Lui Ng

A Subpopulation of CD26+ Cancer Stem Cells with Metastatic Capacity in Human Colorectal Cancer

Recent evidence suggests that a subpopulation of cancer cells, cancer stem cells (CSCs), is responsible for tumor growth in colorectal cancer. However, the role of CSCs in colorectal cancer metastasis is unclear. Here, we identified a subpopulation of CD26(+) cells uniformly present in both the primary and metastatic tumors in colorectal cancer patients with liver metastasis. Furthermore, in patients without distant metastasis at the time of presentation, the presence of CD26(+) cells in their primary tumors predicted distant metastasis on follow-up. Isolated CD26(+) cells, but not CD26(-) cells, led to development of distant metastasis when injected into the mouse cecal wall. CD26(+) cells were also associated with enhanced invasiveness and chemoresistance. Our findings have uncovered a critical role of CSCs in metastatic progression of cancer. Furthermore, the ability to predict metastasis based on analysis of CSC subsets in the primary tumor may have important clinical implication as a selection criterion for adjuvant therapy.

Over-Expression of miR-106b Promotes Cell Migration and Metastasis in Hepatocellular Carcinoma by Activating Epithelial-Mesenchymal Transition Process

Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.

The Enhanced Metastatic Potential of Hepatocellular Carcinoma (HCC) Cells with Sorafenib Resistance

Acquired resistance towards sorafenib treatment was found in HCC patients, which results in poor prognosis. To investigate the enhanced metastatic potential of sorafenib resistance cells, sorafenib-resistant (SorR) cell lines were established by long-term exposure of the HCC cells to the maximum tolerated dose of sorafenib. Cell proliferation assay and qPCR of ABC transporter genes (ABCC1-3) were first performed to confirm the resistance of cells. Migration and invasion assays, and immunoblotting analysis on the expression of epithelial to mesenchymal transition (EMT) regulatory proteins were performed to study the metastatic potential of SorR cells. The expression of CD44 and CD133 were studied by flow cytometry and the gene expressions of pluripotency factors were studied by qPCR to demonstrate the enrichment of cancer stem cells (CSCs) in SorR cells. Control (CTL) and SorR cells were also injected orthotopically to the livers of NOD-SCID mice to investigate the development of lung metastasis. Increased expressions of ABCC1-3 were found in SorR cells. Enhanced migratory and invasive abilities of SorR cells were observed. The changes in expression of EMT regulatory proteins demonstrated an activation of the EMT process in SorR cells. Enriched proportion of CD44+ and CD44+CD133+ cells were also observed in SorR cells. All (8/8) mice injected with SorR cells demonstrated lung metastasis whereas only 1/8 mouse injected with CTL cells showed lung metastasis. HCC cells with sorafenib resistance demonstrated a higher metastatic potential, which may be due to the activated EMT process. Enriched CSCs were also demonstrated in the sorafenib resistant cells. This study suggests that advanced HCC patients with acquired sorafenib resistance may have enhanced tumor growth or distant metastasis, which raises the concern of long-term sorafenib treatment in advanced HCC patients who have developed resistance of sorafenib.

Four and a half LIM protein 2 (FHL2) negatively regulates the transcription of E-cadherin through interaction with Snail1

E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT), which plays a crucial role in cancer metastasis. We previously demonstrated that four and a half LIM protein 2 (FHL2) inhibited E-cadherin expression and promoted invasive potential and EMT in colon cancer. Here, we aim to further define the mechanism underlying the inhibition of E-cadherin by FHL2 in colon cancer. The expression profiles of FHL2 and Snail1 were first observed by Western blot, immunofluorescence and immunohistochemistry. We found that both the protein level and the cellular localisation of Snail1 were quite similar to FHL2 in colon cancer; reciprocal co-immunoprecipitation assay showed that FHL2 was able to bind Snail1 and its intact structure was required. The expression of FHL2 was positively correlated to Snail1 while negatively to E-cadherin and phospho-Snail1. FHL2 over-expression induced the accumulation of Snail1 in the nucleus. Moreover, dual luciferase assay revealed that FHL2 over-expression decreased while FHL2 siRNA increased the transcriptional activities of two E-cadherin promoter constructs which contained E-box sites (Snail1-binding elements). Mutation of E-boxes increased the transcriptional activities and FHL2 expression was involved in the function of mutation. These results suggested that FHL2 negatively regulated E-cadherin transcriptional activity through interaction with Snail1. Our study established a novel regulatory function of FHL2 and revealed a potential mechanism on promoting the process of EMT.

Identification of CD44+ cancer stem cells in human gastric cancer.

The cancer stem cell hypothesis suggests that tumors are initiated and maintained by cancer stem cells capable of self-renewal and differentiation into mature tumor cells. Recent studies have demonstrated the existence of cancer initiating cells in different solid tumors. In this study, we identified a subpopulation of CD44+ cells within the tumor of gastric cancer patients, which, upon treatment by chemotherapeutic agent 5-fluorouracil (5-FU), were markedly enriched. In vitro culture of isolated CD44+ subpopulation from gastric tumors by magnetic beads sorting led to formation of gastric spheroid colonies. These colonies retained CD44+ surface marker expression during culture, and were undifferentiated in nature. Subcutaneous injections of CD44+ gastric cancer cells conferred tumorigenicity in SCID mice. Moreover, implantation of CD44+ cells from these established tumors remained tumorigenic in successive passages. Using CD44+ cells isolated from the gastric cell lines AGS and SGC7901, similar results were obtained. Upon enrichment by 5-FU, CD44+ cells harbored increased ALDH expression as compared with CD44- cells. Our results demonstrated for the first time the existence of CD44+ cells within the tumors of gastric cancer patients that are endowed with stem cells properties, and also provide a plausible explanation for chemo-resistance frequently observed in gastric cancer patients. Such findings provide a basis for further studies on targeting this tumorigenic subpopulation for better treatment of gastric cancer patients.

Prognostic Significance of CD26 in Patients with Colorectal Cancer

Background CD26, dipeptidyl peptidase IV, was discovered firstly as a membrane-associated peptidase on the surface of leukocyte. We previously demonstrated that a subpopulation of CD26+ cells were associated with the development of distant metastasis, enhanced invasiveness and chemoresistance in colorectal cancer (CRC). In order to understand the clinical impact of CD26, the expression was investigated in CRC patients specimens. This study investigated the prognostic significance of tumour CD26 expression in patients with CRC. Examination of CD26+ cells has significant clinical impact for the prediction of distant metastasis development in colorectal cancer, and could be used as a selection criterion for further therapy. Methods Tumour CD26 expression levels were studied by immunohistochemistry using Formalin-fixed paraffin embedded (FFPE) tissues in 143 patients with CRC. Tumour CD26 expression levels were correlated with clinicopathological features of the CRC patients. The prognostic significance of tumour tissue CD26 expression levels was assessed by univariate and multivariate analyses. Result CD26 expression levels in CRC patients with distant metastasis were significantly higher than those in non-metastatic. High expression levels of CD26 were significantly associated with advanced tumour staging. Patients with a high CD26 expression level had significantly worse overall survival than those with a lower level (p<0.001). Conclusions The expression of CD26 was positively associated with clinicopathological correlation such as TNM staging, degree of differentiation and development of metastasis. A high CD26 expression level is a predictor of poor outcome after resection of CRC. CD26 may be a useful prognostic marker in patients with CRC.

Anti-tumor Efficacy of a Recombinant Human Arginase in Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is considered as auxotrophic for arginine and BCT-100, a new recombinant human arginase, has been synthesized for arginine deprivation to inhibit arginine-dependent tumor growth. The aim of the present study was to evaluate the effects of BCT-100 on the inhibition of in vitro cell proliferation of HCC cell lines and in vivo tumor growth. The molecular mechanism involved was also studied. The anti-tumor efficacy of BCT-100 on cell proliferation, cell cycle distribution and cellular apoptosis were determined in human hepatoma HepG2 and PLC/PRF/5 cells. Protein expression in the Wnt/β-catenin and Akt signaling pathways were analyzed by western blotting. Tumors were also established subcutaneously and BCT-100, in combination with oxaliplatin, was administrated i.p. to study the anti-tumor growth of the drugs. Treatment with BCT-100 was found to inhibit cell proliferation and enhance caspasedependent cellular apoptosis. Cell cycle arrest at S phase was observed. Inhibition of Wnt/β-catenin and Akt signaling pathways, with a reduction in survivin and XIAP protein expressions, were also observed. Furthermore, combined treatment of BCT-100 and chemotherapy with oxaliplatin demonstrated synergistic inhibiting effect on tumor growth and the overall survival probability was enhanced as compared with BCT-100 or oxaliplatin treatment alone. These preclinical data demonstrate robust anti-tumor activity of BCT100 in HCC, thus providing the basis for its exploitation as a potential therapeutic agent in arginine-driven tumors. The positive effect of testing BCT100 with oxaliplatin in PLC/PRF/5 tumours also supports the rationale of combining BCT-100 and oxaliplatin in the clinical treatment of HCC.

Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

Background The overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients. Methods This randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay. Results Our findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration. Conclusions The results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.

Osteopontin Overexpression Induced Tumor Progression and Chemoresistance to Oxaliplatin through Induction of Stem-Like Properties in Human Colorectal Cancer

Colorectal cancer (CRC) is one of the most common and fatal malignancies worldwide. The poor prognosis of colorectal cancer patients is due to development of chemoresistance and cancer metastasis. Recently osteopontin (OPN) has been associated with stem-like properties in colorectal cancer. This study further examined the clinicopathological significance of OPN in CRC and its effect on chemoresistance and transcription of stem cell markers. We examined the transcription level of OPN in 84 CRC patients and correlated the expression with their clinicopathological parameters. The associations of OPN overexpression with transcription of stem cell markers and response to chemotherapy in DLD1-OPN overexpressing clones and CRC patients were also investigated. Our results showed that OPN was significantly overexpressed in CRC, and its overexpression correlated with tumor stage and poor prognosis. Overexpression of CRC induced OCT4 and SOX2 expression in vitro and correlated with SOX2 overexpression in CRC patients. In addition, DLD1-OPN overexpressing cells showed enhanced ability to survive upon oxaliplatin treatment, and OPN expression was higher in CRC patients who were resistant to oxaliplatin-involved chemotherapy treatment. Thus, CRC cells overexpressing OPN demonstrated stem-like properties and OPN inhibition is a potential therapeutic approach to combat CRC progression and chemoresistance.

Suppression of actopaxin impairs hepatocellular carcinoma metastasis through modulation of cell migration and invasion

Early reports suggested that actopaxin, a member of the focal adhesion proteins, regulates cell migration. Here we investigated whether actopaxin is involved in hepatocellular carcinoma (HCC) progression and metastasis. We examined actopaxin expression in human HCC samples using immunohistochemistry and western blotting. The functional and molecular effect of actopaxin was studied in vitro by overexpression in a nonmetastatic HCC cell line, as well as repression in a metastatic cell line. The in vivo effect of actopaxin repression was studied in nonobese diabetic and severe combined immunodeficient mice. We found that actopaxin was frequently overexpressed in human HCC patients and its overexpression positively correlated with tumor size, stage, and metastasis. Actopaxin expression also correlated with the metastatic potential of HCC cell lines. Actopaxin overexpression induced the invasion and migration ability of nonmetastatic HCC cells, whereas down‐regulation of actopaxin reverted the invasive phenotypes and metastatic potential of metastatic HCC cells through regulating the protein expression of certain focal adhesion proteins including ILK, PINCH, paxillin, and cdc42, as well as regulating the epithelial‐mesenchymal transition pathway. Furthermore, there was a close association between actopaxin and CD29. HCC cells with stronger CD29 expression showed a higher actopaxin level, whereas actopaxin repression attenuated CD29 activity. Finally, actopaxin down‐regulation enhanced the chemosensitivity of HCC cells towards oxaliplatin treatment by way of a collective result of suppression of survivin protein, β‐catenin, and mammalian target of rapamycin pathways and up‐regulation of p53. Conclusion: This study provides concrete evidence of a significant role of actopaxin in HCC progression and metastasis, by way of regulation of cell invasiveness and motility, an epithelial‐mesenchymal transition process, and chemosensitivity to cytotoxic drugs. (Hepatology 2013;58:667‐679)