DIAPH2 alterations increase cellular motility and may contribute to the metastatic potential of laryngeal squamous cell carcinoma.

Low 5-year survival rate in laryngeal squamous cell carcinoma (LSCC) is to large extent attributable to high rate of recurrences and metastases. Despite the importance of the latter process, its complex genetic background remains not fully understood. Recently, we identified two metastasis-related candidate genes, DIAPH2 and DIAPH3 to be frequently targeted by hemizygous/homozygous deletions, respectively, in LSCC cell lines. They physiologically regulate such processes as cell movement and adhesion, hence we found it as a rationale, to study if tumor LSCC specimens harbor mutations of these genes and whether the mutations are associated with metastasizing tumors. As a proof of concept, we sequenced both genes in five LSCC cell lines derived from lymph node metastases assuming there the highest probability of finding alterations. Indeed, we identified one hemizygous deletion (c.3116_3240del125) in DIAPH2 targeting the FH2 domain. Moreover, we analyzed 95 LSCC tumors (53 N0 and 42 N+) using the Illumina platform and identified three heterozygous single nucleotide variants in DIAPH2 targeting conserved domains exclusively in N+ tumors. By combining these results with cBioPortal data we showed significant enrichment of DIAPH2 mutations (P = 0.036) in N+ tumors. To demonstrate the consequences of DIAPH2 inactivation, CRISPR/Cas9 editing was used to obtain a heterozygous DIAPH2+/- mutant HEK-293T cell line. Importantly, the edited line shows a shift from 'proliferation' to 'migration' phenotype typically observed in metastasizing cells. In conclusion, we report that DIAPH2 alterations are present primarily in metastasizing specimens of LSCC and suggest that they may contribute to the metastatic potential of the tumor.

Recurrent CDK1 overexpression in laryngeal squamous cell carcinoma.

In this study, we analyzed the expression profile of four genes (CCNA2, CCNB1, CCNB2, and CDK1) in laryngeal squamous cell carcinoma (LSCC) cell lines and tumor samples. With the application of microarray platform, we have shown the overexpression of these genes in all analyzed LSCC samples in comparison to non-cancer controls from head and neck region. We have selected CDK1 for further analysis, due to its leading role in cell cycle regulation. It is a member of the Ser/Thr protein kinase family of proven oncogenic properties. The results obtained for CDK1 were further confirmed with the application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, Western blot, and immunohistochemistry (IHC). The observed upregulation of CDK1 in laryngeal squamous cell carcinoma has encouraged us to analyze for genetic mechanisms that can be responsible this phenomenon. Therefore, with the application of array-CGH, sequencing analysis and two methods for epigenetic regulation analysis (DNA methylation and miRNA expression), we tried to identify such potential mechanisms. Our attempts to identify the molecular mechanisms responsible for observed changes failed as we did not observe significant alterations neither in the DNA sequence nor in the gene copy number that could underline CDK1 upregulation. Similarly, the pyrosequencing and miRNA expression analyses did not reveal any differences in methylation level and miRNA expression, respectively; thus, these mechanisms probably do not contribute to elevation of CDK1 expression in LSCC. However, our results suggest that alteration of CDK1 expression on both mRNA and protein level probably appears on the very early step of carcinogenesis.

Polymorphisms of the DNA repair genes XRCC1 and ERCC4 are not associated with smoking- and drinking-dependent larynx cancer in a Polish population.

BACKGROUND: Tobacco smoking and alcohol drinking generate oxidative DNA damage and may contribute to larynx carcinogenesis. The X-ray repair cross complementing 1 (XRCC1) and excision repair cross-complementing rodent repair deficiency, complementation group 4 (ERCC4(XPF)) genes are important components of DNA excision repair systems, which repair DNA damage induced by various factors, including tobacco smoking and alcohol. AIM: To investigate the association between the genotypes of the XRCC1-Arg399Gln (rs25487) and ERCC4-Arg415Gln (rs1800067) polymorphisms and smoking- and drinking-related larynx cancer in a Polish population. METHODS: The polymorphisms were determined by PCR-RFLP method in 253 patients with squamous cell carcinoma of the larynx and 253 sex- and age-matched controls. RESULTS: We did not find any association between the investigated polymorphisms and larynx carcinoma, dependent on either smoking or drinking status. No association was found between these polymorphisms and larynx cancer grade, stage or age at diagnosis. CONCLUSIONS: The results indicated that Arg399Gln polymorphism of XRCC1 gene and Arg415Gln polymorphism of ERCC4 gene may not be associated with smoking- and drinking-related larynx cancer in Polish population.