Chun-Ting Wang

Immunotherapy targeting fibroblast activation protein inhibits tumor growth and increases survival in a murine colon cancer model

Murine studies have shown that immunological targeting of fibroblast activation protein (FAP) can elicit protective immunity in the absence of significant pathology. Fibroblast activation protein is a product overexpressed by tumor‐associated fibroblasts (TAF) and is the predominant component of the stoma in most types of cancer. Tumor‐associated fibroblasts differ from normal adult tissue fibroblasts, and instead resemble transient fetal and wound healing‐associated fibroblasts. Tumor‐associated fibroblasts are critical regulators of tumorigenesis, but differ from tumor cells by being more genetically stable. Therefore, in comparison to tumor cells, TAF may represent more viable therapeutic targets for cancer immunotherapy. To specifically target TAF, we constructed a DNA vaccine directed against FAP. This vaccine significantly suppressed primary tumor and pulmonary metastases primarily through CD8+ T‐cell‐mediated killing in tumor‐bearing mice. Most importantly, tumor‐bearing mice vaccinated against FAP exhibited a 1.5‐fold increase in lifespan and no significant pathology. These results suggest that FAP, a product preferentially expressed by TAF, could function as an effective tumor rejection antigen. (Cancer Sci 2010; 101: 2325–2332)

Phosphorylation and changes in the distribution of nucleolin promote tumor metastasis via the PI3K/Akt pathway in colorectal carcinoma

Here, we investigated the molecular mechanism underlying the changes in the distribution of nucleolin. Our study identified PI3K/Akt signaling as an essential pathway regulating the distribution of nucleolin. Furthermore, nucleolin can interact with phospho‐PI3K‐p55, and changes in the distribution of nucleolin were related to its phosphorylation. Subsequently, we analyzed the correlation of VEGF and nucleolin, and found that distribution of nucleolin related to metastatic potential. Finally, blocking cell surface nucleolin influences the process of epithelial–mesenchymal transitions. This indicates that nucleolin may be a novel cancer therapy target and a predictive marker for tumor migration in colorectal carcinoma.

Intratumoral Expression of Mature Human Neutrophil Peptide-1 Mediates Antitumor Immunity in Mice

Purpose: Human neutrophil peptides (HNP1-3), small molecular antimicrobial peptides, are expressed within tumors and associated with tumor necrosis and inhibition of angiogenesis. Recent investigations have suggested that HNP1-3 are likely to be involved in the host immune responses to tumors. Experimental Design: We used recombinant pSec-HNP1, which expresses a secretable form of HNP1, to obtain expression of HNP1 in the tumor milieu in immunocompetent mice to explore the possible roles of HNP1 in tumor immunity. The antitumor effects were investigated in established CT26 colon cancer and 4T1 breast cancer models. Results: HNP1-mediated chemotactic and activating effects on immature dendritic cells were detected both in vitro and in vivo. Intratumoral expression of HNP1 resulted in not only significant tumor growth inhibition but also increased CTL infiltration within tumors. Adoptive transfer of splenocytes and a 51Cr release assay revealed specific cellular immune responses. Furthermore, increased antibodies were also found in sera from pSec-HNP1treated mice supporting specific humoral immune responses. Increased apoptosis and decreased angiogenesis were also shown in treated tumors. Conclusions: These findings indicate that HNP1 can exert multiple antitumor effects through different mechanisms; more importantly, HNP1 mediates host immune responses to tumors in situ through the recruitment and subsequent activation of immature dendritic cells and thus shows promising potential in cancer therapy. (Clin Cancer Res 2009;15(22):690111)

Downregulation of caspase‐9 is a frequent event in patients with stage II colorectal cancer and correlates with poor clinical outcome

Objective  To evaluate the clinical significance of caspase‐9 mRNA expression and investigate its prognostic value in stage II colorectal cancer.

A whole-cell tumor vaccine modified to express fibroblast activation protein induces antitumor immunity against both tumor cells and cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are common components of the tumor-suppressive microenvironment, and are a major determinant of the poor outcome of therapeutic vaccination. In this study, we modified tumor cells to express the fibroblast activation protein (FAP), which is highly expressed by CAFs, to potentially improve whole-cell tumor vaccines by targeting both tumor cells and CAFs. Tumor cells were transfected with murine FAP plasmids bearing the cationic lipid DOTAP. Its antitumor effects were investigated in three established tumor models. Vaccination with tumor cells expressing FAP eliminated solid tumors and tumors resulting from hematogenous dissemination. This antitumor immune response was mediated by CD8+ T cells. Additionally, we found that CAFs were significantly reduced within the tumors. Furthermore, this vaccine enhanced the infiltration of CD8+ T lymphocytes, and suppressed the accumulation of immunosuppressive cells in the tumor microenvironment. Our results indicated that the FAP-modified whole-cell tumor vaccine induced strong antitumor immunity against both tumor cells and CAFs and reversed the immunosuppressive effects of tumors by decreasing the recruitment of immunosuppressive cells and enhancing the recruitment of effector T cells. This conclusion may have important implications for the clinical use of genetically modified tumor cells as cancer vaccines.

Short hairpin RNA targeting of fibroblast activation protein inhibits tumor growth and improves the tumor microenvironment in a mouse model.

Fibroblast activation protein (FAP) is a specific serine protease expressed in tumor stroma proven to be a stimulatory factor in the progression of some cancers. The purpose of this study was to investigate the effects of FAP knockdown on tumor growth and the tumor microenvironment. Mice bearing 4T1 subcutaneous tumors were treated with liposome-shRNA complexes targeting FAP. Tumor volumes and weights were monitored, and FAP, collagen, microvessel density (MVD), and apoptosis were measured. Our studies showed that shRNA targeting of FAP in murine breast cancer reduces FAP expression, inhibits tumor growth, promotes collagen accumulation (38%), and suppresses angiogenesis (71.7%), as well as promoting apoptosis (by threefold). We suggest that FAP plays a role in tumor growth and in altering the tumor microenvironment. Targeting FAP may therefore represent a supplementary therapy for breast cancer. [BMB Reports 2013; 46(5): 252-257]

Enhancement of cisplatin sensitivity in lewis lung carcinoma by liposome-mediated delivery of a survivin mutant

BackgroundA high concentration of cisplatin (CDDP) induces apoptosis in many tumor cell lines. CDDP has been administered by infusion to avoid severe toxicity. Recently, it has been reported that changes in survivin expression or function may lead to tumor sensitization to chemical and physical agents. The aim of this study was to determine whether a dominant-negative mouse survivin mutant could enhance the anti-tumor activity of CDDP.MethodsA plasmid encoding the phosphorylation-defective dominant-negative mouse survivin threonine 34→alanine mutant (survivin T34A) complexed to a DOTAP-chol liposome (Lip-mS) was administered with or without CDDP in Lewis Lung Carcinoma (LLC) cells and in mice bearing LLC tumors, and the effects on apoptosis, tumor growth and angiogenesis were assessed. Data were analyzed using one-way analysis of variance(ANOVA), and a value of P < 0.05 was considered to be statistically significant.ResultsLLC cells treated with a combination of Lip-mS and CDDP displayed increased apoptosis compared with those treated with Lip-mS or CDDP alone. In mice bearing LLC tumors and treated with intravenous injections of Lip-mS and/or CDDP, combination treatment significantly reduced the mean tumor volume compared with either treatment alone. Moreover, the antitumor effect of Lip-mS combined with CDDP was greater than their anticipated additive effects.ConclusionThese data suggest that the dominant-negative survivin mutant, survivin T34A, sensitized LLC cells to chemotherapy of CDDP. The synergistic antitumor activity of the combination treatment may in part result from an increase in the apoptosis of tumor cells, inhibition of tumor angiogenesis and induction of a tumor-protective immune response.

Inhibition of lymphatic metastases by a survivin dominant-negative mutant.

Metastasis is the most lethal attribute of human malignancy. High-level expression of survivin is involved in both carcinogenesis and angiogenesis in cancer. Previous studies indicate that a mutation of the threonine residue at position 34 (Thr34Ala) of survivin generates a dominant-negative mutant that induces apoptosis, inhibits angiogenesis, and suppresses highly metastatic breast carcinoma in mouse models. We investigated the efficacy of gene therapy with a survivin dominant-negative mutant and possible factors related to lymph node metastasis. The metastasis rate was compared between each group in order to find a survivin-targeted therapy against lymphangiogenesis in its earliest stages. We established lymph node metastasis models and treated animals with H22 tumors with Lip-mSurvivinT34A (Lip-mS), Lip-plasmid (Lip-P), or normal saline (NS). Eight days after the last dose, five randomly chosen mice from each group were sacrificed. We detected the apoptotic index, microvessel density (MVD), lymphatic microvessel density (LMVD), and the expression of VEGF-D with immunohistochemistry. After the remaining animals were sacrificed, we compared the tumor-infiltrated lymph nodes in each group. Administration of mSurvivinT34A plasmid complexed with cationic liposome (DOTAP/chol) resulted in the efficacious inhibition of tumor growth and lymph node metastasis within the mouse H22 tumor model. These responses were associated with tumor cell apoptosis, and angiogenesis and lymphangiogenesis inhibition. Our results suggested that Lip-mSurvivinT34A induced apoptosis and inhibited tumor angiogenesis and lymphangiogenesis, thus suppressing tumor growth and lymphatic metastasis. The mSurvivinT34A survivin mutant is a promising strategy of gene therapy to inhibit lymphatic metastasis.

VEGF-D-induced draining lymphatic enlargement and tumor lymphangiogenesis promote lymph node metastasis in a xenograft model of ovarian carcinoma

BackgroundVascular endothelial growth factor (VEGF)-D has been shown to promote lymph node metastasis in several cancers. Although generally overexpressed in ovarian carcinoma, its role in nodal dissemination of this cancer is unclear. To clarify the role of VEGF-D and the underlying molecular mechanisms, we investigated the function of VEGF-D using a mouse xenograft model of ovarian cancer.MethodsHuman ovarian serous adenocarcinoma SKOV3 cells were transfected with VEGF-D recombinant plasmid DNA, or with control vectors. The cells were injected subcutaneously into the footpads of nude mice. Tumor growth was evaluated weekly. Draining lymphatics were observed grossly with Evan’s blue lymphangiography. Tumoral lymphatics were delineated with both Evan’s blue and LYVE-1 immunostaining. Tumor metastases to lymph nodes were evaluated by H&E and CA125/CD40 staining. Expression of VEGF-D in primary tumors and levels of CA125 in involved lymph nodes were examined by immunohistochemistry. Tumor cell apoptosis was analyzed by Hoechst dyeing.ResultsMice bearing VEGF-D overexpressing xenografts showed a significantly higher rate of lymph node metastasis and markedly greater tumor volume compared with the controls. The functional lymphatic vessels were denser and enlarged in marginal and central tumor portions. Additionally, higher CA125 expression was observed in the involved lymph nodes. Mice bearing VEGF-D overexpressing xenografts also exhibited a markedly lower apoptotic index compared with the controls.ConclusionsOur data demonstrate the important role of VEGF-D in promoting lymph node metastasis by increasing tumor lymphangiogenesis, stimulating draining lymphatic vessel formation, and enhancing tumor invasiveness. Our findings show that VEGF-D can be a promising therapeutic target for ovarian cancer.

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37 degrees C, while it was almost exclusively expressed in soluble form at 20 degrees C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.