Krisztina Kisfalvi

Metformin Disrupts Crosstalk between G Protein–Coupled Receptor and Insulin Receptor Signaling Systems and Inhibits Pancreatic Cancer Growth

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 anchorage-dependent 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 micromol/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.

Crosstalk between insulin/insulin-like growth factor-1 receptors and G protein-coupled receptor signaling systems: a novel target for the antidiabetic drug metformin in pancreatic cancer.

Insulin/insulin-like growth factor 1(IGF-1) receptors and G protein-coupled receptors (GPCR) signaling systems are implicated in autocrine-paracrine stimulation of a variety of malignancies, including ductal adenocarcinoma of the pancreas, one of the most lethal human diseases. Novel targets for pancreatic cancer therapy are urgently needed. We identified a crosstalk between insulin/IGF-1 receptors and GPCR signaling systems in pancreatic cancer cells, leading to enhanced signaling, DNA synthesis, and proliferation. Crosstalk between these signaling systems depends on mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Metformin, the most widely used drug in the treatment of type 2 diabetes, activates AMP kinase (AMPK), which negatively regulates mTORC1. Recent results show that metformin-induced activation of AMPK disrupts crosstalk between insulin/IGF-1 receptor and GPCR signaling in pancreatic cancer cells and inhibits the growth of these cells in xenograft models. Given that insulin/IGF-1 and GPCRs are implicated in other malignancies, a similar crosstalk mechanism may be operative in other cancer cell types. Recent epidemiological studies linked administration of metformin with a reduced risk of pancreatic, breast, and prostate cancer in diabetic patients. We posit that crosstalk between insulin/IGF-1 receptor and GPCR signaling is a mechanism for promoting the development of certain types of cancer and a target for the prevention and therapy of these diseases via metformin administration. Clin Cancer Res; 16(9); 2505–11. ©2010 AACR.

Broad-Spectrum G Protein–Coupled Receptor Antagonist, [D-Arg1,D-Trp5,7,9,Leu11]SP: A Dual Inhibitor of Growth and Angiogenesis in Pancreatic Cancer

Substance P analogues, including [D-Arg(1),D-Trp(5,7,9),Leu(11)]SP (SPA) are broad-spectrum G protein-coupled receptor (GPCR) antagonists that have potential antitumorigenic activities, although the mechanism(s) are not completely understood. Here, we examined the effects of SPA in ductal pancreatic cancers that express multiple GPCRs for mitogenic agonists and also produce proangiogenic chemokines. Using HPAF-II, a well-differentiated pancreatic cancer cell line as our model system, we showed that SPA inhibited multiple neuropeptide-induced Ca(2+) mobilization, DNA synthesis, and anchorage-independent growth in vitro. SPA also significantly attenuated the growth of HPAF-II tumor xenografts in nude mice beyond the treatment period. Interestingly, SPA markedly increased apoptosis but moderately decreased proliferation marker, Ki-67 in the tumor xenografts implying additional mechanism(s) for the significant growth inhibitory effect observed in vivo. HPAF-II cells express ELR(+) CXC chemokines, including IL-8/CXCL8, which bind to CXCR2 (a member of GPCR superfamily) and promote angiogenesis in multiple cancers, including pancreatic cancer. SPA inhibited CXCR2-mediated Ca(2+) mobilization and blocked specifically IL-8/CXCL8-induced angiogenesis in rat corneal micropocket assay in vivo. A salient feature of the results presented here is that SPA markedly reduced tumor-associated angiogenesis in the HPAF-II xenografts in vivo. Our results show that SPA, a broad-spectrum GPCR antagonist attenuates tumor growth in pancreatic cancer via a dual mechanism involving both the antiproliferative and antiangiogenic properties. We conclude that this novel dual-inhibitory property of SPA could be of significant therapeutic value in pancreatic cancer, when used in combination with other antiproliferative and/or antiangiogenic agents.

Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin, Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser473 while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser473 and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.

Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser(79) and Raptor at Ser(792), was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05-0.1 mM) that were 10-100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the α(1) and α(2) catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells

Neurotensin (NT) and epidermal growth factor (EGF) induced rapid extracellular‐regulated protein kinase (ERK) activation through different signaling pathways in the K‐Ras mutated human pancreatic carcinoma cell lines PANC‐1 and MIA PaCa‐2. NT stimulated ERK activation via a protein kinase C (PKC)‐dependent (but EGF receptor‐independent) pathway in PANC‐1 and MIA PaCa‐2 cells, whereas EGF promoted ERK activation through a PKC‐independent pathway in these cells. Concomitant stimulation of these cells with NT and EGF induced a striking increase in the duration of ERK pathway activation as compared with that obtained in cells treated with each agonist alone. Stimulation with NT + EGF promoted synergistic stimulation of DNA synthesis and anchorage‐independent growth. Addition of the MEK inhibitor U0126, either prior to stimulation with NT + EGF or 2 h after stimulation with NT + EGF prevented the synergistic increase in DNA synthesis and suppressed the sustained phase of ERK activation. Furthermore, treatment with the selective PKC inhibitor GF‐1 converted the sustained ERK activation in response to NT and EGF into a transient signal and also abrogated the synergistic increase in DNA synthesis. Collectively, our results suggest that the sustained phase of ERK signaling mediates the synergistic effects of NT and EGF on DNA synthesis in pancreatic cancer cells.

Metformin Inhibits the Growth of Human Pancreatic Cancer Xenografts

Objective Pancreatic ductal adenocarcinoma is a devastating disease, with an overall 5-year survival rate of only 3% to 5%. As the current therapies offer very limited survival benefits, novel therapeutic strategies are urgently required to treat this disease. Here, we determined whether metformin administration inhibits the growth of PANC-1 and MiaPaCa-2 tumor xenografts in vivo. Methods Different xenograft models, including orthotopic implantation, were used to determine whether intraperitoneal or oral administration of metformin inhibits the growth of pancreatic cancer in vivo. Results We demonstrate that metformin given once daily intraperitoneally at various doses (50-250 mg/kg) to nude mice inhibited the growth of PANC-1 xenografts in a dose-dependent manner. A significant effect of metformin was obtained at 50 mg/kg and maximal effect at 200 mg/kg. Metformin administration also caused a significant reduction in the phosphorylation of ribosomal S6 protein and ERK in these xenografts. Metformin also inhibited the growth of pancreatic cancer xenografts when administered orally (2.5 mg/mL) either before or after tumor implantation. Importantly, oral administration of metformin also inhibited the growth of MiaPaCa-2 tumors xenografted orthotopically. Conclusions The studies presented here provide further evidence indicating that metformin offers a potential novel approach for pancreatic ductal adenocarcinoma prevention and therapy.

Induced Overexpression of Protein Kinase D1 Stimulates Mitogenic Signaling in Human Pancreatic Carcinoma PANC-1 Cells

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c‐Jun N‐terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC‐1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC‐1 clones that express wild‐type (WT) or kinase‐dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone‐A (PonA). NT potently stimulated c‐Jun Ser63 phosphorylation in both wild type and clonal derivatives of PANC‐1 cells. PonA‐induced expression of WT, but not K618N PKD1, rapidly blocked NT‐mediated c‐Jun Ser63 phosphorylation either at the level of or upstream of MKK4, a dual‐specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT‐induced JNK/cJun activation in PANC‐1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT‐induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro‐mitogenicERK and pro‐apopotic JNK/c‐Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC‐1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC‐1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly‐(HEMA)]‐coated dishes to eliminate cell adhesion (anchorage‐independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1–10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC‐1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells. J. Cell. Physiol. 223: 309–316, 2010.

Tu1514 Limited Application of New International Consensus Diagnostic Criteria for Autoimmune Pancreatitis in Clinical Practice

G A A b st ra ct s of patients with surgical pathology as the gold standard for outcome. Design: 137 patients underwent diagnostic analysis of EUS aspirated pancreatic cyst fluid. DNA was extracted from cyst fluid with quantity and quality (extent of degradation) measured by optical density and qPCR. Mutational analysis targeted KRAS point mutation and loss of heterogeneity (LOH) mutations of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 18q, 21q, 22q. Molecular findings were integrated with results from EUS, chemistry and cytology. For each marker, presence or absence of mutation and clonality were determined, determined, with high clonality comprising 75% of more of cells affected by LOH and low clonality comprising 50-75% of cells. Interpretation of combined molecular, imaging, and chemistry results were classified and subsequently compared to surgical outcome or followup. Surgical pathology was classified into indolent biological behavior (benign or low grade disease), and aggressive biological behavior (high grade or malignant disease). Results: Surgical pathology was available on 81 of these patients. Outcomes included benign cystic neoplasms with low and high grade dysplasia, malignant ductal disease, endocrine neoplasms and pseudocysts. There were 32 aggressive outcomes and 49 indolent outcomes. Molecular findings, including elevated DNA level, and high numbers and clonality of mutations, correlated strongly with aggressive biological behavior. While KRAS mutation was highly specific for mucinous cyst formation, individual molecular parameters used in isolation showed limited sensitivity for detection of aggressive disease. Combining all molecular parameters together, sensitivity was 94%, and specificity was 100%, with overall accuracy of 97%. Conclusions: The molecular biology of pancreatic cystic disease involves multiple pathways that can each lead to cancer. The majority of pancreatic cysts do not progress to cancer and the diagnosis and biological potential of individual lesions can be effectively characterized by integrating broad panel molecular findings with information from first line testing. The presence or absence of aggressive molecular changes, taken in the context of overall findings provides an effective means to individualize patient management when first line results are unclear.

M1968 Metformin Disrupts Crosstalk Between G Protein-Coupled Receptor and Insulin Receptor Signaling Systems and Inhibits Pancreatic Cancer Growth

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]