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

Abstract

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.