Significant overexpression of DVL1 in Taiwanese colorectal cancer patients with liver metastasis.

Undetected micrometastasis plays a key role in the metastasis of cancer in colorectal cancer (CRC) patients. The aim of this study is to identify a biomarker of CRC patients with liver metastasis through the detection of circulating tumor cells (CTCs). Microarray and bioinformatics analysis of 10 CRC cancer tissue specimens compared with normal adjacent tissues revealed that 31 genes were up-regulated (gene expression ratio of cancer tissue to paired normal tissue > 2) in the cancer patients. We used a weighted enzymatic chip array (WEnCA) including 31 prognosis-related genes to investigate CTCs in 214 postoperative stage I-III CRC patients and to analyze the correlation between gene expression and clinico-pathological parameters. We employed the immunohistochemistry (IHC) method with polyclonal mouse antibody against DVL1 to detect DVL1 expression in 60 CRC patients. CRC liver metastasis occurred in 19.16% (41/214) of the patients. Using univariate analysis and multivariate proportional hazards regression analysis, we found that DVL1 mRNA overexpression had a significant, independent predictive value for liver metastasis in CRC patients (OR: 5.764; 95% CI: 2.588-12.837; p < 0.0001 on univariate analysis; OR: 3.768; 95% CI: 1.469-9.665; p = 0.006 on multivariate analysis). IHC staining of the immunoreactivity of DVL1 showed that DVL1 was localized in the cytoplasm of CRC cells. High expression of DVL1 was observed in 55% (33/60) of CRC tumor specimens and was associated significantly with tumor depth, perineural invasion and liver metastasis status (all p < 0.05). Our experimental results demonstrated that DVL1 is significantly overexpressed in CRC patients with liver metastasis, leading us to conclude that DVL1 could be a potential prognostic and predictive marker for CRC patients.

Differential gene expression profile of MAGE family in taiwanese patients with colorectal cancer.

BACKGROUND: The melanoma-associated antigen (MAGE) gene family consists of different expression patterns in various tumor types. They are considered tumor-specific antigens and are ideal targets for cancer immunotherapy. The purpose of this study is to identify the expression profiles of the MAGE family genes in Taiwanese colorectal cancer patients. METHODS: In this study, a well-constructed chip array platform was used to analyze the expression of the MAGE family genes of 100 colorectal cancer tissues. Statistical analysis of the experimental results and patients' clinical manifestations were also conducted. RESULTS: The results showed MAGE-A2 (87%), -A7 (83%), -A8 (75%), -A12 (71%), -B2 (75%), -B3 (79%), -D2 (75%), -F1 (79%), and -H1 (70%) were significantly overexpressed genes in colorectal cancer tissues. MAGE-A2 was the most highly overexpressed gene among the MAGE family. MAGE-B3 gene expression is statistically correlated with tumor size, lymph node, and UICC stage. In addition, the overexpression of MAGE-D2 and -H1 genes are statistically correlated to the tumor size and depth, respectively (P < 0.05). CONCLUSIONS: This is the first comprehensive report to clarify the differential expression profile of whole MAGE family in CRCs, and it might provide some crucial information about the carcinogenesis and progression in Taiwanese patients with CRC.

GLUT1 gene is a potential hypoxic marker in colorectal cancer patients.

BACKGROUND: Tumor hypoxia is an important factor related to tumor resistance to radiotherapy and chemotherapy. This study investigated molecules synthesized in colorectal cancer cells during hypoxia to explore the possibility of developing molecular probes capable of detecting cell death and/or the efficiency of radiotherapy and chemotherapy. METHODS: At first, we incubated two human colorectal adenocarcinoma cell lines SW480 (UICC stage II) and SW620 (UICC stage III) cells in hypoxic (< or =2% O2, 93% N2, and 5% CO2) and normoxic conditions (20% O2, 75% N2, and 5% CO2) for 24 h and 48 h. The relative expression ratio of GLUT1 mRNA in hypoxic conditions was analyzed by RT-PCR. Ten cancerous tissues collected from human colorectal cancer patients were examined. HIF-1alpha and HIF-2alpha levels were measured to indicate the degree of hypoxia, and gene expression under hypoxic conditions was determined. As a comparison, HIF-1alpha, HIF-2alpha, and GLUT1 levels were measured in the peripheral blood of 100 CRC patients. RESULTS: Hypoxia-induced lactate was found to be elevated 3.24- to 3.36-fold in SW480 cells, and 3.06- to 3.17-fold in SW620 cells. The increased relative expression ratio of GLUT1 mRNA, under hypoxic conditions was higher in SW620 cells (1.39- to 1.72-fold elevation) than in SW480 cells (1.24- to 1.66-fold elevation). HIF-1alpha and HIF-2alpha levels were elevated and GLUT1 genes were significantly overexpressed in CRC tissue specimens. The elevated ratio of GLUT1 was higher in stage III and IV CRC tissue specimens than in the stage I and II (2.97-4.73 versus 1.44-2.11). GLUT1 mRNA was also increased in the peripheral blood of stage II and III CRC patients as compared to stage I patients, suggesting that GLUT1 may serve as a hypoxic indicator in CRC patients. CONCLUSION: In conclusion, this study demonstrated that GLUT1 has the potential to be employed as a molecular marker to indicate the degree of hypoxia experienced by tumors circulating in the blood of cancer patients.

Significance of the glycolytic pathway and glycolysis related-genes in tumorigenesis of human colorectal cancers.

We investigated gene expressions involved in the glycolytic pathways in colorectal cancer. The study was designed to use gene ontology and its relevant bioinformatics tools to analyze the microarray data obtained from CRC tissues and their corresponding normal tissues, in order to explore the correlation between the glycolytic metabolic pathway and possible pathogenesis of this disease. The overexpression of glycolysis-related genes was observed in over 76% of CRC tissues. In addition, we stimulated the SW480 and SW620 CRC cell lines with 15 mM D-(+)-glucose and 10 mM 2-deoxy-D-glucose respectively. The results indicate that the proliferation response of both the SW480 and SW620 cell lines increased remarkably with a time-dependent effect by D-(+)-glucose administration. In contrast, the proliferation response of both the SW480 and SW620 cell lines was significantly inhibited by 2-DG administration. Likewise, further analyses of the expression of related genes triggered by the D-(+)-glucose in vivo show that the activation process of these eight genes - GLUT1, HK1, GPI, GAPD, PGK1, PGK2, ENO2, PKM2 - prominently increased with a time-dependent effect. In conclusion, this study demonstrates that the glycolytic pathway and glycolysis-related genes may play an important role in the tumorigenesis of CRC, but their molecular mechanisms need further investigation to verify this.

Sarco/endoplasmic reticulum calcium-ATPase 2 expression as a tumor marker in colorectal cancer.

Maintaining a high calcium concentration in the endoplasmic reticulum through the action of sarco/endoplasmic reticulum calcium-ATPases (SERCAs) is crucial in many cell functions involved in intracellular signal transduction, control of proliferation, programmed cell death, or the synthesis of mature proteins. Recent studies have found that many SERCAs have altered expression patterns in various malignancies. The purpose of the current study was to quantify the expression of SERCA2 in colorectal cancer (CRC) tissues and the corresponding noncancerous tissues, and to statistically analyze whether the SERCA2 expression levels correlate with the clinico-pathologic features and prognosis of CRC patients. Paired colorectal tissue samples from cancerous and the corresponding noncancerous tissues were obtained from 50 patients who underwent surgical resection. Semiquantitative measurements of SERCA2 messenger RNA (mRNA) expression were done using the multiplex reverse transcriptase-polymerase chain reaction. CRC tissues were analyzed through immunohistochemistry for the SERCA2 protein. SERCA2 mRNA overexpression in cancerous tissues compared with normal counterparts was observed in 45 of 50 (90%) patients. The mean expression level of SERCA2 mRNA in cancerous tissues was significantly higher than that in noncancerous tissues (P = 0.01). Increased SERCA2 protein expression was significantly correlated with serosal invasion (P = 0.012), lymph node metastasis (P = 0.009), and advanced tumor stage (P = 0.004). Furthermore, patients with high SERCA2 expression had a significantly poorer overall survival rate than patients with low SERCA2 (P = 0.032). Multivariate analyses indicated that tumor stage (P = 0.015) and SERCA2 expression were independently correlated with overall survival (P = 0.018). The result of this study indicated that SERCA2 may be a molecular determinant in the development and progression of CRC. The molecular mechanisms underlying the SERCA-dependent calcium accumulation and CRC tumorigenesis are worthy of further investigations.

Molecular detection of circulating cancer cells in the peripheral blood of patients with colorectal cancer by using membrane array with a multiple mRNA marker panel.

The objective of this study was mainly to evaluate the simultaneous detection of expression levels of a multiple mRNA marker panel in the peripheral blood of colorectal cancer (CRC) patients for use in complementary CRC diagnosis. Twenty-seven tumor tissue specimens and 80 peripheral blood specimens were collected from CRC patients. Firstly, the levels of multiple molecular markers in the tumor tissue and blood specimens were evaluated by using real-time quantitative PCR (RT-QPCR) and membrane array. The result of linear regression showed a high degree of correlation (r=0.954, P<0.0001) between the data of these two methods. CK-19 was the marker with the highest detection rate (87.5%) in the peripheral blood, followed by CEA (82.6%), REG4 (80.8%), and then uPA (80.0%) and TLAM1 (80.0%). The levels of the six markers in the peripheral blood were extensively explored. In the 80 patients, the frequency of CK-19, CK-20, CEA, REG4, uPA, and TIAM1 mRNA overexpression was 82.5% (66/80), 78.8% (63/80), 82.5% (66/80), 80.0% (64/80), 78.8% (63/80), and 80.0% (64/80), respectively. Then, a panel combining these 6 mRNA markers was evaluated for its utility in the clinical diagnosis of CRC. The sensitivity, specificity, and accuracy of membrane array-based diagnostic method were 88.8%, 87.8%, and 88.2%, respectively; much higher than those of examinations with single markers. Finally, lymph node metastasis (P=0.024) and TNM stage (P=0.009) were found to be significantly correlated with overexpression of the multiple mRNA marker panel. The detection rates of stage-I and -II CRC by using the multi-marker membrane array were 54.5% (6/11) and 92.0% (23/25), respectively. In conclusion, the results of the present study have shown that this innovative membrane array technique with a multiple mRNA marker panel can significantly improve the diagnosis rate of early colorectal cancer.

DPYD, TYMS, TYMP, TK1, and TK2 genetic expressions as response markers in locally advanced rectal cancer patients treated with fluoropyrimidine-based chemoradiotherapy.

This study is to investigate multiple chemotherapeutic agent- and radiation-related genetic biomarkers in locally advanced rectal cancer (LARC) patients following fluoropyrimidine-based concurrent chemoradiotherapy (CCRT) for response prediction. We initially selected 6 fluoropyrimidine metabolism-related genes (DPYD, ORPT, TYMS, TYMP, TK1, and TK2) and 3 radiotherapy response-related genes (GLUT1, HIF-1α, and HIF-2α) as targets for gene expression identification in 60 LARC cancer specimens. Subsequently, a high-sensitivity weighted enzymatic chip array was designed and constructed to predict responses following CCRT. After CCRT, 39 of 60 (65%) LARC patients were classified as responders (pathological tumor regression grade 2 ~ 4). Using a panel of multiple genetic biomarkers (chip), including DPYD, TYMS, TYMP, TK1, and TK2, at a cutoff value for 3 positive genes, a sensitivity of 89.7% and a specificity of 81% were obtained (AUC: 0.915; 95% CI: 0.840-0.991). Negative chip results were significantly correlated to poor CCRT responses (TRG 0-1) (P = 0.014, hazard ratio: 22.704, 95% CI: 3.055-235.448 in multivariate analysis). Disease-free survival analysis showed significantly better survival rate in patients with positive chip results (P = 0.0001). We suggest that a chip including DPYD, TYMS, TYMP, TK1, and TK2 genes is a potential tool to predict response in LARC following fluoropyrimidine-based CCRT.

MMP13 is a potential prognostic marker for colorectal cancer.

Matrix metalloproteinase 13 (MMP13), a member of the matrix metalloproteinase family, is considered to play a role in the tumor cell proliferation and invasion. The purpose of this study was to verify the expression of MMP13 in colorectal cancer (CRC) in vitro and in vivo, and subsequently analyze whether the MMP13 expression levels correlate with the clinicopathological features and prognosis of CRC patients. We assessed MMP13 mRNA expression profile in human colorectal adenocarcinoma cell lines by quantitative RT-PCR, and further verified if it was a secreted protein or not by Western blot analysis of cell culture medium. By immunohistochemical staining the immunoreactivity of MMP13 showed that MMP13 was localized in the cytoplasm of CRC cells. MMP13 mRNA expression of 80 cancerous tissues collected from UICC stage I to III CRC patients were examined by membrane array. The correlations between MMP13 mRNA expression and patients' clinicopathological features were analyzed. MMP13 was confirmed to be a secreted protein by Western blot analysis. The larger tumor size (P<0.0001), advanced clinical stage (P=0.002), tumor invasive depth (P=0.039), lymph node metastasis (P=0.001) and post-operative relapse (P<0.0001) were significantly correlated with the MMP13 mRNA overexpression. Patients with MMP13 mRNA overexpression have a higher risk of postoperative relapse (P<0.0001; OR=7.989; 95% CI, 2.607-24.481). The results of the present study highly suggest that MMP13 is a secreted protein with a significant correlation to development of postoperative relapse; hence it could be a potential prognostic marker for CRC patients.

PPARgamma-2 and BMPR2 genes were differentially expressed in peripheral blood of SLE patients with osteonecrosis.

Most researchers believe that the peroxisome proliferative activated receptor gamma (PPARgamma-2) and bone morphogenetic protein receptor type II (BMPR2) play important roles in steroid-induced osteonecrosis (ON). However, the molecular mechanism of this process is still unclear. Recent studies indicate that steroid treatments cause adipocyte formation due to differentiation of mesenchymal stem cells, which then prevents osteoblast formation. This study examined PPARgamma-2, bone morphogenetic protein 2 (BMP2), and BMPR2 in patients with systemic lupus erythromatosus (SLE) who eventually developed ON after prolonged steroid treatment. The subjects of this experiment included 220 SLE patients who had undergone steroid treatment for at least 2 years. Fifty-five of the 220 patients were ON patients, and 165 were non-ON patients. Real-time PCR was performed to analyze the expression of the PPARgamma-2, BMP2, and BMPR2 mRNA in the peripheral blood of these patients. The results indicated that the expression of PPARgamma-2 mRNA increased 37% in the ON patients' peripheral blood, but the expression of BMPR2 mRNA decreased 57%. The average expression of the PPARgamma-2 mRNA in the ON patients was significantly higher than that in the non-ON patients (p = 0.044). Conversely, the expression of BMPR2 mRNA was significantly lower than that in non-ON patients (p = 0.036), but the expression of BMP2 mRNA did not significantly differ. This study demonstrated that the PPARgamma-2 and BMPR2 have important roles in the ON process after prolonged steroid administration in SLE patients; however, the detailed molecular mechanisms of this process require further study.