Entamoeba histolytica Trophozoites Interact with the c-Met Receptor at the Surface of Liver Origin Cells through the Gal/GalNAc Amoebic Lectin.

Amoebiasis in humans is caused by the protozoan parasite Entamoeba histolytica, which cytotoxic activity has been demonstrated on a wide variety of target cells. The process involves the adherence of the parasite to the cell, and such adherence is mediated by an amoebic surface lectin, known as Gal/GalNAc lectin. It is composed of heavy, intermediate, and light subunits. The carbohydrate recognition domain (CRD) has been identified within a cysteine-rich region in the lectin heavy subunit and has an amino acid sequence identity to the receptor-binding domain of hepatocyte growth factor (HGF). Recombinant CRD has been previously shown to compete with HGF for binding to the c-Met receptor IgG fusion protein. In the present study, we searched for evidence of interaction between the Gal/GalNAc lectin at the surface of trophozoites with the c-Met receptor expressed at the surface of HepG2 in coculture assays. Immunoprecipitation of the coculture lysate indicated interaction of the c-Met with a 60 kDa peptide recognized by antiamoebic lectin antibody. Colocalization of both molecules was detected by fluorescence confocal microscopy. Incubation of HepG2 cells with HGF before coculture with trophozoites prevents the cytotoxic effect caused by the parasites but not their adherence to the cells. Our results point to Gal/GalNAc lectin as a ligand of the c-Met receptor at the surface of HepG2 cells.

Expression of miR-34a and miR-15b during the progression of cervical cancer in a murine model expressing the HPV16 E7 oncoprotein.

The high-risk human papillomavirus (HR-HPV) E7 oncoprotein appears to be a major determinant for cell immortalization and transformation altering critical processes such as cell proliferation, apoptosis, and immune response. This oncoprotein plays an essential role in cervical carcinogenesis, but other cofactors such as long-term use of hormonal contraceptives are necessary to modulate the risk of cervical cancer (CC). The role of HR-HPVs in the alteration of microRNA (miRNA) levels in persistent viral infections currently remains unclear. The aim of this study was to evaluate the miR-34a and miR-15b expression levels in the murine HPV16K14E7 (K14E7) transgenic model after chronic estrogen (E2) treatment and their involvement in CC. Interestingly, results showed that, although miR-34a expression is elevated by the HPVE7 oncogene, this expression was downregulated in the presence of both the E7 oncoprotein and chronic E2 in cervical carcinoma. On the other hand, miR-15b expression was upregulated along cervical carcinogenesis mainly by the effect of E2. These different changes in the expression levels of miR-34a and miR-15b along cervical carcinogenesis conduced to low apoptosis levels, high cell proliferation and finally, to cancerous cervical tissue development. In this work, we also determined the relative mRNA expression of Cyclin E2 (Ccne2), Cyclin A2 (Ccna2), and B cell lymphoma 2 (Bcl-2) (target genes of miR-34a and miR-15b); Sirtuin 1 (Sirt1), Cmyc, and Bax (miR-34a target genes); and p21/WAF1 (mir15b target gene) and the H-ras oncogene. Given the modifications in the expression levels of miR-34a and miR-15b during the development of cervical cancer, it will be useful to carry out further investigation to confirm them as molecular biomarkers of cancer.

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.].

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.

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.

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.

Urinary microRNA-based signature improves accuracy of detection of clinically relevant prostate cancer within the prostate-specific antigen grey zone.

At present, prostate-specific antigen (PSA) is used as a clinical biomarker for prostate cancer (PCa) diagnosis; however, a large number of patients with benign prostate hyperplasia (BPH) with PSA levels in the 'gray area' (4-10 ng/ml) are currently subjected to unnecessary biopsy due to overdiagnosis. Certain microRNAs (miRs) have been proven to be useful biomarkers, several of which are detectable in bodily fluids. The present study identified and validated a urinary miR­based signature to enhance the specificity of PCa diagnosis and to reduce the number of patients with benign conditions undergoing biopsy. Seventy­three urine samples from Mexican patients with diagnosis of PCa with a Gleason score ≥7 and 70 patients diagnosed with BPH were collected after digital rectal examination (DRE) of the prostate. miR expression profiles were determined using TaqMan Low Density Array experiments, and normalized Ct values for the miRs were compared between PCa and BPH groups. Receiver operating characteristic (ROC) curve analysis was performed to evaluate whether miR detection in urine is suitable for distinguishing patients with PCa from those with BPH. The identified miR­100/200b signature was significantly correlated with PCa. Using a multivariable logistic regression approach, a base model including the clinical variables age, prostate­specific antigen (PSA), the percentage of free PSA and DRE was generated, and a second base model additionally contained the miR­100/200b signature. ROC analysis demonstrated that the combined model significantly outperformed the capacity of PSA (P<0.001) and the base model (P=0.01) to discriminate between PCa and BPH patients. In terms of evaluation of the sub­group of patients in the gray zone of PSA levels, the performance of the combined model for predicting PCa cases was significantly superior to PSA level determination (P<0.001) and the base model (P=0.009). In addition, decision curve analysis demonstrated that the use of the combined model increased the clinical benefit for patients and produced a substantial reduction in unnecessary biopsies across a range of reasonable threshold probabilities (10­50%). Detection of the urinary miR signature identified in the present study as part of clinical diagnostic procedures will enhance the accuracy of PCa diagnosis and provide a clinical benefit for patients with BPH by sparing them from undergoing invasive biopsy. To the best of our knowledge, the present study was the first to describe the profiling of urinary miR100 and miR-200b levels for the clinical diagnosis of PCa.

RXRα deletion and E6E7 oncogene expression are sufficient to induce cervical malignant lesions in vivo.

Cervical cancer is the second leading cause of cancer deaths among women worldwide. High-Risk-Human Papillomaviruses (HR-HPVs) play an important etiologic role in the development of carcinoma of the uterine cervix. However, host factors are important in determining the outcome of genital HPV infection as most cervical precancerous lesions containing HR-HPVs do not progress to invasive carcinomas. Retinoids, acting through nuclear receptors (RARs, RXRs), play a crucial role in cervix development and homeostasis regulating growth and differentiation of a wide variety of cell types; indeed, they can inhibit cell proliferation, and induce cell differentiation or apoptotic cell death. Here we introduce a mouse model that develops spontaneously malignant cervical lesions allowing the study of the cooperative effect between HPV16E6E7 expression and the lack of RXRα in cervical cancer development. This model could be useful to study multistep carcinogenesis of uterine cervix tissue and might improve chemopreventive and chemotherapeutic strategies for this neoplasia.