Implications for Tumor Growth and Response to Tamoxifen

ORIGINAL ARTICLE Identiﬁcation of RBCK1 as a novel regulator of FKBPL: implications for tumor growth and response to tamoxifen

C Donley1,4, K McClelland1,4, HD McKeen1,4, L Nelson1, A Yakkundi1, PV Jithesh2, J Burrows1, L McClements1, A Valentine1, KM Prise3, HO McCarthy1 and T Robson1

FKBPL has been implicated in processes associated with cancer, including regulation of tumor growth and angiogenesis with high levels of FKBPL prognosticating for improved patient survival. Understanding how FKBPL levels are controlled within the cell is therefore critical. We have identiﬁed a novel role for RBCK1 as an FKBPL-interacting protein, which regulates FKBPL stability at the post-translational level via ubiquitination. Both RBCK1 and FKBPL are upregulated by 17-b-estradiol and interact within heat shock protein 90 chaperone complexes, together with estrogen receptor-a (ERa). Furthermore, FKBPL and RBCK1 associate with ERa at the promoter of the estrogen responsive gene, pS2, and regulate pS2 levels. MCF-7 clones stably overexpressing RBCK1 were shown to have reduced proliferation and increased levels of FKBPL and p21. Furthermore, these clones were resistant to tamoxifen therapy, suggesting that RBCK1 could be a predictive marker of response to endocrine therapy. RBCK1 knockdown using targeted small interfering RNA resulted in increased proliferation and increased sensitivity to tamoxifen treatment. Moreover, in support of our in vitro data, analysis of mRNA microarray data sets demonstrated that high levels of FKBPL and RBCK1 correlated with increased patient survival, whereas high RBCK1 predicted for a poor response to tamoxifen. Our ﬁndings support a role for RBCK1 in the regulation of FKBPL with important implications for estrogen receptor signaling, cell proliferation and response to endocrine therapy.

Oncogene (2014) 33, 3441–3450; doi:10.1038/onc.2013.306; published online 5 August 2013 Keywords: RBCK1; FKBPL; estrogen receptor; p21; tamoxifen

INTRODUCTION these data support a role for FKBPL as a tumor suppressor and, The FKBPL gene encodes a protein with a molecular mass of therefore, understanding how this protein is regulated is critical. B39 kDa, with two characteristic regions, the tetratricopeptide RBCK1 (HOIL-1) encodes a 510 amino-acid protein with several repeat domain and a partial peptidyl-prolyl-cis-trans-isomerase distinct functional domains: a RING-IBR domain, B-Box, two coiled- 11 domain. These domains are features of the FKBP/immunophilin coil regions and a ubiquitin-like sequence. The RING-IBR consists family of which FKBPL represents a divergent member. The of two RING fingers and an in-between RING-finger motif making tetratricopeptide repeat domain is required for protein–protein it homologous with E3 ubiquitin ligase enzymes. Functional interactions1 and facilitates interaction with the cellular analysis has identified RBCK1 as an E3 ligase involved in the chaperone, heat shock protein (Hsp)90.2 Through its interaction ubiquitination of PKC, IRP2, SOCS6, BACH1, TAB2/3 and IRF3 12–16 with Hsp90, FKBPL has been shown to regulate the stability proteins. RBCK1 is also involved in the 600-kDa heteromeric of the cyclin-dependent kinase inhibitor, p213,4 and also form linear ubiquitin chain assembly complex17,18 responsible for the co-chaperone complexes with the glucocorticoid receptor, linear polyubiquitination of NF-kappa-B essential modulator androgen receptor and estrogen receptor-a (ERa).5–7 FKBPL (NEMO) resulting in activation of the nuclear factor-kB pathway directly regulates protein levels of ERa and its phosphorylation involved in immune and inflammatory responses.19,20 RBCK1 has at Ser118, sensitizing ER-positive breast cancer cell lines to been implicated in modulating cell growth through regulation of estrogen deprivation and endocrine therapy.6 Overexpression of p53 activation of p21 transcription.21 RBCK1 contains DNA-binding FKBPL inhibited the growth of several tumor cell lines,6,8 and high domains and directly influences the transactivation of numerous FKBPL was also found to correlate with both increased overall genes including, p21, HBv-X and Eya1.21–23 It also positively survival and distant metastasis-free survival in microarray data regulates ERa transcription by enhancing promoter binding, sets.6 This was further supported in a breast cancer tissue resulting in transactivation of ER responsive genes, c-myc, Cyclin microarray where high FKBPL was associated with increased D and pS2.24,25 Here, we show that RBCK1 is a post-transcriptional survival and reduced tumor size and grade,9 highlighting its regulator of FKBPL, stabilizing FKBPL through ubiquitination. prognostic value. FKBPL’s prognostic potential may also be related We further show that RBCK1 acts as a co-chaperone within to its anti-angiogenic activity; secreted FKBPL and a therapeutic Hsp90/FKBPL/ERa complexes, regulating a number of processes, peptide derivative inhibit angiogenesis in vitro and in vivo, result- including ERa transactivation in association with FKBPL, response ing in inhibition of human tumor xenograft growth.10 Together, to endocrine therapy and cell proliferation.

1School of Pharmacy, McClay Research Centre, Queen’s University, Belfast, Northern Ireland; 2Liverpool Cancer Research UK Centre, University of Liverpool, Liverpool, UK and 3Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, Northern Ireland. Correspondence: Professor T Robson, School of Pharmacy, McClay Research Centre, Queen’s University, 97 Lisburn Road, Belfast, Country Antrim BT9 7BL, Northern Ireland. E-mail: [email protected] 4These authors contributed equally to this work. Received 21 January 2013; revised 27 May 2013; accepted 11 June 2013; published online 5 August 2013 RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3442 RESULTS a characteristic ubiquitin smear when co-transfected with either RBCK1 is a novel FKBPL-interacting protein involved in FKBPL full-length or RBCK1DRING mutant; no smear was observed when ubiquitination transfected with the RBCK1DUbl mutant (Figure 1e). This suggests An yeast-2-hybrid screen was used to isolate FKBPL-interacting that the Ubl domain of RBCK1 is required for the ubiquitination of proteins, identified by their ability to induce expression of three FKBPL. To assess the type of ubiquitin chain that RBCK1 attaches independent reporter genes enabling yeast cell growth on amino- to FKBPL, two ubiquitin mutants were utilized, one with all lysine acid drop out media, inducing b-gal activity and inhibiting yeast residues mutated to arginine, except K48, and the other with only growth on media containing 5-fluoroorotic acid (Figure 1a). Three K48 mutated to arginine (Supplementary Figure 1c); wild-type independent clones of RBCK1 were identified as strong interacting ubiquitin was used as a control. FKBPL was ubiquitinated in the partners; these clones spanned the Ubl and RING domains of presence of both mutants and wild-type ubiquitin (Figure 1f), RBCK1. The mammalian two-hybrid assay was used to confirm and suggesting that the ubiquitin chains attached to FKBPL are not map the interacting domains. Co-transfection of full-length FKBPL linked via any of the seven classical lysine residues. RBCK1 has and RBCK1 resulted in a 20-fold induction in firefly luminescence been shown to work in tandem with RNF3 to form linear ubiquitin over the self-activation control, indicating a strong interaction chains that are formed by N to C terminal conjugation rather than 14,16,17,19 (Figure 1b). Deletion of the tetratricopeptide repeat domain in from lysine residues. RBCK1 could therefore modify FKBPL FKBPLD200, FKBPLD151 or FKBPLD86 constructs (Supplementary via this type of ubiquitination. Figure 1a) abrogated the interaction with RBCK1. The interaction was confirmed using pull-down assays; full-length but not trun- RBCK1 regulates FKBPL protein levels cated FKBPLD200 interacted with myc-tagged RBCK1 (Figure 1c). To assess the functional potential of the interaction between Mutational analysis of RBCK1 demonstrated an interaction bet- RBCK1 and FKBPL, RBCK1 was transiently overexpressed in MCF-7 ween FKBPL and the myc-tagged RBCK1/DRING mutant, but not cells at both the mRNA and protein levels (Figure 2a). This the RBCK1/DUbl mutant, confirming that the Ubl was important coincided with a significant increase in FKBPL protein (Po0.05), for its interaction with FKBPL, although the RING domain may still whereas FKBPL mRNA levels remained constant (Figure 2a and be needed to enhance this interaction (Figure 1d and Supplemen- Supplementary Figure 2a). MCF-7 clones stably overexpressing tary Figure 1b). To test whether RBCK1 could alter the ubiquitin RBCK1, 3.1 Â 8 and 3.1 Â 10, had increased levels of RBCK1 at status of FKBPL, we assessed the ability of full-length RBCK1 and the mRNA and protein levels (Figure 2b and Supplementary mutated RBCK1 (lacking either the Ubl or RING domain) to Figure 2b). In support of the transient transfections, FKBPL protein ubiquitinate FKBPL. Immunoprecipitation of FKBPL resulted in levels were significantly elevated in both RBCK1 clones compared

Figure 1. Full-length FKBPL interacts with RBCK1. (A) A yeast two-hybrid positive clone obtained from screening a human fetal brain library with full-length FKBPL. The detection of (a) b-galactosidase activity, (b, c) yeast colony growth on drop-out media (SC-Leu-Trp-His þ 10 mM 3AT or SC-Leu-Trp-Ura) and (d) yeast growth inhibition on SC-Leu-Trp þ 5FOA represented a positive protein interaction. (B) Mammalian two-hybrid assay demonstrating interaction between RBCK1 and full-length FKBPL but not FKBPL-truncated mutants in L132 cells (n ¼ 4). (C) Co-immunoprecipitation in COS-7 lysates immunoabsorbed to protein G-Sepharose beads with antibodies against MYC-tagged RBCK1 and HA-tagged FKBPL. (D) Co-immunoprecipitation in COS-7 lysates immunoabsorbed to protein G-Sepharose beads with antibodies against MYC-tagged RBCK1 and FKBPL. (E) T47D cells transfected with myc-tagged RBCK1 (full-length RBCK1 or truncated mutants), immunoprecipitated with FKBPL antibody and probed for HA-ubiquitin. (F) T47D cells transfected with myc-tagged RBCK1 and full-length or ubiquitin mutants, immunoprecipitated for FKBPL and probed for HA-ubiquitin.

Oncogene (2014) 3441 – 3450 & 2014 Macmillan Publishers Limited RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3443

Figure 2. RBCK1 modulates FKBPL levels. (a) FKBPL and RBCK1 mRNA and protein levels in MCF-7 cells transiently transfected with pcDNA3.1/ RBCK1 or empty vector control, pcDNA3.1 (n ¼ 3). (b) mRNA and protein levels of FKBPL and RBCK1 in RBCK1 stable clones (3.1 Â 10 and 3.1 Â 8) compared with parental or empty vector control, 3.1E21 (n ¼ 3). (c) mRNA and protein levels of FKBPL and RBCK1 in MCF-7 cells transfected with RBCK1 siRNA or non-targeting (NT) control siRNA (n ¼ 3). *Po0.05; **Po0.01. with the empty vector control, 3.1E21 (Po0.05; Figure 2b and (P ¼ 0.007) and 99% (P ¼ 6.6 Â 10 À 13), respectively, compared Supplementary Figure 2b), whereas mRNA levels remained with MCF-7 parental and 3.1E21 cells (Figure 3d), whereas unchanged. When a RBCK1-targeted small interfering RNA (siRNA) RBCK1 siRNA-transfected parental MCF-7 cells showed a threefold approach was used, RBCK1 knockdown resulted in a significant increase in clonogenic survival compared with non-targeting decrease in FKBPL protein (Po0.05) and no significant change at siRNA controls (P ¼ 0.025, Figure 3e). RBCK1 depletion also the mRNA level (Figure 2c and Supplementary Figure 2c), resulted in a significant increase in cell proliferation (Figure 3f). suggesting that RBCK1 is involved in post-translational stabilization of FKBPL. Supplementary Figure 2d shows RBCK1 knockdown following transfection of individual siRNAs rather than the pool. RBCK1 is an estrogen-inducible gene that associates with the FKBPL molecular complex We have previously demonstrated that FKBPL has a role in ER RBCK1 modulates p21 and ER expression with subsequent signaling and is regulated by 17-b-estradiol (E2)6 and have effects on cell proliferation determined that RBCK1 is also involved in this pathway. Previous studies have implicated both FKBPL and RBCK1 involve- To investigate the role of ER in RBCK1 regulation, MCF-7 cells ment in p21 transcription and protein stability.3,4,21 To further were treated with 10 À 8 mol/l E2 for a 48 h time course. characterize the significance of this interaction, we assessed the RBCK1 levels increased 4 h after treatment with E2 and further levels of p21 in the MCF-7 clones stably expressing RBCK1. increased at later time points (Figure 4a). The increase in RBCK1 Quantitative reverse transcriptase-PCR analysis revealed a 1.5-fold expression was reversed following 24 h treatment with the anti- increase in p21 mRNA in the 3.1 Â 10 cell line (Po0.01) and a estrogen, tamoxifen (Figure 4b); this was more dramatic in the 5.5-fold increase in 3.1 Â 8 cell line (Po0.01; Figure 3a and MCF-7 cells compared with the T47D cells. To assess whether Supplementary Figure 3a). Western blot analysis demonstrated an RBCK1 was present in the previously reported Hsp90/ER/FKBPL approximately twofold increase in p21 protein levels in 3.1 Â 10 molecular complex,6 co-immunoprecipitations were performed (Po0.05) and 3.1 Â 8(Po0.001) cell lines compared with the þ / À E2 in MCF-7 and T47D cells. In MCF-7 cells, endogenous 3.1E21 empty vector control (Figure 3a). ERa mRNA and protein RBCK1 did not co-precipitate in the absence of E2 but co- levels were not significantly altered in RBCK1-overexpressing cell precipitated with FKBPL and ERa in the presence of E2 (Figure 4c). lines (Figure 3b and Supplementary Figure 3b); however, RBCK1 In T47D cells, RBCK1 co-precipitated with HSP90 only in the knockdown in MCF7 and T47D cell lines resulted in decreased absence of E2, and co-precipitated with FKBPL, Hsp90 and ER in ERa (Figure 3c and Supplementary Figure 3c). In addition, we the presence of E2 (Figure 4c). These data imply that E2 is required investigated the proliferative/clonogenic capacity of RBCK1 stable for the formation of a robust complex comprising FKBPL/RBCK1/ clones and following RBCK1 knockdown. Clonogenic survival was HSP90/ERa and that the composition of the complex is cell-line significantly inhibited in the 3.1 Â 10 and 3.1 Â 8 clones by 50% dependent.

& 2014 Macmillan Publishers Limited Oncogene (2014) 3441 – 3450 RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3444

Figure 3. RBCK1 regulates p21 and ERa expression with effects on clonogenic growth and proliferation. (a) p21 mRNA and protein levels in RBCK1 stable clones, 3.1 Â 10 and 3.1 Â 8, compared to MCF-7 and 3.1E21 empty vector control (n ¼ 3). (b)ERa mRNA and protein levels in RBCK1 stable clones, 3.1 Â 10 and 3.1 Â 8, compared to 3.1E21 empty vector control (n ¼ 3). (c)ERa expression following RBCK1 siRNA-targeted knockdown in MCF7 and T47D cells, compared to non-targeted (NT) siRNA as a control. (d) Clonogenic assays to determine the surviving fraction of the MCF-7, 3.1E21 control and 3.1 Â 10 and 3.1 Â 8 stable clones (n ¼ 3). (e) Clonogenic analysis of MCF-7 cells transfected with RBCK1 siRNA, compared to non-targeted (NT) siRNA control (n ¼ 3). (f) Real-time cell proliferation in MCF-7 cells transfected with RBCK1 siRNA, compared to non-targeted (NT) siRNA control (f, analyzed by two-way analysis of variance (ANOVA); *Po0.05; **Po0.01; ***Po0.001; n ¼ 3).

RBCK1 is a potential ER coactivator increased in the presence of E2, we hypothesized that both FKBPL Previous studies have shown that RBCK1 is a co-activator of HBv and RBCK1 could be involved in regulation of ER-responsive genes. X-protein-induced transactivation22 and affects ER signaling;24,25 Using chromatin immunoprecipitation, we assessed whether FKBPL therefore, its effect on ER transactivation was assessed in light of and RBCK1 were recruited to the promoter of the ER-responsive evidence that RBCK1 was present in the ER/FKBPL/HSP90 complex. gene, pS2. Following treatment with 10 nM E2, FKBPL, RBCK1 and ERa Using parental and FKBPL stably overexpressing (3.1D2) MCF-7 cells, were bound to the promoter region of the pS2 (Figure 5c); RNA FKBPL overexpression increased ER-mediated transactivation over polymerase II was also recruited and acted as a positive control. parental control in the presence of E2 only (Po0.05). However, Analysis of the pS2 distal region confirmed that this interaction was transfection of the 3.1D2 cell line with RBCK1 caused a 4.5-fold specific to the promoter sequence of pS2 (Figure 5c). We then increase in transcriptional activation in the absence of ligand assessed the effect of RBCK1 on pS2 protein expression and found (P ¼ 0.05), which was not further increased following the addition of that pS2 levels were significantly increased in the RBCK1-over- E2 (Po0.01; Figure 5a). These data suggest that RBCK1 can regulate expressing clone, 3.1 Â 8 compared with the empty vector control, ER transactivity in an FKBPL concentration-dependent manner. As 3.1E21 (Po0.05; Figure 5d). Following treatment with 1 mM tamoxifen, RBCK1 seems to enhance ER transactivation and may represent an pS2 expression was inhibited by 50% in the 3.1E21 control clone and ER transcriptional coactivator, the ability of ER to bind the LxxLL 42% in the 3.1 Â 8 clone. However, pS2 levels in the tamoxifen- consensus-binding motif of ER coactivator proteins in the presence treated 3.1 Â 8 clone were similar to the non-treated 3.1E21 control of increased RBCK1 levels was assessed (Figure 5b). 3.1D2 cells (Figure 5e), suggesting an attenuation of tamoxifen antagonist demonstrated a significant increase in the ability of ER to bind this activity in the presence of high RBCK1 levels. The data support a role motif (P ¼ 0.0127) compared with parental cells. However, increased for FKBPL and RBCK1 in regulating this ER responsive gene. RBCK1 levels resulted in decreased ability of ER to bind the coactivator motif in both parental and FKBPL stable cells, suggesting that RBCK1, in its role as an ER co-activator, is recruited to the LxxLL- High RBCK1 levels promote agonistic effects of tamoxifen binding region of ER, blocking the ability of ER to bind this motif. High levels of ER co-activator proteins have been associated with As we identified that the interaction between FKBPL and RBCK1 was conferring a tamoxifen-resistant phenotype; therefore, we

Oncogene (2014) 3441 – 3450 & 2014 Macmillan Publishers Limited RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3445

Figure 4. RBCK1 is an estrogen-inducible protein involved in Hsp90/FKBPL/ER complexes. (a) RBCK1 levels in MCF-7 cells treated with ethanol vehicle control or 10 nM E2 for the 48 h time course. (b) RBCK1 expression in MCF7 and T47D cells following treatment with ethanol vehicle control, 10 nM E2 or 10 nM E2 plus 1 mM tamoxifen for 24 h. (c) Endogenous co-immunoprecipitation of FKBPL, RBCK1 and ERa in MCF-7 or T47D cells ±10 nM E2.

investigated the effects of RBCK1 expression on tamoxifen high levels of RBCK1 correlated with increase distant metastasis- sensitivity. As pS2 levels remain high in the 3.1 Â 8 clone following free survival (Supplementary Figure 5). As both FKBPL and RBCK1 tamoxifen treatment and FKBPL has been shown to affect were shown to correlate with increased patient survival separately, tamoxifen sensitivity in vitro,6 we assessed whether RBCK1 could we assessed the effect of the two proteins together on patient also affect sensitivity to tamoxifen. Following treatment with survival by using the ratio of the two markers. The GSE22219 data 1–10 mM tamoxifen, clonogenic survival in the 3.1 Â 8 clone set was further separated for low FKBPL/low RBCK1 (Low), low demonstrated resistance to tamoxifen compared with MCF-7 FKBPL/high RBCK1, high FKBPL/low RBCK1 and high FKBPL/high parental and 3.1E21 controls (Po0.001; Figure 6a). Moreover, in RBCK1 (High), and distant relapse-free survival assessed. High this clone, tamoxifen had an agonistic effect, increasing clono- levels of both FKBPL and RBCK1 significantly correlated (Po0.05, genicity at all doses except the highest dose. Following RBCK1 Mantel–Cox log-rank test) with increased distant relapse-free siRNA knockdown, increased tamoxifen sensitivity was detected in survival (Figure 7b). These data suggest that high levels of FKBPL cell lines, MCF7 (1–10 mM tamoxifen, Figures 6b) and T47D (1 mM and RBCK1 are advantageous for patient survival and predict for a tamoxifen, Figure 6c) compared with non-targeted siRNA controls. less aggressive tumor phenotype, supporting our in vitro growth This strongly supports a correlation between RBCK1 levels and inhibitory findings. tamoxifen sensitivity. We assessed how FKBPL and RBCK1 levels had an impact on patient response to tamoxifen. FKBPL and RBCK1 seems to work in tandem to regulate cell growth; however, our in vitro data High RBCK1 expression correlates with improved patient survival suggested that RBCK1 may work in an opposing manner to FKBPL but poor response to tamoxifen with respect to tamoxifen efficacy. This was evaluated further by To assess the clinical relevance of our in vitro findings, several analysis of the GSE12093 data set.29 In support of our in vitro microarray patient data sets (Supplementary Table 1), GSE7390,26 studies and previous data,6 following treatment with tamoxifen, GSE203427 and GSE22219,28 were analyzed for RBCK1 expression high FKBPL/low RBCK1 (FKBPL:RBCK142.478) significantly corre- and correlated with patient survival using the Kaplan–Meier lated with increased distant metastasis-free survival in patients, analysis. The GSE2034 data set showed a significant correlation whereas patients with low FKBPL/high RBCK1 (FKBPL:RBCK1 (Po0.05, Mantel–Cox log-rank test) between high RBCK1 levels p2.478) exhibited poor distant metastasis-free survival (Po0.05; and increased overall patient survival (Figure 7a). The GSE7390 Figure 7c). data set showed a trend in which high RBCK1 levels correlated with increased overall and distant metastasis-free survival, whereas there was a significant correlation with relapse-free survival DISCUSSION (Po0.05, Mantel–Cox log-rank test; Supplementary Figure 4). The FKBPL has demonstrated an increasingly important role in GSE22219 data set showed a trend toward significance, where tumorigenesis by a variety of signaling pathways including

& 2014 Macmillan Publishers Limited Oncogene (2014) 3441 – 3450 RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3446

Figure 5. RBCK1 is an ER coactivator. (a) Transactivation of ER element reporter construct in parental and FKBPL overexpressing MCF-7 cells, 3.1D2±an RBCK1-overexpressing construct±E2 (n ¼ 4). *Po0.05; **Po0.01. (b) LxxLL coactivator sequence-binding assay using nuclear extracts from parental MCF-7 cells or FKBPL overexpressing MCF-7 cells, 3.1D2 þ / À RBCK1 (n ¼ 2 in triplicate). (c) Chromatin immunoprecipitation of MCF-7 cells treated with 10 nM E2 and screened for RNA polymerase II, ERa, FKBPL and RBCK1 binding on the pS2 promoter. (d) pS2 levels in the 3.1 Â 8 clone compared with those of 3.1E21 control clone (n ¼ 3; *Po0.05). (e) pS2 levels in the 3.1 Â 8 stable clone compared with those of 3.1E21 control clone treated with 1 mM tamoxifen (n ¼ 2).

regulation of ER signaling and CD44-mediated angiogenesis.4–10 shown to increase the stability of the TNF-R1 signalling complex The level of FKBPL expression in breast cancer cells is important, (TNF–RSC) complex.18 Our studies indicate that RBCK1 does not with high levels correlating with tumor growth inhibition and ubiquitinate FKBPL through classical lysine residues, and may increased sensitivity to endocrine therapy.6 However, very little is therefore stabilize FKBPL via linear ubiquitination; this would need known of how FKBPL is regulated or which other proteins are to be further validated to support this hypothesis. involved in its signaling. Here we provide evidence that RBCK1 is FKBPL protein stabilization by RBCK1 resulted in increased levels an FKBPL-interacting partner that stabilizes FKBPL expression, of the cyclin-dependent kinase inhibitor, p21, causing a decrease affects ER signaling and response to endocrine therapy. in clonogenic survival and proliferation. Previously, RBCK1 and RBCK1 interacts only with full-length FKBPL and the Ubl domain FKBPL have both been implicated in controlling p21 levels at the of RBCK1 is required for this interaction. This domain is crucial for transcriptional and protein levels, respectively.3,4,6,21 Our data are the E3 ligase activity of RBCK1 and has a role in substrate the ﬁrst to report that FKBPL and RBCK1 may work together to recognition,12 suggesting that FKBPL may be a RBCK1 substrate. regulate p21 and cell cycle progression. Using the clinical data set RBCK1 acts in tandem with another ubiquitin ligase RNF31/HOIP GSE22219, analysis of RBCK1 and FKBPL levels revealed that to form a 600 kDa linear ubiquitin chain assembly complex, which patients with high levels of both FKBPL and RBCK1 demonstrated generates multiple and different ubiquitin chains, including linear increased survival, whereas patients with low FKBPL/high RBCK1 ubiquitination chains.17 Although the functional signiﬁcance is or high RBCK1/low FKBPL had poor survival outcomes. This poorly characterized, linear ubiquitination by RBCK1 has been supports our in vitro data where high RBCK1 increases FKBPL and

Oncogene (2014) 3441 – 3450 & 2014 Macmillan Publishers Limited RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3447

Figure 6. RBCK1 promotes agonistic effects of tamoxifen in vitro.(a) Clonogenic assay to determine surviving fraction after treatment with tamoxifen in parental MCF-7, 3.1E21 control and 3.1 Â 8 clone (n ¼ 3). (b) Clonogenic assay to determine surviving fraction after treatment with tamoxifen in parental MCF-7 cells þ RBCK1 siRNA or NTsiRNA control (n ¼ 3). (c) Clonogenic assay to determine surviving fraction after treatment with 1 mM tamoxifen in parental T47D cells þ RBCK1 siRNA or NTsiRNA control (n ¼ 3; *Po0.05; ***Po0.001, two-way analysis of variance (ANOVA)).

Figure 7. High RBCK1 expression correlates with increased patient survival but poor tamoxifen response. Kaplan-Meier curves of breast cancer patients for (a), relapse-free survival (GSE2034) stratified with RBCK1 expression (b), distant relapse-free survival (GSE 22219) stratified using the ratio of FKBPL and RBCK1 expression (c), distant metastasis-free survival from patients treated with tamoxifen therapy (GSE 12093) and stratified with RBCK1:FKBPL expression.

p21 levels and inhibits tumor growth. FKBPL has previously been molecular complex that binds ERa in the presence of E2. In identiﬁed as a prognostic marker6,9 and we propose that RBCK1 addition, RBCK1 levels are rapidly upregulated by E2 and this was also represents a novel prognostic marker in breast cancer. reversed by tamoxifen; RBCK1 is necessary for ER-mediated We have shown that RBCK1 has an important role in the FKBPL/ transcription in response to E224 and is critical to ER-mediated ER signaling pathway. RBCK1 is a member of the Hsp90/FKBPL transcription, and therefore would need to be rapidly induced by

& 2014 Macmillan Publishers Limited Oncogene (2014) 3441 – 3450 RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3448 E2 to facilitate regulation of this pathway. We provide evidence 50 nM of a pool of RBCK1 siRNA or Non Targeting control (Stealth RNAi, that RBCK1 in conjunction with FKBPL and ERa bind to the Invitrogen) were transfected using Oligofectamine reagent (Invitrogen), promoter of the ER-responsive gene, pS2, and regulate ER according to the manufacturer’s instructions. Cells were assayed 72 h after transactivation. RBCK1 has previously been shown to act as a transfection. transcriptional coregulator and low RBCK1 levels have been associated with decreased transcription of the ER-regulated genes Yeast two-hybrid assay 21–25 c-myc, cyclin D1 and pS2, and this effect was attributed to The ProQuest Yeast Two-Hybrid System (Life Technologies, Paisley, UK) was RBCK1’s regulation of ERa levels. We have also shown that RBCK1 used to identify FKBPL-interacting proteins. Briefly, in-frame cloning of depletion results in a concomitant decrease in ERa expression, full-length FKBPL into the pDBLeu-GAL4 DNA-binding domain plasmid was although high RBCK1 levels did not significantly affect ERa carried out and used to screen a human fetal brain cDNA library expression. We therefore suggest that RBCK1 is an ER coactivator (Invitrogen) constructed in the pPC86-GAL4 activation domain plasmid. as high RBCK1 levels caused increased pS2 expression; increased Plasmids were transformed into MaV203 yeast cells using the polyethylene glycol/lithium acetate method according to the manufacturer’s instructions ER transactivity even in the absence of E2; and reduced ER and transformants were plated onto selective medium to determine the capacity to bind its coactivator consensus sequence, LxxLL, induction of three independent reporter genes. DNA sequencing and suggesting that RBCK1 occupies this binding site on ER in its BLAST sequence analysis (http://www.ncbi.nlm.nih.gov) was performed to role as an ER co-activator. There is evidence that steroid receptor/ identify interacting cDNA. immunophilin complexes exist in the nucleus30,31 and are involved 32 in chromatin regulation and association with DNA. Immuno- Mammalian two-hybrid assay philins have been shown to act as scaffold proteins to facilitate The CheckMate Mammalian-Two Hybrid (Promega, Southhampton, UK) interactions between transcription factors and transcriptional 32 was used to confirm protein interactions in a mammalian cell system. co-activators, and therefore FKBPL may represent an immuno- FKBPL and RBCK1 mutants were engineered using the QuikChange philin important in facilitating RBCK1-mediated transcriptional Site-Directed Mutagenesis (Stratagene, Leicester, UK) method according to activation. We have demonstrated that RBCK1 overexpression the manufacturer’s instructions. L132 cells at 60–80% confluence were increases p21 expression, suggesting that RBCK1-mediated stabili- transfected with pBIND/FKBPL (full-length or mutant) or pACT/RBCK1 zation of FKBPL has subsequent downstream effects on p21 (full-length or mutant) and pG5luc plasmid. After 24 h, cells were analyzed stability.4,6 using the Dual-Glo Luciferase assay (Promega). Firefly luciferase activity RBCK1 overexpression resulted in resistance to tamoxifen and was normalized to Renilla luciferase activity and presented as relative light appeared to promote the agonistic effects of tamoxifen by units. increasing cell growth; RBCK1 knockdown using targeted siRNA reversed these effects. In addition, pS2 levels remained high Clonogenic assay/growth assays following tamoxifen treatment in the RBCK1-overexpressing cell Cells were plated in Dulbecco’s modified Eagle’s medium þ 10% fetal calf line, 3.1 Â 8, suggesting that the antagonist activity of tamoxifen serum and incubated for 16 days at 37 1C. Alternatively, after 24 h, medium was compromised. Elevated levels of the ER co-activator, AIB1, was replaced with Dulbecco’s modified Eagle’s medium þ 10% fetal calf have been shown to increase ER signaling resulting in tamoxifen serum containing 1 mM tamoxifen (Sigma) and incubated under normal 33 conditions for 16 days and stained with 0.4% crystal violet. Using the resistance, and as a potential ER co-activator RBCK1 seems to act XCelligence system (Roche, Welwyn Garden City, UK) to assess growth, in a similar way. Elevated co-activator levels prevent the 1 Â 104 MCF-7 cells were seeded into an E-16 multi-well plate (Roche) recruitment of the NCoR1 and NCoR2 repressor proteins and the in triplicate and cell growth was measured every 15 min for 96 h. inhibition of activation function-2 of ER,34 therefore reversing the therapeutic effect of tamoxifen. To assess the clinical relevance of Co-immunoprecipitation these findings, we examined the GSE12093 data set from patients treated with adjuvant tamoxifen therapy;29 the ratio of FKBPL– To confirm protein interactions, L132 cells were transfected with HA-FKBPL or HA-FKBPLD200 and Myc-RBCK1. Myc-RBCK1DUbl and Myc-RBCK1DRING RBCK1 was assessed. In support of our in vitro data and previous 6 mutants were engineered using the QuikChange Site-Directed Mutagen- work, high levels of FKBPL and low levels of RBCK1 significantly esis (Stratagene) method according to the manufacturer’s instructions. correlated with increased distant metastasis-free survival in COS-7 cells were transfected with FKBPL and Myc-RBCK1DUbl or Myc- patients treated with tamoxifen. RBCK1DRING. All plasmids were transfected using Lipofectamine 2000, In summary, we provide evidence that, in addition to its role according to the manufacturer’s instructions. Following incubation, cells as an ER coactivator, RBCK1 is an important regulator of FKBPL were lysed (lysis buffer; 1% Igepal, 0.1% SDS, 0.05% sodium deoxycholate, and p21 with downstream consequences for tumor growth and 1 protease inhibitor tablet and 1 PhosSTOP tablet (Roche)), precleared and lysates were incubated with antibody-bound Protein G-Sepharose beads response to tamoxifen. RBCK1 may therefore act as a novel 1 prognostic and predictive biomarker in breast cancer. (Cancer Research, London, UK) overnight at 4 C. Beads were washed in lysis buffer and resuspended in 5 Â reducing sample buffer (Pierce, Cramlington, UK).

MATERIALS AND METHODS Ubiquitination assay Cell culture and transfections The ubiquitin constructs, pEFIRES-HA-ubiquitin, pEFIRES-HA-K48R and The MCF-7, T47D and COS-7 cell lines were obtained from ATCC pEFIRES-HA-R48K, were a gift from Professor Yossi Yarden, as previously (Teddington, Middlesex, UK). The L132 cell line was obtained from Gibco described.35 To assess protein ubiquitination, T47D cells were co-trans- (Paisley, Scotland, UK), UK. Cell lines were maintained as monolayers in fected with HA-Ubiquitin, Myc-RBCK1 and either Myc-RBCK1DUbl/Myc- medium supplemented with 10% fetal calf serum (Dulbecco’s modiﬁed RBCK1DRING mutants or HA-Ubiquitin^K48R/HA-Ubiquitin^R48K mutants. Eagle’s medium: Invitrogen, Paisley, UK for MCF-7, T47D and COS-7 cells; Transfections were carried out using Lipofectamine 2000 according to the and minimum essential medium: Invitrogen for L132 cells). Cell lines were manufacturer’s instructions. Samples were immunoprecipitated using authenticated by short tandem repeat proﬁling carried out by the FKBPL antibody and resuspended in non-reducing sample buffer (Pierce). suppliers. Testing revealed that cells were mycoplasma free. All experi- ments were carried out between passages 1 and 20. The MCF-7 FKBPL-overexpressing cell line, 3.1D2, was generated as Western blot analysis previously described.6 Transient and stable transfections were carried out Samples were subjected to SDS–polyacrylamide gel electrophoresis using using empty vector pcDNA3.1 (as a control) or pcDNA3.1/RBCK1 and the XCellSureLock Mini-Cell system (Invitrogen) and transferred onto Lipofectamine Plus according to the manufacturer’s instructions, except nitrocellulose membranes (Millipore, Watford, UK) and probed accordingly. when stated otherwise. Stably transfected MCF-7 cells were selected using Antibody binding was detected using Supersignal West Pico Chemilumi- 750 mg/ml G418 (Sigma, Gillingham, UK). For RBCK1 knockdown analysis, nescent substrate (Pierce) according to the manufacturer’s instructions.

Oncogene (2014) 3441 – 3450 & 2014 Macmillan Publishers Limited RBCK1 modulates FKBPL and affects tumor growth C Donley et al 3449 Antibodies included FKBPL (1:2000; ProteinTech, Manchester, UK), Hsp90 Quantification and statistical analysis (1:1000; BD Transduction Laboratories, Oxford, UK), ERa (1:1000; Millipore), Image acquisition and densitometric analysis was performed using UVP pS2 (1:500; Santa Cruz, Heidelberg, Germany), RBCK1 (1:500; Abcam, VisionWorksLS (Ultra-Violet Products Ltd, Cambridge, UK) and ImageJ Cambridge, UK), GAPDH (1:5000; Sigma), anti-HA and anti-Myc (Co-IP; Cell software (National Institutes of Health, Bethesda, MD, USA). One-way Signalling, Hitchin, UK), anti-Myc (1:1000; WB; SantaCruz). analysis of variance was used for all statistical analyses, unless otherwise indicated. Real-time reverse transcriptase PCR RNA was isolated from cells using STAT60 and transcribed using CONFLICT OF INTEREST Transcriptor First Strand cDNA Synthesis kit (Roche) as per the manufacturer’s instructions. Real-time PCR was performed using the Lightcycler The authors declare no conflict of interest. 480 PCR machine (Roche) to quantify the expression levels of FKBPL, RBCK1, p21 using a validated Taqman mono-hydrolysis PCR primer probes (Roche). Glyceraldehyde 3-phosphate dehydrogenase was used as an ACKNOWLEDGEMENTS internal control to normalize mRNA expression. The financial support for this work was provided by PhD studentships awarded to C Donley and K McClelland from Department of Employment and Learning, Northern Ireland and Action Cancer, Northern Ireland, respectively. HD McKeen was supported ER element transactivation assay by grants from Breast Cancer Campaign, United Kingdom and then the Medical MCF-7 parental and 3.1D2 FKBPL stable cells were transfected with Research Council, United Kingdom and A Yakkundi was supported by a grant from pcDNA3.1/RBCK1, pGL2-ERE luciferase reporter plasmid and the pBINDRe- the Biotechnology and Biological Sciences Research Council, United Kingdom. All nilla plasmid to normalize for transfection efficiency. After 24 h, cells were grants were awarded to T Robson. À 8 treated with dimethyl sulfoxide vehicle control or 10 M E2 for 24 h, and were then analyzed using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity and presented as relative light units. REFERENCES 1 Robson T, Joiner MC, Wilson GD, McCullough W, Price ME, Logan I et al. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2014) 3441 – 3450 & 2014 Macmillan Publishers Limited