Expression Analysis of Sonic Hedgehog in Breast Cancer and its Relationship with Metastasis

Abstract

Expression Analysis of Sonic Hedgehog in Breast Cancer and its Relationship with Metastasis Involvement of Hedgehog pathway during embryogenesis and mammary development is well known. Salient members of this pathway include SHH, DHH, IHH, PTCH1, SMO and GLI1. Considering high prevalence and early onset of breast cancer among Pakistani population, reactivation of this developmental pathway is hypothesized. In the present study, expression profiling of Hedgehog pathway ligands (SHH, DHH, IHH), mediators (PTCH, SMO) and downstream effector (GLI1) in breast tumorigenesis was explored. The study was divided in two phases. The initial phase included screening of Hedgehog pathway molecules at mRNA and protein levels in Pakistani breast cancer patients. In the second phase, in-vitro models were generated for inhibition of SHH/GLI1 axis. Moreover, effect of SHH/GLI1 inhibition on EMT markers and metastatic cascade was investigated. In the first phase, expression of Hedgehog pathway molecules was evaluated using qRT-PCR and immunohistochemistry in tumor tissues (N=250) and adjacent noncancerous healthy tissues (N=250). SHH, DHH, PTCH1 and GLI1 were significantly overexpressed in tumors compared with respective controls. Significant correlation of SHH, DHH and GLI1 expression with advanced stage, grade, nodal involvement and distant metastasis was observed (p<0.05). Patients having high expression of SHH, DHH and GLI1 demonstrated poor overall survival in the cohort. Overexpression of SHH, DHH and GLI1 was observed to be significantly related with patients having early disease onset and pre-menopausal status. Moreover, Hedgehog pathway was frequently upregulated in luminal B or triple negative breast cancer affected patients. In addition, positive correlation was observed among aforementioned members of the pathway and Ki67 (r-value: 0.63-0.78) emphasizing their role towards disease progression. Expression of Vimentin and Snail was upregulated among SHH/GLI1 positive patients. Strong positive correlation of both Vimentin and Snail with SHH/GLI1 positive patients was observed. On the contrary, E-cadherin was downregulated in SHH/GLI1 positive patients and its correlation with SHH/GLI1 was also negative. xiii In the second phase, SHH knockout models were generated using CRISPR/CAS9 in MDA-MB-231 and MCF-7 cells. Additionally, both cancer cell lines were treated with GANT61 (GLI1 inhibitor) and its effect on cell viability and apoptosis was evaluated using CCK-8 and flow cytometry respectively. Effect of SHH knockout and GANT61 treatment was observed on EMT markers and Akt/ERK using qRT-PCR, western blot analysis and immunofluorescence staining. Invasion and migration assays were performed to investigate influence of SHH/GLI1 suppression on metastatic cascade. Treatment with GANT61 reduced cell viability (p<0.01) and induced apoptosis (p<0.01) in breast cancer cell lines. Expression of Vimentin and Snail was reduced in SHH knockout models and GANT61 treated cells. On the contrary, expression of E-cadherin was increased after inhibition of SHH or GLI1. Suppression of SHH/GLI1 axis also inhibited activation Akt/ ERK as well as reduced migration and invasion capacity in breast cancer cells. Based on these findings, SHH/GLI1 may be used as a prognostic marker in breast cancer patients. Moreover, inhibition of SHH/GLI1 axis provides a novel therapeutic target against aggressive breast cancer subtypes.