ABSTRACT
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 mRNA levels and activity in AR-positive (AR+) PCa cell lines concomitant with the upregulation of pro-survival 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 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 towards 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 de-repression 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.
INTRODUCTION
Androgen deprivation is used as a first line therapy for PCa patients, but some patients will inevitably progress to androgen-independent PCa1. Activating mutations, splice variation, or overexpression of AR can render AR ligand-independent or functional under castrate levels of androgen1. Therefore, patients that develop androgen independence are given anti-androgens that directly bind and prevent AR transactivation activity1. PCa tumors can, however, evolve anti-androgen resistance, leading to lethal disease1. Anti-androgens can function as AR agonists resulting in treatment resistance1; but anti-androgen resistance and AR independence can also result from the emergence of PCa tumor cells with low or no AR (ARlow/-) accumulation or activity2.
We and others have found that IL-1 represses AR levels and activity in PCa cells3–7 and one group has shown that chronic IL-1 exposure reduces sensitivity to the anti-androgen, bicalutamide, in the androgen-dependent LNCaP PCa cell line5. While inflammation and IL-1 are associated with reduced AR levels or activity in benign prostatic hyperplasia7 and there is evidence that IL-1 inversely correlates with AR activity in metastatic PCa patient tissue8, it is unknown if IL-1 is sufficient to drive AR independence or treatment resistance during PCa disease progression. We previously reported that concomitant with AR repression, IL-1 induces a suite of genes in LNCaP cells that mimic basal gene expression in AR-independent PCa cell lines6, suggesting that IL-1 exposure could select for viable AR-independent PCa tumor cells. For example, we demonstrated that Sequestome-1 (p62), an autophagy and antioxidant protein from our gene suite6, is required for cell survival in AR-independent PCa cell lines9 and others have recently discovered that p62 overexpression can drive prostate cell tumor formation in vivo10.
To begin to understand how IL-1 drives AR independence, we evaluated IL-1 regulation of AR repression across a disease progression cell line model using LNCaP, C4-2, and C4-2B PCa cell lines. LNCaP is an androgen-dependent cell line originating from a PCa patient lymph node metastasis. LNCaP cells require androgen and AR activity for survival and do not form metastatic tumors in mice11–13. The C4-2 and C4-2B PCa cell lines are LNCaP sublines isolated from a subcutaneous tumor (C4-2) in a castrated mouse that metastasized to bone (C4-2B) when re-grafted in a castrated mouse13. Thus, C4-2 and C4-2B cells represent progressive, metastatic, androgen-independent PCa disease13. As with LNCaP cells, IL-1 represses AR levels in C4-2 and C4-2B cell lines6, 14 and, thus, are relevant cell line models to begin to address IL-1 regulation of AR during disease progression.
Nuclear Factor Kappa B (NF-κB) transcription factor is the canonical mediator of IL-1 signaling15. The NF-κB transcription factor family is comprised of RELA (p65), RELB, c-REL, NFKB1 (p105/p50) and NFKB2 (p100/p52)15. RELA, RELB, and c-REL contain DNA binding and transactivation domains15. NFKB1 and NFKB2 are processed from 105 and 100 kDa proteins to 50 and 52 kDa subunits, respectively, that bind DNA but lack transactivation domains15. The NF-κB family members form homo- and heterodimers to induce or repress transcription15, where the RELA/p50 heterodimer typically regulates gene expression in response to IL-115. Using RNA sequencing pathway analysis of IL-1-treated LNCaP cells, we identified several candidate transcriptional regulators of AR expression. Not surprisingly, our analysis identified RELA as one of the most significantly activated transcriptional regulators of AR expression in IL-1-treated cells predicted by IPA (z-score = 5.58, p-value 1.6E-11) and we show that RELA can indeed mediate IL-1 repression of AR levels and AR activity. Using the LNCaP PCa cell line progression model, our results also suggest that RELA-dependent repression of AR evolves with PCa progression towards androgen independence.
MATERIALS AND METHODS
Cell Culture
LNCaP, C4-2, and C4-2B prostate cancer (PCa) cell lines were maintained in a 37°C, 5.0% (v/v) CO2 growth chamber and cultured in DMEM (Gibco/Thermo Scientific; 1185-092) supplemented with 10% (v/v) fetal bovine essence (fetal bovine serum alternative) (FBE; Seradigm; 3100-500), 0.4mM L-glutamine (L-glut; Gibco/Invitrogen; 25030-081), and 10U/ml penicillin G sodium and 10 mg/ml streptomycin sulfate (pen-strep; Gibco/ Invitrogen; 15140-122).
Cell Treatments
Intereleukin-1 (IL-1): Vehicle control (0.1% bovine serum albumin (BSA) in 1X phosphate buffered saline (PBS)), 0.5 ng/ml, 5 ng/ml, or 25 ng/ml IL-1α (Gold Bio, 1110-01A-10) or IL-1β (Gold Bio, 1110-01B-10) were added to DMEM/10% FBE growth medium. Gene silencing (siRNA): The following siRNA concentrations were used: 70nM non-targeting siRNA (Dharmacon, D-001206-13-05), RELA siRNA (Dharmacon, M-003533-02-0005), NFKB1 siRNA (Dharmacon, M-003520-01-0005) or p62 siRNA (Dharmacon, M-010230-00-0005); 100nM non-targeting siRNA, RELB siRNA (Dharmacon, M-004767-02-0005), c-REL siRNA (Dharmacon, M-004768-01-0005), or NFKB2 siRNA (Dharmacon, M-003918-02-0005); 70nM non-targeting siRNA (Origene, SR30004) or RELA siRNA (Origene, SR 321602A-C). Cells were transfected with siRNA using siTran 1.0 transfection reagent (Origene, TT300003) added to cells in DMEM/10% FBE growth medium for 1 day. siRNA containing medium was then removed and replaced with fresh DMEM/10% FBE plus vehicle control, IL-1α, or IL-1β. Serum starvation: After plating, cells were maintained in DMEM/2.5% FBE for several days prior to replacing medium with DMEM/0% FBE (serum starvation) or DMEM/10% FBE (replete medium control).
Cell Counts
Cells were fixed in 100% cold methanol. The nuclei were stained with DAPI (Roche Diagnostics, 10236276001) and the stained cells imaged and counted using the Cytation3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, Vermont).
Crystal Violet Staining
Cells were fixed in 100% cold methanol at −20C for 30 minutes and stained with 0.25% crystal violet (Acros Organics, AC212120250) in 10% methanol for 5 minutes at room temperature. Excess stain was washed off with deionized water.
RNA Isolation and Reverse Transcription Quantitative PCR (RT-QPCR)
Total RNA was extracted, reverse transcribed, and analyzed by RT-QPCR as previously described6. Primer sequences for genes of interest are listed below. Gene of interest cycle times (CT) were normalized to the β-actin. Relative mRNA levels were calculated using the 2-ΔΔCT method. Primer sequences, 5’-3’: Androgen Receptor (AR), forward AAGACGCTTCTACCAGCTCACCAA, reverse TCCCAGAAAGGATCTTGGGCACTT; Beta actin (β-actin), forward GATGAGATTGGCATGGCTTT, reverse CACCTTCACCGGTCCAGTTT; Prostate Specific Antigen (PSA), forward CACCTGCTCGGGTGATTCTG, reverse ACTGCCCCATGACGTGATAC; Sequestosome-1 (p62), forward AAATGGGTCCACCAGGAAACTGGA, reverse TCAACTTCAATGCCCAGAGGGCTA; Mitochondrial Superoxide Dismutase 2 (SOD2), forward GGCCTACGTGAACAACCTGA, reverse GTTCTCCACCACCGTTAGGG; RELA, forward TGAACCAGGGCATACCTGTG, reverse CCCCTGTCACTAGGCGAGTT; NFKB1, forward TGATCCATATTTGGGAAGGCCTGA, reverse GTATGGGCCATCTGTTGGCAG; NFKB2, forward CCTAAGCAGAGAGGCTTCCG, reverse TCTTTCGGCCCTTCTCACTG; RELB, forward AGCTCTACTTGCTCTGCGAC, reverse AACACAATGGCAATCTGGCG; c-REL, forward ACAGCACAGACAACAACCGA, reverse AGTAGCCGTCTCTGCAGTCT.
RNA Sequencing (RNA-seq) Analysis
Pathway analysis was conducted using QIAGEN’s Ingenuity Pathway Analysis (IPA) tool (http://www.qiagen.com/ingenuity). RNA-seq datasets used for this study are available at GEO NCBI, accession GSE105088. LNCaP samples treated with vehicle control or IL-1β were considered and 3,658 differentially expressed genes with log2 counts per million (CPM) ≥ 0, absolute log2 fold change (FC) > 0.6, and false discovery rate (FDR) ≤ 0.05 were considered for further downstream analysis.
Western Blot
Western blotting was done as previously described6. Briefly, primary and secondary antibodies were diluted in 2.5% BSA in 1X TBST. Protein blot bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific; 34095) or Clarity Western ECL Substrate (Bio-Rad; 1705061) and imaged using the Amersham Imager 600 (GE, Marlborough, MA). Primary antibodies: AR (Cell Signaling; D6F11), p62 (Abnova; H00008878-M01), SOD2 (Abgent; AM7579a), β-actin (Santa Cruz; sc-69879), RELA (Cell Signaling; 6956S), NFKB1 (Cell Signaling; 3035S), PSA (Cell Signaling; 5365S), or c-REL (Cell Signaling; 4727T). Secondary antibodies: Sheep anti-mouse (Jackson ImmunoResearch Laboratories; 515-035-062), goat anti-rabbit (Abnova; PAB10822). Densitometry: Western blot densitometry for protein bands was performed using Image J (National Institutes of Health, Bethesda, Maryland). Protein bands were first normalized to β-actin loading control and then normalized to the treatment control.
Immunofluorescence
Cells were fixed and permeabilized with 100% methanol at −20°C for 30 minutes. Fixed cells were blocked with 2.5% BSA in 1X PBS at room temperature for at least 30 minutes. Antibodies were diluted in 2.5% BSA in 1X PBS. Primary antibody: AR (Cell Signaling; D6F11). Secondary antibody: Alexafluor 568, goat anti-rabbit (Invitrogen; A11061). Nuclei were stained with DAPI (Roche Diagnostics, 10236276001). Immunostained cells were imaged at 20X magnification using the Nikon Eclipse Ti fluorescence microscope and Nikon NIS Elements software (Nikon, Melville, New York).
Statistical Analysis
Statistical significance was determined using unpaired student’s t-test. p-value ≤ 0.05 is considered statistically significant. Error bars, +/- standard deviation (STDEV); n = 3 biological replicates. Experiments were repeated at least 3 times and representative data shown.
RESULTS
RNA Sequencing pathway analysis reveals RELA as a potential regulator of AR expression
We previously performed RNA sequencing on the LNCaP PCa cell line treated with IL-1β and found that AR and AR target genes are down regulated6. Here, we performed upstream regulator analysis using IPA on the vehicle control versus IL-1β-treated LNCaP data sets and identified RELA as a significantly active transcriptional regulator of AR (z-score = 5.58, p-value 1.6E-11). Figure 1 shows selected RELA interactors from our IPA analysis. A complete list of RELA interactors identified based on IPA for IL-1β-treated LNCaP cells and their relationships can be found in Supplemental Table 1. It is worth noting that IPA’s knowledge base indicates RELA to be a positive regulator of AR expression which is inconsistent (indicated by the yellow arrow between RELA and AR, Fig. 1) with our RNA sequencing data that indicates that IL-1β represses AR mRNA levels in LNCaP cells. Therefore, we set out to experimentally test the relationship between RELA activity and AR expression in our model. To do so, we silenced RELA in IL-1-treated LNCaP cells and used Super Oxide Dismutase 2 (SOD2) expression as a positive control for RELA activity.
LNCaP, C4-2 and C4-2B cell lines show differential sensitivity to IL-1
Prior to embarking on our investigation of RELA regulation of AR expression in the LNCaP cells, we had observed that LNCaP cells are more sensitive to IL-1 signaling than C4-2 or C4-2B cells. For example, we find that IL-1-treated LNCaP cells show greater SOD2 upregulation and AR repression than C4-2 or C4-2B cells. Based on RT-QPCR, 25 ng/ml IL-1α or IL-1β induced SOD2 mRNA levels ∼18 fold in LNCaP cells versus ∼6 fold in C4-2B cells (Fig. 2A) and based on western blot, 25 ng/ml IL-1α or IL-1β induced SOD2 protein accumulation ∼5 fold in LNCaP cells versus ∼3 fold in C4-2 and C4-2B cells (Fig. 2B). Furthermore, 25 ng/ml IL-1α or IL-1β repressed AR mRNA levels ∼5 fold in LNCaP cells versus ∼3 fold in C4-2 or ∼2 fold in C4-2B cells (Fig. 2A). In kind, western blot shows that 25 ng/ml IL-1α or IL-1β repressed AR protein accumulation ∼6 fold in LNCaP cells versus ∼4 fold in C4-2 cells or ∼2 fold in C4-2B cells (Fig. 2B). Interestingly, while IL-1 receptor (IL-1R1) mRNA levels are elevated in C4-2 and C4-2B cells and RELA mRNA and protein levels are similar across LNCaP, C4-2, and C4-2B cell lines, basal NFKB1 mRNA and p50 protein levels are lower in C4-2 and C4-2B cells than in LNCaP cells and IL-1-mediated induction of NFKB1 mRNA and p50 protein levels is not as robust in C4-2 or C4-2B cells as in LNCaP cells (Fig. 2A & B). Given that canonical IL-1 signaling is mediated via the RELA/p50 heterodimer15 the lower NFKB1 mRNA and p50 protein levels might underlie C4-2 and C4-2B reduced sensitivity to IL-1.
In addition to reduced sensitivity to IL-1 modulation of target mRNA and protein levels, C4-2 and C4-2B cells are less sensitive to IL-1 induced cytotoxicity. LNCaP, C4-2, and C4-2B cells were grown in 25 ng/ml IL-1α or IL-1β for 1,3, or 5 days and the cell number was recorded (Fig. 2C). Cells were also visualized on day 5 using crystal violet stain (Fig. 2C). C4-2 and C4-2B cells show faster growth rate than LNCaP cells in both vehicle and IL-1 treatment conditions; but when comparing cytotoxicity in IL-1, C4-2 and C4-2B cells show ∼14-23% reduction in cell numbers on day 5, while LNCaP cells show ∼ 58% reduction (Fig. 2C). Thus, compared to LNCaP cells, C4-2 and C4-2B cells have reduced sensitivity to IL-1.
As stated earlier, C4-2 and C4-2B cell lines are less dependent on androgen than LNCaP cells13. For example, compared to LNCaP cells, C4-2 and C4-2B cells have reduced sensitivity to growth arrest under serum-free growth conditions (Supplemental Fig. 1). Interestingly, relative to LNCaP cells, C-42 and C4-2B cells have reduced basal mRNA and protein levels of the AR target gene, Prostate Specific Antigen (PSA) (Fig. 2); but, have higher basal levels of the AR target gene, Kallikrein (KLK2) (Supplemental Fig. 3A). Indicating that the regulation of AR target genes is altered in C4-2 and C4-2B cells. Taken together, cell response to IL-1 signaling may evolve with androgen, including IL-1-RELA regulation of AR levels and activity, which we investigated below.
RELA mediates IL-1 repression of AR levels and activity differentially in LNCaP versus C4-2 and C4-2B cells
To determine if RELA mediates IL-1 repression of AR, we siRNA-silenced RELA in IL-1-treated LNCaP, C4-2, or C4-2B cells and analyzed AR mRNA levels and protein accumulation. We also analyzed PSA or KLK2 mRNA and/or protein levels as surrogates for AR transcriptional activity and we immunostained cells for AR nuclear accumulation. LNCaP, C4-2, and C4-2B cells were treated with vehicle control, 25 ng/mL IL-1α, or 25 ng/mL IL-1β in the presence of non-targeting control siRNA or two different pools of RELA siRNA. RELA siRNA was sufficient to attenuate IL-1 repression of AR, PSA, and KLK2 mRNA and/or protein (Fig. 3, Supplemental Fig. 2 & 3B) and AR nuclear accumulation (Fig. 4) in C4-2 and C4-2B cells treated with 25 ng/ml IL-1. However, RELA siRNA was not sufficient to attenuate IL-1 repression of AR or showed only a slight effect on PSA or KLK mRNA and/or protein in LNCaP cells treated with 25 ng/ml IL-1 (data not show). Given the enhanced sensitivity of LNCaP cells to IL-1, we instead silenced RELA in LNCaP cells treated with a lower IL-1 concentration (0.5 ng/mL) and found that RELA siRNA was sufficient to attenuate IL-1 repression of AR, PSA, and KLK2 mRNA and/or protein levels; however, the LNCaP response to RELA siRNA was not consistent or as robust as for the C4-2 or C4-2B cell lines (Fig. 3, Supplemental Fig. 2 & 3, data not shown). As a positive control for RELA siRNA efficacy, RELA siRNA was sufficient to attenuate SOD2 mRNA and protein levels in IL-1-treated LNCaP, C4-2, and C4-2B cells (Fig. 3, Supplemental Fig. 2) and as a negative control for RELA siRNA specificity, RELA siRNA did not affect KEAP1 mRNA levels (Supplemental Fig. 2). Taken together, our data indicates that RELA mediates IL-1 repression of AR in C4-2 or C4-2B cells and to a lesser extent in LNCaP cells.
IL-1 repression of AR levels or activity is specific to the RELA NF-κB family member
Given that the RELA/p50 heterodimer mediates canonical IL-1 signaling15 and that RELA siRNA downregulates NFKB1 mRNA and p50 protein in IL-1-treated cells (Fig. 3, Supplemental Fig. 2), we expected that loss of NFKB1 would also attenuate IL-1 repression of AR levels and activity. NFKB1 siRNA alone was not sufficient to attenuate IL-1 repression of AR or PSA levels, nor sufficient to block IL-1 induction of SOD2 mRNA or protein levels in LNCaP, C4-2, or C4-2B cells (Fig. 5). Similar results were obtained for RELB, c-REL, and NFKB2 siRNA (Supplemental Fig. 4-6). Taken together, our data suggest that RELA is the NF-κB family member that is necessary and sufficient for IL-1-mediated AR repression.
p62 does not mediate IL-1 repression of AR levels or activity
IL-1 upregulates p62 accumulation in PCa cell lines6, 14, p62 is an IL-1-RELA target gene (Fig. 3), and p62 is a known mediator of IL-1-induced NF-κB activation16–18. To investigate if p62 mediates IL-1-RELA repression of AR, IL-1-treated LNCaP, C4-2, or C4-2B cells were transfected with p62 siRNA. p62 siRNA was not sufficient to attenuate IL-1 repression of AR or PSA levels or to block IL-1 induction of SOD2 levels in LNCaP, C4-2, or C4-2B cells (Fig. 6). Thus, while IL-1-RELA signaling induces p62 mRNA and protein accumulation, p62 does not appear to be necessary for IL-1-RELA repression of AR levels or activity in LNCaP, C4-2, or C4-2B cells. Interestingly, p62 siRNA consistently upregulates PSA mRNA and protein in C4-2 and C4-2B cells under basal growth conditions, but p62 siRNA does not affect PSA or AR levels or AR activity in the presence of IL-1 (Fig. 6). Thus, p62 function includes PSA repression, but independent of IL-1 signaling.
DISCUSSION
IL-1-RELA represses AR mRNA and protein levels
RELA overexpression in LNCaP cells has been shown to induce AR mRNA accumulation and AR nuclear accumulation19, 20; conversely, co-silencing of RELA and p50 reduced AR protein levels in LNCaP cells19. Another study found that p52 overexpression, but not RELA overexpression, induces AR nuclear accumulation and activity in LNCaP cells, while neither p52 nor RELA overexpression affected AR mRNA levels21. Finally, counter to AR activation by RELA or p52 overexpression, c-REL overexpression was shown to block AR activity in LNCaP cells22. Thus, NF-κB regulation of AR expression and activity is complex and context-specific.
Notably, the studies described above mimic constitutive NF-κB activity through forced overexpression. We instead, silenced basal or IL-1-induced NF-κB activity in LNCaP, C4-2, or C4-2B cells. We did not observe a change in AR levels or AR activity under basal growth conditions by any NF-κB family member and, using two different pools of oligos, only RELA repressed AR levels and activity and only in the presence of IL-1. RELA is the canonical mediator of IL-1-induced NF-κB signaling15, so it is not surprising that RELA would mediate IL-1 repression of AR levels and activity.
Like IL-1, Tumor Necrosis Factor Alpha (TNFα) signals through RELA/p5015. TNFα was shown to repress AR expression in a NFκB-dependent23 or RELA-dependent manner24 in LNCaP cells. As we observed for IL-1-treated cells, siRNA loss of NFKB1 did not affect AR mRNA levels in TNFα-treated cells24; however, p50 did bind the AR promoter in TNFα-treated cells, along with RELA and the HDAC and NCOR co-repressors24. It remains to be investigated if IL-1 signaling represses AR expression through a signaling mechanism similar to TNFα and/or if RELA indirectly, for example through micro RNAs, downregulates AR mRNA levels. Indeed, it has been shown that TNFα reduces AR mRNA stability posttranscriptionally25. RELA can also form homodimers to regulate transcription15; thus, it is also possible that RELA mediates IL-1 repression of AR levels and activity independent of p50.
IL-1-RELA signaling differs among androgen-dependent LNCaP versus androgen-independent C4-2 and C4-2B cells
We confirmed that the C4-2 and C4-2B cell lines are less dependent on androgen than the LNCaP cell line and we discovered that C4-2 and C4-2B cells are less sensitive to IL-1 signaling than LNCaP cells, yet C4-2 and C4-2B cells are more dependent on RELA-mediated repression of AR levels and activity in response to IL-1 signaling. Interestingly, C4-2 and C4-2B are also less sensitive to TNFα than LNCaP cells and reduced sensitivity is, in part, dependent on reduced basal levels of the TNFα receptor associated protein, TRADD23 and we find that C4-2 and C4-2B cells have reduced basal levels of the NFκB p50 transcription factor subunit which likely contributes to reduced IL-1 and TNFα sensitivity. Taken together, our and other published results suggest that during PCa disease progression, cell response to inflammation evolves. To better understand the molecular mechanisms underlying IL-1-RELA signaling in AR-dependent versus AR-independent PCa cells, it will be informative to identify the differences in co-factors, chromatin markers, and chromatin structure at the AR locus in IL-1-treated LNCaP, C4-2, and C4-2B cells.
Our results suggest that changes can occur in IL-1-RELA signaling during PCa disease progression that could promote AR independence and resistance to AR-targeting anti-androgens by selecting for cells that adapt to survive with low or no AR accumulation or activity. Indeed, studies show that inflammation, including IL-1 accumulation, inversely correlate with AR levels or activity in PCa disease progression7, 8. Interestingly, while IL-1 has been found to inversely correlate with AR levels or activity, constitutive RELA accumulation appears to positively correlate with biochemical relapse26, 27, presumably due to AR activity, in PCa disease progression. In this context, RELA accumulation may reflect evolved constitutive RELA signaling that is independent of cell response to exogenous IL-1.
p62 silencing does not prevent IL-1-RELA repression of AR level or activity
Using p62 siRNA, we were able to reduce p62 levels to at least the levels observed in the RELA knockdowns of IL-1-treated cells and, thus, we would have expected to detect some attenuation of IL-1-induced AR repression in p62-silenced cells if p62 were required. p62 silencing did not affect AR mRNA levels in IL-1-treated cells. Likewise, the RELA target gene, SOD2, did not change in p62-silenced IL-1-treated cells, further supporting that RELA can function independently of p62 in response to IL-1. Our results were unexpected given that p62 has been shown to mediate IL-1 activation of NF-κB activity in HepG and HEK293 cells16–18; but our results may reflect underlying differences in cell lines and/or treatment conditions.
Interestingly, p62 silencing did significantly upregulate PSA mRNA levels in C4-2 and C4-2B cells under basal growth conditions, suggesting the p62 promotes the repression of basal PSA levels and/or basal AR activity. While we did not detect an effect on PSA mRNA or protein levels in LNCaP cells transfected with p62 siRNA, the sensitivity of RNA sequencing does reveal that p62 siRNA upregulates PSA mRNA levels in LNCaP cells (data not shown). Thus, as observed for IL-1-RELA signaling, LNCaP, C4-2 and C4-2B cells may have differential response to p62 signaling that correlates with castration resistance.
Our RELA siRNA data indicate that RELA mediates IL-1 induction of p62 mRNA and protein accumulation. RELA siRNA, however, had no effect on p62 levels under basal growth conditions. Thus, p62 repression of basal PSA levels and/or basal AR activity is independent of RELA activity. The molecular mechanism and functional significance of p62 repression of PSA is unknown but should be explored given that PSA is prognostic for PCa diagnosis and treatment response28.
CONCLUSION
Cumulative evidence suggests that IL-1 promotes androgen and AR independence in PCa. Elevated IL-1 is associated with disease progression8, 29, IL-1 accumulation inversely correlates with AR activity in PCa patient tissue8, IL-1 signaling down regulates AR levels and activity3–7, and chronic IL-1 exposure promotes anti-androgen resistance5. Androgen deprivation can induce tumor microenvironment inflammation, including IL-1 accumulation, by promoting immune cell infiltration and PCa cell IL-1 secretion30, 31. Thus IL-1 can select for treatment resistant PCa cells, for example following therapy, by selecting for PCa cells that adapt to low or no AR accumulation or activity.
In the era of anti-androgen use, there has been an increase in castration resistant patients with low or no AR activity32; the functional or correlative significance of IL-1-RELA signaling has yet to be determined in these patients. Our data shows that IL-1-induced RELA activity can attenuate AR accumulation and activity in PCa cells and, thus, further exploration and clarification of IL-1-RELA signaling in PCa disease progression and treatment resistance is warranted to identify alternative therapeutic targets to AR.
FIGURE LEGENDS
Supplemental Table 1. RELA Reactome. Complete list of molecules in RELA network (Fig. 1) and their relationships with RELA. The relationships are determined from the Ingenuity Knowledge Data Base.
ACKNOWLEDGEMENTS
For their advice and support throughout this process, we would like to thank all the members of the labs of Drs. Nikki Delk and Chao Xing. We would also like to acknowledge financial support for the Delk lab from the University of Texas at Dallas and financial support from the National Institutes of Health (NIH/NCI R21CA175798 (Delk); NIH/NCI K01CA160602 (Delk); NIH UL1TR001105 (Xing)).
Footnotes
Financial support: NIH/NCI R21CA175798 (Delk); NIH/NCI K01CA160602 (Delk), University of Texas at Dallas (Delk), NIH UL1TR001105 (Xing)
Conflict of Interest: the authors declare no potential conflicts of interest