Published OnlineFirst June 24, 2014; DOI: 10.1158/1078-0432.CCR-14-0305 Clinical Cancer Biology of Human Tumors Research miR-409-3p/-5p Promotes Tumorigenesis, Epithelial-to-Mesenchymal Transition, and Bone Metastasis of Human Prostate Cancer Sajni Josson1, Murali Gururajan1, Peizhen Hu1, Chen Shao1, Gina Chia-Yi Chu1, Haiyen E. Zhau1, Chunyan Liu1, Kaiqin Lao2, Chia-Lun Lu1, Yi-Tsung Lu1, Jake Lichterman1, Srinivas Nandana1, Quanlin Li3, Andre Rogatko3, Dror Berel3, Edwin M. Posadas1, Ladan Fazli4, Dhruv Sareen5, and Leland W.K. Chung1 Abstract Purpose: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in humanprostatecancerbonemetastaticcelllines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. Experimental Design: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. Results: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409- 3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor–treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle–treated cells. Conclusion: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer. Clin Cancer Res; 20(17); 1–11. Ó2014 AACR. Introduction process hijacked by the cancer cells. The role of noncoding Metastasis of cancer cells to distant organs involves epi- RNAs in both the EMT and subsequent bony metastasis is thelial-to-mesenchymal transition (EMT), an embryonic less well understood. Recent studies highlight the role of noncoding RNAs, including miRNAs and lncRNAs, in can- cer progression and metastasis (1–5). The delta-like 1 1Uro-Oncology Research Program, Department of Medicine, Samuel homolog–deiodinase, iodothyronine 3 (DLK1-DIO3) Oschin Comprehensive Cancer Institute, Los Angeles, California. 2Genetic imprinted embryonically cluster contains several large and 3 Systems, Life Technologies Inc., South San Francisco, California. Bio- small noncoding RNA genes, which are deregulated in statistics and Bioinformatics, Samuel Oschin Comprehensive Cancer Institute, Los Angeles, California. 4Vancouver Prostate Cancer Center, cancer development (6, 7). The DLK1-DIO3 gene cluster University of British Columbia, Vancouver, Canada. 5Regenerative Medi- was previously shown to be aberrantly silenced in human cine Institute, Cedars-Sinai Medical Center, Los Angeles, California. and mouse induced pluripotent stem cells (iPSC) but not in Note: Supplementary data for this article are available at Clinical Cancer fully pluripotent embryonic stem cells, indicating the Research Online (http://clincancerres.aacrjournals.org/). importance in the generation of fully functional iPSCs S. Josson and M. Gururajan contributed equally to this article. (8, 9). This suggests that certain miRNAs in this region are Corresponding Authors: Leland W.K. Chung, Uro-Oncology Research involved in totipotency. Several studies show that some Program, Department of Medicine, Samuel Oschin Comprehensive Cancer miRNAs in this cluster are differentially expressed in pros- Institute, Cedars-Sinai Medical Center, 8750 Beverly Boulevard, Atrium 103, Los Angeles, CA 90048. Phone: 310-423-7622; Fax: 310-423-8543; tate, breast, and liver cancer (7, 10, 11). Interestingly, E-mail: [email protected]; Sajni Josson, [email protected]; and miRNA members of the DLK1-DIO3 cluster have been Murali Gururajan, [email protected] shown to be upregulated in the serum of patients with doi: 10.1158/1078-0432.CCR-14-0305 cancer. Specifically, in prostate cancer, miR-409-3p has Ó2014 American Association for Cancer Research. been shown to be upregulated in the serum of patients www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst June 24, 2014; DOI: 10.1158/1078-0432.CCR-14-0305 Josson et al. mRNA analysis. Total RNA was isolated using the Translational Relevance RNeasy Mini Kit (Qiagen). cDNA was made using Super- Currently, there are limited options for targeted treat- scriptIII Reverse Transcriptase (Life Technologies). mRNA ment or biomarkers of cancer bone metastasis. In this study, primers were designed and synthesized at Integrated DNA we have identified a novel role for miR-409-3p/-5p in Technologies. mRNA expression levels were determined by prostate tumor growth, epithelial-to-mesenchymal transi- qRT-PCR assays and SYBR Green Dye (Applied Biosystems). tion, stemness, and bone metastasis. miR-409-3p/-5p is locatedinanembryonically regulated cluster and appears Long noncoding RNA analysis to be activated during metastasis. We demonstrate that both mRNA was extracted as described above. LncRNA expres- miR-409-3p and miR-409-5p are elevated in tumor tissues sion levels were determined as per manufacturer’s instruc- of prostate cancer patients with high Gleason scores. Using tions (System Biosciences) using real-time PCR. Relative a publicly available database, we observed that elevated levels of MEG9 were plotted normalized to GAPDH. miR-409-3p levels correlate with patient disease-free sur- vival. Overexpression of miR-409-3p/-5p in mouse prostate Cytoscape analysis induces tumor growth, and inhibition of miR-409-5p Cytoscape image was created using miR-409-5p and miR- results in decreased bone metastasis in experimental mod- 409-3p target genes from Targetscan v12 software analysis els. Thus, miR-409-3p/-5p, implicated in embryonic devel- and Genecard website (STRING: functional protein associ- opment, has an unexpected oncogenic role in prostate ation networks). cancer bone metastasis, and thus could serve both as a biomarker and as a therapeutic target. In situ hybridization–quantum dots Human Gleason tissue array. A Gleason score tissue array was obtained from Vancouver Prostate Center. The use of specimens in research was approved by the institu- with high-risk prostate cancer compared to patients with tional review board of the Cedars-Sinai Medical Center low-risk prostate cancer (12). miR379 expression was (IRB# Pro21228). The tissues consisted of benign prostatic increased in the tissues of metastatic prostate cancer com- hyperplasia (BPH; N ¼ 14), Gleason 6 (N ¼ 26), and pared with localized prostate cancer (13). Also, miR379 and Gleason 7(N ¼ 35). Each tissue had two cores in the miR154Ã have been shown to be increased in circulating array. These patients had no treatment. The tissue array was exosomes of patients with lung adenocarcinomas versus stained for hematoxylin and eosin (H&E) and graded by a healthy smokers (14). In this study, we manipulated miR- pathologist. Information on Gleason score of the cancer 409-3p/-5p expression in adult normal prostate and in and miR-409 intensity is included in Supplementary Fig. S1. prostate cancer cells and report a surprising and novel The control scramble and miR-409-5p and -3p probes were discovery of transforming effects of this miRNA conferring 50-biotin labeled. The probes were linked to streptavidin- prostate epithelium to undergo EMT, and expressing stem- conjugated quantum dot (QD). Multiplex QD labeling ness and tumorigenic phenotypes in mice. Inhibition of (mQDL) was performed as previously described (16). miR-409-5p in a human prostate cancer cell line resulted in miR-409-5p was labeled with 625 nm QD (red) followed decreased bone metastasis in vivo. by miR-409-3p (green) which was labeled with 565 nm QD (16). The QD fluorescence intensity of each tissue section Materials and Methods was determined and analyzed. Statistical analysis was per- Cell culture formed on the dataset using a Kruskal–Wallis one-way Human androgen-refractory prostate cancer cells (ARCaPE ANOVA and post hoc Tukey method for multiple compar- Neo RANKL and ARCaPM)andLNCaP and LNCaP prostate isons between groups. Data distribution was depicted as cancer cells (15–17) were used. Prostate cancer cells and box plots. 293T cells were cultured in T-medium (GibcoBRL) supple- In vivo animal studies. Mouse tumor and tumor xeno- mented with 5% heat-inactivated FBS (Bio-Whittaker), as grafts were formalin-fixed and paraffin-embedded. miRNA previously mentioned (18). All cells were tested for mycoplas- in situ hybridization (ISH) protocol was followed as per the ma every 3 months and were negative. The embryonic stem manufacturer’s instructions (Exiqon). Single QD labeling cells and iPSC-derived