The effect of inflammatory cytokines on steroidogenesis in colon cancer cells

Abstract

Colorectal cancer (CRC) is the third most prevalent cancer worldwide and has a high incidence and mortality rate. It is known that oestrogens impact CRC incidence and prognosis. Sulfated oestrogens, primarily oestrone sulfate (E1S), are taken into cells via organic anion transporting polypeptides (OATPs). Steroid sulfatase (STS) then desulfates this into oestrone (E1). E1 can be reduced by 17β-hydroxysteroid dehydrogenases (HSD17Bs) to form biologically active oestradiol (E2). Our group previously showed that STS activity and HSD17Bs expression are altered in CRC to favour E2 synthesis. However, what regulates STS activity, HSD17Bs and OATPs expression in CRC is poorly understood. My hypothesis is that inflammatory cytokines drive these changes in E2 synthesis. In this thesis, the Cancer Genome Atlas (TCGA) human colorectal adenocarcinoma RNA-Seq dataset (n = 433, normal=51 and CRC=382) was analysed to generate Kaplan–Meier survival curves and correlation graphs comparing normal and CRC tissue expression of TNFα, HSD17Bs, OATPs, and STS. How inflammatory cytokines impacted oestrogen uptake, enzyme activity and cell proliferation were further assessed through radiolabelled enzyme activity assays, LC-MS/MS, and proliferation assays. In CRC tissue, results demonstrated positive correlations between TNFα and STS (*P=0.036), HSD17B4 (**P=0.0045), HSD17B12 (*P=0.016), OATP2B1 (***P<0.0001), OATP3A1 (***P<0.0001), OATP3A1 (***P<0.0001), and OATP1C1 (***P<0.0001). In CRC cell lines, treatment with TNFα significantly increased STS activity as measured via the conversion of 3H-E1S to 3H-E1. An increase in STS activity was associated with an increase in CRC cell proliferation. HCT116 and Colo205 cells displayed the greatest proliferative responses compared to LoVo and SW620 cells. For HSD17Bs, TNFα treatment decreased HSD17B2 and HSD17B4 expression and increased 2 HSD17B7 and HSD17B12 expression, suggesting greater E2 synthesis. TNFα also significantly upregulated OATP2B1 expression in HCT116 and Colo205 cells. E1S uptake significantly increased in HCT116 and SW620 cells. Finally, CRC oestrogen metabolism, measured by LC-MS/MS, showed that TNFα reduced E2 loss in CRC cell lines and reduced E1 synthesis from E2. Taken together, these data show that TNFα significantly increases STS activity, alters HSD17B expression, and upregulates oestrogen uptake in CRC cells. This suggests that TNFα is an important regulator of oestrogen metabolism in CRC.