c‐Jun N‐terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cells

Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c‐Jun N‐terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), and the proinflammatory cytokines IL‐1β, TNFα, and IL‐1α. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL‐1β, TNFα, and IL‐1α, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT‐transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells. J. Cell. Physiol. 225: 720–733, 2010. © 2010 Wiley‐Liss, Inc.     

Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to <Emphasis Type="Italic">Clostridium perfringens</Emphasis> enterotoxin via claudin-4

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and −4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.     

G protein-coupled receptor signaling in human ductal pancreatic cancer cells: neurotensin responsiveness and mitogenic stimulation

Neuropeptides and their corresponding G protein-coupled receptors (GPCRs) are increasingly implicated in the autocrine/paracrine stimulation of growth of human cancers. We report that neurotensin induced rapid Ca2+ mobilization from intracellular stores followed by Ca2+ influx in five human ductal pancreatic cancer cell lines: HPAF-II, Capan-1, Capan-2, PANC-1, and MIA PaCa-2. In addition, most cell lines exhibited Ca2+ responses to multiple neuropeptides including bombesin, bradykinin, cholecystokinin, and vasopressin and to bioactive lipids, including lysophosphatidic acid (LPA), that also act via GPCRs. The well-differentiated line HPAF-II responded to at least seven independent GPCR agonists. The concentrations of neurotensin required to induce half-maximal effects (EC50) in HPAF-II and PANC-1 cells were 5 and 8nM, respectively. Digital fluorescence image analysis to measure Ca2+ responses in single cells revealed that 90% or more of HPAF-II and PANC-1 cells responded to 10nM neurotensin. Addition of neurotensin to PANC-1 cells also induced rapid and dose-dependent extracellular-regulated protein kinase (ERK-1 and ERK-2) activation and subsequently, stimulated DNA synthesis. The signaling complexity of GPCRs uncovered by these studies reveals a new aspect in the biology of human pancreatic cancer and could offer the basis for new approaches to the treatment of this disease.     

Claudin-18 Is an Early-Stage Marker of Pancreatic Carcinogenesis

Pancreatic ductal neoplasms exhibit gastric epithelium–like characteristics. In this study, we evaluated the expression of claudin-18 (CLDN18), a gastric epithelium–associated claudin, in pancreatic intraepithelial neoplasias (PanINs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and pancreatic ductal adenocarcinomas (PDACs) using immunohistochemistry. We observed a high level of expression of CLDN18 in PanINs (31/32, 97%), IPMNs (61/65, 95%), and MCNs (4/5, 80%) using ordinary tissue section analysis. Furthermore, we observed a high level of CLDN18 expression in PDACs (109/156, 70%) using tissue microarray analysis. However, the normal pancreatic duct or the ductal metaplasia of the acinar cells was not immunoreactive. Comparative analysis of CLDN18 and phenotypic markers in IPMNs revealed that simultaneous expression of CLDN18 and intestinal markers frequently occurred, even in intestinal-type IPMNs. CLDN18 variant 2 mRNA was expressed and was similarly upregulated by phorbol 12–myristate 13–acetate (PMA) treatment in pancreatic cancer cell lines and in a gastric cancer cell line. An inhibitor of pan-PKC (GF109203X) completely suppressed this upregulation in pancreatic cancer cells. These results indicate that CLDN18, a marker for the early carcinogenetic process, is commonly expressed in precursor lesions of PDAC. Activation of the PKC pathway might be involved in CLDN18 expression associated with pancreatic carcinogenesis.     

PKCδ and Tissue Transglutaminase are Novel Inhibitors of Autophagy in Pancreatic Cancer Cells

Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKCδ) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKCδ/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKCδ/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.

Addendum to:

Tissue Transglutaminase Inhibits Autophagy in Pancreatic Cancer Cells

U. Akar, B. Ozpolat, K. Mehta, J. Fok, Y. Kondo and G. Lopez-Berestein

Mol Cancer Res 2007; 5:241-9     

Pharmacological Reversal of Histone Methylation Presensitizes Pancreatic Cancer Cells to Nucleoside Drugs: In Vitro Optimization and Novel Nanoparticle Delivery Studies

We evaluated the potential of an investigational histone methylation reversal agent, 3-deazaneplanocin A (DZNep), in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines by increased apoptosis. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents to produce a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs through the reversal of histone methylation, emphasizing the promising clinical utilities of epigenetic reversal agents in future pancreatic cancer combination therapies.     

Identification and in silico characterization of a novel gene: TPA induced trans-membrane protein

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter with wide ranging, diverse, and sometimes opposite cellular effects. Using oligonucleotide microarray analysis, we have identified a novel gene that is upregulated following treatment with TPA in the pancreatic cancer cell line CD18. Real-time PCR validated the microarray results in CD18 and HeLa cells, and showed that upregulation of the gene is time- and concentration-dependent. In silico analysis showed the gene product to be a single-pass transmembrane protein of 217 residues that is localized to the endoplasmic reticulum, thus the name TPA induced trans-membrane protein (TTMP). A luciferase reporter assay demonstrated that upregulation of TTMP by TPA is triggered at the promoter level.     

Increase in pancreatic exocrine secretion during uncoupling: evidence for a protein kinase C-independent effect

It has been demonstrated that blockade of the normal communication between pancreatic acinar cells leads to an increase in amylase release. Although the physiological mechanisms that regulate the gating of gap junction channels are unknown, the involvement of protein kinase C (PKC) in the inhibition of cell coupling has been reported in various cell lines. Since the activation of PKC also stimulates amylase secretion of pancreatic acinar cells, we sought to determine whether blockers of gap junctions and activators of PKC modify basal secretion by a similar mechanism. Thus, we have studied the effects of heptanol and of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the subcellular distribution of PKC, dye coupling, and amylase release of dispersed pancreatic acini. The data show that TPA activates PKC and stimulates amylase secretion without affecting the extensive dye coupling of acinar cells. By contrast, heptanol inhibits cell-to-cell coupling and increases enzyme output without altering the subcellular distribution of PKC. Heptanol also enhances significantly the secretion evoked by TPA. These results indicate that the stimulation of amylase release caused by uncoupling of acinar cells occurs by a mechanism(s) that does not involve the activation of PKC.     

Novel synthetic oleanane triterpenoid AMR-MeOAc inhibits K-Ras through ERK,Akt and survivin in pancreatic cancer cells

K-Ras activating mutations are a major problem that drives aggressive tumor growth and metastasis in pancreatic cancer. Currently, there are no effective targeted therapies for this genetically defined subset of cancers harboring oncogenic K-Ras mutations that confer drug resistance, aggressive tumor growth, metastasis and poor clinical outcome. We identified a novel synthetic oleanane triterpenoid compound designated AMR-MeOAc that effectively kills K-Ras mutant pancreatic cancer HPAF-II cells. The cytotoxic effects correlated with apoptosis induction, as was evidenced by increase of apoptosis cells upon the treatment of AMR-MeOAc in HPAF-II cells. Our studies revealed that AMR-MeOAc treatment inhibits cancer associated survival gene survivin. Moreover, AMR-MeOAc also led to down regulation of Akt, ERK1/2 and survivin protein levels. Our results indicate that AMR-MeOAc or its active analogs could be a novel class of anticancer agents against K-Ras driven human pancreatic cancer.     

Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells

The small GTPase Cdc42 has been implicated as an important regulator of cell migration. However, whether Cdc42 plays similar role in all cancer cells irrespective of metastatic potential remains poorly defined. Here, we show by using three different breast cancer cell lines with different metastatic potential, the role of Cdc42 in cell migration/invasion and its relationship with a number of downstream signaling pathways controlling cell migration. Small interfering RNA (siRNA)-mediated knockdown of Cdc42 in two highly metastatic breast cancer cell lines (MDA-MB-231 and C3L5) resulted in enhancement, whereas the same in moderately metastatic (Hs578T) cell line resulted in inhibition of intrinsic cellular migration/invasion. Furthermore, Cdc42 silencing in MDA-MB-231 and C3L5 but not Hs578T cells was shown to be accompanied by increased RhoA activity and phosphorylation of protein kinase C (PKC)-δ, extracellular signal regulated kinase1/2 (Erk1/2), and protein kinase A (PKA). Pharmacological inhibition of PKCδ, MEK-Erk1/2, or PKA was shown to inhibit migration of both control and Cdc42-silenced MDA-MB-231 cells. Furthermore, introduction of constitutively active Cdc42 was shown to decrease migration/invasion of MDA-MB-231 and C3L5 but increase migration/invasion of Hs578T cells. This decreased migration/invasion of MDA-MB-231 and C3L5 cells was also shown to be accompanied by the decrease in the phosphorylations of PKCδ, Erk1/2, and PKA. These results suggested that endogenous Cdc42 could exert a negative regulatory influence on intrinsic migration/invasion and some potentially relevant changes in phosphorylation of PKCδ, Erk1/2, and PKA of some aggressive breast cancer cells.     

Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression.

By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.     

Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS ^G12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.     

Proliferative responses of epithelial cells to 8-bromo-cyclic AMP and to a phorbol ester change during breast Pathogenesis

We have explored the relationship of changes in proliferative responses of human mammary epithelial cells to a phorbol ester (TPA) and to 8-Br-cAMP, which modulate the activities of protein kinases A and C (PKA and PKC), with breast tumour progression. Treatment with TPA had no effect on nontumorigenic cell lines established from human fibrocystic biopsies and apparently normal tissue around a tumour. In contrast, TPA strongly inhibited the proliferation of numerous human tumorigenic breast cell lines. Treatment with 8-Br-cAMP decreased the proliferation of all studied nontumorigenic and tumorigenic cell lines. We have also studied the effect of TPA and 8-Br-cAMP on growth of epithelial cells in short-term culture obtained from surgical human mammary biopsies with different states of breast disease. Both drugs enhanced growth of normal breast cells but had no significant effects on cells from biopsies with benign breast disease. In contrast, all examined cuitures from breast cancer biopsies were strongly inhhited by 8-Br-cAMP. Otherwise, TPA had an inhibitory effect only in the case of invasive ductal carcinoma of grade III. Malignant Ha-ras-transformation of nontumorigenic TPA-insensitive breast HBL-100 cells induced an inhibitory effect of TPA. In addition, a TPA-insensitive MCF7 clone was much less tumorigenic in athymic mice than the parental strain shown to be inhibited by TPA. These data suggest that the two intracellular transduction pathways change at different stages of breast pathogenesis. Alterations in the PKA pathway are early events and are probably important to cell immortalization but do not necessarily lead to malignant development. In contrast, changes in PKC pathway are rather later events associated with advanced malignant transformation. © 1994 Wiley-Liss, Inc.     

Quantitative analysis of CD5 antigen modulation by 12-O-tetradecanoylphorbol-13-acetate in T-lymphoblastic leukemia cells: individual response patterns and their relationships with both maturation and protein kinase C content.

Modulation of CD5 expression by TPA was investigated on T-leukemic cell lines corresponding to different stages of ontogeny. These CD5 changes have been analyzed simultaneously with modifications of cell growth, cell cycle, cell surface phenotype, and PKC content. CD5 expression was found 6- to 17-fold increased by TPA in a dose-dependent manner on phenotypically mature T-cells (Jurkat, JM, and T-CLL) while T-cells from earlier stages of differentiation (CEM III, CEM 95, and CEM 44) were found unresponsive. CD5 upregulation on TPA-sensitive JM cells appears correlated with inhibition of cell growth, blockage in G1 phase, and phenotypic maturation (downregulation of CD7 and CD1 antigens) and seemed to be related to PKC activation since DiC8 (a PKC activator) mimicked this TPA effect and H7 (a PKC inhibitor) partially reduced it. On the other hand, on CEM III cells TPA induced no modulation of CD5 antigen, a less dramatic effect on cell growth and cell cycle, but a CD7 downregulation. TPA appeared fully effective in binding and translocating PKC in both CEM III and JM cells, although the PKC activity level was three times higher in the latter. Finally, our study suggests that CD5 expression is at least partially under control of PKC in phenotypically mature neoplastic T-cells while PKC could not be directly involved in the regulation of CD5 antigen in leukemic cells arrested at earlier stages of differentiation.