Chronic inflammation promotes prostate cancer (PCa) initiation and progression. We previously reported that acute intereluekin-1 (IL-1) exposure represses androgen receptor (AR) accumulation and activity, providing a possible mechanism for IL-1-mediated development of androgen- and AR-independent 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. We generated isogenic sublines from LNCaP cells chronically exposed to IL-1α or IL-1β. Cells were treated with IL-1α, IL-1β, TNFα or HS-5 bone marrow stromal cells conditioned medium to assess cell viability in the presence of cytotoxic inflammatory cytokines. Cell viability was also assessed following serum starvation, AR siRNA silencing and enzalutamide treatment. Finally, RNA sequencing was performed for the IL-1 sublines. 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. 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. Our data provide evidence that chronic IL-1 exposure promotes PCa cell androgen and AR independence and, thus, supports CRPCa development.

Chronic inflammation is a cancer hallmark and chronic exposure to interleukin-1 (IL-1) transforms castration-sensitive prostate cancer (PCa) cells into more fit castration-insensitive PCa cells. p62 is a scaffold protein that protects cells from nutrient deprivation via autophagy and from cytotoxic reactive oxygen via NFκB and NRF2 antioxidant signaling. Herein, we report that the LNCaP PCa cell line acquires high basal accumulation of the p62-KEAP1 complex when chronically exposed to IL-1. p62 promotes non-canonical NRF2 antioxidant signaling by binding and sequestering KEAP1 to the autophagosome for degradation. But despite high basal p62-KEAP1 accumulation, only two of several NRF2-induced genes analyzed, GCLC and HMOX1, showed high basal mRNA levels, suggesting that the high basal p62-KEAP1 accumulation does not result in overall high basal NRF2 activity. Nutrient starvation induces NRF2-dependent GCLC upregulation and HMOX1 repression, and we found that chronic IL-1-exposed LNCaP cells show hypersensitivity to serum starvation-induced GCLC and HMOX1 regulation. Thus, chronic IL-1 exposure affects cell response to nutrient stress. While HMOX1 expression remains NRF2/KEAP1-dependent in chronic IL-1-exposed LNCaP cells, GCLC expression is NRF2/KEAP1-independent. Furthermore, the high basal p62-KEAP1 complex accumulation is not required to regulate GCLC or HMOX1 expression, suggesting cells chronically exposed to IL-1 evolve a novel NRF2-independent role for the p62/KEAP1 axis.

Background/Objectives: The milieu of inflammatory cytokines present in the prostate cancer (PCa) tumor microenvironment exerts various effects on cancer progression. Chronic exposure to the inflammatory cytokine interleukin-1 (IL-1) has been shown to impact signaling via the RELA/NF-kB pathway; however, the effects of chronic inflammation on the integration of different inflammatory signaling pathways, such as the interleukin-6 (IL-6)/STAT3 axis, requires further exploration. Methods: We generated in vitro subline models by exposing the C4-2 and LNCaP PCa cell lines to either IL-1α or IL-1β for several months. We then treated the resulting sublines with acute IL-1 alone, IL-6 alone, or IL-1/IL-6 in combination and assessed for sensitivity to cytokine signaling. We observed changes in proliferation and quantified using Ki-67 immunostaining. Cell proliferation was assessed after siRNA silencing RELA or STAT3. Results: IL-1/IL-6 signaling in combination enhanced the signaling effects of either cytokine alone, particularly cytostasis. While the chronic IL-1 sublines maintained sensitivity to acute IL-6 signaling, they lost sensitivity to acute IL-1 signaling and did not show the enhanced IL-1/IL-6 cytostatic response. Inhibition of RELA and STAT3 rescued cytostasis after IL-1/IL-6 treatment in parental PCa cell lines, but only STAT3 inhibition rescued proliferation in the chronic IL-1 sublines. Conclusions: Our work shows that IL-1/RELA and IL-6/STAT3 work in parallel to synergistically induce cytostasis. However, chronic IL-1 exposure selects for cells that attenuate IL-1/RELA signaling, subsequently attenuating IL-1/IL-6 synergy.

Background: Breast (BCa) and prostate (PCa) cancers are hormone receptor (HR)-driven cancers. Thus, BCa and PCa patients are given therapies that reduce hormone levels or directly blocks HR activity; but most patients eventually develop treatment resistance. We have previously reported that interleukin-1 (IL-1) inflammatory cytokine downregulates ERα and AR mRNA in HR-positive (HR ⁺ ) BCa and PCa cell lines, yet the cells can remain viable. Additionally, we identified pro-survival proteins and processes upregulated by IL-1 in HR ⁺ BCa and PCa cells, that are basally high in HR ⁻ BCa and PCa cells. Therefore, we hypothesize that IL-1 confers a conserved gene expression pattern in HR ⁺ BCa and PCa cells that mimics conserved basal gene expression patterns in HR ⁻ BCa and PCa cells to promote HR-independent survival and tumorigenicity. Methods: We performed RNA sequencing (RNA-seq) for HR ⁺ BCa and PCa cell lines exposed to IL-1 and for untreated HR ⁻ BCa and PCa cell lines. We confirmed expression patterns of select genes by RT-qPCR and used siRNA and/or drug inhibition to silence select genes in HR ⁻ BCa cell lines. Finally, we performed Ingenuity Pathway Analysis (IPA) to identify signaling pathways encoded by our RNA-seq data set. Results: We identified 350 genes in common between BCa and PCa cells that are induced or repressed by IL-1 in HR ⁺ cells that are, respectively, basally high or low in HR ⁻ cells. Among these genes, we identified Sequestome-1 ( SQSTM1/p62 ) and SRY ( Sex-Determining Region Y ) -Box 9 ( SOX9 ) to be essential for survival of HR ⁻ BCa and PCa cell lines. Analysis of publicly available data indicates that p62 and SOX9 expression are elevated in HR-independent BCa and PCa sublines generated in vitro , suggesting that p62 and SOX9 have a role in acquired treatment resistance. We also assessed HR ⁻ cell line viability in response to the p62-targeting drug, verteporfin, and found that verteporfin is cytotoxic for HR ⁻ cell lines. Conclusions: Our 350 gene set can be used to identify novel therapeutic targets and/or biomarkers conserved among acquired (e.g. due to inflammation) or intrinsic HR-independent BCa and PCa.

Background: Cancer cells live in a dynamic and favorable ecosystem conducive to their growth and survival, known as the tumor microenvironment (TME). The TME is replete with various proinflammatory cell types that mediate crosstalk via the secretion of cytokines and chemokines to facilitate tumor growth and development. One cardinal proinflammatory cytokine that is present in the TME is interleukin-1 (IL-1). IL-1 promotes tumor angiogenesis and cancer cell metastasis; thus IL-1 receptor antagonist (IL-1Ra) is of clinical interest. Our lab previously reported that chronic exposure to exogenous IL-1 can select for cancer cells that evolve insensitivity to IL-1 signaling, thus rendering IL-1-targeting therapies, like IL-1Ra, irrelevant. While immune cells are the primary source of TME IL-1, cancer cells can also produce and secrete IL-1 to engage in autocrine and/or paracrine signaling. In this context, cancer cell exposure to multiple other sources of exogenous IL-1 beyond autocrine production might also amplify extrinsic IL-1 signaling in these cells, but the consequences of sustained amplified IL-1 signaling on the regulation, function, and therapeutic response of these cancer cells need to be explored. Methods: Using the IL-1-secreting 5637 bladder cancer (BlCa) cell line, we generated chronic IL-1 sublines by spiking the growth medium with additional IL-1α or IL-1β chronically for 6 months. Once established, we assessed subline acute IL-1 sensitivity, paracrine signaling and response to IL-1Ra. Results: Following chronic exposure to elevated exogenous IL-1 levels, the 5637 BlCa cell line retains sensitivity to acute IL-1 and IL-1Ra and maintains the ability to induce IL-1-dependent endothelial cell activation, which is reversed with IL-1Ra. These data suggest that for cancer cells that engage in cell autonomous IL-1 signaling, sustained extrinsic IL-1 exposure does not dampen IL-1 or IL-1Ra sensitivity, supporting the context-dependent use of IL-1 antagonists as rational therapeutics in both acute and chronic inflammatory TMEs.