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Effect of Insulin on Cell Cycle Progression in MCF-7 Breast Cancer Cells

2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês

10.1074/jbc.m104416200

1083-351X

James Chappell, J. Wayne Leitner, Scott R. Solomon, Inga Golovchenko, Marc L. Goalstone, Boris Draznin,

Protein Kinase Regulation and GTPase Signaling

We recently demonstrated that in MCF-7 breast cancer cells, insulin promoted the phosphorylation and activation of geranylgeranyltransferase I (GGTI-I), increased the amounts of geranylgeranylated Rho-A and potentiated the transactivating activity of lysophosphatidic acid (LPA) (Chappell, J., Golovchenko, I., Wall, K., Stjernholm, R., Leitner, J., Goalstone, M., and Draznin, B. (2000)J. Biol. Chem. 275, 31792–31797). In the present study, we explored the mechanism of this potentiating effect of insulin on LPA. Insulin (10 nm) potentiated the ability of LPA to stimulate cell cycle progression and DNA synthesis in MCF-7 cells. The potentiating effect of insulin appears to involve increases in the expression of cyclin E and decreases in the expression of the cyclin-dependent kinase inhibitor p27Kip1. All potentiating effects of insulin were inhibited in the presence of an inhibitor of GGTase I, GGTI-286 (3 μm) or by an expression of a dominant negative mutant of Rho-A. In contrast to its potentiating action, a direct mitogenic effect of insulin in MCF-7 cells involves activation of phosphatidylinositol 3-kinase and increased expression of cyclin D1. We conclude that the ability of insulin to increase the cellular amounts of geranylgeranylated Rho-A results in potentiation of the LPA effect on cyclin E expression and degradation of p27Kip1 and cell cycle progression in MCF-7 breast cancer cells. We recently demonstrated that in MCF-7 breast cancer cells, insulin promoted the phosphorylation and activation of geranylgeranyltransferase I (GGTI-I), increased the amounts of geranylgeranylated Rho-A and potentiated the transactivating activity of lysophosphatidic acid (LPA) (Chappell, J., Golovchenko, I., Wall, K., Stjernholm, R., Leitner, J., Goalstone, M., and Draznin, B. (2000)J. Biol. Chem. 275, 31792–31797). In the present study, we explored the mechanism of this potentiating effect of insulin on LPA. Insulin (10 nm) potentiated the ability of LPA to stimulate cell cycle progression and DNA synthesis in MCF-7 cells. The potentiating effect of insulin appears to involve increases in the expression of cyclin E and decreases in the expression of the cyclin-dependent kinase inhibitor p27Kip1. All potentiating effects of insulin were inhibited in the presence of an inhibitor of GGTase I, GGTI-286 (3 μm) or by an expression of a dominant negative mutant of Rho-A. In contrast to its potentiating action, a direct mitogenic effect of insulin in MCF-7 cells involves activation of phosphatidylinositol 3-kinase and increased expression of cyclin D1. We conclude that the ability of insulin to increase the cellular amounts of geranylgeranylated Rho-A results in potentiation of the LPA effect on cyclin E expression and degradation of p27Kip1 and cell cycle progression in MCF-7 breast cancer cells. geranylgeranyltransferase lysophosphatidic acid retinoblastoma phosphatidylinositol 3-kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase mitogen-activated protein extracellular signal-regulated kinase cyclin-dependent kinase Both hyperinsulinemia and cancer are extremely prevalent pathophysiological conditions associated with major morbidity and mortality. The implication of insulin in the pathogenesis of cancer has been in the English medical literature since the early 1970's (1Kessler I.I. J. Chronic Dis. 1971; 23: 579-600Abstract Full Text PDF PubMed Scopus (66) Google Scholar). 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Sci. 1996; 784: 173-188Crossref PubMed Scopus (58) Google Scholar), and a recent abstract in the American Society of Clinical Oncology (22Goodwin P.J. Ennis M. Pritchard K.I. Trudeau M.E. Koo J. Madaranas Y. Hood N. Hartwick W. Hoffman B. American Society of Clinical Oncology Online. 2000; (asco.org)Google Scholar) indicated that fasting insulin levels predict disease-free survival and overall survival of women with breast cancer who are receiving adjuvant therapy. We have recently identified a novel aspect of insulin action: its ability to stimulate prenylation of small molecular weight GTPases (23Goalstone M.L. Draznin B. J. Biol. Chem. 1996; 271: 27585-27589Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24Goalstone M.L. Leitner J.W. Wall K. Dolgonos L. Rother K.I. Accili D. Draznin B. J. Biol. Chem. 1998; 273: 23892-23896Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Chappell J. Golovchenko I. Wall K. Stjernholm R. Leitner J. Goalstone M. 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Bioessays. 1995; 17: 395-404Crossref PubMed Scopus (193) Google Scholar). Posttranslational modification of these proteins by prenylation appears to be a prerequisite for their subsequent activation (30Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1457) Google Scholar, 31Willumsen B.M. Christensen A. Hubbert N.L. Papageorge A.G. Lowy D.R. Nature. 1984; 310: 583-586Crossref PubMed Scopus (396) Google Scholar). Hyperinsulinemia significantly increases the activities of farnesyltransferase (FTase) and geranylgeranyltransferases I and II (GGTase1 I and II) (23Goalstone M.L. Draznin B. J. Biol. Chem. 1996; 271: 27585-27589Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24Goalstone M.L. Leitner J.W. Wall K. Dolgonos L. Rother K.I. Accili D. Draznin B. J. Biol. Chem. 1998; 273: 23892-23896Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Chappell J. Golovchenko I. Wall K. Stjernholm R. Leitner J. Goalstone M. Draznin B. J. Biol. Chem. 2000; 275: 31792-31797Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 26Goalstone M.L. Leitner J.W. Golovchenko I. Stjernjolm M.R. Cormont M. Le Marchand-Brustel Y. Draznin B. J. Biol. Chem. 1999; 274: 2880-2884Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Increased activity of these prenyltransferases leads to increases in the amounts of farnesylated p21Ras and geranylgeranylated Rho-A in various tissues (23Goalstone M.L. Draznin B. J. Biol. Chem. 1996; 271: 27585-27589Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24Goalstone M.L. Leitner J.W. Wall K. Dolgonos L. Rother K.I. Accili D. Draznin B. J. Biol. Chem. 1998; 273: 23892-23896Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Chappell J. Golovchenko I. Wall K. Stjernholm R. Leitner J. Goalstone M. Draznin B. J. Biol. Chem. 2000; 275: 31792-31797Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 26Goalstone M.L. Leitner J.W. Golovchenko I. Stjernjolm M.R. Cormont M. Le Marchand-Brustel Y. Draznin B. J. Biol. Chem. 1999; 274: 2880-2884Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 32Golovchenko I. Goalstone M.L. Watson P. Brownlee M. Draznin B. Circ. Res. 2000; 87: 746-752Crossref PubMed Scopus (94) Google Scholar). These increases provide more prenylated p21Ras and Rho-A available for activation by other growth promoting agents, thus augmenting cellular mitogenic responses (24Goalstone M.L. Leitner J.W. Wall K. Dolgonos L. Rother K.I. Accili D. Draznin B. J. Biol. Chem. 1998; 273: 23892-23896Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Chappell J. Golovchenko I. Wall K. Stjernholm R. Leitner J. Goalstone M. Draznin B. J. Biol. Chem. 2000; 275: 31792-31797Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 32Golovchenko I. Goalstone M.L. Watson P. Brownlee M. Draznin B. Circ. Res. 2000; 87: 746-752Crossref PubMed Scopus (94) Google Scholar). We have recently demonstrated in a MCF-7 breast cancer cell line, that insulin promoted the phosphorylation and activation of GGTase I (25Chappell J. Golovchenko I. Wall K. Stjernholm R. Leitner J. Goalstone M. Draznin B. J. Biol. Chem. 2000; 275: 31792-31797Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Increases in the GGTase I activity resulted in significant augmentation of the amounts of geranylgeranylated Rho-A in the insulin-treated cells and potentiation of the nuclear effects ofl-α-lysophosphatidic acid (LPA). In the present experiments, we explore the mechanism of the potentiating effect of insulin on LPA-induced cell cycle progression and DNA synthesis in MCF-7 breast cancer cells. Tissue culture media was from Meditech, Inc. (Herndon, VA). Gentamicin was from Life Technologies, Inc. (Gaithersburg, MD). Fetal calf serum was from Gemini Bio-Products, Inc. (Calabasas, CA). Bovine serum albumin, LPA, and other biochemicals were from Sigma. Insulin was from Eli Lilly (Indianapolis, IN). Anti-cyclin E and anti-cyclin D1 mouse monoclonal antibodies and geranylgeranyltransferase inhibitor-286 (GGTI-286) were from Calbiochem (San Diego). Anti-p27 and Anti-retinoblastoma (Rb) mouse monoclonal antibodies, protein G-plus/protein A-agarose and immunoprecipitation reagents were from Oncogene Science, Inc. (Cambridge, MA). Bicinchoninic acid Protein Assay Kit was from Pierce. SDS-polyacrylamide gel electrophoresis supplies and reagents were from Bio-Rad (Hercules, CA). MCF-7 breast cancer cells were a gift from Dr. Carla L. Van Den Berg (University of Colorado Health Science Center, Denver, CO), and a dominant negative mutant of Rho-A was a gift from Dr. J. J. Baldassare (St. Louis University, St. Louis, MO), and BrdUrd Cell Proliferation enzyme-linked immunosorbent assay kit was from Roche Molecular Biochemicals. MCF-7 cells were grown to 80% confluence at 37 °C, 5% CO2 in improved minimal essential media + 5% heat inactivated fetal bovine serum, non-essential amino acids, l-glutamine (200 mm), and insulin (60 pm). The cells were serum- and insulin-starved for 24 h and then preincubated with insulin (10 nm) for 24 h with and without 3 μmGGTI-286, 100 nm wortmannin, or 20 μmPD98059. Cells were then incubated for an additional 24 h with LPA (20 μm). Cells were lifted from the plates using 2 ml Versene 1:5000 (Life Technologies, Inc.), pelleted in 1× phosphate buffered saline, and stained with propidium iodide using the method of Krishan (33Krishan A. J. Cell Biol. 1975; 66: 188-193Crossref PubMed Scopus (1496) Google Scholar). Cell cycle analysis was performed using a Coulter Epics XL flow cytometer (Beckman-Coulter, Hialeah, FL). Alignment of the instrument was verified daily using DNA check beads (Coulter). Peakversus integral gating was used to exclude doublet events from the analysis. Data were collected for 10,000 events. Modfit LT (Verity Software House, Topsham, MA) was used for cell cycle modeling. MCF-7 cells were grown to 80% confluence in 96-well plates as described above. The cells were serum- and insulin-starved for 24 h and then preincubated with insulin (10 nm) for 24 h with and without 3 μm GGTI-286. Cells were then incubated for an additional 8 h with LPA (20 μm) in the presence of 10 μl of BrdUrd (diluted 1:100 in serum-free medium). Labeling medium was removed and BrdUrd incorporation was determined by cell proliferation enzyme-linked immunosorbent assay, visualized, and quantified by colorimetry. MCF-7 cells were grown to 80% confluence as described above. The cells were serum- and insulin-starved for 24 h and then preincubated with insulin (10 nm) for 24 h with and without 3 μmGGTI-286. Cells were then incubated for an additional 24 h with LPA (20 μm). Cells were washed with phosphate-buffered saline and lysed using a Triton X-100-based lysis buffer (50 mm HEPES, pH 7.5, 15 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 1 mmNa2HPO4, 1% Triton X-100, 1 mmdithiothreitol, 1 mm Na vanadate, 0.05% SDS, 10 μg/ml aprotinin, 10 μg/ml leupeptin), sonicated, and centrifuged, and protein concentrations were diluted to 1 mg/ml. Cyclin E and cyclin D1 protein were immunoprecipitated using the respective antibodies. Immunoprecipitates were resolved by polyacrylamide gel electrophoresis, determined by Western blot, and quantitated by densitometry. MCF-7 cells were grown to 80% confluence as described above. The cells were serum- and insulin-starved for 24 h and then preincubated with insulin (10 nm) for 24 h with and without 3 μmGGTI-286. Cells were then incubated for an additional 24 h with LPA (20 μm). Cells were washed with phosphate-buffered saline and lysed as above, sonicated, and centrifuged, and protein concentrations were diluted to 1 mg/ml. p27Kip1 and Rb protein were immunoprecipitated using the respective antibodies. Immunoprecipitates were resolved by polyacrylamide gel electrophoresis, determined by Western blot and quantitated by densitometry. For the assessment of Rb phosphorylation, the Western blot of the Rb immunoprecipitates was performed using anti-phosphoserine and anti-phosphothreonine antibody. Statistics were analyzed by Student's paired or unpaired t test, with p < 0.05 considered significant. Because our recently published observations (35Jackson J. White M. Yee D. J. Biol. Chem. 1998; 273: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) indicated that hyperinsulinemia potentiates the nuclear effects of LPA by increasing the availability of geranylgeranylated Rho-A, the current experiments were performed to further elucidate the mechanism of the potentiating influence of insulin on growth of MCF-7 cells. In the initial experiments, we examined the direct effects of insulin on MCF-7 breast cancer cell proliferation by florescence activated cell cycle analysis. Insulin (10 nm) enhanced cell growth by ∼40% over control when supplemented in serum free medium (Fig.1). The effect of insulin was dose-dependent, with 100 nm insulin enhancing growth by 70% (not shown). Addition of wortmannin (100 nm), an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), completely blocked the mitogenic effect of insulin, whereas the MEK inhibitor PD98059 (20 μm) had no significant effect on insulin-induced growth (Fig. 1). Thus, these experiments confirmed that the direct mitogenic action of insulin in MCF-7 cells is dependent upon the PI 3-kinase branch of insulin's intracellular signaling (34Dufourny B. Alblas J. van Teeffelen H. van Schaik F. van der Burg B. Steenbergh P. Sussenbach J. J. Biol. Chem. 1997; 272: 31163-31171Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 35Jackson J. White M. Yee D. J. Biol. Chem. 1998; 273: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Because the effect of insulin on GGTase I and geranylgeranylation of Rho-A is mediated by the Shc-Ras-MAP kinase pathway and is completely independent of PI 3-kinase signaling (36Goalstone M.L. Leitner J.W. Berhanu P Sharma P.G. Olefsky J.M. Draznin B. J. Biol. Chem. 2001; 276: 12805-12812Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), these cells serve as an excellent model to examine the mechanism of the potentiating influence of insulin independent of its direct mitogenic action, which is PI-3-kinase-dependent. We then examined the potentiating effect of insulin on the mitogenic action of LPA. Insulin (10 nm) alone showed the same 40% increase in growth over control, whereas LPA (20 μm) enhanced MCF-7 cell growth 5% over control (Fig.2). When LPA was added to MCF-7 cells, which were preincubated with 10 nm insulin for 24 h, growth was enhanced to 65% over control. This potentiating effect of insulin was blocked by the addition of the geranylgeranyltransferase inhibitor GGTI-286 (3 μm). GGTI-286 had no effect on insulin alone (data not shown). While addition of wortmannin completely blocked the effect of insulin (Fig. 1), it did not affect the ability of insulin to potentiate LPA action to ∼25% above controlversus 5% with LPA alone (Fig. 2). These findings indicate that the potentiating effect of insulin is not dependent on PI 3-kinase. Finally, addition of GGTI to insulin, LPA, and wortmannin completely blocked the potentiating effect of insulin. BrdUrd incorporation into DNA is another marker of DNA synthesis and cell proliferation. Therefore, we examined the ability of insulin to potentiate the effect of LPA on BrdUrd incorporation in MCF-7 cells. Insulin alone increased BrdUrd incorporation into DNA 18% above control (Fig. 3), whereas LPA alone increased BrdUrd incorporation by 7%. When LPA was added to cells, which were preincubated with 10 nm insulin, BrdUrd incorporation into DNA was enhanced to 27%. If insulin potentiates the effect of LPA by increasing the availability of prenylated Rho-A, then inhibition of geranylgeranylation of Rho-A should block the potentiating effect of insulin. Indeed, addition of GGTI-286 to the preincubation medium blocked the potentiating effect of insulin, significantly decreasing the incorporation of BrdUrd as compared with the cells incubated with insulin and LPA. BrdUrd incorporation in cells incubated with insulin in combination with 3 μm GGTI-286 was not significantly different from that observed in experiments with insulin alone (not shown). To confirm the role of Rho-A in mediating the potentiating effect of insulin, cells were transiently transfected with a dominant negative mutant of Rho-A prior to the insulin and LPA challenges. Expression of a dominant negative Rho-A completely blocked the ability of insulin to potentiate the effect of LPA on BrdUrd incorporation (Fig. 3), without any effect on insulin alone. Because flow cytometry and BrdUrd incorporation represent surrogate measures of mitogenesis, we performed direct cell count of MCF-7 cells exposed to insulin and LPA (Fig.3B). We observed a synergistic effect of insulin and LPA on cell growth that was completely abolished in the presence of either a GGTase I inhibitor or a dominant negative mutant of Rho-A. Taken together, these experiments suggest that the potentiating effect of insulin is related to the ability of insulin to activate GGTase I, while its direct effect is independent of its action on prenylation. To assess whether an introduction of a dominant negative Rho-A could squelch the signal transduction proteins, we examined the effect of insulin on the phosphorylation of ERK 1/2 and Akt in control and transfected MCF-7 cells. Transfection of a dominant negative Rho-A had no effect on the ability of insulin to stimulate the phosphorylation of ERK 1/2 and Akt (not shown), suggesting normal functioning of signaling pathways in these cells. Because insulin increased the progression of MCF-7 cells into cell cycle and potentiated the mitogenic effect of LPA, we examined the effect of 10 nm insulin on cell cycle initiation by evaluating its effect on cyclin D1 and cyclin E expression. The aim of these experiments was to identify the point in the initiation of the cell cycle that is potentiated by insulin. Insulin (10 nm) stimulated cyclin D1 production ∼30% over control, whereas LPA (20 μm) alone did not stimulate cyclin D1 production over control (Fig.4A). However, insulin did not potentiate the effect of LPA on cyclin D1. In cells preincubated with 10 nm insulin and then exposed to 20 μm LPA, cyclin D1 production was not significantly different from that seen in the cells incubated with LPA alone. In contrast to the experiments with cyclin D1, insulin (10 nm) alone showed no significant stimulatory effect on cyclin E production, whereas LPA (20 μm) stimulated cyclin E production almost 2-fold (Fig. 4B). However, preincubation of MCF-7 cells with 10 nm insulin significantly increased the LPA-stimulated cyclin E production to more than 3-fold as compared with controls. This potentiating effect of insulin was completely abolished with the addition of 3 μm GGTI-286 or by a transient transfection of a dominant negative mutant of Rho-A (Fig. 4B), again suggesting that the potentiating influence of insulin is mediated by its effect on GGTase I. Because the function of cyclin E is partly regulated by its interactions with the cyclin-dependent kinase (Cdk) inhibitor p27Kip1(reviewed in Refs. 37Gitig D. Koff A. Meth. Mol. Biol. 2000; 142: 109-123PubMed Google Scholar and 38Sherr C. Roberts J. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5131) Google Scholar) and because Rho-A stimulates p27Kip1 degradation through its regulation of cyclin E-Cdk 2 activity (39Hu W. Bellone C. Baldassare J. J. Biol. Chem. 1999; 274: 3396-3401Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), we examined insulin's effect on p27Kip1expression in MCF-7 cells. Either insulin (10 nm) or LPA (20 μm) alone showed a mild and non-significant inhibitory effect on p27Kip1 production. However, when LPA (20 μm) was added to MCF-7 cells preincubated with 10 nm insulin for 24 h, p27Kip1 production was significantly decreased by 33% (p < 0.04) (Fig.5). The presence of GGTI-286 or transfection of a dominant negative Rho-A completely abrogated a decrease in p27Kip1 that was caused by insulin in combination with LPA (Fig. 5). Regulation of the Rb protein function occurs predominantly though the phosphorylation of serine and threonine residues in the Rb protein predominantly by cyclin D1-Cdk 2/6 (reviewed in Ref. 40Bartek J. Bartkova J. Lukas J. Curr. Opin. Cell Biol. 1996; 8: 805-814Crossref PubMed Scopus (366) Google Scholar). Phosphorylation of these residues results in the dissociation of the Rb protein from the E2F transcription factor thereby resulting in increased RNA transcription and progression of cells through the restriction point into the cell cycle. We therefore examined insulin's effect on the phosphorylation of the Rb protein. Although insulin and LPA both increased Rb phosphorylation, we did not observe a potentiating effect of insulin on LPA-mediated Rb phosphorylation (not shown). Epidemiological studies show that the incidence of breast cancer is increased in women with hyperinsulinemic disorders including Type 2 diabetes and polycystic ovary syndrome (2Giovannucci E. Cancer Causes Control. 1995; 6: 164-179Crossref PubMed Scopus (684) Google Scholar, 3Wideroff L. Gridley G. Mellemkjaer L. Chow W.H. Linet M. Keehn S. Borch-Johnson K. Olsen J.H. J. Natl. Cancer Inst. 1997; 89: 1360-1365Crossref PubMed Scopus (571) Google Scholar, 4Weiderpass E. Gridley G. Persson I. Nyren O. Ekbom A. Adami H.O. Int. J. Cancer. 1997; 71: 360-363Crossref PubMed Scopus (175) Google Scholar, 10Stoll B.A. Breast Cancer Res. Treat. 1996; 38: 239-246Crossref PubMed Scopus (49) Google Scholar, 41Levine W. Dyer A.R. Shekelle R.B. Schoenberger J.A. Stamler J. Am. J. Epidemiol. 1990; 131: 254-262Crossref PubMed Scopus (93) Google Scholar), though only slightly greater than in the general population (∼1.6-fold). Most of these epidemiological studies, however, have been based on populations of patients from diabetic clinics without regard to type of diabetes (Type 1 or Type 2). Data from several animal studies show a protective effect from cancer in Type 1 diabetes with a lower incidence of breast cancer (42Heuson J.C. Legros N. Heimann R. Can. Res. 1972; 32: 233-238PubMed Google Scholar, 43Cocca C. Martin G. Rivera E. Davio C. Cricco G. Lemos B. Fitzsimons C. Gutierrez A. Levin E. Levin R. Croci M. Bergoc R.M. Eur. J. Cancer. 1998; 34: 889-894Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 44Cohen N.D. Hilf R. Proc. Soc. Exp. Biol. Med. 1975; 148: 339-343Crossref PubMed Scopus (10) Google Scholar) and tumor regression (43Cocca C. Martin G. Rivera E. Davio C. Cricco G. Lemos B. Fitzsimons C. Gutierrez A. Levin E. Levin R. Croci M. Bergoc R.M. Eur. J. Cancer. 1998; 34: 889-894Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 44Cohen N.D. Hilf R. Proc. Soc. Exp. Biol. Med. 1975; 148: 339-343Crossref PubMed Scopus (10) Google Scholar, 45Heuson J.C. Legros N. Can. Res. 1972; 32: 226-232PubMed Google Scholar, 46Shafie S.M. Hilf R. Can. Res. 1978; 38: 759-764PubMed Google Scholar) in rats with mammary tumors made diabetic (insulinopenic) using streptozotocin or alloxan. Alternatively, administration of insulin to rats bearing mammary tumors resulted in a significant increase in tumor growth (42Heuson J.C. Legros N. Heimann R. Can. Res. 1972; 32: 233-238PubMed Google Scholar). These findings suggest that the epidemiological studies may have underestimated the risk of breast cancer in the hyperinsulinemic state (Type 2 diabetes). In previous experiments, we have shown insulin's ability to induce prenylation of low molecular weight GTPases, specifically farnesylation of Ras protein and geranylgeranylation of Rho-A protein (23Goalstone M.L. Draznin B. J. Biol. Chem. 1996; 271: 27585-27589Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24Goalstone M.L. Leitner J.W. Wall K. Dolgonos L. Rother K.I. Accili D. Draznin B. J. Biol. Chem. 1998; 273: 23892-23896Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Chappell J. Golovchenko I. Wall K. Stjernholm R. Leitner J. Goalstone M. Draznin B. J. Biol. Chem. 2000; 275: 31792-31797Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 26Goalstone M.L. Leitner J.W. Golovchenko I. Stjernjolm M.R. Cormont M. Le Marchand-Brustel Y. Draznin B. J. Biol. Chem. 1999; 274: 2880-2884Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These increases in the pool of prenylated small GTPases result in augmentation of DNA synthesis in response to other growth factors and augmentation of Rho-A-mediated transcriptional activation of SRE-dependent genes by LPA (25Chappell J. Golovchenko I. Wall K. Stjernholm R. Leitner J. Goalstone M. Draznin B. J. Biol. Chem. 2000; 275: 31792-31797Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) as well as transactivation of nuclear factor κB by angiotensin II, hyperglycemia, and advanced glycosylation end products (32Golovchenko I. Goalstone M.L. Watson P. Brownlee M. Draznin B. Circ. Res. 2000; 87: 746-752Crossref PubMed Scopus (94) Google Scholar). The salient feature of the present investigation is that insulin, in addition to its direct mitogenic effects through the PI 3-kinase signaling cascade, potentiates the effect of LPA-mediated MCF-7 cell transition into cell cycle via the Rho-A-dependent activation of cyclin E. Cells preincubated with insulin and then challenged with LPA exhibited a 65% increase in transition into cell cycle, which was greater than insulin alone (40%), LPA alone (5%), and greater than an additive effect of these two agents. Inhibition of a direct mitogenic effect of insulin with wortmannin did not affect the ability of insulin to potentiate the effects of LPA. The addition of the geranylgeranyltransferase inhibitor GGTI-286 completely abolished insulin's potentiating effect on LPA-mediated transition into cell cycle, both in the absence and in the presence of wortmannin. Our experiments with insulin effect on BrdUrd incorporation into DNA, as a marker for DNA synthesis and cellular growth, mirrored our results form cell cycle analysis. In addition, transfection of a dominant negative mutant of Rho-A also completely prevented the potentiating effect of insulin. Neither GGTI nor the dominant negative Rho-A had any appreciable effect on insulin alone, whereas wortmannin inhibited the direct effect of insulin. The subsequent set of experiments was designed to identify the point of cell cycle initiation at which insulin exerts its potentiating effect. Because overexpression of either cyclin D1 or cyclin E has been shown to shorten the G1 interval in various mammalian cell lines (47Ando K. Ajchenbaum-Cymbalista F. Griffin J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9571-9575Crossref PubMed Scopus (170) Google Scholar, 48Jiang W. Kahn S. Zhou P. Zhang Y. Cacace A. Infante A. Doi S. Santella R. Weinstein I. Oncogene. 1993; 8: 3447-3457PubMed Google Scholar, 49Musgrove E. Lee C. Buckley M. Sutherland R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8022-8026Crossref PubMed Scopus (338) Google Scholar, 50Ohtsubo M. Roberts J. Science. 1993; 259: 1908-1912Crossref PubMed Scopus (662) Google Scholar, 51Quelle D. Ashmun R. Shurtleff S. Kato J. Bar-Sagi D. Roussel M. Sherr C. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (979) Google Scholar, 52Resnitzky D. Gossen M. Bujard H. Reed S. Mol. Cell. Biol. 1994; 14: 1669-1679Crossref PubMed Scopus (989) Google Scholar, 53Wimmel A. Lucibello F. Sewing A. Adolf S. Muller R. 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In contrast to its direct mitogenic effect, insulin also potentiates the effect of LPA, a major serum mitogen. This potentiating influence of insulin appears to involve the prenylation of Rho-A, expression of cyclin E, and inhibition of p27Kip1. These data are in agreement with our previous findings that insulin activates the prenyltransferases via the Shc-MAP kinase signaling pathway (36Goalstone M.L. Leitner J.W. Berhanu P Sharma P.G. Olefsky J.M. Draznin B. J. Biol. Chem. 2001; 276: 12805-12812Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 63Goalstone M.L. Carel K. Leitner J. Draznin B. Endocrinology. 1997; 3: 155-173Google Scholar). We have previously shown that insulin potentiates EGF- and IGF-1-mediated p21Ras-GTP loading (64Leitner J.W. Kline T. Carel K.M.G. Draznin B. Endocrinology. 1997; 138: 2211-2214Crossref PubMed Google Scholar), EGF, IGF-1, and PDGF effects on DNA synthesis (24Goalstone M.L. Leitner J.W. Wall K. Dolgonos L. Rother K.I. Accili D. Draznin B. J. Biol. Chem. 1998; 273: 23892-23896Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) in 3T3-L1 fibroblasts through increases in the amounts of farnesylated p21Ras. Moreover, insulin potentiates platelet-derived growth factor-mediated vascular endothelial growth factor gene expression and thymidine incorporation in vascular smooth muscle cells as well (65Goalstone M.L. Natarajan R. Standley P.R. Walsh M.F. Leitner J.W. Carel K. Scott S. Nadler J. Sowers J.R. Draznin B. Endocrinology. 1998; 139: 4067-4072Crossref PubMed Google Scholar). This effect of insulin was also mediated by the increased amounts of farnesylated p21Ras (65Goalstone M.L. Natarajan R. Standley P.R. Walsh M.F. Leitner J.W. Carel K. Scott S. Nadler J. Sowers J.R. Draznin B. Endocrinology. 1998; 139: 4067-4072Crossref PubMed Google Scholar). Here, we show that insulin potentiates LPA-mediated cell cycle progression and BrdUrd incorporation in MCF-7 breast cancer cells and that the mechanism of the enhanced cell cycle progression is through the Rho-A-dependent up-regulation of cyclin E and down-regulation of the Cdk inhibitor p27Kip1. Because either the presence of GGTI-286 (a geranylgeranyltransferase inhibitor) or the expression of a dominant negative Rho-A completely blocks the potentiating effect of insulin on all aspects of LPA-mediated cell cycle progression, we postulate that insulin potentiates LPA action through its effect on Rho-A prenylation and provision of increased amounts of geranylgeranylated Rho-A. We have previously shown that the inhibitory effects of GGTI-286 on insulin's ability to potentiate nuclear effects of LPA are specific and not toxic (25Chappell J. Golovchenko I. Wall K. Stjernholm R. Leitner J. Goalstone M. Draznin B. J. Biol. 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