PaCa-2 Cells Research Articles

Abstract 3535: A novel tumor-specific oncolytic biological therapy against invasive pancreatic cancer

Abstract Pancreatic cancer is one of the worst prognosis disease all over the world and has an overall 5-year survival rate of less than 5%. At the time of diagnosis, more than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis. Moreover, even after curative surgery, pancreatic cancers still show poor prognosis due to local recurrence and systemic metastasis. Gemcitabine is the standard chemotherapeutic agent to treat advanced pancreatic cancers, but median overall survival ranged from 5.0 to 7.2 months, suggesting that standard chemotherapy is less effective to most of pancreatic cancer patients. Therefore, the development of novel therapeutic strategy is needed for the treatment of pancreatic cancers. We developed a novel oncolytic adenovirus, OBP-702, in which a human telomerase reverse transcriptase gene promoter drives viral E1 gene for virus replication, and armed with the wild-type tumor suppressor p53 gene. Precise antitumor effects of OBP-702 have been evaluated in human pancreatic cancer cells. We used four pancreatic cancer cell lines with different invasion ability, including non-invasive type (MIA PaCa-2, Panc-1) and invasive type (BxPC-3, Capan-1). The antitumor effect of OBP-702 for each cell line was assessed using XTT assay. OBP-702 induced profound anti-tumor effect in all human pancreatic cancer cells, whereas OBP-301 lacking the p53 tumor suppressor gene showed differential effects among the cell types. OBP-301 induced moderate anti-tumor effect in MIA Paca-2, BxPC-3 and Capan-1 cells and strong anti-tumor effect in Panc-1 cells in a dose-dependent manner, suggesting the broad spectrum of OBP-702's efficacy. The molecular mechanism of anti-tumor effect was assessed by Western blot analysis. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. These results suggest that an oncolytic adenovirus, OBP-702, is a promising antitumor agent to induce profound cell death in invasive human pancreatic cancer cells. The clinical trial of intratumoral administration of OBP-702 in patients with invasive pancreatic cancer is warranted. Citation Format: Takeshi Koujima, Hiroshi Tazawa, Naoto Hori, Shuta Tamura, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. A novel tumor-specific oncolytic biological therapy against invasive pancreatic cancer. [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 3535. doi:10.1158/1538-7445.AM2015-3535

Abstract 3586: The use of kinome profiling to determine potential resistance pathways in pancreatic cancer cells treated with PIM kinase inhibitors

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent cancer of the pancreas with a 5-year survival rate of 6%, making it the 4th most common cause of cancer deaths in the United States. One of the challenges related to this cancer is the issue of chemoresistance. Chemoresistance renders most drugs ineffective in the treatment of PDAC, including kinase inhibitors and the chemotherapeutic drug gemcitabine. PIM (Proviral Integration site for the Moloney murine leukemia virus) kinases are a family of proteins shown to play an important role in the chemoresistance of many cancers, including pancreatic cancer. PIMs act downstream of the JAK-STAT cytokine signaling pathway and phosphorylate substrates involved in numerous cell functions including cell cycle progression and apoptosis. One objective of our research was to determine the role of PIM kinases on transformed phenotypes of PDAC cells. We were able to demonstrate that the PIM kinase inhibitor SGI-1776 and TP-3654, a second generation PIM kinase inhibitor, were able to inhibit growth in cell viability assay. Also, the PIM inhibitors were able to decrease various phenotypes such as anchorage-dependent and anchorage-independent growth in MIA PaCa-2 and PANC-1 cells. A second objective was to determine if PIM kinase inhibitors could reduce chemoresistance in PDAC cells. PIM inhibitors in combination with gemcitabine decreased cell viability in PDAC cells significantly more than when administered separately. To investigate resistance pathways activated in PDAC cells after treatment with PIM kinase inhibitors, we applied multiplexed inhibitor beads (MIBs) mass spectrometry (MS) to profile the kinome. We observed numerous kinases that were downregulated or upregulated after treatment. One of the kinases upregulated was IKKα, a key kinase in the anti-apoptotic NF-κB signaling pathway. Our results suggest that PIM kinases are an important target for cell growth and chemoresistance in pancreatic cancer. Also, these results suggest that treating PDAC cells with a PIM inhibitor simultaneously with an IKK inhibitor might prevent the effects of resistance pathways. Overall, our studies demonstrate that kinome profiling is an effective strategy to identify cellular responses to PIM kinase inhibitors as well as other drugs for treating pancreatic and other cancers. Citation Format: Vandana Singh, Brittany Nixon, T.S. Karim Gilbert, Steve Warner, David Bearss, Lee M. Graves, Antonio T. Baines. The use of kinome profiling to determine potential resistance pathways in pancreatic cancer cells treated with PIM kinase inhibitors. [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 3586. doi:10.1158/1538-7445.AM2015-3586

Abstract 1330: The influence of anticancer agents and heat treatment on PD-L1 expression on human and murine pancreatic cancer cell lines

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies. Currently conducted chemotherapy for PDA didn't get the sufficient clinical results, so we need to get new strategy for PDA therapy immediately. For breaking through this situation, the inhibitors of immune checkpoint PD-1/PD-L1 are hopeful candidates because PD-1/PD-L1 inhibitors relatively got the good results for some advanced malignancies including PDA, but the sole therapy doesn't result in good outcome. In murine model, the combination therapy of PD-L1 inhibitor and gemcitabine has been reported to show a synergistic effect on PDA compared to each sole therapy. But the mechanism of the synergistic effect is not well understood. On the other hand, we have previously demonstrated the efficacy of the combined therapy of regional heat treatment and gemcitabine in advanced PDA patients, but the effect of heat treatment on immune checkpoint is also unknown. Then, for future combination therapy of anticancer agents, immune check point inhibitor and heat treatment, we analyzed the influence of some anticancer agents and heat treatment on the expression of PD-L1 on pancreatic cancer cell lines. Methods: Human pancreatic cancer cell lines MIA PaCA-2, AsPC-1 and murine pancreatic cancer cell line Pan02 were used in this study. These cells were adjusted to 1.0 × 105 / ml and incubated with anticancer agents such as gemcitabine, paclitaxel and 5-fluorouracil at 37°C for 24 - 72 hours. For checking the influence of anticancer agents on the expression of PD-L1, their expression of PD-L1 was examined by flow cytometry and qRT-PCR. Next, before exposing cancer cells to gemcitabine, MIA PaCA-2 and AsPC-1 were incubated at 43°C as heat treatment. After the heat treatment, PD-L1 expression was analyzed using flow cytometry. Results: In AsPC-1 and Pan02, PD-L1 expression was enhanced with all three anticancer agents in concentration dependency by both flow cytometry and qRT-PCR. In MIA PaCA-2, PD-L1 expression was enhanced with all three anticancer agents by flow cytometry analysis, but the result of qRT-PCR could not show the difference. PD-L1 expression on MIA PaCA-2 and AsPC-1 with heat treatment was suppressed compared to only anticancer agent treatment. Conclusion: The stimulation of anticancer agents tends to lead to an enhancement of PD-L1 expression on both human and murine pancreatic cancer cell lines. The synergistic effect of PD-L1 inhibitor and gemcitabine has the possibility to be brought about by PD-L1 up-regulation. On the other hand, heat treatment has the possibility to suppress PD-L1 expression and this mechanism might lead to cancel out the immune tolerance caused by anticancer agent administration with up-regulation of PD-L1. For future, we need to further examine the most effective timing and method of combined therapy of anticancer agent, PD-L1 inhibitor and heat treatment. Citation Format: Toshifumi Doi, Tetsuya Okayama, Takeshi Ishikawa, Kaname Oka, Naoyuki Sakamoto, Tomoyo Yasuda, Yuji Naito, Yoshito Itoh. The influence of anticancer agents and heat treatment on PD-L1 expression on human and murine pancreatic cancer cell lines. [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 1330. doi:10.1158/1538-7445.AM2015-1330

ING3 as a novel regulator of pancreatic cancer growth and aggressiveness.

304 Background: Recent studies demonstrate that chromatin regulation by posttranslational means, such as histone methylation or acetylation, is an essential control mechanism in tumorigenesis and tumor progression. Accumulative findings support the notion that pancreatic cancer tumorigenesis is not only governed by genetic alterations but also aberrant epigenetic regulation. The aim of the present study was to identify and assess the role of chromatin regulators in pancreatic cancer. Methods: The gene expression profile of chromatin regulators was assessed using gene expression microarray analysis in pancreatic cancer and uninvolved human tissues. Validation of microarray findings was performed by qRT-PCR in two extended cohorts of patients and by immunohistochemistry in pancreas tumor tissue microarrays. PANC-1, MIA Paca-2, Capan-2, HPAF-II and AsPC-1 cell lines were used for in vitro assays. Efficiency of knockdown experiments, performed by RNAi interference assays and shRNA-expressing lentiviral vectors, was evidenced by qRT-PCR and Western Blot Analysis. Cell proliferation, invasion and colony formation assays were conducted in genetically modified cells. The significance of variability between the results from each group and corresponding control was determined by unpaired t-test. Results: Differential expression analysis of chromatin regulators in pancreatic cancer versus uninvolved tissues demonstrates that 27 epigenetic molecules are significantly de-regulated (>1.5 fold, P<0.05). By hierarchical clustering, the samples are classified into two major groups that reflect the normal and cancer state. The decreased expression of inhibitor of growth family member 3 (ING3), a component of the NuA4 histone acetyl-transferase complex, was further validated by qRT-PCR and immunohistochemistry analysis in tumor tissues. Transient or stable silencing of ING3 caused significant increase of viability, proliferation, colony formation and invasion in several pancreatic cell lines. Conclusions: Our data show that human pancreatic cancer is characterized by loss of ING3 expression and we defined a potential role of the latter in pancreatic cancer cell growth and invasion.

Abstract 2155: Novel combinations of nano-formulated ferulic acid and aspirin show high potential for chemoprevention of pancreatic cancer

Abstract Pancreatic cancer ranks as the fourth most deadly form of cancer in the United States with about 37,000 deaths each year. This study evaluated the chemopreventive potential of combinations of free and nano-encapsulated mixtures of ferulic acid (FA) and aspirin (ASP) in low doses to human pancreatic cancer cells, MIA PaCa-2 and Panc-1. Cell lines were tested according to published protocols wherein cells were seeded in 96-well microplates and incubated with the unmodified and modified combinations for 72 h and analyzed using MTS cell viability assay method. Apoptosis was measured by flow cytometry. Results demonstrated that free FA (200 µM) and ASP (1 mM) combinations on average reduced cell viability by 60% (P<0.001), and also induced cell apoptosis by 47% (P<0.001) on the two cell lines. However, when individual agents (FA 200 μM; ASP 1mM) were tested, they were virtually ineffective in reducing cell viability, thus indicating an additive/synergistic effect of the two agents in combination. Subsequently, nano-formulations of FA and ASP (nano-FA and nano-ASP) were prepared by encapsulating the agents within solid lipid nanoparticles (SLNs) and tested on the same cell lines. Prior to this, nano-FA and nano-ASP were evaluated for particle sizes and encapsulation efficiencies. Initial particle sizes were 183± 46 nm (nano-FA) and 229±67 nm (nano-ASP), respectively. Furthermore, the encapsulation efficiencies were high for both nano-FA (86%) and nano-ASP (78%). The combination mixture of nano-FA (25 µM) and nano-ASP (25 µM) reduced the cell viability of MIA PaCa-2 and Panc-1 cells by 70% (P<0.001), and also induced cell apoptosis by 30% (P<0.01), thus reducing the dosage of FA by 8-fold and that of ASP by 40-fold to elicit a comparable response as the mixture of the free FA and ASP. Finally, a trans-AM NF-κB transcription factor assay kit was used to help understand the mechanism. Our results demonstrate that nano-FA and nano-ASP combinations significantly down-regulated NF-κB DNA binding activity in MIA PaCa-2 and Panc-1 cells. In conclusion, convincing evidence is provided of the chemopreventive potential of combinations of FA and ASP against pancreatic cancer, especially when formulated within a nanotechnology-based delivery system. Citation Format: Arvind Thakkar, Jeffrey Wang, Sunil Prabhu. Novel combinations of nano-formulated ferulic acid and aspirin show high potential for chemoprevention of pancreatic cancer. [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 2155. doi:10.1158/1538-7445.AM2014-2155

Abstract LB-14: Polyisoprenylated cysteinyl amides as targeted small molecule anti-Ras therapies

Abstract Pancreatic cancer harbors the highest reported rates of K-Ras mutations, lacks effective therapies and is the most challenging to treat. It is thus the most deadly neoplasm with a 5-year survival rate of only 6%. We reveal here that polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in 93% of pancreatic duct adenocarcinoma. We further present novel compounds with the potential to serve as effective targeted therapies for pancreatic and other cancers with hyperactive growth signaling pathways mediated by Ras and related G-proteins. The design and synthesis of the polyisoprenylated cysteinyl amide inhibitors (PCAIs) of PMPMEase incorporate three key elements to obtain compounds to selectively disrupt polyisoprenylation-mediated protein-protein interactions; the farnesyl group for high affinity interactions, a substituted amide bioisostere of the scissile ester bond of the endogenous substrates and an ionizable appendage group designed to mitigate the excessive hydrophobicity of the farnesyl cysteinyl amide that constitutes the pharmacophore. The PCAIs inhibited PMPMEase with Ki values ranging from 3.7 to 20 μM. The 48 h EC50 values for pancreatic cancer Mia PaCa-2 and BxPC-3 cell lines were as low as 1.9 μM while Salirasib (farnesylthiosalicylic acid, FTS) and farnesylthiosalicylamide were not effective even at 20 μM. The PCAIs thus have the potential to serve as a novel class of targeted therapies for cancers with hyperactive G-proteins and the growth factor receptors whose signals they transmit. Citation Format: Nazarius S. Lamango, Byron J. Aguilar, Augustine T. Nkembo, Randolph Duverna, Felix Amissah, Rosemary A. Poku, Seth Y. Ablordeppey. Polyisoprenylated cysteinyl amides as targeted small molecule anti-Ras therapies. [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 LB-14. doi:10.1158/1538-7445.AM2014-LB-14

Abstract 4961: Longitudinal bioluminescence imaging of primary versus abdominal metastatic tumor growth in orthotopic pancreatic tumor models in NOD/SCIDγ(-/-) mice

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis and is currently the fourth leading cause of cancer death in the United States. The lethal nature of PDAC is strongly associated with metastases to distant organs. The purpose of the present study was to develop and validate noninvasive bioluminescence imaging methods for differentially monitoring the kinetics of primary and abdominal metastatic tumor growth in mouse orthotopic models of pancreatic cancer. A semiautomated maximum entropy segmentation method was implemented for the primary tumor region-of-interest (ROI), and a rule-based method for manually drawing an ROI for the abdominal metastatic region also was developed. The two ROI methods were first validated by having two observers independently construct ROIs for the tumors of animals implanted orthotopically with Panc-1 cells, and the results compared with the number of mesenteric lymph node metastatic nodules counted upon necropsy. The findings were extended to orthotopic tumors of the more metastatic MIA PaCa-2 and AsPC-1 cells where different groups of animals were implanted with different numbers of cells. When the data were expressed as the total photon flux (Ph/sec) in the ROIs for the primary tumor and metastases, the total flux within the metastasis ROI was larger in magnitude than the total flux from the primary tumor ROI, at times by as much as several orders of magnitude. However, when the data were expressed as the average flux density (Ph/sec*mm2) within the ROIs, the density of the flux within the smaller primary tumor ROI was larger in magnitude than the density of the flux from the larger metastasis ROI, by as much as several orders of magnitude. Interobserver assessments for total flux and flux density from ROIs for both the primary tumors and metastatic region were highly concordant, with correlation coefficients of r > 0.98, coefficients of variation of ≤ 0.02, and limits of agreement within <5%. Further, there were statistically significant differences in the growth kinetics of AsPC-1 and MIA PaCa-2 orthotopic tumors when mice were implanted with different numbers of cells for each cell line. The present results demonstrated that the segmentation methods were highly reliable, reproducible and robust, and allowed statistically significant discrimination in the growth kinetics of primary and abdominal metastatic tumors of different cell lines implanted with different cell numbers. Thus, primary tumors and abdominal metastatic foci in orthotopic pancreatic cancer models can be reliably monitored separately and noninvasively over time with bioluminescence imaging. The novel segmentation methods reported here will facilitate investigations of the biology of primary and metastatic tumor growth, as well as the effects of novel treatments, over time, in individual animals. Citation Format: Harlan E. Shannon, Melissa L. Fishel, Jingwu Xie, Dongsheng Gu, Brian P. McCarthy, Amanda A. Riley, Anthony L. Sinn, Jayne M. Silver, Mark R. Kelley, Helmut Hanenberg, Murray Korc, Karen E. Pollok, Paul R. Territo. Longitudinal bioluminescence imaging of primary versus abdominal metastatic tumor growth in orthotopic pancreatic tumor models in NOD/SCIDγ(-/-) mice. [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 4961. doi:10.1158/1538-7445.AM2014-4961

288 HDAC-1 Inhibition Prevents Smoking Induced PanIN Lesions and EMT in Kras Mouse Model of Pancreatic Cancer

Background: Histone deacetylases (HDAC) inhibitors have been shown to suppress tumor growth in many cancers. HDAC class 1 regulates pancreatic cancer growth and resistance to cell death through a mechanism that involves NF-kB. We have developed a mouse model of pancreatic cancer induced by cigarette smoke. We hypothesized that HDAC-1 mediates progression of the disease induced by smoking and that inhibition of HDAC-1 will prevent both cancer cell survival and EMT/metastasis in vitro and in vivo. Methods: Pdx-Cre;LSLKras mice were exposed to filtered air or to cigarette smoke in smoke chambers for 6 weeks. A set of mice were injected with HDAC1 inhibitor SAHA (50mg/Kg) 5 times per week. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation weremeasured by immunohistochemistry (IHC). Pancreas levels of proliferation, apoptosis, and EMT markers were measured by IHC andWestern. Pharmacological and molecular inhibitors of HDAC1 were applied in MIA PaCa-2 and AsPC-1 cell lines. Inhibition of Zeb1, a regulator of EMT, was achieved by using Zeb1 siRNA. EMT markers and transcription factors as well as proteins acetylation level were measured by Western. Cell survival and apoptosis in cells were measured by MTT assay and DNA fragmentation ELISA assay, respectively. Results: Mice exposed to cigarette smoke showed increased level of PanIN lesions, fibrosis, and inflammation. The three pro-cancer effects were prevented in mice exposed to HDAC-1 inhibitor SAHA. SAHA decreased the levels of PanIN lesions and fibrosis, not only to a level lower than in mice exposed to cigarette smoke, but also lower than in control mice exposed to filtered air. SAHA decreased the protein level of vimentin, zeb1, sox2 and CD133 and increased E-cadherin level in pancreatic tissue indicting that it prevented EMT and stemness. Pharmacological and molecular inhibitions of HDAC-1 also significantly and dose-dependently decreased proliferation and at a lesser extent stimulated apoptosis in pancreatic cancer cells. Zeb1 siRNA decreased the level of vimentin and increased E-cadherin in pancreatic cancer cells. Conclusion: We found that smoking stimulates an increase in PanIN lesions through a mechanism that involves HDAC-1 in Kras transgenic mice. HDAC-1 not only affected proliferation of the cancer cells, but also EMT, which is involved in metastasis and cancer stemness. These data suggest an important role for HDAC-1 in pancreatic carcinogenesis.

290 CUGBP1 and HuR Regulate E-Cadherin Translation by Altering Recruitment of E-Cadherin mRNA to Processing Bodies During Total Parenteral Nutrition

Background: Histone deacetylases (HDAC) inhibitors have been shown to suppress tumor growth in many cancers. HDAC class 1 regulates pancreatic cancer growth and resistance to cell death through a mechanism that involves NF-kB. We have developed a mouse model of pancreatic cancer induced by cigarette smoke. We hypothesized that HDAC-1 mediates progression of the disease induced by smoking and that inhibition of HDAC-1 will prevent both cancer cell survival and EMT/metastasis in vitro and in vivo. Methods: Pdx-Cre;LSLKras mice were exposed to filtered air or to cigarette smoke in smoke chambers for 6 weeks. A set of mice were injected with HDAC1 inhibitor SAHA (50mg/Kg) 5 times per week. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation weremeasured by immunohistochemistry (IHC). Pancreas levels of proliferation, apoptosis, and EMT markers were measured by IHC andWestern. Pharmacological and molecular inhibitors of HDAC1 were applied in MIA PaCa-2 and AsPC-1 cell lines. Inhibition of Zeb1, a regulator of EMT, was achieved by using Zeb1 siRNA. EMT markers and transcription factors as well as proteins acetylation level were measured by Western. Cell survival and apoptosis in cells were measured by MTT assay and DNA fragmentation ELISA assay, respectively. Results: Mice exposed to cigarette smoke showed increased level of PanIN lesions, fibrosis, and inflammation. The three pro-cancer effects were prevented in mice exposed to HDAC-1 inhibitor SAHA. SAHA decreased the levels of PanIN lesions and fibrosis, not only to a level lower than in mice exposed to cigarette smoke, but also lower than in control mice exposed to filtered air. SAHA decreased the protein level of vimentin, zeb1, sox2 and CD133 and increased E-cadherin level in pancreatic tissue indicting that it prevented EMT and stemness. Pharmacological and molecular inhibitions of HDAC-1 also significantly and dose-dependently decreased proliferation and at a lesser extent stimulated apoptosis in pancreatic cancer cells. Zeb1 siRNA decreased the level of vimentin and increased E-cadherin in pancreatic cancer cells. Conclusion: We found that smoking stimulates an increase in PanIN lesions through a mechanism that involves HDAC-1 in Kras transgenic mice. HDAC-1 not only affected proliferation of the cancer cells, but also EMT, which is involved in metastasis and cancer stemness. These data suggest an important role for HDAC-1 in pancreatic carcinogenesis.

289 Tight Junction Protein Claudin-2 Is a Direct Target of Vitamin D Receptor Signaling in Intestinal Epithelium

Background: Histone deacetylases (HDAC) inhibitors have been shown to suppress tumor growth in many cancers. HDAC class 1 regulates pancreatic cancer growth and resistance to cell death through a mechanism that involves NF-kB. We have developed a mouse model of pancreatic cancer induced by cigarette smoke. We hypothesized that HDAC-1 mediates progression of the disease induced by smoking and that inhibition of HDAC-1 will prevent both cancer cell survival and EMT/metastasis in vitro and in vivo. Methods: Pdx-Cre;LSLKras mice were exposed to filtered air or to cigarette smoke in smoke chambers for 6 weeks. A set of mice were injected with HDAC1 inhibitor SAHA (50mg/Kg) 5 times per week. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation weremeasured by immunohistochemistry (IHC). Pancreas levels of proliferation, apoptosis, and EMT markers were measured by IHC andWestern. Pharmacological and molecular inhibitors of HDAC1 were applied in MIA PaCa-2 and AsPC-1 cell lines. Inhibition of Zeb1, a regulator of EMT, was achieved by using Zeb1 siRNA. EMT markers and transcription factors as well as proteins acetylation level were measured by Western. Cell survival and apoptosis in cells were measured by MTT assay and DNA fragmentation ELISA assay, respectively. Results: Mice exposed to cigarette smoke showed increased level of PanIN lesions, fibrosis, and inflammation. The three pro-cancer effects were prevented in mice exposed to HDAC-1 inhibitor SAHA. SAHA decreased the levels of PanIN lesions and fibrosis, not only to a level lower than in mice exposed to cigarette smoke, but also lower than in control mice exposed to filtered air. SAHA decreased the protein level of vimentin, zeb1, sox2 and CD133 and increased E-cadherin level in pancreatic tissue indicting that it prevented EMT and stemness. Pharmacological and molecular inhibitions of HDAC-1 also significantly and dose-dependently decreased proliferation and at a lesser extent stimulated apoptosis in pancreatic cancer cells. Zeb1 siRNA decreased the level of vimentin and increased E-cadherin in pancreatic cancer cells. Conclusion: We found that smoking stimulates an increase in PanIN lesions through a mechanism that involves HDAC-1 in Kras transgenic mice. HDAC-1 not only affected proliferation of the cancer cells, but also EMT, which is involved in metastasis and cancer stemness. These data suggest an important role for HDAC-1 in pancreatic carcinogenesis.

Abstract C287: Efficacy and safety of the TH-302, gemcitabine, and nab-paclitaxel combination in xenograft models of pancreatic cancer.

Abstract Background: Tumors often consist of hypoxic regions which are resistant to chemo- and radiotherapy. TH-302 (T) is an investigational hypoxia-targeted drug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. A randomized Phase 2 clinical trial in pancreatic ductal adenocarcinoma (PDAC) demonstrated a significant increase in progression-free survival from the addition of T to gemcitabine (G) vs. G only (NCT01144455). A Phase 3 trial of G + T compared to G + placebo is currently underway (NCT01746979). G + nab-Paclitaxel (nP) significantly prolonged overall survival in a recent Phase 3 trial (NCT00844649). We investigated the efficacy and safety profile of combining T with G and nP in PDAC xenograft models. Methods: Four PDAC xenograft models were established by s.c. implantation of Hs766t, MIA PaCa-2, PANC-1 and BxPC-3 cells into the flank of nude mice. A ‘same day’ administration of T, nP and G with a Q3Dx5 regimen was employed: T, 50mg/kg, ip; nP: 30mg/kg, iv; and G: 60mg/kg ip. Saline treated animals served as vehicle control (V). Antitumor activity of the G + nP + T triplet was assessed, and compared with the G + nP doublet. Hematological, blood chemistry, and immunohistochemical-based analyses were also performed in PANC-1 bearing nude and/or CD-1 mice. Samples were collected 24 hours after the last treatment. Neuropathy assays were performed in CD-1 mice. Results: Using tumor growth inhibition, and Kaplan-Meier analysis of median time to tumor size of 1000mm3 to evaluate antitumor activity, G + nP + T triplet exhibited enhanced efficacy compared with T-alone or the G + nP in all four models. Of note, the complete response rate (CR) of G + nP + T in the PANC-1 model reached 100%, compared to 10% and 50% in the T-alone and G + nP, respectively. G + nP or T-alone treatment reduced neutrophils or lymphocytes compared with V, but there was no additional decrease in neutrophil or lymphocyte count when T was added to G + nP compared to G + nP. Plasma ALT and AST levels were elevated after G + nP treatment, and G + nP + T did not further increase the levels of these liver function parameters. By immunostaining, Ki67-positive cells (a marker of cell proliferation) were significantly reduced after the drug treatment in the PANC-1 xenograft, and to a greater magnitude in G + nP + T. The necrotic region in G + nP + T was increased compared with all other groups. Employing the von Frey neuropathy assay, G + nP treated mice exhibited mechanical allodynia in the hind paw 9 days after initiation of treatment whereas mice treated with T did not, and there was no additive neuropathy in G + nP + T. Conclusions: The triplet combination of G + nP + T exhibited superior efficacy but additive toxicity was not evident compared to G + nP. These studies provide a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C287. Citation Format: Jessica D. Sun, Qian Liu, Dharmendra Ahluwalia, Wenwu Li, Yan Wang, Ayesha S. Ruprell, Charles P. Hart. Efficacy and safety of the TH-302, gemcitabine, and nab-paclitaxel combination in xenograft models of pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C287.

Abstract 2596: Inhibition of PI3K-mTOR pathway using combination of chloroquine and aspirin regimen leads to apoptosis in pancreatic cancer cells.

Abstract Pancreatic cancer ranks as the fourth most deadly form of cancer in the United States with approximately 37,000 deaths each year. The present study aims to evaluate the combinatorial chemopreventive effects of low doses of chloroquine (CQ) and aspirin (ASP) alone and in combination CQA (CQ+ASP) on three pancreatic cancer cell lines, MIA PaCa-2, Panc-1 and BxPC-3. Results demonstrated that low doses of CQ (6.25μM) and ASP (1mM) alone did not reduce cell viability, whereas combination CQA reduced cell viability by ∼80% (P<0.001). CQA combination blocked cell cycle progression at G0/G1 checkpoint. The CQA treatment for 24 h inhibited the NF-κB p50 DNA binding activity by ∼45% (P<0.01) in MIA PaCa-2 and BxPC-3 cells, but no NF-κB inhibition observed in Panc-1 cells. Mechanistic studies revealed that treatment with CQA combination increased the expression of LC3-II protein, indicates inhibition of autophagy. In order to verify the involvement of PI3K and mTOR pathway in CQA-induced apoptosis, we used PI3K inhibitor (3-MA) and mTOR inhibitor (Rapamycin) and analyzed by cell viability assay. The inhibition of PI3K by 3-MA pretreatment enhanced the cell growth inhibition of CQA treatment, whereas the inhibition of m-TOR by rapamycin pretreatment attenuated CQA induced reduction in cell viability. Overall, our results for the first time, illustrate that low dose CQA combinations impart synergistic suppressive activity than chemopreventive agents alone, thus confirming the importance of novel combination chemoprevention strategies against pancreatic cancer. Citation Format: Arvind Thakkar, B. Karthik Grandhi, Jeffrey Wang, Sunil Prabhu. Inhibition of PI3K-mTOR pathway using combination of chloroquine and aspirin regimen leads to apoptosis 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 2596. doi:10.1158/1538-7445.AM2013-2596

Comparative benefits of nab-paclitaxel over gemcitabine or polysorbate-based docetaxel in experimental pancreatic cancer.

192 Background: Gemcitabine (Gem), a standard cytotoxic therapy for pancreatic cancer, has shown limited clinical benefits. Nanoparticle albumin-bound (nab) paclitaxel (NPT), an approved treatment for breast cancer, has shown efficacy as mono- and combination therapy in multiple tumor types including pancreatic, lung and ovarian cancer. We evaluated the NPT treatment benefits compared with Gem or solvent-based taxane docetaxel (DT) in experimental pancreatic cancer. Methods: In vitro cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Tumor growth and animal survival studies were performed in murine xenografts. Intratumoral proliferative activity was measured using Ki67 nuclear antigen staining. Results: For AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1 cells in vitro, Gem IC50 levels were 23.9 mM, 506 nM, 332 nM and 14.5 nM; DT IC50 levels were 30 nM, 4.6 nM, 37.5 nM and 27 nM; and NPT IC50 levels were 7.6 mM, 208 nM, 519 nM and 526 nM. NPT addition decreased Gem IC50 to 1.7 mM, 189 nM, 123 nM and 913 nM; DT addition decreased Gem IC50 to 436 nM, 470 nM, 124 nM and 0.2 nM in AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1 cells, respectively. NPT and DT treatment increased stathmin phosphorylation and decreased tubulin expression in vitro. In a heterotopic in vivo model, net tumor growth inhibition after Gem, DT and NPT was 67, 31 and 72 percent, while intratumoral proliferative index inhibition was 41, 53 and 68 percent, respectively. In an intraperitoneal model, median animal survival was significantly longer in the NPT treatment group (41 days, p<0.002 vs. control and Gem) compared to Gem (32 days, p=0.005 vs. control), DT (32 days, p=0.005 vs. control) and controls (20 days). Animal survival in NPT-Gem and DT-Gem sequential treatment groups was 43 and 40 days, and thus not superior to NPT alone. Conclusions: Nab-paclitaxel has significantly superior antitumor activity as a single agent in experimental pancreatic cancer compared with gemcitabine or docetaxel. These findings provide a strong rationale for considering nab-paclitaxel as first-line monotherapy in patients with pancreatic cancer.

Abstract A65: Activation of ERK1/2 using combination of aspirin, curcumin and sulforaphane regimen leads to NF-κB inhibition and apoptosis in pancreatic cancer cells

Abstract Pancreatic cancer ranks as the fourth most deadly form of cancer in the United States with approximately 37,000 deaths each year. Due to poor prognosis, attention has recently focused on the chemoprevention of this disease rather than on diagnosis and treatment. The present study aims to evaluate the combinatorial chemopreventive effects of low doses of aspirin (ASP), curcumin (CUR), sulforaphane (SFN) alone and in combination ACS (ASP+CUR+SFN) on two pancreatic cancer cell lines, MIA PaCa-2 and Panc-1. Results demonstrated that low doses of ASP (1mM), CUR (10μM) and SFN (5μM) alone did not reduce cell viability, whereas combination ACS reduced cell viability by ∼70% (P<0.001). ACS treatment also induced cell apoptosis by ∼60% accompanied by activation of caspase- 3 and Poly (ADP-ribose) polymerase (PARP) proteins. The NF-κB DNA binding activity was inhibited by ∼45% (P<0.01) and ∼75% (P<0.001) in MIA PaCa-2 and Panc-1 cells respectively. The reduction in NF-κB activity was accompanied by down-regulation of phospho IκBα and phospho Akt proteins. Mechanistic studies revealed that ACS promoted reduction in cell viability, associated with increased expression of phospho extracellular signal-regulated kinase 1/2 (ERK1/2), phospho c-Jun, phospho p38 MAPK and phospho p53 proteins. Furthermore, to evaluate the significance of activation of ERK1/2 pathway on ACS induced reduction in cell viability and NF-κB, the cells were pretreated with U0126 (ERK1/2 inhibitor). The inhibition of ERK1/2 by U0126 pretreatment abolished the effect of ACS on cell viability and NF-κB DNA binding activity. Overall, our results for the first time, illustrate that low dose ACS combinations impart synergistic suppressive activity than chemopreventive agents alone thus confirming the importance of novel combination chemoprevention strategies against pancreatic cancer. Citation Format: Arvind Thakkar, Dhruvitkumar Sutaria, Karthik Grandhi, Ying Huang, Jeffrey Wang, Sunil Prabhu. Activation of ERK1/2 using combination of aspirin, curcumin, and sulforaphane regimen leads to NF-κB inhibition and apoptosis in pancreatic cancer cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A65.

Exocrine Pancreatic Carcinogenesis and Autotaxin Expression

Exocrine pancreatic cancer is an aggressive disease with an exceptionally high mortality rate. Genetic analysis suggests a causative role for environmental factors, but consistent epidemiological support is scarce and no biomarkers for monitoring the effects of chemical pancreatic carcinogens are available. With the objective to identify common traits for chemicals inducing pancreatic tumors we studied the National Toxicology Program (NTP) bioassay database. We found that male rats were affected more often than female rats and identified eight chemicals that induced exocrine pancreatic tumors in males only. For a hypothesis generating process we used a text mining tool to analyse published literature for suggested mode of actions (MOA). The resulting MOA analysis suggested inflammatory responses as common feature. In cell studies we found that all the chemicals increased protein levels of the inflammatory protein autotaxin (ATX) in Panc-1, MIA PaCa-2 or Capan-2 cells. Induction of MMP-9 and increased invasive migration were also frequent effects, consistent with ATX activation. Testosterone has previously been implicated in pancreatic carcinogenesis and we found that it increased ATX levels. Our data show that ATX is a target for chemicals inducing pancreatic tumors in rats. Several lines of evidence implicate ATX and its product lysophosphatidic acid in human pancreatic cancer. Mechanisms of action may include stimulated invasive growth and metastasis. ATX may interact with hormones or onco- or suppressor-genes often deregulated in exocrine pancreatic cancer. Our data suggest that ATX is a target for chemicals promoting pancreatic tumor development.

Abstract 1294: Suppression of uPA in chemo-sensitization and p53 regulation in pancreatic cancer cells

Abstract Despite new knowledge of the molecular profile of pancreatic cancer and its precursor lesions, survival rates have changed very little over the last 40 years. The ability to predict which patients would benefit most from surgical intervention and/or chemotherapy would be a great clinical asset. Researchers have demonstrated that uPA is frequently present in a higher concentration in the serum of pancreaticobiliary cancer patients. It is known that uPA is involved in ECM degradation and has been correlated with malignant transformation of cancer cells. Emerging data have suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor called cancer stem cells (CSCs) or cancer initiating cells. Under in vitro cell culture conditions, these cells possess sphere-forming ability, which is known to be one of the properties of CSCs. Interestingly, the significance of this sphere formation ability still remains unclear. Recent studies have demonstrated that these sphere-forming cells acquired chemoresistance to cisplatin. In the present study, as observed by FACS analysis, we show that a sub-population of MIA PaCa-2 and PANC-1 pancreatic cancer cells possess cancer stem cell properties. RNAi-mediated suppression of uPA in these cells retarded their tumor forming ability in a nude mouse model. We also observed that uPA-suppressed MIA PaCa-2 and PANC-1 cells, after supplementation with uPA protein regained their cancer stem cell phenotype and showed increased expression of Lhx-2. To determine whether these sub-population cells possess any chemoresistance, we treated these cells with varying concentrations of gemcitabine (0-1000 nM) and observed that these cells were resistant to gemcitabine even at the 1000 nM concentration. Further suppression of uPA using siRNA in these cells sensitized these cells to gemcitabine. In addition, to determine whether uPA has any transcriptional role, we examined whether uPA interacted with any known transcription factors we used a TF-protein array. We observed that uPA interacted with Hoxa-5 very strongly. To further determine whether the interaction of uPA with Hoxa-5 invokes any regulatory response, we used a p53 promoter known to possess Hoxa-5 binding regions to drive a Luciferace gene, both with and without the presence of uPA. We observed that uPA retarded p53 promoter activity. Finally, nude mice implanted with MIA PaCa-2 cancer stem cells were susceptible to gemcitabine treatment after uPA downregulation. Taken together, these results indicate that targeting uPA can sensitize chemoresistant pancreatic cells to gemcitabine. 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 1294. doi:1538-7445.AM2012-1294

Abstract 1470: Enhancing radiation therapy by simultaneous inhibition of thioredoxin- and glutathione-dependent metabolism

Abstract Cancer cells have been shown to be in a constant state of metabolic oxidative stress, concurrent with an elevated level of reactive oxygen species (ROS). In order to facilitate survival in the face of increased steady-sate levels of ROS, cancer cells rely on glutathione (GSH) and thioredoxin (Trx) dependent metabolism. We have recently demonstrated that the simultaneous inhibition of GSH using Buthionine sulphoximine [(BSO), a GHS synthesis inhibitor] and thioredoxin reductase using auronofin [(Au), a thioredoxin reductase inhibitor] effectively chemo-radio-sensitized lung cancer cells via a mechanism involving oxidative stress. In this work we extend this observation to demonstrate that Au 2 mg/kg and BSO 450 mg/kg, given prior to ionizing radiation (6 Gy x 2 doses) can significantly delay tumor growth rate for mice treated with radiation alone in H292 lung cancer xenografts. In addition, we demonstrate through clonogenic survival assays that the combination of Au (250-500 nM) and BSO (100 μM) radio-sensitizes two breast cancer cell lines in vitro, and more importantly the combination of Au and BSO given by intraperitoneal injection prior to radiation decreases the percent of breast cancer stem/progenitor cells in vivo. The rate limiting substrate for GSH synthesis is cysteine, whose level is controlled by the xc- cystine/glutamate antiporter. Sulfasazine (SSZ) has been shown to be a potent inhibitor of the xc- cystine/glutamate antiporter. We demonstrate that SSZ (0.1-0.5 mM) decreases total GSH (up to 90%) levels in a dose dependent manner in both Mia PaCa-2 and PANC-1 human pancreatic cell lines. The combination of Au (250 nM for 3 hours) with SSZ (0.2 mM for 24 hours) demonstrates an 80% and 30% decrease in clonogenic cell survival in MiaPaCa-2 and PANC-1 pancreatic cells that is inhibited by treatment with the thiol antioxidant, 15 mM N-acetylcysteine. These results indicate that inhibition of hydroperoxide metabolism using pharmacologically relevant agents to simultaneously disrupt GSH and Trx system, can sensitize multiple cancer cell lines to radiation. These results support the hypothesis that standard of care radiotherapy protocols could be enhanced using SSZ, Au, and/or BSO. Supported by The Iowa Center for Research by Undergraduates, R21CA139182, R01CA133114 and a Breast Cancer Research Group Seed Grand from the Holden Comprehensive Cancer Center. 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 1470. doi:1538-7445.AM2012-1470

Abstract B63: Synergistic combination between resveratrol and tanshinone I for effective prevention of pancreatic cancer

Abstract Pancreatic cancer is the fourth most common cause of cancer-related mortality in the United States. Pancreatic cancer is often dubbed the silent killer because it seldom causes symptoms until advanced. As a result, few pancreatic cancers are operable at diagnosis. Furthermore, pancreatic cancer is often resistant to conventional radiotherapy or chemotherapy, and the morbidity rate almost equals the mortality rate. Therefore, search for effective regimens with moderate side effects for pancreatic cancer therapy and prevention is an urgent necessity. Chinese herbal medicines have been used for centuries with demonstrated safety profiles, thus their bioactive ingredients may be effective candidates for the prevention and/or treatment of pancreatic cancer. Danshen (Salvia miltiorrhiza) has been widely adopted in the traditional Chinese medicinal preparations for treatment of cardiovascular disease with minimal side effects. In the present study, we evaluated the effect of several major Danshen components, tanshinone IIA (T2A), tanshinone I (T1) and cryptotanshinone (CT) on pancreatic cancer cells. We found that CT, T2A and T1 all showed inhibitory effects on the growth of human pancreatic cancer cell lines MIA PaCa-2 and ASPC-1 with IC50 around 30μM, 3–8μM and 3–5μM, respectively. The drug-resistant human pancreatic cancer cell line PANC-1 showed less sensitivity to CT and T2A treatment but was very sensitive to T1 treatment with IC50 around 3.5μM. The potent T1 activity against pancreatic cancer cells was associated with downregulation of several protein markers related to cell cycle (cyclin D and cyclin B) and anti-apoptosis (Bcl2 and survivin), c-myc and Aurora A and B kinases in both MIA PaCa-2 and PANC-1 cells. To further identify more potent synergistic combination effect of natural bioactive compounds against pancreatic cancer, we determined the anti-growth activity of the combination between T1 and resveratrol, a natural compound found in the skin of red grape and other fruits. T1 and resveratrol combinations consistently showed synergistic effect on inhibiting the growth of MIA PaCa-2, ASPC-1 and PANC-1 cells. Protein assays showed that the synergistic combination effect of T1 and resveratrol was associated with combined downregulation of c-myc and Aurora A protein levels, suggesting that T1 and resveratrol may inhibit the growth of pancreatic cancer cells in part via combined inhibition on the functions of c-myc and Aurora A. Aurora A is the kinase that plays a critical role in controlling mitosis and cell growth. We further confirmed if Aurora A was a functional target for the synergistic combination effect of T1 and resveratrol. Knockdown of Aurora A dramatically inhibited PANC-1 cell growth and decreased the T1 and resveratrol combination activity, suggesting that Aurora A could be one of the targets by the T1 and resveratrol combination effect. Aurora A gene promoter contains the c-myc binding site, suggesting that Aurora A may be regulated by c-myc and the inhibition of c-myc expression may be one of the causes of reduced Aurora A expression following T1 treatment. To confirm this, we treated PANC-1 cells with a c-myc inhibitor and found decreased Aurora A gene expression in a time-dependent manner, indicating the regulation of c-myc on Aurora A gene expression. Our results suggest that the T1 and resveratrol combination may serve as a novel chemopreventive regimen for effective prevention of pancreatic cancer. Supported in part by DOD (PC073988) and NCI/NIH (R21CA127794) Citation Information: Cancer Prev Res 2011;4(10 Suppl):B63.

Abstract 2456: External uPA overexpresses and interacts with Lhx-2 and induces a pancreatic cancer stem cell phenotype

Abstract Although the body of knowledge surrounding the molecular profile of pancreatic cancer has substantially increased, survival rates have not changed for over four decades. Previous studies have demonstrated that urokinase plasminogen activator (uPA) is frequently present in a higher concentration in the serum of pancreatic cancer patients. uPA is involved in extracellular matrix (ECM) degradation and has been correlated with the malignant transformation of cancer cells. Additionally, recent reports have also shown the translocation of uPA to the nucleus without binding to uPAR. However, its role in the nucleus is still unclear. Existing evidence suggest that a hypoxic environment forces cancer cells to acquire stem cell characteristics, causing recurrence after chemotherapy and radiation. In the present study, we have attempted to correlate the roles of uPA and hypoxia in cancer stem cell transformation. Initially, to determine the interaction of uPA with Lhx-2, we conducted immunoprecipitation and immunolocalization studies in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines. Further, to determine the role of exogenous uPA on Lhx-2 expression in an uPA-null background, cells were downregulated for uPA and followed by supplementation with purified uPA under normoxic as well as hypoxic conditions. To further resolve whether Lhx-2 is involved in the activation of CD133 and STRO-1 expression in pancreatic cells, uPA and/or Lhx-2 were knocked down under normoxic or hypoxic conditions and the expression levels of CD133 and STRO-1 were determined. In addition, cell lysates were screened for stem cell markers using stem cell-specific antibody arrays. To determine the role of uPA in tumorigenicity after hypoxia, MIA PaCa-2 or PANC-1 hypoxic cells were subcutaneously implanted in nude mice followed by injection of siRNA for uPA or Lhx-2 and 100 nM of uPA at the tumor site, and tumor growth was determined. From the immunoprecipitation and immunolocalization studies, we observed that uPA interacts with Lhx-2 both in the cytoplasm and the nucleus of MIA PaCa-2 and PANC-1 pancreatic cancer cells. Further, we observed that the addition of external uPA caused an increase in the expression of Lhx-2. The addition of uPA under hypoxic conditions resulted in increased expression of CD133 and STRO-1 whereas suppression of Lhx-2 retarded the overexpression of CD133 and STRO-1. Antibody array analysis indicated that hypoxic cells had increased expression of stem cell markers when supplemented with uPA, which was abolished by suppression of Lhx-2. From the animal studies, we observed that hypoxic MIA PaCa-2 or PANC-1 cells supplemented with external uPA were able to form subcutaneous tumors within 30 days of implantation. Taken together, our results demonstrate that in a hypoxic environment, uPA activates the expression of stem cell markers mediated by the expression of Lhx-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2011-2456

Abstract 3545: Autophagy inhibition as a therapeutic strategy in cancer

Abstract Autophagy is an evolutionarily conserved process involving degradation of cellular components in lysosomes to maintain protein and organelle quality control, and temporarily sustain cell viability in response to stressful stimuli. Several studies have demonstrated the dual role of autophagy in tumorigenesis as both a tumor suppressor and a protector of cancer cell survival under stress. It is well recognized that anticancer therapies, such as chemotherapy and irradiation (RT), induce autophagy as a pro-survival response, likely resulting in therapeutic resistance. Therefore, concurrent autophagy inhibition as a chemo- and RT-sensitizer has generated great interest in cancer therapy. We generated Beclin1+/+ and Beclin1+/- immortalized mouse mammary epithelial cells (iMMECs) stably expressing the apoptosis inhibitor BCL-2 and the autophagy marker EGFP-LC3 to investigate how acute autophagy inhibition and chronic autophagy deregulation impact tumor sensitivity to anticancer drugs commonly used against breast cancer. We also used human cancer cell lines, including MCF-7, PANC-1, MIA PaCa-2 and HCT-116 cells, to explore whether pharmacologic autophagy inhibition or genetic knockdown of autophagy regulators exhibits a synergistic anticancer effect with newer antineoplastic drugs, such as mTOR inhibitors (CCI-779 and RAD001) and novel antimicrotubule agents (ixabepilone). Treatment efficacy was evaluated by clonogenic survival and cytotoxicity assays in 2D- and 3D-epithelial cell culture in vitro and by tumor responsiveness assays in mice in vivo. Autophagy inhibition sensitized Beclin1+/+ apoptosis-defective iMMECs to doxorubicin, but not to paclitaxel, and had an even greater impact on autophagy-deficient cells. Moreover, autophagy inhibition via choloroquine or Beclin1 knockdown enhanced the treatment efficacy of mTOR inhibitors on MCF-7, PANC-1, MIA PaCa-2 and HCT116 cells in vitro and in xenograft tumors in nude mice in vivo. These studies provide insight into how autophagy functional status in tumors impacts treatment responsiveness and into how to best incorporate pharmacologic autophagy modulation in the decision tree for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3545. doi:10.1158/1538-7445.AM2011-3545