Activity In Pancreatic Cancer Research Articles

Synthetic Lethality Screen Identifies RPS6KA2 as Modifier of Epidermal Growth Factor Receptor Activity in Pancreatic Cancer

Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR) inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA)-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2)/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy.

The Winning Formulation: The Development of Paclitaxel in Pancreatic Cancer

Paclitaxel has wide application in anticancer therapy but was never considered an efficacious agent in pancreatic cancer. A review of the experience with the Cremaphor formulation hinted at paclitaxel's activity in pancreatic cancer, but the early development was hampered by significant toxicities such as neutropenia and infection at clinically tolerable doses. However, such efficacy was confirmed in the recently completed phase III Metastatic Pancreatic Adenocarcinoma Clinical Trial (MPACT), in which the addition of nab-paclitaxel to gemcitabine significantly improved the survival of patients with metastatic pancreatic cancer. Several other Cremaphor-free formulations of paclitaxel had also been evaluated in pancreatic cancer, and the reasons for the success of the albumin nanoparticulate are examined here. In the era of biologic and molecularly targeted agents, the success of nab-paclitaxel in recalcitrant pancreatic cancer is a timely reminder of the importance and relevance of pharmacology and novel drug delivery technology in the development of anticancer drugs.

Abstract 4013: S100A4 promotes cell migration and growth through focal adhesion kinase and Src mediated dual signaling pathways in pancreatic cancer cells.

Abstract BACKGROUD: Pancreatic cancer is one of the deadliest types of cancer. Activation and expression of focal adhesion kinase (FAK) and Src kinase is abnormally upregulated in pancreatic cancers. The results of clinical trials targeting either Src or FAK by inhibitors are largely unknown. S100A4 is a calcium-binding protein and aberrant S100A4 expression is an independent biomarker for poor outcomes of pancreatic cancer; however, the involved mechanism is unclear. METHODS: The effects of inhibitors on FAK and Src activation, and on cell migration and growth, were examined in MiaPaca2 and S2VP10 cell lines. To determine whether FAK and Src depend on each other or independently regulate pancreatic cancer cell phenotypes, the effects of FAK or Src mutant expression were determined. The role of S100A4 on FAK and Src activation was evaluated through loss/gain approaches. The association of S100A4 expression with FAK and Src activation was determined by immunostaining on human pancreatic cancer tissue sections. RESULTS: FAK and Src kinase inhibitors blocked activation in a dose dependent manner in both MiaPaca2 and S2VP10 cells. However, it required more inhibitor to inhibit either FAK or Src in S2VP10 cells. Blocking FAK (or Src) does not completely inhibit Src (or FAK) activation. This is confirmed by the finding that FAK mutant expression completely inhibited FAK activation, but not Src activation. Similarly, Src mutant expression did not inhibit FAK activation. Downregulation of S100A4 expression significantly decreased FAK and Src activation. Importantly, increased S100A4 expression is associated with increased FAK and Src activation in pancreatic cancer. CONCLUSIONS: These data support that the Src-FAK axis forms a dual signaling pathway, and each of them contributes independently to the aggressive pancreatic cancer cell phenotype. Also, these data support that S100A4 promotes the Src-FAK dual signaling pathway which possibly leads to a feed-forward loop facilitating pancreatic tumor progression. Citation Format: Guoqiang Cai, Elizabeth H. Kerr, Meng Hu, Youfeng Yang, Donald J. Buchsbaum, William E. Grizzle, Qiang Ding. S100A4 promotes cell migration and growth through focal adhesion kinase and Src mediated dual signaling pathways in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4013. doi:10.1158/1538-7445.AM2013-4013

Abstract 1257: Investigating the protective role of HLA-B35 in pancreatic cancer.

Abstract HLA-B35 has recently been reported as a protective marker for pancreatic cancer development. The goal of the current study was to investigate whether this link between B35 and cancer protection was related to cellular immunity and correlated with the presence of HLA-B35-restricted cytotoxic T cell activity. As a model antigen we chose mesothelin (MSLN), a tumor antigen expressed by >90% of pancreatic tumors and known to be immunogenic to T cells. To activate MSLN-specific T cells we generated autologous DCs from 15 HLA-B35 positive and 12 HLA-B35 negative donors and loaded them with a pepmix (15mers overlapping by 11aas) spanning MSLN in order to use an HLA unbiased approach for antigen stimulation. These were used to stimulate PBMCs in the presence of a cytokine cocktail (IL7, 12, 15, 6), which we have optimized for activating tumor-directed T cells in vitro. We were successfully able to amplify an immune response against MSLN in 12 of 15 (80%) B35+ donors based on IFNg ELIspot analysis (range 23-10108 SFC/2x10e5). In contrast we were successful in only 1 of 12 (8%) B35-ve donors. Since the CTL lines were polyclonal (CD8+, median 69%, range 38-97%; CD4+, median 30%, range 2-55%) we next investigated, using ICS, whether this anti-tumor activity was in the helper or cytotoxic T cell compartments. Eight of 13 MSLN responders had activity exclusively in CD8+ T cells (range 3-64% IFNg+ cells) and 2 had activity in both CD8 and CD4 T cells, while in the only non-B35 responder activity was detected exclusively in the CD4+ T cell compartment. We next performed epitope mapping studies using peptide minipools and detected activity against 3 published CD8+ epitopes [HLA-24 FYP (aa435-443) and LYP (aa475-483); HLA-A2 VLP (aa530-538)] as well as 5 novel B35-restricted epitopes, one of which was detected in 7 of our 12 B35 responders. Finally, to assess whether these novel epitopes were recognized in the context of HLA-B35 we loaded autologous and partially HLA-matched APCs with the individual peptides and assessed cytolytic activity. Autologous peptide-loaded targets were killed (range 52-98% killing, E:T 40:1) as were allogeneic targets exclusively matched at HLA-B35 (range 32-92% killing), confirming that all epitopes were presented by the HLA-B35 allele. These B35-restricted CTLs were also able to kill HLA-matched tumors expressing MSLN - we saw specific lysis of CFPAC-1 (B35+MSLN+) (36% killing E:T 40:1) with no recognition of CAPAN-1 (B35-MSLN+) (3% killing). Thus, our results support a contribution of HLA-B35-restricted T cells and anti-tumor activity in pancreatic cancer and highlight the importance of cellular immunity in protection against cancer. Citation Format: Ryu Yanagisawa, Jacqueline M. Keirnan, Usha L. Katari, Usanarat Anurathapan, Malcolm K. Brenner, Cliona M. Rooney, Juan F. Vera, Ann M. Leen. Investigating the protective role of HLA-B35 in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1257. doi:10.1158/1538-7445.AM2013-1257

Therapeutic Efficacy of Bifunctional siRNA Combining TGF-β1 Silencing with RIG-I Activation in Pancreatic Cancer

Deregulated TGF-β signaling in pancreatic cancer promotes tumor growth, invasion, metastasis, and a potent immunosuppressive network. A strategy for disrupting this tumor-promoting pathway is silencing TGF-β by siRNA. By introducing a triphosphate group at the 5' end of siRNA (ppp-siRNA), gene silencing can be combined with immune activation via the cytosolic helicase retinoic acid-inducible gene I (RIG-I), a ubiquitously expressed receptor recognizing viral RNA. We validated RIG-I as a therapeutic target by showing that activation of RIG-I in pancreatic carcinoma cells induced IRF-3 phosphorylation, production of type I IFN, the chemokine CXCL10, as well as caspase-9-mediated tumor cell apoptosis. Next, we generated a bifunctional ppp-siRNA that combines RIG-I activation with gene silencing of TGF-β1 (ppp-TGF-β) and studied its therapeutic efficacy in the orthotopic Panc02 mouse model of pancreatic cancer. Intravenous injection of ppp-TGF-β reduced systemic and tumor-associated TGF-β levels. In addition, it induced high levels of type I IFN and CXCL10 in serum and tumor tissue, systemic immune cell activation, and profound tumor cell apoptosis in vivo. Treatment of mice with established tumors with ppp-TGF-β significantly prolonged survival as compared with ppp-RNA or TGF-β siRNA alone. Furthermore, we observed the recruitment of activated CD8(+) T cells to the tumor and a reduced frequency of CD11b(+) Gr-1(+) myeloid cells. Therapeutic efficacy was dependent on CD8(+) T cells, whereas natural killer cells were dispensable. In conclusion, combing TGF-β gene silencing with RIG-I signaling confers potent antitumor efficacy against pancreatic cancer by breaking tumor-induced CD8(+) T cell suppression.

Signal transducer and activator of transcription 5 (STAT5) expression and activation in pancreatic cancer

s / Pancreatology 13 (2013) e1–e94 e53 of the mouse pancreas via laparotomy. Two weeks post-cancer cell injection, a second laparotomy was performed to inject drug-loaded MPs or saline into the same tail section of the pancreas. Mouse tissue samples were taken to evaluate the local effects of constant drug release on the pancreatic tumors and to determine the extent of drug escape to the spleen and liver. Positive results of these combined studies will justify additional pre-clinical investigation in a transgenic mouse model of pancreatic cancer.

Phase II consortium (P2C) study of gemcitabine and tanespimycin (17AAG) for metastatic pancreatic cancer.

245 Background: Gemcitabine (GEM) monotherapy for PDA has modest activity. 17-N-Allylamino-17-demthoxygeldanamycin (Tanespimycin/17AAG), is an HSP90 inhibitor, which results in degradation of a number of client proteins such as RAF and Akt. Arlander et al (J Biol Chem. 2003 278:52572-7) have demonstrated that 17AAG targeting of HSP90 leads to Chk1 degradation. Chk1 is upregulated with GEM treatment which affects cell survival (Karnitz et al. Mol Pharmacol. 2005 68:1636-44). In vitro, the combination demonstrated in vitro synergy. Thus the potential clinical activity in pancreatic cancer of the combination was explored. Methods: A phase II multicenter prospective study was performed using GEM 750 mg/m2 d1,8 and 17AAG 154 mg/m2 d2,9 on a 21 day cycle, as defined in a previous phase I trial. Patients with stage IV PDA, with adequate liver, kidney function and counts, ECOG 0-2 and no prior therapy for metastatic disease were eligible. The primary endpoint of 60% overall survival at 6 months was considered of clinical interest. Sixty-six patients were planned for accrual, and an interim analysis was planned after 25 evaluable patients. Informed, written consent was obtained from all pts. Results: Due to lack of drug availability and failure to achieve the planned endpoint, accrual was halted at 21 pts, enrolled from 5/08 to 9/10, of which 20 were evaluable (1 cancelled prior to treatment). Median age was 61.5 yrs., 55% were male, PS 0 (50%), 1 (45%), 2 (5%); 95% were non-Hispanic white, 1 was African American. Two had received adjuvant therapy for PDA. Tolerability was moderate, with 13 pts (65%) having > grade 3 adverse event (AE), with 15% gr 4 AEs and 0% gr 5. Nausea (20%), vomiting (20%), constipation (15%), dehydration (15%), and anorexia (10%), were the most common non-heme AEs. Lymphopenia (15%), leukopenia (10%) and neutropenia (10%) were common grade 3 hematologic AEs. No complete or partial responses were seen. 40% of patients were alive at 6 months, with a median OS of 5.4 months. Conclusions: HSP 90 inhibitor 17AAG in combination with GEM does not show activity for treatment of PDA. Further trials should not be pursued with this combination. Supported by N01-CM-62205. Clinical trial information: NCT00577889.

Parameter identifiability and sensitivity analysis predict targets for enhancement of STAT1 activity in pancreatic cancer and stellate cells.

The present work exemplifies how parameter identifiability analysis can be used to gain insights into differences in experimental systems and how uncertainty in parameter estimates can be handled. The case study, presented here, investigates interferon-gamma (IFNγ) induced STAT1 signalling in two cell types that play a key role in pancreatic cancer development: pancreatic stellate and cancer cells. IFNγ inhibits the growth for both types of cells and may be prototypic of agents that simultaneously hit cancer and stroma cells. We combined time-course experiments with mathematical modelling to focus on the common situation in which variations between profiles of experimental time series, from different cell types, are observed. To understand how biochemical reactions are causing the observed variations, we performed a parameter identifiability analysis. We successfully identified reactions that differ in pancreatic stellate cells and cancer cells, by comparing confidence intervals of parameter value estimates and the variability of model trajectories. Our analysis shows that useful information can also be obtained from nonidentifiable parameters. For the prediction of potential therapeutic targets we studied the consequences of uncertainty in the values of identifiable and nonidentifiable parameters. Interestingly, the sensitivity of model variables is robust against parameter variations and against differences between IFNγ induced STAT1 signalling in pancreatic stellate and cancer cells. This provides the basis for a prediction of therapeutic targets that are valid for both cell types.

The role of the tumor endothelium in leukocyte recruitment in pancreatic cancer

Although pancreatic cancer tissue frequently induces an immune reaction, immunocompetent cells are not able to eliminate the tumor. One potential cause for this ineffective immune response is that a number of active, tumor-cytotoxic T cells are not able to invade into the tumor. A potential barrier for invading leukocytes can be the tumor endothelium, which controls recruitment of leukocytes from circulating blood into the tissue. Although attenuated expression of adhesion molecules on the tumor endothelium has been proposed as a mechanism which suppresses intratumoral leukocyte infiltration, the relevance of adhesion molecules for leukocyte recruitment in tumor tissue is poorly understood. The leukocyte extravasation in normal pancreas during acute pancreatitis follows the "classic" leukocyte recruitment cascade and is controlled by the overexpression of endothelial adhesion molecules, such as selectins, intracellular adhesion molecule-1, and platelet endothelial cell adhesion molecule-1. In contrast to acute inflammation in normal pancreas, leukocyte recruitment in pancreatic cancer is a slow process, which does not show a strong dependence on intracellular adhesion molecule-1. In addition, pancreatic cancer has a high degree of heterogeneity of both immunogenic properties and the distribution of tumor-infiltrating leukocytes, such as CD8(+), CD4(+), or regulatory T cells. Additional studies may clarify whether T cell recruitment and their activity in pancreatic cancer can be enhanced by modulation of endothelial adhesion molecules.

HOTAIR is a negative prognostic factor and exhibits pro-oncogenic activity in pancreatic cancer

HOTAIR is a long intervening non-coding RNA (lincRNA) that associates with the Polycomb Repressive Complex 2 (PRC2) and overexpression is correlated with poor survival for breast, colon and liver cancer patients. In this study, we show that HOTAIR expression is increased in pancreatic tumors compared to non-tumor tissue and is associated with more aggressive tumors. Knockdown of HOTAIR (siHOTAIR) by RNA interference shows that HOTAIR plays an important role in pancreatic cancer cell invasion and as reported in other cancer cell lines. In contrast, HOTAIR knockdown in Panc1 and L3.6pL pancreatic cancer cells that overexpress this lincRNA decreased cell proliferation, altered cell cycle progression, and induced apoptosis, demonstrating an expanded function for HOTAIR in pancreatic cancer cells compared to other cancer cell lines. Results of gene array studies showed that there was minimal overlap between HOTAIR-regulated genes in pancreatic vs. breast cancer cells and HOTAIR uniquely suppressed several interferon-related genes and gene sets related to cell cycle progression in pancreatic cancer cells and tumors. Analysis of selected genes suppressed by HOTAIR in Panc1 and L3.6 pL cells showed by knockdown of EZH2 and chromatin immunoprecipitation assays that HOTAIR-mediated gene repression was both PRC2-dependent and -independent. HOTAIR knockdown in L3.6pL cells inhibited tumor growth in mouse xenograft model, further demonstrating the pro-oncogenic function of HOTAIR in pancreatic cancer.

Abstract 203: Hotair, a long non-coding RNA, exhibits pro-oncogenic activity in pancreatic cancer

Abstract HOTAIR is a long intervening non-coding RNA (lincRNA) that represses specific gene transcription by interaction with the Polycomb Repressive Complex 2 (PRC2) that trimethylates histone H3 lysine 27, leading to reprograming of chromatin states. HOTAIR is a poor prognostic indicator for survival of breast, colon and liver cancer patients and is highly associated with cancer invasion and metastasis. In this study, we show that HOTAIR is more highly expressed in pancreatic tumors than normal tissue and is strongly correlated not only with cell invasiveness but also cell proliferation and death. Overexpression of HOTAIR increased cell invasion in pancreatic cancer cells expressing low levels of HOTAIR and this was previously observed in other cancer cell lines. However, unlike previous reports, HOTAIR knockdown in Panc1 and L3.6pL pancreatic cancer cells that overexpress this lincRNA decreased cell proliferation, altered cell cycle progression, and induced apoptosis, demonstrating an expanded function for HOTAIR in pancreatic cancer cells compared to other cancer cell lines. Results of gene profiling studies after HOTAIR knockdown showed that there was minimal overlap between HOTAIR-regulated genes in pancreatic vs. breast cancer cells and HOTAIR uniquely suppressed several interferon-related genes and genes related to cell cycle progression in pancreatic cancer cells. Consistently, Gene Set Enrichment Analysis (GSEA) shows that HOTAIR regulates gene sets relevant to cell proliferation using HOTAIR Knockdown gene profiling data and pancreatic patient gene expression data. Analysis of selected genes suppressed by HOTAIR in Panc1 and L3.6 pL cells showed by knockdown of EZH2 and chromatin immunoprecipitation assays that HOTAIR-mediated gene repression was both PRC-2-dependent and -independent. HOTAIR knockdown in L3.6pL cells also inhibited tumor growth in mouse xenograft model, further demonstrating the pro-oncogenic function of HOTAIR in pancreatic cancer. These results indicated that HOTAIR is a potential molecular target for inhibiting pancreatic cancer cell invasion, metastasis and proliferation for inducing cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 203. doi:1538-7445.AM2012-203

Abstract 2362: Targeted suppression of TGF-beta2 by trabedersen (AP 12009) in an orthotopic xenograft melanoma mouse model

Abstract Background: TGF-β2 is a mediator of carcinogenesis by regulating key mechanisms such as cell proliferation, metastasis, and immunosuppression. The antisense oligonucleotide trabedersen (AP 12009) specifically inhibits TGF-β2 expression. Clinical studies already demonstrated its antitumor activity in pancreatic cancer, high-grade glioma, and malignant melanoma. Here we examined the anti-tumorigenic and anti-metastatic effects of trabedersen in an orthotopic xenograft melanoma mouse model. Methods: BALB/c nu/nu mice were intradermally injected with 0.5x10E6 cells of the human TGF-β2 expressing melanoma cell line C8161. Intraperitoneal treatment with different doses of trabedersen or vehicle (controls) started 2 days after tumor implantation thrice weekly. Doses of trabedersen were 50/16 mg/kg (i.e. initial dose of 50 mg/kg and subsequent 16 mg/kg), 16 mg/kg, 4 mg/kg, 1 mg/kg, and 0.25 mg/kg. Treatment continued until first tumor reached 2000 mm3 (approx. 32-35 days after inoculation). The incidence of liver and lung metastases as well as growth and weight of the dermal tumors were determined. To evaluate the immunomodulatory effect of trabedersen, NK cells and MDSCs prepared from spleens were analyzed of trabedersen-treated or control mice (flow cytometry; MDSCs: anti-CD11b+, anti-Ly6G, anti-Ly6C; NK cells: CD49+, CD314+). Cellular uptake was examined by using fluorescently labeled trabedersen (5′-FAM-AP 12009) which was intra-tumorally injected 4 h prior to termination of the study. Cryosections were semi-quantitatively evaluated for the presence of fluorescence. Results: Tumor weight was significantly reduced for trabedersen-treated mice (50/16 mg/kg) compared to control (0.395 g vs. 0.715 g, p<0.05). Moreover, a dose dependent response for trabedersen treatment with 1 mg/kg, 4 mg/kg, and 16 mg/kg regarding tumor weight and tumor growth was observed. Trabedersen treatment significantly inhibited lung metastasis. There was no difference for liver metastasis between trabedersen and control. Analysis of spleen cell preparations of trabedersen-treated and control mice revealed a trend to less monocytic MDSCs under trabedersen treatment. There was no difference in the amount of granulocytic MDSCs and NK cells between trabedersen-treated and control mice. Intra-cellular and intra-nuclear fluorescence was clearly detectable in all tumor sections of all mice receiving the intratumoral injection of 5′-FAM-AP 12009, indicating uptake of trabedersen in the tumor cells and nuclear localisation. Conclusions: In an orthotopic xenograft melanoma mouse model trabedersen demonstrated potent antitumor activity as it inhibits the tumor promoting and metastatic effects of TGF-β2 such as tumor cell proliferation and immunosuppression. This is in line with promising results from clinical studies in patients with advanced pancreatic cancer or malignant melanoma (Phase I/II). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2362. doi:1538-7445.AM2012-2362

The activity of Gli transcription factors is essential for Kras-induced pancreatic tumorigenesis

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here we find that transcriptional activation mediated by the Gli family of transcription factors, although dispensable for pancreatic development, is required for Kras-induced proliferation and survival in primary pancreatic epithelial cells in culture and for Kras-driven pancreatic intraepithelial neoplasia and PDAC formation in vivo. Further, ectopic Gli1 activation in the mouse pancreas accelerates Kras-driven tumor formation, underscoring the importance of Gli transcription factors in pancreatic tumorigenesis. Interestingly, we demonstrate Gli-regulated I-kappa-B kinase epsilon (IKBKE) and NF-κB activity in pancreatic cancer cells and show that this activity is a critical downstream mediator for Gli-dependent PDAC cell transformation and survival. Together, these studies demonstrate the requirement for Gli in Kras-dependent pancreatic epithelial transformation, suggest a mechanism of Gli-NF-κB oncogenic activation, and provide genetic evidence supporting the therapeutic targeting of Gli activity in pancreatic cancer.

The RON‐receptor regulates pancreatic cancer cell migration through phosphorylation‐dependent breakdown of the hemidesmosome

The recepteur d'origine nantais (RON) receptor tyrosine kinase is overexpressed and stimulates invasive growth in pancreatic cancer cells, yet the mechanisms that underlie RON-mediated phenotypes remain poorly characterized. To better understand RON function in pancreatic cancer cells, we sought to identify novel RON interactants using multidimensional protein identification analysis. These studies revealed plectin, a large protein of the spectrin superfamily, to be a novel RON interactant. Plectin is a multifunctional protein that complexes with integrin-β4 (ITGB4) to form hemidesmosomes, serves as a scaffolding platform crucial to the function of numerous protein signaling pathways and was recently described as an overexpressed protein in pancreatic cancer (Bausch D et al., Clin Cancer Res 2010; Kelly et al., PLoS Med 2008;5:e85). In this study, we demonstrate that on exposure to its ligand, macrophage-stimulating protein, RON binds to plectin and ITGB4, which results in disruption of the plectin-ITGB4 interaction and enhanced cell migration, a phenotype that can be recapitulated by small hairpin ribosomal nucleic acid (shRNA)-mediated suppression of plectin expression. We demonstrate that disruption of plectin-ITGB4 is dependent on RON and phosphoinositide-3 (PI3) kinase, but not mitogen-activated protein kinase (MEK), activity. Thus, in pancreatic cancer cells, plectin and ITGB4 form hemidesmosomes which serve to anchor cells to the extracellular matrix (ECM) and restrain migration. The current study defines a novel interaction between RON and plectin, provides new insight into RON-mediated migration and further supports efforts to target RON kinase activity in pancreatic cancer.

Vitamin E δ-Tocotrienol Augments the Antitumor Activity of Gemcitabine and Suppresses Constitutive NF-κB Activation in Pancreatic Cancer

The NF-κB transcription factor functions as a crucial regulator of cell survival and chemoresistance in pancreatic cancer. Recent studies suggest that tocotrienols, which are the unsaturated forms of vitamin E, are a promising class of anticancer compounds that inhibit the growth and survival of many cancer cells, including pancreatic cancer. Here, we show that tocotrienols inhibited NF-κB activity and the survival of human pancreatic cancer cells in vitro and in vivo. Importantly, we found the bioactivity of the four natural tocotrienol compounds (α-, β-, δ-, and γ-tocotrienol) to be directly related to their ability to suppress NF-κB activity in vitro and in vivo. The most bioactive tocotrienol for pancreatic cancer, δ-tocotrienol, significantly enhanced the efficacy of gemcitabine to inhibit pancreatic cancer growth and survival in vitro and in vivo. Moreover, we found that δ-tocotrienol augmentation of gemcitabine activity in pancreatic cancer cells and tumors is associated with significant suppression of NF-κB activity and the expression of NF-κB transcriptional targets (Bcl-X(L), X-linked inhibitor of apoptosis, and survivin). Our study represents the first comprehensive preclinical evaluation of the activity of natural vitamin E compounds in pancreatic cancer. Given these results, we are conducting a phase I trial of δ-tocotrienol in patients with pancreatic cancer using pancreatic tumor cell survival and NF-κB signaling components as intermediate biomarkers. Our data also support future clinical investigation of δ-tocotrienol to augment gemcitabine activity in pancreatic cancer.

Experimental study on anti-metastasis effect of emodin on human pancreatic cancer

To investigate the anti-metastasis effect of emodin on the pancreatic cancer in vitro and in vivo. Human pancreatic cancer cell line SW1990 was treated with different concentrations of emodin (10, 20, 40 micromol x L(-1)) for 2 h, the effects of emodin on the migration and invasion of SW1990 cells were examined by using wound assay and matrigel counting. Western blot was used to detect the protein expression of NF-kappaB and MMP-9 in SW1990 cells after various concentrations of emodin (10, 20, 40 micromol x L(-1)) treatment for 48 h. Metastatic model simulating human pancreatic cancer was established by orthotropic implantation of histologically intact human tumor tissue into pancreatic wall of nude mice, and then divided into three groups: control group, low-dose emodin group (L-EMO) and high-dose emodin group (H-EMO). Eight weeks after implantation, the presences of metastasis were evaluated respectively after the mice were sacrificed. Immunohistochemistry was used to detect the positive expression of CD34, NF-kappaB and MMP-9 in the tumors. Emodin suppressed the migration and invasion of SW1990 cells in a dose-dependent manner. Western bolt assay indicated that emodin down-regulated the expression of NF-kappaB and MMP-9 proteins in SW1990 cells. The incidences of metastasis were decreased significantly in L-EMO group and H-EMO group as compared with that in control group. The percentage of CD34, NF-kappaB and MMP-9-positive cells in the tumors were significantly reduced by the administration of emodin. Emodin exerts anti-metastatic activity in pancreatic cancer both in vitro and in vivo, which may be related to down-regulation of NF-kappaB and MMP-9.

Prolyl hydroxylase‐2 (PHD2) exerts tumor‐suppressive activity in pancreatic cancer

Pancreatic cancer is 1 of the most common and poorly treated tumors. In search of new therapeutic approaches, the oxygen sensors prolyl hydroxylases (PHD) are potential targets. PHD2 is considered the key oxygen sensor-regulating hypoxia-inducible factor (HIF). Currently, there is conflicting evidence regarding the exact role of PHD2 in tumorigenesis. The objective of this study was to investigate the role of PHD2 in pancreatic cancer growth and progression. PHD2 expression was analyzed by quantitative real-time polymerase chain reaction analysis and immunohistochemistry in human tissue specimens and cell lines. Knockdown of PHD2 was done by using short-interfering RNAs (siRNAs) specific against PHD2, and PHD2 overexpression was achieved by stable combinational DNA transfection. In vivo, an orthotopic murine model was used. Angiogenic cytokines were assessed with enzyme-linked immunosorbent assays, and invasion was studied with Matrigel assays. PHD2 expression was not altered substantially in cancer tissues and their metastases. Lymph node-negative tissues had higher levels of PHD2 than lymph node-positive tissues. PHD2 was hypoxia-inducible in pancreatic cancer cell lines and regulated cell growth through cyclin D1 down-regulation samples with PHD2 suppression and through p21 up-regulation in samples with of PHD2 overexpression. In vivo, PHD2 caused tumor growth retardation and reduced tumor invasion by inhibiting angiogenesis. This observation was caused by the suppression of angiogenic cytokines and tumor invasion. The current results indicated that PHD2 plays an important role in pancreatic tumorigenesis. In summary, the authors concluded that PHD2 may function as a tumor suppressor gene in pancreatic cancer and, thus, may define a potential target for the treatment of pancreatic cancer.

A phase II study of the halichondrin B analog eribulin mesylate in gemcitabine refractory advanced pancreatic cancer

Eribulin mesylate is a halichondrin B analog that inhibits microtubule dynamics. Pre-clinical studies have suggested anti-tumor activity in pancreatic cancer. This phase II study of eribulin in patients with advanced pancreatic cancer previously treated with gemcitabine was conducted by the Princess Margaret Hospital Phase II consortium. Eligibility criteria included locally advanced or metastatic pancreatic adenocarcinoma and previous treatment with gemcitabine. The study was a single arm phase II trial using a Simon 2-stage design. The primary endpoint was response rate, secondary endpoints included time to progression and overall survival. Fifteen patients were enrolled, 14 received treatment, and 12 were evaluable for response. The median age was 61, and the majority of patients were ECOG performance status 1. Grade 3 or greater adverse events included neutropenia (29%), fatigue (14%), peripheral neuropathy (7%) and thrombosis (7%). There were no complete or partial responses and therefore the study was closed after the first stage. The best response was stable disease in 5/12 (42%) of patients. Of these five patients, three had stable disease for 9 months or greater. Median time to progression was 1.4 months, and median overall survival was 6.1 months. Eribulin was well tolerated but did not result in any objective responses in gemcitabine refractory pancreatic cancer. However, several patients had prolonged stable disease, suggesting that further studies of eribulin in pancreatic cancer may be warranted.

Nafamostat mesilate can prevent adhesion, invasion and peritoneal dissemination of pancreatic cancer thorough nuclear factor kappa‐B inhibition

Constitutive activation of nuclear factor kappa-B (NF-κB) contributes to the aggressive behavior of pancreatic cancer. Over-expression of downstream target genes of NF-κB such as intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) leads to the promotion of cell adhesion, angiogenesis, invasion and metastasis. We previously reported that nafamostat mesilate, a synthetic serine protease inhibitor, blocks NF-κB activation in pancreatic cancer. We hypothesized that nafamostat mesilate may inhibit cell adhesion, angiogenesis, invasion and metastases in peritoneal dissemination of pancreatic cancer. In vitro, we assessed inhibition of NF-κB, phosphorylated IκBα, ICAM-1, VEGF and MMP-9 activity by nafamostat mesilate using human pancreatic cancer cell lines (AsPC-1, BxPC-3 and PANC-1). Changes in adhesion and invasion abilities of cancer cells were then evaluated by nafamostat mesilate treatment. In vivo, the efficacy of nafamostat mesilate treatment was assessed using peritoneal dissemination of pancreatic cancer in mice. In vitro, nafamostat mesilate inhibited activities of NF-κB, phosphorylated IκBα, ICAM-1, VEGF and MMP-9. Moreover, nafamostat mesilate not only inhibited cell adhesion and invasion but also increased the sensitivity of anoikis. In vivo, tumor growth using AsPC-1 cells of the treatment group was significantly slower, and survival rate was significantly better, than those in control group (p < 0.05). Nafamostat mesilate reduced peritoneal metastasis and prolonged survival of pancreatic cancer-bearing mice.

Dihydroartemisinin inhibits angiogenesis in pancreatic cancer by targeting the NF-κB pathway

Dihydroartemisinin (DHA) has recently shown antitumor activity in human pancreatic cancer cells. However, its effect on antiangiogenic activity in pancreatic cancer is unknown, and the mechanism is unclear. This study was aimed to investigate whether DHA would inhibit angiogenesis in human pancreatic cancer. Cell viability and proliferation, tube formation of human umbilical vein endothelial cells (HUVECs), nuclear factor (NF)-κB DNA-binding activity, expressions of vascular endothelial growth factor (VEGF), interleukin (IL)-8, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9 were examined in vitro. The effect of DHA on antiangiogenic activity in pancreatic cancer was also assessed using BxPC-3 xenografts subcutaneously established in BALB/c nude mice. DHA inhibited cell proliferation and tube formation of HUVECs in a time- and dose-dependent manner and also reduced cell viability in pancreatic cancer cells. DHA significantly inhibited NF-κB DNA-binding activity, so as to tremendously decrease the expression of NF-κB-targeted proangiogenic gene products: VEGF, IL-8, COX-2, and MMP-9 in vitro. In vivo studies, DHA remarkably reduced tumor volume, decreased microvessel density, and down-regulated the expression of NF-κB-related proangiogenic gene products. Inhibition of NF-κB activation is one of the mechanisms that DHA inhibits angiogenesis in human pancreatic cancer. We also suggest that DHA could be developed as a novel agent against pancreatic cancer.