Tumor Progression In Pancreatic Cancer Research Articles

MiR-455-3p Functions as a Tumor Suppressor by Restraining Wnt/β-Catenin Signaling via TAZ in Pancreatic Cancer.

BackgroundPancreatic cancer (PC) is a highly invasive tumor with a poor prognosis, short overall survival rate and few chemotherapeutic choices. Despite the importance of finding ways to treat pancreatic cancer, the mechanisms of tumor progression have not been fully elucidated. microRNA-455-3p (miR-455-3p) has been reported to play an important role in several cancers, but its function in pancreatic cancer remains unclear.MethodsTo investigate the biological functions, miRNAs mimics or inhibitors were transfected into pancreatic cancer cells. Flow cytometry was used to detect cell apoptosis. Wound healing and Transwell assays were employed to observe cell invasion and migration abilities. The expression of Bcl-2, Bax, caspase-3, E-cadherin, N-cadherin, Snail, β-Catenin, c-Myc and Cyclin D1 were evaluated by qPCR and Western blot.ResultsWe confirmed that inhibition of miR-455-3p decreases cell apoptosis and increases cell migration, invasion and EMT of pancreatic cancer, whereas forced overexpression of miR-455-3p has the opposite effect. Furthermore, we demonstrated that the tumor suppression effects of miR-455-3p were partially reversed by TAZ overexpression. In addition, miR-455-3p led to inactivation of Wnt/β-catenin signaling in pancreatic cancer cells, and TAZ overexpression restored the inhibition of Wnt/β-catenin signaling.ConclusionTaken together, our data demonstrated that miR-455-3p functions as an important tumor suppressor that suppresses the Wnt/β-catenin signaling pathway via TAZ to inhibit tumor progression in pancreatic cancer. We conclude that the miR-455-3p/TAZ/Wnt axis may be a potential therapeutic target for pancreatic cancer.

The Unfolded Protein Response Causes Prothrombotic Transformation of Pancreatic Cancer Linking Tumor Progression with Cancer-Associated Thrombosis

Thrombosis is a common complication of advanced stage cancer. Yet the underlying mechanisms that link tumor progression to clot formation are poorly understood. The unfolded protein response (UPR) is associated with malignant transformation in pancreatic cancer, but whether or not activation of the UPR is linked to cancer thrombosis has not previously been evaluated. To determine whether UPR signaling functions in the prothrombotic transformation of pancreatic cancer, we exposed pancreatic adenocarcinoma cells (HPAF-II cells) to three UPR inducers (tunicamycin, triptolide or thapsigargin) that act via independent mechanisms. Induction of UPR resulted in the release of thrombogenic material into the supernatant as evidenced by a ~3-fold increase in thrombin generation in pelleted material. Release of thrombogenic material was inhibited by siRNA-mediated knockdown of UPR components including IRE1a (80±3% decrease) or PERK (60±10% decrease). Chemical inhibition of UPR also inhibited release of thrombogenic material from HPAF-II cells. Exposure to the IRE1a inhibitor, MKC-3946, resulted in a 70±10% decrease in thrombin generation in pelleted material, and incubation with the PERK inhibitor, GSK2606414, caused an 80±5% decrease. Characterization of the thrombogenic activity revealed that it was present on extracellular vesicles (EVs) and was inhibited by anti-TF antibodies. Flow cytometry demonstrated on average a 3-fold increase in fluorescence of TF-bearing EVs following UPR induction. Electron microscopy showed that the HPAF II EVs ranged from 100-500 mm and demonstrated increased clustering following UPR induction. Three-color immunofluorescence microscopy of HPAF II cells with labeling of actin, nuclei, and TF showed that induction of the UPR resulted in actin-poor membrane blebs rich in TF. Apoptosis as detected by caspase-3 cleavage was not observed under these conditions. Brefeldin A, which inhibits vesicular transport between the endoplasmic reticulum and the Golgi, inhibited UPR-induced generation of TF-bearing EVs, indicating that UPR-mediated vesicular trafficking contributes to TF-bearing EV formation. To evaluate the possibility of an association between the UPR and cancer thrombosis in the clinical setting, we analyzed plasmas collected from pancreatic cancer patients who were monitored prospectively for the development of venous thromboembolism (including a lower extremity ultrasound performed at baseline and at 2 months). Proteomic analysis was performed using Somalogic technology to evaluate ~1300 analytes in plasmas from nine pancreatic cancer patients who subsequently developed venous thromboembolism and ten patients with similar pancreatic cancer characteristics who remained free of venous thromboembolism. Evaluation of eight UPR markers present in the SOMAscan panel demonstrated significant upregulation in plasmas of patients who developed clots compared to those who did not (p = 0.0001). Our current findings support a model whereby activation of the UPR results in increased vesicular trafficking leading to the release of TF-bearing EVs. These observations indicate a mechanistic link between tumor progression in pancreatic cancer and cancer associated thrombosis. Disclosures Peters: Platelet BioGenesis: Employment. Zwicker:Portola: Consultancy; Parexel: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Daiichi: Consultancy; Quercegen: Research Funding; Incyte: Research Funding. Flaumenhaft:Relay Therapeutics: Consultancy; PlateletDiagnostics: Consultancy, Other: Founder.

Cancer‑associated adipocytes exhibit distinct phenotypes and facilitate tumor progression in pancreatic cancer.

Adipocyte infiltration in pancreatic cancer (PC) has been demonstrated to be independently associated with PC risk and an active contributor to tumor progression. However, to date, little is known about these unique pancreatic tumor-surrounding adipocytes, or their response to cancer cells. The present study utilized an in vitro indirect coculture model in which the phenotypic changes of adipocytes following exposure to PC cells were directly observed. RNA-sequencing was performed on 3T3-L1 adipocytes cultured with or without Panc-1 cancer cells, and significant changes were identified at the transcriptional level. In terms of delipidation and the impaired function of glucose and lipid metabolism, coculture with tumor cells resulted in an altered metabolic phenotype in mature adipocytes. In co-cultured adipocytes, the appearance of fibroblast-like cells was observed, and the mesenchymal cell differentiation pathway was enriched following the integrated analysis into the transcriptome. In addition, reverse transcription-quantitative PCR analyses of co-cultured adipocytes revealed a loss in gene expression of mature adipocyte markers, and a gain in gene expression of fibroblast-specific markers. It was also confirmed that newly generated cancer-associated adipocytes could facilitate the invasive capacities of the tumor, and may contribute to PC stromal remodeling. The present study supports a novel concept that reprogramming of stromal adipocytes orchestrated by PC cells may generate cancer-associated adipocytes with activated phenotypes, which may ultimately drive pancreatic tumor progression.

MiR-223 Regulates Cell Proliferation and Invasion via Targeting PDS5B in Pancreatic Cancer Cells

Emerging evidence has demonstrated that miR-223 is critically involved in the progression of pancreatic cancer (PC); however, the underlying mechanisms are not fully elucidated. In the present study, we explored the molecular basis of miR-223-mediated tumor progression in PC. We performed numerous approaches including MTT, FACS, transfection, RT-PCR, western blotting, Transwell, and animal studies to determine the physiological role of miR-223 in PC cells. We found that sister chromatid cohesion protein PDS5 homolog B (PDS5B) is a direct target of miR-223 in PC. Moreover, PDS5B exhibits tumor-suppressive function in PC cells. Consistently, ectopic overexpression of PDS5B reversed miR-223-mediated tumor progression in PC cells. These results suggest that the miR-223/PDS5B axis regulates cell proliferation and invasion in PC cells. Our findings indicated that downregulation of miR-223 could be a novel therapeutic approach for PC.

Abstract 60: The association between cellular senescence of cancer associated fibroblasts and tumor progression in pancreatic cancer

Abstract Background: In recent days, cellular senescence of cancer associated fibroblasts (CAFs) is gaining increased attention, yet incompletely understood the role in the development of malignant diseases. Previous studies reported that Caveolin-1 plays a major role in cellular senescence, and its expression in CAFs. Therefore, we hypothesized that cellular senescence of CAFs introduces the tumor progression in pancreatic cancer (PC). Methods: A total of 159 consecutive patients with PC who underwent curative resection between January 2004 and December 2016 were enrolled. The patients were divided into two groups according to the expression of Caveolin-1 in CAFs, which were analyzed by immunohistochemistry. First, we investigated the relationship between the expression of Caveolin-1 in CAFs and patients' clinicopathological factors including survival outcomes. Second, we established the CAFs-cell lines from the resected tissues of patient with PC. We stocked CAFs-cell line after 6 times passages at least. Third, we controlled the expression of Caveolin-1in CAFs, and underwent coculture of PC cell lines and CAFs-conditioned medium (CM) to evaluate the effect of cellular senescence in CAFs on invasion of PC cell lines. Results: The high level of Caveolin-1 expression group counts of 49 patients (31%), and the low level of Caveolin-1 expression group counts of 110 patients (69%). As for patients' clinicopathological factors, the serum levels of CA19-9 (p=0.0008) and pathological T factor stage (p=0.014), defined by Japan Pancreas Society as the “General Rules for the Study of Pancreatic Cancer” were significantly higher in the high Caveolin-1 expression group than were those of the low expression group. The high Caveolin-1 expression group had significantly worse outcomes in overall (log-rank p = 0.022) and disease-free survivals (log-rank p = 0.011). We established 10 CAFs cell lines from clinical samples, and evaluated Caveolin-1 expressions. We identified the high Caveolin-1 expression CAFs (CAF-4 and 5), and suppressed its expression by transfection of short interfering (si) Caveolin-1 knockdown. We collected CAF-CM from CAF-4 and CAF-5 with siCaveolin-1suppressed the invasive ability of MiaPaCa-2 compared to that with si control (p<0.0001). Conclusion: The present result suggested that Caveolin-1 expression in CAFs in PC is associated with patients' poor prognosis. The downregulation of Caveolin-1 in CAFs would lead to less invasion of PC cells. Therefore, there is a possibility that Caveoin-1 of CAFs in PC is a new target for treatment. Citation Format: Takanobu Yamao, Yo-ichi Yamashita, Kensuke Yamamura, Naoki Umezaki, Masayo Tsukamoto, Yuki Kitano, Kota Arima, Tatsunori Miyata, Shigeki Nakagawa, Hirohisa Okabe, Katsunori Imai, Hidetoshi Nitta, Akira Chikamoto, Ishiko Takatoshi, Hideo Baba. The association between cellular senescence of cancer associated fibroblasts and tumor progression in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 60.

Elevated expression of Tiam1 is associated with poor prognosis and promotes tumor progression in pancreatic cancer

ObjectiveT-cell lymphoma invasion and metastasis inducing factor 1 (Tiam1) is known to be involved in tumor progression. However, its molecular roles and mechanism in pancreatic ductal adenocarcinoma (PDAC) remain unclear. The purpose of this study is to determine Tiam1 expression levels and investigate its underlying molecular mechanism in PDAC.Materials and methodsTiam1 protein expression levels in PDAC tissues were examined using immunohistochemistry. Tiam1 expression was confirmed in pancreatic cancer (PC) cells by Western blot and immunofluorescence staining. Tiam1-silenced PC cells were created using short interfering RNA. Subsequently, colony formation, scratch, and migration and invasion assays were carried out to explore the molecular mechanisms of Tiam1 in PC cells.ResultsThe results indicated that Tiam1 expression was significantly higher in PDAC tissues than in paired non-tumor tissues, and overexpression of Tiam1 was significantly correlated with histological grade (P=0.040) and lymph node metastasis (P=0.031) in PDAC. The PDAC patients with high Tiam1 expression had significantly lower 5-year overall survival than patients with low Tiam1 expression. More importantly, univariate and multivariate analysis suggested that Tiam1 expression, along with lymph node metastasis, is a significant independent prognostic factor for patients with PDAC. Furthermore, we also demonstrated that the downregulation of Tiam1 was associated with decreased cell proliferation and reduced migratory and invasive capability.ConclusionHigh expression of Tiam1 plays a significant role in the progression of PDAC and may be a potential biomarker of poor prognosis as well as a therapeutic target.

Galectin-1 expressed in pancreatic stellate cells promotes tumor progression in pancreatic cancer via upregulation of SDF-1 and activation of NF-kappaB

Galectin-1 expressed in pancreatic stellate cells promotes tumor progression in pancreatic cancer via upregulation of SDF-1 and activation of NF-kappaB

The utility of hyperthermic intra-abdominal chemotherapy with gemcitabine for the inhibition of tumor progression in an experimental model of pancreatic peritoneal carcinomatosis, in relation to their behavior with pancreatic cancer stem cells CD133+ CXCR4+

ObjectiveThe origin of pancreatic cancer has been identified as a population of malignant pancreatic stem cells CD133+ CXCR4+ immunophenotype. These cells have high capacity for early locoregional invasion, being responsible for early recurrence and high mortality rates of pancreatic cancer. We propose a study for decreasing tumor progression of pancreatic cancer by reducing the volume and neoplastic subpopulation of pancreatic cancer stem cells CD133+ CXCR4+. Therefore, we develop a new therapeutic model, characterized by the application of HIPEC (Hyperthermic Intraperitoneal Chemotherapy) with gemcitabine. DesignPancreatic tumor cell line: human cell line BxPC-3. The animal model involved 18 immunosuppressed rats 5 weeks weighing 150–200 gr. The implantation of 13 × 106 cells/mL was performed with homogeneous distribution in the 13 abdominopelvic quadrants according to the peritoneal carcinomatosis index (PCI) and were randomized into three treatment groups. Group I (4 rats) received intravenous saline. Group II (6 rats) received intravenous gemcitabine. Group III (8 rats) received HIPEC at 41 °C for 30 min with gemcitabine + gemcitabine IV. A histological study confirmed pancreatic cancer and immunohistochemical quantification of pancreatic cancer stem cells CD133+ CXCR4+ tumor cells. ResultsThere was a population decline of pancreatic cancer stem cells CD133+ CXCR4+ in the HIPEC group with respect to the other two groups (p < 0.001). There was a decrease in PCI between treatment groups (p < 0.05). ConclusionThe initial results are encouraging since there is a declining population of cancer stem cells CD133+ CXCR4+ in the HIPEC group and decreased tumor volume compared to the other two treatment groups. All the conclusions are only valid for BxPC3 cell line, and the effects HIPEC on Kras-driven pancreatic tumors remain to be determined.

MiRNA dynamics in tumor-infiltrating myeloid cells modulating tumor progression in pancreatic cancer

ABSTRACTMyeloid cells including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are known as important mediators of tumor progression in solid tumors such as pancreatic cancer. Infiltrating myeloid cells have been identified not only in invasive tumors, but also in early pre-invasive pancreatic intraepithelial precursor lesions (PanIN). The functional dynamics of myeloid cells during carcinogenesis is largely unknown. We aimed to systematically elucidate phenotypic and transcriptional changes in infiltrating myeloid cells during carcinogenesis and tumor progression in a genetic mouse model of pancreatic cancer. Using murine pancreatic myeloid cells isolated from the genetic mouse model at different time points during carcinogenesis, we examined both established markers of macrophage polarization using RT-PCR and FACS as well as transcriptional changes focusing on miRNA profiling. Myeloid cells isolated during carcinogenesis showed a simultaneous increase of established markers of M1 and M2 polarization during carcinogenesis, indicating that phenotypic changes of myeloid cells during carcinogenesis do not follow the established M1/M2 classification. MiRNA profiling revealed distinct regulations of several miRNAs already present in myeloid cells infiltrating pre-invasive PanIN lesions. Among them miRNA-21 was significantly increased in myeloid cells surrounding both PanIN lesions and invasive cancers. Functionally, miRNA-21-5p and -3p altered expression of the immune-modulating cytokines CXCL-10 and CCL-3 respectively. Our data indicate that miRNAs are dynamically regulated in infiltrating myeloid cells during carcinogenesis and mediate their functional phenotype by facilitating an immune-suppressive tumor-promoting micro-milieu.

Prognostic and Functional Significance of MAP4K5 in Pancreatic Cancer.

ObjectivesMAP4K5 plays an important role in regulating a range of cellular responses and is involved in Wnt signaling in hematopoietic cells. However, its functions in human malignancies have not been studied. The major objectives of this study are to examine the expression, functions and clinical significance of MAP4K5 in pancreatic ductal adenocarcinoma (PDAC).Materials and MethodsThe expression levels of MAP4K5, E-cadherin, vimentin, and carboxylesterase 2 (CES2) were examined by immunohistochemistry in 105 PDAC and matched non-neoplastic pancreas samples from our institution. The RNA sequencing data of 112 PDAC patients were downloaded from the TCGA data portal. Immunoblotting and RNA sequencing analysis were used to examine the expression of MAP4K5 and E-cadherin in pancreatic cancer cell lines. The effect of knockdown MAP4K5 using siRNA on the expression of CDH1 and vimentin were examined by Real-time RT-PCR in Panc-1 and AsPC-1 cells. Statistical analyses were performed using IBM SPSS Statistics.ResultsMAP4K5 protein is expressed at high levels specifically in the pancreatic ductal cells of 100% non-neoplastic pancreas samples, but is decreased or lost in 77.1% (81/105) of PDAC samples. MAP4K5-low correlated with the loss of E-cadherin (P = 0.001) and reduced CES2 expression (P = 0.002) in our patient populations. The expression levels of MAP4K5 mRNA directly correlated with the expression levels of CDH1 mRNA (R = 0.2490, P = 0.008) in the second cohort of 112 PDAC patients from The Cancer Genome Atlas (TCGA) RNA-seq dataset. Similar correlations between the expression of MAP4K5 and E-cadherin were observed both at protein and mRNA levels in multiple pancreatic cancer cell lines. Knockdown MAP4K5 led to decreased CDH1 mRNA expression in Panc-1 and AsPC-1 cells. MAP4K5-low correlated significantly with reduced overall survival and was an independent prognosticator in patients with stage II PDAC.ConclusionsMAP4K5 expression is decreased or lost in majority of PDACs. The strong associations between low MAP4K5 expression and loss of E-cadherin, reduced CES2 expression and decreased overall survival may suggest an important role of MAP4K5 in epithelial-to-mesenchymal transition, chemotherapy resistance and tumor progression in pancreatic cancer. Targeting impaired MAP4K5 signaling may represent a new therapeutic strategy for pancreatic cancer.

Abstract 923: Nitric oxide signaling pathway as a pathogenic driver in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is among the most malignant cancers, with 5-year survival of 6%. Chronic inflammation is associated with increased risk of PDAC. Nitric oxide (NO) is an important mediator of inflammatory responses and plays a role in tumorigenesis. The principal source of an increased and sustained level of NO is inducible nitric oxide synthase (NOS2). In this study, we’ve tested the hypothesis that NO enhances tumor progression in pancreatic cancer. We found that higher NOS2 expression in tumors was associated with poor survival in resected PDAC patients (p = 0.011). We then investigated the role of NO by deleting NOS2 gene in a genetically engineered mouse model of pancreatic cancer (KPC mice) with pancreas-specific activation of mutant KRAS and P53, which faithfully recapitulates the development and progression of human pancreatic cancer. NOS2-deficient KPC mice showed longer survival compared to the KPC littermates with wildtype NOS2 (p&amp;lt;0.01). Compared to tumors from KPC mice, KPC/NOS2−/− tumors were less vascularized (p&amp;lt;0.001) and with less macrophage recruitment (p&amp;lt;0.005), indicated by decreased expression of CD31 and F4/80 respectively. NOS2 deficiency led to attenuated proliferation and invasion, and enhanced apoptosis indicated by high level of cleaved caspase-3 (p = 0.02). Consistent with the less malignant phenotype, NOS2 deficiency reduced nuclear pERK accumulation (P = 0.034) and the subsequent phosphorylation of FOXO3 (p = 0.026). Further investigation showed a positive correlation between NOS2 expression and pFOXO level in human PDAC specimen (p = 0.005). In addition, NOS2 deficiency reduced the expression of a well known oncogenic microRNA, miR21 (p = 0.001). In summary, NOS2 gene expression in tumors is a candidate prognostic marker in resected pancreatic cancer patients, and NOS2/NO signaling may contribute to the pancreatic tumor progression by inactivating FOXO3 in KPC mice. In this ongoing study, we are further investigating the mechanistic role of NO in disease progression. Citation Format: Jian Wang, Peijun He, Matthias M. Gaida, Shouhui Yang, Aaron Schetter, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Harris G. Yfantis, Dong H. Lee, Jonathan M. Weiss, Nadar Hanna, H. Richard Alexander, S. Perwez Hussain. Nitric oxide signaling pathway as a pathogenic driver in 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 923. doi:10.1158/1538-7445.AM2015-923

The c-Myc-LDHA axis positively regulates aerobic glycolysis and promotes tumor progression in pancreatic cancer.

The transcription factor c-Myc plays critical roles in cancer development and progression through regulating expression of targeted genes. Lactate dehydrogenase A (LDHA), which catalyzes the conversion of l-lactate to pyruvate in the final step of anaerobic glycolysis, is frequently upregulated in pancreatic cancer. However, little is known about the effects of c-Myc–LDHA axis in the progression of pancreatic cancer. In this study, we found that c-Myc and LDHA are concomitantly overexpressed in pancreatic cancer cell lines and clinical specimens. c-Myc overexpression and LDHA overexpression were correlated with TNM stage and tumor size and indicated poor prognosis in patients with pancreatic cancer. Knockdown of c-Myc reduced the protein expression of LDHA, lactate production and glucose consumption, and silencing of LDHA mimicked this effect. Meanwhile, reduced c-Myc–LDHA signaling resulted in decreased tumor growth and metastasis in pancreatic cancer. Treatment with 2-Deoxy-d-glucose, an inhibitor of anaerobic glycolysis, completely blocked the oncogenic roles of c-Myc–LDHA signaling. Taken together, dysregulated c-Myc–LDHA signaling plays important roles in aerobic glycolysis and facilitates tumor progression of pancreatic cancer.

Tenascin-C induces resistance to apoptosis in pancreatic cancer cell through activation of ERK/NF-κB pathway.

As a glycol-protein located in extracellular matrix (ECM), tenascin-C (TNC) is absent in most normal adult tissues but is highly expressed in the majority of malignant solid tumors. Pancreatic cancer is characterized by an abundant fibrous tissue rich in TNC. Although it was reported that TNC's expression increased in the progression from low-grade precursor lesions to invasive cancer and was associated with tumor differentiation in human pancreatic cancer, studies on the relations between TNC and tumor progression in pancreatic cancer were rare. In this study, we performed an analysis to determine the effects of TNC on modulating cell apoptosis and chemo-resistance and explored its mechanisms involving activation in pancreatic cancer cell. The expressions of TNC, ERK1/2/p-ERK1/2, Bcl-xL and Bcl-2 were detected by immunohistochemistry and western blotting. Then the effects of exogenous and endogenous TNC on the regulation of tumor proliferation, apoptosis and gemcitabine cytotoxicity were investigated. The associations among the TNC knockdown, TNC stimulation and expressions of ERK1/2/NF-κB/p65 and apoptotic regulatory proteins were also analyzed in cell lines. The mechanism of TNC on modulating cancer cell apoptosis and drug resistant through activation of ERK1/2/NF-κB/p65 signals was evaluated. The effect of TNC on regulating cell cycle distribution was also tested. TNC, ERK1/2/p-ERK1/2, and apoptotic regulatory proteins Bcl-xL and Bcl-2 were highly expressed in human pancreatic cancer tissues. In vitro, exogenous TNC promoted pancreatic cancer cell growth also mediates basal as well as starved and drug-induced apoptosis in pancreatic cancer cells. The effects of TNC on anti-apoptosis were induced by the activation state of ERK1/2/NF-κB/p65 signals in pancreatic cell. TNC phosphorylate ERK1/2 to induce NF-κB/p65 nucleus translocation. The latter contributes to promote Bcl-xL, Bcl-2 protein expressions and reduce caspase activity, which inhibit cell apoptotic processes. TNC mediated gemcitabine chemo-resistance via modulating cell apoptosis in pancreatic cancer. TNC resulted in the enrichment of pancreatic cancer cells in S-phase with a concomitant decrease in number of cells in G1 phase. The present study indicated TNC in cellular matrix induces an activation of ERK1/2/NF-κB/p65 signaling cascade and thereby mediates resistance to apoptosis in pancreatic cancer. TNC could serve as a diagnostic marker and predictor of gemcitabine response and potentially as a target for chemotherapy of pancreatic cancer.

Antisense inhibition of microRNA-21 and microRNA-221 in tumor-initiating stem-like cells modulates tumorigenesis, metastasis, and chemotherapy resistance in pancreatic cancer.

Our preliminary studies identified a small population side population (SP) cells in pancreatic cancer cells with stem cell-like properties, which were able to induce fast and aggressive tumor formation in nude mice. Gene expression analysis showed a significant difference in the expression of more than 1,300 genes in SP cells, among which a highly significant difference in microRNA expression of miR-21 and miR-221 between SP and NSP cells was identified. SP cells were identified and characterized by flow cytometry using Hoechst 33342 dye staining from a highly metastatic human pancreatic cancer cell line (L3.6pl). Antagomir transfection was performed using miRNA-21 and miRNA-221 antisense oligonucleotides (ASOs) and followed by detection of cell apoptosis, cell cycle progression, chemosensitivity, and invasion. Sorted SP cells from gemcitabine-resistant L3.6pl cells (L3.6pl(Gres)-SP) cells were orthotopically implanted in nude mice with or without miRNA-21 and miRNA-221 ASOs mono- and combination therapy. The administration of antagomir-21 and antagomir-221 significantly reduced the SP cell fraction, decreased SP cell differentiation, and downstream gene regulation, and thereby induced reduction of L3.6pl cell proliferation, invasion, and chemoresistance against gemcitabine and 5-Fluorouracil. Combination of ASOs therapy against miRNA-21 and miRNA-221 significantly inhibited primary tumor growth and metastasis compared to single antagomir treatment, especially, in L3.6plGres-SP-induced pancreatic tumor growth in vivo. These findings further indicate that the inhibition of miR-21 and miR-221 appear particularly suitable to target stem-like subpopulations and address their specific biological function to promote tumor progression in pancreatic cancer.

Abstract 926: Nitric oxide enhances tumor progression and disease aggressiveness in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is among the most malignant cancer, with 5-year survival of a mere 6%. Chronic inflammation is associated with an increased risk of PDAC. Nitric oxide (NO•) is an important mediator of immune and inflammatory responses and plays a role in tumorigenesis. The principal source of an increased and sustained level of NO• is inducible nitric oxide synthase (NOS2). In this study, we have tested the hypothesis that NO• enhances tumor progression in pancreatic cancer. We found that a higher NOS2 expression in tumors was associated with poor survival in resected PDAC patients (p=0.011). We then used a genetic strategy to investigate the role of NO• in the development and progression of PDAC by deleting NOS2 gene in a genetically engineered mouse model of pancreatic cancer (KPC mice) with pancreas specific activation of mutant KRAS and P53, which faithfully recapitulates the development and progression of human pancreatic cancer. NOS2-deficient KPC mice showed a longer survival as compared to the KPC littermates with wild type NOS2 (p&amp;lt;0.01). Tumors from KPC/NOS2-/- mice showed reduced tumor cell proliferation and increased apoptosis as indicated by a lower expression of Ki67 and a higher level of activated caspase-3, as compared with KPC mice. Furthermore, tumors from KPC/NOS2-/- mice were less vascularized as compared with KPC mice, as indicated by CD31 expression. Additionally, NOS2-deficiency led to a decreased inflammatory response by attenuation of macrophage recruitment in pancreatic tumors as indicated by a significantly lower expression of F4/80. However, in contrast FOXP3+ regulatory T-cells were increased in tumors from KPC/NOS2-/- mice. Further mechanistic analysis revealed a higher expression of ph-FOXO3 and nuclear ph-ERK in tumors from KPC mice as compared with KPC/NOS2-/- mice. Analysis of human PDAC samples showed a positive correlation between NOS2 and ph-FOXO3 expression (P=0.004). In summary, NOS2 gene expression in tumors is a candidate prognostic marker in resected pancreatic cancer patients, and NOS2/NO• signaling may contribute to the pancreatic tumor progression by inactivating FOXO3 in KPC mice. In this ongoing study, we are further investigating the mechanistic role of NO• in disease progression. Citation Format: Jian Wang, Peijun He, Matthias M. Gaida, Shouhui Yang, Aaron Schetter, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Harris G. Yfantis, Dong H. Lee, Jonathan M. Weiss, Jim Stauffer, Nader Hanna, H. Richard Alexander, S. Perwez Hussain. Nitric oxide enhances tumor progression and disease aggressiveness in 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 926. doi:10.1158/1538-7445.AM2014-926

Stem Cell Marker Nestin Is Critical for TGF-β1-Mediated Tumor Progression in Pancreatic Cancer

The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-β1/Smad4 pathway in PDAC. We examined nestin expression after TGF-β1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-β1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-β1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-β1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-β1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis.

Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.

Abstract 4277: Comparative gene expression analysis of proliferating stromal cells from pancreatic ductal adenocarcinoma, pancreatitis and normal pancreas

Abstract Introduction: Stromal cells associated with cancer cells are considered an important component of tumor microenvironment of pancreatic cancer. The intratumoral desmoplasia characteristic of pancreatic cancer is a result of growing carcinoma paracrine action on surrounding normal tissue cells. Selective targeting of tumor stroma cells can markedly increase the effectiveness of currently used chemotherapeutic agents against pancreatic cancer. Therefore, a search for new targets in stromal cells is an important part of developing new combined therapies of this disease. Our work was aimed at comparative studying of gene expression in stromal cells of normal pancreas, pancreatitis and pancreatic ductal adenocarcinoma tissues. Methods: Cultured stromal cells were obtained from samples of normal pancreatic (n=3), pancreatitis (n=3) and ductal adenocarcinoma tissues (n=4). The cell cultures obtained were preliminary characterized by immunofluorescence, RT-PCR and western blotting. A full genome gene expression analysis was performed using a modified SAGE technique. Expression of selected dysregulated genes was analyzed by quantitative PCR. Results: Morphological and immunofluorescent analyses of the obtained primary stromal cultures indicated that they could be assigned to pancreatic stellate cells (PSC). The SAGE analysis allowed to quantitatively estimate expression of 9860 genes in normal PSC, 8744 PSC genes in pancreatitis, and 9311 genes in tumor PSC. A comparison of normal and tumor PSC revealed statistically significant (P&amp;lt;0.001) differences in expression of 270 genes. Of them, 146 genes were upregulated and 124 genes downregulated in tumor PSC as compared to normal PSC. An expression analysis of selected dysregulated genes by RT-PCR detected changes in the activity of some genes involved in tumor progression of pancreatic cancer. In particular, the genes of two chemokines (CXCL12 and CX3CL1) and the JAG1 Notch ligand were upregulated in tumor PSC. A similar comparison of tumor PSC and PSC from pancreatitis tissue detected 68 differentially expressed genes (P&amp;lt;0.001), 40 of which were upregulated and 28 downregulated in tumor PSC as compared to pancreatitis PSC. Expression of 23 genes was upregulated in tumor PSC as compared with PSC both from pancreas and pancreatitis. On the whole, the expression profile of tumor PSC was markedly more similar to that of PSC from pancreatitis tissues than from normal PSC. Conclusions: The results obtained in our work revealed statistically significant changes in the expression of some PSC genes in stroma of ductal pancreatic adenocarcinoma as compared to normal PSC. The similarity of tumor and pancreatitis PSC expression profiles may suggest mutual mechanisms of forming the fibrous tissue of pancreatitis and tumor stroma of pancreatic carcinomas. 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 4277. doi:1538-7445.AM2012-4277

Targeting the Metastasis Suppressor NDRG1: A New Strategy for the Treatment of Pancreatic Cancer

AimsThe metastasis suppressor, N‐myc downstream regulated gene‐1 (NDRG1), is negatively correlated with tumor progression in pancreatic cancer. Moreover, NDRG1 is an iron‐regulated gene that is markedly up‐regulated by cellular iron‐depletion using novel anti‐tumor agents such as the chelator, Dp44mT, in pancreatic cancer cells. However, the exact molecular function(s) of NDRG1 remain to be established.ResultsUsing gene‐array analysis together with NDRG1 over‐expression and silencing, we identified molecular targets of NDRG1 in 3 pancreatic cancer cell lines. For the first time, we demonstrate that NDRG1 up‐regulates neural precursor cell expressed developmentally down‐regulated 4‐like (NEDD4L) and GLI‐similar 3 (GLIS3). Further studies examining the down‐stream effects of NEDD4L led to the discovery that NDRG1 affects the transforming growth factor β (TGF‐β) pathway, leading to up‐regulation of two key tumor suppressor proteins, namely phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and mothers against decapentaplegic homolog 4 (SMAD4) in multiple cell lines. Moreover, NDRG1 inhibited the phosphatidylinositol 3‐kinase (PI3K) and Ras oncogenic pathways.ConclusionThe identified target genes of NDRG1 and their effect on the TGF‐â signaling pathway reveal its molecular function in pancreatic cancer and a novel therapeutic avenue.

Abstract 223: ΔNp63α, a p53 family member protein, promotes tumor progression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in both men and women. Targeted therapies thus far have failed to demonstrate survival advantage in PDAC and novel therapies are needed. Mutations in Ras and p53 are frequently found in PDAC and promote cancer cell survival, invasiveness and chemoresistance. In contrast, p63, an ancestral member of p53 family, is rarely mutated in cancer. ΔNp63α was shown to be overexpressed in squamous cell tumors of the lung and head and neck and thus has been implicated in carcinogenesis. We tested expression of p63 isoforms in five PDAC cell lines and investigated its role in tumor progression. Expression of p63 was determined by immunoblotting, real-time quantitative and semi-quantitative PCR in 5 PDAC cell lines. PANC-1 cells, with low levels of p63, were transfected with ΔNp63α cDNA-expressing vector. Conversely, high endogenous p63 levels in T3M4 and BxPC3 cell lines were suppressed by specific shRNA. We studied cell proliferation in a doubling assay, motility in wound assay and invasiveness in Matrigel chambers. We found that pancreatic cancer cell lines predominantly expressed ΔN isoform of p63, and specifically ΔNp63α variant. ΔNp63α protein and transcript levels were high in T3M4, BxPC3 and Colo-357 cell lines and low in ASPC-1 and PANC-1 cell lines. Interestingly, transcript levels of TAp63, a p63 variant implicated in cellular senescence and suppression of metastases, were near the detection threshold in all cell lines. Overexpression of ΔNp63α in PANC-1 cells resulted in increased proliferation and enhanced migration of those cells but had no effect on invasiveness in serum-free conditions. Incubation of ΔNp63α-expressing PANC-1 cells in presence of epidermal growth factor increased both migration and invasiveness. By contrast, shRNA knockdown of endogenous p63 in BxPC3 cells led to decreased proliferation. Silencing p63 in T3M4 cells resulted in attenuated migration in serum-free conditions and decreased migration and invasiveness in response to epidermal growth factor. Thus, ΔNp63α is a predominantly expressed isoform of p63 in PDAC cell lines. ΔNp63α plays an important role in PDAC progression and thus its associated pathways may potentially be therapeutically targeted in this disease. 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 223. doi:10.1158/1538-7445.AM2011-223