Aiguo Lu

Vascular Endothelial Growth Factors C and D Represent Novel Prognostic Markers in Colorectal Carcinoma Using Quantitative Image Analysis

Background/Aims: Vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor D (VEGF-D) are potent lymphangiogenic and angiogenetic mediators in many kinds of tumors. However, the exact impacts of VEGF-C and VEGF-D on the prognosis of colorectal cancer (CRC) remain elusive. The aims of this study were to demonstrate the expression of VEGF-C and VEGF-D and to correlate their expression levels with clinicopathological factors and long-term survival in patients with CRC. Patients and Methods: Between January 1996 and January 1998, 69 patients with pathologically confirmed CRC who received routine follow-up at the Ruijin Hospital were included in this study. VEGF-C and VEGF-D protein expression and microvessel density of 69 surgical specimens were assessed by immunohistochemistry, with 20 samples of normal colorectal tissues as controls. All patients were followed up for 108 months or until death. The Immunohistochemical stains were quantified and analyzed by means of a Zeiss Axioplan 2 imaging analysis system. Results: The protein expression of VEGF-C and VEGF-D in tumor tissues was much higher than that in normal colorectal tissues (p < 0.01). The VEGF-C expression significantly correlated with lymph node metastasis (p = 0.011) and clinical stages of CRC (p < 0.01). The VEGF-D expression correlated with patient ages (p = 0.013), depth of tumor invasion (p = 0.013), and lymph node metastasis (p = 0.028). The expression of VEGF-C and VEGF-D was significantly correlated with the microvessel density. Both overall survival and disease-free survival at 108 months were significantly lower in the CRC patients with a high VEGF-C and/or a high VEGF-D expression, and the patients with a high expression of both VEGF-C and VEGF-D had the shortest overall survival and disease-free survival when compared with other patients. Conclusion: The VEGF-C or VEGF-D expression was significantly correlated with lymph node metastasis and long-term prognosis and could be applied as prognostic markers in CRC.

Long-term outcome of laparoscopic total mesorectal excision for middle and low rectal cancer

Abstract This study aimed to assess the feasibility and long-term outcome of laparoscopic total mesorectal excision for middle and lower rectal cancer. Retrospective assessment was performed on 612 patients with middle and low rectal cancer in the surgery department of our hospital. Three-hundred and three patients underwent laparoscopic total mesorectal excision (LTME), and 309 patients underwent open TME (OTME). All the data regarding patient details, operative variables and the short- and long-term outcomes were collected and compared. The sphincter-preserving rates of the two groups were similar. The conversion rate in LTME was 2.31% (seven cases). Fourteen cases (6.67%) of protective diverting stoma were fashioned in the LTME group compared with 57 cases (26.64%) in the OTME group. The postoperative morbidity was the same in these two groups, while the postoperative period until bowel movement and hospital discharge was shorter in the LTME group (P < 0.01). The median follow-up period was 34 (6–81) months for the LTME group and 36 (6–81) months for the OTME group. Local recurrence rates, the five-year disease-free survival rate and the five-year overall survival rate showed no difference between the two groups. Laparoscopic surgery is feasible and safe in patients with middle and lower rectal caner and can provide favorable short-term and long-term outcomes.

Tumor-derived CXCL5 promotes human colorectal cancer metastasis through activation of the ERK/Elk-1/Snail and AKT/GSK3β/β-catenin pathways

BackgroundMetastasis is a major cause of death in human colorectal cancer patients. However, the contribution of chemokines in the tumor microenvironment to tumor metastasis is not fully understood.MethodsHerein, we examinined several chemokines in colorectal cancer patients using chemokine ELISA array. Immunohistochemistry was used to detect expression of CXCL5 in colorectal cancer patients tissues. Human HCT116 and SW480 cell lines stably transfected with CXCL5, shCXCL5 and shCXCR2 lentivirus plasmids were used in our in vitro study. Immunoblot, immunofluorescence and transwell assay were used to examine the molecular biology and morphological changes in these cells. In addition, we used nude mice to detect the influence of CXCL5 on tumor metastasis in vivo.ResultsWe found that CXCL5 was overexpressed in tumor tissues and associated with advanced tumor stage as well as poor prognosis in colorectal cancer patients. We also demonstrated that CXCL5 was primarily expressed in the tumor cell cytoplasm and cell membranes, which may indicate that the CXCL5 was predominantly produced by cancer epithelial cells instead of fibroblasts in the tumor mesenchyme. Additionally, overexpression of CXCL5 enhanced the migration and invasion of colorectal cancer cells by inducing the epithelial-mesenchymal transition (EMT) through activation of the ERK/Elk-1/Snail pathway and the AKT/GSK3β/β-catenin pathway in a CXCR2-dependent manner. The silencing of Snail and β-catenin attenuated CXCL5/CXCR2-enhanced cell migration and invasion in vitro. The elevated expression of CXCL5 can also potentiate the metastasis of colorectal cancer cells to the liver in vivo in nude mice intrasplenic injection model.ConclusionIn conclusion, our findings support CXCL5 as a promoter of colorectal cancer metastasis and a predictor of poor clinical outcomes in colorectal cancer patients.

The metastasis suppressor, NDRG1, inhibits "stemness" of colorectal cancer via down-regulation of nuclear β-catenin and CD44.

N-myc downstream-regulated gene 1 (NDRG1), has been identified as an important metastasis suppressor for colorectal cancer (CRC). In this study, we investigated: (1) the effects of NDRG1 on CRC stemness and tumorigenesis; (2) the molecular mechanisms involved; and (3) the relationship between NDRG1 expression and colorectal cancer prognosis. Our investigation demonstrated that CRC cells with silenced NDRG1 showed more tumorigenic ability and stem cell-like properties, such as: colony and sphere formation, chemoresistance, cell invasion, high expression of CD44, and tumorigenicity in vivo. Moreover, NDRG1 silencing reduced β-catenin expression on the cell membrane, while increasing its nuclear expression. The anti-tumor activity of NDRG1 was demonstrated to be mediated by preventing β-catenin nuclear translocation, as silencing of this latter molecule could reverse the effects of silencing NDRG1 expression. NDRG1 expression was also demonstrated to be negatively correlated to CRC prognosis. In addition, there was a negative correlation between NDRG1 and nuclear β-catenin and also NDRG1 and CD44 expression in clinical CRC specimens. Taken together, our investigation demonstrates that the anti-metastatic activity of NDRG1 in CRC occurs through the down-regulation of nuclear β-catenin and suggests that NDRG1 is a significant therapeutic target.

CCR4 promotes metastasis via ERK/NF-κB/MMP13 pathway and acts downstream of TNF-α in colorectal cancer

Chemokines and chemokine receptors are causally involved in the metastasis of human malignancies. As a crucial chemokine receptor for mediating immune homeostasis, however, the role of CCR4 in colorectal cancer (CRC) remains unknown. In this study, we found that high expression of CCR4 in CRC tissues was correlated with shorter overall survival and disease free survival. In vitro and in vivo experiments revealed that silencing CCR4 attenuated the invasion and metastasis of CRC cells, whereas ectopic overexpression of CCR4 contributed to the forced metastasis of these cells. We further demonstrated that matrix metalloproteinase 13 (MMP13) played an important role in CCR4-mediated cancer cell invasion, which is up-regulated by ERK/NF-κB signaling. Positive correlation between CCR4 and MMP13 expression was also observed in CRC tissues. Moreover, our investigations showed that the level of CCR4 could be induced by TNF-α dependent of NF-κB activation in CRC cells. CCR4 might be implicated in TNF-α-regulated cancer cells metastasis. Combination of CCR4 and TNF-α is a more powerful prognostic marker for CRC patients. These findings suggest that CCR4 facilitates metastasis through ERK/NF-κB/MMP13 signaling and acts as a downstream target of TNF-α. CCR4 inhibition may be a promising therapeutic option for suppressing CRC metastasis.

Plk2 promotes tumor growth and inhibits apoptosis by targeting Fbxw7/Cyclin E in colorectal cancer

Polo-like kinase 2 (Plk2) and Polo-like kinase 3 (Plk3) have been documented as a tumor suppressor and are lowly expressed in several types of cancer. However, our results showed that Plk3 was lowly expressed, whereas Plk2 expressed highly in tumor tissues. We therefore aimed to explore the mechanisms governing the role of Plk2 in colorectal cancer (CRC). Our investigation demonstrated that Plk2 was an independent prognostic marker in CRC patients. Plk2 promotes tumor growth and inhibits apoptosis of CRC cells in vitro and in vivo. Moreover, Plk2 binds to Fbxw7 and results in its subsequent degradation, which in turn leads to the stabilization of Cyclin E. The pro-tumor activity of Plk2 could be inverted by restoring Fbxw7 expression and depletion of Cyclin E. In addition, the expressions of Fbxw7 and Cyclin E were significantly associated with Plk2 protein levels in CRC tissues. In conclusion, our data show that Plk2 represents an independent prognostic marker and regulates tumor growth and apoptosis by targeting Fbxw7/Cyclin E pathway in CRC, suggesting Plk2 as a potential therapeutic target.

Cadherin-12 contributes to tumorigenicity in colorectal cancer by promoting migration, invasion, adhersion and angiogenesis

BackgroundCadherin 12 (CDH12), which encodes a type II classical cadherin from the cadherin superfamily, may mediate calcium-dependent cell adhesion. It has been demonstrated that CDH12 could play an important role in the invasion and metastasis of salivary adenoid cystic carcinoma. We decided to investigate the relationship between CDH12 expression level and clinicopathologic variables in colorectal carcinoma (CRC) patients and to explore the functions of CDH12 in tumorigenesis in CRC.MethodsThe expression levels of CDH12 in colorectal carcinoma tissues were detected by immunohistochemistry. Real-time PCR and Western Blot were used to screen CDH12 high-expression cell lines. CCK-8 assay was used to detect the proliferation ability of CRC cells being transfected by shRNAs against CDH12. The wound assay and transwell assay were performed to test migration and invasion ability. The importance of CDH12 in cell-cell junctions was detected by cell adhesion assay and cell aggregation assay. Endothelial tube formation assay was used to test the influence of CDH12 on angiogenesis.ResultsStatistical analysis of clinical cases revealed that the positive rate of CDH12 was higher in the CRC tumor tissues compared with the adjacent non-tumor tissues. The expression levels of CDH12 in CRC patients are significantly correlated with invasion depth. Consistently, the ability of proliferation, migration and invasion were suppressed when CDH12 was decreased in CRC cells transfected with shRNAs. Cell adhesion assay and cell aggregation assay presented that tumor cells tend to disperse with the lack of CDH12. Endothelial tube formation assay showed that down-regulation of CDH12 could obviously inhibit the process of angiogenesis, implying that CDH12 may play an important role in tumor metastasisConclusionOur results showed that CDH12 promotes proliferation, migration, invasion, adhesion and angiogenesis, suggesting that CDH12 may be an oncogene in colorectal cancer. CDH12 is expected to become a new diagnostic and prognostic marker and a novel target of the treatment of colorectal cancer.

Cadherin-12 enhances proliferation in colorectal cancer cells and increases progression by promoting EMT

Cadherin-12 (CDH12) is a subtype of N-cadherin family. In this study, we investigated the expression of CDH12 and the role of CDH12 in prognosis of colorectal cancer (CRC) patients. In addition, we observed the influence of CDH12 on proliferation and progression of CRC cell lines. By using immunohistochemical staining, we analyzed CRC samples and adjacent non-tumor tissues collected from 78 patients who underwent laparoscopic surgery in Shanghai Minimally Invasive Center, China. Statistical analyses were used to analyze relationship between CDH12 and tumor features. Kaplan-Meier method was used to analyze patients’ survival. Proliferation ability of CRC cells was tested by CCK-8 assay, and transwell assays were performed to detect migration and invasion ability. Western blot assay was performed to investigate epithelial-mesenchymal transition (EMT) variants. We found that expression of CDH12 in tumor tissue was higher than in adjacent normal tissue. High expression of CDH12 was associated with tumor invasion depth and predicts poor prognosis of CRC patients. Ectopic/repressing expression of CDH12 increased/decreased the proliferation and migration ability of CRC cells. CDH12 is able to increase cancer cell migration and invasion via promoting EMT by targeting transcriptional factor Snail. These findings may conclude that CDH12 may act as a predictor in CRC patients’ prognosis and an oncogene in CRC cell proliferation and migration. CDH12 may influence CRC cell progression through promoting EMT by targeting Snail. In addition, CDH12 is promoted by MCP1 through induction of MCPIP.

TXNDC9 Expression in Colorectal Cancer Cells and Its Influence on Colorectal Cancer Prognosis

In this study, we analyzed the protein expression of thioredoxin domain containing 9 (TXNDC9) in 116 colorectal cancer (CRC) cases. Among them, 97 were positive in CRC tissues and 60 were positive in normal mucosa. TXNDC9 expression in CRC was correlated with the extent of tumor invasion and the tumor size. TXNDC9-negative patients had longer lifespans. In vitro assays showed the significant suppression of CRC cell proliferation (P < .01) compared with two control groups; the number of invaded cells also decreased (P < .01). These findings suggest that TXNDC9 gene may function in cancer development and may be an effective target for inhibiting the growth and metastasis of CRC cells.

Aberrant expression and demethylation of γ-synuclein in colorectal cancer, correlated with progression of the disease

Recent evidence suggests that γ‐synuclein is abnormally expressed in a high percentage of tumor tissues of diversified cancer types, but rarely expressed in tumor‐matched non‐neoplastic adjacent tissues (NNAT). The molecular mechanism of CpG island demethylation may underlie aberrant γ‐synuclein expression. To fully understand the roles of aberrant γ‐synuclein expression and demethylation in the development of colorectal cancer (CRC), we examined the expression and methylation status of γ‐synuclein in 67 CRC samples, 30 NNAT samples, and five CRC cell lines as well. By using reverse transcription–polymerase chain reaction (RT‐PCR), western blot, and immunohistochemistry analyses, γ‐synuclein expression was detected in both HT‐29 and HCT116 cells, and was much higher in CRC samples than in NNAT samples (P < 0.05). The demethylating agent, 5‐aza‐2¢‐deoxycytidine, can induce re‐expression of γ‐synuclein in COLO205, LoVo, and SW480 cells. Unmethylated γ‐synuclein alleles were detected in HT‐29, HCT116, and LoVo cells by nested methylation‐specific PCR, and the demethylated status of γ‐synuclein was much higher in CRC samples than in NNAT samples by real‐time quantitative methylation‐specific PCR (P < 0.05). The results of genomic bisulfite DNA sequencing further confirmed that the aberrant γ‐synuclein expression in CRC was primarily attributed to the demethylation of CpG island. The protein expression and demethylation status of γ‐synuclein in 67 CRC samples correlated with clinical stage, lymph node involvement, and distant metastasis. These findings suggest an involvement of aberrant γ‐synuclein expression and demethylation in progression of CRC, especially in advanced stages. (Cancer Sci 2008; 99: 1924–1932)