SFRP1 increases TMPRSS2-ERG expression promoting neoplastic features in prostate cancer in vitro and in vivo.

BACKGROUND: Prostate cancer (PCa) is the second cause of cancer related death in North American men. Androgens play an important role in its progression by regulating the expression of several genes including fusion ones that results from structural chromosome rearrangements. TMPRSS2-ERG is a fusion gene commonly observed in over 50% of PCa tumors, and its expression can be transcriptionally regulated by the androgen receptor (AR) given its androgen responsive elements. TMPRSS2-ERG could be involved in epithelial-mesenchymal transition (EMT) during tumor development. ERG has been reported as a key transcriptional factor in the AR-ERG-WNT network where five SFRP proteins, structurally similar to WNT ligands and considered to be WNT pathway antagonists, can regulate signaling in the extracellular space  by binding to WNT proteins or Frizzled receptors. It has been shown that over-expression of SFRP1 protein can regulate the transcriptional activity of AR and inhibits the formation of colonies in LNCaP cells. However, the effect of SFRP1 has been controversial since differential effects have been observed depending on its concentration and tissue location. In this study, we explored the role of exogenous SFRP1 protein in cells expressing the TMPRSS2-ERG fusion. METHODS: To evaluate the effect of exogenous SFRP1 protein on PCa cells expressing TMPRSS2-ERG, we performed in silico analysis from TCGA cohort, expression assays by RT-qPCR and Western blot, cell viability and cell cycle measurements by cytometry, migration and invasion assays by xCELLigance system and murine xenografts. RESULTS: We demonstrated that SFRP1 protein increased ERG expression by promoting cellular migration in vitro and increasing tumor growth in vivo in PCa cells with the TMPRSS2-ERG fusion. CONCLUSIONS: These results suggest the possible role of exogenous SFRP1 protein as a modulator of AR-ERG-WNT signaling network in cells positive to TMPRSS2-ERG. Further, investigation is needed to determine if SFRP1 protein could be a target in against this type of PCa.

Corrigendum: HypoxamiRs Profiling Identify miR-765 as a Regulator of the Early Stages of Vasculogenic Mimicry in SKOV3 Ovarian Cancer Cells.

[This corrects the article DOI: 10.3389/fonc.2019.00381.].

Analysis of the dynamic aberrant landscape of DNA methylation and gene expression during arsenic-induced cell transformation.

Inorganic arsenic is a well-known carcinogen associated with several types of cancer, but the mechanisms involved in arsenic-induced carcinogenesis are not fully understood. Recent evidence points to epigenetic dysregulation as an important mechanism in this process; however, the effects of epigenetic alterations in gene expression have not been explored in depth. Using microarray data and applying a multivariate clustering analysis in a Gaussian mixture model, we describe the alterations in DNA methylation around the promoter region and the impact on gene expression in HaCaT cells during the transformation process caused by chronic exposure to arsenic. Using this clustering approach, the genes were grouped according to their methylation and expression status in the epigenetic landscape, and the changes that occurred during the cellular transformation were identified adequately. Thus, we present a valuable method for identifying epigenomic dysregulation.

HypoxamiRs Profiling Identify miR-765 as a Regulator of the Early Stages of Vasculogenic Mimicry in SKOV3 Ovarian Cancer Cells

Vasculogenic mimicry (VM) is a novel cancer hallmark in which malignant cells develop matrix-associated 3D tubular networks with a lumen under hypoxia to supply nutrients needed for tumor growth. Recent studies showed that microRNAs (miRNAs) may have a role in VM regulation. In this study, we examined the relevance of hypoxia-regulated miRNAs (hypoxamiRs) in the early stages of VM formation. Data showed that after 48 h hypoxia and 12 h incubation on matrigel SKOV3 ovarian cancer cells undergo the formation of matrix-associated intercellular connections referred hereafter as 3D channels-like structures, which arose previous to the apparition of canonical tubular structures representative of VM. Comprehensive profiling of 754 mature miRNAs at the onset of hypoxia-induced 3D channels-like structures showed that 11 hypoxamiRs were modulated (FC>1.5; p < 0.05) in SKOV3 cells (9 downregulated and 2 upregulated). Bioinformatic analysis of the set of regulated miRNAs showed that they might impact cellular pathways related with tumorigenesis. Moreover, overall survival analysis in a cohort of ovarian cancer patients (n = 485) indicated that low miR-765, miR-193b, miR-148a and high miR-138 levels were associated with worst patients outcome. In particular, miR-765 was severely downregulated after hypoxia (FC < 32.02; p < 0.05), and predicted to target a number of protein-encoding genes involved in angiogenesis and VM. Functional assays showed that ectopic restoration of miR-765 in SKOV3 cells resulted in a significant inhibition of hypoxia-induced 3D channels-like formation that was associated with a reduced number of branch points and patterned tubular-like structures. Mechanistic studies confirmed that miR-765 decreased the levels of VEGFA, AKT1 and SRC-α transducers and exerted a negative regulation of VEGFA by specific binding to its 3'UTR. Finally, overall survival analysis of a cohort of ovarian cancer patients (n = 1435) indicates that high levels of VEGFA, AKT1 and SRC-α and low miR-765 expression were associated with worst patients outcome. In conclusion, here we reported a novel hypoxamiRs signature which constitutes a molecular guide for further clinical and functional studies on the early stages of VM. Our data also suggested that miR-765 coordinates the formation of 3D channels-like structures through modulation of VEGFA/AKT1/SRC-α axis in SKOV3 ovarian cancer cells.

A Novel OsteomiRs Expression Signature for Osteoblast Differentiation of Human Amniotic Membrane-Derived Mesenchymal Stem Cells.

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are a potential source of cells for therapeutic applications in bone regeneration. Recent evidence reveals a role for microRNAs (miRNAs) in the fine-tuning regulation of osteogenesis (osteomiRs) suggesting that they can be potential targets for skeleton diseases treatment. However, the functions of osteomiRs during differentiation of hAM-MSCs to osteogenic lineage are poorly understood. In this investigation, we discovered a novel miRNAs expression signature corresponding to the matrix maturation (preosteoblast) and mineralization (mature osteoblast) stages of dexamethasone-induced osteoblastic differentiation of hAM-MSCs. Comprehensive miRNAs profiling using TaqMan Low Density Arrays showed that 18 miRNAs were significantly downregulated, whereas 3 were upregulated in the matrix maturation stage (7 days after osteogenic induction) in comparison to undifferentiated cells used as control. Likewise, 47 miRNAs were suppressed and 25 were overexpressed at mineralization stage (14 days after osteogenic induction) in comparison to osteoprogenitors cells. Five out 93 miRNAs (miR-19b-3p, miR-335-3p, miR-197-3p, miR-34b-39, and miR-576-3p) were regulated at both 7 and 14 days suggesting a role in coordinated guidance of osteoblastic differentiation. Exhaustive bioinformatic predictions showed that the set of modulated miRNAs may target multiple genes involved in regulatory networks driving osteogenesis including key members of BMP, TGF-ß, and WNT/ß-catenin signaling pathways. Of these miRNAs, we selected miR-204, a noncoding small RNA that was expressed at matrix maturation phase and downregulated at maturation stage, for further functional studies. Interestingly, gain-of-function analysis showed that restoration of miR-204 using RNA mimics at the onset of mineralization stage dramatically inhibited deposition of calcium and osteogenic maturation of hAM-MSCs. Moreover in silico analysis detected a conserved miR-204 binding site at the 3'UTR of TGF-ßR2 receptor gene. Using luciferase assays we confirmed that TGF-ßR2 is a downstream effector of miR-204. In conclusion, we have identified a miRNAs signature for osteoblast differentiation of hAM-MSCs. The results from this study suggested that these miRNAs may act as potential inhibitors or activators of osteogenesis. Our findings also points towards the idea that miR-204/TGF-ßR2 axis has a regulatory role in differentiation of hAM-MSCs committed to osteoblastic lineage.

Cooperative multi-targeting of signaling networks by angiomiR-204 inhibits vasculogenic mimicry in breast cancer cells.

RNA-based multi-target therapies focused in the blocking of signaling pathways represent an attractive approach in cancer. Here, we uncovered a miR-204 cooperative targeting of multiple signaling transducers involved in vasculogenic mimicry (VM). Our data showed that invasive triple negative MDA-MB-231 and Hs-578T breast cancer cells, but not poorly invasive MCF-7 cells, efficiently undergoes matrix-associated VM under hypoxia. Ectopic restoration of miR-204 in MDA-MB-231 cells leads to a potent inhibition of VM and reduction of number of branch points and patterned 3D channels. Further analysis of activation state of multiple signaling pathways using Phosphorylation Antibody Arrays revealed that miR-204 reduced the expression and phosphorylation levels of 13 proteins involved in PI3K/AKT, RAF1/MAPK, VEGF, and FAK/SRC signaling. In agreement with phospho-proteomic profiling, VM was impaired following pharmacological administration of PI3K and SRC inhibitors. Mechanistic studies confirmed that miR-204 exerts a negative post-transcriptional regulation of PI3K-α and c-SRC proto-oncogenes. Moreover, overall survival analysis of a large cohort of breast cancer patients indicates that low miR-204 and high FAK/SRC levels were associated with worst outcomes. In conclusion, our study provides novel lines of evidence indicating that miR-204 may exerts a fine-tuning regulation of the synergistic transduction of PI3K/AKT/FAK mediators critical in VM formation.