Gli2对TNBC侵袭中EMT的调控

Despite improvements in survival for patients with primary disease only, breast cancer remains a leading cause of cancer-related death for women in part due to the propensity of breast cancer to metastasize and develop therapy resistance. Triple negative breast cancer (TNBC) has high metastatic potential, and an estimated 30-50% of patients develop tumors that are resistant to chemotherapies. To metastasize, tumors undergo epithelial-to-mesenchymal transition (EMT), in which transcriptional alterations allow tumor cells to become more invasive through reduced adhesion and increased motility. In addition to facilitating the early stages of metastasis and allowing tumors to invade, EMT has been associated with a more plastic, stem-like phenotype that may lead to therapy resistance. Thus, preventing the acquisition of an EMT phenotype may offer a dual role in inhibiting metastatic potential as well as resensitizing therapy-resistant tumors in patients with TNBC. While the Hedgehog (HH) signaling pathway is primarily active during embryonic development, its aberrant activation has been observed to promote tumor growth, invasion, metastasis, and chemoresistance. Previous data have shown that components of the HH pathway are upregulated in clinical samples of breast cancer in patients with greater mesenchymal gene signatures and those with metastatic disease. Additionally, Gli2 (Glioma-associated oncogene 2), a downstream transcriptional regulator of HH signaling, correlates with poorer patient outcomes. Thus, we hypothesize that Gli2 transcriptionally regulates EMT-associated genes, therefore promoting invasive potential in TNBC. We examined the pharmacological inhibition of Gli2 and found a reduction in EMT marker expression in TNBC cell lines. Specifically, we have identified genes such as Cldn1 (FC = 0.382, p = 0.0003), Zeb2 (FC = 0.426 , p = 0.0005), and Spp1 (FC = 0.163, p = 0.0009) as potential targets of Gli2-mediated EMT. We will use Cleavage Under Targets & Release Using Nuclease (CUT&RUN) sequencing to identify regions of DNA bound by Gli2, followed by comparison to genes that have differentially expressed RNA transcripts upon inhibition of Gli2. Following confirmation of Gli2-regulation of EMT-associated genes, we will genetically knockdown these target genes in Gli2-overexpressing TNBC cell lines and overexpress these genes in Gli2-knockdown TNBC cell lines. We will then perform transwell migration and invasion assays as well as ex vivo invasion assays on decellularized extracellular matrix (dECM) scaffolds from mouse mammary fat pads using the above cell models to assess the rescue or depletion of EMT phenotypes. The proposed studies will provide insight into the mechanisms by which Gli2 regulates EMT and invasion in TNBC. Elucidating direct transcriptional regulation of Gli2 on the EMT program may provide insight into potential therapeutic targets to reduce metastatic burden and combat chemoresistance in TNBC patients.