SUMOylation of TBL1 and TBLR1 promotes androgen-independent prostate cancer cell growth

Chronic inflammation is strongly associated with prostate cancer pathogenesis. Transducin β-like protein (TBL1) and Transducin β-like 1X-linked receptor 1 (TBLR1) have been identified recently as a coactivator for NF-κB-mediated transcription; however, the underlying mechanism by which TBL1 and TBLR1 activate NF-κB function during inflammation remains unknown. Here, we demonstrate that cytokine production is significantly elevated in androgen-independent PC-3 prostate cancer cells compared with androgen-dependent LNCaP prostate cancer cells. Elevated cytokine production positively correlates with the TBL1 and TBLR1 SUMOylation level in PC-3 cells. We show that both TBL1 and TBLR1 are SUMOylated in response to TNF-α treatment, and this increases formation of the TBL1-TBLR1-NF-κB complex, which leads to NF-κB-mediated transcriptional activation of cytokine gene expression. Conversely, SENP1-mediated deSUMOylation of TBL1 and TBLR1 inhibits NF-κB-target gene expression by dissociating TBL1 and TBLR1 from the nuclear hormone receptor corepressor (NCoR) complex. TBL1 knockdown substantially suppresses inflammatory signaling and PC-3 cell proliferation. Collectively, these results suggest that targeted SUMOylation of TBL1 and TBLR1 may be a useful strategy for therapeutic treatment of androgen-independent prostate cancer.

and anti-apoptotic factors [8].The mammalian NF-κB family has five members: RelA (p65); NF-κB1 (p50, p105); NF-κB2 (p52, p100); c-Rel; and RelB.The most abundant activated form of NF-κB is a heterodimer that contains p65 and p50 subunits.NF-κB is located in the cytoplasm along with its inhibitor protein IκB.Inflammatory stimuli promote IκB degradation, which enables NF-κB translocation to the nucleus and binding to target gene promoters that regulate transcription of the pro-inflammatory cytokines.NF-κB is highly activated at sites of inflammation in multiple human cancers.There is accumulating evidence that NF-κB activation is correlated with prostate cancer development, prognosis, and castrate-resistant progression [9][10][11][12].Cytokine IL-6 stimulates prostate cancer growth by stimulating the androgen receptor, and is involved in the development of bone metastasis [13].Increased levels of NF-κB and IL-6 are implicated in the development of prostate cancer cell chemoresistance [14].These combined studies suggest that NF-κB may be a promising target for improving chemotherapeutic efficacy in prostate cancer.
TBL1 and TBLR1, which are components of the nuclear hormone receptor corepressor (NCoR)/silencing mediator for retinoid and thyroid hormone receptors (SMRT) corepressor complexes, contain a Lis1 homology domain (LisH)/WD-40 motif, and have approximately 86% identity [15,16].Recent studies report that TBL1 and TBLR1 stabilize the quaternary structure of the NCoR/SMRT corepressor complex and bind to the tails of hypoacetylated histones H2B and H4, which target NCoR/ SMRT complexes to chromatin [17].The LisH domains of both TBL1 and TBLR1 are reported to be required for oligomerization, and contribute to the stable binding of NCoR/SMRT corepressor complexes to chromatin to completely repress target gene expression [18].These results suggest that TBL1 and TBLR1 have important role in class I HDAC corepressor complex-mediated transcriptional repression.However, TBL1 and TBLR1 also were found to function as E3 ubiquitin ligase adaptors.In this capacity, they recruit the ubiquitin-conjugating/19S proteasome complex to the corepressor complex, which leads to corepressor degradation, and mediates the exchange of corepressors for coactivators in response to ligand binding/stimulation.These actions lead to specific nuclear receptor-mediated gene activation events [19,20].TBL1, TBLR1, and β-catenin recruit one another to Wnt target gene promoters to activate transcription in response to canonical Wnt signaling, and reversible SUMOylation of TBL1 and TBLR1 regulates β-catenin-mediated Wnt signaling [21,22].TBL1 is required for p65 recruitment to NF-κB target gene promoters in response to tumor necrosis factor alpha (TNF-α) [23].These results suggest that TBL1 and TBLR1 function as transcriptional coactivators, and may have a role in the transcriptional activation of NF-κB target genes.However, the detailed mechanism of NF-κB-mediated transcriptional activation by TBL1 and TBLR1 corepressors was unknown.
In this study, we found that constitutive cytokine elevation in the androgen-independent prostate cancer (AIPC) cell lines PC-3 and C4-2B correlates with the SUMOylation levels of TBL1 and TBLR1.TBL1 and TBLR1 are SUMOylated in vivo in response to inflammatory stimuli.TBL1 and TBLR1 SUMOylation dissociate TBL1 and TBLR1 from the NCoR/HDAC3 corepressor complexes and induces formation of the TBL1 SUMO -TBLR1 SUMO -NF-κB complex, which ultimately leads to transcriptional activation of NF-κB target genes.Therefore, this study suggests a regulatory mechanism for elevated NF-κB-mediated inflammatory signaling in AIPCs via reversible SUMOylation of TBL1 and TBLR1.

rEsULts tbL1 and tbLr1 sUMOylation and inflammatory cytokines are elevated in AIPC cells
NF-κB is constitutively activated in prostate tumors and cell lines [5].Therefore, we first examined the inflammatory cytokine levels in prostate cancer cell lines by performing cDNA microarrays using the androgendependent prostate cancer (ADPC) cell line LNCaP and the AIPC cell line PC-3.In agreement with a previous report [24], we observed that the pro-inflammatory cytokines IL-8, IL-1β, and IL-6 were strongly elevated in PC-3 cells compared with LNCaP cells (Figure 1A).Quantitative RT-PCR analysis verified the elevated cytokine levels in PC-3 cells (Figure 1B).
A recent study reported that the TBL1 corepressor acts as a cofactor for recruiting p65 to NF-κB target gene promoters, which eventually leads to the transcriptional activation of inflammatory cytokines [23].Therefore, we explored the possibility that TBL1 and TBLR1 are involved in cytokine elevation in AIPC cells.First, we assessed TBL1 and TBLR1 levels in prostate cancer cells by performing western blot analysis.Immunoprecipitation analysis revealed that the interaction between TBL1/ TBLR1 and RelA in PC-3 cells was strongly increased compared with that in LNCaP cells, and the TBL1 and TBLR1 protein levels in PC-3 cells also were higher than those in LNCaP cells (Figure 1C, left panel).TBL1 and TBLR1 SUMOylation caused TBL1 and TBLR1 dissociation from the NCoR corepressor complex [21].Therefore, we next examined the relative association of TBL1 and TBLR1 with NCoR/HDAC3 corepressor complexes in PC-3 and LNCaP cells.TBL1 and TBLR1 association with NCoR/HDAC3 corepressor complexes were significantly lower in PC-3 cells than in LNCaP cells (Figure 1C, left panel).To verify these results, we performed Duo-link in situ proximity ligation assay (PLA) analysis, which enables the detection of protein interactions and modifications, and verified elevated SUMOylation levels of endogenous TBL1 in PC-3 cells (Figure 1D).www.impactjournals.com/oncotarget Due to their high metastatic potential resulting from their androgen-insensitive state, PC-3 cells have been less extensively studied than LNCaP cells for investigating biochemical changes in advanced prostate cancer.PC-3 cell line was established from bone metastasis of prostate cancer.Therefore, we selected the bone metastasis subline C4-2B, which was generated from parental LNCaP cells, to confirm whether these similar cell lines share the same biochemical features as PC-3 cells.The results showed that inflammatory cytokine levels were highly elevated in C4-2B cells compared with those in LNCaP cells, which was similar to the observed cytokine levels in PC-3 cells.The levels of TBL1 and TBLR1 SUMOylation and association of TBL1 and TBLR1 with RelA were higher in C4-2B cells than in PC-3 cells (Figure 1C, right panel).Collectively, these results suggest that constitutive activation of inflammatory signaling in AIPC cells correlates with TBL1 and TBLR1 SUMOylation.

Inflammatory stimulation promotes TBL1 and tbLr1 sUMOylation
Recent work showed that SUMO modification acts as a molecular switch that regulates corepressive and coactive functions of TBL1 and TBLR1 during Wnt signaling activation [21].Therefore, we examined whether TBL1 and TBLR1 SUMOylation increases in response to inflammatory activation.Myc-TBL1 or Myc-TBLR1 was co-transfected with Flag-SUMO1 into PC-3 cells, the cells were treated with TNF-α, and immunoprecipitation assays were performed.In response to treatment, SUMOylation of both TBL1 and TBLR1 increased in response to TNF-α treatment (Figure 2A and Figure S1A).Our previous work showed that TBL1 and TBLR1 were SUMOylated on lysine 560 and lysine 497, respectively, during activation of Wnt signaling [21].Therefore, we examined whether the TNF-α-mediated TBL1 SUMOylation site was identical with the Wnt signaling-induced SUMOylation site.The results indicated that TBL1 K560R and TBLR1 K497R mutants failed to form complexes with SUMO1, whereas wild-type TBL1 and TBLR1 did form SUMO1 complexes (Figure 2B and Figure S1B).These data confirm that TBL1 and TBLR1 are SUMOylated at Lys560 and Lys497, respectively, in response to inflammatory signaling.

TBL1 SUMOylation enhances NF-κB-mediated transcription
Inflammatory cytokines and SUMOylated TBL1/ TBLR1 are highly elevated in PC-3 cells.Therefore, we examined the effect of TBL1 SUMOylation on NF-κB-mediated transcription of cytokine genes.Ectopic expression of SUMO-conjugated TBL1 enhanced TNFα-induced IL-8 and IL-1β gene transcription compared to that of unconjugated TBL1 (Figure 3A).Expression of SENP1 WT efficiently reversed the effect of Sumoconjugated TBL1, whereas expression of inactive SENP1 C603S failed to reverse the effect (Figure 3B).These combined results indicate a crucial role for TBL1 SUMOylation in NF-κB-mediated activation of cytokine gene transcription.
Next, we tested whether TBL1 or TBLR1 knockdown suppresses transcriptional activation of cytokine gene expression in PC-3 cells.Treatment of PC-3 cells with either siTBL1 or siTBLR1 substantially reduced the basal levels of IL-8 and IL-1β (Figure 3C).Treatment of PC-3 cells with TNF-α time-dependently increased IL-8 and IL-1β gene expression; however, TBL1 knockdown greatly reduced TNF-α-dependent cytokine gene expression in PC-3 cells (Figure 3D).Consistently, overexpression of SENP1 WT , but not SENP1 C603S , efficiently suppressed the cytokine transcript levels in PC-3 cells (Figure 3E), indicating that TBL1 and TBLR1 SUMOylation constitutively enhance inflammatory cytokine production in PC-3 cells.

Inflammatory stimulation induces formation of the TBL1 sUMO -TBLR1 sUMO -NF-κB activator complex
SUMOylation induces TBL1 and TBLR1 dissociation from the NCoR corepressor complex, which corresponds with formation of the TBL1-TBLR1-βcatenin coactivator complex [21].Therefore, we examined whether SUMOylation increases formation of the TBL1-TBLR1-NF-κB complex in response to TNF-α treatment.TNF-α treatment increased the association of wild-type TBL1 with NF-kB (greek letter), whereas TNF-α reduced the interaction between TBL1 and HDAC3 or NCoR (Figure 4A). Figure S2 showed same effect of TNF-α on TBLR1 with NF-κB, HDAC3 or NCoR association.These results verify that TBL1 and TBLR1 assembly with NF-κB depends on TBL1 SUMOylation.Mutation of TBL1 SUMOylation sites attenuated TNF-α-induced TBL1 association with RelA, and promoted the strong interaction of mutant TBL1 with NCoR or HDAC3.These results indicate that SUMOylation of TBL1 and TBLR1 at Lys560 and Lys497, respectively, is required for TNFα-induced TBL1 and TBLR1 assembly with NF-κB and NCoR corepressor complex disassembly.
We tested the effect of SENP1 overexpression on formation of the TBL1-NF-κB complex.Active SENP1 substantially induced TBL1 dissociation from NF-κB and association with the NCoR/HDAC3 corepressor complex, whereas inactive SENP1 C603S did not have these effects (Figure 4B).These combined results indicate that TBL1/ TBLR1 SUMOylation has a crucial role in formation of the TBL1 SUMO -TBLR1 SUMO -NF-κB coactivator complex during inflammatory stimulation.

TBL1 and TBLR1 SUMOylation enhances recruitment of the TBL1 sUMO -TBLR1 sUMO -NF-κB complex to the IL-6 and IL-8 promoter
During immune responses, NF-κB is translocated into the nucleus where it binds to specific sites of cytokine genes such as IL-6, IL-8, and IL-1β [24][25][26].Recent work also showed that TBL1 and NF-κB specifically bind to the promoter regions of NF-κB target genes in an inflammation signal-dependent manner.Therefore, we examined whether SUMOylation of TBL1 and TBLR1 enhances recruitment of the TBL1-TBLR1-NF-κB complex to the NF-κB-binding sites of cytokine genes.TNF-α treatment efficiently induced NF-κB and TBL1-TBLR1 binding to the NF-κB-binding region of IL-6 and IL-8.Conversely, reChIP experiments showed that TBL1 SUMO -TBLR1 SUMO formed a complex with NF-κB, and the complex bound to the NF-κB-binding region of IL-6 and IL-8 (Figure 5A).Overexpression of wild-type TBL1 enhanced NF-κB binding to the IL-6 promoter and dramatically increased histone H3 acetylation in this region, whereas SUMOylation-defective TBL1 mutants did not exhibit these effects.We observed the same effects in IL-8 promoter ChIP assay (Figure 5B).These data suggest that TBL1 SUMOylation triggers formation of an NF-κB-TBL1 complex in the promoter regions of NF-κB target genes.ChIP assays indicated that SENP1 WT overexpression induced TBL1-TBLR1-NF-κB complex dissociation from the NF-κB-binding sites of cytokine genes, whereas overexpression of inactive SENP1 C603S did not significantly affect TBL1-TBLR1-NF-κB complex binding to cytokine genes.These combined results indicate that TBL1 and TBLR1 deSUMOylation attenuate cytokine signaling by inducing TBL1-TBLR1-NF-κB complex dissociation from the NF-κB-binding sites of cytokine genes (Figure 5C).

TBL1 and TBLR1 deSUMOylation suppress PC-3 proliferation
PC-3 cell line is a representative model for androgenindependent prostate cancer.NF-κB is constitutively active in PC-3 and DU145 AIPC cell lines, whereas only very low levels of NF-κB activity are detected in the LNCaP ADPC cell line.These observations suggest that constitutive NF-kB activation may have a role in the survival of AIPC cells [10,11].We examined whether reduced TBL1 levels or TBL1-TBLR1 deSUMOylation attenuated anchorage-independent growth, which typically correlates with in vivo tumorigenic phenotype.PC-3 proliferation was substantially lower after TBL1 knockdown than in cells transfected with control short hairpin RNA (shRNA) (Figure 6A).Overexpression of TBL1 WT or TBLR1 WT enhanced PC-3 cell growth compared with overexpression of TBL1 K560R or TBLR1 K497R (Figure 6B, left panel).Expression of TBL1 K560R reduced PC-3 cell growth and colony formation, whereas expression of SUMO-conjugated TBL1 K560R restored PC-3 cell growth and colony formation (Figure 6B).TBLR1 behaved similarly to TBL1 in these assays (Figure 6B, right panel).Overexpression of wild-type SENP1 WT , but not inactive SENP1 C603S , greatly reduced anchorage-independent PC-3 cell growth, indicating the functional relevance of TBL1-TBLR1 SUMOylation for tumorigenic growth of PC-3 cells (Figure 6C, 6D).These combined results indicate that TBL1-TBLR1 SUMOylation enhances NF-κB-mediated inflammatory signaling and proliferation of androgenindependent prostate cancer cells.

DIscUssION
NF-κB, a transcriptional factor, is strongly activated at sites of inflammation, which plays critical role in variety of gene expression including cytokines [24].A recent study reported that TBL1 was required for p65 recruitment to NF-κB-targeted gene promoters in response to TNF-α [23], which suggests an additional role for TBL1 as a transcriptional activator.In that study, TBL1 recruitment to NF-κB-targeted gene promoters was considered a critical step for the transcriptional activation of target genes.However, the molecular mechanism underlying NF-κB activation by the well-known TBL1 corepressor has not been fully elucidated.
In this study, we present evidence that SUMOylation of TBL1 and TBLR1 enhances association with NF-κB in response to TNF-α, and induce dissociation of the NCoR-TBL1-TBLR1 corepressor complex.Our group previously reported that SUMOylation of TBL1 and TBLR1 promotes β-catenin-mediated Wnt signaling via disassembly of NCoR-TBL1 corepressor complex.We observed that TBL1 and TBLR1 are SUMOylated at lysine 560 and lysine 497, respectively, in response to cytokine signaling, and are deSUMOylated by SENP1.SENP1 overexpression suppresses the coactive function of TBL1 and TBLR1 in NF-κB-mediated transcriptional regulation of cytokine gene expression, which is similar to the effects of Wnt signaling.Therefore, this study provides additional evidence that TBL1 and TBLR1 SUMOylation induce the functional exchange of TBL1 and TBLR1 from corepressor to coactivator activity via disassembly of the NCoR corepressor complex.
Chronic inflammation is linked to carcinogenesis in several organ systems [1], and prostate cancer is strongly correlated with chronic inflammation [5].There is accumulating evidence that elevated serum IL-6 levels are associated with advanced tumor stages in patients with prostate cancer [27].Elevated NF-κB and IL-6 levels have been correlated with drug resistance of prostate cancer cells [9,11].For example, IL-6 is an autocrine and paracrine growth factor for prostate cancer cell lines and functions as a resistance factor for cisplatin-mediated cytotoxicity [14].Clinical trials reported that antibodytargeted IL-6 therapy may be useful for improving the performance of other chemotherapy agents during treatment of metastatic castration-resistant prostate cancer [28].These combined results suggest that suppression of IL-6 levels may be a potential therapeutic strategy for treating prostate cancer.We also observed constitutively elevated levels of cytokines IL-6, IL-8, and IL-1β in PC-3 compared with the levels in LNCaP cells, indicating that NF-κB is constitutively activated in PC-3 cells.This result is consistent with previous studies reporting that NF-κB is constitutively activated in androgen-independent PC-3 prostate cancer cells but not in androgen-dependent LNCaP prostate cancer cells.TBL1/TBLR1 SUMOylation is elevated in AIPC PC-3 cells compared with that in LNCaP cells and the LNCaP-derived AIPC cell line C4-2B.SUMOylation of TBL1 and TBLR1 is closely correlated with NF-κB activity.Furthermore, this study shows direct evidence that sumolyated TBL1 and TBLR1 are recruited to promoters of both IL-6 and IL-8 under inflammation, whereas defective form of SUMOylation of TBL1, TBL1K560R, fails to recruit NF-kB to the promoters, which means SUMOylated TBL1 and TBLR1 upregulate the IL-6 and IL-8 promoter through recruiting NF-kB to the kB binding regions of two genes.

Colony formation
Cells were suspended in RPMI-1640 medium containing 10% FBS and 0.3% SeaKem low melting temperature agarose.A total of 1 × 10 3 cells in a volume of 15 ml were plated in six-well plates over a 1.5-ml layer of solidified RPMI-1640 containing 10% FBS and 0.6% agarose.The plates were incubated at 37°C for 3 weeks, and then the colonies formed on each plate were photographed from three different views.The numbers and sizes of colonies on each plate were counted and measured.

Duo-link in situ proximity ligation assay analysis
Duo-link in situ proximity ligation assay (PLA) analysis was performed according to the manufacturer's instructions (Olink Biosciences, Uppsala, Sweden).Briefly, paraformaldehyde-fixed cells were washed with PBS, incubated in 1.5% hydrogen peroxide for 15 min, washed, and blocked with blocking solution.Primary rabbit antibody was applied, cells were incubated with PLUS and MINUS secondary PLA probes against rabbit IgG only, or against both rabbit and mouse IgG, and then samples were subjected to hybridization, ligation, and amplification.Cells were mounted with Duo-link mounting medium and examined using an Olympus FluoView FV1000 Confocal Microscope (Olympus Corp., Tokyo, Japan).

Statistical analysis
Statistical analyses were performed using Student's t-test with Bonferroni for multiple comparisons.A p-value less than 0.05 was considered as statistically significant.

Figure 1 :
Figure 1: SUMOylation of TBL1 and TBLR1 is strongly elevated in androgen-independent prostate cancer cells enriched with inflammatory cytokines.(A) Inflammatory cytokine levels are higher in PC-3 cells than in LNCaP cells.Changes in mRNA expression were evaluated by cDNA microarray analysis using the Illumina HumanRef-8 v3 Expression BeadChip.(b) Validation of cDNA microarray analysis by quantitative real-time PCR.Expression levels of each gene were analyzed by quantitative RT-PCR.Statistical significance was determined using Student's t-test; # P < 0.01 versus LNCaP cell lines.(c) SUMOylation of TBL1 and TBLR1 levels are higher in PC-3 and C4-2B cells than in LNCaP cells.Immunoprecipitation analysis was performed using cell lysates, and immunoblotting was performed using the indicated antibodies.(D) Validation of TBL1 SUMOylation in vivo in LNCaP and PC-3 cells.Duo-link in situ PLA analysis was performed as described in Materials and methods with the indicated antibodies.

Figure 3 :
Figure 3: SENP1 inhibits TNF-α-induced NF-κB activation via deSUMOylation of TBL1 and TBLR1.(A) TBL1 SUMOylation potentiates its coactive function for NF-κB during inflammatory signaling.PC-3 cells were co-transfected with indicated plasmids for 48 hours and treated with 10 ng/ml TNF-α for 2 hours.Target gene mRNA levels were analyzed by qRT-PCR.Cell lysates were immunoblotted with the indicated antibodies.Statistical significance was determined using Student's t-test; *P < 0.05 and # P < 0.01 versus treatment with TNF-α.(b) SENP1 reverses the effect of TBL1 SUMOylation on TNF-α-induced NF-κB activation.Co-transfected PC-3 cells were treated with TNF-α for 2 hours, mRNA levels were analyzed by qRT-PCR.Cell lysates were immunoblotted with the indicated antibodies.Statistical significance was determined using Student's t-test; *P < 0.05 and # P < 0.01 versus treatment with TNF-α.(c) TBL1 or TBLR1 knockdown reduces cytokine levels in PC-3 cells.Cells were transfected with siRNA against TBL1 or TBLR1, and target gene mRNA levels were analyzed by qRT-PCR.Statistical significance was determined using Student's t-test; *P < 0.05 and # P < 0.01 versus transfection with siControl.(D) TBL1 knockdown greatly reduces TNF-α-induced cytokine production in PC-3 cells.Lentiviral shTBL1infected cells were treated with TNF-α, harvested, and mRNA levels were analyzed by qRT-PCR.Statistical significance was determined using Student's t-test; # P < 0.01 versus infection with shControl samples.(E) SENP1 overexpression reduces cytokine levels in PC-3 cells.PC-3 cells were transfected with Flag-SENP1 WT or Flag-SENP1 C603S , and mRNA levels of target genes were analyzed by qRT-PCR.Statistical significance was determined using Student's t-test; *P < 0.05 and # P < 0.01 versus Control transfection (without SENP1 WT and SENP1 C603S ).

Figure 4 :
Figure 4: TBL1 SUMOylation enhances formation of the TBL1 sUMO -NF-κB complex.(A) TNF-α-induced TBL1 SUMOylation at Lys560, is required for enhanced TBL1 association with NF-κB.PC-3 cells were co-transfected with the indicated plasmids, treated with or without TNF-α for 1 hour, and then harvested.Cell lysates were immunoprecipitated and subsequently immunoblotted with the indicated antibodies.(b) SENP1-mediated TBL1 deSUMOylation induces disassembly of the TBL1-NF-κB complex.PC-3 cells were co-transfected with the indicated plasmids.Cell lysates were immunoprecipitated and subsequently immunoblotted with the indicated antibodies.

Figure 5 :
Figure 5: TBL1 and TBLR1 SUMOylation enhance recruitment of the TBL1-TBLR1-NFκB complex to the IL-6 and IL-8 promoter.(A) TNF-α treatment induces recruitment of the TBL1 SUMO -TBLR1 SUMO -NF-κB complex to the IL-6 and IL-8 promoter regions.Cells were treated with 10 ng/ml TNF-α for 1 hour, ChIP and reChIP assays were performed with the indicated antibodies.Error bars represent standard deviation (SD, n = 3).Statistical significance was determined using Student's t-test; # P < 0.01 versus treatment without TNF-α.(b) TBL1 SUMOylation enhances NF-κB binding to chromatin at the IL-6 and IL-8 promoter.PC-3 cells were transfected with the indicated plasmids, followed by ChIP assays with the indicated antibodies.Error bars represent SD (n = 3).Statistical significance was determined using Student's t-test; # P < 0.01 versus Flag-TBL1 without TNF-α treatment.(c) SENP1-mediated TBL1 and deSUMOylation induces dissociation of the TBL1-TBLR1-NFκB complex from promoters of NF-κB target genes in PC-3 cells.PC-3 cells were transfected with plasmids carrying SENP1 WT and/or SENP1 C603S .Cell lysates were immunoprecipitated and then used for performing ChIP assays with the indicated antibodies.Error bars represent SD (n = 3).Statistical significance was determined using Student's t-test; *P < 0.05 and # P < 0.01 versus transfection of Flag-TBL1 without TNF-α treatment.

Figure 6 :
Figure 6: Inhibition of TBL1 and TBLR1 SUMOylation reduces PC-3 cell growth.(A) TBL1 knockdown greatly reduces anchorage-independent PC-3 cell proliferation.PC-3 cells were infected with lentiviral shTBL1 to generate TBL1 knockdown lines.Knockdown efficiency was measured by RT-PCR and qRT-PCR (left panel) compared to shCon cell lines; shCon or shTBL1 cells were seeded with the same cell numbers, and plates were photographed after 3 weeks.(b) TBL1 and TBLR1 SUMOylation enhance tumorigenic growth of PC-3 prostate cancer cells.PC-3 cells were co-transfected with TBL1 (left panel) or TBLR1 (right panel) plasmids, and the numbers of cells in soft agar were determined under a microscope 2 days after transfection.Data represent the means ± SD of at least three independent experiments.Statistical significance was determined using Student's t-test; # P < 0.01 versus Control.(c, D) SENP1 suppresses tumorigenic growth of PC-3 cells.SENP1 knockdown greatly increased anchorage-independent PC-3 cell proliferation (C).PC-3 cells were infected with lentiviral shSENP1 to generate SENP1 knockdown cell lines; shCon or shSENP1 cells were seeded with the same cell numbers onto media with or without TNF-α (changed every day), allowed to grow for 3 weeks, and then plates were photographed.PC-3 cells were transfected with SENP1 WT or SENP1C 603S as indicated, and the numbers of cells in soft agar were determined under a microscope.Data represent the means ± SD of at least three independent experiments.Statistical significance was determined using Student's t-test; *P < 0.05 versus Control (D).