INVESTIGATING THE ROLE OF THE ARYL HYDROCARBON RECEPTOR AND BCL-2 FAMILY PROTEINS IN OVARIAN CANCER STEM CELLS DEVELOPMENT AND CHEMORESISTANCE

Abstract

Gynecological malignancies are a severe threat to female health. Ovarian Cancer (OC), the most lethal gynecological malignancy, is clinically presented with chemoresistance and a higher relapse rate. This could be endorsed to several factors including the presence of a small sub-population of cells termed Cancer Stem Cells (CSC), directing tumor initiation, development, and metastasis. Recently, an association between CSCs development and exposure to environmental pollutants like 2,3,7,8- Tetrachlorodibenzo-p-dioxin (TCDD) through the activation of the aryl hydrocarbon receptor (AhR) is established. Mechanistically, AhR activation causes induction of several genes such as the cytochrome P451A1 (CYP1A1) and CYP1B1 that play a role in bioactivating procarcinogens into ultimate carcinogens. The process of chemoresistance enabling the cancer cells to evade apoptosis in response to chemotherapeutic drugs is reported to involve BCL family of anti-apoptotic proteins. In this context, the correlation between AhR and apoptotic family of proteins such as BCL-2 requires a thorough understanding. We hypothesized that AhR and BCL-2 signaling pathways are controlling CSCs proliferation, development, self-renewal, and chemoresistance in OC and thus be considered as novel biomarkers and therapeutic molecular targets for cancer development and chemoresistance. This study was conducted to identify the crosstalk between AhR and BCL family members in vitro as previous studies have reported that AhR can regulate the expression of BCL family members, and this interaction can lead to health anomalies including cancer. For this purpose, OC cells were treated with AhR ligand, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), AhR inhibitor, α naphthoflavone (αNF) and BCL-2 inhibitor, venetoclax (VCX) and the effects were determined. Real-Time Cell Analyzer, clonogenic assay, flow cytometry, wound healing assay, RT-PCR were used to identify the effect of AhR induction in differentiating OC cells. A comparative proteomic analysis of OC cells (control vs AhR activated) using a mass spectrometry-based labelfree shotgun proteomics approach was performed, and the most significantly dysregulated proteins were validated by Western blot analysis. Furthermore, assessment of cellular content and localization of proteins was done through immunofluorescence assay. Ovarian CSCs were cultured using spheroid formation assay and characterized. The spheroids were further treated with AhR ligand, inhibitor and BCL-2 inhibitor and the expression of regulatory proteins were determined by RT-PCR to identify the crosstalk between AhR and BCL family members. We found that AhR induction resulted in the induction of PI3K/AKT pathway and upregulation of anti-apoptotic proteins BCL- 2, BCL-xl, and MCL-1. Moreover, a noteworthy rise in stemness marker aldehyde dehydrogenase (ALDH1) was also seen. The expression of Wnt transcription factor, - catenin was found to be increased and EMT was also notably induced. Our proteomics study showed that upon AhR induction, several proteins involved in chemoresistance, cancer progression, invasion and metastasis, apoptosis, survival, and prognosis in OC were significantly dysregulated. Ours is the first report that investigated the role and involvement of AhR and its regulatory proteins in OC with comparative proteomic analysis. The spheroids expressed comparatively higher expression levels of both AhR and BCL proteins and the stemness markers also showed an increased expression in comparisonto adherent cancer cells. Intriguingly, in spheroids and in differentiating cancer cells, AhR inhibition also inhibited BCL family proteins and BCL-2 inhibition negatively modulated AhR and its regulatory proteins, showcasing a strong correlation of these two signaling pathways. AhR inhibition also resulted in the reduced expression of stemness marker Nanog in both differentiating cells and spheroids thereby confirming the involvement of AhR in modulating stemness in OC. Gene expression analysis data integrated from various databases were accessed online to extract data of real-time comparison of gene expression changes between tumor, normal, and metastatic tissues amongst different types of platforms across all genes in OC. Data revealed that AhR, CYP1A1, ARNT, and BCL-2 were highly expressed in ovarian tumor tissues in comparison to the normal ovary tissues. Moreover, Kaplan– Meier plot showing both overall survival (OS) and progression-free survival (PFS) with regard to the overall expression of genes showed that patients expressing higher levels of AhR, MCL-1, and ARNT showed a comparatively lower survival rate in comparison to patients who expressed lower levels of the respective proteins. In conclusion, we confirm that AhR mediates OC progression, stemness characteristics, and metastatic potential via activation of PI3K/Akt, Wnt/-catenin, and EMT. The proteomic study moreover confirmed the tumor-promoting as well as chemoresistanceinducing properties of AhR. Altogether, the present study provides a better understanding of the the cross-talk between AhR and its regulatory proteins, BCL family members and several other cell-signaling pathways. In addition, the study also recommends AhR to be a potential therapeutic target for overcoming chemoresistance in OC. The interactions of AhR and BCL-2 family members further justifies the use of a combination of BCL-2 and AhR inhibitors for more efficient management of OC, more precisely for cancer stem cell therapy.