Signaling Pathways In Pancreatic Cancer Research Articles

Crizotinib exhibits antitumor activity by targeting ALK signaling not c-MET in pancreatic cancer.

Crizotinib, a c-MET/ALK inhibitor, has exhibited antitumor efficacy in different types of cancers. However, studies regarding Crizotinib in pancreatic cancer have been limited. Thus, we investigated the effect of Crizotinib on pancreatic cancer and its mechanism of action. Crizotinib strongly suppressed the growth and proliferation of pancreatic cancer cells in a dose-dependent manner. Also, it induced apoptosis by modulating its related factors. In the study, with regard to the mechanism of action, Crizotinib did not inhibit c-MET expression on pancreatic cancer cells; instead, it specifically inhibited the activity of ALK, which was identified to be highly expressed on various pancreatic cancer cells and tissues in our study. In 42 different receptor tyrosine kinase (RTKs) array, Crizotinib also strongly inhibited the expression of activated ALK in pancreatic cancer cells, modulating its downstream mediators such as STAT3, AKT, and ERK. Furthermore, Crizotinib inhibited angiogenesis in a mouse Matrigel plug assay as well as the progression of tumor growth in a mouse xenograft model. Taken together, our investigation shows that Crizotinib inhibits the ALK signaling pathway in pancreatic cancer, resulting in cell growth/angiogenesis inhibition and apoptosis induction. We suggest that Crizotinib might be used as a novel therapeutic drug for treating pancreatic cancer.

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

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

MicroRNA-gene signaling pathways in pancreatic cancer

Pancreatic cancer is the fourth most frequent cause of cancer-related deaths and is characterized by early metastasis and pronounced resistance to chemotherapy and radiation therapy. Despite extensive esearch efforts, there is not any substantial progress regarding the identification of novel drugs against pancreatic cancer. Although the introduction of the chemotherapeutic agent gemcitabine improved clinical response, the prognosis of these patients remained extremely poor with a 5-year survival rate of 3-5%. Thus, the identification of the novel molecular pathways involved in pancreatic oncogenesis and the development of new and potent therapeutic options are highly desirable. Here, we describe how microRNAs control signaling pathways that are frequently deregulated during pancreatic oncogenesis. In addition, we provide evidence that microRNAs could be potentially used as novel pancreatic cancer therapeutics through reversal of chemotherapy and radiotherapy resistance or regulation of essential molecular pathways. Further studies should integrate the deregulated genes and microRNAs into molecular networks in order to identify the central regulators of pancreatic oncogenesis. Targeting these central regulators could lead to the development of novel targeted therapeutic approaches for pancreatic cancer patients.

CCN1 promotes tumorigenicity through Rac1/Akt/NF-κB signaling pathway in pancreatic cancer

Aberrant CCN1 expression has been reported to play an important role in the tumor development. However, the pattern and the role of CCN1 in pancreatic cancer remain largely unknown. Therefore, we further deciphered the role CCN1 played in pancreatic cancer. We first evaluated the CCN1 expression in human pancreatic cancer tissues and pancreatic cancer cells. Then we forced expression and silenced CCN1 expression in pancreatic cancer cell lines MIA PaCa2 and PANC-1 respectively, using lentivirus vectors. We characterized the stable cells in vitro and in vivo using a nude mouse xenograft model. In this study, we found that CCN1 expression was significantly higher in cancer specimens which positively correlated with the expression level of phosphorylated Akt and p65. and poorer outcome. Moreover, our results demonstrated that CCN1 positively regulated pancreatic cell growth in vitro and in vivo and helped cancer cells resist to tumor necrosis factor alpha-induced apoptosis. Furthermore, we disclosed that activation of CCN1/ras-related c3 botulinum toxin substrate 1 (Rac1)/V-akt murine thymoma viral oncogene homolog (Akt)/nuclear factor-kappa B pathway inhibited apoptosis in pancreatic cancer cells. CCN1 is upregulated in pancreatic cancer and promotes the survival of pancreatic cancer cells. Taken together, these results indicate that CCN1 may be a potential target for pancreatic cancer therapy.

Abstract 1269: Activation of diverse signaling pathways in pancreatic cancer revealed by phosphoproteomics

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. A number of genes that have somatic mutations including CDKN2A, SMAD4, TP53 and KRAS have been linked to pancreatic cancer. Recently, a comprehensive genomic analysis was performed in a panel of 14 primary pancreatic cancer cell lines and 10 xenografts, in which ∼1,500 somatic alterations were identified and a core set of 12 activated signaling pathways were defined. We decided to use a phosphoproteomics approach to identify tyrosine kinase signaling pathways that could serve as attractive therapeutic targets. Based on immunoblotting with anti-phosphotyrosine antibodies, we observed a heterogeneous pattern of tyrosine phosphorylation in the 14 primary pancreatic cancer cell lines whose genomes have been sequenced previously, which indicated that different tyrosine kinases were likely activated in different cells. To identify these kinases, phosphotyrosine-containing peptides were enriched by immunoprecipitation by anti-phosphotyrosine antibodies and analyzed on a high resolution Fourier transform mass spectrometer coupled to nanoflow reversed phase liquid chromatography. W employed the SILAC methodology to quantitate tyrosine phosphorylated peptides across the panel of cell lines. Our preliminary data indicates that the approach used in this study is suitable for global profiling of tyrosine kinase pathways that are abnormally activated in cancers. The differential alteration of tyrosine kinases was confirmed from the accurate quantitative tyrosine phosphoproteomic analysis. For example, HPNE, a normal pancreatic cell line showed the lowest level of phosphotyrosine levels, while each pancreatic cancer cell line could be differentiated based on its own set of activated tyrosine kinases. Interestingly, some of tyrosine kinases were activated in a small number of cell lines (e.g. AXL, SYK, DDR2, RON and RET), while other tyrosine kinases were found to be activated more broadly (e.g. YES1, TYK2, EPHA2 and EPHB4). Overall, we conclude that these aberrantly activated tyrosine kinases could be targeted to develop newer therapeutic regimens. 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 1269. doi:1538-7445.AM2012-1269

The dual PI3K/mTOR inhibitor NVP-BGT226 induces cell cycle arrest and regulates Survivin gene expression in human pancreatic cancer cell lines

The phosphatidylinositol-3-kinase (PI3K) pathway is one of the most commonly activated signaling pathways in pancreatic cancer and is a target of interest for new therapeutic approaches. NVP-BGT226 is a novel dual class PI3K/mammalian target of rapamycin (mTOR) inhibitor that has entered Phase I/II clinical trials. We analyzed the effect of NVP-BGT226 (10-100 nM) on the pancreatic cell lines Panc-1, BxPc-3, AsPC-1 and MiaPaCa-2 in regard to cell viability, induction of apoptosis, cell cycle, and expression of the antiapoptotic genes Survivin, MCL-1, BCL-2 and BCL-xL. Cell viability decreased within 24-72 h after exposure to about 50% compared to untreated control cells in a concentration- but not time-dependent manner. Cell cycle analysis revealed that NVP-BGT226 induced predominantly G0/G1 cell cycle arrest. Additionally, real-time RT-PCR and Western blot analysis showed a remarkable decrease of Survivin expression. Originally designed as a dual inhibitor, there was only a significant inhibition of p-mTOR. In summary, the dual PI3K/mTOR inhibitor NVP-BGT226 induces G0/G1 arrest and acts, at least, partially via downregulation of Survivin.

Aberrant signaling pathways in pancreatic cancer: A two compartment view

Pancreatic cancer is a devastating disease with historically limited success in treatment and a poor prognosis. Pancreatic cancer appears to have a progressive pathway of development, initiating from well-described pancreatic intraepithelial neoplasia lesions and concluding with invasive carcinoma. These early lesions have been shown to harbor-specific alterations in signaling pathways that remain throughout this tumorigenesis process. Meanwhile, new alterations occur during this process of disease progression to have a cumulative effect. This series of events not only impacts the epithelial cells comprising the tumor, but they may also affect the surrounding stromal cells. The result is the formation of complex signaling networks of communication between the tumor epithelial cell and the stromal cell compartments to promote a permissive and cooperative environment. This article highlights some of the most common pathway aberrations involved with this disease, and how these may subsequently affect one or both cellular compartments. Consequently, furthering our understanding of these pathways in terms of their function on the tumoral epithelial and stromal compartments may prove to be crucial to the development of targeted and more successful therapies in the future.

The effect of dual PI3K/mTOR inhibitor NVP-BGT226 on cell cycle and survivin and STAT3 gene expression in human pancreatic cancer cell lines.

e14523 Background: The phosphatidylinositol-3-kinase (PI3K) pathway is one of the most commonly activated signaling pathways in pancreatic cancer. The inhibition of this transducing pathway may open new options for the treatment of pancreatic cancer. NVP-BGT226 is a novel dual class PI3K/mammalian target of rapamycin (mTOR) inhibitor that has entered Phase I/II clinical trials. Methods: We have tested the effects of NVP-BGT226 on cell viability (M'TT assay), apoptosis (AnnexinV/PI-FACS and PARP1 cleavage), cell cycle (FACS PI/RNAse-staining) and expression of Survivin, STAT3 and BCL-xL with Real-time RT-PCR and Western blot in the human pancreatic cancer cell lines Panc-1, BxPc-3, AsPC-1 and MiaPaCa-2. Results: The cell viability decreased within 24-72 hours exposure to about 50% compared to untreated control cells in a concentration and but not time dependent manner. Apoptosis was not detected, but cell cycle analysis revealed that NVP-BGT226 induced predominantly a G0/G1-arrest to about 90-95%. Analysis with Real-time RT-PCR and Western blot showed a remarkable reduction of Survivin and STAT3 expression but an increase of the anti-apoptotic BCL-xL gene expression. Conclusions: The PI3K/mTOR inhibitor NVP-BGT226 induces G0/G1-arrest and and acts via downregulation of Survivin and STAT3.

Abstract 1299: A phase I dose-escalation study of the safety, PK, and PD of vitamin E Δ-tocotrienol administered to subjects with resectable pancreatic exocrine neoplasia

Abstract Background: Δ-Tocotrienol, a natural Vitamin-E analog, which significantly inhibits the growth and survival of pancreatic neoplastic cells by inhibiting signal transduction pathways which are altered in pancreatic neoplasia such as those driven by mutant K-ras. It is hypothesized that this micronutrient may have a role in the prevention and treatment of subjects with pancreatic exocrine neoplasia for which no effective chemoprevention treatment strategy currently exists. We have initiated a phase 1 dose escalation trial of Δ-tocotrienol in order to determine the recommended phase 2 dose and to characterize the safety, tolerability and biologically effective dose. Methods: Eligible patients must have a resectable tumor or cyst arising from the exocrine pancreas for which they are about to undergo resection. This pre-surgical trial design was chosen to allow for acquisition of sufficient tissue for detailed analysis of Δ-tocotrienol's biological effect on signaling within the patient's tumor. Between October 2009 and August 2010, 12 subjects were accrued to the first 4 of 6 planned dose escalation levels. Three patients each were treated at a total of 200, 400, 600 and 800 mg/day divided into 2 doses. Each dose is taken with meals. Treatment was daily for 13-15 days. Blood samples were collected for pharmacokinetic (PK) analysis pre-dose on day 1 and then at 30 min., 1,2,4,6,8 and 12 hours after dosing. Additional samples were obtained pre-dose on day 8 and on the day of surgery. Toxicity assessments were on days 1, 8, the day of surgery and 3-6 weeks after surgery. Study endpoints are to determine 1) safety and tolerability, 2) plasma PK, 3) to evaluate pharmacodynamic markers of Δ-tocotrienol activity such as apoptisis, Ki-67 and p27 expression and 4) the distribution of Δ-tocotrienol in normal and neoplastic pancreatic tissue. Results: Thus far no drug related toxicities have been observed. No elevation in the post-operative complication rate has been observed. To date, the first 12 patients enrolled were evaluated for PK parameters. Interpatient variability within and amongst cohorts was observed. This may be due to varying light breakfast intake amongst subjects leading to differing absorption. The data has been modeled by non-compartmental analysis and the dose independent PK parameters are as follows: Terminal half-life – 3.4 (+/-1.7)hrs; Tmax – 4.9 (+/-2.0) hrs; Volume of Distribution – 229.9 (+/-169.6) L; Clearance – 39.1 (+/-22.5) L/hr. Conclusion: Δ-tocotrienol is well tolerated at doses up to 800 mg daily. Further dose escalation to 3200 mg/day is planned. PD biomarker analysis will be presented. This study demonstrates the feasibility of employing a pre-surgical trial design for determination of biologically effective doses to modulate signaling pathways in pancreatic cancer. 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 1299. doi:10.1158/1538-7445.AM2011-1299

Identification of RegIV as a novel GLI1 target gene in human pancreatic cancer.

Background and AimsGLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. RegIV is associated with regeneration, and cell growth, survival, adhesion and resistance to apoptosis. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1.MethodsGLI1 and RegIV expression were evaluated in tumor tissue and adjacent normal tissues of pancreatic cancer patients and 5 pancreatic cancer cell lines by qRT-PCR, Western blot, and immunohistochemistry (IHC), and the correlation between them. The GLI1-shRNA lentiviral vector was constructed and transfected into PANC-1, and lentiviral vector containing the GLI1 expression sequence was constructed and transfected into BxPC-3. GLI1 and RegIV expression were evaluated by qRT-PCR and Western blot. Finally we demonstrated RegIV to be the target of GLI1 by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA).ResultsThe results of IHC and qRT-PCR showed that RegIV and GLI1 expression was higher in pancreatic cancer tissues versus adjacent normal tissues (p<0.001). RegIV expression correlated with GLI1 expression in these tissues (R = 0.795, p<0.0001). These results were verified for protein (R = 0.939, p = 0.018) and mRNA expression (R = 0.959, p = 0.011) in 5 pancreatic cancer cell lines. RegIV mRNA and protein expression was decreased (94.7±0.3%, 84.1±0.5%; respectively) when GLI1 was knocked down (82.1±3.2%, 76.7±2.2%; respectively) by the RNAi technique. GLI1 overexpression in mRNA and protein level (924.5±5.3%, 362.1±3.5%; respectively) induced RegIV overexpression (729.1±4.3%, 339.0±3.7%; respectively). Moreover, CHIP and EMSA assays showed GLI1 protein bound to RegIV promotor regions (GATCATCCA) in pancreatic cancer cells.ConclusionGLI1 promotes RegIV transcription by binding to the RegIV gene promoter in pancreatic cancer.

Signaling Pathways in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by a plethora of molecular alterations that include major and minor driving mutations, the presence of intense desmoplasia exhibiting numerous proliferating pancreatic stellate cells (PSC) and cancer-associated fibroblasts that produce fibronectin and collagens, and foci of inflammatory cells that produce mitogenic cytokines. This review will focus on signaling by tyrosine kinase receptors, and the role of transforming growth factor beta in this malignancy is described briefly. Potential for therapeutic interventions will be discussed in relation to specific pathways.

Abstract A58: Activation of diverse signaling pathways in pancreatic cancer revealed by phosphoproteomics

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Based on genomic studies, a number of genes that have somatic mutations including CDKN2A, SMAD4, TP53 and KRAS have been identified in pancreatic cancers. Recently, a comprehensive genomic analysis was performed in a panel of 14 primary pancreatic cancer cell lines and 10 xenografts, in which ∼1,500 somatic alterations were identified and a core set of 12 signaling pathways were defined. In this study, we characterized the 14 primary pancreatic cancer cell lines whose genomes have been sequenced. From Western blots using anti-phosphotyrosine antibodies, we observed a heterogeneous pattern of tyrosine phosphorylation in these cell lines, indicating that different tyrosine kinases were likely activated in different cells. Phosphotyrosine-containing peptides were enriched by immunoprecipitation by anti-phosphotyrosine antibodies. The enriched phosphopeptides were analyzed on a high resolution Fourier transform mass spectrometer coupled to nanoflow reverse phase liquid chromatography. Our preliminary data indicates that the approach used in this study is suitable for the global profiling of tyrosine kinase pathways that are abnormally activated in pancreatic cancers. For example, lymphocyte-specific protein tyrosine kinase and spleen tyrosine kinase were observed to be activated in one of pancreatic cancer cell lines (Panc 8.13), while insulin receptor was activated in different cell lines (Panc 198). Citation Information: Cancer Prev Res 2010;3(12 Suppl):A58.

Overexpression of Smoothened Activates the Sonic Hedgehog Signaling Pathway in Pancreatic Cancer–Associated Fibroblasts

Accumulating evidence suggests that cancer-associated stromal fibroblasts (CAF) contribute to tumor growth by actively communicating with cancer cells. Our aim is to identify signaling pathways involved in tumor-stromal cell interactions in human pancreatic cancer. We established primary fibroblast cultures from human pancreatic adenocarcinomas and nonneoplastic pancreas tissues. To identify differentially expressed genes in CAFs, we did gene expression profiling of human pancreatic CAFs and nonneoplastic pancreatic fibroblasts. The Hedgehog receptor Smoothened (SMO) was upregulated in CAFs relative to control fibroblasts. CAFs expressing SMO could transduce the Sonic hedgehog signal to activate Gli1 expression, and small interfering RNA knockdown of SMO blocked the induction of Gli1 in these cells. Stromal fibroblasts of human primary pancreatic adenocarcinomas overexpressed Smo compared with normal pancreatic fibroblasts. These findings implicate overexpression of Smo as a mechanism for the activation of Hedgehog signaling in human pancreatic CAFs and suggest that stromal cells may be a therapeutic target for Smo antagonists in pancreatic cancer.

Expression and regulation of hedgehog signaling pathway in pancreatic cancer

Pancreatic cancer is an aggressive malignancy with a poor prognosis. The overall 5-year survival rate of pancreatic cancer is less than 5%, and has not improved significantly for years. Further understanding of the molecular carcinogenesis of pancreatic cancer is critical for designing effective ways to treat this type of malignancy. In this study, we examine expression of hedgehog signaling molecules in 54 surgically removed pancreatic cancer specimens as well as seven available pancreatic cancer cell lines. We find that expression of Ptch is associated with tumor size, tumor differentiation, lymph node metastasis, and clinical stages, whereas expression of Smo is associated with tumor differentiation and lymph node metastasis. Our studies from pancreatic cancer cell lines indicate that targeted inhibition of hedgehog signaling by Smo signaling inhibitor KAAD-cyclopamine causes hedgehog target gene expression (Gli1) suppression, induces P21 expression and G1 cell population, and reduced expression of Cyclin D1 and IGF2. These results indicate that hedgehog signaling activation is a very common event in pancreatic cancer and that targeted inhibition of hedgehog signaling may be effective in treatment of pancreatic cancer.

Gli1 maintains cell survival by up-regulating IGFBP6 and Bcl-2 through promoter regions in parallel manner in pancreatic cancer cells

Background:Aberrant activation of Hedgehog (Hh) signaling pathway has been reported to be related to malignant biological behavior of pancreatic cancer but its mechanism is unclear yet. Since IGF pathway and Bcl-2 family are involved in proliferation and apoptosis of pancreatic cancer cells, we hypothesize that they are possibly associated with Hh pathway.Materials and Methods:We studied the relationship of Shh-Gli1 signaling pathway with proliferation and apoptosis of pancreatic cancer cells and the regulation of transcription factor Gli1 to insulin-like growth factor binding protein 6 (IGFBP6) and Bcl-2 genes at the level of transcription.Results:Sonic hedgehog (Shh), Smoothened (Smo), patched and Gli1 were expressed in pancreatic cancer cells. Cyclopamine inhibited cell proliferation at low concentration and induced apoptosis at high concentration. Effect of RNA interference (RNAi) for Gli1 to cell survival is mainly due to proliferation inhibition though involved in apoptosis. The transcription factor Gli1 bound to promoter regions of Bcl-2 and IGFBP6 genes and the levels of IGFBP6, proliferating cell nuclear antigen (PCNA) and Bcl-2 messenger RNA (mRNA) were decreased as well as Gli1 mRNA significantly by cyclopamine or RNAi in cultured pancreatic cancer cells (p < 0.01). Finally PCNA, IGFBP6 and Bcl-2 mRNA were upregulated as well as Shh or Gli1 in pancreatic cancer tissues (p < 0.01).Conclusions:Our study reveals that Gli1 maintained cell survival by binding the promoter regions and facilitating transcription of IGFBP6 and Bcl-2 genes in a parallel manner in pancreatic cancer cells and suggests it may be one of the mechanisms of Shh-Gli1 signaling pathway in pancreatic cancer.

Targeting core signaling pathways in pancreatic cancer

Targeting core signaling pathways in pancreatic cancer

Proteasome Inhibition Activates Epidermal Growth Factor Receptor (EGFR) and EGFR-Independent Mitogenic Kinase Signaling Pathways in Pancreatic Cancer Cells

In the current study, we investigate the activation of antiapoptotic signaling pathways in response to proteasome inhibitor treatment in pancreatic cancer and evaluate the use of concomitant inhibition of these pathways to augment proteasome inhibitor treatment responses. Pancreatic cancer cell lines and mouse flank xenografts were treated with proteasome inhibitor alone or in combination with chemotherapeutic compounds (gemcitabine, erlotinib, and bevacizumab), induction of apoptosis and effects on tumor growth were assessed. The effect of bortezomib (a first-generation proteasome inhibitor) and NPI-0052 (a second-generation proteasome inhibitor) treatment on key pancreatic mitogenic and antiapoptotic pathways [epidermal growth factor receptor, extracellular signal-regulated kinase, and phosphoinositide-3-kinase (PI3K)/AKT] was determined and the ability of inhibitors of these pathways to enhance the effects of proteasome inhibition was assessed in vitro and in vivo. Our data showed that proteasome inhibitor treatment activates antiapoptotic and mitogenic signaling pathways (epidermal growth factor receptor, extracellular signal-regulated kinase, c-Jun-NH2-kinase, and PI3K/AKT) in pancreatic cancer. Additionally, we found that activation of these pathways impairs tumor response to proteasome inhibitor treatment and inhibition of the c-Jun-NH2-kinase and PI3K/AKT pathways increases the antitumor effects of proteasome inhibitor treatment. These preclinical studies suggest that targeting proteasome inhibitor-induced antiapoptotic signaling pathways in combination with proteasome inhibition may augment treatment response in highly resistant solid organ malignancies. Further evaluation of these novel treatment combinations in clinical trials is warranted.

BCL‐2 functions as an activator of the AKT signaling pathway in pancreatic cancer

BCL-2 is the prototypic anti-apoptotic protein involved in the regulation of apoptosis. Overexpression of BCL-2 is common in pancreatic cancer and confers resistance to the apoptotic effect of chemo- and radiotherapy. Although these cellular effects of BCL-2 are traditionally related to pathways involving the mitochondrial membrane, we sought to investigate whether BCL-2 is involved in other signaling pathways regulating cell survival and focused on AKT. We examined the effect of overexpression of BCL-2 in the MIA-PaCa-2 human pancreatic cancer cell line on the function and subcellular location of AKT. We observed that the stable subclones of MIA-PaCa-2 overexpressing BCL-2 demonstrated increased activity of AKT as well as IKK (a downstream target of AKT), increasing the transcriptional activity of NF-kappaB. Using immunoprecipitation techniques, we observed co-immunoprecipitation of AKT and BCL-2. Immunocytochemistry demonstrated co-localization of BCL-2 and AKT, which was abrogated by treatment with HA14-1, a small molecule inhibitor of BH-3-mediated protein interaction by BCL-2. Furthermore, treatment with HA14-1 decreased phosphorylation of AKT and increased sensitivity to the apoptotic effect of the chemotherapeutic agent, paclitaxel. These results demonstrate an additional mechanism of regulation of cell survival mediated by BCL-2, namely through AKT activation, in the MIA-PaCa-2 pancreatic cancer cell line. Therefore, directed inhibition of BCL-2 may alter diverse pathways controlling cell survival and overcome the apoptotic resistance that is the hallmark of pancreatic cancer.

The role of Notch3 signaling pathway in pancreatic cancer

21049 Background: Given the extremely poor prognosis of patients diagnosed with pancreatic cancer, identifying new targets for therapeutic intervention is crucial in improving clinical outcome. Existing data support a role for Notch3 pathway in human cancer, pancreas cancer in specific and in normal pancreas development. Method: We developed a pancreatic tissue array. An antibody targeting the extracellular domain of Notch3 was used to evaluate expression levels, grading from 0 to 4+ to determine the prevalence of Notch3 in pancreatic cancer. Notch3 expression was used to correlate with clinical data (IRB approved). Using small interfering RNA (siRNA) we tested the downstream effects of Notch3 in pancreatic cancer cell line BxP3 in vitro. Furthermore, to study pharmacologic inhibition of Notch3 processing and activation we exposed Panc-1, HPAF II and BxP3 pancreas cancer cell lines to γ-secretase inhibitors (previously demonstrated to block upstream of Notch3 thereby inhibiting Notch3). Results: Strong staining (+2 or greater) for Notch3 was seen in 50 of 72 (69%) resected specimens. No correlation with survival or tumor grade was seen. Using siRNA, we demonstrated that inhibiting Notch3 downregulated the antiapoptotic protein Bcl-xL but not Bcl-2. When γ-secretase inhibitors (GSI and L-685,458) were used, the previously mentioned pancreatic cell lines demonstrated reduction in proliferation rates. Conclusion: Notch3 overexpression is common in pancreas cancer. While Notch 3 expression does not clearly correlate with survival, our in vitro data suggest that Notch3 affects apoptotic pathways and may represent a potential target for intervention in patients with pancreatic cancer. No significant financial relationships to disclose.

Alterations of cell signaling pathways in pancreatic cancer.

Pancreatic ductal adenocarcinomas continue to have the worst prognosis of any adult malignancy with a five-year survival rate of less than 4%. One approach to improve patient survival from pancreatic cancer is to identify new biological targets that contribute to the aggressive pathogenecity of this disease and to develop reagents that will interfere with the function of these targets. Apart from the identification of the genetic profile of pancreatic cancer, a number of studies have focused on aberrant cell signaling pathways and their role in pancreatic cancer biology and response to therapy. This review, although not comprehensive, will discuss the salient features of several of these pathways. These include the roles of TGF beta signaling in both tumor suppression and tumor promotion and the effects of deregulation of phosphotyrosine kinase receptor signaling pathways in pancreatic cancer.