Pyrosequencing-based DNA methylation profiling of Fanconi anemia/BRCA pathway genes in laryngeal squamous cell carcinoma

Abstract

Abstract. Fanconi anemia (FA) associated genes [FANCA, -B, -C, FANCD1(BRCA2), -D2, -E, -F, -G, -I, -L, -M, FANCN (PALB2), FANCJ(BRIP1)and FA-linked BRCA1] encode proteins of DNA damage response pathways mutated in FA patients. FA is characterized by congenital malformations, chromosomal instability and high cancer susceptibility. FA patients have a 500-700 times higher risk of head and neck squamous cell carcinoma (HNSCC) compared to the non-FA population. As DNA methylation comprises one of the known gene inactivation mechanisms in cancer we have investigated the methylation status of 13 FA and one FA-linked gene in order to assess the role of FA in sporadic laryngeal squamous cell carcinoma (LSCC) tumor samples. Thirteen laryngeal squamous carcinoma cell lines (UT-SCC) and 64 primary laryngeal carcinoma cases were analyzed by bisulfite pyrosequencing. DNA from buccal swabs of 10 healthy volunteers was used as a control group. Promoter regions of FANCA, BRCA1 and BRCA2 displayed recurrent alterations in the methylation levels in cancer samples as compared to buccal swabs controls. For FANCA, hypomethylation was observed in 11/13 cell lines (p<0.0003) and all 64 primary larynx samples (p<0.001) compared to buccal swabs. For BRCA1, 4/13 cell lines (p=0.04) and 3/58 primary laryngeal cases (p=0.22) showed hypomethylation. In BRCA2, all 13 cell lines (p<0.0001) 4/63 primary LSCC (p<0.01) showed hypermethylation as compared to controls. In conclusion, we show recurrent alterations of DNA methylation levels in three Fanconi anemia genes which might contribute to the pathogenesis of LSCC.

Recurrent epigenetic silencing of the PTPRD tumor suppressor in laryngeal squamous cell carcinoma

“…10 PCR products were mixed with binding buffer (Qiagen, Hilden, Germany) and sepharose coated with streptavidin (GE Healthcare, Chicago, USA), shaked for 10 min and cleaned on vacuum pump station in the following buffers: 70% EtOH, 0.2% NaOH, and washing buffer (Qiagen, Hilden, Germany). Single-strand amplicons were then mixed with annealing buffer (Qiagen, Hilden, Germany), the sequencing primer (0.4 µM), and then heated for 2 min at 85°C and cooled for primer hybridization.…”

DNA Repair Capacity and the Risk of Head and Neck Cancer

“…Somatic inactivation of the FA pathway by epigenetic silencing has been observed in several different types of sporadic cancer (Lyakhovich & Surralles, 2006), such as bladder cancer (Neveling et al, 2007), ovarian cancer (Lim et al, 2008), breast cancer (Wu, Shin-ya, & Brosh, 2008) cervical cancer (Narayan et al, 2004) and of course in HNSCC (Marsit et al, 2004;Szaumkessel et al, 2011 ;Wreesmann et al, 2007). Our own study has demonstrated the disruption of DNA methylation of promoter region in FANCA, BRCA1 and BRCA2 in squamous laryngeal carcinoma cell lines and primary tumors (Szaumkessel et al, 2011).…”

“…Our own study has demonstrated the disruption of DNA methylation of promoter region in FANCA, BRCA1 and BRCA2 in squamous laryngeal carcinoma cell lines and primary tumors (Szaumkessel et al, 2011). BRIP1 and FANCB have been shown to be higher and more frequently methylated compared to normal mucosa (Smith et al, 2010).…”

Bases epigenéticas del cáncer gástrico: oportunidades para la búsqueda de nuevos biomarcadores

“…Esta información fue concordante con el concepto de activación oncogénica mediada por hipometilación. A continuación analizamos el perfil de sobreexpresión de p73 y otros genes (BRCA1, HSP90, STAT1, FHIT, EGFR, p53, p16INK4a) asociados a activación por hipometilación [71][72][73][74][75][76][77][78][79] en microarreglos de tejidos (TMA, tissue microarray) de cáncer gástrico (mucosa gástrica tumoral y mucosa adyacente al tumor, n = 91) y gastritis (n = 148). En forma paralela a este análisis evaluamos las característi-cas histológicas de los casos según el sistema de Sydney para gastritis crónica y OLGA (Operative Link on Gastritis Assessment) para la evaluación de atrofia 80,81 .…”