Dage Liu

Wnt5a Expression Is Associated With the Tumor Proliferation and the Stromal Vascular Endothelial Growth Factor—An Expression in Non–Small-Cell Lung Cancer

PURPOSE The Wnt gene family encodes the multifunctional signaling glycoproteins. We performed the present study to investigate the clinical significance of Wnt5a expression in non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS One hundred twenty-three patients with NSCLC who had undergone resection were investigated. Real-time quantitative reverse transcriptase polymerase chain reaction was performed to evaluate the Wnt5a gene expression. Immunohistochemistry was performed to investigate the Wnt5a protein expression, the Ki-67 proliferation index, tumor angiogenesis, and the expression of beta-catenin and vascular endothelial growth factor-A (VEGF-A). RESULTS Wnt5a gene expression in squamous cell carcinoma was significantly higher than that in adenocarcinoma (P < .0001). There was a significant correlation between the normalized Wnt5a gene expression ratio and the intratumoral Wnt5a protein expression (r = 0.729; P < .0001). The intratumoral Wnt5a expression was significantly correlated with the Ki-67 proliferation index (r = 0.708; P < .0001). In contrast, no correlation was observed between the intratumoral Wnt5a expression and tumor angiogenesis. Furthermore, the intratumoral Wnt5a expression was significantly correlated with the stromal expression of beta-catenin (r = 0.729; P < .0001) and VEGF-A (r = 0.661; P < .0001). In addition, the stromal VEGF-A expression was also correlated with Ki-67 proliferation (r = 0.627; P < .0001). Cox regression analyses demonstrated Wnt5a status to be a significant prognostic factor for NSCLC patients (P = .0193), especially for patients with squamous cell carcinomas (P = .0491). CONCLUSION The present study revealed that an overexpression of Wnt5a could produce more aggressive NSCLC, especially in squamous cell carcinomas, during tumor progression.

The tumour–stromal interaction between intratumoral c-Met and stromal hepatocyte growth factor associated with tumour growth and prognosis in non-small-cell lung cancer patients

Immunohistochemical analyses of the effects of hepatocyte growth factor (HGF) and c-Met expression on tumour growth and angiogenesis were performed on 88 patients with non-small-cell lung cancers (NSCLCs). In all, 22 carcinomas (25.0%) were intratumoral HGF-positive, 14 carcinomas (15.9%) were stromal HGF-positive, and 36 carcinomas (40.9%) were intratumoral c-Met-positive. None of the carcinomas were stromal c-Met-positive. Examination of tumour growth revealed that the frequency of tumours with a high Ki-67 index was significantly greater for stromal HGF-positive tumours than for stromal HGF-negative tumours (P=0.0197). The frequency of tumours with a high Ki-67 index was also significantly greater for intratumoral c-Met-positive tumours than for intratumoral c-Met-negative tumours (P=0.0301). However, there was no significant difference in tumour vascularity with relation to intratumoral HGF status, stromal HGF status, and intratumoral c-Met status. The survival rate of patients with intratumoral c-Met-positive tumours was significantly lower than for patients with c-Met-negative tumours (P=0.0095). Furthermore, the survival rate of patients with both intratumoral c-Met-positive and stromal HGF-positive tumours was significantly lower than for patients with either positive tumours, and that of patients with both negative tumours (P=0.0183 and P=0.0011, respectively). A univariate analysis revealed that intratumoral c-Met expression was a significant prognostic factor of NSCLC patients (relative risk=2.642, P=0.0029). The present study demonstrates that tumour–stromal interaction between tumour cell-derived c-Met and stromal cell-derived HGF affects tumour growth and the prognosis of NSCLC patients.

Galectin-9 as a Prognostic Factor with Antimetastatic Potential in Breast Cancer

Purpose: Galectin-9, a member of the β-galactoside–binding galectin family, induces aggregation of certain cell types. We assessed the contribution of galectin-9 to the aggregation of breast cancer cells as well as the relation between galectin-9 expression in tumor tissue and distant metastasis in patients with breast cancer. Experimental Design: Subclones of MCF-7 breast cancer cells with high or low levels of galectin-9 expression were established and either cultured on plastic dishes or transplanted into nude mice. The tumors of 84 patients with breast cancer were tested for galectin-9 expression by immunohistochemistry. The patients were followed up for 14 years. Results: MCF-7 subclones with a high level of galectin-9 expression formed tight clusters during proliferation in vitro, whereas a subclone (K10) with the lowest level of galectin-9 expression did not. However, K10 cells stably transfected with a galectin-9 expression vector aggregated in culture and in nude mice. Ectopic expression of galectin-9 also reduced MCF-7 cell adhesion to extracellular matrix proteins. Tumors of 42 of the 84 patients were galectin-9 positive, and those of 19 of the 21 patients with distant metastasis were galectin-9 negative. None of the 13 patients with galectin-9–positive tumors and lymph node metastasis up to level II manifested distant metastasis. The cumulative disease-free survival ratio for galectin-9–positive patients was more favorable than that for the galectin-9–negative group (P < 0.0001). Multivariate analysis revealed that galectin-9 status influenced distant metastasis independently of and to a greater extent than lymph node metastasis. Conclusions: Galectin-9 is a possible prognostic factor with antimetastatic potential in breast cancer.

Clinical application of biological markers for treatments of resectable non-small-cell lung cancers

We performed a clinical study to identify biological markers useful for the treatment of resectable non-small-cell lung cancers (NSCLCs). In all, 173 patients were studied. By immunohistochemistry, we evaluated the Ki-67 proliferation index, tumour vascularity, thymidylate synthase (TS), vascular endothelial growth factor (VEGF)-A, VEGF-C, and E (epithelial)-cadherin. Concerning the survival of NSCLC patients, tumour vascularity (P<0.01), VEGF-A status (P=0.03), VEGF-C status (P=0.03), and E-cadherin status (P=0.03) were significant prognostic factors in patients with stage I NSCLCs. The Ki-67 proliferation index (P=0.02) and TS status (P<0.01) were significant prognostic factors in patients with stage II–III NSCLCs. In patients with stage II–III NSCLCs, furthermore, the survival of UFT (a combination of tegafur and uracil)-treated patients with TS-negative tumours was significantly better than those of any other patients. Biological markers associated with tumour angiogenesis or metastasis are useful for the detection of aggressive tumours among early-stage NSCLCs. Postoperative chemotherapy might be necessary in such tumours even in stage I. In contrast, tumour proliferation rate and TS status are useful markers for identifying less aggressive tumours in locally advanced NSCLCs. Thymidylate synthase expression is also a useful marker to evaluate responsiveness of UFT-based chemotherapy for these tumours.

E-cadherin expression associated with differentiation and prognosis in patients with non–small cell lung cancer

BACKGROUND E-Cadherin plays a major role in maintaining the intercellular junctions in epithelial tissues. The reduction of E-cadherin expression in cancer cells may be associated with tumor differentiation, metastasis, and a poor prognosis. METHODS Immunohistochemistry for E-cadherin expression was performed on 109 tumors from patients with non-small cell lung cancer who underwent operations. RESULTS With respect to membranous immunostaining, 57 carcinomas were E-cadherin-positive, 39 carcinomas E-cadherin-reduced, and 13 carcinomas E-cadherin-negative. The percentage of poorly differentiated tumors in the impaired E-cadherin expression group was significantly higher than that in the E-cadherin-positive group (p = 0.005). Furthermore, the frequency of lymph node metastases in tumors with impaired E-cadherin expression was significantly higher than that in the E-cadherin-positive tumors (p = 0.011). A Cox regression analysis revealed that E-cadherin expression was a significant factor in the prediction of survival for patients with non-small cell lung cancer (p = 0.002). CONCLUSIONS E-Cadherin expression was associated with tumor differentiation, lymph node metastasis, and prognosis in patients with non-small cell lung cancer.

The clinical significance of lymphangiogenesis and angiogenesis in non-small cell lung cancer patients

BACKGROUND Angiogenesis and lymphangiogenesis have been reported to affect malignant phenotype. METHOD We investigated 147 patients with non-small cell lung cancer (NSCLC). Immunohistochemistry using D2-40 was performed to evaluate lymphatic vessel density (LVD), including Micro-LVD (without lumen), Tubal-LVD (with lumen) and lymphatic vessel invasion (LVI). The intratumoural microvessel density (MVD) was evaluated by CD-34 immunostaining. The expressions of vascular endothelial growth factor-A (VEGF-A) and VEGF-C were also studied. RESULTS Lymphangiogenesis was significantly associated with Micro-LVD (p=0.0003). The VEGF-C expression was significantly associated with the Micro-LVD (p=0.0057). In contrast, the VEGF-A expression was significantly associated with the MVD (p=0.0092). The survival was significantly lower in patients with Micro-LVD-high tumours than in patients with Micro-LVD-low tumours (p=0.0397). Survival was also significantly lower in patients with MVD-high tumours than in patients with MVD-low tumours (p=0.0334). A multivariate analysis demonstrated that the Micro-LVD (p=0.0363) and the MVD (p=0.0232) were independent prognostic factors for NSCLC patients. CONCLUSIONS Lymphangiogenesis, specifically Micro-LVD and angiogenesis are independently associated with a poor prognosis in NSCLC patients.

E2F1 Overexpression Correlates with Thymidylate Synthase and Survivin Gene Expressions and Tumor Proliferation in Non–Small-Cell Lung Cancer

Purpose: We investigated the clinical significance of E2F1 gene expression in relation to its target genes, thymidylate synthase (TS) and Survivin, in case of non–small-cell lung cancer (NSCLC). Experimental Design: One hundred twenty-seven cases of resected NSCLC were analyzed. Quantitative reverse transcription-PCR was done to evaluate the gene expression of E2F1, TS, and Survivin. Immunohistochemistry was done to investigate the protein expression of E2F1, TS, and Survivin. The Ki-67 proliferation index and the apoptotic index using the terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling method were also evaluated. Results:E2F1 gene expression significantly correlated with the Ki-67 proliferation index (r = 0.487; P < 0.0001), although no correlation was observed between E2F1 gene expression and the apoptotic index. With regard to E2F1 target genes, E2F1 gene expression significantly correlated with TS gene expression (r = 0.709; P < 0.0001) and Survivin gene expression (r = 0.403; P < 0.0001). The overall survival rate was significantly lower in patients with high-E2F1 tumors than in those with low-E2F1 tumors (P = 0.0027), especially among patients with stage II to III NSCLCs (P = 0.0188). A Cox regression analysis showed that the E2F1 status was a significant prognostic factor for NSCLC patients (hazard ratio, 2.052; P = 0.0261). Conclusions: The present study revealed that E2F1 gene expression correlates with TS and Survivin gene expressions and tumor proliferation. During the progression of NSCLC, E2F1 overexpression could produce more aggressive tumors with a high proliferation rate and chemoresistance.

MRP-1/CD9 gene transduction downregulates Wnt signal pathways

Motility-related protein-1 (MRP-1/CD9) is a transmembrane glycoprotein that has been implicated in cell adhesion, motility, proliferation, and differentiation. It has a functional role as a tumor metastatic suppressor. During tumor progression, a reduction of MRP-1/CD9 gene expression results in tumor cells with a high metastatic potential. However, the mechanism of action of MRP-1/CD9 is still unclear. We studied changes of gene expression in relation to MRP-1/CD9 gene transduction into tumor cell lines, HT1080 and A549, using microarray assays and real-time PCR. Consequently, we have demonstrated that MRP-1/CD9 gene transduction can downregulate expression of several Wnt family genes, such as Wnt1, Wnt2b1 and Wnt5a, and their target genes, including WISP-1 (Wnt-1 induced secreted protein 1), WISP-3, c-Myc, vascular endothelial growth factor-A, and matrix metalloproteinase-26. Western blot analyses also showed that MRP-1/CD9 gene transduction downregulated expression of Wnt1 protein and its target proteins. In addition, a neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited the downregulation of Wnt signal pathways in MRP-1/CD9-transfected cells. The present study has revealed that the MRP-1/CD9 signal is located upstream of the Wnt signal pathways. Therefore, MRP-1/CD9 could suppress cell transformation including epithelial to mesenchymal transition through downregulation of Wnt1, and might suppress tumor metastasis through downregulation of Wnt5a.

Wnt1 overexpression promotes tumour progression in non-small cell lung cancer

BACKGROUND The Wnt gene family is involved in embryogenesis and tumourigenesis. We investigated the clinical significance of Wnt1 expression in non-small cell lung cancer (NSCLC). METHOD We studied 216 NSCLC patients. Immunohistochemistry was performed to investigate the Wnt1 expression in relation to the expression of beta-catenin and Wnt-targets, including c-Myc, Cyclin D1, VEGF-A and MMP-7. The Ki-67 proliferation index and the intratumoural microvessel density (IMD) were also evaluated. RESULTS The ratio of tumours with an aberrant beta-catenin expression was significantly higher in Wnt1-positive tumours than in Wnt1-negative tumours (p<0.0001). The Wnt1 expression significantly correlated with the expression of c-Myc (p<0.0001), Cyclin D1 (p<0.0001), VEGF-A (p=0.0160), MMP-7 (p<0.0001), the Ki-67 index (p=0.0048) and the IMD (p=0.0267). Furthermore, the Wnt1 status was a significant prognostic factor for NSCLC patients (p=0.0127). CONCLUSIONS The Wnt1 overexpression is associated with the expression of tumour-associated Wnt-targets, tumour proliferation, angiogenesis and a poor prognosis in NSCLCs.

The HAUSP gene plays an important role in non-small cell lung carcinogenesis through p53-dependent pathways

Herpesvirus‐associated ubiquitin‐specific protease (HAUSP) directly stabilizes the tumour suppressor p53 by de‐ubiquitination. Therefore, the HAUSP gene might play an important role in carcinogenesis. In this paper, HAUSP expression and p53 gene status have been studied in relation to the expression of p53 target genes in 131 patients with non‐small cell lung cancer (NSCLC). p53 gene status was evaluated by polymerase chain reaction‐single‐strand conformation polymorphism (PCR‐SSCP) followed by sequencing. Quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) was performed to evaluate the gene expression of HAUSP, p21, and bax. Immunohistochemistry was performed to evaluate the protein expression of p53, HAUSP, mdm2, p21, and bax. Fifty‐nine carcinomas (45.0%) showed reduced expression of HAUSP, and 58 carcinomas (44.3%) had mutations of p53. Concerning tumour histology, HAUSP mRNA expression was significantly lower in adenocarcinomas than in squamous cell carcinomas (p = 0.0038), while the frequency of p53 mutation was significantly higher in squamous cell carcinomas than in adenocarcinomas (p = 0.0461). There was no significant difference in HAUSP mRNA expression according to p53 gene status. In total, 93 carcinomas (71.0%) showed either mutant p53 or reduced HAUSP expression. The down‐regulation of HAUSP was associated with reduced p53 protein expression (p = 0.0593 in tumours with wild‐type p53 and p = 0.0004 in tumours with mutant p53). Furthermore, p21 and bax protein expression was significantly lower in tumours with either mutant p53 or reduced HAUSP expression than in tumours with both wild‐type p53 and positive HAUSP expression (p = 0.0440 and p = 0.0046, respectively). In addition, the simultaneous evaluation of both HAUSP expression and p53 gene status was a significant indicator of poor prognosis in adenocarcinoma patients (hazard ratio 4.840, p = 0.0357). These results suggest that reduction of HAUSP gene expression may play an important role in NSCLC carcinogenesis, especially in adenocarcinomas, through p53‐dependent pathways. Copyright