Gemcitabine In Pancreatic Ductal Adenocarcinoma Research Articles

NEMO peptide inhibits the growth of pancreatic ductal adenocarcinoma by blocking NF-κB activation

NF-κB essential modulator (NEMO) binds and regulates IκB kinase (IKK) and is required for NF-κB activation. The NEMO-binding domain peptide (NBDP) of IKK was found to inhibit NF-κB activation and promote apoptosis in cancer cells. Studies have shown that constitutive NF-κB activation, one of the signature molecular alterations in pancreatic ductal adenocarcinoma (PDAC), is a potential therapeutic target. However, preclinical and therapeutic evidence that supports direct targeting of IKK activation in therapy is lacking. The aim of this study was to determine whether the combination of NBDP and gemcitabine would sensitize pancreatic cancer to the gemcitabine. We confirmed that NBDP inhibited NF-κB activation and found that NBDP indeed promoted chemo-sensitivity to gemcitabine in PDAC. NBDP increased PARP and caspase 3 cleavage in the apoptosis pathway, increased apoptosis of PDAC cells, and suppressed PDAC cell growth in vitro. In addition, NBDP combined with gemcitabine significantly decreased levels of NF-κB activity and inhibited the growth of PDAC in vivo in an orthotopic xenograft mouse model. Mechanistic investigations showed that NBDP effectively competed with NEMO/IKKγ for binding to IKKs and thus inhibited IKK and NF-κB activation, down-regulated expression levels of Erk, and decreased PDAC cell growth. Taken together, our current data demonstrate that NBDP sensitizes human pancreatic cancer to gemcitabine by inhibiting the NF-κB pathway. NBDP is a potential adjuvant chemotherapeutic agent for treating pancreatic cancer.

Fructose-1,6-bisphosphatase Inhibits ERK Activation and Bypasses Gemcitabine Resistance in Pancreatic Cancer by Blocking IQGAP1-MAPK Interaction.

Dysregulation of the MAPK pathway correlates with progression of pancreatic ductal adenocarcinoma (PDAC) progression. IQ motif containing GTPase-activating protein 1 (IQGAP1) is a MAPK scaffold that directly regulates the activation of RAF, MEK, and ERK. Fructose-1,6-bisphosphatase (FBP1), a key enzyme in gluconeogenesis, is transcriptionally downregulated in various cancers, including PDAC. Here, we demonstrate that FBP1 acts as a negative modulator of the IQGAP1-MAPK signaling axis in PDAC cells. FBP1 binding to the WW domain of IQGAP1 impeded IQGAP1-dependent ERK1/2 phosphorylation (pERK1/2) in a manner independent of FBP1 enzymatic activity. Conversely, decreased FBP1 expression induced pERK1/2 levels in PDAC cell lines and correlated with increased pERK1/2 levels in patient specimens. Treatment with gemcitabine caused undesirable activation of ERK1/2 in PDAC cells, but cotreatment with the FBP1-derived small peptide inhibitor FBP1 E4 overcame gemcitabine-induced ERK activation, thereby increasing the anticancer efficacy of gemcitabine in PDAC. These findings identify a primary mechanism of resistance of PDAC to standard therapy and suggest that the FBP1-IQGAP1-ERK1/2 signaling axis can be targeted for effective treatment of PDAC. Cancer Res; 77(16); 4328-41. ©2017 AACR.

N1-guanyl-1, 7-diaminoheptane enhances the sensitivity of pancreatic ductal adenocarcinoma cells to gemcitabine via the inhibition of eukaryotic translation initiation factor 5A2.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy due to its broad resistance to chemotherapy. Gemcitabine is used as a standard chemotherapeutic drug for PDAC treatment, either alone or in combination with other chemotherapeutics. However, in patients with advanced disease, survival is rarely improved. This study aimed to investigate the therapeutic efficacy of N1-guanyl-1, 7-diaminoheptane (GC7) combined with gemcitabine in PDAC therapy. We measured eukaryotic translation initiation factor 5A2 (eIF5A2) expression and gemcitabine sensitivity in different PDAC cell lines (Panc-1, BxPC-3, and T3-M4). The synergistic cytotoxic effects of gemcitabine combined with GC7 were measured using Cell Counting Kit-8 assays. Western blots were performed to measure eIF5A2 and multi-drug resistance 1 (MDR1) protein expression in PDAC cells. The present findings demonstrated that combined treatment with GC7 and gemcitabine significantly inhibited PDAC cell line viability (P<0.05). EdU incorporation assays also indicated that GC7 co-treatment remarkably enhanced gemcitabine sensitivity in PDAC cells. Furthermore, downregulation of eIF5A2 diminished the regulatory role of GC7 in gemcitabine cytotoxicity. Western blotting data indicated that GC7 downregulated the expression of MDR1 while gemcitabine induced MDR1 upregulation. These findings showed that GC7 combination therapy may enhance the therapeutic efficacy of gemcitabine in PDAC by downregulating MDR1 expression.

Abstract 3190: Using a genome-wide shRNA screen to investigate the mechanism of stroma-conferred gemcitabine resistance in pancreatic ductal adenocarcinoma

Abstract The purpose of this study is to identify mechanisms of resistance to gemcitabine in pancreatic ductal adenocarcinoma (PDAC) using an in vitro coculture system. The inherent resistance of PDAC to first line treatments such as gemcitabine has been attributed to the dense fibrotic stroma. Targeting stromal components, in particular cancer-associated fibroblasts (CAFs), the activated form of pancreatic stellate cells (PSCs), is a promising avenue for therapeutic intervention. We have demonstrated previously the induction of gemcitabine resistance when co-culturing KRAS G12D; p53 R172H; Pdx-cre (KPC) mouse PDAC-derived tumour cells with αSMA+ fibroblast-like cells from PDAC tumour (FLCs), in comparison to PDAC cells co-cultured with PSCs or in monoculture. This resistance-inducing effect is specific to cells of mesenchymal phenotype, is transient in nature and a product of cell-cell contact. To investigate the exact mechanism by which PDAC cells become resistant to gemcitabine in coculture, we undertook a genome-wide shRNA depletion viability screen, infecting KPC mouse PDAC cells with a pooled shERWOOD UltramiR shRNA library both in mono- and co-culture with FLCs, in the presence and absence of gemcitabine. Next-generation sequencing and differential expression analysis, using DESeq2, identified shRNAs depleted through 6 cycles of gemcitabine treatment in coculture, indicating they target genes driving the resistance effect. Pathway analysis of differential expression using MetaCore implicated DNA damage repair and protein modification pathways in the gemcitabine resistance effect. Depletion of p97, ATR, or Chk1 all sensitised PDAC cells to gemcitabine in coculture, corroborating findings with translational value in previous studies. Further evaluation is ongoing using validation shRNA dropout assays in combination with targeted literature reviews to collate a candidate list of targetable genes with translational value in preventing and combating chemotherapy resistance in patients with PDAC. Citation Format: Graham D. Mills, Nicolas Erard, Frances M. Richards, Gregory J. Hannon, Duncan I. Jodrell. Using a genome-wide shRNA screen to investigate the mechanism of stroma-conferred gemcitabine resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3190. doi:10.1158/1538-7445.AM2017-3190

Overcoming erlotinib resistance with STAT3 inhibition in pancreatic cancer.

304 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a major therapeutic challenge. Cytotoxic chemotherapy remains the standard approach in PDAC, but results in minimal survival benefit for patients, highlighting a desperate need for novel treatment strategies. Epidermal growth factor receptor (EGFR) is overexpressed in 25-90% of PDACs and has been shown to be an adverse prognostic marker for survival, making it a rational target. To date, the combination of the EGFR tyrosine kinase inhibitor, erlotinib, with gemcitabine remains the only approved targeted therapy based on a significant, yet modest, improved overall survival when compared to gemcitabine alone in a phase III clinical trial. We have previously shown that constitutively activated signal transducer and activator of transcription 3 (STAT3) signaling is a biomarker of resistance to both gemcitabine and erlotinib. Therefore, we hypothesized that combined STAT3 and EGFR inhibition would overcome resistance to erlotinib and gemcitabine in PDAC. Methods: Human pancreatic cancer cell lines MiaPaca2 ( KRASG12C ; TP53mut ; EGFRwt) or BxPC-3 ( KRASwt ; TP53mut ; EGFRwt) were treated in a dose dependent manner with erlotinib in the presence or absence of gemcitabine. PDAC cells were additionally treated with EC50doses of a STAT3 inhibitor (AZD1480), erlotinib, and/or gemcitabine. Lysates were then collected and western blot analysis was performed to detect total and phosphorylated extracellular signal regulated kinase (ERK) or STAT3. Results: EGFR inhibition with erlotinib, with or without gemcitabine, results in decreased ERK activation, however, causes an increased activation of STAT3 signaling in both MiaPaca2 and BxPC-3 cells. Combined STAT3 and EGFR inhibition results in sustained attenuation of both phosphorylated ERK and STAT3 levels. Conclusions: Our study demonstrates that activated STAT3 signaling appears to be a mechanism of resistance to erlotinib and gemicitabine treatment in PDAC. Combining STAT3 inhibition with EGFR inhibition overcomes this resistance.

Multifaceted peptide assisted one-pot synthesis of gold nanoparticles for plectin-1 targeted gemcitabine delivery in pancreatic cancer

An astute modification of the plectin-1-targeting peptide KTLLPTP by introducing a C-terminal cysteine preceded by a tyrosine residue imparted a reducing property to the peptide. This novel property is then exploited to fabricate gold nanoparticles (GNP) via an in situ reduction of gold(iii) chloride in a one-pot, green synthesis. The modified peptide KTLLPTPYC also acts as a template to generate highly monodispersed, spherical GNPs with a narrow size distribution and improved stability. Plectin-1 is known to be aberrantly expressed in the surface of pancreatic ductal adenocarcinoma (PDAC) cells while showing cytoplasmic expression in normal cells. The synthesized GNPs are thus in situ surface modified with the peptides via the cysteine residue leaving the N-terminal KTLLPTP sequence free for targeting plectin-1. The visual molecular dynamics based simulations support the experimental observations like particle size, gemcitabine conjugation and architecture of the peptide-grafted nanoassembly. Additionally, GNPs conjugated to gemcitabine demonstrate significantly higher cytotoxicity in vitro in two established PDAC cell lines (AsPC-1 and PANC-1) and an admirable in vivo antitumor efficacy in a PANC-1 orthotopic xenograft model through selective uptake in PDAC tumor tissues. Altogether, this strategy represents a unique method for the fabrication of a GNP based targeted drug delivery platform using a multifaceted peptide that acts as reducing agent, template for GNP synthesis and targeting agent to display remarkable selectivity towards PDAC.

Tissue Transglutaminase Activates Cancer-Associated Fibroblasts and Contributes to Gemcitabine Resistance in Pancreatic Cancer

Resistance to chemotherapy is a hallmark of pancreatic ductal adenocarcinoma (PDA) and has been partly attributed to the dense desmoplastic stroma, which forms a protective niche for cancer cells. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme, is secreted by PDA cells and cross-links proteins in the tumor microenvironment (TME) through acyl-transfer between glutamine and lysine residues, promoting PDA growth. The objective of the current study was to determine whether secreted TG2 by PDA cells alters the response of pancreatic tumors to gemcitabine. Orthotopic pancreatic xenografts and co-culture of PDA and stromal cells were employed to determine the mechanisms by which TG2 alters tumor-stroma interactions and response to gemcitabine. Analysis of the pancreatic The Cancer Genome Atlas (TCGA) database demonstrated that increased TG2 expression levels correlate with worse overall survival (hazard ratio=1.37). Stable TG2 knockdown in PDA cells led to decreased size of pancreatic xenografts and increased sensitivity to gemcitabine in vivo. However, TG2 downregulation did not increase cytotoxicity of gemcitabine in vitro. Additionally, multivessel density and gemcitabine uptake in pancreatic tumor tissue, as measured by mass spectrometry (MS-HPLC), were not significantly different in tumors expressing TG2 versus tumors in which TG2 was knocked down. Fibroblasts, stimulated by TG2 secreted by PDA cells, secrete laminin A1, which protects cancer cells from gemcitabine-induced cytotoxicity. In all, our results demonstrate that TG2 secreted in the pancreatic TME orchestrates the cross talk between cancer cells and stroma, impacting tumor growth and response to chemotherapy. Our study supports TG2 inhibition to increase the antitumor effects of gemcitabine in PDA.

TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61) negatively regulates the nucleoside transporters hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 increased expression of hENT1 and hCNT3, increased cellular uptake of gemcitabine and sensitized PDAC cells to gemcitabine-induced apoptosis. In PDAC patient samples, expression of hENT1 and hCNT3 negatively correlates with expression of CYR61 . We demonstrate that stromal pancreatic stellate cells (PSCs) are a source of CYR61 within the PDAC tumor microenvironment. Transforming growth factor-β (TGF-β) induces the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad2/3 signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in PDAC cells in an in vitro co-culture assay. Our results identify CYR61 as a TGF-β-induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.

P21-activated kinase 1 (Pak1) signaling influences therapeutic outcome in pancreatic cancer

Therapeutic resistance to gemcitabine in pancreatic ductal adenocarcinoma (PDAC) is attributed to various cellular mechanisms and signaling molecules that influence as a single factor or in combination. In this study, utilizing in vitro p21-activated kinase 1 (Pak1) overexpression and knockdown cell line models along with in vivo athymic mouse tumor xenograft models and clinical samples, we demonstrate that Pak1 is a crucial signaling kinase in gemcitabine resistance. Pak1 kindles resistance via modulation of epithelial-mesenchymal transition and activation of pancreatic stellate cells. Our results from gemcitabine-resistant and -sensitive cell line models showed that elevated Pak1 kinase activity is required to confer gemcitabine resistance. This was substantiated by elevated levels of phosphorylated Pak1 and ribonucleotide reductase M1 levels in the majority of human PDAC tumors when compared with normal. Delineation of the signaling pathway revealed that Pak1 confers resistance to gemcitabine by preventing DNA damage, inhibiting apoptosis and regulating survival signals via NF-κB. Furthermore, we found that Pak1 is an upstream interacting substrate of transforming growth factor β-activated kinase 1-a molecule implicated in gemcitabine resistance. Molecular mechanistic studies revealed that gemcitabine docks with the active site of Pak1; furthermore, gemcitabine treatment induces Pak1 kinase activity both in vivo and in cell-free system. Finally, results from athymic mouse tumor models illustrated that Pak1 inhibition by IPA-3 enhances the cytotoxicity of gemcitabine and brings about pancreatic tumor regression. To our knowledge, this is the first study illustrating the mechanistic role of Pak1 in causing gemcitabine resistance via multiple signaling crosstalks, and hence Pak1-specific inhibitors will prove to be a better adjuvant with existing chemotherapy modality for PDAC.

Synergy of water soluble prodrug triptolide (minnelide) with gemcitabine and nab-paclitaxel in pancreatic cancer.

259 Background: Gemcitabine with nab-paclitaxel is the standard of care for metastatic Pancreatic Ductal Adenocarcinoma (PDAC). Addition of nab-paclitaxel to gemcitabine improves the survival of patients with metastatic pancreatic cancer by only 6 weeks while increasing toxicity. Meaningful combinations that reduce the doses while improving efficacy are required. Water soluble prodrug of triptolide (Minnelide) is effective against PDAC in preclinical studies and has demonstrated activity in the ongoing phase I trial. We have evaluated the combination of low dose triptolide prodrug with lowered doses of Abraxane and gemcitabine in PDAC. Methods: Pancreatic cancer cell lines S2-013 and S2-VP10 were xenografted both subcutaneously as well as orthotopically in athymic nude mice. Efficacy of a combination of triptolide prodrug (Minnelide: 0.21mg/kg) with reduced dose of gemcitabine 100mg/kg + Abraxane 10mg/kg (~40% of the clinically effective dose) was evaluated and compared with both standard of care gemcitabine 250mg/kg + Abraxane 25mg/kg as well as reduced dose of gemcitabine + Abraxane in animal models. We also used the immunocompetent model where murine PDAC cell line KPC001 implanted subcutaneously in C57/bl6 mice. Results: In subcutaneous syngeneic model, combination of triptolide prodrug and paclitaxel significantly inhibited tumor growth (tumor volumes expressed as % of Control, Mean±SEM: triptolide prodrug: 75.4±25, paclitaxel: 50.0 ±3, triptolide prodrug + paclitaxel: 11.0 ±1). In metastasis model combination therapy markedly improved survival (median survival in days: Vehicle-13, triptolide prodrug -20, standard of care -45, reduced dose standard of care-30, and Minnelide + reduced dose standard of care (all mice alive at 90 days). Combination of triptolide prodrug and low dose gemcitabine + nab-paclitaxel decreased tumor size, increased survival, reduced metastases and malignant ascites in orthotopic model of PDAC. Conclusions: Triptolide prodrug synergizes with reduced dose standard of care (gemcitabine and nab-paclitaxel) and helps in reducing the doses of these toxic drugs all the while achieving same or better efficacy in animal models of PDAC.

Abstract A105: Mechanisms of overcoming intrinsic resistance to gemcitabine in pancreatic ductal adenocarcinoma through redox modulation

Abstract Pancreatic ductal adenocarcinoma (PDAC) usually develops intrinsic and extrinsic resistances to gemcitabine, and the mechanisms are not well understood. Here we found gemcitabine treatment induced NOX-derived ROS generation through the increase of p22-phox expression via NF-kappaB activation. As ROS levels increased, nuclear translocation of Nrf2 stimulated transcription of cytoprotective antioxidant genes, specially, those encoding enzymes that catalyze glutathione production to reduce elevated ROS as one of intrinsic resistance counter measures. RNAi depletion of Nrf2 enhanced the sensitivity to gemcitabine in multiple PDAC cells. The addition of beta–phenylethyl isothiocyanate (PEITC) inhibited the ROS detoxification process by reducing glutathione levels; this, in turn, increased the efficacy of gemcitabine in vitro and in vivo. Thus, our results provide novel pre-clinical knowledge in pancreatic cancer chemoresistance, and combination with a redoxmodulating compound to deplete antioxidants can overcome intrinsic gemcitabine resistance. Our study suggests that a redox-mediated pathway that contributes to the intrinsic resistance of PDAC to gemcitabine and provides a basis for developing strategies to preferentially kill PDAC cells through ROS-mediated mechanism. The combination of gemcitabine and PEITC demonstrates a coordinated and selective cytotoxic effect against pancreatic cancer cells and a new avenue for further clinical studies of this deadly disease. Thus, this combination strategy could prove valuable as a cancer treatment. Note: These results were published in Molecular Cancer Therapeutics as a full-length article before the online publication of this abstract. Citation information: Mechanisms of Overcoming Intrinsic Resistance to Gemcitabine in Pancreatic Ductal Adenocarcinoma through the Redox Modulation. Huai-Qiang Ju, Takeshi Gocho, Mitzi Aguilar, Min Wu, Zhuo-Nan Zhuang, Jie Fu, Katsuhiko Yanaga, Peng Huang, and Paul J. Chiao. Mol Cancer Ther March 2015 14:788-798. doi:10.1158/1535-7163.MCT-14-0420. Citation Format: Huaiqiang Ju, Takshi Gocho, Mitzi Aguilar, Zhuo-Nan Zhuang, Jie Fu, Min Wu, Katsuhiko Yanaga, Peng Huang, Paul Chiao. Mechanisms of overcoming intrinsic resistance to gemcitabine in pancreatic ductal adenocarcinoma through redox modulation. [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 A105.

The association of epithelial expression of hENT1 and survival from adjuvant gemcitabine in pancreatic ductal adenocarcinoma (PDAC).

The association of epithelial expression of hENT1 and survival from adjuvant gemcitabine in pancreatic ductal adenocarcinoma (PDAC).

Mechanisms of Overcoming Intrinsic Resistance to Gemcitabine in Pancreatic Ductal Adenocarcinoma through the Redox Modulation.

Pancreatic ductal adenocarcinoma (PDAC) frequently develops therapeutic resistances, which can be divided into extrinsic and intrinsic resistance. The extrinsic resistance that arises from the surrounding dense tumor stroma is much better understood. However, the mechanisms of intrinsic resistance are not well understood. Here, we report that reactive oxygen species (ROS) induced by gemcitabine treatment, a newly discovered cytotoxic activity, served as a probe in our study to reveal the mechanisms of the intrinsic therapeutic resistance. Our results showed that gemcitabine-induced ROS is generated by NOX and through the increase of p22(-phox) expression via NF-κB activation. As a feedback mechanism, nuclear translocation of Nrf2 stimulated the transcription of cytoprotective antioxidant genes, especially genes encoding enzymes that catalyze glutathione (GSH) production to reduce elevated ROS as an intrinsic resistance countermeasure. RNAi-mediated depletion of Nrf2 or addition of β-phenylethyl isothiocyanate inhibited the ROS detoxification process by reducing GSH levels, which, in turn, increased the efficacy of gemcitabine in vitro and in vivo. Thus, our study suggests that a redox-mediated pathway contributes to the intrinsic resistance of PDAC to gemcitabine and provides a basis for developing strategies to preferentially kill PDAC cells through ROS-mediated mechanism. The combination of gemcitabine and PEITC has a selective cytotoxic effect against pancreatic cancer cells in vivo and could thus prove valuable as a cancer treatment.

The association of epithelial expression of hENT1 and survival from adjuvant gemcitabine in pancreatic ductal adenocarcinoma (PDAC).

296 Background: Human equilibrative nucleoside transporter 1 (hENT1) mediates the cellular uptake of gemcitabine. Low tumour hENT1 expression has been associated with reduced survival from gemcitabine in some studies. We analyzed the prognostic effect of hENT1 expression in a cohort of PDAC patients (pts) who underwent surgical resection. Methods: A tissue microarray (TMA) composed of duplicate 0.6 mm cores from 261 resected PDACs was constructed from all available cases derived from the archives of Vancouver General Hospital between 1988 - 2013. TMA sections (4 µm) were stained with an antibody for hENT1 (SP120- Spring Biosciences) and scored using a four-tiered scoring system (negative, weak, moderate and strong staining). The expression of hENT1 in the epithelial component of PDAC was quantified and patient samples were divided into low (negative and weak staining) and high (moderate and strong staining) hENT1-expressing groups. Treatment and survival data were collected. Survival differences were quantified with Kaplan-Meier method and log-rank statistic. Results: Our cohort included pts who received no adjuvant chemotherapy (CT) (n=182) or gemcitabine (n=59). Cases treated with other regimens (n=20) were excluded. There was no significant difference in the median overall survival (OS) between pts with low (14.0 months, n=167) and high hENT1 expression (15.2 months, n=15) in the absence of adjuvant CT. Gemcitabine treated patients with low hENT1 expression (n=51) had a median OS of 20.9 months. Median OS was not reached in the group of pts with high hENT1 expression (n=8) who received gemcitabine, with only 2 deaths at the 24 month time point and 6 censorings (p = 0.045). Pts who received gemcitabine had an increased median OS (24.8 months) compared to pts who did not receive adjuvant CT (14.0 months) regardless of hENT1 expression (p&lt;0.0001). Conclusions: Consistent with the ESPAC-3 data, in our analysis adjuvant gemcitabine was associated with increased OS in PDAC pts with high epithelial hENT1 expression (determined using the SP120 antibody) compared to pts with low hENT1 expression. Prospective evaluation of the predictive value of hENT1 expression in this setting is needed.

Synergistic interaction of novel lactate dehydrogenase inhibitors with gemcitabine against pancreatic cancer cells in hypoxia

Background:Hypoxia is a driving force in pancreatic-ductal-adenocarcinoma (PDAC) growth, metastasis and chemoresistance. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis, ensuring supply of energy and anabolites in hypoxic-environments. Therefore, we investigated the molecular mechanisms underlying the pharmacological interaction of novel LDH-A inhibitors in combination with gemcitabine in PDAC cells.Methods:Lactate dehydrogenase A levels were studied by quantitative RT–PCR, western blot, immunofluorescence and activity assays in 14 PDAC cells, including primary-cell-cultures and spheroids, in normoxic and hypoxic conditions. Cell proliferation, migration and key determinants of drug activity were evaluated by sulforhodamine-B-assay, wound-healing assay, PCR and LC-MS/MS.Results:Lactate dehydrogenase A was significantly increased under hypoxic conditions (1% O2), where the novel LDH-A inhibitors proved to be particularly effective (e.g., with IC50 values of 0.9 vs 16.3 μM for NHI-1 in LPC006 in hypoxia vs normoxia, respectively). These compounds induced apoptosis, affected invasiveness and spheroid-growth, reducing expression of metalloproteinases and cancer-stem-like-cells markers (CD133+). Their synergistic interaction with gemcitabine, with combination index values <0.4 in hypoxia, might also be attributed to modulation of gemcitabine metabolism, overcoming the reduced synthesis of phosphorylated metabolites.Conclusion:Lactate dehydrogenase A is a viable target in PDAC, and novel LDH-A inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours.

Phase I Trial of AEG35156 an Antisense Oligonucleotide to XIAP Plus Gemcitabine in Patients With Metastatic Pancreatic Ductal Adenocarcinoma

AEG35156 is an antisense oligonucleotide (ASO) that targets the X-linked inhibitor of apoptosis mRNA. Preclinical studies showed potent activity of AEG35156 in combination with gemcitabine in pancreatic ductal adenocarcinoma (PDA). A phase I study was conducted to establish the maximum-tolerated dose, safety, and antitumor activity of AEG35156 plus gemcitabine in metastatic PDA. Fourteen patients with metastatic PDA were enrolled. Nine patients were treated at 350 mg IV and 5 patients at 500 mg IV of AEG35156, 3 weeks on/1 week off of a 28-day cycle. Gemcitabine was administered at 1000 mg/m(2) IV over 30 minutes immediately after AEG35156 in both groups. Because of perceived neurotoxicity dose deescalation to 350 mg was recommended. All 14 patients were evaluable for tolerability and toxicity. Toxicities include neutropenia (grade 3/4, 6 patients), thrombocytopenia (grade 3, 2 patients), peripheral neuropathy (grade 3, 2 patients), fatigue (grade 3, 4 patients), ascites (grade 3, 2 patients), and nausea/vomiting (grade 4, 2 patients). Five patients (45%) experienced stable disease with a median progression-free survival of 58 days (95% CI, 52-107 d). The maximum-tolerated dose is AEG35156 500 mg plus gemcitabine 1000 mg/m(2) given on days 1, 8, and 15 every 28 days. AEG35156 plus gemcitabine failed to show significant clinical activity in advanced PDA.

Minnelide induces cell death in pancreatic cancer through regulation of the transcription factor Sp1

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by overexpression of Akt signaling pathway, which has been shown to be associated with aggressive behaviour and chemoresistance. Aims: The aim of this study was to evaluate the therapeutic potential of perifosine, as a novel Akt inhibitor, in combination with gemcitabine in PDAC. Materials &methods: In vitro studieswere performed in 14 pancreaticcancer-cells, including seven primary PDAC cultures. Growth inhibitory effects of perifosine and gemcitabine were evaluated in PDAC cells, whereas modulation of Akt and phospho-Akt was investigated by Western-blotting and ELISA. Cell-cycle perturbation, apoptosis-induction and anti-migratory behaviors of perifosine were studied by flow-cytometry, AnnexinV, membrane potential, and migration assay, while pharmacological interaction with gemcitabine was determined with combination index (CI) method. Results: Akt expression was detected by quantitative-RT-PCR in 14 PDAC cells, including 7 primary cell cultures. Perifosine (IC50s, 2-hourexposure) modulated the expression of Akt, phospho-Akt, mTOR, Bcl-2, Bad and PARP proteins, and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.1 (CFPAC-1), 0.45 (PANC-1) and 0.75 (PP109). The drug combination reduced the percentages of cells in G2/M phase (e.g., from 28 to 16% in PANC-1, P<0.05), and significantly increased apoptosis compared to gemcitabine-alone. Moreover, perifosine decreased cell migration, which was additionally reduced by perifosine/gemcitabine combination (e.g., -20% in PP109, after 8hr exposure, P<0.05). Conclusion: These data show the ability of perifosine to specifically target Akt, interfere with cell-proliferation, induce apoptosis, reduce migration and synergistically interact with gemcitabine, supporting further studies on this novel therapeutic approach for treatment of pancreatic cancer

Impact of AKT/PI3K signaling pathway in pancreatic cancer and synergistic interaction of the novel Akt inhibitor perifosine with gemcitabine in pancreatic cancer cells

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by overexpression of Akt signaling pathway, which has been shown to be associated with aggressive behaviour and chemoresistance. Aims: The aim of this study was to evaluate the therapeutic potential of perifosine, as a novel Akt inhibitor, in combination with gemcitabine in PDAC. Materials &methods: In vitro studieswere performed in 14 pancreaticcancer-cells, including seven primary PDAC cultures. Growth inhibitory effects of perifosine and gemcitabine were evaluated in PDAC cells, whereas modulation of Akt and phospho-Akt was investigated by Western-blotting and ELISA. Cell-cycle perturbation, apoptosis-induction and anti-migratory behaviors of perifosine were studied by flow-cytometry, AnnexinV, membrane potential, and migration assay, while pharmacological interaction with gemcitabine was determined with combination index (CI) method. Results: Akt expression was detected by quantitative-RT-PCR in 14 PDAC cells, including 7 primary cell cultures. Perifosine (IC50s, 2-hourexposure) modulated the expression of Akt, phospho-Akt, mTOR, Bcl-2, Bad and PARP proteins, and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.1 (CFPAC-1), 0.45 (PANC-1) and 0.75 (PP109). The drug combination reduced the percentages of cells in G2/M phase (e.g., from 28 to 16% in PANC-1, P<0.05), and significantly increased apoptosis compared to gemcitabine-alone. Moreover, perifosine decreased cell migration, which was additionally reduced by perifosine/gemcitabine combination (e.g., -20% in PP109, after 8hr exposure, P<0.05). Conclusion: These data show the ability of perifosine to specifically target Akt, interfere with cell-proliferation, induce apoptosis, reduce migration and synergistically interact with gemcitabine, supporting further studies on this novel therapeutic approach for treatment of pancreatic cancer

Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24+CD44+ and ALDH+ tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for further investigation of this combination in PDA.

Development of the SP120 rabbit monoclonal antibody for determining hENT1 status and predicting response to gemcitabine in pancreatic ductal adenocarcinoma.

4041 Background: Human equilibrative nucleoside transporter 1 (hENT1) is the primary membrane channel through which gemcitabine (Gem) enters pancreatic tumor cells. Patients whose tumors have low expression of hENT1 may derive little benefit from Gem therapy. Methods: We have developed and analytically validated a rabbit monoclonal antibody, SP120, immunohistochemistry (IHC) in vitro diagnostic (IVD) assay, used on the VENTANA BenchMark ULTRA automated slide staining platform, for assessing hENT1 expression in pancreatic adenocarcinoma. Results: SP120 was shown to be sensitive and specific for membrane expression of hENT1 with variable expression demonstrated across a panel of primary pancreatic adenocarcinoma samples. Using retrospectively collected primary tumor samples (n = 201) from a randomized controlled clinical adjuvant trial (RTOG 97-04), we developed and verified a hENT1 scoring algorithm and cut-off for identifying patients least likely to benefit from Gem (hENT1-low). We confirmed the predictive value of the hENT1 assay by showing there was no association with clinical outcome in the 5FU-treated control group. The distribution of hENT1 expression was similar between these samples and an independent set of 130 pancreatic adenocarcinoma specimens from the AIO-PK0104 study (77 were confirmed metastatic biopsies). Importantly, 100% concordance in hENT1 status (high vs. low) was demonstrated between 16 primary tumors and paired, contemporaneous metastatic lesions. Overall, using this cut-off, approximately two thirds of pancreatic tumors displayed low-hENT1 expression across the different data sets (n = 363). Conclusions: The hENT1 IHC IVD has been developed and is being used in a pivotal trial of a novel lipid-conjugated Gem (CO-101, which enters tumor cells independently of hENT1), vs. Gem in hENT1-low pancreatic cancer patients (NCT01124786). This study is the first prospective test of the hypothesis that gemcitabine is not active in tumors with low hENT1 expression.