Chronic IL-1 exposure drives LNCaP cells to evolve androgen and AR independence

Abstract

TX 75390RUNNING TITLE: Chronic IL-1 promotes androgen and AR independence CORRESPONDING AUTHOR: Nikki A. ABSTRACT:Background: Chronic inflammation is a known cause of prostate cancer (PCa) initiation and progression. Interleukin-1 (IL-1) is an inflammatory cytokine secreted by tumor cells and bonederived immune cells that contributes to chronic inflammation in the tumor microenvironment. IL-1 is elevated in PCa patients and contributes to PCa progression and treatment resistance. IL-1 has been shown to promote PCa metastasis and bone colonization, shown to recruit mesenchymal stems cells to the tumor to support castration-resistant PCa (CRPCa) development and chronic IL-1 exposure was shown to promote PCa anti-androgen resistance. We previously reported that acute IL-1 exposure represses androgen receptor (AR) accumulation and activity, providing a possible mechanism for IL-1-mediated development of androgen-and ARindependent PCa. Given that acute inflammation is quickly resolved, and chronic inflammation is, instead, co-opted by cancer cells to promote tumorigenicity, we set out to determine if chronic IL-1 exposure leads to similar repression of AR and AR activity observed for acute IL-1 exposure and to determine if chronic IL-1 exposure selects for androgen-and AR-independent PCa cells. Methods:We isolated isogenic LNCaP sublines from LNCaP cells chronically exposed for 3-4 months to IL-1α or IL-1β. LNCaP and the IL-1 sublines were treated with IL-1α, IL-1β, TNFα or conditioned medium from the HS-5 bone marrow stromal cell line to assess cell viability in the presence of cytotoxic inflammatory cytokines. Cell viability was also assessed following serum starvation, AR siRNA silencing and treatment with the anti-androgen, enzalutamide. RNA sequencing and bioinformatics analysis were also performed for the IL-1 sublines.Results: MTT, RT-qPCR and western blot analysis show that the sublines evolved resistance to inflammation-induced cytotoxicity and intracellular signaling and evolved reduced sensitivity to siRNA-mediated loss of AR, serum deprivation and enzalutamide. Furthermore, differential gene expression reveals that canonical AR signaling is aberrant in the IL-1 sublines, where the cells show constitutive PSA repression and basally high KLK2 and NKX3.1 mRNA levels and bioinformatics analysis predicts that pro-survival and pro-tumorigenic pathways are activated in the sublines. Conclusions:Our data provide evidence that chronic IL-1 exposure promotes PCa cell androgen and AR independence and, thus, supports CRPCa development.