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Colorectal cancer (CRC) is widely prevalent and represents a significant contributor to global cancer‑related mortality. There remains a pressing demand for advancements in CRC treatment modalities. The E3 ubiquitin ligase is a critical enzyme involved in modulating protein expression levels via posttranslational ubiquitin‑mediated proteolysis, and it is reportedly involved in the progression of various cancers, making it a target of recent interest in anticancer therapy. In the present study, using comprehensive expression analysis involving spatial transcriptomic analysis with single‑cell RNA sequencing in clinical CRC datasets, the ubiquitin‑associated protein Shank‑associated RH domain interactor ( SHARPIN ) was identified, located on amplified chromosome 8q, which could promote CRC progression. SHARPIN was found to be upregulated in tumor cells, with elevated expression observed in tumor tissues. This heightened expression of SHARPIN was positively associated with lymphatic invasion and served as an independent predictor of a poor prognosis in patients with CRC. In vitro and in vivo analyses using SHARPIN‑overexpressing or ‑knockout CRC cells revealed that SHARPIN overexpression upregulated MDM2, resulting in the downregulation of p53, while SHARPIN silencing or knockout downregulated MDM2, leading to p53 upregulation, which affects cell cycle progression, tumor cell apoptosis and tumor growth in CRC. Furthermore, SHARPIN was found to be overexpressed in several cancer types, exerting significant effects on survival outcomes. In conclusion, SHARPIN represents a newly identified novel gene with the potential to promote tumor growth following apoptosis inhibition and cell cycle progression in part by inhibiting p53 expression via MDM2 upregulation; therefore, SHARPIN represents a potential therapeutic target for CRC.

Tylosis is an inherited disorder characterized by abnormal palmoplantar skin thickening and a highly elevated risk of esophageal squamous cell carcinoma (ESCC). Analyses of tylosis in families have localized the responsible gene locus to a region of chromosome 17q25.1. Frequent loss of heterozygosity (LOH) in 17q25.1 was also observed in the sporadic form of ESCC. A putative tumor suppressor gene for ESCC may exist at this locus. We investigated the expression patterns of genes on 17q25.1 in tumor and corresponding normal tissues from patients with sporadic ESCC using RNA sequence analysis. For candidate genes, quantitative real-time reverse transcription-PCR (qRT-PCR), direct sequence, LOH and methylation analyses were performed using 93 clinical ESCC samples and 10 cell lines. A significant downregulation of ST6GALNAC1 was demonstrated in ESCC tissues compared to its expression in normal tissues by qRT-PCR (n=93, p<0.0001). Frequent LOH (17/27, 62.9%) and hyper‑methylation in ST6GALNAC1 were also observed in all cell lines. Our results indicated that ST6GALNAC1 was downregulated in sporadic ESCC via hyper-methylation and LOH, and it may be a candidate responsible gene for ESCC. Furthermore, recent studies suggest that multiple genes on chromosome 17q25 are involved in ESCC development.

Recent studies have indicated that increased ribosomal activity contributes to cancer progression. Transcription termination factor, RNA polymerase I (TTF1) acts as a transcription factor for RNA polymerase I. However, the role which TTF1 plays in cancer progression still remains unknown. The present study aimed to determine whether TTF1 plays a critical role in the progression of human liver hepatocellular carcinoma (HCC). In the present study, quantitative real-time reverse transcription polymerase chain reaction was conducted to evaluate TTF1 mRNA expression in 60 HCC tissue samples in order to determine the clinicopathological significance of TTF1. To investigate whether the expression levels of TTF1 were associated known gene signatures which represented ribosomal activity, we applied gene set enrichment analysis (GSEA) to HCC cases in The Cancer Genome Atlas (TCGA) a. We also performed in vitro proliferation assays using TTF1‑overexpressing HCC cells. TTF1 expression was significantly higher in HCC tumor tissues than in adjacent liver tissues (P<0.001). The overall survival (OS) of patients with high TTF1 expression levels was significantly shorter than that of patients with low TTF1 expression (P=0.027). Multivariate analysis indicated that TTF1 expression was an independent prognostic factor for OS (P=0.020). GSEA revealed significant associations between TTF1 expression and gene sets involved in ribosomal function. In vitro, cell proliferation and rRNA transcription were significantly promoted by overexpression of TTF1 in the HCC cell lines HuH-7 and HepG2. From these results, it was suggested that TTF1 participate in poor prognoses and play a role in tumor cell growth in HCC, possibly by upregulating ribosomal activity. In conclusion, we first propose that TTF1 may be a novel biomarker and therapeutic target in HCC. Increased expression of TTF1 was significantly associated with poor prognosis in two independent sets of HCC cases. Furthermore, in vitro experiments provided an explanation for clinical data showing that overexpression of TTF1 contributed to the proliferation of cancer cells.

Depressed and flat surface lesions are not easy to identify with routine colonoscopies during screening for colorectal cancer (CRC). Identifying clinically relevant genes that influence tumor shape could be useful when screening for the presence of depressed lesions. Total RNA was extracted from tumor cells collected by laser microdissection from the primary lesions of 146 CRC cases. Microarray analysis was performed to identify genes that were differentially expressed between depressed and elevated tumors. Single nucleotide polymorphism (SNP) analysis of genomic DNA from the peripheral blood of 67 CRC patients was then used to associate polymorphisms with the occurrence of depressed tumors. Microarray analysis revealed significantly higher expression of the fibronectin 1 (FN1) gene in 129 depressed-type tumors and lesions compared to 17 elevated-type tumors. FN1-abundant CRC tumors were large with a significantly higher incidence of lymphatic permeation. SNP analysis indicated that 44 tumors with a GG genotype at SNP rs6707530 showed significantly higher FN1 expression than did 23 tumors with GT/TT genotypes (p<0.05). The product of the FN1 gene (located at 2q34) is involved in cell adhesion, migration and metastasis in mesenchymal tumors. Abundant expression of FN1 may allow cancer cells to invade deeper layers, which would eventually define tumor shape. Identification of this SNP in blood samples may facilitate disease diagnosis and allow prediction of the presence of depressed tumors in the colorectal epithelium before a colon fiberscope examination.

Conventional cancer chemotherapy preferentially destroys non-stem cancer cells within a tumor, and a subpopulation of cancer stem cells (CSCs) is more resistant and survives, leading to relapses and metastasis. Howeve, recent studies suggest that CD24 and susceptibility to epithelial-mesenchymal transition (EMT) can serve as markers of CSCs. We report that CD24(+) cells are susceptible to induction of EMT, a phenotype important for cancer metastasis. We studied the responsiveness of CSC markers to TGF-β , an effective EMT inducer. The data on CD24 demonstrated that CD24(+) cells are susceptible to EMT, a phenotype important for cancer metastasis in two colorectal cancer cell lines, the CaR-1 and CCK81. CD24(+) cells expressed Notch 1 in response to exposure to TGF-β in culture and showed higher tumorigenic activity compared to controls. This evidence shows that CD24(+) cells are susceptible to EMT induction and to cancer progression and is indicative of the candidacy of CD24 as a therapeutic target in CSC.

Cyclin-dependent kinase subunit 2 (CKS2) is a cyclin-dependent kinase subunit (CKS) family member that participates in cell cycle regulation. Few studies have investigated its involvement in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to assess the clinical significance of CKS2 in ESCC. We used immunohistochemistry to study the clinicopathologic significance of CKS2 protein expression in 121 patients with ESCC. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we examined the expression of CKS2 mRNA in tumors and the corresponding normal esophageal tissues that were obtained from 62 patients. Finally, siRNA-mediated attenuation of CKS2 expression was examined in vitro. CKS2 protein expression was significantly correlated with depth of tumor invasion, clinical stage, lymphatic invasion and distant metastasis (p=0.033, 0.028, 0.041 and 0.009, respectively). CKS2 mRNA expression was higher in cancer tissue than in corresponding normal tissue (p<0.001). Patients with positive-CKS2 protein expression had a poorer five year survival frequency than patients who did not express CKS2 protein (p=0.025). In vitro, siRNA-mediated suppression of CKS2 slowed the growth rate of ESCC cells compared to control cells (p<0.001). The evaluation of CKS2 expression is useful for predicting the cause of malignant tumors and the prognosis of patients with ESSC.

Inhibin β A (INHBA) is a member of the transforming growth factor β (TGF-β) superfamily. INHBA expression is associated with several types of human cancers; however, its significance in colorectal cancer (CRC) is not fully understood. INHBA expression was studied in 126 primary CRC samples and 4 CRC cell lines. Cell growth was assessed after inhibition of INHBA expression or after exogenous overexpression of INHBA in CRC tissues. INHBA expression was significantly higher in CRC tissues when compared to that in the corresponding normal tissues (P<0.001). Patients in the high expression group showed a poorer overall survival rate when compared to those in the low expression group (P<0.001); the present study did not evaluate for an independent prognostic factor but showed the significance of lymph node metastasis as an independent prognostic factor. The present study suggests that INHBA is useful as a predictive marker for prognosis in CRC patients.

Previous studies have suggested conflicting roles for the E3 ubiquitin ligase COP1 in tumorigenesis, providing evidence that both the oncoprotein c-Jun and the tumor suppressor p53 may be COP1 targets. In the present study, we focused on the clinical significance of COP1 expression in gastric cancer cases and analyzed the malignant behavior of COP1‑knockdown gastric cancer cells in vitro. We analyzed COP1 expression in cancer lesions and the corresponding normal mucosa to demonstrate the clinical significance of COP1 expression in 133 cases of gastric cancer. We also investigated the relationship between COP1 expression and cell proliferation and the association of COP1 with c‑Jun transcriptional target genes, such as MMP1, MMP7 and MMP10. The expression of COP1 mRNA was significantly lower in gastric cancer tissues compared to the corresponding normal mucosa (P=0.049). In multivariate analysis for overall survival, we found that COP1 expression was an independent prognostic factor in gastric cancer. Knockdown of COP1 expression in the gastric cancer cell lines MKN‑45 and NUGC4 promoted proliferation, and significant associations between COP1 expression and MMP1, MMP7 and MMP10 were also observed in knockdown assays. In conclusion, the present study suggests that loss of COP1 expression may be a novel indicator for the biological aggressiveness in gastric cancer.

The chromodomain helicase DNA-binding (CHD) family comprises a class of chromatin remodeling enzymes. Previous studies suggest that CHD8 may negatively regulate various genes and signaling pathways, such as the Wnt/β‑catenin pathway. However, few studies have investigated the role of CHD8 in cancer cells. We analyzed the expression of CHD8 in cancer lesions and corresponding non-cancerous tissues to demonstrate the prognostic significance of CHD8 expression in 101 cases of gastric cancer. We also investigated the functional implications of aberrant CHD8 expression by conducting gene set enrichment analysis (GSEA). Expression of CHD8 mRNA was significantly lower in gastric cancer tissues compared to that in corresponding normal tissues (P=0.003). In multivariate analysis for overall survival, we found that CHD8 expression was an independent prognostic factor in gastric cancer. Moreover, GSEA revealed that CHD8 was significantly associated with genes involved in the Wnt/β‑catenin pathway and in the cell cycle. In addition, knockdown of CHD8 expression in the gastric cancer cell lines, MKN45 and NUGC4, promoted proliferation. In conclusion, the present study suggests that loss of CHD8 expression may be a novel indicator for biological aggressiveness in gastric cancer.

Tumor size indicates the extent of cell proliferation in most cases of colorectal cancer (CRC), although there are some advanced small tumors with metastases. Lymph node metastasis is a significant factor that greatly impacts disease prognosis in CRC cases. The underlying factors that cause lymph node metastasis in CRC cells are not fully understood. We investigated the mechanism that might induce CRC metastasis by focusing on smaller sized (<2 cm) invasive tumors. We carried out gene expression array analysis for CRC cases; group 1 consisted of 6 cases with tumors <2 cm with metastases, and group 2 consisted of 65 cases with tumors >2 cm without metastases. Results were validated using gene expression array data from an additional 77 cases and another bulk case set of 172 cases. Gene ontology and pathway analysis using microarray data revealed that anti-apoptotic activity had a crucial role in CRC metastasis. High mobility group A1 (HMGA1) was identified as a biomarker for poor prognosis and metastasis formation. HMGA1 expression levels were higher in lymph node-positive cases than in lymph node-negative cases, even in subgroup analysis of submucosal invasive cases. The present study strongly supports the clinical significance of HMGA1 expression as a predictive indicator of lymph node metastasis in CRC cases, even in submucosal invasive cases which could be cured by local resection.

Cancer stem cells (CSCs) also known as cancer-initiating cells (CICs) show high tumorigenic activity and high chemo- and radiation resistance. It is, therefore, important to identify CSCs reliably to develop novel curative cancer treatments. In this study, we re-evaluated CSC markers of colorectal cancer for their cellular differentiation and tumorigenic activity, with the aim to identify reliable CSC markers. The rates of change in CD44, CD133, CD166, CD24, CD49f and CXCR4 expression during sodium butyrate (NaBT)-induced cell differentiation were assessed in HT29 and Caco2 colon cancer cell lines. Expression levels of target markers were assessed in clinical CRC samples. Tumorigenic activity was assessed on isolated cell fractions identified by multicolor flow cytometric analysis. In the cell differentiation assay, the average percent change was higher in CD44 (-98.2%) and CD49f (-74.4%) compared to CD133 (-17.9%) and CD166 (-49.4%). Expression of CD24 and CXCR4 appeared random in HT29 and Caco2. Expression of CD44, CD49f, CD133 and CD166 was confirmed in all four clinical CRC samples. Limiting dilution assay of CD44- and CD133-expressing cells revealed that only the CD133⁺CD44⁺ population possessed tumorigenic activity. Tumorigenesis was not affected by CD166 expression. Highly tumorigenic cells could be enriched in samples with higher CD49f expression; CD49f⁺ cells showed high tumorigenesis, whereas CD133⁺ and CD44⁺ cells that were negative for CD49f exhibited no tumorigenic activity. Multicolor analysis revealed that CD49f⁺ cells localized in CD44⁺ and CD133⁺ cell fractions. These findings demonstrated that CD49f is an important marker for identifying colorectal CSCs and suggest that the CD49f⁺ cell fraction may be the best candidate for colorectal CSCs.

The functions of many long non-coding RNAs (ncRNAs) in human cancers have not yet been elucidated. The long ncRNA HOTAIR is expressed from the developmental HOXC locus located on chromosome 12q13.13. Previous reports have demonstrated that HOTAIR associates with chromatin modifications in cooperation with the Polycomb complex PRC2, and promotes breast and colorectal cancer metastasis. In this study, we examined the clinical significance of HOTAIR expression in patients with hepatocellular carcinoma (HCC). HOTAIR expression was detected in primary HCCs in 13 out of 64 patients. Patients with HOTAIR expression had significantly poorer prognoses and a larger primary tumor size than those without HOTAIR expression, similar to studies in breast and colorectal cancers. Moreover, introduction of human HOTAIR into liver cancer cells revealed that HOTAIR promoted more rapid proliferation compared to control cells. Thus, although the clinical significance of HOTAIR expression in HCC may not be as pronounced as that in breast and colorectal cancers, the current study demonstrates that HOTAIR expression is associated with HCC progression, warranting further studies.

We previously studied hypoxic tumor cells from hepatic metastases of colorectal cancer (CRC) and determined several potential prognostic factors, including expression of ephrin-A1 (EFNA1), which was highly induced by hypoxia. Here, we further evaluated the prognostic impact of EFNA1 expression. Samples from a total of 366 CRC patients from 11 institutes were analyzed by either microarray (n=220) or quantitative reverse-transcriptase polymerase chain reaction (n=146). EFNA1 was an independent prognostic factor for CRC (p<0.05). In vitro assays revealed that loss of EFNA1 following siRNA treatment was associated with reduced proliferative activity and decreased invasion and migration of CRC cell lines. EFNA1 expression is a useful marker for predicting high risk of relapse and cancer-related death in patients who have undergone curative resection for CRC.

miRNAs regulate gene expression at the post-transcriptional level by degradation of mRNA and translational repression. Recent studies have shown that miR-181a is dysregulated in several types of cancer; however, the clinical significance of miR‑181a in colorectal cancer (CRC) remains unclear. We addressed this question by using quantitative real-time PCR (qRT-PCR) to analyze miR-181a expression in 162 CRC patients. There was no significant difference in miR-181a expression in normal colon vs. colorectal cancer tissue. The cancer tissue samples were categorized into a low and high expression group based on miR-181a expression. Comparison of the clinicopathological factors and prognosis in these two groups showed that the high expression group had a significantly poorer prognosis than the low expression group (P=0.011). Multivariate analysis indicated that high miR-181a expression was an independent significant prognostic factor for CRC. However, there no correlation was observed between miR-181a expression and clinicopathological parameters. In vitro analysis revealed that the overexpression of miR-181a repressed the expression of the tumor suppressor, phosphatase and tensin homolog (PTEN) located on chromosome 10, at the mRNA level. These data suggest that miR-181a may be a new independent prognostic factor for CRC patients.

FBXW7 is a tumor suppressor gene that plays a role in cell cycle regulation via Myc degradation. However, the clinical significance of FBXW7 in esophageal squamous cell carcinoma (ESCC) has not been evaluated. The purpose of this study was to assess the clinical significance of FBXW7 for prognosis in human ESCC. Real-time RT-PCR was used to examine the expression of FBXW7 to determine its clinicopathological significance in 75 cases of ESCC. Overall survival rate was calculated using the Kaplan-Meier method, while multivariate survival was analyzed with the Cox hazard model. FBXW7 suppression analysis was performed to examine proliferation potency and Myc expression in the FBXW7 siRNA groups. The relationship between FBXW7 expression and the copy number loss of FBXW7 was examined in clinical samples of ESCC. Finally, FBXW7 copy number loss was linked to prognosis in 42 ESCC patients. FBXW7 expression in cancer was lower compared to non-cancer tissues (P=0.003) and is an independent prognostic factor. The proliferation rates and Myc protein expression were significantly enhanced in FBXW7 siRNA cells compared to the controls. Cases with a loss of FBXW7 copy number had low FBXW7 expression and a poorer prognosis than cases with no loss of copy number. Genetic alterations in esophageal cancer lead to the loss of FBXW7 expression and increased cell proliferation. These genetic alterations of FBXW7 status may provide a prognostic factor for ESCC patients.

Recent reports have demonstrated that another strand of mature microRNA (miRNA), called microRNA* or 3p (5p) strand, which is generated from the same precursor miRNA (Pre-miR), has a crucial role in cellular function. We previously reported the tumor suppressive effect of miR-125a-5p in gastric cancer. The current study was designed to examine the function and clinical significance of miR-125a-3p, a partner strand of miR-125a-5p, in human gastric cancer. Quantitative RT-PCR was used to evaluate miR-125a-3p expression in 70 gastric cancer cases to determine the clinicopathologic significance of miR-125a-3p expression. In addition, the effect of miR-125a-3p on the proliferation of gastric cancer cells was investigated. Low expression levels of miR-125a-3p were associated with indicators of enhanced malignant potential such as tumor size (p=0.0002), tumor invasion (p=0.0149), lymph node metastasis (p=0.018), liver metastasis (p=0.016), peritoneal dissemination (p=0.03), advanced clinical stage (p=0.0037) and poor prognosis (p=0.0083). Multivariate analysis indicated that low miR-125a-3p expression was an independent prognostic factor for survival, while in vitro assays demonstrated that miR‑125a-3p suppressed the proliferation of gastric cancer cells. MiR-125a-3p is a potent prognostic marker in gastric cancer. The clinical significance and tumor suppressive effect of miR‑125a-3p, as well as previously reported miR-125a-5p, suggest that the functional role of another strand of the mature form miRNA cannot be ignored, at least in miR-125a biogenesis.

The aim of this study was to identify and evaluate novel prognostic markers for gastric cancer. Differential mRNA displays comparing paired tumor/normal stomach samples were assessed. Several differentially expressed cDNA fragments of candidate genes were identified, and one of these was further studied using quantitative reverse transcription-PCR in 140 human gastric carcinomas. To evaluate protein expression, immunohistochemical staining was performed in selected cases. One of the genes abundantly expressed in tumor tissue on the differential mRNA displays was identified as histone deacetylase 1 (HDAC). HDAC was overexpressed in the tumor tissue in 77% of the cases as determined by quantitative reverse transcription-PCR. Immunohistochemical staining revealed analogous results, showing strong expression in cancer cells. Patients were then classified into high (n=78) and low (n=62) expression groups according to the mean value of HDAC expression. High frequencies of lymph vessel and vascular vessel permeations, and advanced stage of the disease were recognized in the high expression group compared to the low expression group (p<0.05). Prognosis was significantly worse for the high expression group than for the low expression group (p<0.05), and multi-variate analysis demonstrated that HDAC expression was an independent prognostic factor. Although not significantly different, lymph node metastasis was recognized more frequently in the high expression group (p=0.07). In conclusion, the findings show that HDAC expression is associated with aggressive behavior of primary gastric cancer, and imply that determination of the HDAC expression status is useful for predicting prognosis in patients with gastric cancer.

Claudin1 (CLDN1) plays an important role not only in the intercellular barrier function of tight junctions (TJs) but also in migration and invasiveness of cancer cells. Previous reports show that CLDN1 overexpression is significantly related to the malignant behavior in several cancer types whereas its significance in colorectal cancer (CRC) is not fully understood. The present study comprised 119 patients who underwent surgery for CRC, as well as 3 cell lines derived from human CRC. The correlation of gene expression with clinical parameters in patients was assessed by knockdown experiments using 3 cell lines. Patients with high CLDN1 expression were statistically shown to have a relatively better prognosis, and those with low CLDN1 expression showed poorer overall survival and disease-free survival than those with high expression. The assessment of CLDN1 knockdown in the 3 cell lines demonstrated that the siRNA inhibition resulted in a statistically significant increase in cell invasiveness. In conclusion, the present data strongly suggest that CLDN1 expression is a prognostic factor in CRC patients.

CDC28 protein kinase regulatory subunit 2 (CKS2) is a cyclin-dependent kinase subunit (CKS) family member that participates in cell cycle regulation. Few studies have investigated its involvement in gastric cancer. In this study, we focused on the clinical and biological significance of CKS2 over-expression in gastric cancer. The expression of CKS2 mRNA was studied by real-time quantitative reverse transcription polymerase chain reaction, and the expression status in each tumor was examined to varify whether any correlation existed with clinical and pathological factors. In addition, an immuno-histochemical study was performed in selected samples. Moreover, we examined the impact of CKS2-siRNA in a gastric cancer cell line. A significantly higher expression of CKS2 mRNA was found in tumor tissues compared to paired normal tissues (p<0.01). Immunohistochemical analyses led to similar results. The overall five-year survival rate was significantly higher in the low CKS2 expression group (59.9%) than in the high CKS2 expression group (23.9%) (p<0.01). Univariate and multivariate analysis showed that CKS2 expression status was an independent prognostic factor (relative risk, 1.41; 95% confidence interval, 1.01-1.97; p<0.05). Moreover, the inhibition of cellular proliferation by CKS2-siRNA was observed in a gastric cancer cell line. CKS2 is one of the gastric cancer-related genes that correlates with biological aggressiveness and poor prognosis of gastric cancer. Thus, CKS2 is a possible candidate gene for diagnosis, as well as targeted molecular diagnosis and therapy in gastric cancer.

Cyclin D1 plays important roles in esophageal squamous cell carcinoma (ESCC) cases by amplification of the 11q13.3 locus. FBXO31 is a subunit of the SCF ubiquitin ligase, which targets cyclin D1 for degradation. In this study, we clarified the clinical significance of FBXO31 and characterized the association between cyclin D1 and FBXO31 in ESCC cases. Total RNA was extracted from tumor tissues obtained from 68 ESCC patients who underwent surgical resection. FBXO31 expression levels were determined by quantitative RT-PCR, and both FBXO31 and cyclin D1 protein expression and localization were evaluated by immunohistochemistry (IHC). Furthermore, using CGH and gene expression array data of another subset, we validated the association between cyclin D1 genomic amplification and FBXO31 expression levels. Higher FBXO31 expression levels significantly correlated with depth of tumor invasion and clinical stage (P<0.05). In addition, the FBXO31 high expression group showed a significantly poorer prognosis than the low expression group (P<0.001). Multivariate analysis indicated that FBXO31 expression was an independent prognostic factor [relative risk (RR): 1.79, confidence interval (CI): 1.14-3.01, P=0.01]. Using IHC, concordant expression was observed between cyclin D1 and FBXO31 in the nucleus and cytoplasm, respectively. CGH array indicated that cases having cyclin D1 with increased copy number were significantly associated with elevated FBXO31 expression levels (P<0.05). FBXO31 could be a novel and robust prognostic marker for ESCC.

MicroRNAs (miRNAs) are small, non-coding RNAs that can function as oncogenes or tumor suppressors in human cancer. Recent reports have highlighted the oncogenic aspects of microRNA miR-125b. However, the clinical significance of miR-125b in gastrointestinal cancers has not been sufficiently investigated. To this end, we analyzed miR-125b expression in colorectal cancer cases. Quantitative RT-PCR was used to evaluate miR-125b expression in 89 colorectal cancer cases to determine the clinicopathological significance of miR-125b expression. The high miR-125b expression group showed a greater incidence of advanced tumor size and tumor invasion compared to the low miR-125b expression group (P<0.05). In addition, the high miR-125b expression group had a significantly poorer prognosis compared to the low expression group (P<0.05). Multivariate analysis indicated that high miR-125b expression was an independent prognostic factor for survival. Our analysis of miR-125b focused on the miR-125b/p53 pathway. In vitro assays revealed that overexpression of miR-125b repressed the endogenous level of p53 protein in human colorectal cancer cells. These data show that miR-125b is directly involved in cancer progression and is associated with poor prognosis in human colorectal cancer. Our findings suggest that miR-125b could be an important prognostic indicator for colorectal cancer patients.

Recently, bone marrow has been considered as playing a critical role in the generation of both metastasis and recurrent disease. The accumulation of a single microRNA in the bone marrow has the potential to regulate the translation of multiple genes in cancer metastasis and may therefore serve as a prognostic marker for cancer recurrence. MicroRNA microarray analysis was performed to compare microRNA levels in bone marrow from 4 breast cancer patients with recurrent disease and 4 patients without recurrence. Accumulation of two of these microRNAs, miR-21 and miR-181a, in the recurrent breast cancer cases was validated by RT-PCR in bone marrow from 291 additional breast cancer cases. Expression of a common target gene, PDCD4, was also determined in bone marrow from 291 breast cancer cases. Increased miR-21 and miR-181a levels were significantly associated with shortened disease-free survival (DFS; p=0.0003, 0.0007) and overall survival (OS; p=0.0351, 0.0443), respectively. While low PDCD4 expression was also significantly associated with poorer DFS (p=0.036). Multivariate analysis identified bone marrow miR-21 and mR-181a levels as valuable independent prognostic factors, with correlation coefficients that were significantly higher than that of the transcript of their common target gene. Accumulation of miR-21 and miR-181a in bone marrow appears to be associated with prognosis in breast cancer patients. The much higher significant correlation with microRNA levels and prognosis suggests epistatic effects on multiple target genes in the bone marrow of breast cancer patients.

Cancer immunotherapy is a potential therapeutic strategy, in addition to surgical treatment, radiotherapy, and chemotherapy. Cancer-specific immunotherapy, such as the MAGE peptide vaccine, has been utilized clinically. How-ever, there are inherent limits to the effectiveness of vaccinotherapy using a single antigen because of the expression frequency of cancer-specific antigens on tumor cells. Thus, identification of a new cancer-specific antigen is needed. In this study, we examined the possibility of using cancer-specific immunotherapy based upon mitotic centromere-associated kinesin (MCAK) which was previously identified as a novel cancer/testis antigen. To evaluate the feasibility of developing cancer immunotherapy using MCAK peptides, we studied HLA-A*0201 and *2402 as targets for CTLs in the context of HLA class I molecules. By using a peptide with a sequence of AINPELLQL (amino acid positions 63-71 in MCAK, HLA-A*0201) and FFEIYNGKL (amino acid positions 401-409 in MCAK, HLA-A*2402), CTL responses could be induced from unseparated PBMCs by stimulation of freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells (APCs) and also by using interleukin-7 and keyhole limpet hemocyanin in primary culture. The induced CTLs could lyse HLA-A-*0201/*2402 colon and gastric cancer cells expressing MCAK, as well as the peptide-pulsed target cells, in an HLA class l, and CD8 restricted manner. The identification of the MCAK/HLA-A*0201 and *2402 peptides suggests the possibility of designing peptide-based immunotherapeutic approaches that might prove effective in treating patients with MCAK-positive cancer.

Ankyrin repeat and SOCS box-containing 9 (ASB9) is involved in the negative regulation of cytokine signaling. However, its biological function is largely undefined. The aim of this study was to assess the value and role of ASB9 as an indicator of prognosis in colorectal cancer (CRC). In order to demonstrate the importance of ASB9 expression for predicting the prognosis of CRC, we analyzed the ASB9 mRNA expression in 125 paired cases of CRC and non-cancerous regions and the protein expression by immunohistochemistry. To investigate the role of ASB9 in vitro, we performed proliferation and invasion assay with small interfering RNA against ASB9. ASB9 mRNA expression was higher in CRC tissue than corresponding normal tissue (P=0.0282). Patients expressing low levels of ASB9 had a poorer overall survival rate than those expressing high levels (P=0.0301), indicating that decreased ASB9 expression was an independent prognostic factor. The immunohistochemical study revealed that ASB9 was predominantly expressed in cancer cells. A multivariate analysis showed that ASB9 expression status was an independent prognostic factor of overall survival (relative risk, 4.09; 95% confidence interval, 1.47-11.88; P=0.007). In an invasion assay, ASB9 siRNA-transfected cells showed significantly high invasiveness. The results of the present study suggest that ASB9 is a useful prognostic marker for CRC.

Recent studies have demonstrated that the novel tumor suppressor protein programmed cell death 4 (PDCD4) is downregulated in several human solid cancer types and is suppressed by microRNA-21 (miR-21). The objectives of this study were: i) to establish the clinicopathological and prognostic significance of PDCD4 mRNA, and ii) to elucidate any correlation between PDCD4 mRNA and miR-21 in gastric cancer. The expression status of PDCD4 mRNA was investigated by qRT-PCR and protein expression was analyzed by an immunohistochemical study. We analyzed PDCD4 mRNA expression with respect to various clinicopathological factors in 105 gastric cancers. We also performed an association study comparing PDCD4 mRNA and miR-21 in eight cell lines and 49 gastric cancers. Expression of PDCD4 mRNA in cancer tissues was significantly lower than in non-cancer tissues (P<0.05). Patients with low PDCD4 mRNA expression was significantly correlated with size, depth, lymph node metastasis, venous invasion, advanced stage, and poor clinical prognosis (P<0.05). Expression of miR-21 in cancer tissues was significantly higher than in non-cancer tissues (P<0.05). Elevated miR-21 expression was significantly correlated with size and depth (P<0.05). An inverse correlation between PDCD4 mRNA and miR-21 was found in gastric cancer. This study revealed that low PDCD4 expression correlates with biological aggressiveness and poor prognosis in gastric cancer. Furthermore, our findings suggest that PDCD4 mRNA is negatively regulated by miR-21 in gastric cancer and may serve as a target for effective therapies.

As recent technological innovations make it possible to clarify the concordant relationship between genomic alterations and aberrant gene expression during the progression of colorectal cancer (CRC), we aimed at identifying new overexpressing genes with genomic amplification on the responsible loci in CRC. The candidate gene was found using cDNA microarray and array-based comparative genomic hybridization (CGH) analysis after laser microdissection (LMD) in 132 Japanese CRC. We focused on SUGT1, which is associated with the assembling of kinetochore proteins at the metaphase of the cell cycle, with significant association between genetic alterations and expression. SUGT1 mRNA expression was evaluated in 98 CRC cases to determine the clinicopathological significance of SUGT1 expression. The mean level of SUGT1 mRNA expression in tumor tissue specimens was significantly higher than in non-tumor tissue. The high SUGT1 expression group was characterized by a significantly elevated frequency of recurrence and a significantly poorer prognosis than the low expression group. There was a significant association between poor prognosis of CRC cases and the overexpression of SUGT1 with genomic amplification of the loci concordantly. The amplification of SUGT1 might give rise to promote the transcription of the gene directly subsequent to the progression of CRC cases with worsening prognosis.

Teratocarcinoma-derived growth factor 1 (TDGF1) is a member of the epidermal growth factor-cripto FRL1 cryptic protein family and is involved in the activation of several different signaling pathways during embryonic development and cellular transformation. Previous reports show that TDGF1 regulates the activation of several signaling pathways and controls cellular transformation in embryonic status, whereas its significance in colorectal cancer (CRC) is not yet fully understood. The present study comprised 55 patients who underwent surgery for CRC, as well as two cell lines derived from human CRC. The correlation of gene expression with clinical parameters in patients was assessed. The biological significance of TDGF1 expression was evaluated by knockdown experiments in the cell lines. Seventeen of 55 (30.9%) cases exhibited a higher TDGF1 expression in cancerous regions than in marginal non-cancerous regions. Patients with high TDGF1 expression were statistically susceptible to a recurrence of the disease, and showed poorer disease-free survival than those with low expression. The assessment of TDGF1 knock-down in the 2 cell lines demonstrated that the siRNA inhibition resulted in a statistically significant reduction in cell growth and invasion. In conclusion, the present data strongly suggest the usefulness of TDGF1 as a predictive marker for metachronous metastasis in CRC patients.

The adenosine triphosphate-binding cassette transporter-homologous gene ATP11A belongs to an extended family of adenosine triphosphate-binding cassette transporters. We analysed the ATP11A gene in 7 colorectal cancer cell lines and 95 paired cases of colorectal cancer and non-cancerous regions to demonstrate the importance of ATP11A expression in colorectal cancer prognosis. ATP11A was expressed in the 7 colorectal cancer cell lines. ATP11A mRNA expression was higher in colorectal cancer tissue than in corresponding normal tissue (P<0.001). Patients with high ATP11A expression showed a poorer disease-free survival rate compared to those with low expression (P<0.001), thus indicating that increase in ATP11A expression was an independent predictor of metachronous metastasis of colorectal cancer. The present study suggests that ATP11A is a useful predictive marker of metastasis in colorectal cancer patients.

Whereas the Her2/neu/erbB2 receptor (Her2) could be a molecular target of the receptor-positive breast cancer, the therapeutic targets of Her2-negative cancer largely remain to be established. The expression of Her2 was evaluated in 48 primary breast cancer tumors by immunohistochemistry. The identified Notch pathway was studied in genotoxin-dependent suppression of breast cancer-initiating cell growth. Immunohistochemical assessment of Her2-negative tumors revealed significant association with overexpression of Notch1 and Notch3. Knockdown of Notch pathway resulted in sensitization of breast cancer cells to deionizing radiation, leading to cell death; the effect was more significant in stem marker CD44+ than in CD44- cells, and more profound in the Her2-negative than in positive cancer cells. The present study indicates that inhibition of Notch signaling could antagonize survival signal of Her2-negative breast cancer-initiating cells carrying genomic damage, and suggests that targeted suppression of the Notch pathway may give the rationale for sensitizing Her2-negative cancer-initiating cells to a therapeutic approach.

MicroRNAs (miRNAs) constitute a class of small (21-23 nucleotides) noncoding RNAs that function as post-transcriptional gene regulators. It is becoming increasingly clear that altered miRNA expression correlates with the pathogenesis of cancers. The purpose of this study was to determine the up-regulated miRNAs in human colorectal cancer. Total RNA was isolated from cancer tissues and corresponding noncancerous tissues from surgically resected colorectal cancers. The expression profiles of miRNAs were determined using a miRNA microarray containing 455 human miRNA probes. The expression status of selected miRNAs in paired clinical samples was then investigated by real-time RT-PCR. Twenty-one miRNAs were identified by miRNA array analysis as overexpressed in colorectal cancer tissues compared to normal epithelial tissues. Among them, the expression of miR-31, miR-183, miR-17-5p, miR-18a, miR-20a and miR-92 were confirmed to be significantly higher in cancer tissues than in normal tissues (P<0.05). In contrast, the expression of miR-143 and miR-145 in cancer tissues were significantly lower than in normal tissues (P<0.05). The miR-18a overexpression group tended to have a poorer clinical prognosis than the low expression group (P=0.07). We identified miRNAs that were overexpressed or under-expressed in colorectal cancers and which may be correlated with colorectal carcinogenesis.

ApoE plays a key role in various biological events. The aim of this study is to clarify its clinical significance in gastric cancer. We obtained paired clinical bulk samples of tumor tissue and corresponding normal tissue from 124 gastric cancer patients. To address ApoE mRNA expression clearly, we selected four samples, and differentially dissected gastric cancer and normal epithelium using laser microdissection (LMD) system. ApoE mRNA expression was examined by real-time reverse transcription (RT)-polymerase chain reaction (PCR). ApoE protein expression was assessed by immnunohistochemistry. The relationship between ApoE mRNA expression and clinicopathologic factors was statistically analyzed. RT-PCR assay for 124 bulk samples showed that ApoE mRNA expression was more highly expressed in gastric cancer tissue than in corresponding normal mucosa (p < 0.0001). By RT-PCR assay of four LMD samples, ApoE mRNA was overexpressed in gastric cancer. Immunohistochemistry showed that ApoE was predominantly expressed in gastric cancer. Tumors with high ApoE mRNA expression showed deeper tumor invasion into the muscle layer (p < 0.0001), the serosal layer (p < 0.01), or more positive lymph node metastasis (p < 0.05). When assessed by Kaplan-Meier analysis, patients with high ApoE expression tumor had a shorter survival than those with low ApoE expression tumor (p < 0.05). Moreover, multivariate analysis indicated that high ApoE mRNA expression was an independent indicator for muscular invasion (p < 0.01). ApoE is highly expressed in gastric cancer, contributing to shorter survival. In particular, ApoE was closely correlated with muscular invasion, and may be a possible biomarker predicting muscular invasion of gastric cancer.

Activin A, a member of the transforming growth factor beta (TGF-beta) superfamily, is often overexpressed in solid carcinomas. We have previously reported that the expression of activin A is associated with lymph node metastasis in esophageal cancer. In the current study, our goal was to clarify the molecular mechanisms underlying the aggressive behavior of tumors expressing high levels of activin A. Using cDNA microarrays, the gene expression profile of a human esophageal carcinoma cell line (KYSE170) stably transfected with activin betaA (Act-betaA, a subunit of activin A) was compared with those of control human esophageal carcinoma cell lines. We found that expression of MMP-7 was higher in the Act-betaA transfectants than in the control cells. To reveal the mechanism of expression of MMP-7 mediated by activin A, we evaluated mRNA expression of MMP-7 and Act-betaA with or without activin A neutralizing antibody, using real-time PCR and Northern blot analysis. We also performed promoter analysis of the MMP-7 promoter and assessed c-Jun and Smad2/3 expression. MMP-7 expression in the transfectants was correlated with the level of Act-betaA expression and was reduced by activin A neutralizing antibody. The Act-betaA transfectants had higher MMP-7 promoter activity than control cells. MMP-7 promoter activity was not affected by mutation in the Smad binding site, while mutation of the AP-1 binding site did reduce activity. Moreover, the expression of c-Jun was increased in Act-betaA transfectants. These results indicate that activin A may modulate the expression of MMP-7 via AP-1 and not through Smad2/3.

Somatic mutations of the epidermal growth factor receptor (EGFR) gene may predict the sensitivity of non-small cell lung cancers to gefitinib. In our previous study, we identified somatic mutations in the tyrosine kinase domain of the EGFR gene in 12.1% of colon cancer cases. Herein, we focus on whether the mutations are associated with the sensitivity of colon cancer to gefitinib. The E749K mutation in exon 19 and E762G and A767T mutations in exon 20 were introduced into the full-length EGFR coding sequence in a pBKCMV-hEGFR vector by site-directed mutagenesis and transfected into LS174T cells. The sensitivity to gefitinib was compared between the transfected LS174T and the parental cells by a cytotoxic assay. The LS174T cells with E749K were significantly (p<0.05) more responsive to gefitinib than the parental cells. On the other hand, LS174T cells with E762G or A767T were significantly (p<0.05) more resistant than the parental cells. In conclusion, detection of somatic mutations in the epidermal growth factor receptor may play an important role in predicting sensitivity to gefitinib in colon cancer.

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-beta superfamily and bone morphogenetic protein (BMP) subfamily, plays a role in regulation of development and differentiation. Although some members of BMP subfamily have been reported to correlate with cancer, the significance of GDF11 has not been studied in a clinical oncology setting. The current study explored the clinicopathological significance of GDF11 expression in colorectal cancer. Quantitative real-time reverse transcription-PCR in colorectal cancer specimens obtained from 130 patients showed that GDF11 mRNA expression in cancer tissue was significantly higher than in normal tissue (p=0.001). Tumors were classified as high GDF11 expression (n=65) or low GDF11 expression (n=65). Patients whose tumors had high GDF11 expression showed a high frequency of lymph node metastasis (p=0.049) and had more cancer-related deaths (p=0.040). Furthermore, the patients with high GDF11 expression had significantly poorer overall survival than those with low expression (p=0.0334). Although multivariate analysis showed that GDF11 was not an independent prognostic factor, these findings suggest that GDF11 may be a novel diagnostic and prognostic biomarker in patients with colorectal cancer.

Human Mob as tumor suppressor 1 (MATS1) has been shown to play a crucial role in tumor growth by regulating cell proliferation and apoptosis. We evaluated the expression of MATS1 in colorectal cancer patients and assessed its clinicopathological significance. MATS1 mRNA expression in paired tumor/normal samples from 72 cases of colorectal cancer was evaluated, using real-time RT-PCR analysis. MATS1 protein expression was determined by immunohistochemical staining. MATS1 mRNA expression was significantly lower in tumor tissues than in normal colorectal tissues (p<0.05). Clinicopathologically, factors such as incidence of large tumor size, deep invasion, lymphatic permeation and liver metastasis were significantly more frequent in the low MATS1 expression group (p<0.05). Prognosis of the low expression group tended to be poorer than that of the high expression group. MATS1 protein expression was diminished in tumor cells compared with corresponding non-cancer colon epithelial cells. MATS1 mRNA expression is suppressed in tumor tissue and its low expression is associated with tumor growth, invasion and metastasis of colorectal cancer. Specifically, low MATS1 expression may be a good marker of liver metastasis, a life-threatening indicator for colorectal cancer patients.

We applied oligomicroarray analysis of 17086 genes to identify the genes related to lymph node metastasis in esophageal squamous cell carcinoma (ESCC). The samples of cancer and non-cancerous paired tissue were taken from 16 patients with ESCC who underwent esophagectomy with lymph node dissection. Total ribonucleic acid was extracted from the cancer cells obtained by using laser microdissection and was amplified by T7 based-amplification for the application to the oligomicroarray. The oligomicroarray demonstrated 43 overexpressed genes, such as cell-cycle regulators, cell adhesion related genes, anti-apoptosis related genes, and 138 suppressed genes such as cell differentiation related and apoptosis related genes in ESCC cells with lymph node metastasis. Among them, 5 overexpressed genes (SPP-1, CKS2, CCT5, STMN1, NDUFB9) and one suppressed expression gene (GJB2) were selected in the gene profiles, and then the expressions of those genes were confirmed by real-time semi-quantitative reverse transcriptional polymerase chain reaction (RT-PCR) method for confirmation of the result not only in study cases but also in additional 21 cases. The gene expression by real-time semi-quantitative RT-PCR was in accordance with the microarray data. Although we were able to extract some genes related to nodal metastasis in ESCC, further examination is necessary in other genes as well as the interaction of stromal tissues.

Identification of the genes that are differentially-expressed between radiosensitive and radioresistant cancer cells is important to the ability to predict the clinical effectiveness of radiotherapy. We established radioresistant human pancreatic cancer cell lines using fractionated irradiation in order to identify genes that are differentially-expressed between parental lines and radioresistant cell sublines. Six pancreatic cancer cell lines (PK-1, PK-8, PK-9, T3M4, MiaPaCa2 and PANC-1) were treated with 10 Gy fractionated irradiation at approximately two-week intervals (total dose 150-180 Gy). Five radioresistant sublines (PK-1, PK-8, PK-9, T3M4, and MiaPaCa2) were successfully established. Using oligonucleotide microarrays containing 17,086 genes, we identified 73 up-regulated genes and 55 down-regulated genes common to radioresistant sublines. Subsequent analysis by quantitative RT-PCR confirmed the reliability of our microarray strategy. Up-regulated genes were associated with growth factor (example, amphiregulin), cell-cycle check point (MAPKAPK2), intracellular signaling pathway (regucalcin), and angiogenesis stimulation (angiopoietin 2). Down-regulated genes were associated with apoptosis (caspase 8), retinoid esterification (lecithin retinol acyltransferase), and electron transport (calcium-activated chloride channel 1). Some of these genes have known association with response to radiation, such as caspase 8 and MAPKAPK2, but others are novel. Global gene analysis of radioresistant sublines may provide new insights into the mechanisms underlying clinical radioresistance and to improving the efficacy of radiotherapy for pancreatic cancer.

The clinical significance of isolated tumor cells (ITC) circulating in the blood of patients with colorectal cancer is unclear. In this study, we investigated the relationship between the presence of ITC that express carcinoembryonic antigen (CEA) and/or cytokeratin 20 (CK20) transcripts in the blood and the clinicopathological findings and prognosis using the quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. We studied peripheral blood and tumor drainage blood from 167 patients with colorectal cancer. Quantitative real-time RT-PCR assay was able to detect one tumor cell in 3x10(6) peripheral blood mononuclear cells. Applying a cut-off value, CEA and/or CK20 (CEA/CK20) were detected in 10.2% (17/167) of the patients' preoperative peripheral blood samples and 34.1% (57/167) of the patients' tumor drainage blood samples. In the relationship between the CEA/CK20 of the blood and the clinicopathological factors, a significant correlation was demonstrated between the positivity of marker genes and the depth of invasion, venous invasion, lymph node metastasis, liver metastasis or stage. The disease-free and overall survival of patients with CEA/CK20-positive peripheral or tumor drainage blood was significantly shorter than that of marker gene-negative patients. CEA/CK20 transcripts in tumor drainage blood were independent factors for prognosis in disease-free survival and overall survival. These results suggest that detecting CEA/CK20 mRNA in tumor drainage blood by real-time RT-PCR has prognostic value in patients with colorectal cancer. Large scale and long-term clinical studies are needed to confirm the prognostic value of genetically detecting ITC in the peripheral blood.

Inactivation of tumor suppressor genes may result in clinically aggressive tumors and poor prognoses for cancer patients. This study was conducted to investigate the clinical significance of genomic screens for genes upregulated by demethylation in colorectal cancer. We performed a comprehensive survey of commonly inactivated genes in colorectal cancer through the functional reactivation of epigenetically silenced genes by 5-Azacytidine, using cDNA microarrays containing 12,814 genes. We then investigated the clinical significance of the identified gene in colorectal cancer patients. Among 41 candidate genes identified by this microarray analysis, 31 (76%) harbored CpG islands, and many of the genes were associated with cancer-testis antigens, Wnt inhibitors, growth factors, and cell cycle regulators. Subsequent analysis by quantitative RT-PCR confirmed the reliability of our microarray strategy. In order to elucidate the potential clinical significance of these identified genes, we selected one of these genes, apolipoprotein D (apo D), and investigated its mRNA expression in 63 colorectal cancer patients using quantitative real-time RT-PCR. The mean expression level of Apo D mRNA was significantly lower in cancerous tissues than in non-cancerous tissues (p < 0.01), and a lower expression of Apo D was significantly correlated with lymph node metastasis (p < 0.05), advanced stages (p < 0.05) and poorer overall survival (p < 0.05). These results indicated that a genomic screen for genes upregulated by demethylation may be a useful approach for the identification of genes that are of clinical significance in colorectal cancer patients.

The clinical significance of occult micrometastasis (O.M) remains unknown. We investigated it in peripheral blood (P.B.) and bone marrow (B.M.) in breast cancer patients with surgery. First, we investigated the expression levels of 7 representative molecular markers for detecting O.M (CEA, CK-7, CK-18, CK-19, CK-20, MAM and MUC-1) in 27 cancer and 8 non-epithelial cell lines using quantitative RT-PCR (QRT-PCR), and showed that the expression level of CK-7 was higher in every cancer cell line than in the non-epithelial cell lines. Next, we studied the clinical significance of O.M in P.B. and B.M. by QRT-PCR for CK-7 in breast cancer patients with surgery. Based on comparison with 17 non-cancer controls, 37 (18.0%) and 100 (48.5%) of the 206 patients were positive for CK-7 in P.B. and B.M., respectively. In 98 cases observed over 24 months after surgery, the CK-7-positive group in P.B. had poorer disease-free survival (DFS) than the negative group (p<0.01). The CK-7-positive group in P.B. showed poorer DFS than the negative group in 132 lymph node-negative cases (p=0.01), and moreover, in 61 lymph node-negative cases observed over 24 months after surgery, the CK-7-positive group in P.B. showed poorer DFS than the negative group (p<0.0001). In B.M., no significant difference in DFS was found between the CK-7-positive and CK-7-negative groups. QRT-PCR for CK-7 could be a useful and universal method for detecting O.M, and the quantitative detection of CK-7 in P.B. would have a prognostic value as a marker of early recurrence in breast cancer patients with surgery.

We confirmed that the enzyme activity of thymidine phosphorylase (TP) can substitute for 5'-deoxy-5-fluorouridine (5'-dFUR) sensitivity in clinical colorectal cancer (CRC) tissues. Moreover, the idenfication of susceptible genes other than TP will be desired for prediction or treatment of 5'-dFUR. We initially established conditions for the MTT assay system in vivo to compare the growth inhibition rate and TP activity in 18 cases of CRC. TP activity levels were concordant with 5'-dFUR sensitivity in the CRCs (p<0.05). In 18 CRC cases, 4 cases of high sensitivity and 3 cases of low sensitivity to 5'-dFUR were compared to analyze expression profiles of 9437 genes on cDNA microarray chips. As a result, a cluster of genes related to the sensitivity were identified and another cluster of genes related to the insensitivity of 5'-DFUR were also listed. We determined that TP expression can precisely predict 5'-dFUR sensitivity in CRCs and found susceptibility related genes.

Recently, much interest have been focused on the role of INI-1, a partner gene of transfusion protein All-1 for leukemogenesis of acute leukemia cases. We found the presence of a mutation at codon 152 in INI-1 that resulted in a conservative amino acid change in cDNA and genomic DNA from an AML patient. We also found the known polymorphic nucleotide change at the third letter of codon 299 in exon 7 of INI-1 in 3 of 31 ALL cases and in 4 of 30 AML cases. In conclusion, INI-1, a partner gene for ALL-1, is considered to be highly conserved in acute leukemia cases.

Activin A is a member of the transforming growth factor beta (TGF-beta) superfamily and is a strong differentiation factor of embryonic stem (ES) cells. It is unknown whether activin A has any correlation with carcinoma cell differentiation. We investigated the expression of activin-betaA (Act-betaA) which is a subunit of activin A, its receptor type I and IIb (ActRI, ActRIIb) and its inhibitor, inhibin-alpha (Inh-alpha), which is a subunit of inhibin A in esophageal carcinoma by reverse transcription polymerase chain reaction (RT-PCR) method. Act-betaA was overexpressed in carcinoma tissues significantly (p=0.030). On the other hand, Inh-alpha, ActRI and ActRIIb were neither overexpressed, nor suppressed. In immunohistochemistry and in situ hybridization analysis, Act-betaA expression was mainly derived from carcinoma cells. The mRNA expression of Act-betaA was not associated with carcinoma cell differentiation but lymph node metastasis (n0, 1, 2 vs. n3, 4; p=0.013) and clinical stage (I, II, III vs. IV; p=0.026). Moreover, patients with high mRNA expression of Act-betaA had a tendency to show poor prognosis compared to those with low mRNA expression (p=0.064). The finding indicated that activin A expression might not be associated with carcinoma cell differentiation but tumor aggressiveness such as lymph node metastasis in esophageal carcinoma.