Abstract B40: Decoding the cellular crosstalk mediated by Hedgehog signaling in the breast tumor microenvironment
Authors: Shevde Lalita A. , Das Shamik , Samant Rajeev S. 
Published: May 2015

Abstract
Aberrant activation of the Hedgehog (Hh) signaling pathway is a salient feature of many malignancies. Besides pathway mutations, activation of Hh signaling can be autocrine or paracrine. We hypothesized that breast cancer cells influence other cells in their microenvironment by paracrine activation of Hh signaling in cells comprising the milieu at the primary tumor site as well at the metastatic site. Sonic Hedgehog (SHH)-mediated signaling initiates a cascade of signaling events that culminates in the translocation of activated GLI transcription factors to the nucleus to regulate target gene transcription. We silenced expression of the principal transcription factor GLI1 in tumor cells. The resultant cells displayed blunted malignant attributes of tumor cells characterized by reduced expression of an oncoprotein, osteopontin. Osteopontin is a secreted matricellular protein that promotes metastatic phenotype by signaling via multiple cell surface receptors. Detailed functional analyses allowed us to assign an important role for osteopontin in mediating the pro-malignant effects of Hh signaling.
Breast cancer has a high propensity to metastasize to bone; nearly 60-75% of breast cancer patients develop bone metastases. GLI1 silencing decreased mesenchymal characteristics of tumor cells and also significantly decreased the intensity and incidence of osteolytic bone metastases. We characterized the nature of paracrine Hh signaling between breast cancer cells and osteoclasts and osteoblasts, the predominant cell types in bone. We determined that Hh ligands produced by breast cancer cells cause paracrine activation of Hh signaling in pre-osteoclasts consequently upregulating osteoclastogenesis (TRAP staining) and resorptive activity characterized by increased levels of the essential proteases, cathepsin K and MMP9. Surprisingly, we found that breast cancer cells also cause early activation of osteoblast differentiation (characterized by increased alkaline phosphatase activity) leading to the expression of RANKL and PTHrP that promote osteoclastogenesis while the osteoblasts are eliminated by early apoptosis. We further noted that inhibition of Hh signaling in breast cancer cells resulted in decreased tumor biomass in the femur and tibia of athymic mice injected with breast cancer cells. Cumulatively our studies suggest that breast cancer cells orchestrate a chain of events in which they directly foster osteolytic bone metastasis.
Bone is a hypoxic microenvironment (pO2 between 1–7%). This imposes cells to adapt their molecular functions to respond to the hypoxic niche. We hypothesized that the hypoxic environment in bone exercises an adaptive response in breast cancer cells characterized by an altered profile of Hh-responsive genes. This altered expression profile mediated by the GLI transcription factors enhances the osteolytic activity of breast cancer cells. Utilizing a strategy of inhibiting GLI transcriptional activity in combination with normoxic or hypoxic conditions we investigated the molecular re-programming of Hh signaling that results in an altered GLI transcriptome.
As such, our work addresses the tumor microenvironment conditions (hypoxia) that alter the transcriptional activity of the GLI transcription factors of the Hh pathway. The identification of novel candidates of Hh signaling will identify putative therapeutic targets to intervene in osteolytic metastasis of breast cancer, and identify molecular surrogates that serve to indicate the efficacy of inhibiting Hh signaling in the hypoxic bone environment
Citation Format: Lalita A. Shevde, Shamik Das, Rajeev S. Samant. Decoding the cellular crosstalk mediated by Hedgehog signaling in the breast tumor microenvironment. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B40. doi:10.1158/1538-7445.CHTME14-B40