Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization

Abstract

We reinvestigated rearrangements occurring in region q13 of chromosome 11 aiming to: (i) describe heterogeneity of the observed structural alterations, (ii) estimate amplicon size and (iii) identify of oncogenes involved in laryngeal cancer progression as potential targets for therapy. The study included 17 cell lines derived from laryngeal cancers and 34 specimens from primary laryngeal tumors. The region 11q13 was analyzed by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and gene expression microarray. Next, quantitative real time PCR was used for chosen genes to confirm results from aCGH and gene expression microarray. The observed pattern of aberrations allows to distinguish three ways, in which gain and amplification involving 11q13 region may occur: formation of a homogeneously staining region; breakpoints in/near 11q13, which lead to the three to sevenfold increase of the copy number of 11q13 region; the presence of additional copies of the whole chromosome 11. The minimal altered region of gain and/or amplification was limited to ~1.8 Mb (chr.11:69,395,184-71,209,568) and comprised mostly 11q13.3 band which contain 12 genes. Five, out of these genes (CCND1, ORAOV1, FADD, PPFIA1, CTTN) had higher expression levels in comparison to healthy controls. Apart from CCND1 gene, which has an established role in pathogenesis of head and neck cancers, CTTN, ORAOV1 and FADD genes appear to be oncogene-candidates in laryngeal cancers, while a function of PPFIA1 requires further studies.

The Landscape of Somatic Copy Number Alterations in Head and Neck Squamous Cell Carcinoma

“…The most commonly altered regions in HNSCC included gains of 3q, 5p, 8q, and losses of 3p, 5q, 8p, 13q, 18q, and 21q. The most notable focal CNA was the overrepresentation of 11q13, which may be associated with oncogene CCND1 amplification (44,45). These results were consistent with previous studies (18,(46)(47)(48)(49).…”

Frequent hypermethylation of WNT pathway genes in laryngeal squamous cell carcinomas

“…The other four genes ( SFRP1, SFRP2, PPP2R2B, and WIF‐1 ) showed high frequency of methylation. DNA methylation profiles were compared with gene expression level of SFRP1 , SFRP2 , PPP2R2B, and WIF‐1 in ten LSCC cell lines using our previously described chip data . In the case of SFRP2 , PPP2R2B, and WIF‐1, cell lines characterized by lack of gene methylation generally showed higher expression than cell lines where gene methylation was observed (data not shown).…”

Recurrent CDK1 overexpression in laryngeal squamous cell carcinoma

“…The culture condition for cell lines and primary cultures of epithelial cells were described elsewhere with minor modification [5, 19, 20]. For DNA, RNA, and protein isolation, cell lines were cultured to 80 % confluence and then harvested with 0.1 % trypsin and 0.2 % EDTA.…”

“…The microarrays were performed and analyzed as described elsewhere [5, 24, 25]. The number of tags used to analyze the expression of chosen genes was as follows: two tags for the CCNA2 gene (203418_at and 213226_at), one for CCNB1 (214710_s_at), and one for CCNB2 (202705_at).…”

“…Until now, several oncogenes have been shown to be involved in the development of head and neck cancers including CCND1 , FGF3 , FGF4 , CTTN [3], or EGFR [4]. In our previous work, we have shown also other oncogenes like the ORAOV1 , FADD [5], and CRKL [6] as potentially related to LSCC.…”