Changjin Kim

Development and Clinical Evaluation of a Highly Sensitive DNA Microarray for Detection and Genotyping of Human Papillomaviruses

ABSTRACT Human papillomavirus (HPV) has been found in cervical cancer, tonsillar cancer, and certain types of head and neck cancers. We report on a DNA microarray-based method for the simultaneous detection and typing of HPVs. The genotype spectrum discriminated by this HPV DNA microarray includes 15 high-risk HPV genotypes and 12 low-risk HPV genotypes. The HPV DNA microarray showed high degrees of specificity and reproducibility. We evaluated the performance of the HPV DNA microarray by application to three HPV-positive cell lines (HeLa, Caski, and SiHa cells) and two HPV-negative cell lines (C33A and A549 cells). The HPV DNA microarray successfully identified the known types of HPV present in the cell lines. The detection limit of the HPV DNA microarray was at least 100-fold higher than that of PCR. To assess the clinical applicability of the HPV DNA microarray, we performed the HPV genotyping assay with 73 nonmalignant and malignant samples from 39 tonsillar cancer patients. Twenty-five of the 39 (64.1%) malignant samples were positive for HPV, whereas 3 of 34 (8.8%) nonmalignant samples were positive for HPV. This result shows a preferential association of HPV with tonsillar carcinomas. The correlations of the presence of HPV with the grade of differentiation and risk factors were not significant. Our data show that the HPV DNA microarray may be useful for the diagnosis and typing of HPV in large-scale epidemiological studies.

The role of vimentin as a methylation biomarker for early diagnosis of cervical cancer

Multiple cytosine guanine dinucleotides (CpG island) are found in the VIM promoter region. The levels of VIM promoter methylation and VIM gene expression were investigated in 7 cervical cancer cell lines and 50 human tissue samples with a distinctive degree of malignant trans-formation. While multiple CpG sites in the VIM promoter were highly methylated in CIN III and invasive carcinoma cells, they were rarely methylated in normal cells. Our result shows that methylation in the VIM promoter appears to start from CIN I and CIN II, relatively early stages of multistep carcinogenesis. This epigenetic alteration in VIM promoter suggests the availability as a biomarker for the early diagnosis and prevention of cervical cancer. We also show that hypermethylation in the VIM promoter is responsible for transcriptional silencing of the VIM gene in cervical cancer cells. In addition, our result shows that exogenous overexpression of the VIM gene in SiHa cervical cancer cells slightly activated cell proliferation and migration as shown in soft agar colony formation and migration assays.

Oncogenic function of p34SEI-1 via NEDD4‑1‑mediated PTEN ubiquitination/degradation and activation of the PI3K/AKT pathway

A 34-KD protein encoded by the SEI-1 gene (p34(SEI‑1)), is a relatively recently discovered oncoprotein that has multiple important biological functions. Our data show that p34(SEI-1) enhances cancer cell survival and promotes tumorigenesis by downregulating the tumor suppressor PTEN, a negative regulator of the PI3K/AKT signaling pathway, and therefore activating the PI3K/AKT signaling pathway. In this process, p34(SEI-1) positively affects NEDD4-1 gene expression both at the transcriptional and protein levels. Furthermore, the expression levels of p34(SEI-1) and NEDD4-1 were found to be coordinated in tumor tissues obtained from patients with breast cancer. We also show that p34(SEI-1) affects the subcellular localization of PTEN.

Epigenetic regulation of the potential tumor suppressor gene, hLHX6.1, in human cervical cancer.

It is well known that the Homo sapiens LIM homeobox domain 6 gene (hLHX6), a putative transcription regulator, controls the differentiation and development of neural and lymphoid cells, particularly in the central nervous system. In this study, we investigated hLHX6.1 (an isoform of hLHX6), which functions as a tumor suppressor gene in the cervix. Firstly, the methylation levels of the hLHX6 and hLHX6.1 promoters were investigated in 8 cervical cancer cell lines and human tissue samples with a distinctive degree of malignant transformation. In spite of the presence of multiple cytosine guanine dinucleotides (CpG islands) in 2 proximal promoters of the hLHX6 and hLHX6.1 genes, only the hLHX6.1 promoters were found to be mostly hypermethylated and associated with transcriptional silencing by promoter methylation, whereas the hLHX6 promoters were not. Methylation levels in the hLHX6.1 promoter were also found to be strongly related to cervical cancer development. The level of hLHX6.1 gene expression was found to be relatively high in normal cells, in which the hLHX6.1 promoter was mostly unmethylated. However, the hLHX6.1 gene expression was down-regulated or undetectable in cervical cancer cell lines and cancer tissues, in which the hLHX6.1 promoter was hypermethylated. This epigenetic alteration in the hLHX6.1 promoter begins at a relatively early stage, suggesting its potential as a biomarker for the early diagnosis and prevention of cervical cancer. Moreover, the overexpression of the hLHX6.1 gene in cervical cancer cells suppressed the tumorigenic phenotype, as shown by soft agar colony formation and migration assays, suggesting that hLHX6.1 could be a new tumor suppressor gene in the cervix.

Distinct regulatory effect of the p34SEI-1 oncoprotein on cancer metastasis in HER2/neu-positive and -negative cells

The p34(SEI-1) oncoprotein is involved in a transcriptional regulation, cell cycle regulation, apoptosis, development and many other important cellular functions. Our present study suggests that p34(SEI-1) can promote metastasis by enhancing migration and invasion of cancer cells. Consistently, p34(SEI-1) expression was found to be increased as the tumor invasiveness progressed in human breast tissues. p34(SEI-1) may promote cancer metastasis by activating the PI3K/AKT signaling pathway. In this process, p34(SEI-1) activates two different serine/threonine kinases, AKT or ILK, depending on the expression status of HER2/neu oncogene. In HER2/neu suppressed cancer cells, p34(SEI-1) promoted metastasis mainly by activating AKT via phosphorylation of the 473 serine residue. In HER2/neu expressing cancer cells, p34(SEI-1) overexpression downregulates HER2/neu expression, leading to the activation of another crucial serine/threonine kinase ILK due to phosphorylation of the 178 threonine residue instead of AKT. These results suggest that p34(SEI-1) affects cancer metastasis by regulating two different signaling pathways depending on the HER2/neu expression level, in which AKT and ILK modulation can be stimulated by p34(SEI-1) overexpression.

Feasibility of quantifying SDC2 methylation in stool DNA for early detection of colorectal cancer

BackgroundColorectal cancer (CRC) screening is the most efficient strategy to reduce disease-related mortality. Frequent aberrant DNA methylation is known to occur in selected genes and early during CRC development, which has emerged as a new epigenetic biomarker for early detection of CRC. Previously, we reported that we identified that CpG sites of SDC2 were aberrantly methylated in tumor tissues of most CRC patients through comprehensive methylation analysis and demonstrated a high potential of quantification of SDC2 methylation in blood for early detection of colorectal cancer. In this study, we aim to investigate the feasibility of quantifying SDC2 methylation in stool DNA for the early detection of CRC. The objective of this study was to confirm a high frequency of SDC2 methylation in tumor tissues at various stages of CRC and investigate the feasibility of a quantitative test for SDC2 methylation in fecal DNA by highly sensitive and accurate real-time PCR for early detection of CRC.MethodsBisulfite-pyrosequencing assay was performed to measure the SDC2 methylation status in tissue samples. For methylation analysis in stool DNA, a highly sensitive and accurate method was applied which implements consecutive two rounds of PCR consisting of unidirectional linear target enrichment (LTE) of SDC2 and quantitative methylation-specific real time PCR (qMSP) for SDC2, named as meSDC2 LTE-qMSP assay. Its limit of detection was 0.1% methylation (corresponding to ~ 6 copies in total ~ 6200 genome copies).ResultsPositive SDC2 methylation was observed in 100% of primary tumors, 90.6% of adenomatous polyps, 94.1% of hyperplastic polyps, and 0% of normal tissues. SDC2 methylation level also significantly (P < 0.01) increased according to the severity of lesions. In stool DNA test for SDC2 methylation by LTE-qMSP comparing CRC patients with various stages (I to IV) (n = 50) and precancerous lesions (n = 21) with healthy subjects (n = 22), the overall sensitivity was 90.0% for detecting CRC and 33.3% for detecting small polyps, with a specificity of 90.9%.ConclusionsTaken together, our result indicates that stool DNA-based SDC2 methylation test by LTE-qMSP is a potential noninvasive diagnostic tool for early detection of CRC.