High prevalence of epigenetic inactivation of the human four and a half LIM domains 1 gene in human oral cancer

Abstract

Abstract.The four and a half LIM domains 1 (FHL1) gene has been related to carcinogenesis. However, the expression status of FHL1 in human oral squamous cell carcinoma (OSCC) remains unclear and the detailed mechanism of gene silencing is poorly understood. The aim of this study was to examine the FHL1 expression level and its regulatory mechanism in OSCCs. Quantitative reverse-transcriptase-polymerase chain reaction (PCR) and western blotting showed significant downregulation of FHL1 in all OSCC-derived cell lines (Sa3, HSC-2, HSC-3, HSC-4, HO-1-u-1, HO-1-N-1, KON and Ca9-22) compared to human normal oral keratinocytes. We also found that FHL1 mRNA expression was frequently downregulated (P<0.01) in 51 (86.4%) of 59 primary OSCCs compared with the corresponding normal oral tissues, while there was no significant difference between the status of the FHL1 protein expression in OSCCs and the clinicopathological features. Using methylationspecific PCR, we detected methylated FHL1 in all cell lines and treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine restored the FHL1 expression. However, no significant restoration of FHL1 expression was observed using sodium butyrate, an inhibitor of histone deacetylase and chromatin immunoprecipitation showed that histone H3 lysine 9 in the FHL1 promoter region was significantly acetylated. In addition, no mutation in the entire coding region of the FHL1 gene was found. Therefore, our data suggested that inactivation of the FHL1 gene is a frequent event during oral carcinogenesis and that the mechanism of FHL1 downregulation in OSCCs is through DNA methylation of the promoter region rather than histone deacetylation or mutation. IntroductionHead and neck squamous cell carcinoma (HNSCC) is one of the most frequently occurring malignancies and a major cause of morbidity and mortality (1,2). Oral cancer is the most common among the HNSCCs and the most frequently occurring oral cancer is squamous cell carcinoma (OSCC), which accounts for more than 90% of all oral malignancies (3). The number of OSCC cases that occur worldwide annually exceeds 300,000 (4). The mechanisms behind tumor progression of OSCC are known to a limited extent, indicating a clear need for comprehensive knowledge that can lead to more specific and effective molecular target.Microarray technology facilitates simultaneous evaluation of thousands of genes in a specimen. The results of microarray analysis provide researchers with high throughput screening to study the roles played by specific genes in cancer development and progression. We previously reported gene expression profiling of OSCC to identify cancer-related genes (5,6).The four and a half LIM domains (FHL) family of genes is characterized by LIM domains, a term derived from the first letters of three transcription factors: Lin-11, Isl-1 and Mec-3. Because the LIM domains provide protein-protein binding interfaces, the FHL genes play an important role in cellular events such as focal adhesion and differentiation by repressing or ac...

FHL1 inhibits the growth of tongue squamous cell carcinoma cells via G1/S cell cycle arrest

“…These results suggest that FHL1 functions as a tumor suppressor in TSCC and that FHL1 may be a useful target for TSCC gene therapy. FHL1 does not affect TSCC cell migration and invasion, which is consistent with a previous study demonstrating that expression of FHL1 does not correlate with node metastasis (13). These findings indicate that FHL1 may be important in the development of TSCC, but not in the progression of TSCC.…”

“…It has been reported that FHL1 is frequently downregulated in primary OSCC tissues compared with the corresponding normal oral tissues (13). One of the mechanisms of FHL1 downregulation in OSCC is hypermethylation of CpG islands within FHL1 gene promoter regions, which is also found in bladder cancer (13,25).…”

“…One of the mechanisms of FHL1 downregulation in OSCC is hypermethylation of CpG islands within FHL1 gene promoter regions, which is also found in bladder cancer (13,25). Due to the importance of FHL1 in the regulation of TSCC cell growth, it would be interesting to investigate whether the FHL1 promoter is methylated in TSCC and to determine other mechanisms underlying FHL1 downregulation in TSCC.…”

“…Previously, FHL1 has been demonstrated to be important in carcinogenesis. FHL1 expression is downregulated in various types of malignancy, including breast cancer, liver cancer, kidney cancer, prostate cancer, gastric cancer, lung cancer and oral squamous cell carcinoma (OSCC) (8)(9)(10)(11)(12)(13). FHL1 exerts its tumor suppressive role via multiple mechanisms.…”

The functional role of long non-coding RNAs and epigenetics

“…Chromatin is in charge of DNA packaging, gene expression and DNA replication [103,104]. The mechanism of epigenomic control is generally considered at the level of chromatin [105-107]. Histones proteins can be chemically modified by as the process such as acetylation, methylation, sumoylation and ubiquitylation.…”