Molecular rationale and preclinical data to support combined Src and aromatase inhibitor therapy for ER positive breast cancer

AACR Centennial Conference: Translational Cancer Medicine-- July 20-23, 2008; Monterey, CA PL07-01 Estrogen plays an important role in human breast cancer development and progression. It is a mitogen for estrogen receptor (ER) positive breast cancers. About seventy percent of newly diagnosed breast cancers express the estrogen receptor (ER) and of these, two thirds will respond to antiestrogen therapies. Aromatase inhibitors (AI) are used to block estrogen synthesis as an important treatment for postmenopausal women with estrogen receptor positive breast cancer. Millions of breast cancer patients are treated with Tamoxifen or aromatase inhibitors (AI) worldwide. In spite of the initial efficacy of hormonal therapies, both de novo and acquired resistance to antiestrogens limit therapeutic efficacy. A better understanding of how antiestrogens arrest breast cancer growth is a first step in the development of drugs that can overcome hormone resistance. Tamoxifen, fulvestrant and estrogen withdrawal all cause ER positive breast cancer cells to arrest in the G1 phase of the cell cycle. We and others have shown that the cell cycle inhibitors, p27 and p21, are essential for the clinical efficacy of antiestrogens1. p27 levels increase following treatment with antiestrogen drugs, Tamoxifen or fulvestrant, leading to G1 arrest. When p27 expression was inhibited by antisense, cells whose proliferation had been blocked by Tamoxifen or ICI182780 re-entered the cell cycle. p27 levels are reduced in up to 60% of human breast cancers and this is prognostic of poor outcome, particularly in ER positive cancers2-4. Loss of p27 in human breast cancers may cause antiestrogens resistance and targeted therapies that reverse this may restore response to antiestrogens in resistant cells. Up to 30% of human breast cancers overexpress the Her2/ErbB2 and/or epidermal growth factor receptor (EGFR) receptor tyrosine kinases at their cell surface5. Antiestrogen resistant breast cancers often show increased expression of the epidermal growth factor receptor family members, Her2/ErbB2 or EGFR. Aberrant oncogenic activation of these receptors turns on additional signaling pathways including the 60kDa tyrosine kinase, cSrc. cSrc and EGFR family members are over-expressed in up to 70% of primary human breast cancers6. Our data indicate that >70% of primary human breast cancers show Src activation, with 40% at high levels. Src is activated by EGFR and Her2 and further phosphorylates and activates these receptor tyrosine kinases to stimulate cell proliferation, motility and survival. Estrogen bound ER recruits and activates cSrc, leading to Shc, and MAPK activation7. We showed that overexpression of Her2 or cSrc in ER positive breast cancer cells alters p27 phosphorylation, impairs its Cdk2 inhibitory function and increases p27 degradation. We recently showed that Src binds and phosphorylates p27 at Y74 and Y88, leading to loss of p27’s Cdk2 inhibitory function and facilitating p27 proteolysis8. In 482 primary breast cancers, Src activation correlated with reduced p27. Furthermore, a Src inhibitor drug restored p27 dependent G1 arrest by tamoxifen in resistant cells8. We have tested the potential for synergy between the Src inhibitor AZD0530 and anastrozole in cell culture and in xenograft tumors in vivo. Our pre-clinical data show that the Src inhibitor, AZD0530, can cooperate with anastrozole to inhibit proliferation of the aromatase transfected MCF-7 derivative MCF-AROM5. The AI, anastrozole, alone caused an incomplete cell cycle arrest in ER positive human breast cancer cells stably transfected with the aromatase gene (MCF-Arom5). Although the Src inhibitor, AZD0530 alone had no effect on cell cycle, it enhanced the antiproliferative effect of anastrozole on cultured MCF-AROM5. Using a dose of AZD0530 that on its own did not affect cell proliferation, it took 10 fold less anastrozole to arrest these cells when the anastrozole was used together with AZD0530. Treatment with anastrozole alone led to activation of Src and MAPK activity, whereas treatment with anastrozole and AZD0530 together inhibited their activities. The two drugs together caused a greater p27 increase and cyclin E-Cdk2 inhibition than observed with either drug alone reflecting a more profound cell cycle arrest with the combination therapy. We also provide evidence for synergy between anastrozole and AZD0530 in vivo in MCF-AROM5 xenograft tumors in nude mice. Without androstenedione, tumor growth was minimal through the whole experiment. In contrast, tumor volume increased rapidly with androstenedione treatment, reflecting the mitogenic effect of intracellular aromatase producing estrogen in this model. Daily oral AZD0530 did not reduced tumor growth. Anastrozole alone suppressed tumor growth, with 0.25mg/kg anastrozole yielding a treated/control (%) of 62.1%. The combination therapy significantly delayed tumor growth, yielding a treated/control (%) of 46.7%. Our calculations indicate synergism between AZD0530 and anastrozole on xenograft tumors in vivo. All animals survived and showed no signs of toxicity or weight loss throughout the whole experiment. These data show that anastrozole cooperates with AZD0530 to inhibit ER positive breast cancer xenograft growth and support further Over a ten fold range of anastrozole concentrations, the growth inhibitory effect of anastrozole was increased by addition of a dose of AZD0530 that only modestly affected cell growth on its own. Plans for a Phase I-randomized Phase II clinical trial of a novel Src inhibitor AZD0530 used together with anastrozole for treatment of receptor positive breast cancer in post-menopausal women will be discussed. Treatment of ER+ breast cancers with Src inhibitors may prevent resistance to anastrozole and augment its efficacy by increasing p27 levels and restoring p27 function. 1. Cariou,S. et al. Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proc. Natl. Acad. Sci. U. S. A 97 , 9042-9046 (2000). 2. Porter,P.L. et al. p27(Kip1) and cyclin E expression and breast cancer survival after treatment with adjuvant chemotherapy. J. Natl. Cancer Inst. 98 , 1723-1731 (2006). 3. Pohl,G. et al. High p27Kip1 expression predicts superior relapse-free and overall survival for premenopausal women with early-stage breast cancer receiving adjuvant treatment with tamoxifen plus goserelin. J. Clin. Oncol. 21 , 3594-3600 (2003). 4. Catzavelos,C. et al. Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer. Nat. Med. 3 , 227-230 (1997). 5. Pegram,M.D., Pauletti,G., & Slamon,D.J. HER-2/neu as a predictive marker of response to breast cancer therapy. Breast Cancer Research and Treatment 52 , 65-77 (1998). 6. Ishizawar,R. & Parsons,S.J. c-Src and cooperating partners in human cancer. Cancer Cell 6 , 209-214 (2004). 7. Migliaccio,A. et al. Tyrosine kinas/p21ras/MAP-kinase pathway activation by estradiol receptor complex in MCF-7 cells. EMBO J. 15 , 1292-300 (1996). 8. Chu,I. et al. p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2. Cell 128 , 281-294 (2007).