A rapid increase in the synthesis of lipid-derived second messengers is an important mechanism for transducing extracellular signals across the plasma membrane (1-3). The phospholipase C-mediated hydrolysis of inositol phospholipids is known to produce two second messengers: inositol 1,4,5-trisphosphate, which induces mobilization of calcium from intracellular stores (3), and diacylglycerol (DAG), which activates protein kinase C (PKC)-originally described as a Ca2+-activated, phospholipid-dependent protein kinase (4). The early findings that the potent tumor promoters of the phorbol ester family can substitute for DAG in PKC activation and that the phorbol ester receptor and PKC copurify supported the hypothesis that the cellular target of the phorbol esters is PKC (4). Subsequent studies revealed the diversity of the individual components of the DAG-PKC signal transduction pathway.

Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase-PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of ␣ subunits of G proteins implicated in intracellular taste signal transduction, namely G␣gust, and G␣t-2, also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase-PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca 2؉ concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.stomach ͉ intestine ͉ gustducin ͉ transducin T he gustatory system has been selected during evolution to detect nutritive and beneficial compounds as well as harmful or toxic substances (1, 2). In particular, bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances (3). Recently, a large family of bitter taste receptors (T2Rs) expressed in specialized neuroepithelial taste receptor cells organized within taste buds in the tongue has been identified in humans and rodents (4-6). These putative taste receptors, which belong to the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily characterized by seven putative transmembrane domains, are distantly related to V1R vomeronasal receptors and opsins (5). Genetic and biochemical evidence indicate that specific G␣ subunits, gustducin (G␣ gust ) and transducin (G␣ t ), mediate bitter and sweet gustatory signals in the taste buds of the lingual epithelium (7-11).Outside the tongue, expression of G␣ gust also has been localized to gastric (12) and pancreatic (13) cells, suggesting that a taste-sensing mechanism also may exist in the gastrointestinal (GI) tract. However, not all cells that express G␣ gust also coexpress members of the T2R family of receptors (5). For example, most G␣ gust -positive taste receptor cells in the lingual fungiform papillae are T2R-negative, implying that G␣ gust also could mediate signaling through other receptors (9). To establish that the gastric an...

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour‐promoting phorbol esters, membrane‐permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat‐1 cells), in secondary cultures of mouse embryo fibroblasts and in COS‐7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31–8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co‐transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS‐7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC‐dependent signal transduction pathway.

Recently, we identified a novel crosstalk between insulin and G protein-coupled receptor (GPCR) signaling pathways in human pancreatic cancer cells. Insulin enhanced GPCR signaling through a rapamycin-sensitive mTOR-dependent pathway. Metformin, the most widely used drug in the treatment of type 2 diabetes, activates AMP kinase (AMPK), which negatively regulates mTOR. Here, we determined whether metformin disrupts the crosstalk between insulin receptor and GPCR signaling in pancreatic cancer cells. Treatment of human pancreatic cancer cells (PANC-1, MIAPaCa-2, and BxPC-3) with insulin (10 ng/mL) for 5 minutes markedly enhanced the increase in intracellular [Ca 2+ ] induced by GPCR agonists (e.g., neurotensin, bradykinin, and angiotensin II). Metformin pretreatment completely abrogated insulin-induced potentiation of Ca 2+ signaling but did not interfere with the effect of GPCR agonists alone. Insulin also enhanced GPCR agonist-induced growth, measured by DNA synthesis, and the number of cells cultured in adherent or nonadherent conditions. Low doses of metformin (0.1-0.5 mmol/L) blocked the stimulation of DNA synthesis, and the anchoragedependent and anchorage-independent growth induced by insulin and GPCR agonists. Treatment with metformin induced striking and sustained increase in the phosphorylation of AMPK at Thr 172 and a selective AMPK inhibitor (compound C, at 5 Mmol/L) reversed the effects of metformin on [Ca 2+ ] i and DNA synthesis, indicating that metformin acts through AMPK activation. In view of these results, we tested whether metformin inhibits pancreatic cancer growth. Administration of metformin significantly decreased the growth of MIAPaCa-2 and PANC-1 cells xenografted on the flank of nude mice. These results raise the possibility that metformin could be a potential candidate in novel treatment strategies for human pancreatic cancer. [Cancer Res 2009; 69(16):6539-45]

Bombesin is shown to be a potent mitogen for Swiss 3T3 cells. At nanomolar concentrations the peptide markedly enhances the ability of fresh serum to stimulate DNA synthesis in confluent and quiescent cultures of these cells. In the presence of a low concentration (3.5%) of serum, bombesin stimulates 3T3 cell proliferation. In serum-free medium, bombesin induces DNA synthesis in the absence of any other added growth factor; halfmaximal effect is obtained at 1 nM. The mitogenic effect of bombesin is dependent on dose and time, is mimicked by litorin, and is markedly potentiated by insulin, colchicine, platelet-derived growth factor, and fibroblast-derived growth factor. These mitogens increase the maximal response elicited by bombesin and decrease the bombesin concentration required to produce halfmaximal effect (from 1 nM to 0.3 nM). In contrast, vasopressin, phorbol esters, or cAMP increasing agents fail to enhance the maximal level of DNA synthesis induced by bombesin. Bombesin and litorin may provide useful model peptides for studies on the mechanism(s) by which extracellular ligands control cell proliferation.In recent years, a considerable number of new regulatory peptides have been identified in the brain, gastrointestinal tract, and other tissues (1-3). Bombesin, a tetradecapeptide originally isolated from frog skin (4), and bombesin-like peptides have been detected in mammalian brain (5, 6), gut (5, 7), and lung (8,9). This peptide has potent pharmacological effects on the central nervous system (10-12) and elicits the release of other peptide hormones including insulin (13,14), glucagon (13,14), gastrin (5,7,14), cholecystokinin (14), and prolactin and growth hormone (15,16). Bombesin binds to specific surface receptors in pancreatic acinar cells (17) and stimulates ion fluxes (17,18) and enzyme secretion (17,18) Recently, several reports demonstrated the presence of high concentrations of bombesin in human pulmonary tumors (19)(20)(21). This observation and the report that repeated administration of bombesin induced pancreatic hyperplasia in the rat (22) raises the possibility that bombesin could participate in the control of cell proliferation, a proposition that hitherto remained unproven. Indeed, it is difficult to obtain unambiguous evidence for a direct growth-promoting activity of bombesin in vivo because the administration of this peptide stimulates the release of many other biologically active peptides (see above) which could act as proximal effectors of the action of bombesin.Cultured cells provide a useful experimental system for elucidating the extracellular factors that control cell proliferation without the many complexities of whole animal experimentation. Many mammalian cells in culture, and Swiss 3T3 cells in particular, cease to proliferate and become arrested in the G1/ Go phase of the cell cycle when they deplete the nutrient medium of its growth-promoting activity (23). Addition of fresh serum or defined growth-promoting factors to such quiescent cells stimulates reinitiati...

A novel protein kinase (named PKD) with an NH2-terminal region containing two cysteine-rich motifs has been expressed in COS-7 cells and identified as a receptor for phorbol esters. COS-7 cells transfected with a PKD cDNA construct (pcDNA3-PKD) exhibit a marked (4.8-fold) increase in [3H]phorbol 12,13-dibutyrate binding. An antiserum raised against the COOH-terminal 15 amino acids of PKD specifically recognized a single 110-kDa band in PKD-transfected cells. PKD prepared by elution from immunoprecipitates with the immunizing peptide efficiently phosphorylated the synthetic peptide syntide-2. The enzyme only poorly phosphorylated a variant syntide-2 where arginine 4 has been replaced by an alanine. The addition of [3H]phorbol 12,13-dibutyrate, 1-oleoyl-2-acetylglycerol, or 1,2-dioctanoyl-sn-glycerol in the presence of dioleoylphosphatidylserine stimulated the syntide-2 kinase activity of PKD in a synergistic fashion (4-6-fold). Furthermore, the autophosphorylation of PKD was strikingly stimulated by the same lipid activators (14-24-fold). Similar properties were found with PKD isolated from mouse lung. The substrate specificity of PKD is different from that of previously identified members of the protein kinase C family since it does not efficiently phosphorylate histone III-S, protamine sulfate, or a synthetic peptide based upon the conserved pseudosubstrate region of the protein kinase C family. Taken together, these data unambiguously establish PKD as a phorbol ester receptor and as a novel phospholipid/diacylglycerol-stimulated protein kinase.

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-κB activation; and abrogated the expression of NF-κB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 μmol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67-positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (P < 0.05), and abrogated the expression of NF-κB-dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer. Mol Cancer Ther; 9(5); 1136-46. ©2010 AACR.

The development of drug resistance by cancer cells is recognized as a major cause for drug failure and disease progression. PI3K/Akt/mTOR pathway is aberrantly stimulated in many cancer cells and thus it has emerged as a target for therapy. However, mTORC1 and S6K also mediate potent negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feedback loops causes over-activation of upstream pathways, including PI3K, Akt and ERK that potentially oppose the anti-proliferative effects of mTOR inhibitors and lead to drug resistance. A corollary of this concept is that release of negative feedback loops and consequent compensatory over-activation of pro-mitogenic pathways in response to signal inhibitors can circumvent the mitogenic block imposed by targeting only one pathway. Consequently, the elucidation of the negative feedback loops that regulate the outputs of signaling networks has emerged as an area of fundamental importance for the rational design of effective anticancer combinations of inhibitors. Here, we review pathways that undergo compensatory over-activation in response to inhibitors that suppress feedback inhibition of upstream signaling and underscore the importance of unintended pathway activation in the development of drug resistance to clinically relevant inhibitors of mTOR, Akt, PI3K or PI3K/mTOR.