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Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid gland and early stages are curable. However, a subset of PTCs shows an unusually aggressive phenotype with extensive lymph node metastasis and higher incidence of locoregional recurrence. In this study, we investigated a large cohort of PTC cases with an unusual aggressive phenotype using a high-throughput RNA sequencing (RNA-Seq) to identify differentially regulated genes associated with metastatic PTC. All metastatic PTC with mutated BRAF (V600E) but not BRAF wild-type expressed an up-regulation of R-Spondin Protein 4 (RSPO4) concomitant with an upregulation of genes involved in focal adhesion and cell-extracellular matrix signaling. Further immunohistochemistry validation confirmed the upregulation of these target genes in metastatic PTC cases. Preclinical studies using established PTC cell lines support that RSPO4 overexpression is associated with BRAF V600E mutation and is a critical upstream event that promote activation of kinases of focal adhesion signaling known to drive cancer cell locomotion and invasion. This finding opens up the potential of co-targeting B-Raf, RSPO and focal adhesion proteins as a pharmacological approach for aggressive BRAF V600E PTC. [ABSTRACT FROM AUTHOR]

Regional metastasis is the single most important prognostic factor in oral squamous cell carcinoma (OSCC). Abnormal expression of N-myc downstream-regulated genes (NDRGs) has been identified to occur in several tumor types and to predict poor prognosis. In OSCC, the clinical significance of deregulated NDRG expression has not been fully established. In this study, NDRG1 relevance was assessed at gene and protein levels in 100 OSCC patients followed up by at least 10 years. Survival outcome was analyzed using a multivariable analysis. Tumor progression and metastasis was investigated in preclinical model using oral cancer cell lines (HSC3 and SCC25) treated with epidermal growth factor (EGF) and orthotopic mouse model of metastatic murine OSCC (AT84). We identified NDRG1 expression levels to be significantly lower in patients with metastatic tumors compared with patients with local disease only (P = 0.001). NDRG1 expression was associated with MMP-2, -9, -10 (P = 0.022, P = 0.002, P = 0.042, respectively) and BCL2 (P = 0.035). NDRG1 lower expression was able to predict recurrence and metastasis (log-rank test, P = 0.001). In multivariable analysis, the expression of NDRG1 was an independent prognostic factor (Cox regression, P = 0.013). In invasive OSCC cells, NDRG1 expression is diminished in response to EGF and this was associated with a potent induction of epithelial–mesenchymal transition phenotype. This result was further confirmed in an orthotopic OSCC mouse model. Together, this data support that NDRG1 downregulation is a potential predictor of metastasis and approaches aimed at NDRG1 signaling rescue can serve as potential therapeutic strategy to prevent oral cancer progression to metastasis. [ABSTRACT FROM AUTHOR]

Quantitative real-time PCR can detect variations in gene expression. The identification of the stable reference genes (RGs) is necessary to evaluate the expression of specific genes of interest under various conditions in many cell types, including human adipose-derived stromal cells (hASCs). In this study, we used the algorithms BestKeeper, NormFinder, geNorm, and RefFinder to investigate the stability of 15 potential RGs (B2M, eEF1A1, GAPDH, H2AFZ, HMBS, HPRT1, PGK1, PPIA, RPL5, SDHA, TBP, TKT, TRFC, TUBB, and UBC) in hASCs during control, adipo-, chondro-, and osteogenic differentiation for 28 days. RPL5, GAPDH, H2AFZ, and HPRT1 were the most stable RGs, while B2M and UBC were the least stable RGs for the majority of group analyses (tri-lineage differentiation and control analyzed combined or individually; each lineage combined with the control). These RGs were used to normalize adipo- (FABP4, LPL, and PPARG), chondro- (COMP and SOX9), and osteogenic gene expression markers (BMP4, COL1A1, and RUNX2). Each marker showed a similar expression when normalized by H2AFZ, HPRT1, or RPL5, confirming that these RGs exhibit stable expression. However, GAPDH, B2M, and UBC exhibited high standard deviation (SD), downregulated and/or up-regulated differentiation gene expression markers when compared with stable RGs, demonstrating that these RGs are unstable. [ABSTRACT FROM AUTHOR]

Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid gland and early stages are curable. However, a subset of PTCs shows an unusually aggressive phenotype with extensive lymph node metastasis and higher incidence of locoregional recurrence. In this study, we investigated a large cohort of PTC cases with an unusual aggressive phenotype using a high-throughput RNA sequencing (RNA-Seq) to identify differentially regulated genes associated with metastatic PTC. All metastatic PTC with mutated BRAF (V600E) but not BRAF wild-type expressed an up-regulation of R-Spondin Protein 4 (RSPO4) concomitant with an upregulation of genes involved in focal adhesion and cell-extracellular matrix signaling. Further immunohistochemistry validation confirmed the upregulation of these target genes in metastatic PTC cases. Preclinical studies using established PTC cell lines support that RSPO4 overexpression is associated with BRAF V600E mutation and is a critical upstream event that promote activation of kinases of focal adhesion signaling known to drive cancer cell locomotion and invasion. This finding opens up the potential of co-targeting B-Raf, RSPO and focal adhesion proteins as a pharmacological approach for aggressive BRAF V600E PTC.

Quantitative real-time PCR can detect variations in gene expression. The identification of the stable reference genes (RGs) is necessary to evaluate the expression of specific genes of interest under various conditions in many cell types, including human adipose-derived stromal cells (hASCs). In this study, we used the algorithms BestKeeper, NormFinder, geNorm, and RefFinder to investigate the stability of 15 potential RGs (B2M, eEF1A1, GAPDH, H2AFZ, HMBS, HPRT1, PGK1, PPIA, RPL5, SDHA, TBP, TKT, TRFC, TUBB, and UBC) in hASCs during control, adipo-, chondro-, and osteogenic differentiation for 28 days. RPL5, GAPDH, H2AFZ, and HPRT1 were the most stable RGs, while B2M and UBC were the least stable RGs for the majority of group analyses (tri-lineage differentiation and control analyzed combined or individually; each lineage combined with the control). These RGs were used to normalize adipo- (FABP4, LPL, and PPARG), chondro- (COMP and SOX9), and osteogenic gene expression markers (BMP4, COL1A1, and RUNX2). Each marker showed a similar expression when normalized by H2AFZ, HPRT1, or RPL5, confirming that these RGs exhibit stable expression. However, GAPDH, B2M, and UBC exhibited high standard deviation (SD), down-regulated and/or up-regulated differentiation gene expression markers when compared with stable RGs, demonstrating that these RGs are unstable.