P4-01-07: Combining Hedgehog Inhibitor, GDC-0449 with Tamoxifen Overcomes Tamoxifen-Resistance in In-Vitro and In-Vivo Studies of Estrogen-Receptor Positive Breast Cancer.
Authors: Majumder S , Lu Y , Teng K-Y , Kaffenberger T , Zhang X , Nuovo G , Love RR , Ramaswamy B 
Published: June 2012

Aims: Approximately 30–40% of patients with estrogen receptor positive (ER+) invasive breast cancer relapse despite targeted endocrine therapy with tamoxifen. Dependency of ER+ breast tumors shifts from estrogen to alternative cell survival mechanisms aberrantly activated over time facilitating drug-resistance and survival. We have identified Hedgehog (Hhg) signaling pathway as one such alternative pathway of survival activated in ER+ tumors that are resistant to tamoxifen. We sought to determine if treating such tumors with Hhg inhibitor and tamoxifen would improve outcomes.

Methods: Using MCF7 cell lines that are either sensitive (parental) or resistant to tamoxifen (4-hydroxy tamoxifen resistant-OHTR) and other ER+ cell lines, expression of Hhg markers (SHH, PTCH, SMO, GLI1, GLI2 and GLI3) and Hhg targets (SNAIL, MYC, BMI1 and FOXM1) was analyzed by Real-time RT-PCR, Western blotting and immunocytochemistry. GLI1 transactivation potential was measured using 8XGLI-Luciferase reporter plasmid. GLI1 was depleted in OHTR and T47D cells using GLI1siRNA. The GLI1 depleted cells were exposed to tamoxifen and assessed for i) cell viability by MTT assay, ii) extent of apoptosis by TUNEL assay and PARP/Caspase cleavage (Western blot), iii) cell cycle profile by flow cytometry and iv) presence of tumor initiating cells (TICs) by mammosphere formation assay. Effect of GDC-0449, a small molecule inhibitor of SMO was tested on OHTR cell induced xenografts in athymic nude mice by monitoring tumor growth and distant metastasis. Correlation of GLI1 expression in primary breast tumor tissues (assessed by immunohistochemistry) and disease-free interval was studied in women enrolled on an ongoing clinical trial (OSU 0483) in premenopausal women with ER+ breast cancer undergoing adjuvant oophorectomy and tamoxifen.

Results: Expression of Hhg signaling molecules and the targets are significantly activated in OHTR cells resistant to 0.5 and 1μM tamoxifen. Serial passage of the resistant cells in mice resulted in aggressive transformation of tumors that micro-metastasized to lung and liver. Concurrent increase in Hhg marker expression, TICs and acquisition of epithelial mesenchymal transition were observed in the cells derived from these tumors. Depletion of GLI1 in OHTR cells resulted in reduced proliferation, colony formation and mammosphere formation and increased apoptosis in presence of tamoxifen. Treatment of OHTR cells with GDC-0449 and tamoxifen inhibited cell proliferation in vitro and supressed tumor growth and distant metastasis of tamoxifen resistant xenografts in mice. Primary human breast tumors expressing higher level of GLI1 demonstrated a trend towards increased risk of recurrence (p= 0.08) after tamoxifen therapy.

Conclusions: Our work has demonstrated for the first time that activated Hhg signaling is an alternate survival mechanism for tamoxifen resistant breast cancer. Furthermore, targeting these tumors with a combination of tamoxifen and anti-Hhg therapy inhibited tumor growth and development of distant metastases demonstrating efficacy of this approach. Clinical trials using this combination in patients with advanced ER+ breast cancer are warranted and are under development.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-07.