Promoter hypermethylation-mediated down-regulation of LATS1 and LATS2 in human astrocytoma

Abstract

LATS1 and LATS2 are tumor suppressor genes implicated in the regulation of cell cycle, but their methylation statuses are still unknown in human astrocytoma. Here, we found that the promoter hypermethylation frequencies of LATS1 and LATS1 were 63.66% (56/88) and 71.5% (63/88) in 88 astrocytomas by methylation-specific PCR. But no methylation of LATS1 and LATS2 promoter was detected in the 10 normal brain tissues. There was an increased methylation frequency of LATS1 and LATS2 with the malignant development of astrcytoma. By real-time PCR, the mRNA expression of LATS1 or LATS2 was detected significantly decreased in different pathological grade astrocytomas (P < 0.05). And the mRNA levels of LATS1 and LATS2 in astrocytomas with hypermethylation were both significantly (P < 0.01) lower than those without methylation. The methylation of LATS1 and LATS2 was detected in U251 and SHG-44 cell lines, and 5-aza-deoxycytidine could restore their expression to induce cell apoptosis. Our results suggested that LATS1 and LATS2 mRNA was down-regulated in astrocytoma by hypermethylation of the promoter. The methylation and mRNA expression of LATS1 and LATS2 may provide useful clues to the development of the diagnostic assays for astrocytoma. Our results also suggested that LATS1 and LATS2 may be a useful target for astrocytoma therapy.

Introduction

Diffusely infiltrating astrocytoma is a leading group of the primary central nervous system tumors, accounting for more than 60% of all primary brain tumors (Kleihues and Cavenee, 2000). It may arise aggressively from the normal astrocytes, or evolve stepwise from its benign precursors. Owing to the difficulties with its early diagnosis and surgical removal of all residue diseased tissues, rapid progression, and frequent reoccurrence, the most advanced form of astrocytoma, glioblastoma (WHO grading IV) represents an extremely life-threatening intracranial malignant tumor both inside and outside of China (China Publishing House of Medical Sciences and Technologies, 1998). In China, there are about 35,000 new glioma patients every year, and most of them are astrcytoma patients. It is therefore important to identify new diagnostic approaches and therapeutic targets for this deadly disease.

It has emerged that epigenetic events can lead to tumor suppressor gene (TSG) inactivation as an alternative mechanism to genetic events, such as gene mutation, deletion or rearrangement. Hypermethylation is a regional event that occurs frequently in GC-rich sequence, called CpG islands and often located within the 5′ regulatory non-transcribed regions of genes. Aberrant methylation of CpG islands of the promoter region of genes, an epigenetic event, which plays a role in the development of various cancers, has become an important area of investigation in assessing the mechanisms of tumor suppressor and regulatory gene inactivation (Toyota and Issa, 1999, Jones and Takai, 2001). It has shown that TSGs can be transcriptionally silenced when their promoter region CpG islands contain methylated cytosines located 5′ to an adjacent guanine (Jones and Baylin, 2002, Baylin and Herman, 2000). The recent advent of better methods to assess this epigenetic gene-silencing event, such as utilization of methylation-specific PCR (MSP), has simplified the amount of specimen DNA (Herman et al., 1996). Meanwhile, the methylation status of TSG promoter regions has been investigated and profiled for a number of cancers (Esteller et al., 2001, Sidransky, 2002).

In astrocytomas, methylation associated silencing has been reported in some genes, including p16, RASSF1A, p73, AR, MGMT, and CDH1 which may be involved in the carcinogenesis of gliomas (Costello, 2003, Yu et al., 2004). The lats (large tumor suppressor) gene has been identified as a tumor suppressor gene in Drosophila and it encodes a putative serine/threonine kinase (Justice et al., 1995, Xu et al., 1995). Two mammalian homologues of the Drosophila lats, LATS1 (large tumor suppressor, homolog 1) and LATS2 (large tumor suppressor, homolog 2), have been identified to play important roles in carcinogenesis (Xia et al., 2002, Li et al., 2003). The human LATS1 gene has been localized to chromosome 6q24–25 (Nishiyama et al., 1999). Because LATS1-deficient mice develop soft tissue sarcomas or ovarian stromal cell tumors, LATS1 has been considered as a tumor suppressor gene (St John et al., 1999). Overexpression of LATS1 causes G2-M arrest through the inhibition of CDC2 kinase activity in breast cancer cell line in vitro and significantly suppresses the tumorigenicity in vivo by inducing apoptosis (Yang et al., 2001, Xia et al., 2002). LATS2 (also known as KPM) was isolated as a second mammalian homologue of the lats tumor suppressor family and the human LATS2 gene was mapped to chromosome 13q11–12 (Yabuta et al., 2000, Hori et al., 2000). Overexpression of LATS2 causes G1-S arrest through the inhibition of cyclin E/CDK2 in vitro as well as suppresses the tumorigenicity of NIH/v-ras-transformed cells in vivo, suggesting that LATS2 is a tumor suppressor gene (Li et al., 2003). Hisaoka's report (Hisaoka et al., 2002) showed that the hypermethylation of the promoter region of the LATS1 gene in six of seven soft tissue sarcomas with a decreased expression of LATS1 mRNA. Jose (Roman-Gomez et al., 2004) and Jimenez (Jimenez-Velasco et al., 2005) reported that hypermethylation of the LATS1 and LATS2 were found in acute lymphoblastic leukemia with the decreased expression of mRNA. Takahashi (Takahashi et al., 2005) also indicated that hypermethylation of the promoter regions of LATS1 and LATS2 likely plays an important role in the down-regulation of their mRNA levels in breast cancers.

All the research obtained on LATS1 and LATS2 strongly indicate that both genes serve as a tumor suppressor gene and hypermethylation might play a significant role in the inactivation of these genes in various human tumors. But the methylation statuses of LATS1 and LATS2 genes are still unknown in human astrocytoma. So in the present study, we have analyzed the methylation status of LATS1 and LATS2 genes as well as its correlation with their mRNA levels in human astrocytoma.