CHARACTERIZATION OF PROMOTER DNA METHYLATION SILENCED TUMOR SUPPRESSOR GENE CANDIDATES DLC1 AND NPAS4 IN NASOPHARYNGEAL CARCINOMA AND OTHER TUMORS

Abstract

Nasopharyngeal Carcinoma (NPC) is the 6th most common cancer in Singapore males with a striking geographical and ethnic distribution. The molecular basis of NPC development is still poorly understood, but a strong linkage to the Epstein-Barr virus (EBV) has been shown. Other contributing factors include various genetic, epigenetic and environmental factors which together drive the development of this tumor. The identification of tumor suppressor genes (TSG) silenced by promoter DNA methylation uncover mechanisms of tumorigenesis and identifies new epigenetic tumor markers for early cancer detection. In this thesis we describe our efforts in the elucidation and characterization of novel tumor suppressor gene candidates epigenetically silenced via promoter DNA hypermethylation and relevant in NPC and multiple other tumors.

In our first study, we described the identification and characterization of a new DLC1 isoform designated as DLC1-isoform 4 (DLC1-i4). This novel isoform encodes an 1125-aa (amino acid) protein with a distinct N-terminus as compared with other known DLC1 isoforms. Similar to other isoforms, DLC1-i4 is expressed ubiquitously in normal tissues and immortalized normal epithelial cells, suggesting a role as a major DLC1 transcript. DLC1-i4 is also found to be significantly down-regulated in multiple carcinoma cell lines, including 2/4 nasopharyngeal (50%), 8/16 (50%) esophageal, 4/16 (25%) gastric, 6/9 (67%) breast, 3/4 (75%) colorectal, 4/4 cervical (100%) and 2/8 (25%) lung carcinoma cell lines. We also found that CpG methylation of the DLC1-i4 promoter is associated with its silencing in tumor cells.

In the second study, we described how we identified the Neuronal PAS domain protein 4 (NPAS4) as a novel candidate tumor suppressor gene in NPC and other tumors. From literature searches, a chromosome 11q13 1.8Mb tumor suppressor critical region was identified and predicted to harbour novel tumor suppressor gene candidates. It was also predicted that this tumor suppressor might play a role in transcriptional regulation. We interrogated the 1.8 Mb tumor suppressive region for candidate transcriptional regulators and identified NPAS4 as the only one down-regulated by promoter DNA hypermethylation in NPC and other tumors. NPAS4 was further found to have tumor inhibitory properties and is an indirect target of the known tumor suppressor TP53 after DNA damage treatments and thus suggested to play a role in the cellular DNA damage response.

In summary, we report the characterization of an epigenetics promoter DNA methylation silencing mechanism responsible in the down-regulation of DLC1-i4 and NPAS4 in NPC and other tumors. The high incidences of epigenetic inactivation of these genes in multiple tumor types strongly indicate that these genes may serve as potential epigenetic markers for the diagnosis and prognosis of tumors.