Cesar Seigi Fuziwara

Insights into Regulation of the miR-17-92 Cluster of miRNAs in Cancer.

Overexpression of the miR-17-92 cluster is a key oncogenic event in various cancer types. The oncogenic effect of the miR-17-92 cluster is enhanced by cooperation between its members in targeting tumor-suppressive proteins and pathways such as PTEN and TGFβ signaling. However, in the case of miR-19a and miR-19b, these have been shown to have a preponderant role in the cluster’s oncogenicity. Important studies have revealed the influence of the Myc proto-oncogene family in the transcriptional regulation of miR-17-92. Recent findings show that other oncogenic signaling pathways, such as those of Notch and Sonic Hedgehog, activate miR-17-92 in cancer. Notwithstanding, another layer of complexity has been added by the influence of the relevant primary miR-17-92 tertiary structure during processing to mature miRNA. In this review, we attempt to integrate current transcriptional and post-transcriptional knowledge to enhance our global understanding of the coordinated up-regulation of miR-17-92 in cancer.

MicroRNAs miR-146-5p and let-7f as prognostic tools for aggressive papillary thyroid carcinoma: a case report

Papillary thyroid cancer (PTC) is the most incident histotype of thyroid cancer. A certain fraction of PTC cases (5%) are irresponsive to conventional treatment, and refractory to radioiodine therapy. The current prognostic factors for aggressiveness are mainly based on tumor size, the presence of lymph node metastasis, extrathyroidal invasion and, more recently, the presence of the BRAFT1799A mutation. MicroRNAs (miRNAs) have been described as promising molecular markers for cancer as their deregulation is observed in a wide range of tumors. Recent studies indicate that the over-expression of miR-146b-5p is associated with aggressiveness and BRAFT1799A mutation. Furthermore, down-regulation of let-7f is observed in several types of tumors, including PTC. In this study, we evaluated the miR146b-5p and let-7f status in a young male patient with aggressive, BRAFT1799A-positive papillary thyroid carcinoma, with extensive lymph node metastases and short-time recurrence. The analysis of miR-146b-5p and let-7f expression revealed a distinct pattern from a cohort of PTC patients, suggesting caution in evaluating miRNA expression data as molecular markers of PTC diagnosis and prognosis.

MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells

BackgroundTumor invasiveness is directly related to the ability of tumor cells to migrate and invade surrounding tissues, usually degrading extracellular matrix. Despite significant progress in the knowledge about migration and invasion, there is much more to elucidate about their regulatory mechanisms, especially in cancer cells. MicroRNAs (miRs) were recently described as important regulators of migration. Differential expression of miRs in cancer is frequently associated with progression, invasion and metastasis. In papillary thyroid carcinoma (PTC), miR-146b-5p is highly expressed and positively correlated to the degree of malignancy.MethodsThis study aimed to investigate the role of miR-146b-5p on the migratory and invasive behaviors of thyroid cells, using a non tumor rat thyroid follicular cell line (PCCl3) transfected with the miR-146b-5p genomic region, and two PTC cell lines (TPC-1 and BCPAP, bearing distinct oncogenic backgrounds), which express high levels of miR-146b-5p, after miR-146b inhibition by antagomiR and miR-146b overexpression by mimics-miR. Migration and invasion were studied by time-lapse and transwell assays (with and without Matrigel®). Gelatin degradation assays were also employed, as well as F-actin staining.ResultsMigration and invasion of PCCl3 were increased 2-3x after miR-146b-5p overexpression (10X) and large lamellipodia were evident in those cells. After miR-146b-5p inhibition, TPC-1 and BCPAP migration and invasion were significantly reduced, with cells showing several simultaneous processes and low polarity. Gelatin degradation was inhibited in TPC-1 cells after inhibition of miR-146b-5p, but was unaffected in BCPAP cells, which did not degrade gelatin. The inhibition of miR-146b-5p in PCCl3 also inhibited migration and invasion, and additional (exogenous) overexpression of this miR in TPC-1 and BCPAP cells increased migration and invasion, without effects on cell morphology or gelatin degradation. The overexpression of SMAD4 in BCPAP cells, a validated target of miR-146b-5p and key protein in the TGF-β signaling pathway, inhibited migration similarly to the effects observed with the antagomiR 146b-5p.ConclusionsmiR-146b-5p positively regulates migration and invasion of thyroid normal and tumor follicular cells (independently from their original mutation, either BRAF or RET/PTC), through a mechanism that involves the actin cytoskeleton but not an increased capacity of matrix degradation.

Identification of Long Noncoding RNAs Deregulated in Papillary Thyroid Cancer and Correlated with BRAF V600E Mutation by Bioinformatics Integrative Analysis

Papillary Thyroid Cancer (PTC) is an endocrine malignancy in which BRAFV600E oncogenic mutation induces the most aggressive phenotype. In this way, considering that lncRNAs are arising as key players in oncogenesis, it is of high interest the identification of BRAFV600E-associated long noncoding RNAs, which can provide possible candidates for secondary mechanisms of BRAF-induced malignancy in PTC. In this study, we identified differentially expressed lncRNAs correlated with BRAFV600E in PTC and, also, extended the cohort of paired normal and PTC samples to more accurately identify differentially expressed lncRNAs between these conditions. Indirectly validated targets of the differentially expressed lncRNAs in PTC compared to matched normal samples demonstrated an involvement in surface receptors responsible for signal transduction and cell adhesion, as well as, regulation of cell death, proliferation and apoptosis. Targets of BRAFV600E-correlated lncRNAs are mainly involved in calcium signaling pathway, ECM-receptor interaction and MAPK pathway. In summary, our study provides candidate lncRNAs that can be either used for future studies related to diagnosis/prognosis or as targets for PTC management.

Osteoglycin inhibition by microRNA miR-155 impairs myogenesis

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80–90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3’UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3’UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.

Modulation of Deregulated MicroRNAs for Target Therapy in Thyroid Cancer

As life expectancy increases, the occurrence of cancer as a cause of mortality and morbidity becomes increasingly more relevant. Thyroid cancer is the most frequent endocrine malignancy, and worldwide incidence is rapidly increasing. Among different follicular thyroid cell-derived cancers, the papillary histotype is the most frequent, as well as being curable, followed by the follicular histotype, and finally, the rare and lethal anaplastic thyroid cancer histotype. However, some thyroid cancer histotypes, such as poorly-differentiated and anaplastic thyroid cancer, are refractory to the conventional therapies of surgical resection and radiotherapy. Currently available molecular therapy involves chemotherapy and treatment with inhibitors of the mitogen-activated protein kinase (MAPK) signaling but is not effective for blocking tumor progression and patient death. It is therefore important to develop an innovative molecular therapy to improve the available thyroid cancer therapies. MicroRNAs (miRNAs) are small endogenous non-coding RNAs (~22 nt) that act in the post-transcriptional regulation of mRNA levels through binding to mRNA 3′-untranslated region. Since miRNA deregulation is a feature of cancer, miRNAs emerge as molecules with intrinsic therapeutic properties, as they are able to exert a regulatory action over a plethora of cancer-related signaling pathways. In thyroid cancer, there is a common signature of deregulated miRNAs in the different histotypes of cancer. A set of over-expressed miRNAs such as miR-146b, miR-221, miR-222 and miR-17-92, and a set of under-expressed miRNAs of the let-7 family, may compose a miRNA signature associated with tumor aggressiveness that could be modulated as an innovative therapeutical approach. Indeed, in vitro studies show that ectopic modulation these miRNAs exerts important suppressive effects in thyroid cancer cell growth, affecting crucial pathways involved in proliferation, migration and invasion. Modulation of deregulated miRNAs in animal models has shown a promising perspective for a new targeted therapy, applicable for adjuvant molecular intervention in aggressive and refractory thyroid cancer, to try to improve the overall survival of these patients.

Nucleic acid recovery from thyroid fine-needle cytology slides.

M uch interest has been focused in nucleic acid isolation from tissue samples stored for long periods and their utilization on molecular biomarkers research. However, sample fixation induces chemical modifications in macromolecules (1) that result in a laborious and difficult protocol to extract nucleic acid for molecular analysis. In this context, the article recently published by Kizys and cols. (2) contributed to show efficient methods of nucleic acid recovery from archived formalin-fixed/ paraffin-embedded (FFPE) and fine-needle aspiration (FNA) samples of thyroid tissue. Despite the efforts, the issue that remains a challenge in the most effective protocols is to obtain better yield and quality of DNA/RNA due to the damaging effect of the fixation process, particularly for RNA analysis. In contrast with the difficulty in obtaining conserved messenger RNA from archived samples, the fraction of microRNA (miRNA) is less affected by fixation and storage time (3,4). Mature miRNA is a single-stranded noncoding small RNA of ~19-22 nucleotide length that regulates gene expression at the post-transcriptional level (5). These molecules pair to 3’UTRs of target mRNA and thereby cause their silencing or degradation. The miRNA might be a promising prognostic and diagnostic biomarker for thyroid cancer (6). In this context, we have performed, in archived FNA-stained slides, the analysis of BRAFT1799A mutation (7) along with miRNAs expression, miR-146b (8) and let7-f (9), which were modulated in papillary thyroid carcinoma (PTC) (10,11). The acid guanidinium thiocyanate-phenol-chloroform method (12) using TRIzolTM enabled the RNA/DNA co-isolation from the same archived FNA, despite the reduced number of cells in the cytological slides. From the extracted RNA, we performed real-time PCR to amplify miRNA, and observed high expression of miR-146b in PTC compared with goiter (normalized expression miR-146b/RNU6B: PTC 21.55 ± 5.83* vs. goiter 0.18 ± 0.06, * P < 0.05), and no change in let7-f expression (normalized expression let7-f /RNU6B: PTC 0.30 ± 0.13 vs. goiter 0.07 ± 0.02, ns). In addition, DNA obtained was suitable for BRAF mutation analysis by RFLP-PCR (Restriction Fragment Length Polymorphism -PCR) in all FNA slides tested. Using this approach, we observed that it is possible to access both nucleic acids, RNA and DNA, from the same archived thyroid FNA-stained slide. Furthermore, DNA was adequate to perform mutation analysis, and the quality of total RNA and its amount was satisfactory to investigate several miRNAs from the same sample. In conclusion, this procedure could be useful in detecting molecular biomarkers and drawing correlations with cytological analysis, which might improve the accuracy of FNA as a diagnostic tool of thyroid nodule. 1 Department of Cell and Developmental Biology, Institute of Biomedical Sciences, Universidade de Sao Paulo (USP), Sao Paulo, SP, Brazil

Functional Toll-like Receptor 4 Overexpression in Papillary Thyroid Cancer by MAPK/ERK-induced ETS1 Transcriptional Activity

Emerging evidence suggests that unregulated Toll-like receptor (TLR) signaling promotes tumor survival signals, thus favoring tumor progression. Here, the mechanism underlying TLR4 overexpression in papillary thyroid carcinomas (PTC) mainly harboring the BRAFV600E mutation was studied. TLR4 was overexpressed in PTC compared with nonneoplastic thyroid tissue. Moreover, paired clinical specimens of primary PTC and its lymph node metastasis showed a significant upregulation of TLR4 levels in the metastatic tissues. In agreement, conditional BRAFV600E expression in normal rat thyroid cells and mouse thyroid tissue upregulated TLR4 expression levels. Furthermore, functional TLR4 expression was demonstrated in PTC cells by increased NF-κB transcriptional activity in response to the exogenous TLR4-agonist lipopolysaccharide. Of note, The Cancer Genome Atlas data analysis revealed that BRAFV600E-positive tumors with high TLR4 expression were associated with shorter disease-free survival. Transcriptomic data analysis indicated a positive correlation between TLR4 expression levels and MAPK/ERK signaling activation. Consistently, chemical blockade of MAPK/ERK signaling abrogated BRAFV600E-induced TLR4 expression. A detailed study of the TLR4 promoter revealed a critical MAPK/ERK–sensitive Ets-binding site involved in BRAFV600E responsiveness. Subsequent investigation revealed that the Ets-binding factor ETS1 is critical for BRAFV600E-induced MAPK/ERK signaling-dependent TLR4 gene expression. Together, these data indicate that functional TLR4 overexpression in PTCs is a consequence of thyroid tumor-oncogenic driver dysregulation of MAPK/ERK/ETS1 signaling. Implications: Considering the participation of aberrant NF-κB signaling activation in the promotion of thyroid tumor growth and the association of high TLR4 expression with more aggressive tumors, this study suggests a prooncogenic potential of TLR4 downstream signaling in thyroid tumorigenesis. Mol Cancer Res; 16(5); 833–45. ©2018 AACR.

Osteoglycin post-transcriptional regulation by miR-155 induces cellular architecture changes in H9c2 cardiomyoblasts

Several studies have demonstrated dysregulated cardiac microRNAs (miRNAs) following cardiac stress and development of cardiac hypertrophy and failure. miRNAs are also differentially expressed in the inflammation that occurs in heart failure and, among these inflammatory-related miRNAs, the miR-155 has been implicated in the regulation of cardiac hypertrophy. Despite these data showing the role of miRNA-155 in cardiomyocyte hypertrophy under a hypertrophic stimulus, it is also important to understand the endogenous regulation of this miRNA without a hypertrophic stimulus to fully appreciate its function in this cell type. The first aim of the present study was to determine whether, without a hypertrophic stimulus, miR-155 overexpression induces H9c2 cardiac cells hypertrophy in vitro. The second objective was to determine whether osteoglycin (Ogn), a key regulator of heart mass in rats, mice, and humans, is post-transcriptionally regulated by miR-155 with a potential role in inducing H9c2 cells hypertrophy. Here, we show that, without a hypertrophic stimulus, miR-155 significantly repressed Ogn protein levels, but induce neither alteration in morphological phenotype nor in the expression of the molecular markers that fully characterize pathological hypertrophy of H9c2 cells. However, most importantly, Ogn silencing in H9c2 cells mimicked the effects of miR-155 overexpression in inducing cellular architecture changes that were characterized by a transition of the cell shape from fusiform to rounded. This is a new role of the post-transcriptional regulation of Ogn by miR-155 in the maintenance of the cardiac cell morphology in physiological and pathological conditions.

Abstract 5210: MicroRNA miR-17-92 impairs TGFβ signaling responsiveness in BRAF oncogene-activated thyroid cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Thyroid follicular cells proliferation is regulated by TGFβ anti-mitogenic signal. However, thyroid cancer cells become refractory to this inhibitory signal. TGFβ signaling deregulation is observed in thyroid cancer, which shows high frequence of MAPK genetic alteration. BRAF mutation (BRAFT1799A), the most prevalent alteration in thyroid cancer, induces MAPK activtion and thyroid oncogenesis. In this process, deregulation of microRNAs potentiates oncogene activation. MicroRNAs are small non-coding RNAs that regulate protein levels by pairing to 3-UTR of mRNA and blocking translation. miR-17-92 cluster of microRNAs, miR-17-5p/3p, miR-18a, miR-19a/b, miR20a and miR-92a, regulates several tumor-suppressor genes and is predicted to target TGFβ pathway components. Therefore, we aim at analyzing the influence of miR-17-92 in TGFβ signaling during oncogene activation in thyroid follicular cells and its implication for thyroid cancer biology. Methods: The BRAF9-6 cells, derived from normal rat thyroid follicular cell PCCl3, express BRAFT1799A under doxicyclin (dox) stimulation. The PCCl3-miR-17-92 cells, derived from PCCl3 cells, over-express miR-17-92 cluster (pCDNA3.1-Neo). PCCl3- is the control cell transfected with empty plasmid. MiR-17-92 expression was detected by real-time qPCR. Cell viability was measured using MTT assay and proliferation by cell counting (24-72h). TGFβ signaling components, Smad4 and Tgfbr2, protein levels was analyzed by Western-blotting. TGFβ responsiveness assay was analyzed by luciferase gene reporter assay after transfection of 3TP-lux plasmid and treatment with 1-2ng/mL recombinant TGFβ for 24 hours. Results: BRAF-oncogene activation (dox) in thyroid follicular cells induced a high expression of miR-17-92 cluster components and reduced Smad4 and Tgfbr2 protein levels. In order to analyze the particular influence of miR-17-92 in TGFβ signaling, we constructed a cell line, PCCl3-miR-17-92, that over-expressed miR-17-92 in normal thyroid follicular cells. Over-expression of miR-17-92 inhibited Tgfbr2 and Smad4 protein levels that reflected in impaired responsiveness to recombinant TGFβ inhibitory signal. Using the 3TP-lux reporter gene assay, that contains TGFβ signaling activation responsive sequences, we observed that recombinant TGFβ treatment in PCCl3- cells resulted in a 8.0 and 12.3-fold increase in luciferase activity at concentrations of 1ng/ml and 2ng/mL of recombinant TGFβ, respectively, while a 4.3 and 5.6-fold increase is observed in PCCl3-miR-17-92 cells. Moreover, PCCl3-miR-17-92 showed increases cell proliferation and cell viability in 18% and 9%, respectively, in comparison to PCCL3-. Conclusion: miR-17-92 over-expression impairs normal thyroid follicular cells responsiveness to TGFβ and increases cell proliferation and viability, which could imply in TGFβ inhibitory signal refractoriness observed in thyroid cancer cells. Citation Format: Cesar Seigi Fuziwara, Felipe E. Martins, Edna T. Kimura. MicroRNA miR-17-92 impairs TGFβ signaling responsiveness in BRAF oncogene-activated thyroid cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5210. doi:10.1158/1538-7445.AM2014-5210