Differentiation‐associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic‐metabolizing activity of “luminal” muscle‐invasive bladder cancers

Abstract

Extra‐hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self‐defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro‐carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier‐forming differentiated states in vitro. However, ethoxyresorufin O‐deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP‐DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain‐of‐function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle‐invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into “luminal” and “basal” groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over‐expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1‐activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over‐expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP‐function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies.

The impact of chemotherapeutic drugs on the CYP1A1-catalysed metabolism of the environmental carcinogen benzo[a]pyrene: Effects in human colorectal HCT116 TP53(+/+), TP53(+/−) and TP53(−/−) cells

“…Two bands were detected on the Western blot for CYP1A1; the top band is the correct molecular weight (58 kDa), and thus, the lower band is assumed to be nonspecific. This is consistent with other studies using this antibody to detect human CYP1A1 in other cultured BaP-treated human cells (Hamouchene et al, 2011; Wohak et al, 2016; Baker et al, 2018). Previous investigations in our laboratory have shown that the top band increases with higher BaP concentrations used and also that only the top band is diminished when BaP-treated cells have been transfected with CYP1A1 siRNA (Kucab & Arlt, unpublished data).…”

Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells

“…CYP1A1 and CYP1B1 activities, but not CYP1A2 activity, were detected in RT4 cells. These data are in agreement with previous reports on CYP1A1 and CYP1B1 protein (Plottner et al 2016), and enzyme activity (Baker et al 2018;Reshetnikova et al 2016) in RT4 cells. The complete inhibition of CYP1A1 and CYP1B1 by α-NF resulted in a 50% decrease in the levels of AαC DNA adducts in RT4 cells, signifying that these isoforms prominently carry out the bioactivation of AαC in RT4 cells.…”

Identification of miR-200c and miR141-Mediated lncRNA-mRNA Crosstalks in Muscle-Invasive Bladder Cancer Subtypes

“…Subtype-related pathways presented in this study ( Tables 1 , 2 ) are largely consistent with the previously identified ( Choi et al, 2014a ; Hurst and Knowles, 2014 ; McConkey et al, 2016 ; Ochoa et al, 2016 ; Hau et al, 2017 ; Seiler et al, 2017 ; Baker et al, 2018 ). In general, pathways that are involved in the EMT, metastasis, and immune system process, are upregulated in the basal subtype, whereas, metabolic-related pathways are upregulated in the luminal subtype.…”