Tzu-Ching Chang

Rho Kinases Regulate the Renewal and Neural Differentiation of Embryonic Stem Cells in a Cell Plating Density–Dependent Manner

Background Rho kinases (ROCKs) mediate cell contraction, local adhesion, and cell motility, which are considered to be important in cell differentiation. We postulated that ROCKs are involved in controlling embryonic stem (ES) cell renewal and differentiation. Methodology/Principal Findings CCE, a murine ES cell, was treated with Y-27632 for 48 to 96 hours and colony formation was evaluated. Y-27632 blocked CCE colony formation and induced CCE to grow as individual cells, regardless of the initial seeding cell density either at 104/cm2 (“high” seeding density) or 2×103/cm2 (“low” density). However, at high seeding density, Y-27632–treated cells exhibited reduction of alkaline phosphatase (AP) staining and Oct3/4 expression. They expressed SOX-1, nestin, and MAP2c, but not βIII-tubulin or NG-2. They did not express endoderm or mesoderm lineage markers. After removal of Y-27632, the cells failed to form colonies or regain undifferentiated state. Silencing of ROCK-1 or ROCK-2 with selective small interference RNA induced CCE morphological changes similar to Y-27632. Silencing of ROCK-1 or ROCK-2 individually was sufficient to cause reduction of AP and Oct3/4, and expression of SOX-1, nestin, and MAP2c; and combined silencing of both ROCKs did not augment the effects exerted by individual ROCK siRNA. Y-27632–treated CCE cells seeded at 2×103 or 6.6×103 cells/cm2 did not lose renewal factors or express differentiation markers. Furthermore, they were able to form AP-positive colonies after removal of Y-27632 and reseeding. Similar to ROCK inhibition by Y-27632, silencing of ROCK-1 or ROCK-2 in cells seeded at 2×103/cm2 did not change renewal factors. Conclusions/Significance We conclude that ROCKs promote ES cell colony formation, maintain them at undifferentiated state, and prevent them from neural differentiation at high seeding density. ROCK inhibition represents a new strategy for preparing large numbers of neural progenitor cells.

Overexpression of 14-3-3ε predicts tumour metastasis and poor survival in hepatocellular carcinoma.

Ko B‐S, Chang T‐C, Hsu C, Chen Y‐C, Shen T‐L, Chen S‐C, Wang J, Wu K K, Jan Y‐J & Liou J‐Y
(2011) Histopathology58, 705–711
Overexpression of 14‐3‐3ε predicts tumour metastasis and poor survival in hepatocellular carcinoma

Bortezomib suppresses focal adhesion kinase expression via interrupting nuclear factor-kappa B.

AIMS Bortezomib is a potent proteasome inhibitor currently used to treat various malignancies with promising results. To explore the role of bortezomib in reducing cancer cell migration and inducing apoptosis, we evaluated the effects of bortezomib on the expression of focal adhesion kinase (FAK). MAIN METHODS Various types of cancer cells including lung cancer A549, H1299; a breast cancer MCF7; a hepatocellular carcinoma Huh7, and a tongue squamous cell carcinoma SCC-25 were treated with different concentrations of bortezomib or MG-132 as indicated for 24h. Protein and mRNA levels were determined by Western blotting and real-time PCR. Apoptosis was analyzed by caspase 3 cleavage and activity. FAK promoter and NFkappaB binding activities were measured by luciferase-reporter method. NFkappaB subunit p65 binding capacity was determined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. KEY FINDINGS Both bortezomib and another proteasome inhibitor, MG-132, significantly reduced FAK expression, suppressed cancer cell migration and increased cell apoptosis. Results of real-time PCR and promoter activity assay revealed that bortezomib decreased FAK expression through transcriptional inactivation. Results of FAK promoter activity and ChIP assays in A549 and H1299 cells indicated that bortezomib suppressed FAK activity through a p53-independent pathway. Furthermore, reduction of NFkappaB binding capacity demonstrated by EMSA and ChIP assay suggested that NFkappaB plays an important role in bortezomib suppressing FAK expression. SIGNIFICANCE These results suggested that FAK is downregulated by bortezomib through a proteasome-dependent NFkappaB inhibitory mechanism. Thus, FAK could be a potential molecular target of bortezomib for therapeutic strategy.

An Efficient Transfection Method for Mouse Embryonic Stem Cells

Embryonic stem (ES) cells are considered to have potentials for tissue regeneration and treatment of diverse human diseases. ES cells are capable of indefinite renewal and proliferation, which can be induced to differentiate into tissues of all three germ lines. Despite these exciting potential, it remains unclear as to how the renewal and differentiation programs are operated and regulated at the genetic level. Genetic manipulation such as delivery of exogenous gene expression or knockdown with small interfering RNA (siRNA) is commonly used in most of cancer or transformed cells but relatively rare in ES cells. In this study, we compare the transfection efficacies of several liposome-based transfection methods by introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into mouse ES (mES) cells. Our results show that transfection by Effectene achieves the efficiency of >98% in CCE and >80% in D3 cells. The optimal ratio of DNA:Effectene for EGFP transfection is between 1:4 and 1:8. Transient-expressed EGFP or endogenous protein kinase A (PKA) were significantly knocked down by Effectene transfection of specific siRNA. High EGFP level expression and accumulation in mES cells induces minor cytotoxicity but can be reduced by introducing siRNA of EGFP. Further, this transfection method did not significantly affect mES properties of proliferation or differentiation. Our results provide an optimal protocol to achieve an efficient transfection for mES cells.

Overexpressed focal adhesion kinase predicts a higher incidence of extrahepatic metastasis and worse survival in hepatocellular carcinoma

Focal adhesion kinase plays a critical role in cancer progression, invasion, and metastasis. Although focal adhesion kinase overexpression indicates poor prognoses for hepatocellular carcinoma, its role in hepatocellular carcinoma metastasis has not been well investigated. In this study, 55 hepatocellular carcinoma patients were enrolled, and their primary liver tumors as well as 18 matched metastases were subjected to semiquantitative immunohistochemistry analysis of focal adhesion kinase expression. Overexpression of focal adhesion kinase was observed in 34 (61.8%) of 55 primary tumors and significantly predicted subsequent extrahepatic metastases (P = .04). Metastatic tumors expressed higher focal adhesion kinase than their matched primaries (P = .010). Focal adhesion kinase overexpression indicated both worse overall 5-year survival rate (51.5% +/- 8.7% versus 90.2% +/- 6.6%; P = .004) and 5-year progression-free survival rate (51.5% +/- 8.7% versus 90.2% +/- 6.6%; P = .041). Taken together, we demonstrated here that focal adhesion kinase expression is significantly related to subsequent hepatocellular carcinoma metastasis. Focal adhesion kinase is thus considered as a reasonable target for novel therapies against hepatocellular carcinoma progression and metastasis.

Involvement of 14-3-3γ overexpression in extrahepatic metastasis of hepatocellular carcinoma.

The 14-3-3γ protein is an important regulator of various cellular and physiologic functions. Overexpression promotes cell proliferation and induces cancer cell polyploidization. Production is up-regulated in human hepatocellular carcinoma. However, the clinical significance of 14-3-3γ for human hepatocellular carcinoma metastasis and survival has not been clarified. In this study, 55 patients with human hepatocellular carcinoma were enrolled; and 18 of them were identified as having extrahepatic metastases. Expression of 14-3-3γ in these primary and metastatic samples was measured with semiquantitative immunohistochemistry analysis. Overexpression of 14-3-3γ was observed in 38 (69.1%) of the primary tumors, correlated significantly with a high α-fetoprotein concentration (P = .003), and predicted a higher probability of extrahepatic metastasis (cumulative probabilities at 5 years: 42.2% ± 8.0% versus 5.9% ± 5.7%, 14-3-3γ positive versus negative; P = .012). Furthermore, 14-3-3γ overexpression was associated with a worse 5-year overall survival rate (81.6% ± 9.6% versus 59.5% ± 8.1%, respectively) and a worse 5-year progression-free survival rate (75.6% ± 10.6% versus 48.6% ± 8.2%, respectively). Elevated expression of 14-3-3γ in human hepatocellular carcinoma predicts extrahepatic metastasis and worse survival. The protein thus is a candidate biomarker and a potential target for novel therapies against human hepatocellular carcinoma progression and metastasis.

14-3-3σ regulates β-catenin-mediated mouse embryonic stem cell proliferation by sequestering GSK-3β.

Background Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear. Methodology and Principal Findings In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding. Significance Our findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.

Upregulation of focal adhesion kinase by 14-3-3ε via NFκB activation in hepatocellular carcinoma.

Focal adhesion kinase (FAK) is implicated in cancer cell survival, proliferation and migration. Expression of FAK expression is elevated and associated with tumor progression and metastasis in various tumors, including hepatocellular carcinoma (HCC). Increased 14-3-3ε expression is shown to be a potential prognostic factor to predict higher risk of distant metastasis and worse overall survival in HCC. The aim of this study is to investigate whether FAK is associated or regulated by 14-3-3ε to modulate tumor progression in HCC. In this study, 114 primary HCC tumors including 34 matched metastatic tumors were subjected to immunohistochemistry analysis of FAK and 14-3-3ε expression. Overexpression of FAK was significantly associated with increased risk of extrahepatic metastasis (p=0.027) and reduced 5-year overall survival rate (p=0.017). A significant correlation of FAK and 14-3-3ε expression was observed in primary tumor (p < 0.001) and also metastatic tumors. Furthermore, overexpression of 14-3-3 ε induced FAK expression and promoter activity which were determined by Western blotting analysis and luciferase-reporter assay. Moreover, 14-3-3ε enhanced NFκB activation and increased nuclear translocation of NFκB. Results from chromatin immunoprecipitation assay revealed that 14-3-3ε induced NFκB binding on FAK promoter region. These findings suggest that FAK expression is correlated with and upregulated by 14-3-3ε via activation of NFκB. Target to suppress or inactivate FAK alone, or combine with 14-3-3ε is thus considered as the potential therapeutic strategy for preventing HCC tumor progression.

14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma

Background14-3-3σ is implicated in promoting tumor development of various malignancies. However, the clinical relevance of 14-3-3σ in hepatocellular carcinoma (HCC) tumor progression and modulation and pathway elucidation remain unclear.MethodsWe investigated 14-3-3σ expression in 109 HCC tissues by immunohistochemistry. Overexpression and knockdown experiments were performed by transfection with cDNA or siRNA. Protein expression and cell migration were determined by Western blot and Boyden chamber assay.ResultsIn this study, we found that 14-3-3σ is abundantly expressed in HCC tumors. Stable or transient overexpression of 14-3-3σ induces the expression of heat shock factor-1α (HSF-1α) and heat shock protein 70 (HSP70) in HCC cells. Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion. Results of an in vitro migration assay indicate that 14-3-3σ promotes cell migration and that 14-3-3σ-induced cell migration is impaired by siRNA knockdown of HSP70. Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β.ConclusionsOur findings indicate that 14-3-3σ participates in promoting HCC cell migration and tumor development via β-catenin/HSF-1α/HSP70 pathway regulation. Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC.

An efficient transfection method for mouse embryonic stem cells.

Embryonic stem (ES) cells are an important source of stem cells in tissue engineering and regenerative medicine because of their high self-renewal capacities and differentiation potentials. However, the detailed molecular mechanisms controlling the differentiation and renewal programs in ES cells remained unclear. One of the difficulties in understanding these mechanisms substantially results from the low efficacies of gene manipulation by delivering exogenous gene expression or knockdown of endogenous gene expression with small interfering RNA (siRNA) in ES cells. Here we describe an optimized protocol for efficiently transfecting mouse ES cells by Effectene, a liposome-based method. The high transfection efficiency in mouse ES cells is demonstrated in this chapter by (1) achieving a percentage of enhanced green fluorescence protein (EGFP) expression in >98% embryoid bodies after introducing plasmids encoding the protein and (2) decreased SOX-2 and Oct-3/4 expression and subsequent morphological evidence of cell differentiation after introducing siRNA expression for suppressing SOX-2 and Oct-3/4, which are known to be essential for maintenance of stem cell properties in mouse ES cells.