Szu-Ta Chen

Connective Tissue Growth Factor Activates Pluripotency Genes and Mesenchymal–Epithelial Transition in Head and Neck Cancer Cells

The epithelial-mesenchymal transition (EMT) is a key mechanism in both embryonic development and cancer metastasis. The EMT introduces stem-like properties to cancer cells. However, during somatic cell reprogramming, mesenchymal-epithelial transition (MET), the reverse process of EMT, is a crucial step toward pluripotency. Connective tissue growth factor (CTGF) is a multifunctional secreted protein that acts as either an oncoprotein or a tumor suppressor among different cancers. Here, we show that in head and neck squamous cell carcinoma (HNSCC), CTGF promotes the MET and reduces invasiveness. Moreover, we found that CTGF enhances the stem-like properties of HNSCC cells and increases the expression of multiple pluripotency genes. Mechanistic studies showed that CTGF induces c-Jun expression through αvβ3 integrin and that c-Jun directly activates the transcription of the pluripotency genes NANOG, SOX2, and POU5F1. Knockdown of CTGF in TW2.6 cells was shown to reduce tumor formation and attenuate E-cadherin expression in xenotransplanted tumors. In HNSCC patient samples, CTGF expression was positively correlated with the levels of CDH1, NANOG, SOX2, and POU5F1. Coexpression of CTGF and the pluripotency genes was found to be associated with a worse prognosis. These findings are valuable in elucidating the interplay between epithelial plasticity and stem-like properties during cancer progression and provide useful information for developing a novel classification system and therapeutic strategies for HNSCC.

IL-6 induces AGS gastric cancer cell invasion via activation of the c-Src/RhoA/ROCK signaling pathway

Interleukin‐6 (IL‐6) is a multifunctional cytokine that is associated with the disease status and outcomes of gastric cancer. Nonetheless, the underlying mechanism of how IL‐6 promotes the spread of gastric cancer is still unclear. In this study, we used a modified Boyden chamber assay to test the invasion ability of different gastric cancer cell lines. Liposome‐mediated transfection was used to introduce an IL‐6 expression vector into AGS cells, and the transfectants were further examined for the expression of active RhoA and phosphorylated Src using a pull‐down assay and coimmunoprecipitation/Western blot analysis. Furthermore, RhoA expression in gastric adenocarcinoma specimens was investigated immunohistochemically. We documented that IL‐6 could promote AGS cell motility and invasiveness, and inhibition of RhoA expression by dominant negative RhoA, C3 transferase, or dominant negative Src expressing plasmids could effectively decrease the invasiveness of IL‐6 transfectants. We also documented an interaction between active RhoA and phosphorylated‐Src following IL‐6 treatment. Gastric cancers displaying high expression of RhoA are highly correlated with aggressive lymph node metastasis, more advanced tumor stage, histologically diffuse type and poorer survival. In conclusion, IL‐6 induces AGS gastric cancer cell invasion via activation of the c‐Src/RhoA/ROCK signaling pathway and RhoA expression could be used as a prognostic factor in patients with gastric adenocarcinoma.

Cyr61Induces Gastric Cancer Cell Motility/Invasion via Activation of the Integrin/Nuclear Factor-KB/Cyclooxygenase-2 Signaling Pathway

Purpose: Cysteine-rich 61 (Cyr61/CCN1) is involved in many different types of tumor development and progression. Nonetheless, the role of Cyr61 in human gastric cancer has not yet been fully characterized. Experimental design: We addressed the issue by immunohistochemical staining of 81 gastric adenocarcinoma specimens. Liposome-mediated transfection was used to introduce a Cyr61 expression vector into gastric cancer AGS cell lines. Transfectants were tested in invasion assay by a Boyden chamber. Furthermore, a cyclooxygenase-2 (COX-2) reporter assay and gel mobility shift assay were done to investigate the potential signal pathway of Cyr61. Results: Patients with gastric adenocarcinoma whose tumor displayed high expression of Cyr61 correlated well with aggressive lymph node metastasis, more advanced tumor stage, histologic diffuse type, and early recurrence. Stable transfection of Cyr61 into the AGS cell line strongly enhanced its invasive activity. The overexpression of Cyr61 into AGS cells significantly increased the expression of COX-2 mRNA, protein, and enzymatic activity. Gel mobility shift assays further showed that the nuclear factor-κB (NF-κB) pathway was evidently activated in Cyr61-expressing AGS cells. Function-neutralizing antibody to αvβ3 but not αvβ5 effectively suppressed Cyr61-mediated NF-κB activation, COX-2 gene expression, and cell invasiveness. Conclusions: Cyr61 may contribute to the malignant progression of gastric cancer by promoting tumor cell motility/invasion through up-regulation of the functional COX-2 via an integrin αvβ3/NF-κB-dependent pathway.

HDAC2 promotes cell migration/invasion abilities through HIF-1α stabilization in human oral squamous cell carcinoma

BACKGROUND Histone deacetylase 2 (HDAC2) expressions in oral squamous cell carcinoma (OSCC) had been implicated in advanced stage and poor prognosis. It suggests a possible link between the migration/invasion potential of oral cancer cells and the prevalent expression of HDAC2. METHODS Five head and neck cancer (HNC) cell lines, including Ca9-22, Cal-27, HSC-3, SAS, and TW2.6, were used. Cells stably overexpressing HDAC2 and shRNA against HDAC2 were established to investigate migration/invasion ability in vitro and tumorigenesis and progression in vivo. RESULTS We found that alterations in the HDAC2 level in OSCC cell lines modulated their invasive ability with a positive correlation. Animal model also showed that knockdown of HDAC2 expression in SAS cells, originally containing high endogenous HDAC2 expression, resulted in decrease in tumor initiation and progression. Using high-throughput transcriptome analysis, numerous genes involved in HIF-1α-associated pathways were found. At the mechanism levels, using agents to block de novo protein synthesis or prevent protein degradation by ubiquitination, we found the stability of hypoxia inducible factor 1α (HIF-1α) protein was maintained in OSCC cells with HDAC2 overexpression. In addition, co-immunoprecipitation assay also revealed that HDAC2-mediated HIF-1α protein stability is because of direct interaction of HIF-1α with von Hippel-Lindau (VHL) protein. CONCLUSIONS Our work demonstrates that HDAC2 maintains HIF-1α stability, probably at the level of protein modification, which in turn leads to the increase in cell invasion/migration ability in oral cancer progression. These findings implicate the potential of HDAC inhibitors for oral cancer therapy.

Changes in tumor growth and metastatic capacities of J82 human bladder cancer cells suppressed by down-regulation of calreticulin expression.

Bladder cancer is a common urothelial cancer. Through proteomic approaches, calreticulin (CRT) was identified and proposed as a urinary marker for bladder cancer. CRT is a multifunctional molecular chaperone that regulates various cellular functions such as Ca(2+) homeostasis and cell adhesion. CRT is overexpressed in various cancers, but its mechanism of action in the development of bladder tumors remains unclear. We generated J82 bladder cancer cells lines that either stably overexpressed or knocked down CRT to investigate the physiological effects of CRT on bladder tumors. Compared with the transfected control vector cells, the knockdown of CRT suppressed cell proliferation, migration, and attachment, whereas overexpression of CRT enhanced cell migration and attachment. We further demonstrated that the phosphorylation status of focal adhesion kinase and paxillin, important regulators of the focal adhesion complex, was also regulated in these cells. In contrast, phosphorylation of Src, a protein tyrosine kinase reported to be affected by CRT, was not significantly different between the control and CRT-RNAi groups. Most importantly, we observed that tumors derived from J82 CRT-RNAi cells were significantly smaller and had fewer metastatic sites in the lung and liver in vivo than did transfected control vector cells. In conclusion, our results suggest that alteration of CRT expression levels might affect bladder cancer progression in vitro and in vivo.

The Influence of Donor Characteristics on Survival After Heart Transplantation

With improved immunosuppressive regimens, transplantation techniques, and postoperative care, heart transplantation (HTx) has been established as a definite therapy for end-stage heart disease. Because of a donor shortage, we have accepted marginal individuals. In this study, we identified donor-related factors influencing survival after HTx by retrospective analysis of recipient data after primary HTx from February 2002 to December 2006. The Cox regression model was used to examine the effects of the following variables on survival of 112 heart transplant recipients: demographic data of gender, age, body weight, donor-recipient body weight ratio; history of smoking, alcohol drinking, diabetes mellitus, hypertension, hepatitis B surface antigen, anti-hepatitis C virus antibody; donor condication before transplantation including catecholamine doses, hypotension, cardiopulmonary resuscitation, creatine MB isoenzyme of creatine kinase (CK-MB), tropinin I, and cold ischemic time of the allograft. Catecholamines and smoking showed significant influences on HTx survival. In our series, the percentage of donors receiving inotropic support before donation was 88% (n = 99), and the percentage of donors with a history of smoking was 25% (n = 28). There was no influence of donor status of diabetes, hypertension, or hepatitis B or C infection on postoperative survival. Our results showed that inotropic support of and a history of smoking by the donor were significant factors influencing posttransplant survival.

Insulin-like growth factor II mRNA-binding protein 3 expression predicts unfavorable prognosis in patients with neuroblastoma

Insulin‐like growth factor II mRNA‐binding protein 3 (IMP3) has been reported to enhance proliferation and invasion in various cancers. The role of IMP3 on neuroblastoma (NB) is unknown. We aimed to clarify the prognostic significance of IMP3 expression in patients with NB. By microarray analysis, high IMP3 expression was found in patients with poor outcome. IMP3 expression in 90 NB samples was analyzed by immunohistochemical staining to correlate with clinical stages, histology, and patient outcome. Positive IMP3 expression was detected in 52 of 90 patients, and was significantly correlated with undifferentiated histology, advanced stages, MYCN amplification, and poor outcome. In subgroups, positive IMP3 expression could predict an even worse prognosis in patients with advanced disease, with normal MYCN status, or with MYCN amplification (P = 0.005, P = 0.001, and P = 0.033, respectively). The IMP3 expression decreased by induction of differentiation with retinoid acid treatment in SK‐N‐DZ and SK‐N‐SH cells in vitro. The invasion ability of NB cells also decreased as IMP3 knockdown by using RNA interference in vitro. In summary, high expression of IMP3 in NB might contribute to the undifferentiated phenotype and invasive behaviors, leading to a poor prognosis. Determining IMP3 expression in NB could help to improve a personalized therapy. (Cancer Sci 2011; 102: 2191–2198)

Prenatal diagnosis of progressive familial intrahepatic cholestasis type 2

Background and Aim:  Progressive familial intrahepatic cholestasis type 2 (PFIC2) results from genetic defects of the hepatobiliary bile salt export pump (BSEP, ABCB11) at chromosome 2q24. Patients with progressive cholestasis and liver cirrhosis usually need liver transplantation in the first decade. Mutations in ABCB11 are also associated with benign recurrent intrahepatic cholestasis type 2 and intrahepatic cholestasis of pregnancy in adult patients. We aimed to make the prenatal diagnosis of PFIC2.

HMGCS2 enhances invasion and metastasis via direct interaction with PPARα to activate Src signaling in colorectal cancer and oral cancer.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) is the rate-limiting enzyme of ketogenesis. Growing evidence indicates that HMGCS2 may be involved in cancer progression, but its exact role is largely unknown. In this study, we demonstrate that HMGCS2 mRNA expression is associated with poor clinical prognosis and outcomes in patients with colorectal cancer (CRC) and oral squamous cell carcinoma (OSCC). In vitro, ectopic expression of HMGCS2 enhanced cancer cell motility in a ketogenesis-independent manner. Moreover, HMGCS2 promoted Src activity by directly binding to peroxisome proliferator-activated receptor alpha (PPARα), a transcriptional activator of Src. Taken together, these results suggest that HMGCS2 may serve as a useful prognostic marker and vital target for future therapeutic strategies against advanced cancer.

Prognostic Significance of TWEAK Expression in Colorectal Cancer and Effect of Its Inhibition on Invasion

BackgroundTumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been implicated in tumor development and progression. The aim of this study was to investigate the role of TWEAK in colorectal cancer (CRC) progression.MethodsTo investigate the involvement of TWEAK in the progression of human CRC, normal, and tumor specimens from 174 patients were analyzed immunohistochemically for the expression of TWEAK. TWEAK recombinant protein treatment, transfection of expression plasmids, and small interfering RNA to knockdown TWEAK expression were performed to test invasive ability with a Boyden chamber. The mRNA expression profile in recombinant TWEAK treatment was compared to a control group by microarray analysis. To identify downstream effectors, Raf kinase inhibitor (RKIP) and its correlation with TWEAK in vitro and in vivo were examined by quantitative real-time polymerase chain reaction and invasion assays.ResultsCRC patients whose tumors displayed high TWEAK expression had a statistically significantly higher overall survival and a disease-free advantage over those with a low TWEAK expression. In in vitro invasion assays, alterations in TWEAK expression in CRC cell lines inversely modulated their invasive ability. By means of integrated genomics, we identified RKIP as a downstream effector in TWEAK-mediated invasion inhibition. Knockout of RKIP expression in HCT116 cells by short hairpin RNA (shRKIP) resulted in increased invasiveness. Clinically, RKIP and TWEAK mRNA expression showed strong positive correlations in CRC patient samples.ConclusionsOur results implicate TWEAK as a key regulator of CRC invasion, and it appears to be a useful prognostic factor for patients with CRC.