Wei-Hong Wen

Overexpression of YAP and TAZ is an independent predictor of prognosis in colorectal cancer and related to the proliferation and metastasis of colon cancer cells.

Background and Objective Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are nuclear effectors of the Hippo pathway. Although they are abundantly expressed in the cytoplasm and nuclei of human colorectal cancer (CRC), and related to tumor proliferation status, there have been few studies on the predictive role of YAP and TAZ expression on the overall survival of patients with CRC. This study investigated YAP and TAZ expression in both CRC patients and colon cancer cell lines, and assessed their prognostic value. Methods Paraffin-embedded specimens from 168 eligible patients were used to investigate YAP and TAZ expression by immunohistochemistry, and compared with experimental results in colon cancer HCT116 cell line to explore their clinical significance in CRC. Results Statistically significant positive correlations were found between protein expression of YAP and TAZ in CRC tissues. Patients with higher YAP or TAZ expression showed a trend of shorter survival times; more importantly, our cohort study indicated that patients with both YAP and TAZ overexpression presented the worst outcomes. This was supported by multivariate analysis. In HCT116 colon cancer cells, the capacity for proliferation, metastasis, and invasion was dramatically reduced by knockdown of YAP and TAZ expressions by siRNA. Conclusions Co-overexpression of YAP and TAZ is an independent predictor of prognosis for patients with CRC, and may account for the higher proliferation, metastasis, and poor survival outcome of these patients.

B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells

Background And Objective The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells. Methods Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells. Results Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition. Conclusions Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.

Human activated CD4(+) T lymphocytes increase IL-2 expression by downregulating microRNA-181c.

MicroRNAs, a large family of small regulatory RNAs, are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner, thereby controlling diverse aspects of cell function, including immune reaction. In this study, we screened and identified a group of differentially expressed miRNAs in naive and activated CD4(+) T cells. Among the miRNAs studied, miR-181c was proven to have the potential to regulate CD4(+) T cell activation. miR-181c was downregulated in the process of CD4(+) T cell activation, and transfection of miR-181c mimics partially repressed the activation of both Jurkat cells and human peripheral blood mononuclear cells (PBMC) CD4(+) T cells. We further showed that miR-181c can bind to the IL-2 3 UTR and repress its expression by inhibiting translation. Moreover, miR-181c mimics reduced activated CD4(+) T cell proliferation. Taken together, our results show that miR-181c serves as a negative regulator that modulates the activation of CD4(+) T cells.

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

RNA interference is highly effective at inhibiting HBV gene expression and replication. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody‐mediated delivery is a novel approach for targeting siRNA to appropriate cells. In this report, we asked whether this siRNA delivery strategy would be effective against HBV. Of 5 candidates, a specific siRNA that effectively inhibited HBV gene expression and replication was determined. Two fusion proteins, s‐tP and sCκ‐tP, were constructed to contain a single chain of the human variable fragment, scFv, against hepatitis B surface antigen (HBsAg), a truncated protamine (tP), and in the case of sCκ‐tP, a constant region of the κ chain (Cκ). S‐tP and sCκ‐tP were developed to provide targeted delivery of the siRNA, siRNA expressing cassettes (SEC), and siRNA‐producing plasmids. Fluorescein isothiocyanate‐siRNA, fluorescein isothiocyanate‐SEC, and plasmid DNA were specifically delivered into HBsAg‐positive cells using the sCκ‐tP fusion protein, and effectively inhibited HBV gene expression and replication. HBV gene expression was also inhibited by siRNA or siRNA‐producing plasmids in HBV transgenic mice. Conclusion: Our results describe a potential method for the targeted delivery of siRNA or siRNA‐producing plasmids against HBV, using anti‐HBsAg fusion proteins. (HEPATOLOGY 2007;46:84–94.)

Recombinant immunoproapoptotic proteins with furin site can translocate and kill HER2-positive cancer cells.

We previously reported the selective killing of HER2-positive tumor cells by a class of immunoproapoptotic proteins containing single-chain antibody, translocation domain of Pseudomonas exotoxin A (domain II; PEA II), and constitutively active human apoptotic molecules. In this study, a novel class of antitumor immunoproapoptotic proteins was explored to mediate tumor-specific apoptosis both in vitro and in vivo. Three furin cleavage sequences, including a synthetic polyarginine tract, and two furin cleavable sequences from PEA and diphtheria toxin were respectively used to replace PEA II in the previously constructed immunoproapoptotic protein. When produced and secreted by the genetically modified Jurkat cells, the novel targeted proapoptotic proteins selectively bound to HER2, which is often overexpressed on tumor cell surface. Followed by receptor-mediated endocytosis and furin cleavage in the endosome, the recombinant proteins could translocate into the cytosol, leading to irreversible cell death. Moreover, delivery of these proteins by either i.m. plasmid injection or i.v. injection of plasmid-expressing Jurkat cells led to tumor regression and prolonged animal survival in a nude mouse xenograft tumor model, indicating in vivo antitumor activity of the recombinant proteins. We conclude that the new class of immunoproapoptotic proteins show comparable activity with PEA II-containing counterpart and provide an attractive therapeutic alternative as they contain much less exogenous fragments.

A survivin double point mutant has potent inhibitory effect on the growth of hepatocellular cancer cells

Survivin is an attractive target in cancer therapy. Previous studies have demonstrated that survivin dominant-negative mutants T34A and C84A were able to induce apoptosis in cancer cells. Given that they had different mechanisms in inducing apoptosis, our study was undertaken to determine whether a survivin double point mutant (TC34,84AA) could achieve more potent inhibitory effect on the growth of hepatocellular cancer cells. Adenoviruses expressing survivin mutants were constructed and transduced into hepatocellular cancer cells. The inhibitory effect of the survivin mutants on cancer cell growth was measured. Transduction of cancer cells with all three survivin mutants resulted in significant apoptosis. Compared with survivin mutants T34A or C84A alone, the cancer killing effect of survivin TC34,84AA was much stronger. In addition, the survivin mutants were more sensitive than wild type survivin to the degradation in the ubiquitin-proteasome pathway. Our results suggest that adenovirus-delivered dominant-negative survivin TC34,84AA promotes apoptosis-mediated hepatocellular carcinoma suppression, and could potentially be a promising candidate for cancer therapies.

Selective proapoptotic activity of a secreted recombinant antibody/AIF fusion protein in carcinomas overexpressing HER2

Apoptosis-inducing factor (AIF) represents a caspase-independent apoptotic pathway in the cell, and a mitochondrial localization sequence-truncated AIF (AIFΔ1–120) can be relocated from the cytoplasm to the nucleus and exhibit a constitutive proapoptotic activity. Here, we generated a chimeric immuno-AIF protein, which comprised an HER2 antibody, a Pseudomonas exotoxin translocation domain and AIFΔ1–120. Human Jurkat cells transfected with the immuno-AIF gene could express and secrete the chimeric protein, which selectively recognized HER2-overexpressing tumor cells and was endocytosed. Subsequent cleavage of truncated AIF from immuno-AIF and its release from the internalized vesicles resulted in apoptosis of tumor cells. Intramuscular injection of the immuno-AIF gene caused significant suppression of tumors and substantially prolonged mice survival in an HER2-overexpressing xenograft tumor model. Our study demonstrates the feasibility of the immuno-AIF gene as a novel approach to treating cancers that overexpress HER2.

Potent killing of HBV-related hepatocellular carcinoma by a chimeric protein of anti-HBsAg single-chain antibody and truncated Bid.

Targeted therapy is needed for hepatitis B virus (HBV)-mediated hepatocellular carcinoma (HCC) which shows overexpression of HBV surface antigen (HBsAg). We previously developed scFv15, a human single-chain antibody against HBsAg. Here we tested the strategic feasibility of scFv15-mediated delivery of apoptotic effectors for HBsAg-targeted HCC therapy and application of HA2 motif of influenza hemagglutinin to enhance endosome escape and antitumor effect. A class of HBsAg-targeted immunoproapoptotic molecule was generated by sequentially fusing scFv15, the furin-cleavable motif from diphtheria toxin (Fdt), HA2 and a truncated apoptotic protein Bid (tBid). The resulting scFv15-Fdt-HA2-tBid was prokaryotically expressed and functionally characterized for HBsAg-binding capacity, endosome escape activity and antitumor effect as compared with scFv15-Fdt-tBid. Both scFv15-Fdt-HA2-tBid and scFv15-Fdt-tBid retained affinity and specificity for HBsAg, and bound and selectively killed HBsAg-positive HCC cells via apoptosis. Notably, the IC50 of scFv15-Fdt-HA2-tBid in HBsAg-positive PLC/PRF/5 cells was 10 times lower than that of scFv15-Fdt-tBid. In vivo imaging of antitumor activity demonstrated 95% growth inhibition of orthotopic HCC by scFv15-Fdt-HA2-tBid compared with 75% suppression by scFv15-Fdt-tBid. This study represents an extended application of the immunoproapoptotic strategy in the treatment of HBsAg-positive HCC and shows significant potential of HA2 as a functional enhancer for endosome-encapsulated antibody-conjugates.

Ectopically Expressed Perforin-1 Is Proapoptotic in Tumor Cell Lines by Increasing Caspase-3 Activity and the Nuclear Translocation of Cytochrome c

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell’s plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.