Signaling In Pancreatic Cancer Cells Research Articles

Abstract B04: IKBKE integrates Gli and mTOR-AKT activation during Kras-induced pancreatic tumorigenesis

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest malignancies lacking effective therapeutic strategies. Here we show that the non-canonical IκB-related kinase, IKBKE, acts downstream of the Gli transcription factors and engages in reciprocal regulation of Gli signaling in pancreatic cancer cells. We find that loss of IKBKE inhibits the initiation and progression of pancreatic tumors in mice carrying pancreatic specific KRAS activation. Mechanistically, IKBKE does not play a major role in NF-κB activation in pancreatic cancer; but is critical for AKT activation in vivo. Moreover, we find that IKBKE promotes reactivation of AKT post-inhibition of mTOR in PDAC cells, and that combined inhibition of IKBKE and mTOR synergistically blocks pancreatic tumor growth. Together, our findings highlight the functional interaction of IKBKE with Hh/Gli and mTOR/AKT signaling in pancreatic cancer, and suggest IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic. Citation Format: Mihir Rajurkar, Martin Fernandez-Zapico, Brian Lewis, Junhao Mao. IKBKE integrates Gli and mTOR-AKT activation during Kras-induced pancreatic tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B04.

Epidermal Growth Factor Receptor Signaling to the Mitogen Activated Protein Kinase Pathway Bypasses Ras in Pancreatic Cancer Cells.

Epidermal growth factor (EGF) receptor (EGFR/HER1) is overexpressed in human pancreatic cancers. However, anti-EGFR therapy does not exhibit significant therapeutic activity with oncogenic K-ras mutation. We sought to assess the signaling relationship between EGFR and mutant K-ras, which is commonly detected in pancreatic cancer. Pancreatic cancer cells harboring mutated K-ras were treated with EGF to assess signaling from EGFR to mitogen-activated protein kinase (MAPK) pathway. The role of Ras family of proteins in transducing EGFR signals was assessed using short interfering RNA. Other components of MAPK and PI3K (phosphoinositide 3-kinase) pathways were examined for their roles in EGFR signaling. First, EGF signaling in pancreatic cancer cells occurs selectively through HER1. Second, knockdown of all Ras isoforms failed to block EGF-mediated phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of Raf was observed to partially abrogate ERK phosphorylation, whereas MEK inhibition resulted in complete attenuation of EGF-mediated ERK phosphorylation. Finally, inhibition of phosphoinositide 3-kinase/AKT and CDC42/PAK pathways did not block EGFR signaling. Our study results demonstrate that EGFR-mediated signaling in mutant K-ras pancreatic cancer cells does not follow canonical MAPK signaling. Our novel findings suggest the existence of alternate signaling pathways to downstream MAPK in the presence of mutant K-ras.

Abstract B21: Protein phosphatase 2A (PP2A) activation functions synergistically with kinase inhibition in pancreatic cancer

Abstract The 5-year survival rate for pancreatic cancer patients is only 6%, the lowest of all major cancers, indicating a critical need for increased understanding of pancreatic cancer development and identification of new therapeutic targets. Several genetic mutations are associated with the progression of human pancreatic ductal adenocarcinoma, including KRAS, which is mutated in ~95% of all pancreatic ductal adenocarcinomas (PDAC). Targeted therapies, such as kinase inhibitors, provide a means to reduce oncogenic signaling pathways that drive pancreatic cancer; however, the presence of resistance mechanisms, through extensive feedback loops, reduces the clinical efficacy of single agent treatments. Protein Phosphatase 2A (PP2A) is a critical tumor suppressor that negatively regulates several key oncogenic pathways implicated in mediating therapeutic resistance, including the PI3K/Akt, RAS/ERK, and Myc pathways. The goal of this research is to identify the therapeutic benefit of combined phosphatase activation and kinase inhibition, as a means to attenuate resistance mechanisms, reduce pancreatic tumor growth, and improve therapeutic efficacy in pancreatic cancer patients. We have determined that SET, an endogenous inhibitor of PP2A, is overexpressed in pancreatic cancer cell lines and primary patient samples, suggesting that suppression of PP2A contributes to pancreatic oncogenic signaling. As a result, we have started investigating the therapeutic efficacy of two novel therapeutic agents that activate PP2A: OP449, a SET antagonist, and DT1154, a small molecule direct PP2A activator. Treatment of pancreatic cancer cell lines with these compounds results in increased PP2A activity, reduced MYC levels, and reduced tumorigenic potential both in vitro and in vivo. Since these compounds reduce oncogenic signals known to contribute to pancreatic cancer, we hypothesize that phosphatase activation will function synergistically with select kinase inhibitors, resulting in sustained attenuation of oncogenic signaling in pancreatic cancer cells. In order to identify pathways that function synergistically with PP2A activation, a panel of pancreatic cancer cell lines was plated with and without OP449 into a 384-well kinase inhibitor (KI) screen, which evaluates the efficacy of over 130 kinase inhibitors at seven serial dilutions spanning the predicted IC50. Several signaling nodes were found to be synergistic with OP449, with the PI3K/AKT/mTOR pathways being particularly susceptible. Specifically, the combination of OP449 with Ink128, an mTOR1/2 inhibitor, reduced pancreatic cancer cell survival, oncogenic signaling, and transformed phenotypes, over either drug alone. These results provide new insight into our understanding of the regulation of pancreatic oncogenic signaling by phosphatases and identify new combination therapies that reduce resistance mechanisms. Citation Format: Brittany L. Allen-Petersen, Amy S. Farrell, Zina P. Jenny, Colin J. Daniel, Zhiping Wang, Charles D. Lopez, Dale J. Christensen, Goutham Narla, Brett C. Sheppard, Rosalie C. Sears. Protein phosphatase 2A (PP2A) activation functions synergistically with kinase inhibition in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B21.

Nanoformulation of Geranylgeranyltransferase-I Inhibitors for Cancer Therapy: Liposomal Encapsulation and pH-Dependent Delivery to Cancer Cells.

Small molecule inhibitors against protein geranylgeranyltransferase-I such as P61A6 have been shown to inhibit proliferation of a variety of human cancer cells and exhibit antitumor activity in mouse models. Development of these inhibitors could be dramatically accelerated by conferring tumor targeting and controlled release capability. As a first step towards this goal, we have encapsulated P61A6 into a new type of liposomes that open and release cargos only under low pH condition. These low pH-release type liposomes were prepared by adjusting the ratio of two types of phospholipid derivatives. Loading of geranylgeranyltransferase-I inhibitor (GGTI) generated liposomes with average diameter of 50–100 nm. GGTI release in solution was sharply dependent on pH values, only showing release at pH lower than 6. Release of cargos in a pH-dependent manner inside the cell was demonstrated by the use of a proton pump inhibitor Bafilomycin A1 that Increased lysosomal pH and inhibited the release of a dye carried in the pH-liposome. Delivery of GGTI to human pancreatic cancer cells was demonstrated by the inhibition of protein geranylgeranylation inside the cell and this effect was blocked by Bafilomycin A1. In addition, GGTI delivered by pH-liposomes induced proliferation inhibition, G1 cell cycle arrest that is associated with the expression of cell cycle regulator p21CIP1/WAF1. Proliferation inhibition was also observed with various lung cancer cell lines. Availability of nanoformulated GGTI opens up the possibility to combine with other types of inhibitors. To demonstrate this point, we combined the liposomal-GGTI with farnesyltransferase inhibitor (FTI) to inhibit K-Ras signaling in pancreatic cancer cells. Our results show that the activated K-Ras signaling in these cells can be effectively inhibited and that synergistic effect of the two drugs is observed. Our results suggest a new direction in the use of GGTI for cancer therapy.

Abstract 1028: Prostaglandin E2 activates the mTORC1 pathway through an EP4/cAMP/PKA and EP1/calcium-mediated mechanisms in human pancreatic carcinoma cells

Abstract Obesity, a known risk factor for pancreatic cancer, is associated with inflammation and insulin resistance. The pro-inflammatory prostaglandin E2 (PGE2) that signals via EP receptors and cAMP, and insulin-like growth factor 1 (IGF-1) that is elevated in insulin resistant states and activates the Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling module, are both shown to play critical roles in pancreatic cancer progression. We aimed at exploring the potential signaling crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 pathways in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In multiple human pancreatic cancer cell lines, PGE2 exposure increased intracellular cAMP levels, indicating the activation of Gαs-coupled receptors EP2 and/or EP4. In PANC-1 cells, which showed the greatest cAMP response, PGE2 dose- and time-dependently increased the phosphorylation of mTORC1 downstream targets p70S6 kinase (p70S6K) and S6 ribosomal protein (S6rp), suggesting a crosstalk between PGE2/cAMP and mTORC1. Accordingly, the mTORC1 inhibitor rapamycin suppressed PGE2-induced p70S6K and S6rp phosphorylation (Ser235/236 and Ser240/244). Also, the effect of PGE2 on p-S6rp was mimicked by forskolin, a cAMP stimulator. Interestingly, PGE2 and forskolin had no effect on p-Akt, suggesting a link downstream of Akt. Subsequent analysis using EP2 and EP4 antagonists/ agonists revealed that the PGE2-induced responses are mainly mediated by EP4. Further, PGE2-induced mTORC1 activation paralleled an increase in the phosphorylation of cAMP response element-binding protein (CREB), a substrate of protein kinase A (PKA). Pharmacological and genetic inhibition of PKA reduced baseline and PGE2-activated p-S6rp, indicating a role of PKA in the crosstalk. Additionally, calcium signaling seemed to be also involved in PGE2-induced mTORC1 activation, as p-S6rp (Ser235/236 and Ser240/244) was significantly decreased in cells pre-incubated with BAPTA-AM, a cell permeable Ca2+ chelator. Moreover, knockdown of EP1, a receptor known to mediate PGE2-induced Ca2+ response, attenuated PGE2-stimulated mTORC1 activation. Importantly, PGE2 enhanced the effect of IGF-1 on mTORC1 activation, suggesting a positive reinforcement by the interaction between the two pathways. Together, our data reveal a previously unrecognized mechanism of PGE2-stimulated mTORC1 activation mediated by EP4/cAMP/PKA and EP1/Ca2+ signaling in pancreatic cancer cells, which may be of great importance in elucidating the promoting effects of obesity in pancreatic cancer. Ultimately, a precise understanding of these molecular links may provide novel targets for efficacious interventions devoid of adverse effects. Citation Format: Hui-Hua Chang, Steven Young, James Sinnett-Smith, Caroline Ei Ne Chou, Oscar Joe Hines, Enrique Rozengurt, Guido Eibl. Prostaglandin E2 activates the mTORC1 pathway through an EP4/cAMP/PKA and EP1/calcium-mediated mechanisms in human pancreatic carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1028. doi:10.1158/1538-7445.AM2015-1028

Abstract A104: Phosphatase activation and kinase inhibition as a novel therapeutic strategy for pancreatic cancer

Abstract The 5-year survival rate for pancreatic cancer patients is only 6%, the lowest of all major cancers, indicating a critical need for increased understanding of pancreatic cancer development and identification of new therapeutic targets. Several genetic mutations are associated with the progression of human pancreatic ductal adenocarcinoma, including KRAS, which is mutated in ~95% of all pancreatic ductal adenocarcinomas (PDAC). Targeted therapies, such as kinase inhibitors, provide a means to reduce oncogenic signaling pathways that drive pancreatic cancer; however, the presence of resistance mechanisms, through extensive feedback loops, reduces the clinical efficacy of single agent treatments. Protein Phosphatase 2A (PP2A) is a critical tumor suppressor that negatively regulates several key oncogenic pathways implicated in mediating therapeutic resistance, including the PI3K/Akt, RAS/ERK, and Myc pathways. The goal of this research is to identify the therapeutic benefit of combined phosphatase activation and kinase inhibition, as a means to attenuate resistance mechanisms, reduce pancreatic tumor growth, and improve therapeutic efficacy in pancreatic cancer patients. We have determined that SET, an endogenous inhibitor of PP2A, is overexpressed in pancreatic cancer cell lines and primary patient samples, suggesting that suppression of PP2A contributes to pancreatic oncogenic signaling. Therapeutic inhibition of SET utilizing a novel SET antagonist, OP449, leads to increased PP2A activity and significant cytotoxic activity in pancreatic cancer cells in vitro and in vivo. Importantly, these results correlate with reduced activation of pathways known to contribute to therapeutic resistance, including c-Myc and Akt. I hypothesize that phosphatase activation will function synergistically with select kinase inhibitors, resulting in sustained attenuation of oncogenic signaling in pancreatic cancer cells. In order to identify pathways that function synergistically with PP2A activation, a panel of pancreatic cancer cell lines was plated with and without OP449 into a 384-well kinase inhibitor (KI) screen, which evaluates the efficacy of over 130 kinase inhibitors at seven serial dilutions spanning the predicted IC50. Several signaling nodes were found to be synergistic with OP449, with the PI3K/AKT/mTOR pathways being particularly susceptible. Specifically, the combination of OP449 with Ink128, an mTOR1/2 inhibitor, reduced pancreatic cancer cell survival, oncogenic signaling, and transformed phenotypes, over either drug alone. These results provide new insight into our understanding of the regulation of pancreatic oncogenic signaling by phosphatases and identify new combination therapies that reduce resistance mechanisms. Citation Format: Brittany L. Allen-Petersen, Amy S. Farrell, Colin J. Daniel, Zhiping Wang, Charles D. Lopez, Dale J. Christensen, Brett C. Sheppard, Rosalie C. Sears. Phosphatase activation and kinase inhibition as a novel therapeutic strategy for pancreatic cancer. [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 A104.

Quercetin 3-O-glucoside suppresses epidermal growth factor-induced migration by inhibiting EGFR signaling in pancreatic cancer cells.

Pancreatic cancer is one of the most dangerous cancers and is associated with a grave prognosis. Despite increased knowledge of the complex signaling networks responsible for progression of pancreatic cancer, many challenging therapies have fallen short of expectations. In this study, we examined the anti-migratory effect of quercetin 3-O-glucoside in epidermal growth factor-induced cell migration by inhibiting EGF receptor (EGFR) signaling in several human pancreatic cancer cell lines. Treatment with quercetin, quercetin 3-O-glucoside, and quercetin 7-O-glucoside differentially suppressed epidermal growth factor-induced migration activity of human pancreatic cancer cells. In particular, quercetin 3-O-glucoside strongly inhibited the infiltration activity of pancreatic cancer cells in a dose-dependent manner. Furthermore, quercetin 3-O-glucoside exerted the anti-migratory effect even at a relatively low dose compared with other forms of quercetin. The anti-tumor effects of quercetin 3-O-glucoside were mediated by selectively inhibiting the EGFR-mediated FAK, AKT, MEK1/2, and ERK1/2 signaling pathway. Combinatorial treatment with quercetin 3-O-glucoside plus gemcitabine showed the synergistic anti-migratory effect on epidermal growth factor-induced cell migration in human pancreatic cancer cell lines. These results suggest that quercetin 3-O-glucoside has potential for anti-metastatic therapy in human pancreatic cancer.

Decoy receptor 3 suppresses FasL-induced apoptosis via ERK1/2 activation in pancreatic cancer cells

Resistance to Fas Ligand (FasL) mediated apoptosis plays an important role in tumorigenesis. Decoy receptor 3 (DcR3) is reported to interact with FasL and is overexpressed in some malignant tumors. We sought to investigate the role of DcR3 in resistance to FasL in pancreatic cancer. We compared expression of apoptosis related genes between FasL-resistant SW1990 and FasL-sensitive Patu8988 pancreatic cell lines by microarray analysis. We explored the impact of siRNA knockdown of, or exogenous supplementation with, DcR3 on FasL-induced cell growth inhibition in pancreatic cancer cell lines and expression of proteins involved in apoptotic signaling. We assessed the level of DcR3 protein and ERK1/2 phosphorylation in tumor and non-tumor tissue samples of 66 patients with pancreatic carcinoma. RNAi knockdown of DcR3 expression in SW1990 cells reduced resistance to FasL-induced apoptosis, and supplementation of Patu8988 with rDcR3 had the opposite effect. RNAi knockdown of DcR3 in SW1990 cells elevated expression of caspase 3, 8 and 9, and reduced ERK1/2 phosphorylation (P < 0.05), but did not alter phosphorylated-Akt expression. 47 tumor tissue specimens, but only 15 matched non-tumor specimens stained for DcR3 (χ2 = 31.1447, P < 0.001). The proliferation index of DcR3 positive specimens (14.26 ± 2.67%) was significantly higher than that of DcR3 negative specimens (43.58 ± 7.88%, P < 0.01). DcR3 expression positively correlated with p-ERK1/2 expression in pancreatic cancer tissues (r = 0.607, P < 0.001). DcR3 enhances ERK1/2 phosphorylation and opposes FasL signaling in pancreatic cancer cells.

Caveolin-1 is Associated with Tumor Progression and Confers a Multi-Modality Resistance Phenotype in Pancreatic Cancer.

Caveolin-1 (Cav-1) is a 21 kDa protein enriched in caveolae, and has been implicated in oncogenic cell transformation, tumorigenesis, and metastasis. We explored roles for Cav-1 in pancreatic cancer (PC) prognostication, tumor progression, resistance to therapy, and whether targeted downregulation could lead to therapeutic sensitization. Cav-1 expression was assessed in cell lines, mouse models, and patient samples, and knocked down in order to compare changes in proliferation, invasion, migration, response to chemotherapy and radiation, and tumor growth. We found Cav-1 is overexpressed in human PC cell lines, mouse models, and human pancreatic tumors, and is associated with worse tumor grade and clinical outcomes. In PC cell lines, disruption/depletion of caveolae/Cav-1 reduces proliferation, colony formation, and invasion. Radiation and chemotherapy up-regulate Cav-1 expression, while Cav-1 depletion induces both chemosensitization and radiosensitization through altered apoptotic and DNA repair signaling. In vivo, Cav-1 depletion significantly attenuates tumor initiation and growth. Finally, Cav-1 depletion leads to altered JAK/STAT, JNK, and Src signaling in PC cells. Together, higher Cav-1 expression is correlated with worse outcomes, is essential for tumor growth and invasion (both in vitro and in vivo), is responsible for promoting resistance to therapies, and may serve as a prognostic/predictive biomarker and target in PC.

Homeostatic Restoration of Desmoplastic Stroma Rather Than Its Ablation Slows Pancreatic Cancer Progression

Homeostatic Restoration of Desmoplastic Stroma Rather Than Its Ablation Slows Pancreatic Cancer Progression

The Role of Nutraceuticals in Pancreatic Cancer Prevention and Therapy: Targeting Cellular Signaling, MicroRNAs, and Epigenome.

Pancreatic cancer is one of the most aggressive malignancies in US adults. Experimental studies have found that antioxidant nutrients could reduce oxidative DNA damage, suggesting that these antioxidants may protect against pancreatic carcinogenesis. Several epidemiologic studies showed that dietary intake of antioxidants was inversely associated with the risk for pancreatic cancer, demonstrating the inhibitory effects of antioxidants on pancreatic carcinogenesis. Moreover, nutraceuticals, the anticancer agents from diet or natural plants, have been found to inhibit the development and progression of pancreatic cancer through the regulation of cellular signaling pathways. Importantly, nutraceuticals also up-regulate the expression of tumor-suppressive microRNAs (miRNAs) and down-regulate the expression of oncogenic miRNAs, leading to the inhibition of pancreatic cancer cell growth and pancreatic cancer stem cell self-renewal through modulation of cellular signaling network. Furthermore, nutraceuticals also regulate epigenetically deregulated DNAs and miRNAs, leading to the normalization of altered cellular signaling in pancreatic cancer cells. Therefore, nutraceuticals could have much broader use in the prevention and/or treatment of pancreatic cancer in combination with conventional chemotherapeutics. However, more in vitro mechanistic experiments, in vivo animal studies, and clinical trials are needed to realize the true value of nutraceuticals in the prevention and/or treatment of pancreatic cancer.

Risk factors for pancreatic cancer: underlying mechanisms and potential targets.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths among both men and women in the United States and remains essentially without effective therapies. The 5-year survival of patients with pancreatic cancer is a dismal 6% or less. Environmental risk factors such as smoking, diabetes, obesity, and alcoholism play major roles in the promotion of PDAC. However, currently we have limited understanding of how these risk factors promote the disease. The papers presented in this topic illustrate the latest knowledge regarding the mechanisms of pancreatic cancer induction and promotion by major risk factors such as smoking, pancreatitis, alcohol abuse, obesity and diabetes (Kolodecik et al., 2013), and less studied factors such blood group types (Pelzer et al., 2013) and the genetic mutations (Kong et al., 2013; Reznik et al., 2014; Weiss, 2014). Indeed, an inherited predisposition to PDAC is believed to present in familial cancer syndromes such as the Peutz-Jeghers Syndrome, which is associated with germline mutations in the STK11/LKB1 gene, Familial Atypical Multiple Mole Melanoma syndrome, which results due to germline mutations in the p16/CDKN2A gene, Hereditary Breast-Ovarian Cancer syndrome (BRCA1/2 genes), Hereditary Non-polyposis Colorectal Cancer (mismatch repair genes), and Familial Adenomatous Polyposis syndrome (Reznik et al., 2014). Hereditary causes of pancreatitis, such as the autosomal dominant form caused by germline mutations of the cationic trypsinogen gene, PRSS1, have been indirectly linked to PDAC through early onset chronic pancreatitis with an associated 53-fold increased incidence and approximately 40% of hereditary pancreatitis patients noted to develop pancreatic cancer by age 70 (Weiss, 2014). Finally, more patients with blood group A suffer from PDAC whereas blood group O was less frequent in patients with PDAC (Pelzer et al., 2013). Kras mutations remain the most abundant genetic alteration found in pancreatic cancer patients. Kras mutations may lead to reducing power for ROS detoxification, leading to low ROS levels in pancreatic pre-neoplastic cells and in cancer cells. In adult stem cells and cancer stem cells, low ROS levels have been associated with the formation of a proliferation-permissive intracellular environment and with perseverance of self-renewal capacities. Therefore, it is conceivable that low intracellular ROS levels may contribute significantly to oncogenic Kras-mediated PDAC formation (Kong et al., 2013). Specific gene-based, gene-product, and marker-based testing for the early detection of pancreatic cancer are currently being developed, with the potential for these to be useful as potential therapeutic targets as well. Today, the role of stromal cells is highly appreciated for pancreatic cancer development. Immune cell infiltration into the tumor not only fail to contribute to disease eradication but rather due to exhibiting a Th2-type inflammation and immunosuppression is associated with more rapid disease progression, cachexia induction, and reduced survival. Polarization of macrophages toward M2-type correlates with a poor prognosis after surgery in resected patients. High CD163+ and CD204+ cell counts correlate with metastasis and poor prognosis in PDAC patients (Protti and De Monte, 2013; Tan et al., 2014). Src kinase might serve as a critical mechanistic link between inflammation and cancer, mediating and propagating a cycle between immune and tissue cells that can ultimately lead to the development and progression of cancer (Liu et al., 2013). In addition, there is now compelling evidence that pancreatic stellate cells interact not only with cancer cells themselves, but with several other cell types in the stroma (endothelial cells, immune cells, and possibly neuronal cells) to promote cancer progression. Strategies to target the tumor microenvironment cells are proposed (Wilson et al., 2014). The central role of the mammalian target of rapamycin (mTOR) in mediating a crosstalk between the insulin/IGF-1 and GPCR signaling in pancreatic cancer cells is discussed in depth and strategies, including the use of metformin, to target this signaling pathway in PDAC cells are proposed (Rozengurt, 2014). Finally, a comprehensive review of the latest animal models of pancreatic cancer are discussed, proposing novel tools to study the mechanism of pancreatic cancer initiation and promotion by major risk factors.

Abstract 4115: GS-19, a novel GSK inhibitor suppresses the growth of pancreatic cancer cells by inhibiting EGFR/AKT/STAT-3 signaling

Abstract Pancreatic cancer remains the fourth leading cause of cancer related deaths in the United States. Therefore, identification of novel strategies to control and treat pancreatic cancer is of great importance. GS-19 is a novel compound recently found to be a GSK inhibitor. In this study, we determined the anti-cancer effects of GS-19 in pancreatic cancer cells. GS-19 treatment inhibited the growth of Panc-1 and BxPC-3 cells in a concentration-dependent manner. Both the cell lines treated with GS-19 exhibited G-1 cell cycle arrest as well as apoptosis. Western blot analysis of both BxPC-3 and Panc-1 cells treated with GS-19 inhibited the activation (phosphorylation) and expression of key survival signaling proteins such as EGFR, STAT3 and AKT. The apoptosis induced by GS-19 was associated with cleavage of caspase-3 and PARP. Taken together, our results suggest that GS-19 suppress pancreatic cancer cell growth by inhibiting EGFR/AKT/STAT-3 signaling. However, further mechanistic studies are in progress to establish the role of EGFR/AKT/STAT-3 signaling in pancreatic cancer cells. [The studies in part were supported by R01 grant CA129038 awarded by NIH to S.K.S.] Citation Format: Alok Ranjan, Ashlee Birkenfeld, Jianyu Lu, Duy Hua, Sanjay K. Srivastava. GS-19, a novel GSK inhibitor suppresses the growth of pancreatic cancer cells by inhibiting EGFR/AKT/STAT-3 signaling. [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 4115. doi:10.1158/1538-7445.AM2014-4115

A TrxR inhibiting gold(I) NHC complex induces apoptosis through ASK1-p38-MAPK signaling in pancreatic cancer cells.

BackgroundCancer cells in the advanced stage show aberrant antioxidant capacity to detoxify excessive ROS resulting in the compensation for intrinsic oxidative stress and therapeutic resistance. PDAC is one of the most lethal cancers and often associated with a high accumulation of ROS. Recent studies identified gold(I) NHC complexes as potent TrxR inhibitors suppressing cell growth in a wide spectrum of human malignant cell lines at the low micromolar concentration. However, the mechanism of action is not completely elucidated yet.MethodsTo understand the biological function of gold(I) NHC complexes in PDAC, we used a recently published gold(I) NHC complex, MC3, and evaluated its anti-proliferative effect in four PDAC cell lines, determined by MTT and SRB assays. In further detailed analysis, we analyzed cellular ROS levels using the ROS indicator DHE and mitochondrial membrane potential indicated by the dye JC-1 in Panc1. We also analyzed cell cycle arrest and apoptosis by FACS. To elucidate the role of specific cell signaling pathways in MC3-induced cell death, co-incubation with ROS scavengers, a p38-MAPK inhibitor and siRNA mediated depletion of ASK1 were performed, and results were analyzed by immunoblotting, ELISA-microarrays, qRT-PCR and immunoprecipitation.ResultsOur data demonstrate that MC3 efficiently suppressed cell growth, and induced cell cycle arrest and apoptosis in pancreatic cancer cells, in particular in the gemcitabine-resistant cancer cells Panc1 and ASPC1. Treatment with MC3 resulted in a substantial alteration of the cellular redox homeostasis leading to increased ROS levels and a decrease in the mitochondrial membrane potential. ROS scavengers suppressed ROS formation and rescued cells from damage. On the molecular level, MC3 blocked the interaction of Trx with ASK1 and subsequently activated p38-associated signaling. Furthermore, inhibition of this pathway by using ASK1 siRNA or a p38 inhibitor clearly attenuated the effect of MC3 on cell proliferation in Panc1 and ASPC1.ConclusionsOur results confirm that MC3 is a TrxR inhibitor and show MC3 induced apoptosis in gemcitabine-resistant PDACs. MC3 mediated cell death could be blocked by using anti-oxidants, ASK1 siRNA or p38 inhibitor suggesting that the Trx-ASK1-p38 signal cascade played an important role in gold(I) NHC complexes-mediated cellular damage.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-221) contains supplementary material, which is available to authorized users.

Mechanistic target of rapamycin (mTOR): a point of convergence in the action of insulin/IGF-1 and G protein-coupled receptor agonists in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is one of the most lethal human diseases. PDAC is now the fourth leading cause of cancer mortality in both men and women and deaths due to PDAC are projected to increase dramatically. Novel targets and agents for chemoprevention are urgently needed and will most likely arise from a more detailed understanding of the signaling mechanisms that stimulate the promotion and progression of sub-malignant cells into pancreatic cancer cells and from the identification of modifiable risk factors for PDAC. Many epidemiological studies have linked obesity and long-standing type 2 diabetes mellitus (T2DM) with increased risk and worse clinical outcomes for developing PDAC. These diet-related metabolic disorders are multifaceted but characterized by peripheral insulin resistance, compensatory overproduction of insulin and increased bioavailability of insulin-like growth factor-1 (IGF-1). Mounting evidence indicates that the insulin/IGF-1 receptor system plays a critical role in PDAC development and multiple studies support the notion that crosstalk between the insulin receptor and heptahelical G protein-coupled receptor (GPCR) signaling systems is an important element in the biological responses elicited by these signaling systems, including cell proliferation. This article highlights the central role of the mechanistic target of rapamycin (mTOR) in mediating crosstalk between insulin/IGF-1 and GPCR signaling in pancreatic cancer cells and proposes strategies, including the use of metformin, to target this signaling system in PDAC cells.

TRAIL mediated signaling in pancreatic cancer.

Research over the years has progressively shown substantial broadening of the tumor necrosis factor alpha- related apoptosis-inducing ligand (TRAIL)-mediated signaling landscape. Increasingly it is being realized that pancreatic cancer is a multifaceted and genomically complex disease. Suppression of tumor suppressors, overexpression of oncogenes, epigenetic silencing, and loss of apoptosis are some of the extensively studied underlying mechanisms. Rapidly accumulating in vitro and in vivo evidence has started to shed light on the resistance mechanisms in pancreatic cancer cells. More interestingly a recent research has opened new horizons of miRNA regulation by DR5 in pancreatic cancer cells. It has been shown that DR5 interacts with the core microprocessor components Drosha and DGCR8, thus impairing processing of primary let-7. Xenografting DR5 silenced pancreatic cancer cells in SCID-mice indicated that there was notable suppression of tumor growth. There is a paradigm shift in our current understanding of TRAIL mediated signaling in pancreatic cancer cells that is now adding new layers of concepts into the existing scientific evidence. In this review we have attempted to provide an overview of recent advances in TRAIL mediated signaling in pancreatic cancer as evidenced byfindings of in vitro and in vivo analyses. Furthermore, we discuss nanotechnological advances with emphasis on PEG-TRAIL and four-arm PEG cross-linked hyaluronic acid (HA) hydrogels to improve availability of TRAIL at target sites.

Sonic hedgehog paracrine signaling activates stromal cells to promote perineural invasion in pancreatic cancer.

Pancreatic cancer is characterized by stromal desmoplasia and perineural invasion (PNI). We sought to explore the contribution of pancreatic stellate cells (PSC) activated by paracrine Sonic Hedgehog (SHH) in pancreatic cancer PNI and progression. In this study, the expression dynamics of SHH were examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of carcinomatous and nonneoplastic pancreatic tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of PSCs activated by paracrine SHH signaling in pancreatic cancer PNI and progression. We show that SHH overexpression in tumor cells is involved in PNI in pancreatic cancer and is an important marker of biologic activity of pancreatic cancer. Moreover, the overexpression of SHH in tumor cells activates the hedgehog pathway in PSCs in the stroma instead of activating tumor cells. These activated PSCs are essential for the promotion of pancreatic cancer cell migration along nerve axons and nerve outgrowth to pancreatic cancer cell colonies in an in vitro three-dimensional model of nerve invasion in cancer. Furthermore, the coimplantation of PSCs activated by paracrine SHH induced tumor cell invasion of the trunk and nerve dysfunction along sciatic nerves and also promoted orthotropic xenograft tumor growth, metastasis, and PNI in in vivo models. These results establish that stromal PSCs activated by SHH paracrine signaling in pancreatic cancer cells secrete high levels of PNI-associated molecules to promote PNI in pancreatic cancer.

The Prolyl Peptidases PRCP/PREP Regulate IRS-1 Stability Critical for Rapamycin-induced Feedback Activation of PI3K and AKT

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells.

Hedgehog acyltransferase as a target in pancreatic ductal adenocarcinoma.

Sonic Hedgehog (Shh) is abnormally expressed in pancreatic cancer and is associated with disease onset and progression. Inhibition of Shh signaling is thus an attractive clinical target for therapeutic intervention. Most efforts to block Shh signaling have focused on inhibitors of Smoothened, which target the canonical Shh signaling pathway. These approaches have met with limited success, in part due to development of resistance-conferring mutations and contributions from non-canonical signaling pathways. Here, we show that Hedgehog acyltransferase (Hhat), the enzyme responsible for the attachment of palmitate onto Shh, is a novel target for inhibition of Shh signaling in pancreatic cancer cells. Depletion of Hhat with lentivirally delivered small hairpin RNA decreased both anchorage-dependent and independent proliferation of human pancreatic cancer cells. In vivo, Hhat knockdown led to reduction of tumor growth in a mouse xenograft model of pancreatic cancer. RU-SKI 43, a small molecule inhibitor of Hhat recently developed by our group, reduced pancreatic cancer cell proliferation and Gli-1 activation through Smoothened-independent non-canonical signaling. In addition, RU-SKI 43 treatment inhibited two key proliferative pathways regulated by Akt and mTOR. This work demonstrates that Hhat has a critical role in pancreatic cancer and that a small molecule inhibitor of Hhat can successfully block pancreatic cancer cell proliferation. It also highlights the importance of developing optimized Hhat inhibitors to be used as therapeutics in pancreatic cancer, as well as in other malignancies characterized by Shh overexpression.

Novel Mechanism of MDA-7/IL-24 Cancer-Specific Apoptosis through SARI Induction

Subtraction hybridization combined with induction of cancer cell terminal differentiation in human melanoma cells identified melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) and SARI (suppressor of AP-1, induced by IFN) that display potent antitumor activity. These genes are not constitutively expressed in cancer cells and forced expression of mda-7/IL-24 (Ad.mda-7) or SARI (Ad.SARI) promotes cancer-specific cell death. Ectopic expression of mda-7/IL-24 induces SARI mRNA and protein in a panel of different cancer cells, leading to cell death, without harming corresponding normal cells. Simultaneous inhibition of K-ras downstream extracellular signal-regulated kinase 1/2 signaling in pancreatic cancer cells reverses the translational block of MDA-7/IL-24 and induces SARI expression and cell death. Using SARI-antisense-based approaches, we demonstrate that SARI expression is necessary for mda-7/IL-24 antitumor effects. Secreted MDA-7/IL-24 protein induces antitumor "bystander" effects by promoting its own expression. Recombinant MDA-7/IL-24 (His-MDA-7) induces SARI expression, supporting the involvement of SARI in the MDA-7/IL-24-driven autocrine loop, culminating in antitumor effects. Moreover, His-MDA-7, after binding to its cognate receptors (IL-20R1/IL-20R2 or IL-22R/IL-20R2), induces intracellular signaling by phosphorylation of p38 MAPK, leading to transcription of a family of growth arrest and DNA damage inducible (GADD) genes, culminating in apoptosis. Inhibition of p38 MAPK fails to induce SARI following Ad.mda-7 infection. These findings reveal the significance of the mda-7/IL-24-SARI axis in cancer-specific killing and provide a potential strategy for treating both local and metastatic disease.