- [Show abstract] [Hide abstract] ABSTRACT: Context: Long non-coding RNAs (lncRNAs) regulate pathological processes, yet their potential roles in papillary thyroid carcinoma (PTC) are poorly understood. Objective: To profile transcriptionally dysregulated lncRNAs in PTC and identify lncRNAs associated with clinicopathological characteristics. Design: We performed RNA sequencing of 12 paired PTC tumors and matched noncancerous tissues and correlated the expression of lncRNAs with clinical parameters. The 2 most significantly dysregulated lncRNAs were studied in an Ohio PTC cohort (n=109) and in PTC data (n=497) from TCGA. Setting: A combination of laboratory-based studies and computational analysis using clinical data and samples and a publically available database. Main outcome measures: Correlation between expression values and clinical parameters. Results: We identified 218 lncRNAs showing differential expression in PTC (fold change ≥2.0, p value <0.01). Significant correlation was observed between the expression of two lncRNAs (XLOC_051122 and XLOC_006074) and (i) lymph node metastasis (N stage) and (ii) BRAF (V600E) mutation. Among patients with wild type BRAF, the expression of these 2 lncRNAs showed significantly higher levels in the patients with lymph node metastasis. In silico analysis of these lncRNAs pinpointed cell movement and cellular growth and proliferation as targeted functions. Conclusions: Comprehensive expression screening identified 2 novel lncRNAs associated with risk factors of adverse prognosis in PTC patients. These lncRNAs may be novel players in PTC carcinogenesis.
- [Show abstract] [Hide abstract] ABSTRACT: Chromosomal aberrations and multiple genome-wide association studies (GWASs) have established a major hematopoietic quantitative trait locus in chromosome 6q23.3. The locus comprises an active enhancer region, in which some of the associated SNPs alter transcription factor binding. We now identify microRNA-3662 as a new functional driver contributing to the associated phenotypes. The GWAS SNPs are strongly associated with higher miR-3662 expression. Genome editing of rs66650371, a three base pair deletion, suggests a functional link between the SNP genotype and the abundance of miR-3662. Increasing miR-3662's abundance increases colony formation in hematopoietic progenitor cells, particularly the erythroid lineage. In contrast, miR-3662 is not expressed in acute myeloid leukemia cells and its overexpression has potent anti-leukemic effects in vitro and in vivo. Mechanistically, miR-3662 directly targets NF-ĸB-mediated transcription. Thus, miR-3662 is a new player of the hematopoietic 6q23.3 locus.
- [Show abstract] [Hide abstract] ABSTRACT: Background: Papillary thyroid cancer (PTC) is reported to be highly heritable in epidemiological studies. Genome-wide association studies (GWAS) have uncovered several variants associated with PTC predisposition. It remains unknown whether these variants might contribute to better clinical stratification of PTC patients. Methods: In order to assess the usefulness of germline genetics analyses in the management of PTC patients we determined the genotypes of 5 variants (rs965513, rs944289, rs116909374, rs2439302 and rs966423) in 1216 PTC patients and 1416 controls. Additionally, we measured the expression of 7 genes located close to GWAS variants (PTCSC3, MBIP, NKX2-1, FOXE1, DIRC3, PTCSC2 and NRG1) in 73 PTC paired tumor/normal tissues, respectively. Next, we analyzed the association between the genotypes of the germline variants and the levels of gene expression with clinical/pathological features such as age, gender, TNM staging, multifocality status, extrathyroidal expansion and MACIS score. Results: The risk allele of rs965513 was associated with larger tumor size (p=0.025) and extrathyroidal expansion (OR=1.29, p=0.045). The variant rs2439302 showed association with lymph node metastasis (OR=1.24, p=0.016, and multifocality status of the tumor (OR=1.24, p=0.012). The expression of MBIP was associated with T stage (p=0.010). MBIP and PTCSC3 displayed lower expression in PTC tissue in males than in females (p=0.025 and p=0.036, respectively). NKX2-1 displayed lower expression in patients with N1 stage (p=0.040). Conclusions: The risk alleles of germline variants predisposing to PTC were associated with a more aggressive course of the disease reflected by larger tumor diameter, higher multifocality rate and more advanced N stage at the time of diagnosis. Our results show that germline variants not only predispose to PTC but also might impact its clinical course. However, the germline-clinical associations were only moderate and further large multi-ethnic studies are required to evaluate the usefulness of germline variant assessment in the clinical stratification of PTC patients.
- [Show abstract] [Hide abstract] ABSTRACT: Skeletal muscle growth immediately following birth is critical for proper body posture and locomotion. However, compared with embryogenesis and adulthood, the processes regulating the maturation of neonatal muscles is considerably less clear. Studies in the 1960s predicted that neonatal muscle growth results from nuclear accretion of myoblasts preferentially at the tips of myofibers. Remarkably, little information has been added since then to resolve how myoblasts migrate to the ends of fibers. Here, we provide insight into this process by revealing a unique NF-κB-dependent communication between NG2+ interstitial cells and myoblasts. NF-κB in NG2+ cells promotes myoblast migration to the tips of myofibers through cell-cell contact. This occurs through expression of ephrinA5 from NG2+ cells, which we further deduce is an NF-κB target gene. Together, these results suggest that NF-κB plays an important role in the development of newborn muscles to ensure proper myoblast migration for fiber growth.
Dataset: S2 Fig[Show abstract] [Hide abstract] ABSTRACT: qPCR reaction in paired tumor and unaffected tissue from 8 PTC patients. (PDF)
- [Show abstract] [Hide abstract] ABSTRACT: The main nonmedullary form of thyroid cancer is papillary thyroid carcinoma (PTC) that accounts for 80-90% of all thyroid malignancies. Only 3-10% of PTC patients have a positive family history of PTC yet the familiality is one of the highest of all cancers as measured by case control studies. A handful of genes have been implicated accounting for a small fraction of this genetic predisposition. It was therefore of considerable interest that a mutation in the HABP2 gene was recently implicated in familial PTC. The present work was undertaken to examine the extent of HABP2 variant involvement in PTC. The HABP2 G534E variant (rs7080536) was genotyped in blood DNA from 179 PTC families (one affected individual per family), 1160 sporadic PTC cases and 1395 controls. RNA expression of HABP2 was tested by qPCR in RNA extracted from tumor and normal thyroid tissue from individuals that are homozygous wild-type or heterozygous for the variant. The variant was found to be present in 6.1% familial cases, 8.0% sporadic cases (2 individuals were homozygous for the variant) and 8.7% controls. The variant did not segregate with PTC in one large and 6 smaller families in which it occurred. In keeping with data from the literature and databases the expression of HABP2 was highest in the liver, much lower in 3 other tested tissues (breast, kidney, brain) but not found in thyroid. Given these results showing lack of any involvement we suggest that the putative role of variant HABP2 in PTC should be carefully scrutinized.
Dataset: S1 Table[Show abstract] [Hide abstract] ABSTRACT: Clinical and demographic information on cases and controls. (PDF)
Dataset: S3 Fig[Show abstract] [Hide abstract] ABSTRACT: qPCR reaction in normal kidney, liver, breast and brain. (PDF)
Dataset: S4 Fig[Show abstract] [Hide abstract] ABSTRACT: qPCR reaction using 2 different qPCR assays in normal kidney, liver, breast and brain. (PDF)
Dataset: S1 Fig[Show abstract] [Hide abstract] ABSTRACT: Pedigrees of 7 families genotyped for the HABP2 G534E (c.1601G>A) variant in available samples. (PDF)
- [Show abstract] [Hide abstract] ABSTRACT: Background Traditionally, the CD56dimCD16+ subset of Natural Killer (NK) cells has been thought to mediate cellular cytotoxicity with modest cytokine secretion capacity. However, studies have suggested that this subset may exert a more diverse array of immunological functions. There exists a lack of well-developed functional models to describe the behavior of activated NK cells, and the interactions between signaling pathways that facilitate effector functions are not well understood. In the present study, a combination of genome-wide microarray analyses and systems-level bioinformatics approaches were utilized to elucidate the transcriptional landscape of NK cells activated via interactions with antibody-coated targets in the presence of interleukin-12 (IL-12). Methods We conducted differential gene expression analysis of CD56dimCD16+ NK cells following FcR stimulation in the presence or absence of IL-12. Next, we functionally characterized gene sets according to patterns of gene expression and validated representative genes using RT-PCR. IPA was utilized for biological pathway analysis, and an enriched network of interacting genes was generated using GeneMANIA. Furthermore, PAJEK and the HITS algorithm were employed to identify important genes in the network according to betweeness centrality, hub, and authority node metrics. Results Analyses revealed that CD56dimCD16+ NK cells co-stimulated via the Fc receptor (FcR) and IL-12R led to the expression of a unique set of genes, including genes encoding cytotoxicity receptors, apoptotic proteins, intracellular signaling molecules, and cytokines that may mediate enhanced cytotoxicity and interactions with other immune cells within inflammatory tissues. Network analyses identified a novel set of connected key players, BATF, IRF4, TBX21, and IFNG, within an integrated network composed of differentially expressed genes in NK cells stimulated by various conditions (immobilized IgG, IL-12, or the combination of IgG and IL-12). Conclusions These results are the first to address the global mechanisms by which NK cells mediate their biological functions when encountering antibody-coated targets within inflammatory sites. Moreover, this study has identified a set of high-priority targets for subsequent investigation into strategies to combat cancer by enhancing the anti-tumor activity of CD56dimCD16+ NK cells.
- [Show abstract] [Hide abstract] ABSTRACT: The B-Raf proto-oncogene serine/threonine kinase (BRAF) gene is the most frequently mutated gene in malignant melanoma (MM) and papillary thyroid cancer (PTC) and is causally involved in malignant cell transformation. Mutated BRAF is associated with an aggressive disease phenotype, thus making it a top candidate for targeted treatment strategies in MM and PTC. We show that BRAF mutations in both MM and PTC drive increased expression of oncomiR-3151, which is coactivated by the SP1/NF-κB complex. Knockdown of microRNA-3151 (miR-3151) with short hairpin RNAs reduces cell proliferation and increases apoptosis of MM and PTC cells. Using a targeted RNA sequencing approach, we mechanistically determined that miR-3151 directly targets TP53 and other members of the TP53 pathway. Reducing miR-3151's abundance increases TP53's mRNA and protein expression and favors its nuclear localization. Consequently, knockdown of miR-3151 also leads to caspase-3-dependent apoptosis. Simultaneous inhibition of aberrantly activated BRAF and knockdown of miR-3151 potentiates the effects of sole BRAF inhibition with the BRAF inhibitor vemurafenib and may provide a novel targeted therapeutic approach in BRAF-mutated MM and PTC patients. In conclusion, we identify miR-3151 as a previously unidentified player in MM and PTC pathogenesis, which is driven by BRAF-dependent and BRAF-independent mechanisms. Characterization of TP53 as a downstream effector of miR-3151 provides evidence for a causal link between BRAF mutations and TP53 inactivation.
- [Show abstract] [Hide abstract] ABSTRACT: We previously showed that a long non-coding RNA (lncRNA) gene (PTCSC3) located close to the variant rs944289 that predisposes to papillary thyroid carcinoma (PTC) might target the S100A4 gene. The aim was to investigate the impact of PTCSC3 on S100A4 expression and its role in cancer development. S100A4 abundance was analyzed by qPCR in unaffected and tumor tissue from n=73 PTC patients. The expression of PTCSC3 and S100A4 was studied in BCPAP and TPC-1 cell lines with forced expression of PTCSC3 by quantitative PCR (qPCR). Expression of S100A4 target genes (VEGF and MMP-9) was studied in the BCPAP cell lines with forced expression of PTCSC3 by qPCR, reverse transcriptase PCR and Western blot. The impact of PTCSC3 on BCPAP motility and invasiveness was analyzed by the Transwell and Matrigel assays, respectively. This was a laboratory-based study using cells from clinical samples and thyroid cancer cell lines. Evidence for a link between the expression of PTCSC3 and thyroid carcinogenesis. Expression data from PTC cell lines pinpointed S100A4 as the most significantly downregulated gene in the presence of PTCSC3. S100A4 was upregulated in tumor tissue (p=9.33x10(-7)) while PTCSC3 was strongly downregulated (p=2.2x10(-16)). S100A4 transcription was moderately correlated with PTCSC3 expression in unaffected thyroid tissue (r=0.429, p=0.0001), and strongly in unaffected tissue of patients with the risk allele of rs944289 (r=0.685, p=7.88x10(-5)). S100A4, VEGF and MMP-9 were suppressed in the presence of PTCSC3 (p=0.0051, p=0.0090 and p=0.0037, respectively). PTC cells expressing PTCSC3 showed reduction in motility and invasiveness (p=4.52x10(-5) and p=1.0x10(-4), respectively). PTCSC3 downregulates S100A4 leading to a reduction in cell motility and invasiveness. We propose that PTCSC3 impacts PTC predisposition and carcinogenesis through the S100A4 pathway.
- [Show abstract] [Hide abstract] ABSTRACT: Papillary thyroid carcinoma (PTC) displays strong but so far largely uncharacterized heritability. Here we studied genetic predisposition in a family with six affected individuals. We genotyped all available family members and conducted whole exome sequencing of blood DNA from two affected individuals. Haplotype analysis and other genetic criteria narrowed our list of candidates to a germline variant in the serine/arginine repetitive matrix 2 gene (SRRM2). This heterozygous variant, c.1037C > T (Ser346Phe or S346F; rs149019598) cosegregated with PTC in the family. It was not found in 138 other PTC families. It was found in 7/1,170 sporadic PTC cases and in 0/1,404 controls (p = 0.004). The encoded protein SRRM2 (also called SRm300) is part of the RNA splicing machinery. To evaluate the possibility that the S346F missense mutation affects alternative splicing, we compared RNA-Seq data in leukocytes from three mutation carriers and three controls. Significant differences in alternative splicing were identified for 1,642 exons, of which a subset of 7 exons was verified experimentally. The results confirmed a higher ratio of inclusion of exons in mutation carriers. These data suggest that the S346F mutation in SRRM2 predisposes to PTC by affecting alternative splicing of unidentified downstream target genes.
- [Show abstract] [Hide abstract] ABSTRACT: The [A] allele of SNP rs965513 in 9q22 has been consistently shown to be highly associated with increased papillary thyroid cancer (PTC) risk with an odds ratio of ∼1.8 as determined by genome-wide association studies, yet the molecular mechanisms remain poorly understood. Previously, we noted that the expression of two genes in the region, forkhead box E1 (FOXE1) and PTC susceptibility candidate 2 (PTCSC2), is regulated by rs965513 in unaffected thyroid tissue, but the underlying mechanisms were not elucidated. Here, we fine-mapped the 9q22 region in PTC and controls and detected an ∼33-kb linkage disequilibrium block (containing the lead SNP rs965513) that significantly associates with PTC risk. Chromatin characteristics and regulatory element signatures in this block disclosed at least three regulatory elements functioning as enhancers. These enhancers harbor at least four SNPs (rs7864322, rs12352658, rs7847449, and rs10759944) that serve as functional variants. The variant genotypes are associated with differential enhancer activities and/or transcription factor binding activities. Using the chromosome conformation capture methodology, long-range looping interactions of these elements with the promoter region shared by FOXE1 and PTCSC2 in a human papillary thyroid carcinoma cell line (KTC-1) and unaffected thyroid tissue were found. Our results suggest that multiple variants coinherited with the lead SNP and located in long-range enhancers are involved in the transcriptional regulation of FOXE1 and PTCSC2 expression. These results explain the mechanism by which the risk allele of rs965513 predisposes to thyroid cancer.
- [Show abstract] [Hide abstract] ABSTRACT: Context: By genome-wide association studies (GWAS) the risk allele [A] of SNP rs965513 predisposes strongly to papillary thyroid carcinoma (PTC). It is located in a gene-poor region of 9q22 some 60kb from the FOXE1 gene. The underlying mechanisms remain to be discovered. Objective: Our objective was to identify novel transcripts in the 9q22 locus and correlate gene expression levels with the genotypes of rs965513. Design: We performed 3' and 5' RACE and RT-PCR to detect novel transcripts. One novel transcript was forcibly expressed in a cell line followed by gene expression array analysis. We genotyped rs965513 from PTC patients and measured gene expression levels by real time RT-PCR in unaffected thyroid tissue and matched tumor. Setting: This was a laboratory-based study using cells from clinical tissue samples and a cancer cell line. Main Outcome Measures: We detected previously uncharacterized transcripts and evaluated the gene expression levels and the correlation with the risk allele of rs965513, age, gender, chronic lymphocyte thyroiditis (CLT), and TSH levels. Results: We found a novel long intergenic noncoding RNA (lincRNA) gene and named it papillary thyroid cancer susceptibility candidate 2 (PTCSC2). Transcripts of PTCSC2 are downregulated in PTC tumors. The risk allele [A] of rs965513 was significantly associated with low expression of unspliced PTCSC2, FOXE1 and TSHR in unaffected thyroid tissue. We also observed a significant association of age and CLT with PTCSC2 unspliced transcript levels. The correlation between the rs965513 genotype and the PTCSC2 unspliced transcript levels remained significant after adjusting for age, gender, and CLT. Forced expression of PTCSC2 in the BCPAP cell line affected the expression of a subset of non-coding and coding transcripts with enrichment of genes functionally involved in cell cycle and cancer. Conclusions: Our data suggest a role for PTCSC2, FOXE1, and TSHR in the predisposition to PTC.
- [Show abstract] [Hide abstract] ABSTRACT: Neuroblastoma rat sarcoma (RAS) viral oncogene homolog (NRAS), a small GTPase, is one of the most thoroughly studied oncogenes that controls cell growth, differentiation, and survival by facilitating signal transduction. Here, we identify four novel naturally occurring NRAS isoforms (isoforms 2-5) in addition to the canonical isoform (isoform 1). Expression analyses performed on a panel of several different human malignancies and matching normal tissue revealed distinct isoform expression patterns. Two of the novel isoforms were found in the nucleus and cytoplasm, whereas the others were exclusively cytoplasmic. The isoforms varied in their binding affinities to known downstream targets and differentially regulated the RAS signaling pathway. Strikingly, forced expression of isoform 5, which encodes only a 20-aa peptide, led to increased cell proliferation and to transformation by activation of known NRAS targets. These discoveries open new avenues in the study of NRAS.