Xinying Wang

CD24-dependent MAPK pathway activation is required for colorectal cancer cell proliferation

CD24 is a glycosylphosphatidylinositol‐anchored membrane protein reported to be overexpressed in human tumorigenesis and progression. Our purpose was to determine the role of CD24 in the proliferation of colorectal cancer cells and the potential mechanisms in this process. Our data showed that CD24 promoted cell growth and induced activation of extracellular signal‐regulated kinases, Raf‐1, and p38 mitogen‐activated protein kinase. Furthermore, suppression of extracellular signal‐regulated kinases and p38 mitogen‐activated protein kinase activity by their specific inhibitors, U0126 and SB203580, abrogated CD24‐induced proliferation in vitro. By tumorigenicity assay in female BALB/c nude mice, we further demonstrated that CD24 promoted tumor growth in vivo. Immunohistochemical analysis revealed that CD24 expression occurred in 92.5% of human colorectal cancer tissue, and increased with tumor progression. More importantly, the stainings of phospho‐extracellular signal‐regulated kinases and phospho‐p38 mitogen‐activated protein kinase were strongly correlated with CD24 expression. Taken together, our data suggest that CD24‐dependent extracellular signal‐regulated kinases and p38 mitogen‐activated protein kinase activations are required for colorectal cancer cell proliferation in vitro and in vivo. The linkage of CD24 and the mitogen‐activated protein kinase pathway may unravel a novel mechanism in the regulation of colorectal cancer proliferation. (Cancer Sci 2009; 00: 000–000)

Identification of Kininogen-1 as a Serum Biomarker for the Early Detection of Advanced Colorectal Adenoma and Colorectal Cancer

Background Serum markers represent potential tools for the detection of colorectal cancer (CRC). The aim of this study was to obtain proteomic expression profiles and identify serum markers for the early detection of CRC. Methods Proteomic profiles of serum samples collected from 35 healthy volunteers, 35 patients with advanced colorectal adenoma (ACA), and 40 patients with CRC were compared using Clinprot technology. Using enzyme-linked immunosorbent assays (ELISAs), 366 sera samples were additionally analyzed, and immunohistochemistry studies of 400 tissues were used to verify the expression of kininogen-1 and its value in the early detection of CRC. Results Predicting models were established among the three groups, and kininogen-1 was identified as a potential marker for CRC using Clinprot technology. ELISAs also detected significantly higher serum kininogen-1 levels in ACA and CRC patients compared to controls (P<0.05). Furthermore, the area under the receiver operating characteristic curve (AUC) for serum kininogen-1 in the diagnosis of ACA was 0.635 (P = 0.003), and for serum carcinoembryonic antigen (CEA) was 0.453 (P = 0.358). The sensitivity, specificity, and accuracy of serum kininogen-1 for diagnosing Duke’s stage A and B CRC was 70.13%, 65.88%, and 67.90%, respectively, whereas serum CEA was 38.96%, 85.88%, and 63.58%, respectively. Moreover, immunohistochemistry showed that expression of kininogen-1 was significantly higher in CRC and ACA tissues than in normal mucosa (48.39% vs. 15.58% vs. 0%, P<0.05). Conclusions These results suggest that Clinprot technology provides a useful tool for the diagnosis of CRC, and kininogen-1 is a potential serum biomarker for the early detection of advanced colorectal adenoma and CRC.

PAK1-dependent MAPK pathway activation is required for colorectal cancer cell proliferation

P21-activated protein kinase1 (PAK1), a main downstream effector of small Rho GTPases, Rac1, and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis, and angiogenesis. Despite its importance, the molecular mechanisms of PAK1 that contributed to colorectal carcinogenesis remain unclear. Our immunohistochemistry showed that PAK1 expression was increased with colorectal cancer (CRC) progression through the adenoma to carcinoma sequence. Furthermore, our results suggested a relationship between PAK1 nuclear localization and the Dukes staging. In the present study, we showed that PAK1 knockdown decreased proliferation and delayed the G1/S cell-cycle transition, and increased apoptosis in vivo and in vitro. In addition, PAK1 knock-down downregulated c-Jun amino terminal kinases (JNK) activity and the levels of cyclinD1, CDK4/6. Inhibition of the JNK activity by chemical inhibitor (SP600125) significantly reduced the effects of PAK1 on CRC proliferation via accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In conclusion, our results demonstrate that knockdown of PAK1 could enhance the chemosensitivity of CRCs to 5-fluorouracil through G1 arrest. The mechanism by which PAK1 induced cancer growth might involve activation of JNK as well as downregulation of PTEN. Targeting PAK1 may represent a novel treatment strategy for developing novel chemotherapeutic agents.

Co-Expression of XIAP and Cyclin D1 Complex Correlates with a Poor Prognosis in Patients with Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Despite improved diagnosis and treatment, the prognosis for HCC patients remains poor. The goal of this study was to identify key regulatory proteins and signaling pathways important for cell apoptosis and proliferation as biomarkers for prognostication and targeted therapy. Protein Pathway Array was applied to screen 38 signaling proteins and phosphoproteins in 12 paired HCC tumors and surrounding benign tissues and found that 20 of them, including XIAP, CDK4, CDK6, and Cyclin D1, were overexpressed in HCC tissues. Immunostaining results of XIAP, CDK4, and Cyclin D1 in an additional 59 HCC tissues showed that the expression of XIAP correlated with the expression of CDK4/Cyclin D1, and that the increased expression of these proteins correlated with poor overall survival in these patients. Further studies using the HCC Huh7 cell line transfected with XIAP siRNA or expression vector demonstrated that XIAP regulated the expression of CDK4, CDK6, and Cyclin D1 via NF-êB and PTEN pathways. Finally, inhibition of XIAP using embelin, a XIAP-specific small molecule, leads to an increased apoptosis and decreased cell proliferation via arrest at G1 phase. Taken together, XIAP is a central modulator regulating cell apoptosis and cell cycle progression. Therefore, XIAP together with cell cycle regulatory proteins can be used as prognostic markers and therapeutic targets.

Diagnostic value of stool DNA testing for multiple markers of colorectal cancer and advanced adenoma: a meta-analysis.

BACKGROUND AND OBJECTIVES The diagnostic value of stool DNA (sDNA) testing for colorectal neoplasms remains controversial. To compensate for the lack of large-scale unbiased population studies, a meta-analysis was performed to evaluate the diagnostic value of sDNA testing for multiple markers of colorectal cancer (CRC) and advanced adenoma. METHODS The PubMed, Science Direct, Biosis Review, Cochrane Library and Embase databases were systematically searched in January 2012 without time restriction. Meta-analysis was performed using a random-effects model using sensitivity, specificity, diagnostic OR (DOR), summary ROC curves, area under the curve (AUC), and 95% CIs as effect measures. Heterogeneity was measured using the χ(2) test and Q statistic; subgroup analysis was also conducted. RESULTS A total of 20 studies comprising 5876 individuals were eligible. There was no heterogeneity for CRC, but adenoma and advanced adenoma harboured considerable heterogeneity influenced by risk classification and various detection markers. Stratification analysis according to risk classification showed that multiple markers had a high DOR for the high-risk subgroups of both CRC (sensitivity 0.759 [95% CI 0.711 to 0.804]; specificity 0.883 [95% CI 0.846 to 0.913]; AUC 0.906) and advanced adenoma (sensitivity 0.683 [95% CI 0.584 to 0.771]; specificity 0.918 [95% CI 0.866 to 0.954]; AUC 0.946) but not for the average-risk subgroups of either. In the methylation subgroup, sDNA testing had significantly higher DOR for CRC (sensitivity 0.753 [95% CI 0.685 to 0.812]; specificity 0.913 [95% CI 0.860 to 0.950]; AUC 0.918) and advanced adenoma (sensitivity 0.623 [95% CI 0.527 to 0.712]; specificity 0.926 [95% CI 0.882 to 0.958]; AUC 0.910) compared with the mutation subgroup. There was no significant heterogeneity among studies for subgroup analysis. CONCLUSION sDNA testing for multiple markers had strong diagnostic significance for CRC and advanced adenoma in high-risk subjects. Methylation makers had more diagnostic value than mutation markers.

p21-activated kinase 5 inhibits camptothecin-induced apoptosis in colorectal carcinoma cells

Abstractp21-activated kinase 5 (PAK5) is a recently identified member of the group B PAK family. The PAK proteins are effectors of the small GTPase Cdc42 and Rac1 and are known to regulate cell motility and activate cell-survival signaling pathways. Especially, the mitochondrial localization of PAK5 is vital to its effects on apoptosis and cell survival. Previously, we demonstrated that PAK5 expression increased significantly during the malignant progression of colorectal carcinoma (CRC) and that PAK5 promoted CRC metastasis by regulating CRC cell adhesion and migration. In the present study, we aim to investigate the role of PAK5 in camptothecin-induced apoptosis and its potential mechanism of action. Our results showed that overexpression of PAK5 inhibited camptothecin-induced apoptosis by inhibiting the activity of caspase-8 in CRC cells. Accordingly, knockdown of PAK5 in LoVo cells resulted in increased apoptosis. Mechanistically, we found that PAK5 directly phosphorylated Bad on serine 112 and indirectly led to phosphorylation of serine 136 via the Akt pathway. In conclusion, our study revealed previously unappreciated inhibitory role of PAK5 in camptothecin-induced apoptosis, thus suggesting PAK5 as a novel therapeutic target in CRC.

BRD4 Inhibitor Inhibits Colorectal Cancer Growth and Metastasis

Post-translational modifications have been identified to be of great importance in cancers and lysine acetylation, which can attract the multifunctional transcription factor BRD4, has been identified as a potential therapeutic target. In this paper, we identify that BRD4 has an important role in colorectal cancer; and that its inhibition substantially wipes out tumor cells. Treatment with inhibitor MS417 potently affects cancer cells, although such effects were not always outright necrosis or apoptosis. We report that BRD4 inhibition also limits distal metastasis by regulating several key proteins in the progression of epithelial-to-mesenchymal transition (EMT). This effect of BRD4 inhibitor is demonstrated via liver metastasis in animal model as well as migration and invasion experiments in vitro. Together, our results demonstrate a new application of BRD4 inhibitor that may be of clinical use by virtue of its ability to limit metastasis while also being tumorcidal.

Increased Expression of β-Catenin, Phosphorylated Glycogen Synthase Kinase 3β, Cyclin D1, and c-myc in Laterally Spreading Colorectal Tumors

Laterally spreading tumors (LSTs) are considered a special subtype of superficial colorectal tumor. This study was performed to characterize the clinicopathological features and examine activation of the Wnt/β-catenin pathway in LSTs and protruded-type colorectal adenomas (PAs). Fifty LSTs and 54 PAs were collected, and their clinicopathological characteristics were compared. The expression of E-cadherin, β-catenin, glycogen synthase kinase-3β (GSK-3β), phosphorylated GSK-3β, (phospho-GSK-3β), cyclin D1, and c-myc was investigated by immunohistochemical staining on serial sections. Patients with LSTs were significantly older than those bearing PAs (63.4 vs 47.4 years old; p<0.001). The mean size of LSTs was significantly larger than that of PAs (27.0 mm vs 14.6 mm; p<0.01). Forty-eight percent of LSTs were located in the proximal colon, which was significantly higher than that of PAs (18.5%; p<0.05). Expression of β-catenin, phospho-GSK-3β, cyclin D1, and c-myc was significantly increased in LSTs compared with PAs (p<0.05). However, E-cadherin and total GSK-3β expression was not significantly different between the two groups. The level of β-catenin expression correlated strongly with phospho-GSK-3β, cyclin D1, and c-myc expression in LSTs but not in PAs. Our findings suggest that activation of the Wnt/β-catenin pathway is more prevalent in LSTs than in PAs, suggesting that phosphorylation-dependent inactivation of GSK-3β may be involved in LST carcinogenesis.

Lyn is involved in CD24-induced ERK1/2 activation in colorectal cancer

CD24 expression is associated with human colorectal cancer (CRC). Our previous data indicated that CD24 promoted the proliferation and invasion of colorectal cancer cells through the activation of ERK1/2. Since Src family kinases are frequently deregulated in CRC and closely related to the MAPK signaling pathway, we investigated the impact of Lyn, an important member of SFKs, on CD24-induced ERK1/2 activation in CRC. The interaction of CD24 and Lyn was identified by co-immunoprecipitation (Co-IP) and ectopic expression of CD24-induced Lyn activation. Inhibition of Lyn activation by phosphatase PP2 in SW480CD24cells abrogated CD24-induced invasion. The results of the Co-IP and immunofluorescence assay revealed that overexpression of CD24 enhanced the interaction of Lyn and ERK1/2 and induced the nuclear translocation of Lyn. However, inhibition of Lyn activity attenuated CD24-induced ERK1/2 activation, and depletion of CD24 disrupted Lyn-ERK1/2 interaction. Immunohistochemistry analysis for 202 cases of CRC showed that the expression of both CD24 and Lyn was positively correlated with tumor grade, stage, lymph node and distant metastasis. Patients with lower expression of CD24 or Lyn had a higher survival rate. The Cox multivariate analysis showed that CD24 expression, but not Lyn expression, was an independent prognostic factor of CRC. Our results suggest that Lyn is involved in CD24-induced ERK1/2 activation in CRC. The expression of CD24 is associated with activation of Lyn and ERK1/2, which might be a novel mechanism related to CD24-mediated regulation of CRC development.

The comparison of the clinical manifestations and risk factors of colorectal cancer and adenomas: results from a colonoscopy-based study in southern Chinese

Background and aimsColorectal cancer (CRC) is one of the most common gastrointestinal tumors in the world. This study aimed to compare the clinical manifestations and risk factors of CRC and adenomas in native patients of Guangzhou.MethodsPatients who underwent colonoscopy for the first time at Nanfang Hospital between July 2008 and July 2009 were recruited. Data on demographic information, main clinical manifestations, results of endoscopies and pathology, and possible risk factors of colorectal tumor were collected. Chi-square test and logistic regression were used to compare the clinical characteristics and risk factors for CRC and adenomas.ResultsHematochezia and body weight loss were more frequent in proximal and distal CRC groups, respectively (P ≤ 0.05). Older age [odds ratio (OR), 1.079; 95% confidence interval (CI), 1.065–1.093], smoking status (OR, 1.712; 95% CI, 1.158–2.531), BMI = 18.5–24.9 and ≥ 25.0 (OR, 2.384; 95% CI, 1.250–4.549; OR, 2.162; 95% CI, 1.044–4.478, respectively) were significant risk factors for advanced adenoma, while female (OR, 0.638; 95% CI, 0.429–0.949) and using aspirin (OR, 0.188; 95% CI, 0.042–0.845) were significant protective factors. Hyperlipemia (OR, 0.109; 95% CI, 0.013–0.886) was identified as a protective factor for proximal CRC. Smoking (OR, 1.717; 95% CI, 1.093–2.696), drinking (OR, 1.817; 95% CI, 1.145–2.883), DM history (OR, 2.204; 95% CI, 1.044–4.652) were identified as independent risk factors for distal CRC, and using aspirin (OR, 0.190; 95% CI, 0.043–0.840) was a protective factor. Drinking (OR, 3.288; 95% CI, 1.546–6.994; OR, 1.862; 95% CI, 1.037–3.343, respectively) was an independent risk factor for both poorly to moderately differentiated CRC and well-differentiated CRC. Besides, DM (OR, 3.761; 95% CI, 1.615–8.762) and hypertension (OR, 0.384; 95% CI, 0.178–0.828) were identified as independent risk factor and protective factor for well-differentiated CRC, respectively.ConclusionsHematochezia and body weight loss were representative manifestations for distal and proximal CRC, respectively. For southern Chinese the most important influential factors for colorectal tumor are age, smoking, drinking, nutritional state, DM, hypertension, and the use of aspirin.