Gli2 increases Osteopontin expression to alter cancer associated fibroblast behavior

Breast cancer is known to often metastasize into the bone. Among the subtypes, triple negative is the most aggressive. Once the cancer spreads to the bone it activates a positive feedback loop known as the vicious cycle of bone metastasis. In this cycle, Parathyroid hormone-Related protein (PTHrP) is released from tumor cells which activate osteoblasts inducing their expression of Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL). RANKL binds to Receptor Activator of Nuclear factor Kappa-B Ligand (RANK) expressed on osteoclasts, thereby activating the osteoclasts to induce bone resorption subsequently releasing growth factors that fuel the tumor. This can lead to increased risk in bone fractures from the tumor indirectly activating osteoclasts. Chemotherapy is commonly given to patients with breast cancer such as paclitaxel, however, drug resistance often occurs, and treatment becomes less effective, particularly in metastatic disease. Fibroblasts are cells that are responsible for wound healing in normal circumstances. However, in cancer they can transform into Cancer Associated Fibroblasts (CAFs) capable of producing extracellular matrix proteins that surround the tumor which prevents drugs and immunotherapies from reaching the tumor. Our recent studies indicate that a transcription factor from hedgehog signaling known as GLI2 binds is upregulated in response to the chemotherapeutic, paclitaxel. RNA-sequencing analyses showed that increased expression of Gli2 increases genes associated with CAFs including osteopontin (OPN), a secreted glycoprotein. Furthermore, using the JASPAR transcription binding software, we found that Gli2 is predicted to bind to the SPP1 promoter (gene name for OPN) on chromosome 4 at a potential Gli2 binding element. Therefore, we hypothesize that osteopontin secretion is directly regulated by GLI2 in the tumor cells through direct binding of the SPP1 promoter. To test this, we knocked out GLI2 expression in bone metastatic breast cancer cell lines and showed a reduction in OPN secretion. Additional studies with a Gli2 small molecule inhibitor Hedgehog Pathway Inhibitor 1 (HPI-1) showed a 75% reduction in SPP1 by QPCR and a significant reduction of OPN secretion by ELISA. Furthermore, we showed direct changes in the production of collagens and other matrix proteins both by QPCR and immunohistochemistry. In the future, we will directly examine changes in the surrounding fibroblast phenotypes and investigate the impact on drug resistance that CAFs may play in bone metastatic tumors. Taken together, this suggests that Gli2 expressing tumors can increase OPN secretion and this may alter the production of extracellular matrix proteins by the surrounding CAFs.