathway. Methods: We probed pancreatic cancer cell lines (L3.6pl, PANC-1, Mia-PaCa-2) for innate gemcitabine chemoresistance with cell viability MTT assay and western blot analysis of PARP cleavage programmed cell death. Gemcitabine-sensitive cells were exposed to rising gemcitabine concentrations to establish a resistant subtype, L3.6plGemRes. Protein analysis and mRNA expression were determined by western blot analysis and RT-PCR respectively. Induction of Src phosphorylation or Id1 expression was performed with nicotine (1 μM). Results: Inhibition of c-Src expression was performed with short-interfering RNA (siRNA). Nicotine-induced Src phosphorylation and Id1 expression. Inhibition of Src by siRNA resulted in decreased nicotine-induced Id1 expression. Inhibition of Src and Id1 expression by siRNA in innate or established gemcitabine resistant pancreatic cancer cells resulted in gemcitabine sensitization. To determine if nicotine contributes to gemcitabine chemoresistance, we exposed gemcitabine-sensitive cells to nicotine with subsequent exposure to gemcitabine IC50, 250 ng/ml, and cell viability assays confirmed a 2-fold increase in cell prolilferation and a 4.5-fold reduction in apoptosis. Further, nicotine induced phosphorylation of key signaling enzymes involved in proliferation and apoptosis, Erk1/2 and Akt respectively. Conclusions: In summary, we demonstrate that Id1, through a nicotine-promoting Src-dependent pathway, is necessary for establishment of a chemoresistant phenotype in pancreatic cancer cells. Understanding the signaling pathways involved in pancreatic tumor chemoresistance will lead to therapies resulting in improved tumor responses. No significant financial relationships to disclose.

Highlights of This Issue

Once developed, the progression of pancreatic cancer metastasis is virtually unstoppable. Villarroel and colleagues provide first clinical evidence of integrating personalized xenografts with global exomic sequencing, having the potential to customize cancer therapy tailored to the genetic environment of patient's tumor. Contrary to the expected median survival of three months for pancreatic cancer patients who progress on gemcitabine, a patient with advanced, gemcitabine-resistant pancreatic cancer later treated with DNA damaging agents is virtually symptom-free for three years after progression to gemcitabine. The strategies used here have the potential to improve patient outcome and to make personalized medicine a reality for cancer patients.ENMD-2076 is a novel orally active, small molecule kinase inhibitor with a mechanism of action involving several pathways key to tumor growth and survival, angiogenesis, proliferation, and the cell cycle. In this study, Fletcher and colleagues have characterized the properties of ENMD-2076. They show that ENMD-2076 inhibits a unique profile of tyrosine kinase targets. These results indicate that ENMD-2076 is a welltolerated, orally active multitarget kinase inhibitor with a unique antiangiogenic/antiproliferative profile and provides strong preclinical support for use as a therapeutic for human cancers. Several Phase 1 studies involving ENMD-2076 have been recently completed, and the compound is currently being evaluated in a Phase 2 clinical trial in patients with platinum-resistant ovarian cancer.Rituximab, directed against the CD20 antigen on B cells, was the first commercially available monoclonal antibody for the treatment of lymphoma. It is the current treatment of choice for a variety of lymphoproliferative disorders including low and high grade B-cell non-Hodgkin's lymphomas. Follicular lymphoma is the most common subtype of indolent lymphoma. Rituximab is now widely used either alone or in combination with multi-agent chemotherapy for the treatment of follicular lymphoma, either at diagnosis, at relapse, or for maintenance therapy. However, despite its well-established clinical efficacy, a subpopulation of patients does not initially respond to rituximab and most patients will relapse after rituximab therapy. In this study, Dalle and colleagues compared the effect of GA101 and rituximab on the human follicular RL lymphoma model, both in vitro and in vivo.Previous studies have shown that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has significant apoptosis-inducing activity in some glioma cell lines, although many lines are either moderately or completely resistant, which has limited the therapeutic applicability of this agent. Because recent studies by Jane and colleagues showed that inhibition of proteasomal function may be independently active as an apoptosis-inducing stimulus in these tumors, they investigated the sensitivity of a panel of glioma cell lines (U87, T98G, U373, A172, LN18, LN229, LNZ308, and LNZ428) to TRAIL alone and in combination with the proteasome inhibitor bortezomib. Jane and colleagues show that the differential sensitivity correlated with activation status of NF-κB. In addition, bortezomib exhibited potent antiglioma activity and dramatically sensitized even highly resistant glioma cells to TRAIL cytotoxicity, suggesting that this may be a promising combination strategy for glioma therapeutics.

A phase I/II study of gemcitabine-based chemotherapy plus curcumin for patients with gemcitabine-resistant pancreatic cancer

Curcumin, a plant-derived natural polyphenol, could be a promising anti-cancer drug and shows synergic effects with cytotoxic agents. We evaluated the safety and feasibility of combination therapy using curcumin with gemcitabine-based chemotherapy. Gemcitabine-resistant patients with pancreatic cancer received 8g oral curcumin daily in combination with gemcitabine-based chemotherapy. The primary endpoint was safety for phase I and feasibility of oral curcumin for phase II study. Twenty-one patients were enrolled. No dose-limiting toxicities were observed in the phase I study and oral curcumin 8g/day was selected as the recommended dose for the phase II study. No patients were withdrawn from this study because of the intolerability of curcumin, which met the primary endpoint of the phase II study, and the median compliance rate of oral curcumin was 100% (Range 79-100%). Median survival time after initiation of curcumin was 161days (95% confidence interval 109-223days) and 1-year survival rate was 19% (4.4-41.4%). Plasma curcumin levels ranged from 29 to 412ng/ml in five patients tested. Combination therapy using 8g oral curcumin daily with gemcitabine-based chemotherapy was safe and feasible in patients with pancreatic cancer and warrants further investigation into its efficacy.

Integrated preclinical and clinical development of mTOR inhibitors in pancreatic cancer

Background:The purpose of this work was to determine the efficacy of inhibiting mammalian target of rapamycin (mTOR) in pancreatic cancer preclinical models and translate preclinical observations to the clinic.Methods:Temsirolimus (20 mg Kg−1 daily) was administered to freshly generated pancreatic cancer xenografts. Tumour growth inhibition was determined after 28 days. Xenografts were characterised at baseline by gene expression and comparative genomic hybridisation. Patients with advanced, gemcitabine-resistant pancreatic cancer were treated with sirolimus (5 mg daily). The primary end point was 6-month survival rate (6mSR). Correlative studies included immunohistochemistry assessment of pathway expression in baseline tumours, drug pharmacokinetics (PKs), response assessment by FDG-PET and pharmacodynamic effects in peripheral-blood mononuclear cells (PBMCs).Results:In all, 4 of 17 xenografts (23%) responded to treatment. Sensitive tumours were characterised by gene copy number variations and overexpression of genes leading to activation of the PI3K/Akt/mTOR pathway. Activation of p70S6K correlated with drug activity in the preclinical studies. Sirolimus was well tolerated in the clinic, showed predictable PKs, exerted pathway inhibition in post-treatment PBMCs and resulted in a 6mSR of 26%. No correlation, however, was found between activated p70S6K in tumour tissues and anti-tumour effects.Conclusion:Sirolimus activity in pancreatic cancer was marginal and not predicted by the selected biomarker.

Establish a gemcitabine-resistant pancreatic cancer cell line SW1990/GZ and research the relationship between SW1990/GZ and pancreatic cancer stem cell

To establish a gemcitabine-resistant pancreatic cancer cell line SW1990/GZ, and to explore the relationship between drug-resistant cell line SW1990/GZ and pancreatic cancer stem cell. Gemcitabine-resistant pancreatic cancer cell line SW1990/GZ was obtained by treating parental cell line SW1990 in vitro with increasing dosage of gemcitabine in culture medium intermittently for 24 weeks. Stable cultures were obtained which were 77.2-fold increased in resistance relative to parental cells. Gene expressions of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP were determined by real-time PCR. Tumorigenic potential was performed by nude mice xenograft transplant experiments. Side population analysis and CD24CD44 positive cells explore were determined by flow cytometry to examine cancer stem cell proportion. Gemcitabine-resistant cell line SW1990/GZ underwent obvious morphological and functional changes. Compared with the parental cell line, SW1990/GZ cell was small and turned into round shape. SW1990/GZ had a higher gene expression level of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP than SW1990 (P < 0.01). Nude mice xenograft transplant experiments showed that only 1 × 10(5) SW1990/GZ cells were sufficient for tumor formation, whereas an injection of 1 × 10(5) SW1990 cells did not initiate tumors. Flow cytometry analysis showed that SP proportion in SW1990/GZ was (11.0 ± 1.0)%, whereas in parental SW1990 it was (4.6 ± 0.9)%, CD44CD24 positive cells was (8.73 ± 0.81)% in SW1990/GZ, whereas (1.1 ± 0.4)% in SW1990. Gemcitabine-resistant cell line SW1990/GZ has a higher proportion of pancreatic cancer stem cells compared to its parental cell line SW1990. CD44 is mainly responsible for acquired drug resistance, which can be a potential target to overcome acquired drug resistance in pancreatic cancer.

Molecular mechanisms underlying gemcitabine resistance in pancreatic cancer and restoration of sensitivity using lenalidomide.

e13630 Background: Gemcitabine is the leading therapeutic for the treatment of pancreatic cancer, despite emergence of resistance. Understanding the mechanisms of gemcitabine resistance and discove...

Phase I and II studies of intravenous Rexin-G as monotherapy for stage IVb gemcitabine-resistant pancreatic cancer.

4149 Background: To evaluate (1) the safety and antitumor activity of Rexin-G, and (2) the cumulative toxicity and sustained efficacy of Rexin-G continuance in gemcitabine-resistant Stage IVb pancreas cancer. Methods: Fifteen patients were given escalating doses of 1, 2 or 3 × 10e11 cfu of Rexin-G i.v. 2 to 3 times a week for 4 weeks. A phase II component was incorporated by adaptive design, and additional cycles were given if there was ≤ grade 1 toxicity. The estimated tumor burden was determined by PET-CT scan using the formula: S [Target Lesions (cm) + (No. of Non- Target Lesions + (20*))] × 10e9. Note: (20*) indicates ascites, pleural effusion, and/or

Gemcitabine Sensitivity Can Be Induced in Pancreatic Cancer Cells through Modulation of miR-200 and miR-21 Expression by Curcumin or Its Analogue CDF

Curcumin induces cancer cell growth arrest and apoptosis in vitro, but its poor bioavailability in vivo limits its antitumor efficacy. We have previously evaluated the bioavailability of novel analogues of curcumin compared with curcumin, and we found that the analogue CDF exhibited greater systemic and pancreatic tissue bioavailability. In this study, we evaluated the effects of CDF or curcumin alone or in combination with gemcitabine on cell viability and apoptosis in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer (PC) cell lines. Mechanistic investigations revealed a significant reduction in cell viability in CDF-treated cells compared with curcumin-treated cells, which were also associated with the induction of apoptosis, and these results were consistent with the downregulation of Akt, cyclooxygenase-2, prostaglandin E(2), vascular endothelial growth factor, and NF-kappaB DNA binding activity. We have also documented attenuated expression of miR-200 and increased expression of miR-21 (a signature of tumor aggressiveness) in gemcitabine-resistant cells relative to gemcitabine-sensitive cells. Interestingly, CDF treatment upregulated miR-200 expression and downregulated the expression of miR-21, and the downregulation of miR-21 resulted in the induction of PTEN. These results prompt further interest in CDF as a drug modality to improve treatment outcome of patients diagnosed with PC as a result of its greater bioavailability in pancreatic tissue.

Abstract 3933: Collagen-proteinase cross-talk promotes gemcitabine resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a 5-year survival of ∼5%. A hallmark of PDAC is desmoplastic reaction (DR), the pronounced stromal reaction composed of extracellular matrix proteins along with activated fibroblasts, immune cells and endothelial cells. Although it has been suggested that the DR may be responsible for drug resistance, the mechanism is not well deciphered. We now show that collagen type I, the major component of DR, helps PDAC cells override the cell cycle checkpoint arrest induced by gemcitabine, the most commonly used chemotherapy for PDAC. Relative to cells grown on tissue culture plastic, PDAC cells grown in 3D type I collagen gels demonstrated decreased activating phosphorylation of the checkpoint protein Chk1 following gemcitabine treatment. Interestingly, PDAC cells grown atop 3D collagen gels were more sensitive to gemcitabine treatment and demonstrated higher phospho-Chk1 levels compared to cells growing within 3D collagen gels, suggesting that collagen functions as a barrier to drug delivery. As we had previously shown that collagen could induce expression of the key collagenase membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) by PDAC cells, we examined the contribution of MT1-MMP to drug resistance in our model system. Downregulating MT1-MMP with siRNA attenuated the effect of collagen on gemcitabine-induced checkpoint arrest. In contrast, MT1-MMP overexpression further protected the cells from gemcitabine-induced checkpoint arrest. Overall, our results demonstrate the complex interplay between the proteinase MT1-MMP and its substrate type I collagen in regulating chemotherapy resistance, and suggests that targeting MT1-MMP could help overcome PDAC drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3933.

Abstract 2561: Genistein restored chemosensitivity to oxaliplatin in gemcitabine resistant pancreatic cancer cells in vitro and in orthotopic xenograft mouse model

Abstract Chemotherapy remains the main stay for pancreatic cancer (PC) treatment with Gemcitabine showing modest benefit. Oxaliplatin has been used as a second line therapy in combination with gemcitabine but the beneficial effect was mitigated due to ‘off target’ toxicity as well as due to de novo and acquired resistance. However, effort toward restoration of sensitivity of PC cells to low concentrations of oxaliplatin is an emerging area of research. Here we report our in vitro and in vivo preclinical evidence in support of chemo-sensitization of gemcitabine resistant PC cells (Panc-1, Panc-28 and MiaPaCa-2) by a non-toxic chemopreventive agent known as genistein. Genistein pretreatment of PC cells exhibiting moderate (Panc-1, Panc-28) to less gemcitabine sensitivity (MiaPaCa-2) using low concentrations of oxaliplatin resulted in a significant reduction in the viability of cells concomitant with increased apoptosis (p&amp;lt;0.01) occurred in a synergistic manner as assessed by colony formation experiments. Since drug-resistance of PC is allegedly linked with constitutive activation of NF-κB as well as further activation by conventional chemotherapeutic agents including oxaliplatin, our mechanistic showed that inactivation of NF-κB by genistein prior to treatment of cell with oxaliplatin is required for the killing of drug resistant cells, which indeed was consistent with the down- regulation of NF-κB and its downstream anti-apoptotic genes (Bcl-2 XIAP's, survivin) leading to the appearance of cleaved PARP and induction of apoptosis. Most importantly, our in vivo experiments using orthotopic mouse model of pancreatic cancer showed significant reduction in tumor size (p&amp;lt;0.01) and reduction of locoregional lymph node metastasis when genistein was given in combination with oxaliplatin compared to monotherapy. These results were also consistent with inactivation of NF-κB and the down-regulation of NF-kB downstream targets (Bcl-xL, survivin and XIAP), all of which was consistent with in vitro findings. Both in vitro as well as in vivo findings were also consistent with immunohistochemistry findings on proliferation marker (Ki-67), apoptosis (TUNEL) and phospho p-65 immunostaining. From these results, we conclude that our results provide strong in vivo evidence in support of our hypothesis showing that genistein sensitizes drug-resistant pancreatic cancer to oxaliplatin, which is mechanistically linked with inactivation of NF-κB, which could be a useful strategy for the treatment of PC in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2561.

Abstract 3920: EphA2 transcription is regulated by activation of Ras / MEK2 / cytoplasmic ERK pathway in pancreatic cancer

Abstract Background: We have previously shown that the EphA2 receptor is overexpressed in pancreatic cancer. Silencing of EphA2 receptor in combination with gemcitabine decreases tumor growth and metastases in a mouse model of gemcitabine resistant pancreatic cancer. However, regulation of EphA2 expression in pancreatic cancer is unknown. The objective of this study was to determine a possible relationship between Ras/MAPK pathway and EphA2. Methods: Pancreatic cancer cell lines, MPanc96 and MiaPaca-2, were used in this study. Ras was silenced with 2 different siRNA constructs; EphA2 protein and RNA expression levels were measured. ERK involvement in EphA2 regulation was analyzed by treating cells with 2 inhibitors for MEK (U0126 and PD 98059) and siRNA specific for either MEK1 or MEK2. Effect of MEK inhibition on EphA2 mRNA stability was examined by blocking mRNA synthesis with actinomycin D and measuring mRNA degradation by Q-PCR. In order to investigate the role of subcellular localization of ERK in regulation of EphA2 expression, we overexpressed cytoplasmic anchor protein PEA-15, which binds to ERK and sequesters it to the cytoplasm. Results: Silencing of mutant K-Ras decreased EphA2 mRNA and protein levels. EphA2 mRNA and protein levels decreased in a time- and dose-dependent manner after treatment with MEK chemical inhibitors. Inhibition of MEK activity did not affect EphA2 mRNA stability. Silencing of MEK2 but not MEK1 decreased EphA2 mRNA levels. Overexpression of PEA-15 depleted nuclear ERK, indicated by the absence of phosphorylated Fra-1. Depletion of the nuclear ERK did not affect EphA2 transcription, suggesting that EphA2 is regulated through the cytoplasmic ERK. Conclusions: These data suggest that EphA2 levels are regulated by the RAS / MEK2 / cytoplasmic ERK pathway via regulation of EphA2 mRNA transcription. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3920.

Abstract 1605: A possible target key molecule for treatment of pancreatic cancer, screened as cancer-specific immortalization associated gene

Abstract [Background] Pancreatic caner shows a poor prognosis, and thus development of an efficient therapy is needed. Last year, we have reported that 13 genes were screened in relation to an event “cancer cell immortalization” by means of comprehensive gene expression analyses of normal, mortal and immortal cells and tissue specimen, and that these might be potent candidates for novel targets of anticancer chemotherapy. [Purpose] GTSE1 gene has been selected as one of the 13 genes that expressed at higher levels in immortal cancer cells, though its biological function relative to growth regulation of cancer cells are yet to be determined. To clarify its roles, we performed in vitro experiments to validate its possibility as a target key molecule for treatment of cancer, specifically pancreatic one. [Materials and Methods] Quantitative analyses of gene expression levels were conducted by real-time RT-PCR method using gene specific-primers and Universal Probe Library probes (Roche). Whole genome gene expression analysis was conducted with 4×44K Whole Human Genome Microarray (Agilent). Knockdown experiments were performed in various cancer cells using Stealth RNAi and RNAiMax reagent (Invitrogen). Cell proliferation was measured with MTT assay. Caspase activities were analyzed using ApoAlert Caspase Profiling Plate (Clontech). Cell cycle profile was obtained by measuring PI-stained cells with FACS “Calibur” (BD Biosciences). [Results] GTSE1 expressed at much higher levels in primary pancreatic and endometrial cancer tissue specimens as compared with matched normal ones: it expressed at higher levels in pancreatic, esophageal, and lung cancer cell lines than normal cells: higher expression levels of GTSE1 were observed in transformants of lung fibroblast TIG-1 cells with SV40 and TERT as compared with TIG-1 cells harboring TERT alone. Treatment with GTSE1 siRNA revealed a significant inhibition of growth of various cancer cells including paclitaxel-resistant ovarian cancer KFr13Tx, cisplatin-resistant lung cancer PC-9/CDDP, and gemcitabine-resistant pancreatic cancer MIAPaCa-2 cells. GTSE1 knockdown induced alteration of cell cycle profile and apoptosis of pancreatic cancer cells: it enhanced caspase 2 and 3 activities. Moreover, to seek for downstream genes, we performed microarray analysis of KLM-1, MIAPaCa-2 and DLD-1 cells treated with GTSE1 siRNA for 72 hrs: We found a gene A that showed less than 1/8 gene expression levels as compared with negative control ones in all of the tested cells by using microarray: This was confirmed by real-time RT-PCR. Furthermore, knockdown of the selected gene also altered cell cycle profile and apoptosis of pancreatic cancer cells. [Conclusion] These results indicate that GTSE1 may be involved in cancer-specific immortalization and may be useful as a molecular target for cancer therapy including pancreatic one. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1605.

Phase II study of S-1 in patients with gemcitabine-resistant advanced pancreatic cancer

The primary objective of this study was to assess the efficacy and safety of S-1 in patients with gemcitabine-resistant advanced pancreatic cancer. Patients with histologically or cytologically proven, advanced pancreatic cancer who had received first-line chemotherapy with gemcitabine were eligible for this study. S-1 was administered orally at a dose of 40 mg/m(2) twice daily for 28 days, followed by 14 days' rest. Treatment was repeated every 6 weeks until disease progression. Twenty-one patients were enrolled in this study. Grade 3 and 4 toxicities included anorexia in 14% of the patients, abdominal pain in 4.8% and infection without neutropenia in 4.8%. S-1 was discontinued in two patients because of toxicity. Of the 21 eligible patients, 2 (9.5%) achieved a partial response and 9 (43%) had stable disease. A marked decrease (≥50%) in tumor marker (CA19-9) was observed in 5 (28%) of the 18 evaluable patients. The median progression-free survival and the median survival time from the first day of S-1 therapy were 4.1 months (95% CI, 1.3-6.9 months) and 6.3 months (95% CI, 3.6-8.9 months), respectively. Second-line chemotherapy with S-1 was tolerated with acceptable toxicity and resulted in a relatively high disease control rate in patients with gemcitabine-resistant advanced pancreatic cancer. As an oral agent, S-1 may be a feasible treatment option for this patient population.

A Fine-Needle Aspirate–Based Vulnerability Assay Identifies Polo-Like Kinase 1 as a Mediator of Gemcitabine Resistance in Pancreatic Cancer

This work aimed to discover targets for combination treatment with gemcitabine in pancreatic cancer. We selected 11 tumors from our live collection of freshly generated pancreatic cancer xenografts with known degrees of varying gemcitabine sensitivity. We briefly (6 h) exposed fine-needle aspiration material to control vehicle or gemcitabine (1 mumol/L) and compared the gene expression of the treated and untreated samples using a reverse transcription-PCR-based, customized low-density array with 45 target genes of therapeutic interest. The gene expression of the untreated sample (which can be considered a baseline/static readout) was not predictive of gemcitabine efficacy in these tumors. Altogether, the only gene that differentiated sensitive versus resistant cases was polo-like kinase 1 (Plk1), showing >50% downregulation in sensitive cases and no change in the resistant cases. Inhibition of Plk1 by either small interfering RNA gene knockdown or with the Plk1 pathway modulator (ON 01910.Na) synergized with gemcitabine in gemcitabine-refractory in vitro models providing mechanistic proof of concept. In vivo experiments in gemcitabine-resistant xenografts showed synergistic activity decreasing cell proliferation and tumor regressions. A quantitative gene expression-based vulnerability assay identified Plk1 as a relevant target dictating the susceptibility of pancreatic cancer to gemcitabine. Dynamic interrogation of cancer has the potential to provide key information about mechanisms of resistance and to enhance individualization of treatment.

Advanced Phase I/II Studies of Targeted Gene Delivery In Vivo: Intravenous Rexin-G for Gemcitabine-resistant Metastatic Pancreatic Cancer

Rexin-G, a nonreplicative pathology-targeted retroviral vector bearing a cytocidal cyclin G1 construct, was tested in a phase I/II study for gemcitabine-resistant pancreatic cancer. The patients received escalating doses of Rexin-G intravenously from 1 × 1011 colony-forming units (cfu) 2–3× a week (dose 0–1) to 2 × 1011 cfu 3× a week (dose 2) for 4 weeks. Treatment was continued if there was less than or equal to grade 1 toxicity. No dose-limiting toxicity (DLT) was observed, and no vector DNA integration, replication-competent retrovirus (RCR), or vector-neutralizing antibodies were noted. In nine evaluable patients, 3/3 patients had stable disease (SD) at dose 0–1. At dose 2, 1/6 patients had a partial response (PR) and 5/6 patients had SD. Median progression-free survival (PFS) was 3 months at dose 0–1, and >7.65 months at dose 2. Median overall survival (OS) was 4.3 months at dose 0–1, and 9.2 months at dose 2. One-year survival was 0% at dose 0–1 compared to 28.6% at dose 2, suggesting a dose–response relationship between OS and Rexin-G dosage. Taken together, these data indicate that (i) Rexin-G is safe and well tolerated, and (ii) Rexin-G may help control tumor growth, and may possibly prolong survival in gemcitabine-resistant pancreatic cancer, thus, earning US Food and Drug Administration's (FDA) fast-track designation as second-line treatment for pancreatic cancer.

Abstract B70: Histone deacetylase inhibitors reverse EMT phenotype and chemoresistance in pancreatic cancer cell lines

Abstract Background: The epithelial-to-mesenchymal transition (EMT) is a cell development program charyyacterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell motility regulated by transcription factors and noncoding microRNAs. Recently we reported that EMT may contribute to gemcitabine resistance in pancreatic cancer. Histone deacetylase inhibitors (HDACi) are a new class of antineoplastic agents that have been shown to have a number of antitumor effects including enhanced differentiation of cancer cells. The aims of this study were to determine the potential role of HDACi in regulating EMT and in overcoming gemcitabine resistance in pancreatic cancer cell lines. Methods: SAHA (HDACi) and gemcitabine were tested against a panel of 7 human pancreatic cancer cell lines. Apoptotic cell death was measured by flow cytometry/propidium iodine staining (PIFACS). Cell lines were considered resistant if &amp;lt;50% cell death occurred. To elucidate the mechanism of drug responsiveness, transcriptome analysis was performed before and after drug treatment. Microarray findings were confirmed by western blot analysis and RT-PCR. MicroRNA 200c (miR200c) levels were analyzed by RTPCR. Resistant cell lines were treated with SAHA followed by gemcitabine. Cell death was evaluated by PIFACS. Results: 4 cell lines were identified as resistant. Unsupervised hierarchical analysis of all cell lines demonstrated accurate clustering of cell lines based on gemcitabine sensitivity. E-cadherin and Zeb-1 expression were highly correlated with gemcitabine sensitivity. E-cadherin was increased in chemosensitive and decreased in chemoresistant cell lines; zeb-1 was inversely correlated to E-cadherin. As a potential regulator of zeb-1 expression, miR 200c was evaluated and found to closely correlate inversely with Zeb-1 level and drug sensitivity. Silencing of Zeb-1 led to increased E-cadherin expression and increased sensitivity to gemcitabine. Silencing of E-cadherin in chemosensitive cells did not induce chemoresistance. Pre-treatment of resistant pancreatic cancer cell lines with SAHA led to decreased expression of Zeb-1, increased expression of E-cadherin and mir200c. Importantly it increased sensitivity to gemcitabine. Microarray analysis of resistant pancreatic cell lines after treatment with SAHA demonstrated that these lines now clustered with the sensitive pancreas cell lines. Conclusion: We identified a close association between the expression of miR200c, E-cadherin level, Zeb-1 level, epithelial phenotype and chemosensitivity to gemcitabine in pancreatic cancer cell lines. Interestingly, pre-treatment of resistant cells with SAHA increased miR200c and E-cadherin levels and increased sensitivity to gemcitabine. It appears that combined therapy with HDAC inhibitors and gemcitabine increases chemosensitivity in resistant cell lines and may be a target for overcoming chemoresistance in pancreatic cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B70.