kt-5720 and Breast-Neoplasms

Voltage-gated Na(+) channels (VGSCs) are expressed in excitable cells (e.g. neurons and muscles), as well as in some classically 'non-excitable' cells (e.g. fibroblasts), and in carcinomas. In general, functional expression of VGSCs in plasma membrane (PM) is hierarchical and dynamic. Previously, we have shown that an activity-dependent positive feedback mechanism involving cAMP-dependent protein kinase A (PKA) plays a significant role in upregulation of VGSCs in strongly metastatic rat prostate cancer Mat-LyLu cells expressing Nav1.7. Here, we investigated the possible role of PKA in VGSC regulation and its functional consequences in strongly metastatic human breast cancer (BCa) MDA-MB-231 cells, where the neonatal splice form of Nav1.5 (nNav1.5) is the predominant VGSC present. Treatment with the PKA activator forskolin for 24h increased mRNA and PM protein levels of nNav1.5, without changing the total VGSC protein level. Opposite effects were obtained by application of the PKA inhibitor KT5720 or the highly specific VGSC blocker tetrodotoxin (TTX), the latter implying activity-dependent upregulation. We tested the possibility, therefore, that the activity dependence of VGSC (nNav1.5) expression involved PKA. Indeed, TTX pretreatment reduced the level of phosphorylated PKA and eliminated basal and PKA-stimulated cellular migration. These data suggested that activity-dependent positive feedback mediated by PKA plays an important role in the functional expression of nNav1.5 in BCa, and in turn, this enhances the cells' metastatic potential.

Topics: Animals; Breast Neoplasms; Carbazoles; Cell Line, Tumor; Cell Movement; Colforsin; Cyclic AMP-Dependent Protein Kinases; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Humans; Infant, Newborn; Muscle Proteins; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Phosphorylation; Pyrroles; Rats; Sodium Channels; Stromal Cells; Tetrodotoxin; Up-Regulation

Previously, we have shown that leptin potentiates the antiproliferative action of cAMP elevating agents in breast cancer cells and that the protein kinase A (PKA) inhibitor KT-5720 prevented the antiproliferative effects induced by the leptin plus cAMP elevation. The present experiments were designed to gain a better understanding about the PKA role in the antitumor interaction between leptin and cAMP elevating agents and on the underlying signaling pathways. Here we show that exposure of MDA-MB-231 breast cancer cells to leptin resulted in a strong phosphorylation of both ERK1/2 and STAT3. Interestingly, intracellular cAMP elevation upon forskolin pretreatment completely abrogated both ERK1/2 and STAT3 phosphorylation in response to leptin and was accompanied by a consistent CREB phosphorylation. Notably, leptin plus forskolin cotreatments resulted in a strong decrease of both PKA regulatory RIα and catalytic subunits protein levels. Importantly, pretreatment with the PKA inhibitor KT-5720 blocked the forskolin-induced CREB phosphorylation and prevented both the inhibition by forskolin of leptin-induced ERK1/2 and STAT3 phosphorylation and the PKA subunits down-regulation induced by the combination of leptin and forskolin. Altogether, our results indicate that leptin-dependent signaling pathways are influenced by cAMP elevation and identify PKA as relevantly involved in the pharmacological antitumor interaction between leptin and cAMP elevating drugs in MDA-MB-231 cells. We propose a molecular model by which PKA confers its effects. Potential therapeutic applications by our data will be discussed.

Topics: Antineoplastic Agents; Breast Neoplasms; Carbazoles; Cell Line, Tumor; Cell Proliferation; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Enzyme Activators; Female; Humans; Leptin; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Protein Kinase Inhibitors; Pyrroles; Recombinant Proteins; Signal Transduction; STAT3 Transcription Factor; Time Factors; Up-Regulation

Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the anti-proliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signalling might represent a simple novel way for therapeutic intervention in breast cancer.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Apoptosis; Breast Neoplasms; Carbazoles; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclin-Dependent Kinase Inhibitor p27; Female; G1 Phase; Humans; Leptin; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Signal Transduction

Estrogen triggers rapid yet transient activation of the MAPKs, extracellular signal-regulated kinase (Erk)-1 and Erk-2. We have reported that this estrogen action requires the G protein-coupled receptor, GPR30, and occurs via Gbetagamma-subunit protein-dependent transactivation of the epidermal growth factor (EGF) receptor through the release of pro-heparan-bound EGF from the cell surface. Here we investigate the mechanism by which Erk-1/-2 activity is rapidly restored to basal levels after estrogen stimulation. Evidence is provided that attenuation of Erk-1/-2 activity by estrogen occurs via GPR30-dependent stimulation of adenylyl cyclase and cAMP-dependent signaling that results in Raf-1 inactivation. We show that 17beta-E2 represses EGF-induced activation of the Raf-to-Erk pathway in human breast carcinoma cells that express GPR30, including MCF-7 and SKBR3 cells which express both or neither, ER, respectively. MDA-MB-231 cells, which express ERbeta, but not ERalpha, and low levels of GPR30 protein, are unable to stimulate adenylyl cyclase or promote estrogen-mediated blockade of EGF-induced activation of Erk-1/-2. Pretreatment of MDA-MB-231 cells with cholera toxin, which ADP-ribosylates and activates Galphas subunit proteins, results in G protein-coupled receptor (GPCR)-independent adenylyl cyclase activity and suppression of EGF-induced Erk-1/-2 activity. Transfection of GPR30 into MDA-MB-231 cells restores their ability to stimulate adenylyl cyclase and attenuate EGF-induced activation of Erk-1/-2 by estrogen. Moreover, GPR30-dependent, cAMP-mediated attenuation of EGF-induced Erk-1/-2 activity was achieved by ER antagonists such as tamoxifen or ICI 182, 780; yet not by 17alpha-E2 or progesterone. Thus, our data delineate a novel mechanism, requiring GPR30 and estrogen, that acts to regulate Erk-1/-2 activity via an inhibitory signal mediated by cAMP. Coupled with our prior findings, these current data imply that estrogen balances Erk-1/-2 activity through a single GPCR via two distinct G protein-dependent signaling pathways that have opposing effects on the EGF receptor-to-MAPK pathway.

Topics: Adenylyl Cyclases; Breast Neoplasms; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; ErbB Receptors; Estradiol; Estrogen Antagonists; Estrogens; Fulvestrant; Humans; Indoles; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-raf; Pyrroles; Receptors, Cell Surface; Receptors, Estrogen; Receptors, G-Protein-Coupled; Tamoxifen; Tumor Cells, Cultured

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of 32P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723-20729, 1989; Mol Cell Biol 10:3020-3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca++, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr(R) and murine melanoma B16/Adr(R) cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.

Topics: Alkaloids; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Calcium; Carbazoles; Carrier Proteins; Clone Cells; Cyclic AMP; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Humans; Indole Alkaloids; Indoles; KB Cells; Melanoma, Experimental; Membrane Glycoproteins; Mice; Neoplasm Proteins; Phosphatidylserines; Phosphorylation; Phosphothreonine; Phosphotyrosine; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Pyrroles; Staurosporine; Transfection; Tumor Cells, Cultured; Tyrosine