Epidermal growth factor receptor signaling promotes metastatic prostate cancer through microRNA-96-mediated downregulation of the tumor suppressor ETV6

Cancer Letters 384 (2017) 1e8

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Article

Epidermal growth factor receptor signaling promotes metastatic prostate cancer through microRNA-96-mediated downregulation of the tumor suppressor ETV6 Yuan-Chin Tsai a, Wei-Yu Chen b, c, Man Kit Siu a, d, Hong-Yuan Tsai a, Juan Juan Yin e, Jiaoti Huang f, Yen-Nien Liu a, * a

Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Department of Pathology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan d Department of Anesthesiology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan e Laboratory of Genitourinary Cancer Pathogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA f Department of Pathology, Duke University Medical Center, Durham, NC, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 August 2016 Received in revised form 4 October 2016 Accepted 4 October 2016

It has been suggested that ETV6 serves as a tumor suppressor; however, its molecular regulation and cellular functions remain unclear. We used prostate cancer as a model system and demonstrated a molecular mechanism in which ETV6 can be regulated by epidermal growth factor receptor (EGFR) signaling through microRNA-96 (miR-96)-mediated downregulation. In addition, EGFR acts as a transcriptional coactivator that binds to the promoter of primary miR-96 and transcriptionally regulates miR96 levels. We analyzed two sets of clinical prostate cancer samples, confirmed association patterns that were consistent with the EGFR-miR-96-ETV6 signaling model and demonstrated that the reduced ETV6 levels were associated with malignant prostate cancer. Based on results derived from multiple approaches, we identified the biological functions of ETV6 as a tumor suppressor that inhibits proliferation and metastasis in prostate cancer. We present a molecular mechanism in which EGFR activation leads to the induction of miR-96 expression and suppression of ETV6, which contributes to prostate cancer progression. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: ETV6 Epidermal growth factor receptor (EGFR) microRNA (miR)-96 Prostate cancer Bone metastasis

Introduction Dysregulation of epidermal growth factor receptor (EGFR) signaling is crucial to tumor progression [1e3]. Importantly, perturbation of microRNAs (miRs) is one mechanism whereby EGFR signaling contributes to metastatic prostate cancer [2,3]. We have demonstrated a role of EGFR translocation to the nucleus in regulating the transcription of miR-1 [3]; however, it is not yet clear whether EGFR is involved in the regulation of other miRs using a similar mechanism. miRs are short RNA molecules that target selected genes for downregulation by interaction with their 30 untranslated regions (30 UTRs) [4], and there is emerging evidence that miRs play multiple roles in metastasis [5]. miR-96 is

* Corresponding author. Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan. Fax: þ886 2 2655 8562. E-mail address: [email protected] (Y.-N. Liu). http://dx.doi.org/10.1016/j.canlet.2016.10.014 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

considered an oncomir and metamir (a miR that is associated with tumors and metastatic tumors) based on findings that some tumor suppressors were targeted by miR-96 [6,7] and that miR-96 has a role in promoting metastatic prostate cancer [8]. We have reported that miR-96 is regulated by TGFb signaling using adenocarcinomas derived from prostate-specific Pten/TP53 double knockout mice [8,9]. The involvement of other oncogenic pathways in the regulation of miR-96 remains unclear. ETS variant gene 6 (ETV6) belongs to one E26 transformationspecific (ETS) subfamily; it has been suggested that ETV6 functions as a tumor suppressor due to its observed deletion in metastatic prostate cancer [10e12]. However, its association with advanced prostate cancer could be complicated by other factors that may be deleted in the same region, and direct evidence showing the function of ETV6 in metastasis is missing. In the current study, we confirmed the regulation of miR-96 through nuclear EGFR-mediated transcription. We determined that one AT-rich

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consensus sequence (ATRS1) in the promoter region of pri-miR-96 (primary miR-96) is crucial to the physical interaction with nuclear EGFR and the ATRS1 sequence-dependent regulation of miR-96 in response to EGFR activities, consistent with a translocationdependent function of EGFR. We next demonstrated a sequencespecific regulation of ETV6 by miR-96, as the downregulation of ETV6 is dependent on a specific miR-96 binding site in the 30 UTR. We further confirmed the inverse correlation between ETV6 and miR-96 in clinical tissue samples in which nuclear EGFR, not cytoplasmic EGFR, is associated with lower levels of ETV6. Using another set of clinical tissue samples containing more prostate cancer cases, reduced ETV6 levels were indeed associated with malignant prostate cancer, consistent with previous findings [11]. Finally, based on results derived from multiple approaches including cell line proliferation/migration/invasion assays and a mouse metastasis model, we concluded that ETV6 serves as a tumor suppressor. This study supports the idea that dysfunction of ETV6, either through genomic deletion or perturbed EGFR activities, is important to prostate cancer progression. Materials and methods Cell lines and reagents All the cell lines except RasB1 were obtained from the American Type Culture Collection (ATCC, MD, USA). The metastatic RasB1 cell line, which was provided by Dr. Kathleen Kelly (NCI/NIH, MD, USA), was derived from DU-145 cells containing an active Ras mutation [13] and selected from a bone metastasis [14,15]. All cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. Stable cell lines expressing ETV6 were established by transfection with an ETV6-expressing vector (pCDH-CMV-MCS-EF1-Puro vector, System Biosciences, CA, USA), with empty vector as a control, followed by selection with puromycin for one month. Transient transfections of plasmids and siRNAs were carried out using X-tremeGENE HP DNA transfection reagent or Lipofectamine RNAiMAX, respectively. Treatment of the EGFR inhibitor (CI1033, 10 ng/ml for 24 h) was performed in 10% serum-containing medium, while EGF treatment (100 ng/ml) was followed by starvation for 24 h under serum-free conditions. EGF was obtained from R&D Systems (MN, USA), and EGFR inhibitor (CI1033) was obtained from Selleck (TX, USA). Anti-miR inhibitors (control and anti-miR-96) and miR precursors (control and miR-96 precursor) were from GeneCopoeia (MD, USA). The miR-96 mimic was obtained from Thermo Scientific Dharmacon (MA, USA). siRNAs (control, siETV6, and siEGFR) were obtained from ON-TARGETplus siRNA. Reporter constructs containing the human ETV6 30 UTR miR response elements were constructed using the psiHECKTM-2 vector (Promega, WI, USA). Point mutations were performed using a Site-Directed Mutagenesis System Kit. MicroRNA mimics were synthesized individually. Tissue samples and immunohistochemistry (IHC) We employed two independent sets of clinical tissue samples from patients with prostate cancer; study protocols were approved by the Taipei Medical UniversityJoint Institutional Review Board and Taipei Medical University-Wan Fang Hospital Institutional Review Board. All patients provided informed consent, and the samples were re-evaluated for grade and histological type by two independent pathologists. Procedures for performing IHC are detailed in the Supplementary methods. Proliferation assay PC3 cells selected for the expression of ETV6 or empty vector were seeded at a density of 2  103 cells per well in a 96-well plate. Similarly, 22RV1 cells treated with ETV6 and scramble siRNA were seeded in a 96-well plate. Cells were harvested and proliferation was spectrophotometrically determined. In brief, cells were stained with 0.5% crystal violet fixative solution for 15 min, rinsed in distilled water, and allowed to air-dry. Then, the crystal violet was dissolved in 50% ethanol containing 0.1 M sodium citrate and quantified at a wavelength of 550 nm. Colony formation assay PC3 or RasB1 cells expressing ETV6 or empty vector were seeded at a density of 5  104 cells per well in a 12-well plate in 0.3% soft agar. Assays were performed in hexaplicate and incubated for 2 days at 37  C in a humidified incubator. After 2 days, half of the medium was gently aspirated and replaced with fresh medium in soft agar. On day 7, colonies were stained with trypan blue and counted if the size was more than 50 mm in diameter. Invasion and migration assays The Boyden chamber was used for both assays. For the invasion assay, RasB1 and PC3 cells expressing ETV6 or empty vector were prepared in serum-free medium at

a concentration of 2.5  105 cells/ml and plated on a Matrigel coating in the upper chamber. The lower chamber was filled with serum-containing medium or serumfree medium plus EGF. Cells that had passed through the Matrigel-coated wells after 12 h were fixed and stained with a 0.5% crystal violet for 15 min, followed by quantification (OD 550 nm) in triplicate. The migration assay was performed similarly, except Matrigel was not used. Metastasis and survival assays in mice Animal work was performed in accordance with a protocol approved by the Taipei Medical University Animal Care and Use Committee (Taiwan). To analyze metastasis, 5-week-old male nude mice (NLAC, Taiwan; 8 mice/group) were subjected to intracardiac injections with 105 RasB1 or PC3 cells expressing ETV6 or empty vector. Both cell lines also harbor a luciferase expression vector for bioluminescence imaging (BLI) as previously described [15]. For survival studies, mice were euthanized upon 10% loss of initial body weight. Gene set enrichment analysis (GSEA) We also used the clinical dataset [12] deposited in the Memorial-Sloan Kettering Cancer Center (MSKCC) Cancer Genomics data portal containing both mRNA and microRNA expression data from 98 primary and 13 metastatic prostate cancer samples. For gene set enrichment analysis (GSEA), software was downloaded from the Broad Institute [16], and the prostate cancer dataset was assigned to two groups based on the median of ETV6 expression. The number of permutations was set to “1000,” and the permutation type was set to “phenotype.” The normalized enrichment score (NES) and false discovery rate q-value (FDR q-value) were calculated by the program. Statistical analysis Statistical calculations were performed with GraphPad Prism analysis tools. Differences between individual groups were primarily determined by Student's ttest. The analyses for the clinicopathologic features and ETV6 levels were performed using the chi-square test. A log-rank test was used for the survival curve analysis. The correlation between ETV6 and miR-96 levels was determined using Pearson's test. P values < 0.05 were considered statistically significant.

Results Primary miR-96 is transcriptionally regulated by nuclear EGFR Although miR-96 was identified as a key factor in metastatic prostate cancer and a downstream target of TGFb signaling [8], we questioned whether it can be regulated by other important pathways involved in prostate cancer progression. Our recent findings showing that dysregulation of miR levels by EGFR signaling is a key mechanism [2,3] underlying metastatic prostate cancer prompted us to examine the effects of EGFR on miR-96 regulation. As shown in Fig. 1A, endogenous miR-96 levels were induced by EGF (veh vs. EGF) but reduced by an EGFR inhibitor (DMSO vs. CI1033), suggesting EGFR-dependent regulation. Based on the effects of nuclear EGFR observed in various cancers [17e20] and our recent findings [3], we examined whether EGFR can serve as a transcriptional coactivator and directly regulate miR96 expression. It has been shown that AT-rich minimal consensus sequences (ATRSs) are crucial to the function of nuclear EGFR [17,21]. Indeed, we identified two ATRSs within the promoter region of the primary miR-96 (pri-miR-96, Fig. 1B). Following ChIP analysis, we found that only ATRS1, not ATRS2, showed enrichment of both p-EGFR (phosphorylated EGFR) and EGFR after EGF treatment (Fig. 1C), suggesting a site-specific interaction. Next, we demonstrated that the binding between nuclear EGFR and ATRS1 is dependent on the EGFR activities in RasB1, a metastatic prostate cancer cell line [13] (Fig. 1D). When we performed a reporter assay using the ATRS1-containing promoter (bottom, Fig. 1B), we observed that the reporter signals were also dependent on the activities of EGFR as evidenced by treatment with its ligand (EGF) or an inhibitor (CI1033) (Fig. 1E). However, mutation of the sequence at ATRS1 (Mut.) disrupted this EGFR-dependent regulation, supporting the specificity between nuclear EGFR and the primary miR96 promoter (Fig. 1E). Furthermore, we showed that EGFR

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Fig. 1. miR-96 is transcriptionally regulated by nuclear EGFR. (A) Relative levels of miR-96 following EGFR activation (veh vs. EGF) or inactivation (DMSO vs. CI1033) in RasB1 cells (n ¼ 3). Both veh and DMSO served as solvent controls. (B) Schematics of the AT-rich consensus sequences (ATRS1 and ATRS2) within the promoter of human pri-miR-96 (top) and the EGFR-96 reporter construct containing ATRS1 only (bottom). The sequences of the original (WT) and mutated (Mut.) ATRS1 are listed. RFP: red fluorescent protein. (C) ChIP assays in RasB1 cells following EGF treatment for 24 h. Specific antibodies (p-EGFR, EGFR, and GAPDH) were used to precipitate chromatin for ATRS DNA (ATRS1 and ATRS2) quantification by real-time PCR (n ¼ 3). (D) ChIP assays following EGFR activation (veh vs. EGF) or inactivation (DMSO vs. CI1033) in RasB1 cells (n ¼ 3). (E) The role of ATRS1 was tested in an EGFR-96 reporter construct containing the WT or Mut sequence. The activities were measured in RasB1 cells following regulation of EGFR activities (n ¼ 3). (F) The role of EGFR in miR-96 was tested by siRNA-mediated knockdown (scr. vs. siEGFR). Endogenous miR-96 levels were measured by real-time PCR following treatment with an EGFR inhibitor (DMSO vs. CI1033) (n ¼ 3). Knockdown efficiency was measured by Western blotting assay (bottom). Significance was calculated by Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

knockdown (siEGFR) prevented the suppressive effects of the EGFR inhibitor on miR-96 levels, consistent with the mechanism of EGFRmediated regulation (Fig. 1F). In summary, our results are consistent with a molecular mechanism by which EGFR regulates miR-96 through a physical interaction at the promoter region of primary miR-96. ETV6 is negatively regulated by EGFR-miR-96 signaling It has been suggested that ETV6 is involved in metastatic prostate cancer [11]; however, important issues relating to its regulation and biological functions are still poorly understood. As shown in Fig. 2A, we found that ETV6 was subject to miR-96 regulation, as endogenous ETV6 mRNA levels were reduced by the miR-96 precursor but increased by a miR-96 inhibitor (anti-96). To further address whether the effects are specific to miR-96, we identified two miR-96 response elements (RE1 and RE2) in the ETV6 30 UTR and monitored the expression of reporter constructs containing these elements (Fig. 2B). As shown in Fig. 2C, we observed that the reporter activities were similar to the regulation patterns on endogenous ETV6 mRNA levels (Fig. 2A). In addition, mutation at one of the miR-96 response elements (RE1, not RE2) resulted in insensitivity to the inhibitory effects of the miR-96 precursor, supporting a physical interaction between miR-96 and the ETV6 30 UTR (Fig. 2D). We further examined the effects on ETV6 protein levels by using synthetic miR-96 mimics (miR-96) and a miR-96 inhibitor (anti-96). As shown in Fig. 2E, miR-96 mimics

significantly reduced the steady-state levels of ETV6 in RasB1 cells, while anti-96 slightly increased those levels in RasB1 cells. To evaluate whether EGFR signaling can exert miR-96dependent regulation of ETV6, we examined the reporter activities of constructs containing miR-96 response elements (Fig. 2B) following treatments with the EGFR ligand (EGF) or an inhibitor (CI1033) (Fig. 2F). We found that EGFR activities tightly regulated the reporter activity (WT) and that mutation at RE1 (RE1/Mut.), but not RE2, disrupted this response (Fig. 2F). Consistent with the results using the miR-96 precursor (Fig. 2D), we concluded that RE1 is the key element responsible for miR-96-mediated downregulation of ETV6. In addition, we found that EGFR negatively controlled the endogenous ETV6 mRNA levels (Fig. 2G) and that the ETV6 protein levels were decreased following EGF treatment in a timedependent manner in RasB1 cells (Fig. 2H). Furthermore, we evaluated the association between the EGFR-regulated genes and ETV6 using a clinical prostate cancer dataset [12]. The dataset was separated into 2 groups based on the relative ETV6 mRNA levels and analyzed by GSEA (ETV6_high vs. ETV6_low, Fig. 2I). We chose the gene set that was suppressed when EGFR was activated [22] and found that the gene set was enriched in the population containing higher ETV6 levels (Fig. 2I). Following analysis with the clinical dataset, we identified two other miRs (486-5p and 548c-3p) showing inverse correlation with ETV6 expression and confirmed their regulation by EGFR signaling (Supplementary Figs. S1A and S1B). To examine the individual effects of the three miRs (96, 486-5p and 548c-3p) on ETV6, we

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performed experiments using synthetic mimics that represent the mature forms of the miRs. We measured the steady-state levels of the miRs (Supplementary Fig. S1C) and the relative protein levels of ETV6 (Supplementary Fig. S1D) after transfection with each miR mimic. The relative knockdown efficiencies (KD.Ef.) were derived from (1-ratio)/(relative miR level) and normalized to the value of miR-96 (Supplementary Fig. S1D). We concluded that miR-96 has the best knockdown efficiency, indicating its specificity to ETV6 downregulation. In summary, our results support the hypothesis that ETV6 is negatively regulated by EGFR signaling through miR96-mediated downregulation. ETV6 is inversely associated with EGFR-miR-96 signaling in clinical tissue samples Following our results from in vitro and database analyses, we sought to examine the EGFR-miR-96-ETV6 signaling model using clinical samples. However, we first analyzed the levels of both miR96 and ETV6 in a panel of prostate cancer cell lines. We found that miR-96 levels were the highest and ETV6 levels were the lowest in two metastatic cell lines (PC3 and RasB1) compared with other non-metastatic cell lines (Supplementary Figs. S1EeS1G). Therefore, there was a negative association between ETV6 and miR-96. Next, we analyzed the mRNA levels in the 14 prostate cancer samples collected from the Taipei Medical University Joint human biological database (Taiwan) and found an inverse correlation between the expression of miR-96 and ETV6 (Fig. 3A). The 14 samples were divided into two groups based on miR-96 levels and analyzed by IHC (Fig. 3B). We found that phosphorylated EGFR (p-EGFR) was associated with increased miR-96 levels (miR96 high, Fig. 3C), while ETV6 was primarily detected in samples with lower miR-96 levels (miR96 low, Fig. 3C). Because the levels of p-EGFR correlate with EGFR activation [23], these results are consistent with the idea that EGFR activation induces miR-96 levels followed by ETV6 downregulation. Furthermore, our results have shown that activated EGFR can translocate to the nucleus and bind to the promoter of pri-miR-96 to regulate miR-96 levels (Fig. 1); therefore, we analyzed the nuclear and cytoplasmic localization of EGFR in IHC samples categorized into 2 groups based on ETV6 levels (Fig. 3D). Consistent with the proposed model, we observed a negative association between ETV6 and nuclear EGFR (left, Fig. 3E). Conversely, cytoplasmic EGFR was positively associated with ETV6 (right, Fig. 3E). Taken together, the results of the IHC analysis further support the EGFR-miR-1-ETV6 axis in the clinical tissue samples.

Fig. 2. ETV6 is negatively regulated by EGFR-miR-96 signaling. (A) Detection of endogenous ETV6 mRNA levels in two cell lines (RasB1 and PC3) following treatments

that increase (EV vs. 96 pre) or decrease (ctrl vs. anti-96) miR-96 levels (n ¼ 3). (B) Schematics of the ETV6 reporter construct containing its 30 UTR with miR-96 response elements (RE1 and RE2). The original (WT) and mutated (Mut.) sequences of the two miR-96 REs are listed. The bicistronic luciferases (Rluc: Renilla, Fluc: firefly) were driven by different promoters (SV40 and HSV-TK). (C) The 96 RE-ETV6 reporter signal ratio (RL/FL) in RasB1 cells following treatments with agents that regulate miR-96 levels (n ¼ 3). (D) Effects of the miR-96 precursor on the 96 RE-ETV6 reporter signal ratio (RL/FL) with original (WT) or mutated (RE1/Mut. & RE2/Mut.) sequences (n ¼ 3). (E) Western blotting analyses of ETV6 levels in response to miR-96 mimics and antimiR-96 in RasB1 cells. (F) Effects of EGFR activity on the 96 RE-ETV6 reporter signal ratio (RL/FL) with original (WT) or mutated (RE1/Mut. & RE2/Mut.) sequences (n ¼ 3). (G) Endogenous ETV6 mRNA levels in response to regulation of EGFR activities in RasB1 and PC3 cell lines. (H) Western blotting analyses monitoring the time course of EGF treatment in RasB1 cells. (I) GSEA analysis with a clinical prostate cancer dataset (n ¼ 111) separated into 2 groups based on the relative ETV6 mRNA levels. The gene set that showed downregulation following EGFR activation was used for measuring enrichment scores. NES: normalized enrichment score; FDR q: false discovery rate qvalue; Hits: members of the gene set identified in the ranked list of the clinical dataset; Ranking metric score: correlation levels with ETV6; EV: empty vector; 96 pre: miR-96 precursor; miR-96: miR-96 mimics; Ctrl: control mimic; NC: scramble negative control; anti-96: miR-96 inhibitor; CI1033: EGFR inhibitor. Both veh and DMSO served as solvent controls. Significance was calculated by Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

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ETV6 is involved in proliferation regulation and is negatively associated with cancer progression Based on the observed deletion of ETV6 in metastatic prostate cancer [11], it has been suspected that ETV6 plays a role as a tumor suppressor; however, its biological functions in prostate cancer have not been examined. To address this issue, we established a metastatic cell line (PC3) that stably expresses ETV6 and found that overexpression of ETV6 inhibited the proliferation rate of these cells (Fig. 4A). Using another approach, we observed the same inhibitory effects by performing a colony formation assay in two metastatic cell lines (Fig. 4B). To test whether reducing ETV6 levels has the opposite effect, we performed siRNA-mediated ETV6 knockdown in the nonmetastatic 22RV1 cell line [24]. As shown in Fig. 1C, ETV6 knockdown (siETV6) resulted in an elevated proliferation rate. The ETV6 levels under either stable expression (PC3 and RasB1) or downregulation (22RV1) conditions were confirmed by Western blotting assays (Fig. 4D). Next, we performed IHC to examine ETV6 protein patterns in tissues with different differentiation/malignancy statuses (Fig. 4E). We found that ETV6 staining was enriched in the nuclei of normal prostate samples, whereas samples with increased Gleason scores showed weak ETV6 staining (Fig. 4E). We measured the ETV6 levels in another set of tissues containing 55 cases of prostatic adenocarcinoma (using a protocol approved by Taipei Medical UniversityWan Fang Hospital, Taiwan) and analyzed the ETV6 distribution in samples with different malignant statuses as indicated by Gleason scores. The samples were classified into 2 groups based on ETV6 levels measured by IHC (ETV6 low and ETV6 high). We found that the samples with poorly differentiated statuses (Gleason score 8) had an evident increase in the number of cases (19 in 25) with lower ETV6 levels compared to samples with lower Gleason scores, which had 0 cases of low ETV6 (Fig. 4F). When we performed a Kaplan-Meier survival analysis in the clinical prostate cancer dataset [12], we found that the patients with higher ETV6 levels had better survival rates than those with lower ETV6 levels (Fig. 4G). In summary, our results show that ETV6 acts as a tumor suppressor in prostate cancer. ETV6 suppresses metastatic prostate cancer To confirm the role of ETV6 in metastasis, we analyzed the two metastatic prostate cancer lines (PC3 and RasB1) and found that stable expression of ETV6 inhibits their invasion abilities (Fig. 5A; selected images in Supplementary Fig. S1H and S1I). We have reported that EGFR signaling promotes prostate cancer metastasis [2,3]; however, stable expression of ETV6 can reduce the invasion abilities even under EGF treatment conditions (EGF, Fig. 5A). In contrast, ETV6 knockdown in the nonmetastatic 22RV1 (Fig. 5B; selected images in Supplementary Fig. S1J and S1K) and DU145 (Supplementary Fig. S1J-S1L) cell lines can enhance both migration and invasion abilities. Furthermore, we found that the levels of

Fig. 3. ETV6 is inversely associated with EGFR-miR-96 signaling in the clinical samples. (A) Pearson correlation analysis of ETV6 mRNA and miR-96 in the clinical tissue samples (n ¼ 14). (B) Immunohistochemistry analyses with antibodies against nuclear p-EGFR (Y1068) and ETV6 in the clinical tissue samples. The samples were separated into two groups based on miR-96 levels (n ¼ 7 per group). Scale bars represent 100 mm. (C) Quantification of IHC signals derived from panel B. (D) Selected images of IHC with an antibody against nuclear p-EGFR (Y1068) in samples with different ETV6 levels. The clinical tissue samples were separated into two groups based on ETV6 levels measured by IHC (n ¼ 7 per group). Scale bars represent 100 mm. (E) Distribution of nuclear EGFR (left) and cytoplasmic EGFR (right) signals in different ETV6 groups. The percentage in each sample was acquired by counting at least 500 cells as described in the Materials and Methods section. Significance was calculated by Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

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several EMT markers were elevated after downregulation of ETV6 by siRNA in the nonmetastatic DU145 and LNCaP cell lines, which is consistent with the role of ETV6 in suppressing metastasis (Supplementary Fig. S1M). To assess the suppressive effects in vivo, we monitored the metastasis of RasB1, a metastatic cell line, through intracardiac injection in mice. A cell line containing either control (EV) or ETV6expressing (ETV6) vector was also engineered to stably express luciferase for in vivo visualization. Surprisingly, we found that ETV6 markedly suppressed metastasis as evidenced by reduced luciferase signals (Fig. 5C) and by histopathological examinations (arrow heads, Fig. 5D). After statistical analysis, mice injected with ETV6-expressing RasB1 cells showed significant reductions in both bone and brain metastases compared to mice injected with cells expressing the control vector alone (ETV6 vs. EV, Fig. 5E). In addition, mice injected with RasB1 cells experienced cachexia-like symptoms and weight loss. Mice were sacrificed when weight loss exceeded 10% of initial body weight by regulation guidelines. Therefore, we utilized weight loss as an indicator of survival ability and found that mice injected with ETV6-expressing RasB1 cells maintained better survival rates compared to those injected with cells containing the empty vector (left, Fig. 2F). In parallel, we set up a similar experiment using another metastatic cell line (PC3) and monitored the mice over time for the development of bone metastases. As shown in Fig. 5G, mice injected with PC3 cells stably expressing ETV6 exhibited delayed progression of bone metastasis (Fig. 2G). Based on these results, we concluded that ETV6 plays a key role in suppressing metastatic prostate cancer. Discussion

Fig. 4. ETV6 is involved in the regulation of proliferation and negatively associated with cancer progression. (A) Proliferation rate analyses of the PC3 stable cell line containing different vectors (EV vs. ETV6; black: EV, blue: ETV6, n ¼ 4). EV: empty vector, ETV6: ETV6-expressing vector. (B) Colony formation analyses for PC3 and RasB1 stable cell lines containing different vectors (n ¼ 6). (C) Proliferation analyses of 22RV1 cells treated with scramble (scr.) or ETV6-specific (siETV6) siRNA (n ¼ 4). Significance was calculated by Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Expression levels of ETV6 in stable cell lines containing different vectors (top) or in 22RV1 cells transfected with siRNA (bottom) by Western blotting assays. (E) Selected immunohistochemistry images of prostate tissue samples stained with ETV6 antibody. Scale

Based on our data, we propose a molecular mechanism in which activation of EGFR by its specific ligands (e.g., EGF) leads to its phosphorylation and nuclear translocation (Fig. 6). Nuclear EGFR induces miR-96 expression followed by the miR-96-mediated downregulation of ETV6. Because ETV6 acts as a tumor suppressor and suppresses metastasis, decreased ETV6 levels facilitate tumor progression (Fig. 6). This study demonstrates the biological functions of ETV6 in prostate cancer and supports the idea that dysfunction of ETV6 either through genomic deletion or perturbed EGFR activities is important to prostate cancer progression. We have shown that miR-96 can promote bone metastasis in prostate cancer partly through transforming growth factor-beta (TGFb)-Smad-mediated regulation [8]. Therefore, key players contributing to prostate cancer progression (e.g., miR-96) could be regulated by multiple signaling pathways. This finding might provide a secondary explanation for the lack of effectiveness of some therapies targeting EGFR/and HER2 [25], in addition to mutations (e.g., ras, PTEN loss, EGFR itself) that bypass the mitogendependent activation of EGFR/and HER2. Therefore, targeting the factors (e.g., miR-96) that overlap in the key pathways leading to prostate cancer progression could provide a new direction for therapy. Based on our IHC analyses, the proportion of ETV6 deficiency was clearly increased in tumor samples with higher Gleason scores, which indicate a poorly differentiated histologic pattern [26]. Whether this ETV6 deficiency is due to genomic deletion [11] or dysregulated EGFR activities as demonstrated in the current study, these results suggest that ETV6 plays a key role in the late stages of bars represent 100 mm. (F) Association profiles between the ETV6 levels and Gleason scores. Statistical analysis was performed by chi-square test (n ¼ 55). (G) Kaplan-Meier survival analysis with the clinical prostate cancer dataset. The data were grouped based on the ETV6 mRNA levels.

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Fig. 6. Molecular mechanism of miR-96-mediated downregulation of ETV6 following EGFR activation. Working model: Extracellular EGFR ligands (e.g., EGF) activate EGFR followed by phosphorylation and translocation to the nucleus. The nuclear EGFR serves as a transcriptional coactivator to induce miR-96 levels, which recognizes the 30 UTR of the ETV6 transcript and leads to its downregulation (yellow box). Reduction of ETV6 levels inhibits its tumor suppressor functions and promotes prostate cancer progression. RNAP: RNA polymerase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

prostate cancer. However, we did not exclude the possibility that ETV6 is active in preventing the initiation of prostate cancer due to the fact that ETV6 can inhibit proliferation (Fig. 4) and even overcome the mitogenic stimulation from EGF (Fig. 5A). ERG is another ETS gene frequently identified as the TMPRSS2:ERG fusion in prostate cancer and its overexpression, together with PTEN loss, can contribute to cancer initiation [27e29]. Because the loss of PTEN is a common genetic alteration in prostate cancer [9,30], it is of great value to further examine whether disruption of ETV6 can facilitate tumorigenesis in this type of genetic background.

Fig. 5. ETV6 suppresses metastatic prostate cancer. (A) Invasion analyses in response to EGF treatment in the two stable cell lines containing different vectors (: EV; þ:

ETV6; n ¼ 3); veh served as solvent control. (B) Invasion and migration analyses in 22RV1 cells with siRNA-mediated knockdown of ETV6 levels (scr. vs. siETV6, n ¼ 3). (C) Selected bioluminescent images of mice following intracardiac injection of the RasB1 cells stably expressing different vectors. (D) Representative images of bone marrow in mice injected with different RasB1 stable cell lines. Tumor metastases are marked by arrows. Scale bar: 100 mm. (E) Quantitation of bioluminescent signals in mice following intracardiac injection of RasB1 stable cell lines. Signals representing metastases were recorded on day 28 for different organs: bone (left) and brain (right) (n ¼ 8). (F) Mouse survival analyses following intracardiac injection of the RasB1 cells. Mice were euthanized when the body weight loss was more than 10% of the initial weight. (G) Time course of bone metastases in mice following intracardiac injection of the PC3 cell lines containing different vectors. The bioluminescent signals were monitored at different times to trace the occurrence of metastases. EV: empty vector; ETV6: ETV6expressing vector.

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Author contributions YCT, WYC, MKS, HYT, and IPC performed the experiments. YCT, JJY, JH, and YNL developed methodology. YCT and YNL conceived the project and designed the experiments. WYC provided the human prostate cancer samples and performed histomorphometric analysis. MKS performed animal experiments and metastatic analysis. JJY provided cell lines. All the authors analyzed and interpreted of data. YCT, JH and YNL wrote, revised, and edited the manuscript. Acknowledgements We thank Dr. Susan Spence (MD, USA) and Dr. Ji-Hshiung Chen (Tzu Chi University, Taiwan) for reading the manuscript and for comments and helpful suggestions. This work was supported by the Ministry of Science and Technology of Taiwan (MOST103-2314B-038-051, MOST104-2314-B-038-045-MY3) to YNL, (MOST 1042320-B-038-055-MY3) to YCT, and by the National Health Research Institutes of Taiwan (NHRI-EX105-10308BC) to YNL. Conflicts of interest The authors declare no potential conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2016.10.014. References [1] F. Ciardiello, G. Tortora, EGFR antagonists in cancer treatment, N. Engl. J. Med. 358 (2008) 1160e1174. [2] M.K. Siu, W. Abou-Kheir, J.J. Yin, Y.S. Chang, B. Barrett, F. Suau, O. Casey, W.Y. Chen, L. Fang, P. Hynes, Y.Y. Hsieh, Y.N. Liu, J. Huang, K. Kelly, Loss of EGFR signaling regulated miR-203 promotes prostate cancer bone metastasis and tyrosine kinase inhibitors resistance, Oncotarget 5 (2014) 3770e3784. [3] Y.S. Chang, W.Y. Chen, J.J. Yin, H. Sheppard-Tillman, J. Huang, Y.N. Liu, EGF receptor promotes prostate cancer bone metastasis by downregulating miR-1 and activating TWIST1, Cancer Res. 75 (2015) 3077e3086. [4] S.L. Ameres, P.D. Zamore, Diversifying microRNA sequence and function, Nat. Rev. Mol. Cell Biol. 14 (2013) 475e488. [5] M.S. Nicoloso, R. Spizzo, M. Shimizu, S. Rossi, G.A. Calin, MicroRNAsethe micro steering wheel of tumour metastases, Nat. Rev. Cancer 9 (2009) 293e302. [6] A. Fendler, M. Jung, C. Stephan, A. Erbersdobler, K. Jung, G.M. Yousef, The antiapoptotic function of miR-96 in prostate cancer by inhibition of FOXO1, PLoS One 8 (2013) e80807. [7] H. Lin, T. Dai, H. Xiong, X. Zhao, X. Chen, C. Yu, J. Li, X. Wang, L. Song, Unregulated miR-96 induces cell proliferation in human breast cancer by downregulating transcriptional factor FOXO3a, PLoS One 5 (2010) e15797. [8] M.K. Siu, Y.C. Tsai, Y.S. Chang, J.J. Yin, F. Suau, W.Y. Chen, Y.N. Liu, Transforming growth factor-beta promotes prostate bone metastasis through induction of microRNA-96 and activation of the mTOR pathway, Oncogene 34 (2015) 4767e4776. [9] P. Martin, Y.N. Liu, R. Pierce, W. Abou-Kheir, O. Casey, V. Seng, D. Camacho, R.M. Simpson, K. Kelly, Prostate epithelial Pten/TP53 loss leads to transformation of multipotential progenitors and epithelial to mesenchymal transition, Am. J. Pathol. 179 (2011) 422e435.

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