miR-409-3p/-5p promotes tumorigenesis, epithelial-to-mesenchymal transition, and bone metastasis of human prostate cancer

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 human prostate cancer bone metastatic cell lines; 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.

Figure 1

MicroRNA miR-409-3p/-5p in the imprinted DLK1-DIO3 cluster is overexpressed in bone metastatic EMT models of human PCa. A, miR-409-3p/-5p in bone metastatic PCa models (mesenchymal cells ARCaP_M compared to ARCaP_E) and (LNCaP^Neo verses LNCaP^RANKL PCa cells). All miRNA and RNA analysis was performed by qRT-PCR analysis. B, mRNA levels of MEG9 of ARCaP_E and ARCaP_M PCa cells. C, miR-409-3p/-5p expression in H9 embryonic stem cells and, D, iPSCs. *: p<0.05 were considered to be statistically significant by t-test.

Figure 2

miR-409 inhibits tumor suppressor genes in prostate cancer. A, mRNA targets of miR-409-5p: STAG2, RBL2, RSU1 and NPRL2 and mRNA targets of miR-409-3p: RSU1, PHC3 and TUSC1, assayed by triplicate wells in qRT-PCR of ARCaP_E and ARCaP_M cells. The representative RT-PCR is shown. The experiment was repeated twice. B, Western analysis of STAG2 and RSU1 in ARCaP_E and ARCaP_M PCa cells. C and D, Cytoscape images of miR-409-3p and miR-409-5p signaling pathways. *: p<0.05 were considered to be statistically significant by t-test.

Figure 3

Human prostatic tissues with higher Gleason score and prostate cancer bone metastasis tissues express elevated levels of miR-409. A, Quantitative analysis of miR-409-3p and miR-409-5p expression in tumor tissues with Gleason grade. B, Representative image of miR-409-3p (green) and miR-409-5p (red) expression in tumor tissues and H&E staining (40x). The tissue array consisted of BPH (N=14), Gleason 6 (N=26) and Gleason ≥ 7 (N=35), data analyzed by Kruskal-Wallis one way analysis of variance- Tukey method. C, miR-409-3p expression in Gleason_high (N=29) and Gleason_low (N=78) based on MSKCC dataset. D, Kaplan-Meier disease free survival (DFS) curves for the prostate cancer patients, based on miR-409-3p expression in the MSKCC dataset. The y-axis is disease free survival probability, and the x-axis is survival in months. Blue line represents the DFS of patients with miR-409-3p lower than the median of the normal individuals (n=78). Red line represents the DFS of patients with miR-409-3p higher than the median of the normal individuals (n=29). Data was analyzed using log-rank test (p=4.3e-05). *: p<0.05 were considered to be statistically significant.

Figure 4

Ectopic expression of miR-409 leads to increased invasiveness and aggressiveness of prostate cancer cells and conversely inhibition of miR-409 results in increased cell death in PCa cells. A, miR-409-5p and -3p expression by qRT-PCR in ARCaP_E-C and ARCaP_E-409 expressing PCa cells. B, RNA expression of miR-409-5p/-3p targets in ARCaP_E-C and ARCaP_E-409 expressing PCa cells assayed by real time PCR. (miR-409-5p mRNA targets: STAG2, RBL2, NPRL2 and RSU1and miR-409-3p mRNA targets: RSU1, PHC3 and TUSC1). C, Invasion and migration assay of in ARCaP_E-C and ARCaP_E-409 expressing PCa cells. D, Cell viability in ARCaP_M PCa cells in response to a miR-409-5p inhibitor. Growth curve of ARCaP_M-C and ARCaP_M-409-5pi PCa cells. E, Expression of miR-409-5p assayed by qRT- PCR in ARCaP_M-C control PCa and ARCaP_M-409-5pi (miR-409-5p inhibitor transfected cells). F, RNA expression of miR-409-5p targets in ARCaP_M-C control and ARCaP_M-409-5pi cells assayed by qRT- PCR. (miR-409-5p mRNA targets: NPRL2 and STAG2). G, Protein expression of STAG2 and RSU1 in ARCaP_M-C cells and ARCaP_M-409-5pi cells. *: p<0.05 were considered to be statistically significant by t-test.

Figure 5

Ectopic expression of miR-409 in the prostate gland transforms normal prostate epithelia, promotes tumorigenecity, EMT and stemness in vivo. A, Comparison of normal prostate and miR-409 expressing prostates. Top row represents green fluorescence for cells containing control GFP plasmid or miR-409 GFP expressing plasmid. Bottom panel represent tumor specific NIR dye (IR783) uptake in control or miR-409 expressing prostates. B, H&E staining of normal control prostate and adenocarcinoma lesions of miR-409 overexpressing prostates (40x), followed by miRNA detection of scramble miRNA and miR-409-5p/-3p of control and miR-409 expressing tissues by ISH and QD detection (40x). C, IHC staining of Ki-67, STAG2, RSU1, vimentin and p-AKT in control prostate and miR-409 expressing prostate tissues (20x).

Figure 6

Inhibition of miR-409-5p results in decreased bone metastasis in PCa in vivo. A, Morphological EMT changes in miR-409-5p inhibited ARCaP_M cells; magnification 10x. RNA expression assayed by qRT-PCR of EMT markers, E-cadherin and N-cadherin. Migration and invasion assay of ARCaP_M-C and ARCaP_M-409-5pi PCa cells (n=3). B, Metastatic lesions observed by luciferase imaging of tumors of ARCaP_M–C cells and ARCaP_M-409-5pi cells in SCID/Beige mice following intra-cardial injections (N=5). C, Kaplan Meier survival curve of mice injected with ARCaP_M–C cells and ARCaP_M-409-5pi cells mice. D, X-ray image of metastatic bone lesion from ARCaP_M–C bone tumors. E, Tumor dye (IR-783 dye) uptake by ARCaP_M–C metastatic tumors from a representative mouse.