Abstract
Laryngeal squamous cell carcinoma is a major medical problem worldwide. Although our understanding of genetic changes and their consequences in laryngeal cancer has opened new therapeutic pathways over the years, the diagnostic as well as treatment options still need to be improved. In our previous study, we identified CRKL (22q11) as a novel putative oncogene overexpressed and amplified in a subset of LSCC tumors and cell lines. Here we analyze to what extent CRKL DNA copy number gains correlate with the higher expression of CRKL protein by performing IHC staining of the respective protein in LSCC cell lines (n = 3) and primary tumors (n = 40). Moreover, the importance of CRKL gene in regard to proliferation and motility of LSCC cells was analyzed with the application of RNA interference (siRNA). Beside the physiological cytoplasmic expression, the analysis of LSCC tumor samples revealed also nuclear expression of CRKL protein in 10/40 (25%) cases, of which three (7.5%), presented moderate or strong nuclear expression. Similarly, we observed a shift towards aberrantly strong nuclear abundance of the CRKL protein in LSCC cell lines with gene copy number amplifications. Moreover, siRNA mediated silencing of CRKL gene in the cell lines showing its overexpression, significantly reduced proliferation (p < 0.01) as well as cell migration (p < 0.05) rates. Altogether, these results show that the aberrantly strong nuclear localization of CRKL is a seldom but recurrent phenomenon in LSCC resulting from the increased DNA copy number and overexpression of the gene. Moreover, functional analyses suggest that proliferation and migration of the tumor cells depend on CRKL expression.
Highlights
Laryngeal squamous cell carcinoma (LSCC) belongs to the highly heterogeneous group of head and neck squamous cell carcinomas (HNSCC)
Thereafter, in order to analyse if the copy number alterations of the CRKL gene result in differences in the expression of CRKL protein we have performed Immunohistochemical staining (IHC) staining on LSCC cell lines (n = 3) and LSCC tumors (n = 40) (Fig. 1A,C)
The UT-SCC-6A cell line with 143 gene copies was found with aberrantly strong nuclear abundance of the CRKL protein (+++)
Summary
Laryngeal squamous cell carcinoma (LSCC) belongs to the highly heterogeneous group of head and neck squamous cell carcinomas (HNSCC). The first, Cetuximab is based on a monoclonal antibody directed against the epidermal growth factor receptor (EGFR) found recurrently amplified in head and neck cancer cells[4]. Comparative Genomic Hybridization) platforms we identified CRKL (V-crk avian sarcoma virus CT10 oncogene homolog-like, 22q11) as a novel putative oncogene amplified and overexpressed in a subset of LSCC tumors and cell lines[8]. The CRKL protein belongs to the adaptor cell signaling proteins which are classified into two groups based on their function and structure[10]. The second group, without membrane localization, comprises adaptor proteins containing the SH2 and SH3 domains known to be involved in multiple signal transduction pathways[11]. Based on immunohistochemical analyses, we propose an explanation, how the oncogenic potential of CRKL is triggered by copy number gains
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