Abstract S3-8: RNAi Screening Identifies the Insulin/Insulin-Like Growth Factor-I Receptor Pathway as a Mechanism of Escape from Hormone Dependence in Estrogen Receptor-Positive Breast Cancer

Abstract

Abstract Estrogen receptor-positive (ER+) breast cancers are treated with strategies aimed at inducing estrogen deprivation such as aromatase inhibitors (AIs). However, many ER+ tumors adapt to hormone deprivation and acquire resistance to AIs. The purpose of this study was to identify kinases essential for the growth of endocrine-resistant ER+ breast cancer cells. To model acquired resistance to AIs, we maintained four ER+, estrogen-dependent human breast cancer cell lines (MCF-7, MDA-361, HCC-1428, and ZR75- 1) under hormone-depleted conditions for several months until hormone-independent populations emerged (termed long-term estrogen-deprived, LTED). We then performed an RNAi screen targeting 779 protein kinases to identify molecules required for growth of MCF-7/LTED cells in hormonefree conditions. Individual knockdown of 42 kinases significantly inhibited MCF-7/LTED cell growth ≥33% (p≥0.05) in at least 3/4 independent experiments. Of these 42 hits, knockdown of the insulin receptor (InsR) inhibited MCF-7/LTED growth (n=4 experiments) compared to control siRNA. Knockdown of InsR expression using independent siRNA oligonucleotides significantly inhibited growth of MCF-7/LTED, ZR75-1/LTED, and HCC-1428/LTED cells. The InsR can heterodimerize with the highly homologous insulin-like growth factor-I receptor (IGF-IR) to potently activate the phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway. Immunoblot analysis revealed that siRNA knockdown of InsR or IGF-IR caused a compensatory upregulation of IGF-IR and InsR expression, respectively. Knockdown of InsR or IGF-IR inhibited growth of 3/4 LTED lines, but knockdown of both InsR and IGF-IR was required for maximal inhibition of PI3K/AKT. Inhibition of these receptors by treatment with the InsR/IGF-IR tyrosine kinase inhibitor (TKI) OSI-906 decreased PI3K signaling and disrupted the basal association between p85 and the IGF-IR/InsR effectors IRS-1 and IRS-2 in MCF-7/LTED cells. Further, OSI-906 inhibited the hormone-independent growth of all four LTED lines and prevented the emergence of hormone-independent ER+ parental cells following estrogen deprivation. Treatment with the TKI or the neutralizing IGF-IR monoclonal antibody MK-0646 induced feedback upregulation and phosphorylation of the ErbB3/HER3 receptor tyrosine kinase (RTK). Induction of P-HER3 was blocked by the EGFR/HER2 TKI lapatinib. Combined treatment of lapatinib and OSI-906 prevented the emergence of hormone-independent (HER2+/ER+) MDA-361 cells more effectively than either drug alone. Finally, treatment of ovariectomized nude female mice with OSI-906 suppressed the growth of established MCF-7 xenografts. In addition, treatment with OSI-906 decreased PI3K signaling as measured by immunoblot analysis of tumor extracts using P-AKT antibodies. These results suggest that 1) InsR/IGF-IR signaling is required for PI3K/AKT signaling and hormone-independent growth of ER+ breast cancer cells; 2) these cells compensate for the inhibition of InsR/IGF-IR by upregulating alternative RTK pathways such as HER3; 3) combined targeting of InsR/IGF-IR and ER signaling may be effective for the treatment of endocrine-resistant breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S3-8.