Therapeutic Strategy For Pancreatic Cancer Research Articles

Stathmin, Interacting with Nf-κB, Promotes Tumor Growth and Predicts Poor Prognosis of Pancreatic Cancer

Stathmin (STMN) has been known as a p53-regulated protein and has been shown to play an oncogenic role in a range of human malignancies. Paradoxically, most recent studies demonstrated that stathmin has a dual function as both an oncogene and a metastasis suppressor. Stathmin is a member of microtubule dynamic destabilizing proteins and stathmin-regulated microtubule disruption could lead to a variety of cell dysfunctions such as enhanced chronic hypoxia in pancreatic cancer. In this study, we identified that stathmin promotes proliferation of pancreatic cancer cells by an underlying nuclear factor kappa B (Nf-κB) interacting mechanism. In human specimens, stathmin was significantly overexpressed in pancreatic cancer tissues and high expression of stathmin was correlated with vascular emboli (p=0.028), tumor size (p=0.019), and overall survival (p=0.031). Functional assays showed that knockdown of stathmin significantly reduced pancreatic cancer cell viability, colony formation, and arrested the cell cycle at the G2/M phase. Furthermore, silence of stathmin could reduce pancreatic tumor growth in nude mice. For the mechanism, Western blot analyses demonstrated that Nf-κB (p65) was significantly down-regulated when stathmin was silenced. In addition, co-immunoprecipitation (CoIP) assay suggested that stathmin was able to interact with Nf-κB (p65). Our findings indicate that stathmin might play its oncogenic role by an interaction with Nf-κB pathway, which may reveal a novel mechanism to uncover the role of microtubule-destabilizing stathmin in pancreatic cancer environment as well as provide a potential therapeutic strategy for pancreatic cancer.

Suppression of Tregs by anti‐glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon‐α gene therapy for pancreatic cancer

We have reported that interferon (IFN)-α can attack cancer cells by multiple antitumor mechanisms including the induction of direct cancer cell death and the enhancement of an immune response in several pancreatic cancer models. However, an immunotolerant microenvironment in the tumors is often responsible for the failure of the cancer immunotherapy. Here we examined whether the suppression of regulatory T cells (Tregs) within tumors can enhance an antitumor immunity induced by an intratumoral IFN-α gene transfer. First we showed that an intraperitoneal administration of an agonistic anti-glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model. The anti-GITR mAb was then combined with the intratumoral injection of the IFN-α-adenovirus vector. The treatment with the antibody synergistically augmented the antitumor effect of IFN-α gene therapy not only in the vector-injected tumors but also in the vector-uninjected tumors. Immunostaining showed that the anti-GITR mAb decreased Foxp3+ cells infiltrating in the tumors, while the intratumoral IFN-α gene transfer increased CD4+ and CD8+ T cells in the tumors. Therefore, the combination therapy strongly inclined the immune balance of the tumor microenvironment in an antitumor direction, leading to a marked systemic antitumor effect. The CCR5 expression on Tregs was downregulated in the antibody-treated mice, which may explain the decrease of tumor-infiltrating Tregs. The combination of Treg-suppression by GITR mAb and the tumor immunity induction by IFN-α gene therapy could be a promising therapeutic strategy for pancreatic cancer.

Identification of Critical Biomarkers Responsive to Anti-Autophagy Therapies for Pancreatic Ductal Adenocarcinoma through a Performance Analysis of miRNA Platforms

Background: Autophagy is a fundamental catabolic pathway that involves the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. The role of autophagy in cancer is complex, exhibiting tumor suppressive or tumor proliferative effects depending on the cancer type. Pancreatic ductal adenocarcinoma has constitutively activated autophagy that results in the selective proliferation of its tumor cells. Understanding the mechanisms by which the inhibition of autophagy occurs could shape our current therapeutic strategies for pancreatic cancer. Recent research has implicated microRNAs as important regulators of autophagy, but how miRNAs influence this inhibition is largely unexplored. Results: It is known that there exists inter-platform variability across miRNA platforms and therefore in order to identify bona fide miRNAs that are involved with the inhibition of autophagy we carried out a performance analysis of miRNA arrays from three vendors. In this paper we report our findings from this cross-platform analysis of miRNA arrays and the identity of several critical biomarkers of response to anti-autophagy therapies in the immortalized human pancreatic ductal adenocarcinoma cells 8988T as compared to normal human pancreatic ductal epithelial cells. Our cross-platform analysis of three different miRNA arrays revealed that RT-PCR-based technologies provide higher inter-platform reproducibility than hybridization-based technologies, a greater sensitivity indicated by larger dynamic ranges and reduced processing time. We also discovered two miRNAs, miRNA 720 and miRNA 29b to play a role in the inhibition of autophagy in 8988T cells and found miRNA 30a to be involved with the inhibition of autophagy in normal human pancreatic ductal epithelial cells. Conclusion: This cross-platform analysis of miRNA arrays contributed significant information about miRNA expression systems and led to the first report of miRNAs implicated in the inhibition of autophagy in pancreatic ductal adenocarcinoma. Our findings should provide new insights for developing therapeutic solutions for this extremely intractable disease.

Subtype-Specific MEK-PI3 Kinase Feedback as a Therapeutic Target in Pancreatic Adenocarcinoma

Mutations in the KRAS oncogene are dominant features in pancreatic ductal adenocarcinoma (PDA). Because KRAS itself is considered "undruggable," targeting pathways downstream of KRAS are being explored as a rational therapeutic strategy. We investigated the consequences of MAP-ERK kinase (MEK) inhibition in a large PDA cell line panel. Inhibition of MEK activated phosphoinositide 3-kinase in an EGF receptor (EGFR)-dependent fashion and combinations of MEK and EGFR inhibitors synergistically induced apoptosis. This combinatorial effect was observed in the epithelial but not mesenchymal subtype of PDA. RNA expression analysis revealed predictors of susceptibility to the combination, including E-cadherin, HER3, and the miR200-family of microRNAs, whereas expression of the transcription factor ZEB1 was associated with resistance to the drug combination. Knockdown of HER3 in epithelial-type and ZEB1 in mesenchymal-type PDA cell lines resulted in sensitization to the combination of MEK and EGFR inhibitors. Thus, our findings suggest a new, subtype-specific, and personalized therapeutic strategy for pancreatic cancer.

Chronic pancreatitis: A path to pancreatic cancer

Chronic pancreatitis predisposes to pancreatic cancer development and both diseases share a common etiology. A central role has been proposed for the digestive enzyme-secreting acinar cell that can undergo ductal metaplasia in the inflammatory environment of pancreatitis. This metaplastic change is now a recognised precursor of pancreatic cancer. Inflammatory molecules also foster tumour growth through autocrine and paracrine effects in the epithelium and the stroma.These insights have raised new opportunities such as the manipulation of inflammation as a preventive and/or therapeutic strategy for pancreatic cancer. Finally, we address the need for an in-depth study of the pancreatic acinar cells.

Cavin-1 is essential for the tumor-promoting effect of caveolin-1 and enhances its prognostic potency in pancreatic cancer

Caveolin-1 exhibits a stage-dependent, functional fluctuation during pancreatic cancer development, but the underlying mechanisms remain unclear. Here, we report that cavin-1, a structural protein of caveolae, modulates the oncogenic function of caveolin-1 and cooperates with caveolin-1 to enhance pancreatic cancer aggressiveness. Cavin-1 expression is associated with caveolin-1 in pancreatic cancer tissue samples and cell lines, and predicts the metastatic potential of pancreatic cancer. Interactome analyses further revealed the physical interaction of cavin-1 and caveolin-1 and their colocalization in pancreatic cancer cells. Cavin-1 stabilizes caveolin-1 expression or activity by inhibiting its internalization and subsequent lysosomal degradation. More in-depth functional experiments showed that caveolin-1-enhanced aggressiveness of pancreatic cancer cells is dependent on the presence of cavin-1. In contrast, cavin-1 depletion inhibited the invasion and metastasis of pancreatic cancer cells, which could not be restored by caveolin-1-rescue construct. Tissue microarray analyses in two independent clinic cohorts also supported the augment of cavin-1 on the prognostic potency of caveolin-1, and showed that combination of cavin-1 with caveolin-1 predicted worse survival in pancreatic cancer patients. Of note, the phenotypes because of cavin-1 could not be achieved by other cavins such as cavin-2, and the tumor-promoting role of cavin-1 in pancreatic cancer was found to be largely dependent on caveolin-1 expression, which highlights the critical role of cavin-1/caveoin-1 in pancreatic cancer progression, and suggests that the interruption of cavin-1/caveolin-1 interaction is a promising therapeutic strategy for pancreatic cancer.

Abstract 3836: A 3D high throughput RNA1 screen identifies the TLR4 kinase which alters spheroid architecture: a promising therapeutic strategy for pancreatic cancer.

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDA) ranks fourth among cancer-related deaths in the United States. The poor prognosis of PDA is due to resistance to current therapies. Contributing to this resistance is the tumor architecture comprising dense desmoplastic stroma, extracellular matrix (ECM), tumor cells and tumor hypoxia. We hypothesized that destabilizing the compact tumor architecture by weakening the interactions between tumor cells alone or tumor cells along with the ECM may result in more effective therapies for PDA. Methods and Results: Using the pancreatic cell line Panc1, we developed a three dimensional (3D) spheroid model system, that exhibits oxygen gradients and hypoxia that mimics the tumor microenvironment. We used the pancreatic cancer cell line PANC1-HRE that stably expresses HRE-luciferase and detects HIF-1α activation, and a platform comprising a non-matrix nano-structured scaffold, to generate compact spheroids with hypoxic inner cores and HIF-1α activation. A high throughput siRNA screen using the kinome siRNA library and our 3D spheroid modal system identified kinases whose silencing reduced both the level of hypoxia and activity of HIF-1α. The positive control, siRNA against HIF-1α, reduced activity of HIF-1α with no effect on the levels of hypoxia within the spheroids. We identified the kinases Toll-like receptor 4 (TLR4) and its downstream target spleen tyrosine kinase (SYK) as our lead candidates following target validation. Image analysis of spheroids transfected with siRNA against both TLR4 and SYK resulted in the loss of compactness of the spheroids. In addition, these spheroids also demonstrated a significant reduction in the levels of hypoxia determined by the hypoxia probe Lox-1. These findings suggest that the alteration of the spheroid architecture resulted in the reduction in hypoxia. Previous studies have shown that SYK is a direct target of TLR4. Therefore to determine the mechanism through which TLR4 dependent signaling regulates the integrity of the spheroidal architecture we are generating stable cell lines in Panc1 of shRNA targeting TLR4 under an inducible system. We will use our inducible shRNA targeting TLR4 and the two 3D model systems, the hanging drop system (analogous to a primary tumor) and the nanoculture plate system (analogous to the progression of a metastatic loci) to further confirm our findings. Conclusion: TLR4 mediated signaling regulates the integrity of spheroid architecture. Inhibiting this pathway destabilized the tumor architecture altering the hypoxic state of the spheroid. Our long-term goals are to use known pharmacological inhibitors of TLR4 to study the mechanisms by which this kinase regulates tumor architecture, determine the pre-clinical significance of targeting TLR4 utilizing autochtonous mouse models for PDA and develop TLR4-targeted therapy for PDA. Citation Format: Geoffrey A. Bartholomeusz, Alex Campos, Geoffrey Grandjean, Garth Powis. A 3D high throughput RNA1 screen identifies the TLR4 kinase which alters spheroid architecture: a promising therapeutic strategy for pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3836. doi:10.1158/1538-7445.AM2013-3836

Abstract 2320: Oncogenic K-ras and loss of Smad4 mediate invasion by activating a NF-kappaB/EGFR axis that induces expression of MMP9/uPA in human pancreas progenitor cells.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely fatal and highly metastatic in nature. Several pathways and mutations contribute to the development and pathogenesis of PDAC. Activating K-ras mutations and inactivating mutations of Smad4 are two common genetic alterations that occur in the development and progression of PDAC. To further study the role of these two mutations in the pathogenesis of PDAC, immortalized human pancreas nestin postive cells (HPNE) were genetically modified by expressing oncogenic K-ras (GD12) and /or by shRNA knock down of Smad4. Present studies show that these two mutations caused increase in expression of EGFR and erbB2 growth factor receptors and increased expression and nuclear translocation of NF-κB. The increase in EGFR expression and nuclear translocation of NF-κB was associated with development of an invasive phenotype as measured by matrigel assays. Our data also demonstrated that MMP9 and uPA were substantially up regulated in HPNE cells that express K-ras or along with loss of Smad4 activities. MMP-9 and uPA are two extracellular matrix (ECM)-degrading proteinases known to play important roles in driving invasion and metastasis. Zymography studies confirm the expression and enzymatic activities of uPA and MMP-9 in the progenitor cells. The mechanism underlying this uPA/MMP9 induction is linked with NF-κB activation. Immuno-fluorescence, Western blot and chromatin immuno-precipitation (ChIP) analysis confirm the nuclear translocation of NF-κB RelA (p65) sub-unit, by induction of K-ras. Following signaling cascade, EGFR activated by the K-ras mediated NF-κB nuclear localization and further activation induced by loss of Smad4. Our results also suggest that the EGFR pathway as a critical mediator connecting Ras to NF-κB in proteolytic enzyme secretion as well as cancer invasion. In conclusion, the present study reveals a new insight in pancreatic progenitor cell invasion which includes signal transduction of K-ras activity and Smad4 inactivation in regulation of NF-κB/EGFR axis to induce expression of uPA and MMP9. Therefore, induction of K-ras and loss of Smad4 could play important roles in PDAC invasion through NF-κB/EGFR activation and subsequent induced expression of MMP9 and uPA, and might be suitable molecular candidates for the development of new therapeutic strategies for pancreatic cancer. Citation Format: Alakesh Bera, Shujie Zhao, Lin Cao, James W. Freeman. Oncogenic K-ras and loss of Smad4 mediate invasion by activating a NF-kappaB/EGFR axis that induces expression of MMP9/uPA in human pancreas progenitor 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 2320. doi:10.1158/1538-7445.AM2013-2320

MicroRNA-34b Inhibits Pancreatic Cancer Metastasis Through Repressing Smad3

Pancreatic cancer is characterized by extremely poor prognosis because of early recurrence and metastasis, and increasing evidence supports the critical role of microRNA in cancer progression. Here we identified that microRNA-34b functioned as a tumor-suppressing microRNA by targeting oncogenic Smad3 in pancreatic cancer. As a hypovascular tumor with a potential endoplasmic reticulum stress microenvironment, miR-34b was silenced after ER stress inducer thapsigargin (Tg) treatment and negatively regulated by ER stress chaperone glucose regulated protein 78 (GRP78) in pancreatic cancer cells. In human specimens, we found that miR-34b was down-regulated in pancreatic cancer tissues and low level of miR-34b expression was positively correlated with tumor-node-metastasis (TNM) stage, lymph-node metastasis and overall survival. Functional assays showed that over-expression of miR-34b inhibited pancreatic cancer progression in vitro and in vivo. In addition, Smad3 was demonstrated as a direct target of miR-34b and negatively regulated by miR- 34b at mRNA and protein levels. Luciferase assays confirmed that miR-34b could directly bind to the 3'untranslated region of Smad3. An inverse correlation between miR-34b and Smad3 was observed in 64 pancreatic cancer tissues. Our findings indicate that miR-34b acts as a tumor metastasis suppressor through negatively modulating Smad3, which may provide a potential therapeutic strategy for pancreatic cancer.

Hedgehog signaling pathway regulates human pancreatic cancer cell proliferation and metastasis

Pancreatic cancer is one of the most aggressive and devastating malignancies. The Hedgehog (Hh) pathway has been reported to play an important role in pancreatic cancer development and progression. The aim of this study was to examine the activation of the Hh pathway in human pancreatic cancer tissue samples and pancreatic cancer cell lines, and the molecular mechanisms involved in the Hh pathway mediated effects on pancreatic cancer cell proliferation and invasion. The expression levels of Hh molecules in human pancreatic cancer tissue samples and pancreatic cancer cell lines were evaluated using RT-PCR. The role of the Hh pathway in cell proliferation and invasion was evaluated using flow cytometry, MTT, colony formation assays and transwell invasion assays, and the expression of cancer stem cell markers and epithelial-mesenchymal transition(EMT) were evaluated using flow cytometry and RT-PCR. Tumorigenicity assays were used to further investigate the role of the Hh pathway invivo. Hh molecules were highly expressed in human pancreatic cancer tissue samples and pancreatic cancer cell lines. Inhibition of the Hh pathway notably decreased cell proliferation and induced apoptosis through inhibition of the PI3K/AKT pathway and cancer stem cells. Furthermore, inhibition of the Hh signaling pathway significantly inhibited EMT by suppressing the activation of transcription factors Snail and Slug, which are correlated with significantly reduced pancreatic cancer cell invasion, suggesting that the Hh signaling pathway is involved in early metastasis. These results indicate that activation of the Hh pathway is a common event. Inhibition of the Hh pathway may be a potential molecular target of new therapeutic strategies for pancreatic cancer.

A Novel EPAC-Specific Inhibitor Suppresses Pancreatic Cancer Cell Migration and Invasion

Exchange protein directly activated by cAMP (EPAC) and cAMP-dependent protein kinase (PKA) are two intracellular receptors that mediate the effects of the prototypic second messenger cAMP. Identifying pharmacological probes for selectively modulating EPAC activity represents a significant unmet need within the research field. Herein, we report the identification and characterization of 3-(5-tert-butyl-isoxazol-3-yl)-2-[(3-chloro-phenyl)-hydrazono]-3-oxo-propionitrile (ESI-09), a novel noncyclic nucleotide EPAC antagonist that is capable of specifically blocking intracellular EPAC-mediated Rap1 activation and Akt phosphorylation, as well as EPAC-mediated insulin secretion in pancreatic β cells. Using this novel EPAC-specific inhibitor, we have probed the functional roles of overexpression of EPAC1 in pancreatic cancer cells. Our studies show that EPAC1 plays an important role in pancreatic cancer cell migration and invasion, and thus represents a potential target for developing novel therapeutic strategies for pancreatic cancer.

Transforming Growth Factor β and Ras/MEK/ERK Signaling Regulate the Expression Level of a Novel Tumor Suppressor Lefty

The objectives of the present study were (i) to identify a novel tumor suppressor gene whose expression level was regulated by transforming growth factor (TGF-β) and (ii) to evaluate the effect of Ras/MEK/ERK signaling on TGF-β-dependent Lefty up-regulation. Human pancreatic cancer cell lines were used. The effect of Ras/MEK/ERK pathway on TGF-β-mediated Lefty up-regulation was tested by adding K-ras small interfering RNA, MEK inhibitor U0126, or extracellular signal-regulated kinase (ERK) inhibitor LY294002. Transforming growth factor β upregulated Lefty messenger RNA levels within 6 of the 7 cell lines. Lefty exerts an antagonistic effect against the tumor-promoting molecule, Nodal, as recombinant Lefty suppressed Nodal-mediated proliferation. Interestingly, inhibition of the Ras/MEK/ERK pathway dramatically enhanced TGF-mediated Lefty up-regulation, suggesting that Ras/MEK/ERK signaling suppresses TGF-β-Lefty pathway. Our data suggest that Lefty is a novel TGF-β target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-β-Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-β.

MicroRNA-375 Is Downregulated in Pancreatic Cancer and Inhibits Cell Proliferation In Vitro

MicroRNAs (miRNAs) have emerged as important regulators in the development of pancreatic cancer and may be a valuable therapeutic application. Aberrant expression of microRNA-375 (miR-375) has been reported to be involved in development and progression in various types of cancers, but few studies have been conducted to determine its relationship with pancreatic cancer. Quantitative RT-PCR was used to detect the levels of miR-375 expression in pancreatic cancer tissue samples and cells. The cell growth rate of pancreatic cancer cells transfected with pre-miR-375 was examined by CCK8 assay. The effects of miR-375 on cell cycle and apoptosis were assessed by flow cytometry analyses. In this study, we found that the expression levels of miR-375 was significantly lower in pancreatic cancer tissues compared with nontumorous tissues. We found that miR-375 level in pancreatic cancer was associated with lymph nodes metastasis and clinical stage, and did not correlated with any other factors such as sex, age, position, tumor size, or histological grading. The CCK8 assay showed that that cells transfected with pre-miR-375 inhibited cell proliferation in Panc-1 and SW1990 cells. Flow cytometry analysis indicated that upregulation of miR-375 led to an increase in the percentage of cells in G0/G1 phase in the cell cycle distribution and induced cell apoptosis. Our research suggested that miR-375 has potential as a novel suppressor gene in pancreatic cancer and its downregulation may promote the progression of pancreatic cancer. Overexpression of miR-375 impacts cell proliferation, cell cycle distribution, and apoptosis of pancreatic cancer cells, miR-375 may play an important role in the novel therapeutic strategy for pancreatic cancer.

The oncolytic adenovirus AdΔΔ enhances selective cancer cell killing in combination with DNA-damaging drugs in pancreatic cancer models

Pancreatic adenocarcinomas are aggressive and frequently develop resistance to all current therapies. Replication-selective adenoviruses can overcome resistance to chemotherapeutics through their sensitizing effects on drug-induced cell killing. We previously found that adenovirus deleted in the anti-apoptotic E1B19K gene enhanced gemcitabine-induced apoptotis. Here we demonstrate that our engineered double-deleted AdΔΔ mutant (deleted in the pRb-binding E1ACR2 region and E1B19K) selectively replicates and enhances cell killing in combination with DNA-damaging cytotoxic drugs in pancreatic cancer cells. Combinations of AdΔΔ with gemcitabine, irinotecan or cisplatin resulted in two- to fourfold decreases in EC(50) (half maximal effective concentration) values and was more efficent than similar combinations with wild-type virus, the dl1520 (ONYX-015) and dl922-947 mutants. AdΔΔ replication was impaired in normal bronchial human epithelial cells and did not sensitize the cells to drugs. Gemcitabine-insensitive AsPC-1, BxPC-3 and PANC-1 cells were efficiently killed by irinotecan in combination with AdΔΔ. Suboptimal doses of AdΔΔ and gemcitabine significantly prolonged time to tumor progression in two human pancreatic tumor xenograft in vivo models, PT45 and SUIT-2. We conclude that AdΔΔ has low toxicity to normal cells while potently sensitizing pancreatic cancer cells to DNA-damaging drugs, and holds promise as an improved therapeutic strategy for pancreatic cancer.

Autophagy suppression promotes apoptotic cell death in response to inhibition of the PI3K—mTOR pathway in pancreatic adenocarcinoma

Targeting of pathways downstream of RAS represents a promising therapeutic strategy for pancreatic cancer, the fourth leading cause of cancer-related death in the USA, since activation of the Raf-MEK-ERK and PI3K-AKT pathways is found frequently in this disease and is associated with poor prognosis. Taking advantage of a panel of human PDAC cell lines and specific inhibitors of PI3K and/or mTOR, we systematically address the question whether dual-targeted inhibition of the PI3K and mTOR pathways offers advantages over single-targeted inhibition of PI3K in PDAC. We observe greater overall susceptibility of cell lines to dual inhibition compared to targeting PI3K alone. However, we find that dual inhibition of PI3K and mTOR induces autophagy to a greater extent than inhibition of each target alone. In agreement with this, we show that combined administration of PI3K/mTOR and autophagy inhibitors results in increased anti-tumor activity in vitro and in vivo in models of pancreatic adenocarcinoma. XL765, a PI3K/mTOR inhibitor used in our in vivo studies, is currently undergoing clinical evaluation in a variety of cancer types, while the autophagy inhibitor chloroquine is a widely used anti-malaria compound. Thus, our studies provide rationale for clinical development of combinations of these compounds for the treatment of pancreatic adenocarcinoma.

β2-adrenoceptor blocker synergizes with gemcitabine to inhibit the proliferation of pancreatic cancer cells via apoptosis induction

The stimulation of β2-adrenoceptor, which is a major mediator for chronic stress-induced cancers, has been implicated in the progression in the number of cancer cells, including pancreatic cancer, which remains one of the most aggressive and lethal diseases worldwide. Whether β-adrenoceptor antagonists potentiate gemcitabine, a standard first-line treatment for advanced pancreatic cancer that offers only modest benefit due to acquired chemoresistance, has not been elucidated. Thus, we studied the antiproliferative and apoptotic effects and the underlying mechanisms of gemcitabine combined with the β2-adrenoceptor blocker ICI 118551 (1-[2,3-(dihydro-7-methyl-1H-iden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol), in human pancreatic cancer BxPC-3 and MIA PaCa-2 cells. Results show that ICI 118551 significantly synergized the antiproliferative and pro-apoptotic effects induced by gemcitabine in both BxPC-3 and MIA PaCa-2 cells ( P < 0.05 combination vs. control or gemcitabine alone). When cells were treated with the combination of gemcitabine and ICI 118551, NF-κB activation was blocked; the expression of Bax protein was substantially increased; and Bcl-2 protein was downregulated. Taken together, the data suggest that ICI 118551 potentiates the antiproliferative effects of gemcitabine by inducing apoptosis in pancreatic cancer cells. Our study implies that this combination may be an effective therapeutic strategy for pancreatic cancer.

β2-adrenergic antagonists suppress pancreatic cancer cell invasion by inhibiting CREB, NF-κB and AP-1

Smoking and chronic stress are well-documented risk factors that are associated with β-adrenoceptors in the development of pancreatic cancer. Stimulation of β-adrenoceptors can activate cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) pathways in pancreatic cancer cells. Many recent studies have focused on the function of β-adrenoceptors in cancer invasion. Thus, we hypothesized that β-adrenoceptors may play a role in pancreatic cancer invasion, and β-blockers may suppress the pancreatic cancer invasion and proliferation. MIA PaCa-2 and BxPC-3 cell lines express mRNA and protein of both β1 and β2-adrenoceptors. β2-adrenergic antagonist ICI118,551 and β1/2-adrenergic antagonist propranolol significantly suppressed cell invasion and proliferation in comparison to β1-adrenergic antagonist metoprolol and control in a Matrigel invasion assay and subrenal capsular assay. Treatment with β2-adrenoceptor antagonists inhibited activation of transcription factors nuclear factor κB (NF-κB), activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as demonstrated by electrophoretic mobility shift assays and Western blotting. β2-adrenoceptor antagonists also significantly altered vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), matrix metalloproteinase 2 (MMP-2) and MMP-9 expression. The β2-adrenergic antagonists suppressed invasion and proliferation by inhibiting both cAMP/PKA and Ras, which regulate activation of the MAPK pathway and transcription factors, such as NF-κB, AP-1 and CREB, as well as expression of its target genes, MMP-9, MMP-2 and VEGF. However, β1-adrenergic antagonists suppressed invasion by inhibiting only the cAMP/PKA pathway, suggesting that they may be useful as novel preventive and therapeutic strategies for pancreatic cancer.

Gemcitabine combined with gum mastic causes potent growth inhibition and apoptosis of pancreatic cancer cells

To investigate the antiproliferative and apoptotic effects of gemcitabine combined with gum mastic and the underlying mechanisms in human pancreatic cancer cell lines. Cell proliferation and apoptosis were examined using the methyl thiazolyl tetrazolium (MTT) assay and propidium iodine staining, respectively. The expression of Bcl-2, Bax, NF-kappaB p65 subunit, and IkappaBalpha protein was measured using Western blotting. Gemcitabine 0.01-100 microg/mL inhibited cell proliferation and induced apoptosis in both pancreatic cancer BxPC-3 and COLO 357 cells. Gum mastic 40 microg/mL significantly potentiated the antiproliferative and apoptotic effects of gemcitabine 10 microg/mL after 72-h treatment. When cells were treated with gemcitabine in combination with gum mastic, the IkappaBalpha level was increased, whereas NF-kappaB activation was blocked; the expression of Bax protein was substantially increased, but Bcl-2 protein was down-regulated. Gemcitabine combined with gum mastic causes potent apoptosis in pancreatic cancer cells. The combination may be an effective therapeutic strategy for pancreatic cancer.

Involvement of the Tubulin Tyrosine Ligase-Like Family Member 4 Polyglutamylase in PELP1 Polyglutamylation and Chromatin Remodeling in Pancreatic Cancer Cells

Polyglutamylation is a new class of posttranslational modification in which glutamate side chains are formed in proteins, although its biological significance is not well known. Through our genome-wide gene expression profile analyses of pancreatic ductal adenocarcinoma (PDAC) cells, we identified the overexpression of tubulin tyrosine ligase-like family member 4 (TTLL4) in PDAC cells. Subsequent reverse transcription-PCR and Northern blot analyses confirmed its upregulation in several PDACs. TTLL4 belongs to the TTLL family which was reported to have polyglutamylase activity. Knockdown of TTLL4 by short hairpin RNA in PDAC cells attenuated the growth of PDAC cells and exogenous introduction of TTLL4 enhanced cell growth. We also found that TTLL4 expression was correlated with polyglutamylation levels of a glutamate stretch region of the proline, glutamate, and leucine-rich protein 1 (PELP1) that was shown to interact with various proteins such as histone H3, and was involved in several signaling pathways through its function as a scaffold protein. PELP1 polyglutamylation could influence its interaction with histone H3 and affect histone H3 acetylation. We also identified the interaction of PELP1 with LAS1L and SENP3, components of the MLL1-WDR5 supercomplex involving chromatin remodeling. Our findings imply that TTLL4 could play important roles in pancreatic carcinogenesis through its polyglutamylase activity and subsequent coordination of chromatin remodeling, and might be a good molecular candidate for the development of new therapeutic strategies for pancreatic cancer.

Abstract A243: SCH727965, a novel potent cyclin dependent kinase inhibitor, inhibits pancreatic tumor growth and Ras-mediated tumorigenic signaling

Abstract We have previously shown activation of cyclin-dependent kinase 5 (CDK5), a kinase associated with neuronal migration, in human pancreatic cancer. Abrogation of CDK5 function using a dominant-negative protein, RNA interference, or pharmacological disruption significantly mitigated invasion, migration and anchorage independent growth in vitro, and orthotopic tumor formation and systemic metastases in vivo. CDK5 blockade resulted in inhibition of RalA, a key effector pathway downstream of oncogenic Ras, central to tumorigenesis in pancreatic cancer. Restitution of Ral function rescued the effects of CDK5 inhibition in pancreatic cancer cells. Thus, CDK5 is important for Ras signaling in pancreatic cancer, suggesting that CDK5 inhibition may be an effective therapeutic strategy in pancreatic cancer. To begin to explore the therapeutic potential of CDK5 inhibitors in pancreatic cancer, we have examined the effect of the semi-selective CDK5 inhibitor SCH727965 (Schering-Plough). SCH727965 is a potent inhibitor of CDK1, CDK2, CDK5 and CDK9, with in vitro IC50 values of 3, 1, 1 and 4 nM, respectively. In this study, we evaluated the effect of SCH727965 on pancreatic tumor growth and migration. MTT assay results demonstrated that the IC50 for 2 pancreatic tumor cell lines, MiaPaCa-2 and Panc1.98, was 12nM and 24nM respectively. Migration and anchorage independent growth were significantly more sensitive to SCH727965. Thus, Boyden chamber assay, wound-healing assay and soft agar assays indicated 50% inhibition of migration and soft agar growth inhibition at 5nM, and complete inhibition at 10nM drug concentration — concentrations at which the effects on anchorage dependent growth were relatively minor. Tumors from low passage pancreatic tumor xenografts, or from pancreatic cell line orthotopic xenografts, showed 40–75% reduction of tumor growth after treatment with SCH727965, with significant reduction of metastases. SCH727965 treatment resulted in inhibition of RalA activation. Ral activity could be restored by enforced RalA activation, via expression of Rgl2-CAAX, a constitutively active form of a RalGEF (Ral guanine exchange factor) gene. Our results show that SCH727965 is very effective against pancreatic cancer growth and metastasis. These results also suggest that SCH727965 blocks pancreatic tumor growth, migration and metastasis, at least in part by inhibition of dysregulated Ras signaling, by inhibiting CDK5. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A243.