miR-204 Shifts the Epithelial to Mesenchymal Transition in Concert with the Transcription Factors RUNX2, ETS1, and cMYB in Prostate Cancer Cell Line Model
Epithelial to mesenchymal transition is an essential step in advanced cancer development. Many master transcription factors shift their expression to drive this process, while noncoding RNAs families like miR-200 are found to restrict it. In this study we investigated how the tumor suppressor miR-204 and several transcription factors modulate main markers of mesenchymal transformation like E- and N-cadherin, SLUG, VEGF, and SOX-9 in prostate cancer cell line model (LNCaP, PC3, VCaP, and NCI-H660). We found that SLUG, E-cadherin, and N-cadherin are differentially modulated by miR-204, using miR-204 specific mimics and inhibitors and siRNA gene silencing (RUNX2, ETS-1, and cMYB). The genome perturbation associated TMPRSS2-ERG fusion coincided with shift from tumor-suppressor to tumor-promoting activity of this miRNA. The ability of miR-204 to suppress cancer cell viability and migration was lost in the fusion harboring cell lines. We found differential E-cadherin splicing corroborating to miR-204 modulatory effects. RUNX2, ETS1, and cMYB are involved in the regulation of E-cadherin, N-cadherin, and VEGFA expression. RUNX2 knockdown results in SOX9 downregulation, while ETS1 and cMYB silencing result in SOX9 upregulation in VCaP cells. Their expression was found to be also methylation dependent. Our study provides means for understanding cancer heterogeneity in regard to adapted therapeutic approaches development. 1. Introduction Prostate cancer is one of the most common malignancies and the second leading cause of death from cancer in men. Androgen receptor (AR) is paramount for the lineage-specific differentiation of the prostate, inducing the expression of prostate-specific genes, such as PSA and TMPRSS2, and maintaining the differentiated prostate epithelial phenotype [1]. Cellular dedifferentiation and epithelial to mesenchymal transition (EMT), by contrast, are a hallmark of malignant transformation and metastatic disease. In this process the reexpression of conserved developmental programs plays a key role [2]. Chromosomal rearrangements fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG occur in approximately 50% of prostate cancers and more than 90% of them overexpress ERG [3]. TMPRSS2-ERG is crucial for cancer progression by disrupting lineage-specific differentiation of the prostate and potentiating the histone methyltransferase EZH2-mediated dedifferentiation program. ERG disrupts AR signalling by inhibiting AR expression, binding to and inhibiting AR activity at gene-specific loci, and inducing repressive
References
[1]
M. E. Wright, M.-J. Tsai, and R. Aebersold, “Androgen receptor represses the neuroendocrine transdifferentiation process in prostate cancer cells,” Molecular Endocrinology, vol. 17, no. 9, pp. 1726–1737, 2003.
[2]
Z. Huang, P. J. Hurley, B. W. Simons et al., “Sox9 is required for prostate development and prostate cancer initiation,” Oncotarget, vol. 3, no. 6, pp. 651–663, 2012.
[3]
S. A. Tomlins, B. Laxman, S. Varambally et al., “Role of the TMPRSS2-ERG gene fusion in prostate cancer,” Neoplasia, vol. 10, no. 2, pp. 177–188, 2008.
[4]
J. Yu, D. R. Rhodes, S. A. Tomlins et al., “A polycomb repression signature in metastatic prostate cancer predicts cancer outcome,” Cancer Research, vol. 67, no. 22, pp. 10657–10663, 2007.
[5]
V. Davalos, C. Moutinho, A. Villanueva et al., “Dynamic epigenetic regulation of the microRNA-200 family mediates epithelial and mesenchymal transitions in human tumorigenesis,” Oncogene, vol. 31, no. 16, pp. 2062–2074, 2012.
[6]
M. M. Shen and C. Abate-Shen, “Molecular genetics of prostate cancer: New prospects for old challenges,” Genes and Development, vol. 24, no. 18, pp. 1967–2000, 2010.
[7]
C. A. Heinlein and C. Chang, “Androgen receptor in prostate cancer,” Endocrine Reviews, vol. 25, no. 2, pp. 276–308, 2004.
[8]
C. E. Massie, B. Adryan, N. L. Barbosa-Morais et al., “New androgen receptor genomic targets show an interaction with the ETS1 transcription factor,” EMBO Reports, vol. 8, no. 9, pp. 871–878, 2007.
[9]
Y. Shiozawa, E. A. Pedersen, A. M. Havens et al., “Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow,” Journal of Clinical Investigation, vol. 121, no. 4, pp. 1298–1312, 2011.
[10]
N. C. Nicolaides, R. Gualdi, C. Casadevall, L. Manzella, and B. Calabretta, “Positive autoregulation of c-myb expression via Myb binding sites in the 5′ flanking region of the human c-myb gene,” Molecular and Cellular Biology, vol. 11, no. 12, pp. 6166–6176, 1991.
[11]
F. Kalkbrenner, S. Guehmann, and K. Moelling, “Transcriptional activation by human c-myb and v-myb genes,” Oncogene, vol. 5, no. 5, pp. 657–661, 1990.
[12]
M. Lagos-Quintana, R. Rauhut, A. Yalcin, J. Meyer, W. Lendeckel, and T. Tuschl, “Identification of tissue-specific MicroRNAs from mouse,” Current Biology, vol. 12, no. 9, pp. 735–739, 2002.
[13]
J. Lin, Q. Cao, J. Zhang et al., “MicroRNA expression patterns in indeterminate inflammatory bowel disease,” Modern Pathology, vol. 26, no. 1, pp. 148–154, 2013.
[14]
R. Narayanan, J. Jiang, Y. Gusev et al., “Micrornas are mediators of androgen action in prostate and muscle,” PLoS ONE, vol. 5, no. 10, Article ID e13637, 2010.
[15]
M. Karaivanov, K. Todorova, A. Kuzmanov, and S. Hayrabedyan, “Quantitative immunohistochemical detection of the molecular expression patterns in proliferative inflammatory atrophy,” Journal of Molecular Histology, vol. 38, no. 1, pp. 1–11, 2007.
[16]
K. Todorova, S. Zoubak, M. Mincheff, and S. Kyurkchiev, “Biochemical nature and mapping of PSMA epitopes recognized by human antibodies induced after immunization with gene-based vaccines,” Anticancer Research, vol. 25, no. 6, pp. 4727–4732, 2005.
[17]
K. Todorova, I. Ignatova, S. Tchakarov et al., “Humoral immune response in prostate cancer patients after immunization with gene-based vaccines that encode for a protein that is proteasomally degraded,” Cancer Immunity, vol. 5, article 1, 2005.
[18]
K. Todorova, M. Mincheff, S. Hayrabedyan et al., “Fundamental role of microRNAs in androgen-dependent male reproductive biology and prostate cancerogenesis,” American Journal of Reproductive Immunology, vol. 69, no. 2, pp. 100–104, 2013.
[19]
M. Korpal, E. S. Lee, G. Hu, and Y. Kang, “The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2,” The Journal of Biological Chemistry, vol. 283, no. 22, pp. 14910–14914, 2008.
[20]
J. Huang, L. Zhao, L. Xing, and D. Chen, “MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation,” Stem Cells, vol. 28, no. 2, pp. 357–364, 2010.
[21]
Z. Gibas, R. Becher, E. Kawinski, J. Horoszewicz, and A. A. Sandberg, “A high-resolution study of chromosome changes in a human prostatic carcinoma cell line (LNCaP),” Cancer Genetics and Cytogenetics, vol. 11, no. 4, pp. 399–404, 1984.
[22]
M. E. Kaighn, K. S. Narayan, Y. Ohnuki, J. F. Lechner, and L. W. Jones, “Establishment and characterization of a human prostatic carcinoma cell line (PC-3),” Investigative Urology, vol. 17, no. 1, pp. 16–23, 1979.
[23]
S. Korenchuk, J. E. Lehr, L. McLean et al., “VCaP, a cell-based model system of human prostate cancer,” In Vivo, vol. 15, no. 2, pp. 163–168, 2001.
[24]
S.-L. Lai, H. Brauch, T. Knutsen et al., “Molecular genetic characterization of neuroendocrine lung cancer cell lines,” Anticancer Research, vol. 15, no. 2, pp. 225–232, 1995.
[25]
M. Ishiyama, H. Tominaga, M. Shiga, K. Sasamoto, Y. Ohkura, and K. Ueno, “A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet,” Biological and Pharmaceutical Bulletin, vol. 19, no. 11, pp. 1518–1520, 1996.
[26]
M. A. Markus, R. B. Gerstner, D. E. Draper, and D. A. Torchia, “Refining the overall structure and subdomain orientation of ribosomal protein S4 Δ41 with dipolar couplings measured by NMR in uniaxial liquid crystalline phases,” Journal of Molecular Biology, vol. 292, no. 2, pp. 375–387, 1999.
[27]
P. O. Krutzik and G. P. Nolan, “Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events,” Cytometry A, vol. 55, no. 2, pp. 61–70, 2003.
[28]
J. M. Irish, R. Hovland, P. O. Krutzik et al., “Single cell profiling of potentiated phospho-protein networks in cancer cells,” Cell, vol. 118, no. 2, pp. 217–228, 2004.
[29]
Y. Sun, S. Koo, N. White et al., “Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs,” Nucleic Acids Research, vol. 32, no. 22, p. e188, 2004.
[30]
D. P. Turner, V. J. Findlay, O. Moussa et al., “Mechanisms and functional consequences of PDEF protein expression loss during prostate cancer progression,” Prostate, vol. 71, no. 16, pp. 1723–1735, 2011.
[31]
J. S. Imam, J. R. Plyler, H. Bansal et al., “Genomic loss of tumor suppressor miRNA-204 promotes cancer cell migration and invasion by activating AKT/mTOR/Rac1 signaling and actin reorganization,” PLoS ONE, vol. 7, no. 12, Article ID e52397, 2012.
[32]
Y. Lee, X. Yang, Y. Huang et al., “Network modeling identifies molecular functions targeted by miR-204 to suppress head and neck tumor metastasis,” PLoS Computational Biology, vol. 6, no. 4, Article ID e1000730, 2010.
[33]
S. Sharma and A. Lichtenstein, “Aberrant splicing of the E-cadherin transcript is a novel mechanism of gene silencing in chronic lymphocytic leukemia cells,” Blood, vol. 114, no. 19, pp. 4179–4185, 2009.
[34]
I. M. Shapiro, A. W. Cheng, N. C. Flytzanis et al., “An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype,” PLoS Genetics, vol. 7, no. 8, Article ID e1002218, 2011.
[35]
C. Cai, H. Wang, H. H. He et al., “ERG induces androgen receptor-mediated regulation of SOX9 in prostate cancer,” Journal of Clinical Investigation, vol. 123, no. 3, pp. 1109–1122, 2013.
[36]
H.-F. Yuen, W.-K. Kwok, K.-K. Chan et al., “TWIST modulates prostate cancer cell-mediated bone cell activity and is upregulated by osteogenic induction,” Carcinogenesis, vol. 29, no. 8, pp. 1509–1518, 2008.
[37]
J. Gavard, V. Patel, and J. S. Gutkind, “Gutkind, Angiopoietin-1 prevents VEGF-induced endothelial permeability by sequestering Src through mDia,” Developmental Cell, vol. 14, no. 1, pp. 25–36, 2008.
[38]
A. Courboulin, R. Paulin, N. J. Giguère et al., “Role for miR-204 in human pulmonary arterial hypertension,” Journal of Experimental Medicine, vol. 208, no. 3, pp. 535–548, 2011.
[39]
E. Lambertini, G. Lisignoli, E. Torreggiani et al., “Slug gene expression supports human osteoblast maturation,” Cellular and Molecular Life Sciences, vol. 66, no. 22, pp. 3641–3653, 2009.
[40]
J. Akech, J. J. Wixted, K. Bedard et al., “Runx2 association with progression of prostate cancer in patients: mechanisms mediating bone osteolysis and osteoblastic metastatic lesions,” Oncogene, vol. 29, no. 6, pp. 811–821, 2010.
[41]
S. K. Baniwal, O. Khalid, Y. Gabet et al., “Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis,” Molecular Cancer, vol. 9, article 258, 2010.
[42]
E. Lambertini, T. Franceschetti, E. Torreggiani et al., “SLUG: a new target of lymphoid enhancer factor-1 in human osteoblasts,” BMC Molecular Biology, vol. 11, article 13, 2010.
[43]
G. H. Little, S. K. Baniwal, H. Adisetiyo et al., “Differential effects of RUNX2 on the androgen receptor in prostate cancer: synergistic stimulation of a gene set exemplified by SNAI2 and subsequent invasiveness,” Cancer Research, vol. 74, no. 10, pp. 2857–2868, 2014.
[44]
T. Shirakihara, M. Saitoh, and K. Miyazono, “Differential regulation of epithelial and mesenchymal markers by EF1 proteins in epithelial-mesenchymal transition induced by TGF-,” Molecular Biology of the Cell, vol. 18, no. 9, pp. 3533–3544, 2007.
[45]
A. Cheng and P. G. Genever, “SOX9 determines RUNX2 transactivity by directing intracellular degradation,” Journal of Bone and Mineral Research, vol. 25, no. 12, pp. 2680–2689, 2010.
[46]
Z. Gao, G. H. Kim, A. C. Mackinnon et al., “Ets1 is required for proper migration and differentiation of the cardiac neural crest,” Development, vol. 137, no. 9, pp. 1543–1551, 2010.
[47]
E. Matalová, M. Buchtová, A. S. Tucker et al., “Expression and characterization of c-Myb in prenatal odontogenesis,” Development Growth and Differentiation, vol. 53, no. 6, pp. 793–803, 2011.
[48]
V. Oralova, M. Buchtova, E. Janeckova, A. Tucker, and E. Matalova, “Modulation of c-Myb during chondrogenesis,” Bone Abstracts, vol. 1, Article ID PP252, 2013.
