Robert K. Kui

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.

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to Gq-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3–10 nm) or with vasopressin, a different Gq-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser744 and Ser748. Transphosphorylation targeted Ser744, whereas autophosphorylation was the predominant mechanism for Ser748 in cells stimulated with Gq-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to Gq-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of Gq-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.

CID755673 enhances mitogenic signaling by phorbol esters, bombesin and EGF through a protein kinase D-independent pathway

Recently, CID755673 was reported to act as a highly selective inhibitor of protein kinase D (PKD). In the course of experiments using CID755673, we noticed that it exerted unexpected stimulatory effects on [(3)H]thymidine incorporation and cell cycle progression in Swiss 3T3 cells stimulated by bombesin, a Gq-coupled receptor agonist, phorbol 12,13-dibutyrate (PDBu), a biologically active tumor promoting phorbol ester and epidermal growth factor (EGF). These stimulatory effects could be dissociated from the inhibitory effect of CID755673 on PKD activity, since enhancement of DNA synthesis was still evident in cells with severely down-regulated PKD1 after transfection of siRNA targeting PKD1. A major point raised by our study is that CID755673 can not be considered a specific inhibitor of PKD and it should be used with great caution in experiments attempting to elucidate the role of PKD family members in cellular regulation, particularly cell cycle progression from G(1)/G(o) to S phase.

Protein Kinase D1 Mediates Stimulation of DNA Synthesis and Proliferation in Intestinal Epithelial IEC-18 Cells and in Mouse Intestinal Crypts

We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the Gq-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with Gq-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser744 and Ser748 and on the autophosphorylation site, Ser916. To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.

Abstract 4007: Different patterns of Akt and ERK feedback activation in response to allosteric and active-site mTOR inhibitors in comparison to metformin in pancreatic cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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 feedback 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 allosteric mTOR inhibitors (rapamycin and everolimus) or active-site mTOR inhibitors (KU63794 and PP242) suppressed p70S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin and everolimus caused a striking increase in Akt phosphorylation at Ser473 while the active-site inhibitors of mTOR completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin and everolimus did not have any stimulatory effect on ERK activation. Exposure to the compound A66, a selective inhibitor of the 110α catalytic subunit of PI3K, did not prevent enhancement of ERK activation in response to exposure to either KU63794 or PP242, implying that KU63794 and PP242 enhance ERK via a novel PI3K-independent pathway. These results suggest that active-site inhibitors, in contrast to mTORC1 allosteric inhibitors, enhanced ERK through a PI3K-independent pathway. In contrast, metformin which potently inhibited the mTORC1/S6K axis did not cause over-stimulation of Akt phosphorylation on Ser473 and inhibited mitogen-stimulated ERK activation in PDAC cells. 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 mTORC1/S6K. Metformin is emerging as a potential novel agent in cancer chemoprevention. We propose that the favorable effects of metformin on preventing over-activation of pro-oncogenic pathways, such as Akt and ERK, may contribute to its beneficial anticancer effects. Citation Format: Heloisa P. Soares, Ni Yang, James Sinnett-Smith, Robert Kui, Enrique Rozengurt. Different patterns of Akt and ERK feedback activation in response to allosteric and active-site mTOR inhibitors in comparison to metformin in pancreatic cancer 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 4007. doi:10.1158/1538-7445.AM2013-4007

751 Transgenic Protein Kinase D1 Stimulates the Rate of Epithelial Cell Proliferation in Intestinal Crypts

Background: The sequential proliferation, lineage-specific differentiation, crypt-villus migration, and cell death of the epithelial cells of the intestinal mucosa is tightly regulated by regulatory peptides, differentiation signals, and luminal stimuli, including nutrients and pathogenic/commensal organisms. Despite its importance for understanding normal homeostasis and pathogenesis of disease states, the intracellular signal transduction mechanisms involved remain incompletely understood. Here, we tested the hypothesis that PKD1 plays a key role in intestinal crypt cell proliferation In Vivo. Results: To clarify the role of PKD1 in intestinal epithelial cell proliferation In Vivo, we generated transgenic mice that express elevated PKD1 protein in the distal small intestinal and proximal colonic epithelium. The catalytic activity and multi-site phosphorylation (Ser744, Ser748 and Ser916) of PKD1 was strikingly higher in extracts from ileal mucosa of transgenic mice as compared with non-transgenic littermates. These results indicate that transgenic PKD1 is functional in the intestinal epithelium. PKD1 signaling stimulated intestinal cell proliferation, as shown by detection of 5-bromo-2-deoxyuridine (BrdU) incorporated into the cell nuclei of crypt cells of the ileum and proximal colon, where PKD1 protein is maximally expressed. Our results demonstrated a highly statistically significant increase (p<0.005) in DNA synthesizing cells in the crypts of the PKD1 transgenic mice as compared with non-transgenic littermates. In the intestine, normal cell numbers are maintained by balancing rates of cell proliferation, differentiation, migration and apoptosis. Consequently, we determined whether transgenic PKD1 leads to a change in tissue architecture, manifested by an increase in the size and total number of epithelial cells in the crypts. We measured crypt height (in micrometer and cell number) and crypt circumference (in micrometer and cell number) in histological sections of control and PKD1 transgenic mice. The data was used to calculate the size of individual cells and the total number of cells per crypt. Our results show a significant increase in the depth (either in μm or in number of cells) and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the nontransgenic littermates (276 total cells per ileal crypt in transgenic mice versus 192 in nontransgenic mice; p< 0.005). These results indicate that the expression of the PKD1 transgene led to a marked increase (44%) in the total number of intestinal epithelial cells per crypt. Conclusion: Transgenic PKD1 expression increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. These results support the hypothesis that PKD1 signaling plays a role in a pathway leading to proliferation in intestinal epithelial cells.

M1604 Transgenic Expression of PKD Stimulates Intestinal Epithelial Cell Proliferation

intestinal crypts and villi. Since defective Hh signaling causes IEC hyperproliferation, we hypothesized that the increased growth factor receptor expression and signaling may modulate Hh expression. Inhibitors of EGFR, MEK-1 and PI3K caused increased Shh & Ihh mRNA and protein levels, while EGF had the opposite effects. CONCLUSIONS: Our studies suggest that Hhs are major effectors of β1 integrin signaling during intestinal epithelial development. This β1 integrin-Hh signaling pathway represents a novel mechanism by which the extracellular matrix regulates IEC proliferation and intestinal development. β1 integrins may regulate Hh expression through crosstalk with growth factor signaling pathways in IECs.

S1691 Endogenous Protein Kinase D Contributes to Gq-Coupled Receptor Induction of DNA Synthesis in Swiss 3T3 Cells

overexpressing prostaglandin dehydrogenase (18.PGDH) comprise a model with abolished basal PG production. Pyk2 expression, nearly absent in these cells, was reintroduced by transient transfection either as wild type or a kinase deficient (KD) mutant form. Pyk2 immunoprecipitates were used to phosphorylate a FAK regulatory domain segment with either wild type Y397 or mutant Y397F fused to GST. Total 32P incorporation In Vitro from 32P-ATP was measured by autoradiography. Y397 phosphorylation of GST-FAK In Vitro, and endogenous FAK in cell extracts, was detected by Western blot analysis with phosphospecific FAK Y397 antibody. Results: IEC-18 cells with siRNA-reduced Pyk2 expression exhibited similar levels of total FAK protein, but very low basal FAK Y397 phosphorylation in cell extracts. 18.PGDH cells, in comparison with parental IEC-18 cells, also exhibited selectively reduced constitutive FAK Tyr397 phosphorylation. Transfected Pyk2, but not Pyk2-KD, partially restored constitutive FAK Y397 phosphorylation in these cells. Pyk2, but not Pyk2-KD immunoprecipitates incorporated 32P into both GST-FAK and, to a lesser extent, GST-FAK(Y397F), but not GST. In particular, phospho-Y397Western blot confirmed that Pyk2 catalyzed dramatically increased FAK Y397 phosphorylation in GST-FAK. Conclusions: Taken together, these studies extend the observed correlation between Pyk2 downregulation and FAK Y397 phosphorylation in intestinal cells deprived of basal PG, indicating that Pyk2 can directly phosphorylate FAK Y397 as well as additional, as yet unidentified site(s) within the FAK regulatory domain.

Modulation of gastric mucosal paracellular permeability by secretin: Role of src family tyrosine kinase

Background: Previous studies found that physiological concentrations of secretin increase transepithelial resistance (TER) in gastric mucosa in a fashion synergistic with EGF, indicating that spacially limited receptors and distinct signal transduction pathways are involved in the regulation of paracellular permeability. Pretreatment with PP2, a selective inhibitor of sm family of tyrosine kinase, significantly attenuated the increase in TER activated by secretio, but not by EGF, suggesting the src family kinases mediates secretin action on paracelloiar permeability. Aim and Methods: In order to identify the mechanism(s) by which secretio receptors lead to src family kinase activation, we examined the stimulation of src Idnsse activity measured by the autophosphorylation of Tyr416 and the inhibition of the overall profile of cellular protein tyrosine phosphorylation in primary canine gastric epithelial cell monoiayors by PP2(5/~M) after secretin (lnM) activation. Results: Primary gastric epithelial cell cultures showed consistent amount of phosphorylation of src Tyr416 at the basal levels and the stimulation by secretin only caused a maximal 2-fold activation of src activity after 10 to 15 min. However, this activation was not blocked by pretreatment of PP2. When total lysates were blotted with tyrosine phosphorylation antibody (4G10), several tyrosine phosphoryluted proteins activated by secretin were blocked by pretreatment with PP2 in a dose-dependent manner. The secretin-activated tyrosine phosphorylations were not inhibited by pretreatment with PP3(inactive src kinase inhibitor), nor with AG1478, a specific EGFR tyrosine kinase inhibitor, suggesting that these tyrosine pbosphofylated proteins serve either as src-family kinases or as substrates for these kinases. Conclusion: Our results showed that secretin activated specific tyrosine phosphorylated proteins via src-family tyrosine kinases. This finding suggests a novel function of src-family tyrosine kinases in regulating gastric paracelluiar permeability.