Seon-Ah Ha

The bone morphogenetic protein antagonist gremlin 1 is overexpressed in human cancers and interacts with YWHAH protein

BackgroundBasic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of our study was to identify an unique gene that shows cancer-associated expression, and characterizes its function related to human carcinogenesis.MethodsWe used the differential display (DD) RT-PCR method using normal cervical, cervical cancer, metastatic cervical tissues, and cervical cancer cell lines to identify genes overexpressed in cervical cancers and identified gremlin 1 which was overexpressed in cervical cancers. We determined expression levels of gremlin 1 using Northern blot analysis and immunohistochemical study in various types of human normal and cancer tissues. To understand the tumorigenesis pathway of identified gremlin 1 protein, we performed a yeast two-hybrid screen, GST pull down assay, and immunoprecipitation to identify gremlin 1 interacting proteins.ResultsDDRT-PCR analysis revealed that gremlin 1 was overexpressed in uterine cervical cancer. We also identified a human gremlin 1 that was overexpressed in various human tumors including carcinomas of the lung, ovary, kidney, breast, colon, pancreas, and sarcoma. PIG-2-transfected HEK 293 cells exhibited growth stimulation and increased telomerase activity. Gremlin 1 interacted with homo sapiens tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide (14-3-3 eta; YWHAH). YWHAH protein binding site for gremlin 1 was located between residues 61–80 and gremlin 1 binding site for YWHAH was found to be located between residues 1 to 67.ConclusionGremlin 1 may play an oncogenic role especially in carcinomas of the uterine cervix, lung, ovary, kidney, breast, colon, pancreas, and sarcoma. Over-expressed gremlin 1 functions by interaction with YWHAH. Therefore, Gremlin 1 and its binding protein YWHAH could be good targets for developing diagnostic and therapeutic strategies against human cancers.

Cancer-Associated Expression of Minichromosome Maintenance 3 Gene in Several Human Cancers and Its Involvement in Tumorigenesis

Purpose: The purpose of our study was to identify an unique gene that shows cancer-associated expression, evaluates its potential usefulness in cancer diagnosis, and characterizes its function related to human carcinogenesis. Experimental Design: We used the differential display reverse transcription-PCR method with normal cervical, cervical cancer and metastatic tissues, and cervical cancer cell line to identify genes overexpressed in cancers. Results: We identified a minichromosome maintenance 3 (MCM3) gene that was overexpressed in various human cancers, including leukemia, lymphoma, and carcinomas of the uterine cervix, colon, lung, stomach, kidney and breast, and malignant melanoma. Western blot and immunohistochemical analyses also revealed that MCM3 protein was elevated in most of human cancer tissues tested. We compared the MCM3 protein expression levels in human cancers with conventional proliferation markers, Ki-67 and proliferating cell nuclear antigen. MCM3 antibody was the most specific for multiple human cancers, whereas proliferating cell nuclear antigen was relatively less effective in specificity, and Ki-67 failed to detect several human cancers. The down-regulation of MCM3 protein level was examined under serum starvation in both normal and cancer cells. Interestingly, MCM3 protein was stable in MCF-7 breast cancer cells even up to 96 hours after serum starvation, whereas it was gradually degraded in normal BJ fibroblast cells. Nude mice who received injections of HEK 293 cells stably transfected with MCM3 formed tumors in 6 weeks. Conclusions: Our study indicates that determination of MCM3 expression level will facilitate the assessment of many different human malignancies in tumor diagnosis, and MCM3 is involved in multiple types of human carcino-genesis.

The Human Cervical Cancer Oncogene Protein Is a Biomarker for Human Hepatocellular Carcinoma

Human cervical cancer oncogene (HCCR) was identified and appeared to function as a negative regulator of p53 gene. The objective of this study was to validate HCCR expression as a candidate marker for human hepatocellular carcinoma. HCCR epitope was identified as Y355LGTRR360. According to immunofluorescence study, HCCR was predominantly localized in the plasma membrane and cytoplasm of hepatocellular carcinoma. HCCR proteins were overexpressed in the tumorous compared with the nontumorous cirrhosis tissues. However, HCCR was not detected in normal liver tissue. Concentration of HCCR protein in the serum was measured in a total of 570 subjects, and comparisons were made to α-fetoprotein. Serological studies revealed 78.2% sensitivity of HCCR (cutoff value, 15 μg/ml), which was significantly higher than 64.6% of α-fetoprotein (P = 0.0098) and 95.7% specificity for hepatocellular carcinoma. Forty of 52 (76.9%) patients with carcinoma negative for α-fetoprotein showed positive values for HCCR. A positive rate of 69.2% in carcinoma patients with tumor sizes <2 cm was found to be a higher rate than measurement of α-fetoprotein. Furthermore, HCCR expression was also detected in liver cirrhosis at an intermediate level between carcinoma and normal groups, which gave 88.1% sensitivity and 79.0% specificity using 8 μg/ml as a cutoff value. In summary, the HCCR assay may have an advantage over the α-fetoprotein assay in that it is elevated according to disease progression from liver cirrhosis to carcinoma, and it is more frequently positive in patients with early, small hepatocellular carcinoma.

p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance.

BackgroundCancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells.MethodsDoxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated.ResultsYB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics.ConclusionOur work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

Fibrinogen gamma-A chain precursor in CSF: a candidate biomarker for Alzheimer's disease

BackgroundCerebrospinal fluid (CSF) may be valuable for exploring protein markers for the diagnosis of Alzheimers disease (AD). The prospect of early detection and treatment, to slow progression, holds hope for aging populations with increased average lifespan. The aim of the present study was to investigate candidate CSF biological markers in patients with mild cognitive impairment (MCI) and AD and compare them with age-matched normal control subjects.MethodsWe applied proteomics approaches to analyze CSF samples derived from 27 patients with AD, 3 subjects with MCI and 30 controls. The AD group was subdivided into three groups by clinical severity according to clinical dementia rating (CDR), a well known clinical scale for dementia.ResultsWe demonstrated an elevated level of fibrinogen gamma-A chain precursor protein in CSF from patients with mild cognitive impairment and AD compared to the age-matched normal subjects. Moreover, its expression was more prominent in the AD group than in the MCI and correlated with disease severity and progression. In contrast, fibrinogen gamma-A chain precursor protein was detected very low in the age-matched normal group.ConclusionThese findings suggest that the CSF level of fibrinogen gamma-A chain precursor may be a candidate biomarker for AD.

Minichromosome maintenance protein 3 is a candidate proliferation marker in papillary thyroid carcinoma.

The proliferative capacity of tumor cells is a characteristic feature in the whole growing tumors. Many pathologists and clinicians have used the estimation of cell proliferation for prognostic information. Minichromosome maintenance protein 3 (MCM3) is known to have a role on the initiation and regulation of DNA replication during cell cycle. The aim of this study was to evaluate the potential applicability of one of the MCM proteins, MCM3, as a proliferation marker in papillary thyroid carcinoma (PTC) with correlation to clinicopathological parameters. We performed the immunohistochemical analysis for MCM3 and Ki-67 in 60 cases of PTC and Western blot analysis for MCM3 expression in 6 PTCs and normal thyroid tissues. The comparison of MCM3 labeling index (LI) to tumor size (P=0.031) and extrathyroidal extension (P=0.037) was statistically significant while that of Ki-67 LI to them was not. Moreover, a significant association was not observed between MCM3 and Ki-67, but the MCM3 LI was considerably higher. Western blot analyses revealed that the MCM3 protein expression levels were overexpressed in all PTCs. On the contrary, the levels of MCM3 were very low or absent in all normal thyroid tissues. Our results indicate that MCM3 may be a more reliable proliferation marker than Ki-67 in accessing the growth of tumor and evaluating tumor aggressiveness of PTC.

Combined analysis of AFP and HCCR-1 as an useful serological marker for small hepatocellular carcinoma: A prospective cohort study

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors in the world. The only serological marker widely used for the diagnosis of HCC is alpha-fetoprotein (AFP). Despite that AFP is widely used for the diagnosis of HCC, it has a limit as a serological marker due to its low sensitivity and specificity. The human cervical cancer proto-oncogene 1 (HCCR-1) was previously reported as a new biomarker for HCC. To further evaluate the HCCR-1 as a biomarker for HCC, we conducted the prospective cohort study. We evaluated the significance of simultaneous measurement of 2 tumor markers in the diagnosis of HCC in China, Japan and Korea. Two markers for HCC, AFP and HCCR-1, were measured in the sera obtained from 1,338 patients at the time of initial diagnosis of HCC. Of the 1338 HCC patients, 616 (46%) and 686 (51.3%) were sero-positive for AFP and HCCR-1, respectively. The positive rate for HCC was increased up to 74.1% in combined use of AFP and HCCR-1. Many cases (54%) for AFP-negative HCC were positive for HCCR-1 and vice versa. More importantly, the diagnostic rate for small HCC (< 2 cm) was significantly improved in the combined analysis of AFP and HCCR-1 to 56.9% although it was only 40.1% and 23.4% in the single analysis of HCCR-1 and AFP, respectively. Our result suggests that the HCCR-1 could be an useful biomarker for HCC while the diagnostic rate could be significantly improved in the combined use of HCCR-1 and AFP.

Transdifferentiation-inducing HCCR-1 oncogene

BackgroundCell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules.ResultsWe report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell.ConclusionsTherefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.

HCCR-1, a novel oncogene, encodes a mitochondrial outer membrane protein and suppresses the UVC-induced apoptosis.

BackgroundThe Human cervical cancer oncogene (HCCR-1) has been isolated as a human oncoprotein, and has shown strong tumorigenic features. Its potential role in tumorigenesis may result from a negative regulation of the p53 tumor suppressor gene.ResultsTo investigate the biological function of HCCR-1 in the cell, we predicted biological features using bioinformatic tools, and have identified a LETM1 homologous domain at position 75 to 346 of HCCR-1. This domain contains proteins identified from diverse species predicted to be mitochondrial proteins. Fluorescence microscopy and fractionation experiments showed that HCCR-1 is located in mitochondria in the COS-7, MCF-7 and HEK/293 cell lines, and subcompartamentally at the outer membrane in the HEK/293 cell line. The topological structure was revealed as the NH2-terminus of HCCR-1 oriented toward the cytoplasm. We also observed that the D1-2 region, at position 1 to 110 of HCCR-1, was required and sufficient for posttranslational mitochondrial import. The function of HCCR-1 on mitochondrial membrane is to retard the intrinsic apoptosis induced by UVC and staurosporine, respectively.ConclusionOur experiments show the biological features of HCCR-1 in the cell, and suggest that uncontrolled expression of HCCR-1 may cause mitochondrial dysfunction that can result in resisting the UVC or staurosporine-induced apoptosis and progressing in the tumor formation.

Oncoprotein HCCR-1 expression in breast cancer is well correlated with known breast cancer prognostic factors including the HER2 overexpression, p53 mutation, and ER/PR status.

BackgroundOncoprotein HCCR-1 functions as a negative regulator of the p53 and contributes breast tumorigenesis. The serum HCCR-1 assay is useful in diagnosing breast cancer and mice transgenic for HCCR developed breast cancers. But it is unknown how HCCR-1 contributes to human breast tumorigenesis.MethodsOncogene HCCR-1 expression levels were determined in normal breast tissues, breast cancer tissues and cancer cell lines. We examined whether HCCR-1 protein expression in breast cancer is related to different biological characteristics, including ER, PR, p53 genotype, and HER2 status in 104 primary breast cancer tissues using immunohistochemical analyses.ResultsHCCR-1 was upregulated in breast cancer cells and tissues compared with normal breast tissues. In this study, overexpression of HCCR-1 was well correlated with known breast cancer prognostic markers including the presence of steroid receptors (ER and PR), p53 mutation and high HER2 overexpression. HCCR-1 was not detected in the ER-negative, PR-negative, p53 negative and low HER2 breast cancer tissues. These data indicate that the level of HCCR-1 in breast cancer tissues is relatively well correlated with known breast cancer factors, including the HER2 overexpression, p53 mutation, and ER/PR status.ConclusionDetermination of HCCR-1 levels as options for HER2 testing is promising although it needs further evaluation.