Dual Inhibition of mTOR and Estrogen Receptor Signaling In vitro Induces Cell Death in Models of Breast Cancer

Purpose: RAD001 (everolimus), a mammalian target of rapamycin (mTOR) pathway inhibitor in phase II clinical trials in oncology, exerts potent antiproliferative/antitumor activities. Many breast cancers are dependent for proliferation on estrogens synthesized from androgens (i.e., androstenedione) by aromatase. Letrozole (Femara) is an aromatase inhibitor used for treatment of postmenopausal women with hormone-dependent breast cancers. The role of the mTOR pathway in estrogen-driven proliferation and effects of combining RAD001 and letrozole were examined in vitro in two breast cancer models. Experimental Design: The role of the mTOR pathway in estrogen response was evaluated in aromatase-expressing MCF7/Aro breast cancer cells by immunoblotting. Effects of RAD001 and letrozole (alone and in combination) on the proliferation and survival of MCF7/Aro and T47D/Aro cells were evaluated using proliferation assays, flow cytometry, immunoblotting, and apoptosis analyses. Results: Treatment of MCF7/Aro cells with estradiol or androstenedione caused modulation of the mTOR pathway, a phenomenon reversed by letrozole or RAD001. In MCF7/Aro and T47D/Aro cells, both agents inhibited androstenedione-induced proliferation; however, in combination, this was significantly augmented (P < 0.001, two-way ANOVA, synergy by isobologram analysis). Increased activity of the combination correlated with more profound effects on G1 progression and a significant decrease in cell viability (P < 0.01, two-way ANOVA) defined as apoptosis (P < 0.05, Friedman test). Increased cell death was particularly evident with optimal drug concentrations. Conclusion: mTOR signaling is required for estrogen-induced breast tumor cell proliferation. Moreover, RAD001-letrozole combinations can act in a synergistic manner to inhibit proliferation and trigger apoptotic cell death. This combination holds promise for the treatment of hormone-dependent breast cancers.

[1]  S. Johnston.  Combinations of endocrine and biological agents: present status of therapeutic and presurgical investigations. , 2005, Clinical Cancer Research.

[2]  Manuel Hidalgo,et al.  Inhibition of mTOR Activity Restores Tamoxifen Response in Breast Cancer Cells with Aberrant Akt Activity , 2004, Clinical Cancer Research.

[3]  H. Lane,et al.  40 RAD001 sensitizes tumor cells to cisplatin-induced apoptosis in an mTOR dependent manner by inhibition of p53-induced p21 protein expression , 2004 .

[4]  M. Ellis.  Overcoming endocrine therapy resistance by signal transduction inhibition. , 2004, The oncologist.

[5]  T. Golub,et al.  mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways , 2004, Nature Medicine.

[6]  M. Bjornsti,et al.  The tor pathway: a target for cancer therapy , 2004, Nature Reviews Cancer.

[7]  Pier Paolo Pandolfi,et al.  Aberrant mRNA translation in cancer pathogenesis: an old concept revisited comes finally of age , 2004, Oncogene.

[8]  K. Nilsson,et al.  Rapamycin sensitizes multiple myeloma cells to apoptosis induced by dexamethasone. , 2004, Blood.

[9]  G. Mills,et al.  Determinants of Rapamycin Sensitivity in Breast Cancer Cells , 2004, Clinical Cancer Research.

[10]  Jiang Shou,et al.  Cross-Talk between Estrogen Receptor and Growth Factor Pathways as a Molecular Target for Overcoming Endocrine Resistance , 2004, Clinical Cancer Research.

[11]  H. Lane,et al.  Antitumor Efficacy of Intermittent Treatment Schedules with the Rapamycin Derivative RAD001 Correlates with Prolonged Inactivation of Ribosomal Protein S6 Kinase 1 in Peripheral Blood Mononuclear Cells , 2004, Cancer Research.

[12]  L. E. Faber,et al.  Estrogen and rapamycin effects on cell cycle progression in T47D breast cancer cells , 2001, Breast Cancer Research and Treatment.

[13]  E. Perez,et al.  A randomized trial of letrozole in postmenopausal women after five years of tamoxifen therapy for early-stage breast cancer. , 2004, The New England journal of medicine.

[14]  A. Brodie,et al.  Signaling pathways of apoptosis activated by aromatase inhibitors and antiestrogens. , 2003, Cancer research.

[15]  M. Conaway,et al.  Adaptive hypersensitivity following long-term estrogen deprivation: involvement of multiple signal pathways , 2003, The Journal of Steroid Biochemistry and Molecular Biology.

[16]  Mitch Dowsett,et al.  Aromatase inhibitors in breast cancer. , 2003, The New England journal of medicine.

[17]  J. Carroll,et al.  Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells. , 2003, Endocrine-related cancer.

[18]  C. Boni,et al.  Phase III study of letrozole versus tamoxifen as first-line therapy of advanced breast cancer in postmenopausal women: analysis of survival and update of efficacy from the International Letrozole Breast Cancer Group. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[19]  M. Dowsett,et al.  Enhanced Estrogen Receptor (ER) α, ERBB2, and MAPK Signal Transduction Pathways Operate during the Adaptation of MCF-7 Cells to Long Term Estrogen Deprivation* , 2003, Journal of Biological Chemistry.

[20]  H. Kurokawa,et al.  ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanisms. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[21]  Christopher G. Proud,et al.  Does phosphorylation of the cap‐binding protein eIF4E play a role in translation initiation? , 2002, European journal of biochemistry.

[22]  R. Tallarida.  The interaction index: a measure of drug synergism , 2002, Pain.

[23]  C. Sawyers,et al.  The phosphatidylinositol 3-Kinase–AKT pathway in human cancer , 2002, Nature Reviews Cancer.

[24]  O. Stål,et al.  Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients , 2002, British Journal of Cancer.

[25]  R. McPherson,et al.  Linkage of Rapid Estrogen Action to MAPK Activation by ER-Shc Association and Shc Pathway Activation , 2001 .

[26]  A. Bilancio,et al.  PI3‐kinase in concert with Src promotes the S‐phase entry of oestradiol‐stimulated MCF‐7 cells , 2001, The EMBO journal.

[27]  M. Ellis,et al.  Preoperative treatment of postmenopausal breast cancer patients with letrozole: A randomized double-blind multicenter study. , 2001, Annals of oncology : official journal of the European Society for Medical Oncology.

[28]  P. Frost,et al.  mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer. , 2001, Endocrine-related cancer.

[29]  Hong Wu,et al.  Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Boni,et al.  Superior efficacy of letrozole versus tamoxifen as first-line therapy for postmenopausal women with advanced breast cancer: results of a phase III study of the International Letrozole Breast Cancer Group. , 2001, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[31]  B. Geoerger,et al.  Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor/medulloblastoma models as single agent and in combination chemotherapy. , 2001, Cancer research.

[32]  John M. Daly,et al.  ErbB2 Potentiates Breast Tumor Proliferation through Modulation of p27Kip1-Cdk2 Complex Formation: Receptor Overexpression Does Not Determine Growth Dependency , 2000, Molecular and Cellular Biology.

[33]  G. Palumbo,et al.  17β-Estradiol Inhibits Apoptosis in MCF-7 Cells, Inducing bcl-2 Expression via Two Estrogen-Responsive Elements Present in the Coding Sequence , 2000, Molecular and Cellular Biology.

[34]  L. Mazzolini,et al.  Interconnections between E2-dependent regulation of cell cycle progression and apoptosis in MCF-7 tumors growing on nude mice. , 2000, Experimental cell research.

[35]  M. Dowsett,et al.  Time‐related effects of estrogen withdrawal on proliferation‐ and cell death‐related events in MCF‐7 xenografts , 1999, International journal of cancer.

[36]  D. Campos,et al.  Letrozole, a new oral aromatase inhibitor: randomised trial comparing 2.5 mg daily, 0.5 mg daily and aminoglutethimide in postmenopausal women with advanced breast cancer. Letrozole International Trial Group (AR/BC3). , 1998, Annals of oncology : official journal of the European Society for Medical Oncology.

[37]  B K Slinker,et al.  The statistics of synergism. , 1998, Journal of molecular and cellular cardiology.

[38]  P. Dombernowsky,et al.  Letrozole, a new oral aromatase inhibitor for advanced breast cancer: double-blind randomized trial showing a dose effect and improved efficacy and tolerability compared with megestrol acetate. , 1998, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[39]  Shiuan Chen,et al.  Autocrine and paracrine actions of breast tumor aromatase. A three-dimensional cell culture study involving aromatase transfected MCF-7 and T-47D cells , 1997, The Journal of Steroid Biochemistry and Molecular Biology.

[40]  R. Sutherland,et al.  Estrogen-induced Activation of Cdk4 and Cdk2 during G1-S Phase Progression Is Accompanied by Increased Cyclin D1 Expression and Decreased Cyclin-dependent Kinase Inhibitor Association with Cyclin E-Cdk2* , 1997, The Journal of Biological Chemistry.

[41]  J. Harper,et al.  Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. , 1997, Molecular biology of the cell.

[42]  R. Santen,et al.  Effect of androstenedione on growth of untransfected and aromatase-transfected MCF-7 cells in culture , 1993, The Journal of Steroid Biochemistry and Molecular Biology.

[43]  Anthony Howell,et al.  Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75,000 women. Early Breast Cancer Trialists' Collaborative Group. , 1992 .

[44]  Early Breast Cancer Trialists' Collaborative Group.  Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy 133 randomised trials involving 31 000 recurrences and 24 000 deaths among 75 000 women , 1992, The Lancet.

[45]  A. D. Schryver,et al.  Systemic treatment of early breast-cancer by hormonal, cytotoxic, or immune therapy: 133 randomized trials involving 31000 recurrences and 24000 deaths among 75000 women: 1 , 1992 .

[46]  M C Berenbaum,et al.  Criteria for analyzing interactions between biologically active agents. , 1981, Advances in cancer research.