Bartosz Wojtas

Differential miRNA expression defines migration and reduced apoptosis in follicular thyroid carcinomas.

The objective of the study was to identify microRNAs (miRs) characteristic for follicular thyroid carcinoma (FTC) and to define their role in tumorigenesis. A miR-microarray study was conducted to identify miRs differentially expressed between FTCs and their surrounding tissues. Selection was further reinforced by a literature review. Four miRs were selected and confirmed by RT-qPCR: miR-146b, -183, -221 were up-regulated, whereas miR-199b down-regulated in FTCs. The influence of these miRs on cell proliferation, cell cycle, apoptosis and migration was studied in HTori and FTC-133 cells. Functional characterization suggests an impact of miR-183 and miR-146b in FTC development. Overexpression of both miRs significantly induces migration. Moreover, overexpression of miR-183 significantly represses apoptosis. MiR-199b and -221 do not have significant effects on proliferation, cell cycle, apoptosis or migration in HTori and FTC-133 cells. Our data suggest that miR-146b and miR-183 may influence FTC development through the induction of migration and apoptosis inhibition.

Inverse correlation of miRNA and cell cycle-associated genes suggests influence of miRNA on benign thyroid nodule tumorigenesis.

CONTEXT The molecular etiology of cold and benign thyroid nodules (CBTNs) is largely unknown. Increased thyroid epithelial cell proliferation is a hallmark of CBTNs. MicroRNAs (miRNAs) are prominent regulators of cell proliferation. OBJECTIVE Our objective was to assess the influence of miRNAs on the increased proliferation and thus the molecular etiology of CBTNs. DESIGN By using microarrays, we defined the molecular pattern of increased proliferation of CBTNs as a differential expression of cell-cycle-associated genes and miRNAs. In silico integration of differentially expressed miRNAs and mRNAs showed an inverse correlation between the expression of 59 miRNAs and 133 mRNAs. Inverse correlations between cell-cycle-associated genes such as CDKN1C and miR-221, CCND1 and miR-31, GADD45A and miR-130b, or CDKN1A and let-7f suggest a modulation of proliferation in CBTNs by miRNAs. Their expression was validated using quantitative RT-PCR and functionally characterized in cell line models. RESULTS Comparative quantitative RT-PCR of 20 samples of CBTNs and their surrounding tissue revealed an 11-fold down-regulation of miR-31 with a 2.6-fold up-regulation of CCND1, and a 2.6-fold up-regulation of miR-130b with a 2.3-fold down-regulation of its target GADD45A. Using HTori and FTC-133 cell lines, we analyzed proliferation, cell cycle, and apoptosis after transfection of miRNA-31 and miRNA-130b mimic and inhibitors. Overexpression of miR-31 and the resultant down-regulation of CCND1 led to an arrest in the cell cycle phase G1. Overexpression of miR-130b led to an increase of apoptosis and necrosis within 72 h. CONCLUSION miR-31 and miR-130b may have an effect on tumorigenesis of CBTNs by regulating proliferation and apoptosis and the cell cycle through cyclin D1.

BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells

Glioblastoma (GBM) contains rare glioma stem-like cells (GSCs) with capacities of self-renewal, multi-lineage differentiation, and resistance to conventional therapy. Drug-induced differentiation of GSCs is recognized as a promising approach of anti-glioma therapy. Accumulating evidence suggests that unique properties of stem cells depend on autophagy. Here we demonstrate that BIX01294, an inhibitor of a G9a histone methyltransferase (introducing H3K9me2 and H3K27me3 repressive marks) triggers autophagy in human glioma cells. Pharmacological or genetic inhibition of autophagy decreased LC3-II accumulation and GFP-LC3 punctation in BIX01294-treated cells. GSCs-enriched spheres originating from glioma cells and GBM patient-derived cultures express lower levels of autophagy related (ATG) genes than the parental glioma cell cultures. Typical differentiation inducers that upregulate neuronal and astrocytic markers in sphere cultures, increase the level of ATG mRNAs. G9a binds to the promoters of autophagy (LC3B, WIPI1) and differentiation-related (GFAP, TUBB3) genes in GSCs. Higher H3K4me3 (an activation mark) and lower H3K9me2 (the repressive mark) levels at the promoters of studied genes were detected in serum-differentiated cells than in sphere cultures. BIX01294 treatment upregulates the expression of autophagy and differentiation-related genes in GSCs. Pharmacological inhibition of autophagy decreases GFAP and TUBB3 expression in BIX01294-treated GSCs suggesting that BIX01294-induced differentiation of GSCs is autophagy-dependent.

Molecular differential diagnosis of follicular thyroid carcinoma and adenoma based on gene expression profiling by using formalin-fixed paraffin-embedded tissues

BackgroundDifferential diagnosis between malignant follicular thyroid cancer (FTC) and benign follicular thyroid adenoma (FTA) is a great challenge for even an experienced pathologist and requires special effort. Molecular markers may potentially support a differential diagnosis between FTC and FTA in postoperative specimens. The purpose of this study was to derive molecular support for differential post-operative diagnosis, in the form of a simple multigene mRNA-based classifier that would differentiate between FTC and FTA tissue samples.MethodsA molecular classifier was created based on a combined analysis of two microarray datasets (using 66 thyroid samples). The performance of the classifier was assessed using an independent dataset comprising 71 formalin-fixed paraffin-embedded (FFPE) samples (31 FTC and 40 FTA), which were analysed by quantitative real-time PCR (qPCR). In addition, three other microarray datasets (62 samples) were used to confirm the utility of the classifier.ResultsFive of 8 genes selected from training datasets (ELMO1, EMCN, ITIH5, KCNAB1, SLCO2A1) were amplified by qPCR in FFPE material from an independent sample set. Three other genes did not amplify in FFPE material, probably due to low abundance. All 5 analysed genes were downregulated in FTC compared to FTA. The sensitivity and specificity of the 5-gene classifier tested on the FFPE dataset were 71% and 72%, respectively.ConclusionsThe proposed approach could support histopathological examination: 5-gene classifier may aid in molecular discrimination between FTC and FTA in FFPE material.

Differences in the transcriptome of medullary thyroid cancer regarding the status and type of RET gene mutations

Medullary thyroid cancer (MTC) can be caused by germline mutations of the RET proto-oncogene or occurs as a sporadic form. It is well known that RET mutations affecting the cysteine-rich region of the protein (MEN2A-like mutations) are correlated with different phenotypes than those in the kinase domain (MEN2B-like mutations). Our aim was to analyse the whole-gene expression profile of MTC with regard to the type of RET gene mutation and the cancer genetic background (hereditary vs sporadic). We studied 86 MTC samples. We demonstrated that there were no distinct differences in the gene expression profiles of hereditary and sporadic MTCs. This suggests a homogeneous nature of MTC. We also noticed that the site of the RET gene mutation slightly influenced the gene expression profile of MTC. We found a significant association between the localization of RET mutations and the expression of three genes: NNAT (suggested to be a tumour suppressor gene), CDC14B (involved in cell cycle control) and NTRK3 (tyrosine receptor kinase that undergoes rearrangement in papillary thyroid cancer). This study suggests that these genes are significantly deregulated in tumours with MEN2A-like and MEN2B-like mutations; however, further investigations are necessary to demonstrate any clinical impact of these findings.

Differences in miRNA and mRNA Profile of Papillary Thyroid Cancer Variants

Papillary thyroid cancer (PTC) can be divided into classical variant of PTC (cPTC), follicular variant of PTC (fvPTC), and tall cell variant (tcPTC). These variants differ in their histopathology and cytology; however, their molecular background is not clearly understood. Our results shed some new light on papillary thyroid cancer biology as new direct miRNA-gene regulations are discovered. The Cancer Genome Atlas (TCGA) 466 thyroid cancer samples were studied in parallel datasets to discover potential miRNA-mRNA regulations. Additionally, miRNAs and genes differentiating PTC variants (cPTC, fvPTC, and tcPTC) were indicated. Putative miRNA regulatory pairs were discovered: hsa-miR-146b-5p with PHKB and IRAK1, hsa-miR-874-3p with ITGB4 characteristic for classic PTC samples, and hsa-miR-152-3p with TGFA characteristic for follicular variant PTC samples. MiRNA-mRNA regulations discovery opens a new perspective in understanding of PTC biology. Furthermore, our successful pipeline of miRNA-mRNA regulatory pathways discovery could serve as a universal tool to find new miRNA-mRNA regulations, also in different datasets.

Somatic mutation profiling of follicular thyroid cancer by next generation sequencing

The molecular etiology of follicular thyroid tumors is largely unknown, rendering the diagnostics of these tumors challenging. The somatic alterations present in these tumors apart from RAS gene mutations and PAX8/PPARG translocations are not well described. To evaluate the profile of somatic alteration in follicular thyroid tumors, a total of 82 thyroid tissue samples derived from 48 patients were subjected to targeted Illumina HiSeq next generation sequencing of 372 cancer-related genes. New somatic alterations were identified in oncogenes (MDM2, FLI1), transcription factors and repressors (MITF, FLI1, ZNF331), epigenetic enzymes (KMT2A, NSD1, NCOA1, NCOA2), and protein kinases (JAK3, CHEK2, ALK). Single nucleotide and large structural variants were most and least frequently identified, respectively. A novel translocation in DERL/COX6C was detected. Many somatic alterations in non-coding gene regions with high penetrance were observed. Thus, follicular thyroid tumor somatic alterations exhibit complex patterns. Most tumors contained distinct somatic alterations, suggesting previously unreported heterogeneity.

Two-miRNA classifiers differentiate mutation-negative follicular thyroid carcinomas and follicular thyroid adenomas in fine needle aspirations with high specificity

Diagnosis of thyroid by fine needle aspiration is challenging for the “indeterminate” category and can be supported by molecular testing. We set out to identify miRNA markers that could be used in a diagnostic setting to improve the discrimination of mutation-negative indeterminate fine needle aspirations. miRNA high-throughput sequencing was performed for freshly frozen tissue samples of 19 RAS and PAX8/PPARG mutation-negative follicular thyroid carcinomas, and 23 RAS and PAX8/PPARG mutation-negative follicular adenomas. Differentially expressed miRNAs were validated by quantitative polymerase chain reaction in a set of 44 fine needle aspiration samples representing 24 follicular thyroid carcinomas and 20 follicular adenomas. Twenty-six miRNAs characterized by a significant differential expression between follicular thyroid carcinomas and follicular adenomas were identified. Nevertheless, since no single miRNA had satisfactory predictive power, classifiers comprising two differentially expressed miRNAs were designed with the aim to improve the classification. Six two-miRNA classifiers were established and quantitative polymerase chain reaction validated in fine needle aspiration samples. Four out of six classifiers were characterized by a high specificity (≥94 %). The best two-miRNA classifier (miR-484/miR-148b-3p) identified thyroid malignancy with a sensitivity of 89 % and a specificity of 87 %. The high-throughput sequencing allowed the identification of subtle differences in the miRNA expression profiles of follicular thyroid carcinomas and follicular adenomas. While none of the differentially expressed miRNAs could be used as a stand-alone malignancy marker, the validation results for two-miRNA classifiers in an independent set of fine needle aspirations are very promising. The ultimate evaluation of these classifiers for their capability of discriminating mutation-negative indeterminate fine needle aspirations will require the evaluation of a sufficiently large number of fine needle aspirations with histological confirmation.

The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma.

Osteopontin (SPP1, a secreted phosphoprotein 1) is primarily involved in immune responses, tissue remodelling and biomineralization. However, it is also overexpressed in many cancers and regulates tumour progression by increasing migration, invasion and cancer stem cell self-renewal. Mechanisms of SPP1 overexpression in gliomas are poorly understood. We demonstrate overexpression of two out of five SPP1 isoforms in glioblastoma (GBM) and differential isoform expression in glioma cell lines. Up-regulated SPP1 expression is associated with binding of the GLI1 transcription factor to the promoter and OCT4 (octamer-binding transcription factor 4) to the first SPP1 intron of the SPP1 gene in human glioma cells but not in non-transformed astrocytes. GLI1 knockdown reduced SPP1 mRNA and protein levels in glioma cells. GLI1 and OCT4 are known regulators of stem cell pluripotency. GBMs contain rare cells that express stem cell markers and display ability to self-renew. We reveal that SPP1 is overexpressed in glioma initiating cells defined by high rhodamine 123 efflux, sphere forming capacity and stemness marker expression. Forced differentiation of human glioma spheres reduced SPP1 expression. Knockdown of SPP1, GLI1 or CD44 with siRNAs diminished sphere formation. C6 glioma cells stably depleted of Spp1 displayed reduced sphere forming capacity and downregulated stemness marker expression. Overexpression of the wild type Spp1, but not Spp1 lacking a Cd44 binding domain, rescued cell ability to form spheres. Our findings show re-activation of the embryonic-type transcriptional regulation of SPP1 in malignant gliomas and point to the importance of SPP1-CD44 interactions in self-renewal and pluripotency glioma initiating cells.

Gene Expression (mRNA) Markers for Differentiating between Malignant and Benign Follicular Thyroid Tumours

Distinguishing between follicular thyroid cancer (FTC) and follicular thyroid adenoma (FTA) constitutes a long-standing diagnostic problem resulting in equivocal histopathological diagnoses. There is therefore a need for additional molecular markers. To identify molecular differences between FTC and FTA, we analyzed the gene expression microarray data of 52 follicular neoplasms. We also performed a meta-analysis involving 14 studies employing high throughput methods (365 follicular neoplasms analyzed). Based on these two analyses, we selected 18 genes differentially expressed between FTA and FTC. We validated them by quantitative real-time polymerase chain reaction (qRT-PCR) in an independent set of 71 follicular neoplasms from formaldehyde-fixed paraffin embedded (FFPE) tissue material. We confirmed differential expression for 7 genes (CPQ, PLVAP, TFF3, ACVRL1, ZFYVE21, FAM189A2, and CLEC3B). Finally, we created a classifier that distinguished between FTC and FTA with an accuracy of 78%, sensitivity of 76%, and specificity of 80%, based on the expression of 4 genes (CPQ, PLVAP, TFF3, ACVRL1). In our study, we have demonstrated that meta-analysis is a valuable method for selecting possible molecular markers. Based on our results, we conclude that there might exist a plausible limit of gene classifier accuracy of approximately 80%, when follicular tumors are discriminated based on formalin-fixed postoperative material.