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Oral squamous cell carcinoma (OSCC) is the most frequent type of cancer of the head and neck area accounting for approx. 377,000 new cancer cases every year. The epithelial-to-mesenchymal transition (EMT) program plays an important role in OSCC progression and metastasis therefore contributing to a poor prognosis in patients with advanced disease. Transforming growth factor beta (TGF-ß) is a powerful inducer of EMT thereby increasing cancer cell aggressiveness. Here, we aimed at identifying RNA-binding proteins (RBPs) that affect TGF-ß-induced EMT. To this end we treated oral cancer cells with TGF-ß and identified a total of 643 significantly deregulated protein-coding genes in response to TGF-ß. Of note, 19 genes encoded RBPs with NANOS1 being the most downregulated RBP. Subsequent cellular studies demonstrated a strong inhibitory effect of NANOS1 on migration and invasion of SAS oral cancer cells. Further mechanistic studies revealed an interaction of NANOS1 with the TGF-ß receptor 1 (TGFBR1) mRNA, leading to increased decay of this transcript and a reduced TGFBR1 protein expression, thereby preventing downstream TGF-ß/SMAD signaling. In summary, we identified NANOS1 as negative regulator of TGF-ß signaling in oral cancer cells.

Abstract Background Cancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy. Methods We used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis. Results We identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation. Conclusion Analysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.

Abstract Background The human leucine-rich, repeat-containing G protein-coupled receptor 5 (LGR5) is a stem cell marker in numerous adult tissues and is overexpressed in a large number of human carcinoma including colon cancer, breast cancer and oral squamous cell carcinomas (OSCC). The role of the full length transcript (LGR5FL) in progression and prognosis of several cancers was reported. However, the biological function of three splice variants of LGR5 (LGR5Δ5, LGR5Δ8 and LGR5Δ5–8) has yet to be thoroughly investigated. Methods Seventy-eight frozen tumor samples from adult OSCC patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of full length LGR5, the splice variant of LGR5 lacking exon 5 (LGR5Δ5), the splice variant of LGR5 lacking exon 8 (LGR5Δ8) and the mRNA level of all known transcript variants together (LGR5all) were quantified and correlated to overall and disease-specific survival of OSCC patients, clinical parameters and the mRNA level of different tumor-associated markers. Results An elevated level of tumoral LGR5Δ5 mRNA, but not LGR5FL, LGR5Δ8 or LGR5all mRNA was significantly associated with a poor prognosis for the overall and disease-specific survival of OSCC patients (hazard ratio (HR) = 2.0; p = 0.02; 95% CI: 1.1–3.7; HR = 3.2; p = 0.01; 95% CI: 1.3–8.0; multivariable Cox regression), respectively. Additionally, a higher tumoral level of LGR5Δ5 mRNA in primary tumors was associated with the occurrence of regional lymph node metastases in OSCC patients (odds ratio (OR) = 3.1; p = 0.022; 95% CI: 1.2–7.9; binary logistic regression). Furthermore, the mRNA levels of all investigated LGR5 transcript variants were significantly correlated with the mRNA expression of Wnt-target genes and markers of epithelial-to-mesenchymal transition (EMT). Conclusion The mRNA level of the LGR5 splice variant LGR5Δ5 is an independent negative prognostic marker for overall and disease-specific survival and metastasis in OSCC patients. Additionally, we suggest, all LGR5 transcript variants are involved in the EMT process mainly through activating the Wnt-signalling pathway.