Functional Analysis And Methylation Regulation Of Dermol And Twist Transcription Factors In Human Ovary, Breast And Gastric Cancers

Part I Expression and functional analysis ofDermo1 and Twist in human cancersDermo1 and Twist are highly conserved transcription factors that belong to the basic Helix-loop-helix (b-HLH) protein family. These two proteins share structural homology and functional similarity. The b-HLH proteins acting as positive or negative regulators play key roles during developmental process such as neurogenesis and myogenesis.Recent studies indicated that Twist can promote cell survival, block nonmalignant cell differentiation, induce tumorgenesis and enhance malignant progression such as tumor metastasis. The observation of Twist activation in human cancers might have important clinical implications. Twist may be used as a prognostic and/or predictive factor as well as a novel therapeutic target. Recently, Twist overexpression was shown to be an indicator of poor prognosis in breast cancer, melanomas and other cancers. Forced expression of Twist protein triggers the resistance of human cancer cells to microtubule-damaging agents such as taxol and vincristine. Several observations strongly support a regulatory role of Twist protein on programmed cell death. However, the function of Dermo1 in development and tumor progression is less clear. In this study, we analyzed the role of Dermo1 in cancer progression, and compared the functional difference between Demo1 and twist in tumor growth, survival, invasion and metastasis.We have characterized the expression pattern of Dermo1/Twist in human ovary cancer, breast cancer and gastric cancer using immunohistochemical staining. The results indicated that Dermo1 expression is increased in cytoplasma and membrane in ovary cancer cells; it is also upregulated in breast cancer cells mainly in cytoplasma and partly in nucleus; but is downregulated in cytoplasma in gastric cancer cells compared with the adjancent non-cancer or normal cells. Twist expression was not detected in ovary tumor tissues. But Twist is overexpressed in breast cancer, mainly in nucleus. Although Twist expression was detected in gastic tissue, we found no significant difference between tumor area and the adjancent non-tumor area. These data suggested that Dermo1 and Twist proteins display differential expression pattern in varied cancer tissues.Since Dermo1 expression is elevated in ovary tumor samples obtained from patients, we further examined the impact of Dermo 1 overexpression in ovary cancer cell line on cellular behaviors in vitro and in vivo. Human Dermo1 expression vector was constructed for stable overexpression in ovary cancer cell line, HO-8910. The expression of Dermo1 was detected in nucleus of HO-8910 ovary cell. Overexpression of Dermo 1 in the ovary cancer cells did not significantly alter cell proliferation and cell cycle profile. However, in vivo animal experiments demonstrated that Dermo1 overexpression promoted tumor growth and metastasis. To understand the underlying mechanism by which Dermo 1 enhances tumor progression, we examined whether Dermo 1 overexpression can protect cells from apoptosis induced by stress such as hypoxia. We found that Dermo1 expression increased Bcl-2, but decreased Bad to antagonize apoptosis through activation of PI-3K-Akt pathway. In addition, we also found that Dermo1 overexpression increased the colony formation, cell motility and cell invasion potential in vitro, but did not affect cell-matrix adhesion. Moreover, the HO-8910 cells overexpressing Dermo 1 showed changes in cellular morphology and appeared to be fibroblast-like shape. These cells exhibited protein markers for an epithelial-mesenchymal transformation (EMT).Taken together, these data demonstrated that Dermo1 overexpression can promote cell survival, reduce stress-induced apoptosis, and induce an EMT-like transition to augment aggressive progression of ovary cancer cells in vivo. Part IIMethylation Regulation of dermo1 and twist genes in cancer cellsHypermethylation of CpG islands in nuclear DNA is very common in cancers. Because this stable molecular alteration can be detected easily with very small amount of DNA, specific DNA methylation is a potential molecular biomarker for cancer diagnosis. Recent studies have used DNA methylations of specific genes known to be genetically disrupted in tumors as biomarkers for early cancer detection, classification of malignancies, response to drug treatment and prediction of outcome. Dermo1 is a basic Helix-loop-helix (bHLH) transcription factor involved in either positive or negative transcriptional regulation during cell type determination and differentiation. This protein plays critical roles in developmental process. Abnormal regulation of Dermo1 may contribute to disease development such as oncogenesis. To study the epigenetic regulation of this novel cancer-associated gene, we applied Bisulfite PCR, DNA sequencing, Methylation-specific PCR (MSP) to analyzed the specific methylation on promoter region of Dermo1 gene in several cancer cell lines. A pair of primers was designed to amplify a 450 bp fragment that contains 16 CpG sites in the core promoter region of the dermo1 gene. PCR products of the bisulfite-modified CpG islands were analyzed by DNA sequencing. Then 2 pairs of primers were designed to distinguish methylation and unmethylation status according to the BSP sequence results. The methylation status of the Dermo1 promoter in 14 human tumor cell lines (HL60, Molml4, MV4-11, RS4:11, HepG2, Huh7, Skhep1, BT-549, MDM-BA231, MCF7, skBr3, H358, H1299 and Hela) were detected. The expression level of the dermo1 gene in these samples was also examined by RT-PCR. In addition, we further investigated the twist promoter methylation and gene expression because of homology and functional overlap between these two genes.The change in DNA methylation patterns was used to explore the underlying mechanism by which gene expression is regulated in cancer cells. The 5'CpG island methylation patterns and the gene expression status in a pair of bHLH transcription factor, twist (twist1) and dermo1 (twist2), were examined and compared. We used bisulfit-sequencing PCR (BSP) to map the methylation status of the CpG sites on the promoter of dermo1 gene in several human cancer cell lines including leukemia, breast cancer, lung cancer, and cervical cancer. Bisulfite restriction analysis indicated there are varied digested methylated DNA fragments in different cell lines. According to the difference of the methylation pattern among these cell lines, we have performed the methylation-specific PCRs (MSP) analysis. Bisulfite genome sequencing analysis has confirmed the MSP results. Since dermo1 has significant homology and functional overlap with twist, we also analyzed the methylation status of twist promoter by MSP and RT-PCR.We found that CpG sites on dermo1 gene promoter in various cancer cell lines displayed different methylation patterns as measured by BSP. Two sets of MSP primer were designed to distinguish the methylated and un-methylated status. MSP analysis showed that dermo1 was partially methylated in 12/14(85.71%) cell lines; totally methylated in 2/14 (14.29%) cell lines. We next analyzed Dermo1 expression in these cell lines by RT-PCR, and found that most of these cell lines (12/14, 85.71%) showed expression of dermo1 mRNA. The common methylation of Dermo1 promoter in these cancer cell lines suggests a potential epigenetic regulation of this gene. We next analyzed the methylation status of the twist promoter by MSP and RT-PCR. MSP analysis showed that Twist was totally methylated in 7/14 (50%) of these cell lines; partially methylated in 4/14 (28.57%) of these cell lines, unmethylated in 1/14(7.14%) these line. But no signal was detected in 2 cell lines. RT-PCR analysis indicated that more than a half of these cell lines (57.14%) do not express twist mRNA, but 4/14 of these lines (28.57%) showed significant twist expression, and 2/14 of these cell lines(14.29%) express low level of Twist RNA. These data indicated that promoter methylation is involved in the regulation of both dermo1 and twist mRNA expression in human cancer cells.