Androgen receptor (AR) is a major survival factor for prostate cancer. Inflammation is implicated in many cancer types, including prostate cancer. Activation of MAP3K7 (also termed TAK1) and downstream IκB kinase β (IKKβ) by proinflammatory cytokines such as TNFα stimulates NF-κB survival pathways. Paradoxically, MAP3K7 is often deleted in human prostate cancer. Here, we demonstrate that AR protein expression is lower in inflammatory tumor areas compared with non-inflammatory tissues in patients with prostate cancer. Map3k7 knockout increased AR protein levels and activity in the mouse prostate, and MAP3K7 and AR protein levels were inversely correlated in prostate cancer patient specimens. TNFα treatment increased AR protein ubiquitination and proteasomal degradation. Mechanistically, activation of IKKβ by TNFα induced phosphorylation and TRCP1/2 E3 ligase–mediated polyubiquitination and degradation of AR protein. TNFα suppressed prostate cancer proliferation, which could be rescued by blockade of AR degradation. These findings reveal a previously unrecognized tumor suppressive function of the inflammation-activated MAP3K7–IKKβ axis in degrading AR protein. Moreover, they suggest that aberrant elevation of AR protein could be a prognostic biomarker and therapeutic target for MAP3K7-deficient prostate cancer.
This study identifies that MAP3K7–IKKβ signaling plays a tumor-suppressive role in prostate cancer by degrading AR, revealing potential prognostic and therapeutic strategies for MAP3K7-deficient tumors.
Prostate cancer is the most common male malignancy and the second leading cause of male cancer-related death in western countries. Inflammation is implicated in prostate cancer pathogenesis (1, 2). Increasing evidence suggests that the production of the pro-inflammatory cytokines such as TNFα from inflammatory environment and tumor cells promotes activation of the downstream oncogenic NF-κB and survival of cancerous cells (3). Given the importance of inflammation in prostate cancer development and progression (2), the signaling pathways mediating the activation of NF-κB have been attractive targets for chemo-prevention and -therapy of prostate cancer. Although rationalized chemoprevention trials using anti-inflammation drugs have been conducted, the outcomes have been mixed (2), stressing the importance of investigating the mechanism of action of inflammation in prostate cancer.
MAP3K7, a serine/threonine protein kinase (also termed TGFβ-activated kinase 1, TAK1), is an important component of MAPK signaling pathways (4–6). MAP3K7 is also one of the key mediators of the inflammatory signaling and can be activated by TNFα, TGFβ, and lipopolysaccharides. With the stimulation of these cytokines, MAP3K7 can form a complex with TAK1-binding protein 1 (TAB1), TAB2, and TRAF6 to promote the activation of IκB kinase (IKK) complex and its downstream target NF-κB. The IKK complex contains two catalytic subunits, IKKα and IKKβ and one regulatory subunit IKKγ (also known as NEMO). Activation of IKK by proinflammatory cytokines such as TNFα leads to phosphorylation and subsequent poly-ubiquitination and degradation of IκB mediated by E3 ubiquitin ligases. Recent studies show that IKK can also phosphorylate other cancer-related proteins, including BAD, FOXO3, BCL-10, and A20 and promote ubiquitination and degradation of these proteins (7–11). MAP3K7 gene is also implicated in human cancers such as prostate cancer because it is deleted in more than 10% of prostate cancer in patients (12, 13). However, the precise role of MAP3K7 loss in prostate cancer pathogenesis remains largely unclear.
Androgen receptor (AR), a member of the steroid hormone nuclear receptor family, plays a critical role in initiation and progression of prostate cancer. As a key regulator of prostate homeostasis, AR protein level and its activity are precisely regulated by posttranslational modifications including phosphorylation, acetylation, sumoylation, methylation, and ubiquitination (14, 15). Similar to other transcription factors, AR protein is tightly regulated by the ubiquitin–proteasome pathway. The E3 ubiquitin ligases such as MDM2, C-terminal HSP-interacting protein, and Siah have been implicated in the control of AR stability and activity under basal or stress conditions (16–18).
In the present study, we demonstrated that the proinflammatory cytokine TNFα induces AR protein polyubiquitination and proteasomal degradation. By generating prostate-specific Map3k7 gene knockout (KO) mice, we demonstrated that loss of Map3k7 increased AR protein level and activity in the mouse prostate. We also showed that IKKβ, a downstream effector of MAP3K7, interacts with AR and enhances AR degradation in a manner dependent on β-TRCP E3 ligases. We further demonstrated that blockade of AR degradation enhanced AR activity and prostate cancer cell growth in vitro and in castrated mice under the inflammatory condition.
Materials and Methods
Cell lines, cell culture, and transfection
LNCaP, LAPC-4, C4–2, 22Rv1, PC-3, DU145, RWPE-1, and 293T cell lines were purchased from the ATCC and authenticated periodically via STR profiling (IDEXX BioResearch). LNCaP, LAPC-4, C4–2, 22Rv1, PC-3, and DU145 cells were cultured in RPMI-1640 medium supplemented with 10% FBS. RWPE-1 cells were cultured in K-SFM medium supplemented with bovine pituitary extract and human recombinant EGF. 293T cells were maintained in DMEM supplemented with 10% FBS. Mycoplasma contamination was regularly examined using Lookout Mycoplasma PCR Detection Kit (Sigma-Aldrich). Plasmocin (InvivoGen) was routinely added to the cell culture medium to prevent or eliminate Mycoplasma contamination. Transfections were performed by electroporation using an Electro Square Porator ECM 830 (BTX; ref. 19) or by using Lipofectamine 2000 (Thermo Fisher Scientific). Approximately 70%–90% transfection efficiencies were routinely achieved.
Generation of prostate-specific map3k7 KO mice
Map3k7 (Tak1) loxp/loxp (Map3k7P/P) mice were kindly provided by Dr. Jiwang Zhang from the Loyola University Medical Center (Maywood, IL). Exon-2 of the Map3k7 gene was flanked by two loxp sites (20). Probasin (Pb)-Cre4 transgenic mice were acquired from the National Cancer Institute (NCI) Mouse Repository, which were originally generated in the laboratory of Dr. Pradip Roy-Burman at University of Southern California (Los Angeles, CA; ref. 21). Pb-Cre+;Map3k7P/P mice were obtained by cross breeding Pb-Cre4 males with Map3k7P/P females. All mice were maintained under standard conditions of food, water, light, and temperature. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic.
PCR-based genotyping of mice
Genotyping of “wild-type” and Pb-Cre+;Map3k7P/P mice was performed following standard PCR protocol with primers listed in Supplementary Table S1.
Generation of xenografts in mice
The mouse study was conducted according to the NIH guidelines and was approved by the IACUC at Mayo Clinic. Age-matched 6-week-old male SCID mice were castrated before cell injection and randomly separated into different groups. For C4–2 xenograft study, C4–2 cell lines (3 × 106) infected with indicated lentivirus were suspended in 50 μL of PBS and mixed with 50 μL of Matrigel (BD Biosciences) and administered into subcutaneous layer of the left flank of mice. The caliper was used to determine the xenograft size every three day for 27 days. The xenograft volume was determined using the formula (length × width2)/2. After 27-day observation, xenografts were harvested. For LNCaP cell xenograft study, LNCaP cell lines (3 × 106) expressing EV, WT AR or degradation-resistant mutant were suspended in 50 μL of PBS and mixed with 50 μL of Matrigel (BD Biosciences) before they were inoculated into subcutaneous layer of the left flank of mice. Mice were treated with TNFα (10 ng/kg/d) six days after cell injection. A caliper was used to determine the xenograft size every three day for 27 days and xenografts were harvested at the end of measurement.
Tissue section and hematoxylin and eosin staining
4-μm-thick sections were cut from formalin-fixed paraffin-embedded (FFPE) blocks of human prostate cancer specimens, mouse prostate tissues, and xenograft tumors and mounted onto slides. All slides were deparaffinized in xylene for three times and rehydrated through graded ethanol. After stained with hematoxylin and washed with water and ethanol, slides were staining with 1% eosin. Slides were dehydrated through different concentration ethanol washes and xylene washes, and finally sealed over the tissue sections with coverslips.
IHC of human and mouse prostate cancer tissues
A tissue microarray (TMA) was constructed from the FFPE samples of metastatic prostate cancer, identified after a search of pathologic and clinical databases of archival tissues collected originally with written informed consent from patients (22, 23). The Mayo Clinic institutional review board approved the experimental protocols for retrieving pathology blocks/slides and for accessing electronic medical records. 4-μm-thick sections were cut from FFPE blocks of human prostate cancer specimens, mouse prostate tissues, and xenograft tumors and mounted onto slides. IHC was performed as described previously (24). The human TMA contained 157 cores (16 0.6-mm cores and 141 1.0-mm cores) resulting from 53 samples (20 bone metastases and 33 non-bone metastases) of 51 patients. 154 cores were used for IHC data analysis after excluding cores with lost tissue greater than 50%. Staining index (SI) was calculated as follows: Staining intensity (intensity: 0 = no staining, 1 = low staining, 2 = medium staining, and 3 = strong staining) and staining percentage was graded accordingly. A final SI score for each staining was obtained by multiplying values of staining percentage and intensity.
Organoids derived from the prostate tissues of “wild-type” control and Map3k7 KO mice (Pb-Cre+; Map3k7P/P) or 22Rv1 cells were harvested, mounted or seeded on the slides and fixed with ice-cold methanol/acetone (1:1 dilution, stored at −20°C). After washing with IF buffer and blocking with 10% of goat serum, slides were incubated with the primary antibody at 4°C overnight. The next day, slides were incubated with the second antibody and mounted with mounting medium containing DAPI. Immunofluorescence was visualized and photos were taken using LSM700 confocal microscope (Carl Zeiss). The OD values of staining in each cell were measured. 50 cells from each organoid line generated from each mouse were randomly selected and five organoid lines were used for quantitative analysis.
Smart pools of siRNAs for human IKKβ, β-TRCP2, and nonspecific control siRNAs were purchased from Thermo Fisher Scientific—Dharmacon. Sh-AR, sh-MAP3K7, sh-MAPK14, sh-JNK, and nonspecific control shRNAs were purchased from Sigma-Aldrich (a list of shRNAs is provided in Supplementary Table S2). siRNA and shRNA transfections of cells were performed following the manufacturer's instruction.
Total RNA was isolated from cells or mouse prostate tissues and cDNA was synthesized using the Super-Script kit from Thermo Fisher Scientific. Two-step RT-PCR was performed using the SYBR Green Mix (Bio-Rad) and an iCycler iQTM detection system (Bio-Rad) according to the manufacturer's instructions. Both forward and reverse primers were used at a final concentration of 200 nmol/L. Expression of GAPDH gene in each sample was used as an internal control. Information for primers used is provided in Supplementary Table S1.
Experiments were carried out with three or more replicates. Statistical analyses were performed using the Student t test unless otherwise stated. Values with P < 0.05 are considered statistically significant.
Details related to other methods are provided in Supplementary Information.
TNFα promotes AR protein degradation in prostate cancer cells
De Marzo and colleagues (25) made a seminal observation that AR protein levels are much lower in luminal cells within the proliferative inflammation atrophy (PIA) lesion compared with tissues outside PIA. We were interested to determine whether there is any association between low AR protein expression and inflammation in prostate cancer. AR protein IHC analysis in prostate cancer patient specimens showed that AR protein level was much lower in inflammatory areas compared those without inflammation (Fig. 1A). Quantitative analysis further confirmed the negative correlation between inflammation and AR protein expression in prostate cancer (Fig. 1B).
To determine whether inflammation plays any causal role in downregulating AR protein expression in prostate cancer, we examined the effect of proinflammation cytokines on AR protein expression in prostate cancer cell lines. TNFα is one of the important pro-inflammation cytokines released by the inflammatory environment or cancer cells and plays an essential role in prostate cancer progression (26, 27). We treated AR-positive prostate cancer cell lines LNCaP and LAPC-4 with TNFα and harvested cells for Western blot analysis. We demonstrated that TNFα treatment induced downregulation of AR protein in a dose-dependent manner in both cell lines (Fig. 1C), suggesting that there may be a common mechanism mediating TNFα-induced reduction of AR protein. Next, we examined whether TNFα-induced inhibition of AR protein expression is mediated through the ubiquitination and proteasomal degradation pathway. We treated LNCaP cells with TNFα in the presence or absence of the 26S proteasome inhibitor MG132. TNFα-induced decrease in AR protein level was almost completely blocked by MG132 (Fig. 1D). In agreement with this result, TNFα treatment markedly enhanced AR poly-ubiquitination in LNCaP cells (Fig. 1E). Furthermore, we measured the stability of AR protein in LNCaP cells treated with or without TNFα. The AR protein half-life was approximately 4 hours in vehicle-treated LNCaP cells (Fig. 1F and G), consistent with the previous report (28). Notably, the AR protein half-life was drastically shortened in TNFα-exposed cells (Fig. 1F and G). These results indicate that the proinflammatory cytokine TNFα induces a decrease in AR protein level and this effect is mediated through the ubiquitination and proteasomal degradation pathway in prostate cancer cells.
MAP3K7 KO increases AR protein level and activity in the mouse prostate
MAP3K7 is one of the key factors mediating signaling activated by proinflammatory cytokines such as TNFα (5, 6). Similar to previous reports (12, 13, 29–31), meta-analysis of patient data confirmed that the MAK3K7 gene is often (ranging from 10% to 14% of cases) deleted in prostate cancer specimens (Supplementary Fig. S1A). To determine whether MAP3K7 deletion has any impact on AR protein expression and prostate cancer pathogenesis, we generated prostate-specific Map3k7 gene KO mice (Pb-Cre+;Map3k7P/P) by cross-breeding probasin (Pbsn or termed Pb) promoter-driven Cre males (21) with Map3k7loxp/loxp (Map3k7P/P) conditional females (Fig. 2A; ref. 20). IHC analysis showed that Map3k7 protein was well expressed in the prostate of Cre-negative “wild-type” control mice whereas it was effectively eliminated in the prostate of Map3k7 KO littermates (Fig. 2B). Histological (hematoxylin and eosin) analysis indicated that there were no indications of prostate intraepithelial neoplasia (PIN) lesion, including enlarged nucleus, decreased cytoplasm staining, prominent nucleoli or nuclear atypia in the prostate of Map3k7 KO mice at 12 months of age (Fig. 2B). Hyperplasia or dysplasia was detectable, but only in a very small portion of acini in the ventral prostate of Map3k7 KO mice (Fig. 2B). In agreement with these observations, there was no obvious difference in the size and weight of the prostates between control and Map3k7 KO mice (Fig. 2C and D). Ki-67 expression was slightly increased in the prostate of Map3k7 KO mice compared with control mice, but the difference was not statistically significant (Fig. 2E and F).
We also performed IHC analysis of murine AR protein and found that the AR protein level was much higher in the prostate of Map3k7 KO mice compared with WT mice (Fig. 2G and H). In agreement with increased AR protein level, expression of AR target genes, including Pbsn, Nkx3.1, and Tmprss2 was significantly higher in the prostate of Map3k7 KO mice compared with control mice (Supplementary Fig. S1B), which is consistent with the results in MAP3K7 knockdown (KD) prostate cancer cells as reported recently (32). We further generated organoids from the prostate tissues of both "wild-type" and Map3k7 KO mice. Similar to the results in the prostate tissues, AR protein was higher in Map3k7 KO organoids compared with “wild-type” counterparts (Fig. 2I and J). Importantly, TNFα treatment decreased AR protein expression in “wild-type” organoids, a result similarly seen in prostate cancer cell lines (Fig. 1C); however, such effect was abolished by Map3k7 KO (Fig. 2I and J).
That MAP3K7 modulates AR protein level in the mouse prostate prompted us to explore whether this also occurs in human prostate cancer cell lines and patient samples. To this end, we infected 22Rv1 and LNCaP cell lines with lentivirus-expressing nonspecific control small hairpin RNA (sh-NS) or MAP3K7-specific shRNAs and treated cells with TNFα. Similar to the results observed in murine prostate organoids, TNFα-induced downregulation of AR protein was completely abolished by MAP3K7 KD by shRNAs in both 22Rv1 and LNCaP cell lines (Supplementary Fig. S1C and S1D).
To further verify whether MAP3K7 is involved in the regulation of AR protein by proinflammatory signaling, we examined the expression of AR and phosphorylated MAP3K7 (p-MAP3K7) proteins in inflammatory and noninflammatory regions of prostate cancer patient specimens with proficient or deficient expression of MAP3K7. We demonstrated that in MAP3K7-proficient prostate cancer tissues, AR protein level was lower in areas with high infiltration of cells positive for CD3, a T-cell marker and high expression of p-MAP3K7 compared with the areas with little or no infiltration of CD3-positive cells and limited expression of p-MAP3K7 (Supplementary Fig. S1E). In contrast, in MAP3K7-deficient samples AR expression levels in CD3-positive areas were comparable with those in CD3-negative areas (Supplementary Fig. S1E). These data indicate that MAP3K7 activation by proinflammatory signaling plays an important role in mediating AR protein downregulation induced by inflammation in prostate cancer tissues. These data further suggest that although deletion of Map3k7 gene alone is not sufficient to promote prostate tumorigenesis in mice, at least within a one-year timeframe, loss of MAP3K7 induces elevation of AR protein level and activity in both mouse and human prostate.
MAP3K7 loss enhances prostate cancer cell growth in vitro and in mice
Consistent with the results observed in LNCaP prostate cancer cells cultured in charcoal-stripped (TNFα-low/none) medium as reported recently (32), KD MAP3K7 had minimal effect on AR protein expression in RWPE1 cells cultured in serum-free (TNFα-free) media (Supplementary Fig. S2A and S2B); however, MAP3K7 KD largely increased AR protein levels in various cell lines treated with TNFα (Supplementary Fig. S2A–S2C). In addition, KD of MAP3K7 significantly increased AR polyubiquitination in LNCaP cells cultured in regular (TNFα-containing) media (Supplementary Fig. S2D). However, coimmunoprecipitation (co-IP) experiments showed that there was no interaction between MAP3K7 and AR (Supplementary Fig. S2E), suggesting that MAP3K7 regulation of AR protein is mediated through indirect mechanisms.
Castration-resistant prostate cancer (CRPC) often metastasizes to lymph node and bone, two organs heavily enriched with lymphocytes. To explore the clinical relevance of MAP3K7-mediated downregulation of AR protein, we examined the expression of MAP3K7 and AR proteins in CRPC patient samples. We performed MAP3K7 and AR IHC on a TMA, which contains 154 TMA elements derived from 53 samples (20 bone metastases and 33 non-bone metastases) of 51 patients as reported previously (22, 23). We revealed that MAP3K7 protein expression negatively correlated with AR protein level in this cohort and the correlation is statistically significant (Pearson correlation test, r = −0.329, P = 3.0e−5; Fig. 3A–C). Moreover, it has been reported that AR activity is enhanced in TMPRSS2–ERG gene fusion-positive prostate cancer due to increased expression of the ETS transcription factor ERG (33). We found that tumors with MAP3K7 deletions, which had almost no overlap with ERG gene fusions in the The Cancer Genome Atlas (TCGA) database (Supplementary Fig. S2F; refs. 13, 34), exhibited much greater AR activity than ERG fusion-positive tumors in primary prostate cancer specimens (Supplementary Fig. S2G). These data indicate that loss of MAP3K7 associates with increased AR protein expression and activity in prostate cancer patient samples.
Next, we sought to determine the importance of MAP3K7 in regulating prostate cancer cell growth in androgen-depleted conditions. Consistent with recent report (32), MAP3K7 KD significantly enhanced growth of 22Rv1 and LAPC-4 cells cultured in charcoal-stripped media (Supplementary Fig. S2H). Also, we knocked down MAP3K7 and AR individually or together in C4–2 and LAPC-4 cells and cultured cells in charcoal-stripped media. Western blot analysis confirmed the effectiveness of MAP3K7 and AR KD in C4–2 and LAPC-4 cells (Fig. 3D and Supplementary Fig. S2I). As expected, AR KD inhibited C4–2 and LAPC-4 cell growth in vitro (Fig. 3E and Supplementary Fig. S2J). Although MAP3K7 KD substantially increased cell growth, this effect was abolished in AR KD cells (Fig. 3E and Supplementary Fig. S2J). Similar to the findings in the Map3k7 KO mouse prostate (Fig. 2I), MAP3K7 KD alone significantly increased expression of AR target genes, including KLK3 (PSA), NKX3.1 and TMPRSS2 in C4–2 cells, a result similar to the recent report (32), but this effect was suspended in AR KD counterparts (Supplementary Fig. S2K).
To assess the effect of MAP3K7 loss on prostate cancer cell growth in vivo, an additional set of C4–2 stable cell lines generated as in Fig. 3E were cultured in androgen-deprived media for 4 weeks before they were injected subcutaneously into castrated SCID mice. We demonstrated that MAP3K7-KD tumors grew much faster than control tumors, but the effect was blocked in AR KD tumors (Fig. 3F and G). IHC analysis showed that depletion of MAP3K7 significantly increased Ki-67 expression; however, this effect was abolished in AR KD tumors (Fig. 3H; Supplementary Fig. S2L). We also examined the effect of MAP3K7 and/or AR KD on expression of cleaved Caspase-3. We found that MAP3K7 KD alone had no obvious effect on the level of cleaved Caspase-3 in these tumors (Fig. 3I; Supplementary Fig. S2L). In contrast, AR KD largely increased Caspase-3 cleavage in both control and MAP3K7 KD tumors (Fig. 3I; Supplementary Fig. S2L). We further examined the expression of AR target genes including KLK3 (PSA), NKX3.1 and TMPRSS2 in these tumors. We found that knocking down MAP3K7 increased expression of these AR target genes without having significant effects on AR mRNA expression (Supplementary Fig. S2M). Expression of these genes was downregulated by AR co-KD (Supplementary Fig. S2M), similar to the results reported recently (32). Collectively, we demonstrate that deletion of MAP3K7 enhances castration-resistant growth of prostate cancer cells under both in vitro and in vivo conditions and this effect is ablated in the absence of AR protein expression.
IKKβ activation by TNFα induces AR protein degradation in prostate cancer cells
Given that there is no interaction between MAP3K7 and AR (Supplementary Fig. S2E), we sought to determine whether its downstream factors, including MAPK14, JNK, and IKKβ (35), play any role in TNFα-induced AR degradation. We found that KD of endogenous IKKβ (IKBKB) increased the level of endogenous AR protein in LNCaP cells cultured in FBS-supplemented (TNFα-contained) medium (Fig. 4A) whereas neither MAPK14 nor JNK KD had any obvious effect on AR protein expression (Supplementary Fig. S3A and S3B). These data suggest that IKKβ is one of the key downstream effectors of MAP3K7-mediating TNFα-induced AR degradation. To further test this hypothesis, we transfected IKKβ expression plasmid into LNCaP cells and demonstrated that ectopic expression of IKKβ decreased AR protein levels, but this effect was completely abolished by MG132 treatment (Fig. 4B), suggesting that IKKβ promotes AR protein degradation through the proteasomal pathway. Moreover, different from the WT IKKβ, the kinase-dead mutant (IKKβ-KD) failed to decrease AR protein expression in LNCaP cells (Fig. 4C). Treatment of LNCaP cells with the IKKβ inhibitor wedelolactone blocked TNFα-induced AR protein destruction (Fig. 4D). These data support the notion that the kinase activity is essential for IKKβ-mediated degradation of AR protein in LNCaP cells. Because MG132 treatment blocked IKKβ-induced AR destruction, we examined whether IKKβ promotes AR protein polyubiquitination. Expression of WT IKKβ enhanced polyubiquitination of ectopically expressed or endogenous AR, but no such effect was observed for the IKKβ-KD mutant (Fig. 4E; Supplementary Fig. S3C). In contrast, genetic silencing of IKKβ by siRNAs or pharmacological inhibition of IKKβ by wedelolactone decreased AR polyubiquitination (Fig. 4F and G). Protein half-life analysis showed that the AR protein stability was increased upon depletion of IKKβ by siRNAs (Fig. 4H–J). IKKβ is widely studied in different cancer types in various conditions (36). Consistent with the previous report that depletion of IKKβ inhibits proliferation of AR-negative prostate cancer cells (37), shRNA-mediated KD of IKKβ also inhibited growth of PC-3 and DU145, two AR-negative prostate cancer cell lines (Supplementary Fig. S3D). In contrast, we demonstrated that IKKβ KD stimulated growth of AR-positive 22Rv1and LNCaP cell lines (Supplementary Fig. S3D). Furthermore, we demonstrated that TNFα-induced downregulation of AR protein was abolished by depletion of MAP3K7 or IKKβ alone or together (Supplementary Fig. S3E). Collectively, all these data indicate that IKKβ promotes polyubiquitination and proteasomal degradation of AR, an effect similar to its upstream activator MAP3K7.
Β-TRCP E3 ubiquitin ligases mediate AR protein poly-ubiquitination in prostate cancer cells
SKP1-CULLIN1-F-Box protein (SCF) E3 ubiquitin ligases, including β-TRCP, bind the phosphodegron(s) in substrates and mediate target proteins for polyubiquitination and proteasomal degradation (38). Increasing evidence indicates that IKK phosphorylation sequences often serve as the phosphodegrons recognized by β-TRCP (39, 40). We noticed that there are two putative IKK-β-TRCP–targeting phosphodegrons in AR protein that are similar to those identified in several known β-TRCP degradation substrates, including NF-κB1 protein p105, IκB, FOXO3, and β-Catenin (Fig. 5A). We therefore sought to determine whether β-TRCP1 and β-TRCP2 are involved in regulation of AR protein levels. We found that ectopic expression of wild-type β-TRCP1 decreased AR protein level, and a similar result was observed for β-TRCP2, although co-expression of β-TRCP1 and β-TRCP2 resulted in an additive reduction in AR protein level (Fig. 5B). The F box of β-TRCP is essential for its association with the scaffold protein CULLIN1 (CUL1) via the interaction with the adaptor protein SKP1 (38). Apart from the wild-type β-TRCPs, neither the F-box deletion mutant of β-TRCP1 (TRCP1ΔF-box) nor the mutant of β-TRCP2 (TRCP2ΔF-box) was able to decrease AR protein level (Fig. 5B). Given that β-TRCP2 (BTRC2) expression level was much higher than β-TRCP1 (BTRC) in prostate cancer patient samples in the TCGA dataset (Fig. 5C), we chose to focus on β-TRCP2 in further studies. Co-IP assays revealed that AR and β-TRCP2 proteins interacted with each other at the endogenous level in LNCaP cells (Fig. 5D). Their interaction was substantially enhanced by TNFα treatment (Fig. 5D), but this effect was abolished due to the inhibition of IKKβ by wedelolactone (Fig. 5E), suggesting that IKKβ activation is required for TNFα-mediated interaction between β-TRCP2 and AR. In support of this notion, ectopic expression of IKKβ WT, but not the kinase-dead mutant increased β-TRCP2–AR interaction (Fig. 5F). Consistent with this result, depletion of both endogenous IKKβ and β-TRCP2 by siRNAs resulted in an increase of AR protein (Fig. 5G). Furthermore, depletion of both endogenous IKKβ and β-TRCP2 largely attenuated polyubiquitination of both ectopically expressed and endogenous AR (Fig. 5H; Supplementary Fig. S4A). Overexpression of wild-type β-TRCP2 augmented AR polyubiquitination and no such effect was observed for β-TRCP2ΔF-box, the F box deletion mutant of β-TRCP2 (Fig. 5I). Consistent with a role for β-TRCP2 in control of AR stability, the half-life of the endogenous AR protein was extended from approximately 8 to 24 hours after depletion of the endogenous β-TRCP in LNCaP cells (Fig. 5J–L). In addition, TNFα treatment enhanced β-TRCP2–AR interaction, but this effect was abolished by MAP3K7 KD (Supplementary Fig. S4B). Co-IP experiments confirmed that full-length AR and AR V7 variant can both bind to β-TRCP2 and IKKβ (Supplementary Fig. S4C). Similar to the effect of IKKβ KD (Supplementary Fig. S3E), KD of MAP3K7 resulted in the elevation of AR protein as co-KD of both MAP3K7 and β-TRCP2 did (Supplementary Fig. S4D). Taken together, these data indicate that β-TRCP E3 ligases play a critical role in mediating AR protein polyubiquitination and degradation induced by TNFα-mediated activation of MAP3K7 and IKKβ.
The 297DSAGKS302 motif in AR acts as a phosphodegron for AR destruction
Phosphorylation of the substrate plays a key role in recognition, ubiquitination, and subsequent proteolysis by the E3 ubiquitin enzyme β-TRCP. The IKKβ phosphorylation consensus sequence DSψXXS/T, where ψ represents the hydrophobic amino acid and X represents any amino acid, often functions as a phosphodegron in β-TRCP degradation substrates, such as β-catenin and IκB (41–44). We identified two putative β-TRCP–targeting phosphodegrons 157DSAAPS162 and 297DSAGKS302 in AR protein (Figs. 5A and 6A). To determine whether these sites are required for β-TRCP–mediated AR degradation, we constructed two AR mutants, S158A/S162A and S298A/S302A, by mutating two serine (S) residues in each motif (Ser158/Ser162 and Ser298/Ser302) into alanine (A) (Fig. 6A). Western blotting analysis revealed that coexpression of IKKβ and β-TRCP2 decreased protein level of both WT AR and S158A/S162A, but not the S298A/S302A mutant (Fig. 6B). Analogous to this finding, co-IP assays showed that β-TRCP2 interacted with WT AR and the S158A/S162A mutant, but not the S298A/S302A mutant (Fig. 6C). Ubiquitination assays showed that coexpression of IKKβ and β-TRCP2 induced polyubiquitination of WT AR and the S158A/S162A mutant, but not the S298A/S302A mutant (Fig. 6D). Moreover, we demonstrated that TNFα treatment increased phosphorylation of both WT AR and the S158A/S162A mutant, but not the S298A/S302A mutant became resistant in 293T cells (Fig. 6E). Furthermore, we found that the half-life of the S298A/S302A mutant was much longer than those of WT AR and the S158A/S162A mutant (Fig. 6F and G). Together, these data indicate that the 297DSAGKS302 motif functions as a phosphodegron that is essential for AR destruction induced by IKKβ and β-TRCP2 in prostate cancer cells.
Degradation-resistant AR mutant enhances AR activity and prostate cancer growth
We further examined whether AR degradation mediated by MAP3K7, IKKβ, and β-TRCP also affects AR transcriptional activity and AR-dependent prostate cancer growth. Luciferase reporter assays showed that the activities of both PSA promoter/enhancer (PSA-Luc) and composite three-copy androgen response element (3X ARE-Luc)–based luciferase reporters were enhanced by KD of IKKβ, β-TRCP1/2 or together in LNCaP cells cultured in androgen-depleted TNFα-containing medium, and similar results were obtained in cells treated with the synthetic androgen mibolerone (Fig. 7A). KD of IKKβ, β-TRCP1/2 or together also substantially increased expression of AR target genes including KLK3 (PSA), TMPRSS2, NKX3.1 and KLK2 (HK2) in LNCaP cells treated with or without mibolerone (Fig. 7B). Although MAP3K7 KD significantly increased AR target gene expression, this effect was abolished by overexpression of S177/181E, a phospho-mimicking mutant of IKKβ (Supplementary Figs. S2K, S5A, and S5B). We also infected LNCaP cells with lentivirus-expressing empty vector (EV), wild-type AR (AR-WT), S158A/S162A or S298A/S302A mutant. Ectopic expression of both AR-WT and the two mutants significantly enhanced proliferation in LNCaP cells without TNFα treatment (Fig. 7C, left); however, TNFα treatment largely inhibited the growth of LNCaP cells expressing EV, WT AR, and the S158A/S162A mutant, but not S298A/S302A mutant (Fig. 7C, right). To validate our finding in vivo, LNCaP cells expressing EV, WT AR or the S298A/S302A mutant were subcutaneously injected into SCID mice followed by TNFα treatment. We demonstrated that tumors expressing the S298A/S302A mutant grew much faster than tumors expressing EV or WT AR (Fig. 7D–F). These results indicate that AR degradation induced by IKKβ and β-TRCP suppresses AR activity and prostate cancer cell growth in vitro and in mice.
It is well established that AR plays essential roles in prostate cancer survival (45, 46). Acute inflammation is critical for host defense against pathogen exposure and disease conditions (3). In the present study, we identify AR protein as a degradation target of inflammatory signaling. We first noticed that decreased expression of AR associates with inflammation in prostate cancer patient specimens. This result is corroborated by our findings that the treatment of prostate cancer cells with the proinflammatory cytokine TNFα induces proteasomal degradation of AR protein and such effects are mediated by the activation of the MAP3K7–IKKβ inflammatory signaling axis (Fig. 7G, left). This observation provides a possible explanation for the seminal discovery that the AR protein level is lower in the majority of prostatic cells within the PIA lesion compared with areas without inflammation (25). We further show that genetic deletion of MAP3K7 or pharmacological inhibition of IKKβ blocks inflammation signaling-induced AR degradation, thereby providing a plausible explanation for the mixed outcome of the rationalized anti-inflammation chemoprevention trials in prostate cancer (2) because inhibition of inflammation may lead to elevation of AR protein level that thereby antagonizes the potential inhibition of prostate cancer growth and progression imposed by anti-inflammation drugs.
It has long been recognized that activation of the NF-κB pathway due to the aberrant activation of the MAP3K7-IKKβ inflammatory signaling is important for the survival of cells exposed with inflammatory cytokines (47). Surprisingly, in both TRAMP and myc-CaP prostate cancer mouse models conditional deletion of Ikbkb (encoding murine Ikkβ) in prostate epithelial cells failed to trigger tumor regression in both intact and castrated mice, highlighting the relevance of non-cell autonomous pathways in driving castration-resistant progression of prostate cancer (48). However, an alternative explanation could be that although loss of IKKβ results in the inhibition of the NF-κB signaling, it may also augment other survival pathways that can compensate NF-κB inhibition in a cell autonomous manner. We demonstrate that IKKβ binds to AR and promotes AR protein polyubiquitination and proteasomal degradation and this process is mediated by β-TRCP E3 ubiquitin ligases. We provide evidence that depletion of IKKβ by shRNAs or its inhibition by pharmacological inhibitor induces AR stabilization. Importantly, we further show that blocking AR degradation mediated by MAP3K7 and IKKβ promotes prostate cancer cell growth in vitro and in mice. Thus, our findings identify a previously unrecognized tumor-suppressive activity of MAP3K7 and IKKβ in mediating AR protein destruction and prostate cancer cell growth (Fig. 7G, left).
It is generally accepted that activation of the NF-κB pathway by the MAP3K7–IKKβ signaling axis promotes cancer survival and progression. It seems counterintuitive that the MAP3K7 gene is frequently deleted in prostate cancer patient specimens and that MAP3K7 deletion associates with high-grade prostate cancer (13, 29). Given the predominant role of AR in prostate cancer cell growth and survival, our findings support a model that loss of MAP3K7 favors prostate cancer progression due to enhanced AR activity, thereby compensating the inhibition of the NF-κB survival pathway (Fig. 7G, right), a discovery consistent with the findings in a recent report (32). Consistent with this notion, it has been shown previously that loss of MAP3K7 increases proliferation, migration, and invasion of prostate cancer cells (30). Moreover, deletion of the MAP3K7 gene often co-occurs with deletion of the chromatin modifier gene CHD1 in prostate cancer (13, 31, 49), and coordinate loss of MAP3K7 and CHD1 promotes aggressive prostate cancer (31). Intriguingly, it has been shown recently that CHD1 loss induces AR cistrome reprogramming (50). Thus, it is possible that increased AR expression due to loss of MAP3K7 and aberrant AR cistrome due to deletion of CHD1 may cooperate to promote aggressive phenotypes in prostate cancer and further investigation is warranted.
We have shown previously that treatment of prostate cancer cells with TNFα decreased AR expression and that the TNFα receptor-associated death domain (TRADD) protein plays an important role in mediating TNFα inhibition of AR expression (51). However, the downstream signaling pathway that mediates TNFα-induced downregulation of AR expression was unclear. Our present study not only uncovers a pivotal role of the MAP3K7–IKKβ signaling cascade in regulation of AR protein degradation and stability, but also suggests that the TNFα receptor–adaptor proteins such as TRADD may also play a role in TNFα-induced AR protein degradation.
In summary, we demonstrate that the proinflammatory cytokines such as TNFα promote AR protein degradation in prostate cancer cells and this effect is mediated by the MAP3K7–IKKβ signaling. Mechanistically, we show that IKKβ binds to AR and triggers phosphorylation-dependent interaction of AR with the E3 ubiquitin ligases β-TRCP1/2 and subsequent polyubiquitination and proteasomal degradation of AR. Our findings reveal a previously uncharacterized tumor-suppressive role of the MAP3K7–IKKβ signaling in inducing the destruction of AR, a pro-cancer protein in prostate cancer. The relevance of this discovery is supported by the observations in the clinical specimens that the MAP3K7 gene is often deleted in patients with prostate cancer. Thus, identification of IKKβ-mediated β-TRCP–dependent AR degradation enhances our understanding of how AR activity is influenced by tumor microenvironment especially inflammation. These results also suggest that aberrantly elevated AR protein can not only serve as a valuable prognostic factor for the progression of MAP3K7-deficient prostate cancer, but also is a viable therapeutic target of this subtype of prostate cancer.
R.J. Karnes reports other support from Decipher Biosciences outside the submitted work. No disclosures were reported by the other authors.
Z. Huang: Data curation, formal analysis, validation, investigation, methodology, writing–original draft. B. Tang: Resources, data curation, investigation, writing–original draft, writing–review and editing. Y. Yang: Resources, data curation, investigation, methodology. Z. Yang: Resources, data curation, software, formal analysis, investigation, methodology. L. Shi: Investigation, methodology. Y. Bai: Investigation, methodology. B. Yan: Investigation, methodology. R.J. Karnes: Investigation, methodology. J. Zhang: Investigation, methodology. R. Jimenez: Investigation, methodology. L. Wang: Software, methodology. Q. Wei: Data curation, methodology. J. Yang: Resources, data curation, methodology. W. Xu: Conceptualization, resources. Z. Jia: Conceptualization, resources. H. Huang: Conceptualization, supervision, funding acquisition, writing–review and editing.
The authors thank Dr. Jiwang Zhang from the Loyola University Medical Center for kindly providing Map3k7 (Tak1) loxp/loxp conditional knockout mice. This work was supported by funding from the Mayo Clinic Foundation (to H. Huang).
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