Fig 1.
LNCaP cells lose sensitivity to IL-1-mediated AR repression over time.
(A) RT-qPCR and (B) western blot analyses were performed for LNCaP cells treated with vehicle control, 12.5 ng/ml IL-1α or IL-1β for 3 days (D3) or 1 (W1), 2 (W2) or 3 (W3) weeks. mRNA and/or protein accumulation were determined for AR and AR target genes, KLK2, PSA and NKX3.1. IL-1 repressed (A) mRNA and (B) protein for AR and AR target genes, KLK2, PSA and NKX3.1 at 3 days. AR and AR target gene levels began to re-emerge after 1–2 weeks of chronic IL-1 exposure. Error bars, ± STDEV of 3 biological replicates; p-value, *≤0.05, **≤0.005, ***≤0.0005. mRNA fold change is normalized to day 3 vehicle control. Western blot band densitometry is normalized to β-actin and fold change normalized to day 3 vehicle control.
Fig 2.
LNas1 and LNbs1 IL-1 sublines are resistant to IL-1-induced cytotoxicity and intracellular signaling.
(A) Cell viability was determined using MTT for LNCaP, LNas1 and LNbs1 for 5 days with vehicle control, 25 ng/ml IL-1α or IL-1β. IL-1 reduces LNCaP cell viability; however, LNas1 and LNbs1 cells remain viable. (B) LNCaP, LNas1 and LNbs1 cells were treated for 3 days with vehicle control or 25 ng/ml IL-1α or IL-1β and analyzed for protein accumulation by western blot. Western blot analyses were performed for IL-1 target gene, SOD2, to determine treatment efficacy and activation of IL-1 intracellular signaling, PARP cleavage to determine activation of apoptosis, and AR, PSA, and NKX3.1 to determine AR activity. p65 and p50 are NFκB transcription factor subunits. IL-1 induced SOD2 protein accumulation and PARP cleavage in LNCaP cells, but IL-1 had less or no effect on SOD2 protein levels or PARP cleavage in LNas1 or LNbs1 cells. IL-1 downregulated AR, PSA and NKX3.1 protein accumulation in LNCaP cells, but IL-1 had less or no effect on the protein levels in LNas1 and LNbs1 cells. IL-1 had no effect on p65 protein levels in LNCaP, LNas1 or LNbs1. IL-1 induced p50 protein in LNCaP cells, but not in LNas1 or LNbs1 cells. (C, D) LNCaP, LNas1 and LNbs1 cells were treated for 3 days with vehicle control or 25 ng/ml IL-1α or IL-1β and analyzed for mRNA levels by RT-qPCR for SOD2, AR, PSA, KLK2, NKX3.1, IL-1R1, RELA (p65) and NFKB1 (p50). IL-1 significantly induced SOD2 and NFKB1, significantly repressed AR, PSA, KLK2, and slightly repressed NKX3.1 mRNA levels in LNCaP cells, but IL-1 had no significant effect on the mRNA levels in LNas1 or LNbs1 cells. IL-1 had little or no effect on IL-1R1 or RELA mRNA levels in LNCaP, LNas1 or LNbs1 cells. Finally, compared to LNCaP cells, LNas1 and LNbs1 have lower basal PSA and (p50) mRNA or protein levels and higher basal KLK2 and NKX3.1 mRNA or protein levels. Error bars, ± STDEV of 3 biological replicates; p-value, *≤0.05, **≤0.005. For each individual cell line, fold MTT optical density (OD) is normalized to the vehicle control. Western blot band densitometry is normalized to β-actin and fold change normalized to LNCaP vehicle control. PARP cleavage densitometry shows the ratio of cleaved to uncleaved PARP. Fold mRNA levels are normalized to LNCaP vehicle control for IL-1 treatments in order to also compare basal levels between the cell lines.
Fig 3.
LNas1 and LNbs1 IL-1 sublines are resistant to HS-5-induced cytotoxicity and intracellular signaling.
(A) LNCaP cells were pre‐treated for 1 day with vehicle control or 400 ng/ml human recombinant IL‐1RA and the following day the medium was replaced with treatment control (DMEM) or HS‐5 conditioned medium (CM) plus an additional 400 ng/ml IL‐1RA or vehicle control for 3 additional days. Western blot analyses were performed for SOD2 to determine treatment efficacy, PARP cleavage to determine activation of apoptosis, and AR, PSA, and NKX3.1 to determine AR activity. IL-1RA was sufficient to attenuate HS-5 CM-induced SOD2 upregulation, PARP cleavage, and repression of AR, PSA and NKX3.1. (B) Cell viability was determined using MTT for LNCaP, LNas1, LNbs1 and C4-2B cells treated for 5 days with treatment control or HS-5 CM. HS-5 CM reduced LNCaP and C4-2B cell viability; however, LNas1 and LNbs1 cells remained viable. LNCaP, LNas1, LNbs1 and C4-2B cells treated for 3 days with treatment control or HS-5 CM and analyzed for protein accumulation by western blot (C) or mRNA levels by RT-qPCR (D, E). Protein and/or RNA analyses were performed for IL-1 target gene, SOD2, to determine treatment efficacy and activation of IL-1 intracellular signaling, PARP cleavage to determine activation of apoptosis, and AR, PSA, KLK2 and NKX3.1 to determine AR activity. HS-5 CM induced PARP cleavage and SOD2 mRNA and protein levels and repressed AR, PSA, KLK2 and NKX3.1 mRNA and protein levels in LNCaP and C4-2B cells, but HS-5 CM had less effect in LNas1 or LNbs1 cells. Error bars, ± STDEV of 3 biological replicates; p-value, *≤0.05, **≤0.005, ***≤0.0005. For each individual cell line, fold MTT optical density (OD) is normalized to the treatment control (DMEM). Western blot band densitometry is normalized to β-actin and fold change normalized to LNCaP treatment control (DMEM). PARP cleavage densitometry shows the ratio of cleaved to uncleaved PARP. For each individual cell line, fold mRNA levels are normalized to the cell line treatment control (DMEM); basal levels are not compared between the cell lines.
Fig 4.
LNas1 and LNbs1 IL-1 sublines are resistant to TNFα-induced cytotoxicity and intracellular signaling.
(A) Cell viability was determined using MTT for LNCaP, LNas1, LNbs1 and C4-2B cells treated for 5 days with vehicle control or 25 ng/ml TNFα. TNFα reduced LNCaP cell viability; however, LNas1, LNbs1 and C4-2B cells remain viable. LNCaP, LNas1, LNbs1 and C4-2B cells were treated for 3 days with treatment control or 25 ng/ml TNFα and analyzed for mRNA levels by RT-qPCR (B, C) or protein accumulation by western blot (D). RNA and/or protein analyses were performed for TNFα target gene, SOD2, to determine treatment efficacy and activation of TNFα intracellular signaling and for AR, PSA, KLK2 and NKX3.1 to determine AR activity. TNFα induced SOD2 and repressed AR, PSA, KLK2 and NKX3.1 mRNA and protein levels in LNCaP cells, but TNFα had less or no effect on mRNA or protein levels in LNas1, LNbs1 or C4-2B cells. Error bars, ± STDEV of 3 biological replicates; p-value, *≤0.05, **≤0.005, ***≤0.0005. For each individual cell line, fold MTT optical density (OD) is normalized to the vehicle control. For each individual cell line, fold mRNA levels are normalized to the cell line vehicle control; basal levels are not compared between the cell lines. Western blot band densitometry is normalized to β-actin and fold change normalized to LNCaP vehicle control.
Fig 5.
LNas1 and LNbs1 IL-1 sublines have active AR signaling but constitutively low PSA levels.
LNCaP, LNas1, LNbs1 and C4-2B cells were transfected with 70 nM AR siRNA or siRNA control in DMEM/2.5% FBE or treated with 10 nM R1881 or vehicle control in DMEM/0% FBE and analyzed on day 4 for (A, B) RNA levels by RT-qPCR or (C, D) protein accumulation by western blot. AR siRNA reduced and R1881 increased the mRNA and/or protein accumulation of AR target genes PSA, KLK2 and NKX3.1 in LNCaP, LNas1 and LNbs1 cells. (E) LNCaP cells were treated with 25 ng/ml IL-1α or IL-1β for 3 days and LNas1 and LNbs1 cells were left untreated. Conditioned medium from the LNCaP, LNas1 and LNbs1 cells was analyzed for secreted PSA by western blot. LNCaP cells secreted PSA and IL-1 treatment reduced PSA secretion in LNCaP cells. LNas1 and LNbs1 secreted low levels of PSA compared to LNCaP cells. * indicates BSA on the ponceau stain. (F) LNCaP, LNas1 and LNbs1 were grown in DMEM/10% serum for 1 month and analyzed for PSA protein accumulation. PSA levels remain repressed in LNas1 and LNbs1 cells. Error bars, ± STDEV of 3 biological replicates; p-value, *≤0.05, **≤0.005, ***≤0.0005. For each individual cell line, fold mRNA levels are normalized to the cell line vehicle control; basal levels are not compared between the cell lines. Western blot band densitometry is normalized to β-actin and fold change normalized to LNCaP vehicle control.
Fig 6.
LNas1 and LNbs1 IL-1 sublines are insensitive to cytotoxicity induced by loss of AR or AR activity.
(A) LNCaP, LNas1, LNbs1 and C4-2B cells were transfected with 70 nM AR siRNA or siRNA control in DMEM/2.5% FBE for 4 days, treated with 10 nM R1881 or vehicle control in DMEM/0% FBE for 4 days, grown in 10% versus 0% FBE (serum) for 5 days or treated with 50 μM enzalutamide (ENZA) for 5 days and analyzed for cell viability by MTT. LNas1, LNbs1 and C4-2B cells showed reduced sensitivity to AR siRNA, R1881, serum starvation or enzalutamide. (B) LNCaP, LNas1, LNbs1 and C4-2B cells were grown in 10% versus 0% FBE (serum) for 5 days and analyzed for protein accumulation of AR target genes, NKX3.1 and PSA. Western blot shows that serum starvation (0% serum) reduced NKX3.1 and PSA accumulation, suggesting a reduction in AR activity. (C) LNCaP, LNas1 and LNbs1 cells were treated with 50 μM enzalutamide (ENZA) for 3 days and analyzed for protein accumulation of AR target genes, NKX3.1 and PSA. Western blot shows that enzalutamide reduced NKX3.1 and PSA accumulation. Error bars, ± STDEV of 3 biological replicates; p-value, *≤0.05, **≤0.005, ***≤0.0005. For each individual cell line, fold MTT optical density (OD) is normalized to the vehicle control. Western blot band densitometry is normalized to β-actin and fold change normalized to LNCaP vehicle control.
Fig 7.
IL-1 is secreted by immune and cancer cells in the tumor microenvironment. Acute IL-1 exposure is largely cytotoxic for PCa cells, but concomitantly causes the emergence of an ARlow/- PCa subpopulation that is androgen- and AR-independent. If left unresolved acute inflammation becomes chronic inflammation. Chronic IL-1 selects from the ARlow/- PCa subpopulation for PCa cells that evolve resistance to IL-1 (and TNFα) inflammatory cytokine cytotoxicity, restore AR, and acquire reduced androgen and AR dependence. Acute IL-1 responses can be mitigated with IL-1 antagonists, such as IL-1RA, to maintain or restore androgen and AR dependence and sensitivity to ADT and anti-androgens in PCa cells. Chronic IL-1-induced phenotypes are not reversible and, therefore, require the identification of alternative therapeutic targets. Image created with BioRender.com.