Afshan Fathima Nawas

Chronic inflammation is a hallmark of the breast cancer tumor microenvironment and is also known to be associated with disease progression and therapeutic response. Interleukin-1 (IL-1) signaling has been widely studied in breast cancer biology; however, the long-term effect of sustained IL-1 exposure on hormone receptor-positive breast cancer cells remain poorly understood. In this study, we investigated how chronic IL-1 exposure influences inflammatory response, hormone dependency, and therapeutic sensitivity in ERα+/PR+ breast cancer models, MCF7 and T47D. Chronic IL-1 exposure attenuated response to subsequent acute IL-1 treatment, but the chronically exposed cells remained sensitive to serum deprivation, retained dependence on estrogen or progesterone receptor signaling, and responded robustly to endocrine and chemotherapeutic treatments. Extensive changes in basal gene expression and histone modification revealed that chronic IL-1 exposure alters transcriptional reprogramming and chromatin remodeling. Together, these findings demonstrate that chronic IL-1 signaling drives selective inflammatory response in hormone receptor-positive MCF7 and T47D breast cancer cells. This work underscores the continued therapeutic relevance of hormone receptor-targeted strategies in chronically inflamed tumors and provides insight into how sustained inflammatory stress shapes tumor behavior and gene regulation predicted to promote tumor progression.

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.

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 block HR activity; but most patients eventually develop treatment resistance. We have previously reported that interleukin-1 (IL-1) inflammatory cytokine downregulatesERαandARmRNA 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 the BCa and PCa cell lines. Finally, we performed Ingenuity Pathway Analysis (IPA) and used the gene ontology web-based tool, GOrilla, 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 identifiedSequestome-1(SQSTM1/p62) andSRY(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 thatp62andSOX9expression are elevated in HR-independent BCa and PCa sublines generated in vitro, suggesting thatp62andSOX9have a role in acquired hormone receptor independence and 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: The androgen receptor (AR) nuclear transcription factor is a therapeutic target for prostate cancer (PCa). Unfortunately, patients can develop resistance to AR‐targeted therapies and progress to lethal disease, underscoring the importance of understanding the molecular mechanisms that underlie treatment resistance. Inflammation is implicated in PCa initiation and progression and we have previously reported that the inflammatory cytokine, interleukin‐1 (IL‐1), represses AR messenger RNA (mRNA) levels and activity in AR‐positive (AR⁺) PCa cell lines concomitant with the upregulation of prosurvival biomolecules. Thus, we contend that IL‐1 can select for AR‐independent, treatment‐resistant PCa cells.Methods: To begin to explore how IL‐1 signaling leads to the repression of AR mRNA levels, we performed comprehensive pathway analysis on our RNA sequencing data from IL‐1‐treated LNCaP PCa cells. Our pathway analysis predicted nuclear factor kappa B (NF‐κB) p65 subunit (RELA), a canonical IL‐1 signal transducer, to be significantly active and potentially regulate many genes, including AR. We used small interfering RNA (siRNA) to silence the NF‐κB family of transcription factor subunits, RELA, RELB, c‐REL, NFKB1, or NFKB2, in IL‐1‐treated LNCaP, C4‐2, and C4‐2B PCa cell lines. C4‐2 and C4‐2B cell lines are castration‐resistant LNCaP sublines and represent progression toward metastatic PCa disease, and we have previously shown that IL‐1 represses AR mRNA levels in C4‐2 and C4‐2B cells.Results: siRNA revealed that RELA alone is sufficient to mediate IL‐1 repression of AR mRNA and AR activity. Intriguingly, while LNCaP cells are more sensitive to IL‐1‐mediated repression of AR than C4‐2 and C4‐2B cells, RELA siRNA led to a more striking derepression of AR mRNA levels and AR activity in C4‐2 and C4‐2B cells than in LNCaP cells.Conclusions: These data indicate that there are RELA‐independent mechanisms that regulate IL‐1‐mediated AR repression in LNCaP cells and suggest that the switch to RELA‐dependent IL‐1 repression of AR in C4‐2 and C4‐2B cells reflects changes in epigenetic and transcriptional programs that evolve during PCa disease progression.

Estrogen receptor α (ERα)^low/− tumors are associated with breast cancer (BCa) endocrine resistance, where ERα low tumors show a poor prognosis and a molecular profile similar to triple negative BCa tumors. Interleukin‐1 (IL‐1) downregulates ERα accumulation in BCa cell lines, yet the cells can remain viable. In kind, IL‐1 and ERα show inverse accumulation in BCa patient tumors and IL‐1 is implicated in BCa progression. IL‐1 represses the androgen receptor hormone receptor in prostate cancer cells concomitant with the upregulation of the prosurvival, autophagy‐related protein, Sequestome‐1 (p62/SQSTM1; hereinafter, p62); and given their similar etiology, we hypothesized that IL‐1 also upregulates p62 in BCa cells concomitant with hormone receptor repression. To test our hypothesis, BCa cell lines were exposed to conditioned medium from IL‐1‐secreting bone marrow stromal cells (BMSCs), IL‐1, or IL‐1 receptor antagonist. Cells were analyzed for the accumulation of ERα, progesterone receptor (PR), p62, or the autophagosome membrane protein, microtubule‐associated protein 1 light chain 3 (LC3), and for p62‐LC3 interaction. We found that IL‐1 is sufficient to mediate BMSC‐induced ERα and PR repression, p62 and autophagy upregulation, and p62‐LC3 interaction in ERα⁺/PR⁺ BCa cell lines. However, IL‐1 does not significantly elevate the high basal p62 accumulation or high basal autophagy in the ERα⁻/PR⁻ BCa cell lines. Thus, our observations imply that IL‐1 confers a prosurvival ERα⁻/PR⁻ molecular phenotype in ERα⁺/PR⁺ BCa cells that may be dependent on p62 function and autophagy and may underlie endocrine resistance.

Background: In immunosurveillance, bone‐derived immune cells infiltrate the tumor and secrete inflammatory cytokines to destroy cancer cells. However, cancer cells have evolved mechanisms to usurp inflammatory cytokines to promote tumor progression. In particular, the inflammatory cytokine, interleukin‐1 (IL‐1), is elevated in prostate cancer (PCa) patient tissue and serum, and promotes PCa bone metastasis. IL‐1 also represses androgen receptor (AR) accumulation and activity in PCa cells, yet the cells remain viable and tumorigenic; suggesting that IL‐1 may also contribute to AR‐targeted therapy resistance. Furthermore, IL‐1 and AR protein levels negatively correlate in PCa tumor cells. Taken together, we hypothesize that IL‐1 reprograms AR positive (AR⁺) PCa cells into AR negative (AR⁻) PCa cells that co‐opt IL‐1 signaling to ensure AR‐independent survival and tumor progression in the inflammatory tumor microenvironment.Methods: LNCaP and PC3 PCa cells were treated with IL‐1β or HS‐5 bone marrow stromal cell (BMSC) conditioned medium and analyzed by RNA sequencing and RT‐QPCR. To verify genes identified by RNA sequencing, LNCaP, MDA‐PCa‐2b, PC3, and DU145 PCa cell lines were treated with the IL‐1 family members, IL‐1α or IL‐1β, or exposed to HS‐5 BMSC in the presence or absence of Interleukin‐1 Receptor Antagonist (IL‐1RA). Treated cells were analyzed by western blot and/or RT‐QPCR.Results: Comparative analysis of sequencing data from the AR⁺ LNCaP PCa cell line versus the AR⁻ PC3 PCa cell line reveals an IL‐1‐conferred gene suite in LNCaP cells that is constitutive in PC3 cells. Bioinformatics analysis of the IL‐1 regulated gene suite revealed that inflammatory and immune response pathways are primarily elicited; likely facilitating PCa cell survival and tumorigenicity in an inflammatory tumor microenvironment.Conclusions: Our data supports that IL‐1 reprograms AR⁺ PCa cells to mimic AR⁻ PCa gene expression patterns that favor AR‐targeted treatment resistance and cell survival.