Abstract P3-01-25: Hedgehog signaling-mediated adaptation to hypoxic microenvironment promotes osteoclastogenesis in advanced breast cancer

Primary tumors often experience hypoxic microenvironment due to increased demand for oxygen in the proliferating cells. Moreover, the proportions of hypoxic tumor cells in the primary tumor strongly correlate to the incidence of metastasis, with bone presenting as the preferred site for breast cancer metastasis. Hence, studying how breast cancer adapts to its hypoxic microenvironment is crucial to understanding the process of breast cancer metastasis to bones. Previous studies in our lab have shown that the developmental signaling pathway, Hh pathway promotes osteopontin (OPN) and parathyroid hormone-related (PTHrP) expression in breast cancer. Additionally, activation of the Hh signaling in breast cancer enables an increase in osteoclastic activity facilitating osteolysis invivo. Hence, we hypothesized that inhibition of Hh signaling diminishes the ability of breast cancer cells to adapt to hypoxia and consequently their metastatic potential. Hh signaling can be canonically activated through smoothened (SMO) dependent activation of the transcriptional regulator GLI. Hence, we inhibited Hh signaling using small molecule inhibitor GANT61 (GLI inhibitor) and pharmacological inhibitor BMS833923 (SMO inhibitor). Since hypoxic microenvironment is prevalent in the bone, we investigated the effect of conditioned medium from breast cancer cells in hypoxic condition on bone marrow-derived macrophages (BMDMs). We found that hypoxia increases osteoclastogenesis and osteoclastic activity, whereas abrogating GLI1 and GLI2 dampens this effect as evidenced by TRAP and toluidine blue staining. Hypoxia-dependent increase in glucose consumption and lactate production were also dampened in Hh inhibited breast cancer cells. Additionally, we found increased nuclear localization of GLI1 and activation of Hh signaling in breast cancer cells in hypoxic condition compared to the control indicating hypoxia-dependent Hh signaling upregulation. To understand the importance of the Hh activation in hypoxic condition, we inhibited Hh signaling pathway with BMS and GANT61. Interestingly, our results indicate that cellular adaptation to hypoxia is dependent on Hh signaling-mediated stabilization of hypoxia-inducible factors (HIF-1α) and its transcriptional activity. This crosstalk suggests a feed-forward mechanism between Hh signaling and HIF-1α. In the future, we will further investigate the mechanistic role of Hh signaling-mediated modulation of HIF-1α. This study will possibly yield new therapeutic targets in controlling hypoxia-mediated cancer metabolism and tumorigenesis of breast cancer. This work is supported by NIH grant R01CA169202 Citation Format: Tshering Dolma Lama-Sherpa, Shamik Das, Ann Hanna, Sarah Kammerud, Lalita A Shevde. Hedgehog signaling-mediated adaptation to hypoxic microenvironment promotes osteoclastogenesis in advanced breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-25.