Oncogenic and Osteolytic Functions of Histone Demethylase NO66 in Castration-Resistant Prostate Cancer

Oncogene https://doi.org/10.1038/s41388-019-0774-x

ARTICLE

Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer

1,2 1 1 3 4 2 Krishna M. Sinha ● Rozita Bagheri-Yarmand ● Sharmistha Lahiri ● Yue Lu ● Miao Zhang ● Sarah Amra ● 1 4 4 5 4 1 Yasmeen Rizvi ● Xinhai Wan ● Nora Navone ● Bulent Ozpolat ● Christopher Logothetis ● Robert F. Gagel ● Johnny Huard2,6

Received: 19 June 2018 / Revised: 7 December 2018 / Accepted: 16 February 2019 © Springer Nature Limited 2019

Abstract Epigenetic changes that cause dysregulated gene expression during progression of androgen-independent prostate cancer (PCa) and metastatic skeletal lesions remain elusive. Here, we explored the role of histone demethylase NO66 in the pathogenesis of PCa and bone metastasis-related skeletal lesions. Tissue and cDNA microarrays of PCa were analyzed for NO66 mRNA and protein levels. We examined the effects of gain and loss of NO66 function on cell viability, colony formation, migration, invasion, and tumor-induced skeletal lesions in femoral bone. RNAseq and ChIPseq were performed

1234567890();,: 1234567890();,: to elucidate NO66-target genes in PCa. We report that NO66 levels were upregulated in advanced primary prostate tumors compared to normal tissue or tumors with low Gleason scores. Forced expression of NO66 promoted cell survival and invasion of PCa cells; whereas, knockdown of NO66 resulted in decreased cell survival and increased sensitivity to docetaxel. NO66-overexpressing PC3 cells implanted into the femoral bone of male SCID mice caused massive bone loss and stimulation of mouse osteoclast-promoting genes, including Dickkopf1, Cathepsin K, Nf-kβ,; and Calcr, suggesting a role for NO66 in tumor growth in bone and osteoclast activity. Combined RNAseq and ChIP-seq revealed that NO66 activates the survival gene MCL1, the invasion-associated genes IGFBP5 and MMP3, the pro-oncogenic genes CTNNB1 and CCND1, and the epigenetic modiﬁer gene KMT2A in androgen-independent PCa. Our ﬁndings uncover the role of NO66 as a key oncogenic driver in PCa, causing osteolytic lesions through upstream epigenetic regulation of key genes for survival, invasion and metastasis, and pro-osteoclastic factors.

Supplementary information The online version of this article (https:// doi.org/10.1038/s41388-019-0774-x) contains supplementary Introduction material, which is available to authorized users. Metastatic and castration-resistant PCa (mCRPC) invades * Krishna M. Sinha [email protected] primarily to bones and develops focal skeletal lesions, thus – * Johnny Huard resulting in severe bone pain and fragility fractures [1 3]. [email protected] Several molecular prognostic factors regulated by epigenetic mechanisms play a key role in the recurrence and 1 Endocrine Neoplasia and Hormonal Disorders, The University of metastases of PCa and skeletal lesions [4]. Aberrant Texas MD Anderson Cancer Center, Houston, TX, USA expression of histone demethylases in PCa induces 2 Department of Orthopaedic Surgery, McGovern Medical School, expression of many genes through epigenetic mechanism to The University of Texas Health Science Center at Houston, contribute to resistance to a variety of drugs and to function Houston, TX, USA independently of androgen receptor signaling, thus allowing 3 Epigenetics and Molecular Carcinogenesis, The University of progression of mCRPC [4, 5]. Enhancer of zeste homolog 2 Texas MD Anderson Cancer Center, Houston, TX, USA (EZH2) is a biomarker in advanced PCa, contributes to the 4 Genitourinary Medical Oncology, The University of Texas MD activation of AR-target genes in mCRPC, and promotes Anderson Cancer Center, Houston, TX, USA tumor progression [6–8]. MLL1 (KMT2A) is highly elevated 5 Department of Experimental Therapeutics, The University of and translocated in mCRPC, and activates several onco- Texas MD Anderson Cancer Center, Houston, TX, USA genes independently of AR signaling [9]. These epigenetic 6 Steadman Philippon Research Institute, Vail, CO, USA modifiers in PCa also regulatethe expression of growth K. M. Sinha et al. factors and cytokines during metastasis and promoting Negative staining for NO66 was shown in samples with a skeletal lesions [6, 10, 11]. Gleason score of 6. As shown in Fig. 1b, the percentage of PCa cells invade bone tissue, proliferate, and release PCa specimens with high immunostaining intensity for factors, such as transforming growth factor-beta (TGF-β), nuclear NO66 increased for samples with higher Gleason bone morphogenetic protein (e.g., BMP2), insulin-like scores (p = 0.0168, chi-square, df = 12.08, 4) suggesting a growth factor (IGF), fibroblast growth factor (e.g., FGF9), role for NO66 in advanced stages of PCa. Using protein and receptor activator of nuclear factor kappa-B ligand RNA arrays, we also observed that NO66 protein levels are (RANKL), and Wnt proteins, all of which are potent reg- higher in lung, colon, pancreas, and prostate tumors, but ulators of bone homeostasis [12–14]. Bone marrow stromal lower in melanoma tumors than in healthy tissue (Fig. 1c). cells then produce pro-angiogenic factors, growth factors, NO66 mRNA levels were higher in primary prostate tumors and RANKL, which supports tumor growth as well as reg- than in healthy prostate tissue (Fig. 1d). Independent public ulates osteoblast and osteoclast formation [15–17]. Over- data set from TCGA and the Oncomine database indicate expression of noggin in PC3 cells inhibits cell migration, genomic alteration (deletion or amplification) of the NO66 invasion, and osteolytic lesions, indicating that BMPs play a gene in a subset of prostate adenocarcinoma (Fig. 1e) and pathological role in promoting PCa-induced skeletal lesions that NO66 levels in PCa tumors associates with patient poor at metastatic sites [18, 19]. We previously reported that the overall survival (Fig. 1f). histone demethylase NO66, specific for H3K4me3 and H3K36me3, inhibits osteoblasts differentiation and bone NO66 knockdown inhibits cell survival and formation through regulating gene expression [20–22]. potentiates docetaxel-induced cytotoxicity NO66 also acts as hydroxylase for ribosomal proteins [23]. We also showed that NO66 interacts with the polycomb To investigate NO66 function in PCa cell survival, we proteins PHF19, EZH2, and SUZ12, and inhibits the knocked-down NO66 levels in an androgen-independent expression of the PRC2-target genes in mouse embryonic (AI) DU145 cell line by lentivirus NO66 shRNAs (DU-sh) cells [24]. These studies indicate that NO66 serves as an and control shRNA (DU-Nsh). Knockdown efficiency was epigenetic regulator through histone methylation and con- confirmed by measuring the NO66 mRNA levels in those trols chromatin activity. NO66 expression at the RNA and shRNA-expressing clones (Fig. 2a). With clonogenic and protein levels is upregulated in many cancers, including lung MTT assays, we showed that NO66 depletion led to the cancer, renal carcinoma, and colorectal cancer [25, 26]. formation of fewer colonies in DU-sh1 and DU-sh2 than However, the role of NO66 in PCa pathogenesis has not yet control (DU-Nsh) cells and also decreased cell survival in been reported. shRNA-expressing cells (Fig. 2b, c). In addition, the inva- We show that NO66 promotes survival and invasion of sive and migration abilities of NO66-depleted cells were cancer cells and is highly expressed in advanced PCa also reduced as analyzed by Boyden chamber assay (Suppl. tumors compared to normal or low Gleason score tumors. Figure 1). Docetaxel has been an important treatment option NO66-expressing cells implanted into mouse femurs induce for patients with metastatic CRPC, but nearly all patients severe osteolytic lesions through activation of osteoclast eventually develop resistance. To determine whether NO66 formation. Finally, combined RNAseq and ChIPseq alters docetaxel sensitivity, we examined the sensitivity of experiments reveal that NO66 activates a large cohort of NO66-depleted DU145 cells to docetaxel-induced cyto- genes with known functions of cell survival, migration, and toxicity. Indeed, DU-NO66sh cells were more sensitive to drug resistance during PCa progression. docetaxel-induced cell death than DU-Nsh cells, as shown by an MTT assay (Fig. 2d). Furthermore, fluorescence- activated cell sorting (FACS) showed that the fraction of Results subG1 population was increased in NO66-depleted cells (54%) treated with 4 nM docetaxel when compared with NO66 is highly expressed in clinical PCa samples control cells treated with docetaxel (24%) (Fig. 2e), suggesting that NO66 expression may cause resistance to To study the clinical significance of NO66 in PCa, we first docetaxel. Additionally, levels of cleaved PARP increased analyzed the expression of NO66 in patient samples using in NO66-depleted cells compared to control cells, when immunohistochemistry with tissue microarray. We found treated with docetaxel (Fig. 2f). We next tested the level of that NO66 was expressed in more than 70% (63 out of total expression of known candidate genes involved in apoptosis. 90) of the tumors analyzed with predominantly nuclear The expression levels of BBC3, CASP2, PLAUR, and localization and its protein levels were higher in tissue from PUMA genes were significantly upregulated in DU-sh1 and patients with advanced PCa with Gleason scores of 8–10 DU-sh2 cells (Fig. 2g). We then reproduced the oncogenic (Fig. 1a) than in samples with Gleason scores of 6 or 7. role of NO66 in another AI cell line, PC3, in which NO66 Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer

Fig. 1 Expression of NO66 in subset of patient specimens. a Immu- mRNA levels of NO66 in patient samples using cDNA array plate. e nohistochemistry analysis with NO66-specific antibody in PCa patient Genomic alteration of NO66 in interrogated database (Cbio.portal. samples. Representative images with ×10 ×and 20 magnification are org). f Interrogated TCGA data for the NO66 levels in survival ana- shown with negative, medium, and high staining with Gleason Scores. lysis of PCa patients (https://www.proteinatlas.org/ b Quantification of NO66 expression in tissue microarray. c Dot blot ENSG00000170468-RIOX1/pathology/tissue/prostate+cancer) showing levels of NO66 in normal and tumor lysate dot array, and d

was depleted by lenti-shRNA (Fig. 2h). Based on clono- Transcriptome analysis of the NO66-target genes genic and soft agar assays, NO66 depletion decreased both the size and number of colonies (Fig. 2i, j), which suggests To identify the NO66-target genes, we performed RNAseq- a role for NO66 in cellular transformation. based transcriptome analysis of DU-Nsh cells and three DU-sh clones (sh1, 2, and 3). We identified a total of 32 Gain of NO66 function promotes survival, migration, differentially expressed genes; 18 genes were down-regu- and invasion lated, and 14 genes were upregulated (Table 1) in all three shRNA clones compared to control cells. Among the genes In contrast to the effects of NO66 depletion in PCa cells, with low expression in all DU-sh clones, IGFBP5, overexpression of NO66 in PC3 cells (Fig. 3a), NO66 AKR1C3, CEMIP, MTRNR2L2/8/9, and S100A9 are known increased their cell survival compared with control having to play a role in the progression of various cancers, vector alone (PC3-Vec) cells, as indicated with the MTT including prostatic tumors: IGFBP5 (in tumor progression), assay (Fig. 3b). With Boyden chamber assay to study the AKR1C3 (androgen synthesis in metastatic PCa [28–30]), invasion and migration abilities, our data indicate that more CEMIP [31, 32] (tumor cell migration and proliferation), PC3-NO66 cells than control cells migrated to the matrigel MTRNR2L2/8/9 (neuroprotective and anti-apoptotic), and barrier (Fig. 3c, d) while NO66 depletion in DU145 cells S100A9 (inflammation process). Normalized expression inhibited their invasion and migration (Suppl. Figure 1). tags for IGFBP5 and AKR1C3 were obtained by IGV Levels of the invasion marker MMP2 were significantly (Integrated Genome Viewer, MIT) from RNAseq data (Fig. increased in PC3-NO66 cells, thereby indicating that NO66 4a). In addition, with low stringency criteria (FDR > 0.05), promotes invasion of PCa cells (Fig. 3a). RUNX2 levels several other important genes with altered expression levels were also increased in PC3-NO66 cells, which promotes were identified, and a few of them were validated by PCa proliferation and invasion through activation of quantitative RT-PCR in control cells and DU-sh1 and 2 metastases-related genes [27]. clones (Fig. 4b). We showed that genes with oncogenic K. M. Sinha et al.

Fig. 2 Knockdown of NO66 in DU145 and PC3 cells inhibits cell by staining with propidium iodide. f Western blot showing the levels proliferation and induces apoptotic gene expression. a NO66 mRNA of cleaved PARP in docetaxel treated cells (4 nM) using cleaved PARP levels by qRT-PCR in DU-Nsh and DU-sh clones. b Clonogenic assay antibody. g Quantitative gene expression analysis in DU-Nsh and DU- with DU-Nsh and DU-sh cells (n = 3). c MTT assay for cell pro- sh clones. h Western blot for NO66 levels in stably expressing NO66- liferation ability of DU-sh clones at different days. d MTT assay for shRNA clones with NO66-speciﬁc antibody. i Clonogenic assay and j docetaxel-induced toxicity in DU-Nsh (black line) and DU-sh1 cells Anchorage-independent growth in PC3-Nsh and PC3-sh1 cells. Error (red line). e Cell cycle analysis by FACS of untreated and treated cells bars indicate “mean ± SEM”; P < 0.05 with 4 nM docetaxel, as indicated in the panel, for 24 h cells followed function, β-Catenin (CTNNB1), IGFBP5, MMP3, AKR1C3, PC3-sh1 cells showed less bone loss than PC3-Vec cells. CXCL5 [33], and KISS1R [34, 35], were signiﬁcantly down- Both trabecular and cortical bones were decreased in regulated in DU-sh cells compared to control DU-Nsh cells; xenografts with NO66-overexpressing cells (Fig. 5b, c, right whereas, genes with anti-proliferative activity, IL32 [36], panel). X-ray data clearly indicated that bone loss was ESM1 [37, 38], and RGS2 [39, 40], were remarkably apparently greater in the xenograft with PC3-NO66 than upregulated in DU-sh cells. In fact, both β-catenin mRNA with control PC3-Vec cells (Fig. 5d). Bone volume and and protein levels were decreased in NO66-depleted (DU- bone mineral density (BMD) were decreased in NO66- sh1/2) cells compared with control DU-Nsh cells (Fig. 4b, overexpressing cells compared to either control or shRNA- d). Taken together, our transcriptome analysis shows that expressing (sh1) cells (Fig. 5e, f). We reasoned that the NO66 regulates expression of genes associated with pro- increased bone loss in femurs with NO66-overexpressing liferation, survival, and invasion in PCa. xenografts was most likely due to increased osteolytic activity. Fig. 6a shows that expression levels of Wnt inhi- NO66 protein levels correlate with the severity of bitor (Dkk1, Dickkopf1), osteoclast inducers (CtsK, Cathe- osteolytic bone lesions spin K), Nf-kβ; and Calcr (calcitonin receptor) were indeed more stimulated in the femur with PC3-NO66 xenografts. To investigate the role of NO66 in the development of focal Though Nfatc levels were higher in xenograft with control skeletal lesions by PCa in the bone, we injected PC3-NO66, and PC3-NO66 cells than in the femurs with no cells PC3-sh1, and PC3-Vec cells into the distal end of the right injected. We then asked whether NO66 promotes the levels femur of SCID mice. The left femur served as a control of osteolytic factors. Co-culture of murine primary osteo- (with no injection of cells). The microCT analyses indicated blasts with control PC3-Vec and PC3-NO66 cells clearly that injection of PC3-NO66 cells caused more massive showed that the expression levels of Dkk1, Nf-kβ, and Sost femoral bone loss than did no injection (control) or femur genes, which are inducers of osteoclasts, were strongly injected with PC3-Vec cells (Fig. 5a), however, injection of upregulated in primary osteoblasts (Fig. 6b). Furthermore, Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer

Fig. 3 Stably-expressed NO66 in PC3 cells increases their cell proliferation, invasive ability and levels of invasion markers. a Western blot analysis for the levels of NO66 in PC3 cells expressing FlagNO66 with a Flag antibody and levels of MMP2, and RUNX2 with their respective antibodies. Vinculin serves as loading control. b MTT assay in PC3-Vec and PC3-NO66 cells. c Representative image of invaded cells through matrigel barrier. d Quantiﬁcation of the invaded cells (n = 5) for (c). Error bars indicate “mean ± SEM”; P < 0.001

levels of bone forming factor, osteoprotegerin (OPG), were binding of NO66 at gene promoters, co-localized with increased; whereas, levels of osteoclast factor, RANKL, H3K9AC, has a pivotal role in the activation of these genes were decreased in PC3-sh1 cells (Fig. 6c), which supports through histone acetylation and chromatin remodeling the reduced bone loss by PC-sh1 cells (Fig. 5a). These activity. ﬁndings suggest that high levels of NO66 promote the osteolytic lesions in PCa by promoting the osteoclast NO66-target genes play key roles in androgen- activity. independent prostate cancer

Genome wide distribution of the NO66 interactions Our analysis revealed that most of the genes bound with in PC3 cells by ChIP sequencing NO66 and H3K9AC play key roles in the progression and metastasis of PCa, independently of AR signaling. Fig. 7c To gain insights into the molecular mechanism of NO66 in and Suppl. Figure 2 show a snapshot of few genes including gene activation during AI PCa, we performed chromatin miRNAs and long non-coding RNAs with co-localization of immunoprecipitation sequencing (ChIPseq) using NO66- NO66 and H3K9AC at the promoter regions. Expression and H3K9AC-speciﬁc antibodies in PC3-NO66 cells. These levels of these genes were further validated in PC3-NO66 experiments allowed us to identify the NO66-bound genes and PC3-Vec cells (Fig. 7d). The mRNA levels of and their activation in AI-PC3 cells. Bioinformatics analysis CTNNB1, RICTOR, CCND1, DVL (Disheveled), FOXA1, with the peak-calling program MACS was used to identify and the epigenetic modiﬁer MLL1 (KMT2A) were more the NO66 peaks in the genomic loci (criteria- cutoff with p- induced in PC3-NO66 cells than control (PC3-Vec) cells. value < 1.00E-07 and FDR <0.1). We obtained a total of However, expression of a dominant negative histone 897 unique peaks corresponding to the NO66 binding demethylase mutant NO66 in PC3-NO66(AKA) cells also throughout the genome and most of them (51%) lies at the stimulated the transcription of these NO66-target genes, promoter regions and rest of them lies within upstream, which indicates that NO66 regulates transcription of these intronic, exon, and enhancer regions (Fig. 7a). A list of genes independently of its histone demethylase activity NO66 peaks distribution is provided in Suppl. Section (Fig. 7d). Protein levels of MLL1, EZH2, and β-Catenin (NO66 Peak Distribution). Interestingly, 88% of the NO66 were highly induced in PC3-NO66 cells, while that of peaks overlapped with the peaks of H3K9AC (an active CyclinD1 was only partially induced in NO66-expressing histone marker) mostly in the promoter region, as shown PC3 cells (Suppl. Figure 3A). Studies have shown that with a heat map (Fig. 7b). Our analysis suggests that KMT2A, FOXA1, and RICTOR are key inducers in the K. M. Sinha et al.

Table 1 List of differentially Gene symbol DU-Nsh DU-sh log2 ratio p value FDR expressed genes in DU145-Nsh and DU145-sh clones IGFBP5 73.13156 13.15432 −2.474957183 5.91E-05 0.030506 (combined data from Sh1, sh2 − and sh3 cells) from RNAseq TNFSF10 109.6973 28.18546 1.960505661 5.53E-05 0.030506 analysis AKR1C1 858.1532 271.0526 −1.662662217 2.98E-11 1.11E-07 SUSD2 3946.819 1388.889 −1.506758526 8.64E-13 6.46E-09 AKR1C2 385.0834 137.4634 −1.486123077 8.16E-07 0.001356 CEMIP 1888.851 675.3351 −1.483833545 2.46E-11 1.11E-07 C14orf169 1426.065 511.4414 −1.479399279 8.45E-11 2.53E-07 S100A9 831.8715 301.6834 −1.463325584 4.01E-09 1.00E-05 AKR1B10 482.2112 190.5686 −1.339354685 1.75E-06 0.002181 MTRNR2L9 2059.111 931.7874 −1.143948587 1.83E-07 0.000392 NAPSA 812.4459 382.7838 −1.085742018 1.14E-05 0.007397 AKR1C3 1070.692 507.9355 −1.075825994 6.53E-06 0.004882 MTRNR2L8 1463.774 701.2553 −1.061681094 2.66E-06 0.002655 BCAM 2492.186 1234.53 −1.013450101 3.03E-06 0.002706 MTRNR2L2 6522.421 3323.872 −0.972542613 2.47E-06 0.002644 CELSR1 1570.043 817.0929 −0.942232207 2.35E-05 0.014658 AQP3 3826.837 2127.476 −0.847009447 5.66E-05 0.030506 SIK1 3314.917 1859.458 −0.83409034 9.66E-05 0.045189 Upregulated EREG 3562.878 6707.531 0.912738544 7.45E-05 0.035959 CLDN1 628.4744 1283.88 1.030584409 4.84E-05 0.028938 DUSP6 418.2211 872.8554 1.061476812 6.91E-05 0.034484 ITGA2 2454.478 5233.681 1.092409697 3.07E-06 0.002706 CDK6 938.1408 2027.434 1.111778422 6.46E-06 0.004882 NAV3 861.5812 1862.239 1.111979413 7.03E-06 0.005011 SFTA1P 1036.411 2301.623 1.151054707 2.42E-06 0.002644 DUSP5 1172.39 2694.776 1.200712425 8.07E-07 0.001356 RGS2 366.8005 845.6678 1.205095449 8.15E-06 0.005544 CXCL8 754.1692 1743.266 1.208832675 1.39E-06 0.001886 CEACAM6 197.6838 536.7808 1.441138748 1.34E-06 0.001886 HMGA2 122.2668 352.8471 1.529010117 3.76E-06 0.003125 MMP1 322.2359 2239.132 2.79675011 7.38E-24 1.10E-19 ESM1 6.856084 59.59615 3.119762468 5.74E-05 0.030506 List of down- and upregulated genes with log2 ratio, p value and false discovery rate (FDR, <0.05). Downregulation of human NO66 gene in DU-sh clones is indicated by underlined (C14orf169) progression of mCRPC [9, 41–44], while CTNNB1 pro- for the KEGG PCa pathway obtained from the IGV portal, motes PCa proliferation and progression through activation 23 genes from our data set are included in the KEGG of Wnt signaling [45–47]. The occupancy of NO66 at the prostate pathway (Suppl. Table 1). We show the peaks for close proximity of DDIT3 (ER stress gene), MCL1 (survival NO66 and H3K9AC for the corresponding genes which are gene), and CTNNB1 genes was conﬁrmed by manual ChIP present in the PCa pathway (Suppl. Figure 2). Based on PCR in NO66-expressing cells (Fig. 7e). ChIPseq analysis Ingenuity Pathway Analysis (IPA) and DAVID GO, ana- also revealed the NO66 binding peaks in other genes and lysis of the NO66-target genes revealed the network of miRNAs which are known for promoting PCa progression these genes converges to support cancer progression, as and metastasis (Suppl. Figure 2). These genes are EZH2, well as signaling pathways including Wnt/βcatenin, Notch, TGIF1, ANAX2R, MIR21, MIR23A, and MIR100HG in and TGF-β (Suppl. Table 2). We also tested the levels of which interactions of NO66 and H3K9AC are co-localized NO66 in androgen-sensitive C42B cells treated with in their promoters and have an oncogenic role during androgen analog (R1881). At the lower dose of R1881 (1 malignancies [6, 48–55]. Further, out of the 89 genes listed nM), an induction in NO66 and AR levels was observed, Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer

Fig. 4 Validation of RNAseq analysis in DU145 cells. a An IGV showing the mRNA levels of differentially expressed genes. Error bars snapshot of transcriptome analysis for IGFBP5 and AKR1C3 genes indicate “mean ± SEM”; P < 0.05. d Western blot showing the levels between DU-Nsh and DU-sh clones. b, c Quantitative RT-PCR of β-catenin in these cells as indicated above the panel

Fig. 5 NO66 induces bone lysis in xenografts. a Representative microCT of control left femur (no cell injection) and right femur of SCID mice injected with PC3-Vec, PC3-FlagNO66, or PC3-sh1 cells (n = 5, 0.5 million cells/femur) at 21 days after cell injection. b MicroCT showing the trabecular bone. c MicroCT showing cortical bone. d X-ray image taken before microCT. a, d Arrows and bracket indicate lesions. e Bone volume and f bone mineral density (BMD) in femurs with no cells control (−) and injected PC3 cells (−Vec, −NO66, and −sh1). Error bars indicate “mean ± SEM”; P < 0.05 from 5 animal per group

but the higher concentration of R1881 had an inhibitory Discussion effect (Suppl. Figure 3B). Indeed, R1881 stimulated the mRNA levels of NO66 in C42B cells (Suppl. Figure 3C), In this study, we present several lines of evidence that suggesting a role of NO66 in AR-dependent progression of NO66 promotes PCa cell proliferation and migration, and PCa. Consistent with other reports on the role of NO66 in skeletal osteolytic lesions through activation of expression colorectal and lung cancers, our ﬁndings support the of genes associated with cell survival, invasiveness, and oncogenic role of NO66 in PCa and induction of skeletal osteoclast activation. We also show that NO66 is upregu- lesions through activation of the Wnt/β-catenin pathway and lated in high-grade tumors compared to low-grade tumors epigenetic modiﬁers, including MLL1. and normal tissues. K. M. Sinha et al.

Fig. 6 NO66 promotes bone loss through activation of osteolytic factors secreted from PCa cells. a Gene expression levels of mouse genes in femoral bone injected with no cells, PC3-Vec (control) cells, and PC3- FlagNO66 cells. b Gene expression levels of mouse genes in primary murine osteoblasts co-cultured with PC3-Vec and PC3-NO66 cells. Representative data of three independent experiments is shown. Error bars indicate “mean ± SEM”; P < 0.05. c Western blot analysis for levels of OPG and RANKL in lysates of PC3-Nsh and PC3-sh

Fig. 7 Bioinformatics analysis for the genome wide distribution of the defective histone demethylase activity by substitution of catalytic NO66 interactions. a Percentages of the interaction sites in the gen- histidine to alanine [20]. Error bars indicate “mean ± SEM”; P < 0.05. ome. b Representation via heat map for interaction sites after ChIPseq e ChIP analysis for the occupancy of NO66 in the chromatin of PC3- for NO66 and H3K9AC using respective antibodies. c An IGV Vec and PC3-NO66 cells shows the NO66 interaction in the promoter snapshot of the peaks for NO66 and H3K9AC in target genes. d region (Prox) and coding region (Int) of the genes DDIT3, MCL1, and Quantitative RT-PCR for gene expression in PC3-Vec, PC3- CTNNB1 FlagNO66, and PC3-NO66 (AKA) cells. NO66 (AKA) is a mutant for Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer

Expression of NO66 has recently been reported in small PTHLH, that regulate chemo-attraction, interaction with cell lung cancer, colorectal cancer, and renal carcinoma [25, stroma cells, migration, and osteoclast formation. 26, 56]; however, the mechanisms of NO66 function in Taken together, our findings indicate that NO66 is an these cancers and its role in PCa are unknown. A positive epigenetic regulator of AR-independent PCa and osteolytic correlation between NO66 levels and tumors with high lesions, through controlling the expression of a cohort of Gleason scores suggests a clinical significance for NO66 in downstream target genes, including oncogenes and PCa progression. Aberrant expression of histone demethy- cytokine-expressing genes that are associated with PCa lases, including KDM1A, KDM5/6A, and JMJD2A has a progression and metastasis. Since we and others have pro-oncogenic role through deregulation of gene expression reported on the crystal structure of NO66 [62, 63], devel- in various cancers [4, 5, 57, 58]. Our findings, including opment of small molecule inhibitors targeting NO66 may be RNAseq-based transcriptome and ChIPseq analyses, sup- an effective therapeutic approach for treating PCa and other port a pro-oncogenic and anti-apoptotic role for NO66 in malignancies. Furthermore, NO66 levels in prostatic tumors PCa. Knockdown of NO66 decreased cell proliferation, may be a useful molecular marker to predict disease stage most likely due to inhibition of expression levels of pro- and response of patients to chemotherapeutics. oncogenic genes, including IGFBP5, MMP3, CXCL5, and β-catenin; and activation of pro-apoptotic genes, including BBC3, CASP2, and PUMA. It also appears that NO66 Materials and methods regulates expression of genes for androgen synthesis, since levels of AKR1C1, AKR1C2, and AKR1C3 were decreased Cell culture, reagents, and antibodies in NO66-depleted DU145 cells. These genes are involved in androgen synthesis and promote drug resistance in CRPC PCa cell lines PC3, DU145, and C42B (ATCC) were rou- [59]. Furthermore, depletion of NO66 resulted with more tinely cultured in RPMI 1640 media supplemented with sensitivity to docetaxel-mediated cell toxicity, suggesting 10% FBS and 1% penicillin and streptomycin, and peri- that NO66 may stimulate the expression of drug resistance odically tested for mycoplasma contamination. NO66—a genes. Indeed, interactions of NO66 with the promoter of peptide polyclonal antibody was developed previously and the gene RICTOR and strong stimulation of expression of reported by Sinha et al. [20]. Primer sequence and anti- this gene in NO66-overexpressing PC3 cells corroborate a bodies are described in Suppl. Section. positive role for NO66 in drug resistance. Notably, RICTOR promotes cytotoxic drug resistance through the mTOR Generation of cell lines for NO66-shRNA and NO66- pathway in many cancers [60]. Moreover, NO66 interac- overexpression tions with MLL1 (KMT2A) and EZH2, as well as some miRNAs including miR21, miRNA23, and lncRNA The FlagNO66 cDNA was cloned into a pLenti-Vector miR100HG, which are involved in the progression and (Origene, Rockville, MD, USA) and lentiviral particles metastasis of PCa, suggest that NO66 acts as an upstream were produced in HEK293T cells using packaging kit regulator of these epigenetic modifiers, which are associated (TR30037, Origene, Rockville, MD, USA). The virus par- with the progression of AR-independent PCa through gene ticles containing NO66-shRNA (three different shRNAs), activation. Taken together, our data suggest that the mole- control (scrambled) shRNA, pLenti-vector, and pLenti- cular mechanisms of the NO66 oncogenic function are via FlagNO66 were transduced in PC3 or DU145 cells, and its interactions with chromatin and activation of genes for transduced cells were selected with media containing 2 μg/ proliferation, migration, and transcriptional regulation. mL puromycin (Sigma, St Louis, MO, USA) for 1 week. The pathological and clinical features of skeletal metastases from PCa include osteoblastic (bone forming) and Cell proliferation, migration and invasion, and osteoclastic (bone lysing), or mixed, lesions [61]. In animal anchorage-independent growth assays and in vitro co-culture studies, we demonstrated that NO66 expression promoted massive osteolytic lesions and Cell survival/proliferation was studied with an MTT assay increased expression levels of osteoclastic genes (DKK1, in a 96-well plate. Briefly, cells were cultured for the Ctsk, and Nf-kβ), thereby supporting a role for NO66 in the indicated time, incubated with 200 μl of 0.6 mg/ml MTT in secretion of pro-osteoclastic factors which cause osteolytic serum-free medium for 4 h, and then further incubated in lesions in AR-independent PCa. The secretion of these dimethyl sulfoxide for 4 h, followed by a colorimetric factors by PCa cells allows their invasion, migration, and reading at 595 nm with a microplate reader (Biotek,VT, interactions with stromal cells in bones. Consistent with this USA). The invasion assay was performed in Matrigel- notion, ChIPseq analysis identified several NO66-target coated transwell invasion chambers with an 8.0-μm pore genes, including CXCL1, CXCL2, ANAXA2R, RUNX2, and size (BD Biosciences, San Jose, CA). For this assay, 1 × 105 K. M. Sinha et al. cells were added into the top chamber containing 250 μlof procedure using immunoperoxidase procedure (ABC-Elite; serum-free DMEM. Another 750 μL of NIH3T3- Vector Laboratories, Burlingame, CA). The scoring of conditioned media was added into the bottom chamber, NO66 staining in the tumor sections was performed by a and the cells were allowed to invade for 24 h. The cells that pathologist based on nuclear and cytoplasmic localization of invaded through the filter into the bottom chamber were NO66. We quantitated NO66 protein expression using a stained with a three-step process and counted. Each three-tiered scoring system as published previously [65]as experiment was performed in triplicate, and mean values are follows: no detectable staining in more than 70% of tumor presented. For clonogenic assays, cells were plated on 35- cell nuclei was scored as negative, 30% or more tumor cell mm plates, stained with 0.2% crystal violet, and counted nuclei weakly stained (discernable nucleoli) as weak, and under a bright-field light microscope. Cell cycle distribution more than 30% of nuclei strongly stained (invisible was determined by a standard procedure of propidium nucleoli) as strong. Normal and tumor protein blot, and iodide stained cells followed by FACS analysis. Anchorage- cDNA arrays were purchased from Origene (Rockville, independent colony formation assay was performed as MD, USA) (Cat# CSRT101) and were used for detection of described previously [64]. NO66 protein and mRNA levels respectively.

RNASeq, ChIP-Seq, and data analysis Xenograft studies and bone analysis

For RNA sequencing, total RNA was isolated from DU145 Stable cell lines, and PC3-Vec, PC3-NO66sh1, and PC3- cell lines, control shRNA (DU-Nsh), and three different NO66 cells (5 × 105/5 μl/femur) were injected into the distal clones of NO66-shRNA-expressing cells (DU-sh1, DU-sh2, femur of 6-week-old male SCID mice (Charles River, MA, and DU-sh3). For ChIP-Seq experiments, chromatins were USA) (n = 5 mice each group). At 3 weeks post-injection, prepared from PC3-NO66 cells and immunoprecipitation micro CT and X-ray were performed on anesthetized ani- was done with NO66 and H3K9AC (C15410004, Diag- mals and then mice were sacrificed to remove femurs for enode, Denville, NJ, USA) antibodies as described pre- isolation of total RNA and gene expression analysis. Ani- viously by Sinha et al. [20]. Library preparation, adaptor mal studies were performed under approved animal protocol ligation, and sequencing of RNA and DNA samples were by Institutional Animal Care and Utilization Committee of done by the core facility at the University of Texas MD MDACC. Anderson Cancer Center (UTMDACC) Sequencing and Microarray Core Facility. The raw data were analyzed by Statistical analysis bioinformaticians at UTMDACC using appropriate soft- ware programs. Bar graphs and statistical analysis were performed using GraphPad program. One way ANOVA for multiple com- Cell lysates, western blot, and quantitative gene parison were used for p values and Student's t-test was expression performed for Boyden chamber assay. A p value of <0.05 was considered statistically significant, and statistical sig- Cell extracts and Western blot were performed as described nificance was indicated as *p < 0.05, **p < 0.01, and ***p previously [20]. For gene expression analysis, total RNA < 0.001. Clonogenic, boyden chamber assay, MTT, and was extracted with a Zymo DNAse-free RNA isolation kit FACS experiments were repeated at least three times. All (Zymo, CA, USA) and cDNA was prepared with a ViLO kit data were expressed as means ± SEM of representative (Invitrogen, Waltham, MA, USA). Gene-specific primers experiments. were used for quantitative PCR on a StepOne Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Acknowledgements We thank Dr. Mary A. Hall for editorial assis- Details of primers are included in the Supplemental Section. tance, Ling Zhong for microscopy, and Jun Yang for technical assistance. Gene expression values were normalized to 18S rRNA. Funding This work was supported by National Institutes of Health Immunohistochemistry with PCa patient tissue Grant P50 CA140388 (NCI-SPORE- Developmental Research Pro- microarray gram) to CL and KMS, Center for Epigenetics at MDACC allowance for Next-Gen sequencing to KMS, the Bone Diseases Program of Texas to RFG, and by UTHealth startup funding to JH. PCa tissue microarrays obtained from US Biomax (Cat # HProA100PG02, Rockville, MD, USA) contained 90 Author’s contribution KMS and RB-Y designed and performed patient samples with low to high Gleason grades, including experiments, analyzed data and wrote manuscript. YL performed normal prostate tissue samples. Immunohistochemsitry with bioinformatics analysis, MZ conducted histological analysis; SA per- NO66 antibody (1:200 dilution) was done with a standard formed histology; SL and YR provided technical assistance for Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer experiments; NN and XW for cell lines and animal studies; BO, CL, 16. Zhou HJ, Yan J, Luo W, Ayala G, Lin SH, Erdem H, et al. SRC-3 RFG, and JH for administrative and material support; KMS, RB-Y, is required for prostate cancer cell proliferation and survival. BO, and JH discussed and reviewed the manuscript. Cancer Res. 2005;65:7976–83. 17. Verras M, Sun Z. Roles and regulation of Wnt signaling and beta- – Compliance with ethical standards catenin in prostate cancer. Cancer Lett. 2006;237:22 32. 18. Feeley BT, Krenek L, Liu N, Hsu WK, Gamradt SC, Schwarz EM, et al. Overexpression of noggin inhibits BMP-mediated growth of fl fl Con ict of interest The authors declare that they have no con ict of osteolytic prostate cancer lesions. Bone. 2006;38:154–66. interest. 19. Feeley BT, Gamradt SC, Hsu WK, Liu N, Krenek L, Robbins P, et al. Influence of BMPs on the formation of osteoblastic lesions Publisher’s note: Springer Nature remains neutral with regard to in metastatic prostate cancer. J Bone Miner Res. 2005;20:2189– jurisdictional claims in published maps and institutional affiliations. 99. 20. Sinha KM, Yasuda H, Coombes MM, Dent SY, de Crombrugghe References B. Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase. EMBO J. 2010;29:68–79. 1. Logothetis CJ, Lin SH. Osteoblasts in prostate cancer metastasis 21. Sinha KM, Zhou X. Genetic and molecular control of osterix in – to bone. Nat Rev Cancer. 2005;5:21 8. skeletal formation. J Cell Biochem. 2013;114:975–84. 2. Logothetis CJ, Navone NM, Lin SH. Understanding the biology 22. Sinha KM, Yasuda H, Zhou X, deCrombrugghe B. Osterix and of bone metastases: key to the effective treatment of prostate NO66 histone demethylase control the chromatin of Osterix target – cancer. Clin Cancer Res. 2008;14:1599 602. genes during osteoblast differentiation. J Bone Miner Res. 3. Akech J, Wixted JJ, Bedard K, van der Deen M, Hussain S, Guise 2014;29:855–65. TA, et al. Runx2 association with progression of prostate cancer in 23. Ge W, Wolf A, Feng T, Ho CH, Sekirnik R, Zayer A, et al. patients: mechanisms mediating bone osteolysis and osteoblastic Oxygenase-catalyzed ribosome hydroxylation occurs in prokar- – metastatic lesions. Oncogene. 2010;29:811 21. yotes and humans. Nat Chem Biol. 2012;8:960–2. 4. Crea F, Sun L, Mai A, Chiang YT, Farrar WL, Danesi R, et al. 24. Brien GL, Gambero G, O’Connell DJ, Jerman E, Turner SA, Egan The emerging role of histone lysine demethylases in prostate CM, et al. Polycomb PHF19 binds H3K36me3 and recruits PRC2 cancer. Mol Cancer. 2012;11:52. and demethylase NO66 to embryonic stem cell genes during 5. Ellinger J, Kahl P, von der Gathen J, Rogenhofer S, Heukamp LC, differentiation. Nat Struct Mol Biol. 2012;19:1273–81. fi Gutgemann I, et al. Global levels of histone modi cations predict 25. Pires-Luis AS, Vieira-Coimbra M, Vieira FQ, Costa-Pinheiro P, – prostate cancer recurrence. Prostate. 2010;70:61 9. Silva-Santos R, Dias PC, et al. Expression of histone methyl- 6. Xu K, Wu ZJ, Groner AC, He HH, Cai C, Lis RT, et al. EZH2 transferases as novel biomarkers for renal cell tumor diagnosis and oncogenic activity in castration-resistant prostate cancer cells is prognostication. Epigenetics. 2015;10:1033–43. – Polycomb-independent. Science. 2012;338:1465 9. 26. Nishizawa Y, Nishida N, Konno M, Kawamoto K, Asai A, Koseki 7. Yang YA, Yu J. EZH2, an epigenetic driver of prostate cancer. J, et al. Clinical significance of histone demethylase NO66 in – Protein & Cell. 2013;4:331 41. invasive colorectal cancer. Ann Surg Oncol. 2017;24:841–9. 8. Yu J, Yu J, Rhodes DR, Tomlins SA, Cao X, Chen G, et al. A 27. Ge C, Zhao G, Li Y, Li H, Zhao X, Pannone G, et al. Role of polycomb repression signature in metastatic prostate cancer pre- Runx2 phosphorylation in prostate cancer and association with – dicts cancer outcome. Cancer Res. 2007;67:10657 63. metastatic disease. Oncogene. 2016;35:366–76. 9. Malik R, Khan AP, Asangani IA, Cieslik M, Prensner JR, Wang 28. Yepuru M, Wu Z, Kulkarni A, Yin F, Barrett CM, Kim J, et al. X, et al. Targeting the MLL complex in castration-resistant Steroidogenic enzyme AKR1C3 is a novel androgen receptor- – prostate cancer. Nat Med. 2015;21:344 52. selective coactivator that promotes prostate cancer growth. Clin 10. Saha B, Kaur P, Tsao-Wei D, Naritoku WY, Groshen S, Datar Cancer Res. 2013;19:5613–25. RH, et al. Unmethylated E-cadherin gene expression is sig- 29. Adeniji AO, Chen M, Penning TM. AKR1C3 as a target in cas- fi ni cantly associated with metastatic human prostate cancer cells trate resistant prostate cancer. J Steroid Biochem Mol Biol. – in bone. Prostate. 2008;68:1681 8. 2013;137:136–49. 11. Tamada H, Kitazawa R, Gohji K, Kitazawa S. Epigenetic reg- 30. Zeng CM, Chang LL, Ying MD, Cao J, He QJ, Zhu H, et al. Aldo- ulation of human bone morphogenetic protein 6 gene expression keto reductase AKR1C1-AKR1C4: functions, regulation, and – in prostate cancer. J Bone Miner Res. 2001;16:487 96. intervention for anti-cancer therapy. Front Pharmacol. 2017;8:119. 12. Li ZG, Mathew P, Yang J, Starbuck MW, Zurita AJ, Liu J, et al. 31. Liang G, Fang X, Yang Y, Song Y. Silencing of CEMIP sup- Androgen receptor-negative human prostate cancer cells induce presses Wnt/beta-catenin/Snail signaling transduction and inhibits osteogenesis in mice through FGF9-mediated mechanisms. J Clin EMT program of colorectal cancer cells. Acta Histochem. – Invest. 2008;118:2697 710. 2018;120:56–63. 13. Wan X, Liu J, Lu JF, Tzelepi V, Yang J, Starbuck MW, et al. 32. Liang G, Fang X, Yang Y, Song Y. Knockdown of CEMIP Activation of beta-catenin signaling in androgen receptor-negative suppresses proliferation and induces apoptosis in colorectal cancer – prostate cancer cells. Clin Cancer Res. 2012;18:726 36. cells: downregulation of GRP78 and attenuation of unfolded 14. Lee YC, Cheng CJ, Bilen MA, Lu JF, Satcher RL, Yu-Lee LY, protein response. Biochem Cell Biol. 2017;96:332–41. et al. BMP4 promotes prostate tumor growth in bone through 33. Begley LA, Kasina S, Mehra R, Adsule S, Admon AJ, Lonigro – osteogenesis. Cancer Res. 2011;71:5194 203. RJ, et al. CXCL5 promotes prostate cancer progression. Neoplasia 15. Liu XH, Kirschenbaum A, Yao S, Liu G, Aaronson SA, Levine . 2008;10:244–54. AC. Androgen-induced Wnt signaling in preosteoblasts promotes 34. Ji K, Ye L, Mason MD, Jiang WG. The Kiss-1/Kiss-1R complex the growth of MDA-PCa-2b human prostate cancer cells. Cancer as a negative regulator of cell motility and cancer metastasis – Res. 2007;67:5747 53. (Review). Int J Mol Med. 2013;32:747–54. K. M. Sinha et al.

35. Wang H, Jones J, Turner T, He QP, Hardy S, Grizzle WE, et al. 52. Yang B, Liu Z, Ning H, Zhang K, Pan D, Ding K, et al. Clinical and biological significance of KISS1 expression in MicroRNA-21 in peripheral blood mononuclear cells as a novel prostate cancer. Am J Pathol. 2012;180:1170–8. biomarker in the diagnosis and prognosis of prostate cancer. 36. Park MH, Song MJ, Cho MC, Moon DC, Yoon DY, Han SB, Cancer Biomark. 2016;17:223–30. et al. Interleukin-32 enhances cytotoxic effect of natural killer 53. Sheth S, Jajoo S, Kaur T, Mukherjea D, Sheehan K, Rybak LP, cells to cancer cells via activation of death receptor 3. Immunol- et al. Resveratrol reduces prostate cancer growth and metastasis ogy. 2012;135:63–72. by inhibiting the Akt/MicroRNA-21 pathway. PLoS ONE. 37. Rebollo J, Geliebter J, Reyes N. ESM-1 siRNA knockdown 2012;7:e51655. decreased migration and expression of CXCL3 in prostate cancer 54. Wang S, Ke H, Zhang H, Ma Y, Ao L, Zou L, et al. LncRNA cells. Int J Biomed Sci. 2017;13:35–42. MIR100HG promotes cell proliferation in triple-negative breast 38. Bettin A, Reyes I, Reyes N. Gene expression profiling of prostate cancer through triplex formation with p27 loci. Cell Death Dis. cancer-associated genes identifies fibromodulin as potential novel 2018;9:805. biomarker for prostate cancer. Int J Biol Markers. 2016;31:e153–62. 55. Shang C, Zhu W, Liu T, Wang W, Huang G, Huang J, et al. 39. Cacan E. Epigenetic regulation of RGS2 (Regulator of G-protein Characterization of long non-coding RNA expression profiles in signaling 2) in chemoresistant ovarian cancer cells. J Chemother. lymph node metastasis of early-stage cervical cancer. Oncol Rep. 2017;29:173–8. 2016;35:3185–97. 40. Wolff DW, Xie Y, Deng C, Gatalica Z, Yang M, Wang B, et al. 56. Suzuki C, Takahashi K, Hayama S, Ishikawa N, Kato T, Ito T, Epigenetic repression of regulator of G-protein signaling 2 pro- et al. Identification of Myc-associated protein with JmjC domain motes androgen-independent prostate cancer cell growth. Int J as a novel therapeutic target oncogene for lung cancer. Mol Cancer. 2012;130:1521–31. Cancer Ther. 2007;6:542–51. 41. Chowdry RP, Ledet E, Ranasinghe L, Sartor AO. MLL translo- 57. Vieira FQ, Costa-Pinheiro P, Ramalho-Carvalho J, Pereira A, cation in two castration-resistant prostate cancer patients. Can J Menezes FD, Antunes L, et al. Deregulated expression of selected Urol. 2016;23:8483–6. histone methylases and demethylases in prostate carcinoma. 42. Jin HJ, Zhao JC, Ogden I, Bergan RC, Yu J. Androgen receptor- Endocr Relat Cancer. 2014;21:51–61. independent function of FoxA1 in prostate cancer metastasis. 58. Kim TD, Jin F, Shin S, Oh S, Lightfoot SA, Grande JP, et al. Cancer Res. 2013;73:3725–36. Histone demethylase JMJD2A drives prostate tumorigenesis through 43. Bragina O, Njunkova N, Sergejeva S, Jarvekulg L, Kogerman P. transcription factor ETV1. J Clin Invest. 2016;126:706–20. Sonic Hedgehog pathway activity in prostate cancer. Oncol Lett. 59. Stanbrough M, Bubley GJ, Ross K, Golub TR, Rubin MA, Pen- 2010;1:319–25. ning TM, et al. Increased expression of genes converting adrenal 44. Chen X, Cheng H, Pan T, Liu Y, Su Y, Ren C, et al. mTOR androgens to testosterone in androgen-independent prostate can- regulate EMT through RhoA and Rac1 pathway in prostate cancer. Cancer Res. 2006;66:2815–25. cer. Mol Carcinog. 2015;54:1086–95. 60. Morrison Joly M, Hicks DJ, Jones B, Sanchez V, Estrada MV, 45. Nandana S, Tripathi M, Duan P, Chu CY, Mishra R, Liu C, et al. Young C, et al. Rictor/mTORC2 drives progression and ther- Bone metastasis of prostate cancer can be therapeutically targeted apeutic resistance of HER2-Amplified breast cancers. Cancer Res. at the TBX2-WNT signaling axis. Cancer Res. 2017;77:1331–44. 2016;76:4752–64. 46. Sottnik JL, Hall CL, Zhang J, Keller ET. Wnt and Wnt inhibitors 61. Ortiz A, Lin SH. Osteolytic and osteoblastic bone metastases: two in bone metastasis. Bone Rep. 2012;1:101. extremes of the same spectrum? Recent Results Cancer Res 47. Chen G, Shukeir N, Potti A, Sircar K, Aprikian A, Goltzman D, Fortschr der Krebsforsch Progres dans Les Rech sur Le Cancer. et al. Upregulation of Wnt-1 and beta-catenin production in patients 2012;192:225–33. with advanced metastatic prostate carcinoma: potential pathogenetic 62. Tao Y, Wu M, Zhou X, Yin W, Hu B, de Crombrugghe B, et al. and prognostic implications. Cancer. 2004;101:1345–56. Structural insights into histone demethylase NO66 in interaction 48. Xiang G, Yi Y, Weiwei H, Weiming W. TGIF1 promoted the with osteoblast-specific transcription factor osterix and gene growth and migration of cancer cells in nonsmall cell lung cancer. repression. J Biol Chem. 2013;288:16430–7. Tumour Biol. 2015;36:9303–10. 63. Chowdhury R, Sekirnik R, Brissett NC, Krojer T, Ho CH, Ng SS, 49. D’Souza S, Kurihara N, Shiozawa Y, Joseph J, Taichman R, et al. Ribosomal oxygenases are structurally conserved from Galson DL, et al. Annexin II interactions with the annexin II prokaryotes to humans. Nature. 2014;510:422–6. receptor enhance multiple myeloma cell adhesion and growth in 64. Bagheri-Yarmand R, Mandal M, Taludker AH, Wang RA, the bone marrow microenvironment. Blood. 2012;119:1888–96. Vadlamudi RK, Kung HJ, et al. Etk/Bmx tyrosine kinase activates 50.CaiS,ChenR,LiX,CaiY,YeZ,LiS,etal.Downregulationof Pak1 and regulates tumorigenicity of breast cancer cells. J Biol microRNA-23a suppresses prostate cancer metastasis by targeting Chem. 2001;276:29403–9. the PAK6-LIMK1 signaling pathway. Oncotarget. 2015;6:3904–17. 65. Theurillat JP, Udeshi ND, Errington WJ, Svinkina T, Baca SC, 51. Wang Z, Wei W, Sarkar FH. miR-23a, a critical regulator of Pop M, et al. Prostate cancer. Ubiquitylome analysis identifies “migR“ation and metastasis in colorectal cancer. Cancer Discov. dysregulation of effector substrates in SPOP-mutant prostate 2012;2:489–91. cancer. Science. 2014;346:85–9.