Reinforcing Antimelanoma Efficacy Of B16F10/GPI-IL-21 Tumor Vaccine By Regulating Expression Of MiR200c, ZEB1 And TGF-β1

Melanoma is the most aggressive type of skin cancer and has a very highly death rate in skin tumors. In early phase of disease, the symptom of patients with melanoma is insidious; however, once the disease was diagnosed most tumor cells have widely metastasized to other tissues, such as lungs and brain. Therefore, the melanoma is difficult to be ablated by surgical ablation, and has very high rate of mortality l’J. As the progress of tumor immunology, the understanding to tumor antigens, tumor immune escape mechanism and the tumor microenvironment is more profound; an immunotherapy gradually becomes the focus of cancer treatment and research, that means reinforcing antitumor immunity in the tumor microenvironment is to control and kill tumor cells by stimulating the function of immune system, especially in application of antibodies for targeting the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) receptors, which have proven to be breakthrough for immunotherapy of melanoma.Cytokine therapy for tumor immunotherapy is one of important ways, which is of increasing levels of cytokines by some ways for full playing the antitumor functions of cytokine in treatment and prevention of diseases. A abundant clinical data suggests that although the application of immunotherapy makes the curative effect of melanoma has improved significantly, there are still some melanoma patients becoming the malignant recurrence. One of the reasons for the failure of treatment is due to melanoma cells secreting abundant transforming growth factor β (TGF-β), which can suppress immune function and promote the metastasis of cancer cellsIL-21 is a new members of cytokines IL-2 family, was discovered in 2000 and produced by activated CD4+T helper cells, whose receptor is expressed in a variety of immune cells (B, T, NK cells), so it can biologically regulate a variety of immune cells. IL-21 can enhance the proliferation of B lymphocytes, increase the generation of IgG1, thereby regulating the body’s humoral immunity. IL-21 can also enhance the proliferation and cytotoxicities of NK cells and T lymphocytes, thereby enhancing the body’s cellular immunity (Figl, Fig2). Whether the recombinant IL-21 or the expression of IL-21 tumor vaccine, whether monotherapy or combined application, IL-21 have shown a strong antitumor effect in tumor immunotherapy.Our Lab has been engaged in the study of IL-21 since 2001, and first amplified the IL-21 encoding gene in mice in China, and the gene sequence of IL-21 has been submitted to Gene Bank (accession number:AY428162). In our previous study, we found that IL-21 can induce a strong antitumor effect in a variety of mouse tumors. The melanoma vaccine B16F10/GPI-IL-21 can enhance NK cells and cytotoxic CTL function in vaccinated mice, inhibit Treg cells and its inhibitory effect on CD8+T cells; meanwhile increasing the ability of T cells to secrete IFN-y. IFN-y has a direct anti-tumor activity, and can reduce the expression of TGF-β, thereby inhibiting TGF-β induced phosphorylation of Smad3, that is the combination of Smad3 and Smad4, the nuclear accumulation of Smad3, as well as the activation of TGF-β response elements. Consequently, the comprehensive effects significantly reduced the invasiveness of tumor cells. However, GPI-IL-21 can not act directly on the TGF-β/Smad signaling pathway, so it can not completely block the metastasis of melanom.MicroRNAs (miRNAs) are non-coding RNA molecules encoded by the genome, with a length of approximate 22 nucleotides. They are single-stranded noncoding RNAs that bind primarily to the 3’untranslated region of target mRNAs to repress their translation and stability, to regulate the transcription and translation function of mRNA by inhibiting ribosome function and targeting gene mRNA. It is well known that miRNAs are important regulators of malignant transformation and metastasis .Epithelial-mesenchymal transition (EMT) refers to epithelial cells under physiological and pathological conditions specific to mesenchymal transition phenomena. Tumors of epithelial origin can be transferred to beyond by EMT. Melanoma is not of epithelial origin, but tumor cells with epithelioid features, such as prone EMT and metastasis. Zinc finger enhancer binding protein 1 (zinc-finger E-box binding homeobox 1, ZEB1) are epithelial cadherin (E-cadherin) transcriptional repressor, with typical zinc finger domain, can bind to the E-box of target gene E-cadherin promoter, to directly inhibit the transcription of E-cadherin, and down-regulate its expression, resulting in reduced intercellular adhesion, enhanced mobility and transferability.It has been shown that miR200c can inhibit the expression of ZEB1 and ZEB2. Conversely, ZEB1 and ZEB2 can also inhibit the expression of miR200c at the transcriptional level. MiR200c and ZEB family constitutes a two-way feedback control loop, which plays an important role in the development of colon cancer, breast cancer, pancreatic cancer and other tumors. Moreover, the overexpression of miR200c can block EMT induced by TGF-beta. Therefore, investigation on this area has become the focus for cancer targeted therapy.Previous studies have shown that cytokines IL-2, IL-12, IL-15, and IL-21 as an immune reinforcing agent of tumor vaccine, just limited to the activation of immune cells. However, the research on the activation of tumor cells and their associated signaling pathways is poorly understood The regulation of tumor cell signaling pathways themselves and effect on its biological characteristics have an important influence on immune responses that was induced by tumor vaccine, and will plays key function in immune therapy for melanoma. Thus, we assumed if we can directly decrease the expression of TGF-β1 in the melanoma cells, and regulate the expression of miR-200 and ZEB1 through different ways; maybe produce a synergistic effect on enhancement efficacy of vaccine B16F10GPI-IL-21. This comprehension effects will improve the immune effects of immune system against tumor cells on the one hand, and will reduce or prevent the invasion and metastasis of melanoma on the other hand, thereby playing a better antitumor effects. Therefore, in this study, we carried out the following series of experiments.1. Objective:Reinforcing antimelanoma efficacy of B16F10/glycosylphospha-tidylinositol-interleukin 21 (B16F10/GPI-IL-21) tumor vaccine by regulating expression of miR200c, ZEB1 and TGF-β1, and providing an experiment data and a novel thread for immunotherapy of melanoma.2. Methods:1) Study of the antitumor efficacy and mechanisms of B16F10/GPI-IL-21 vaccine combined with miR200c over expression and knockdown of ZEB1(1) The previously constructed recombinants pSUPER-EGFβ1-shZEB1(shZEB1) and pcDNA3.1/IL-21-gpi were respectively transfected into B16F10 cells by Lipo-fectamine 2000; the positively clones of stable transfection were selected with G418, and labeled "B16F10/shZEB1"and "B16F10/GPI-IL21", and identified by RT-PCR and Western-blot. To generate the miR200c expression lentivirus vector, we amplified an insert (full-length mouse miR200c) by PCR from B16F10 DNA. The lentivirus miR200c was produced from the transient transfection of the HEK293T cells with pHAGE-CMV-miR200c-IZsGreen, psPAX2, and pMD2.G plasmid DNAs plus Lipofectamine 2000. Forty-eight hours after the co-transfection, the lentivirus-bearing supernatants were collected. The B16F10 cells were infected with the pHAGE-CMV-miR200c-IzsGreen lentivirus, and were selected by the IzsGreen expression. The stable expression colonies were selected by limiting the dilution assay, and labeled "B16F10/miR200c". The expression of miR200c and ZEB1 was respectively first detected by RT-PCR. The expression of ZEB1, E-Cadherin, N-Cadherin, Vimentin, Smad7 as well as EMT relevant molecules were analyzed by Western-blot in wild type B16F10 cells, B16F10/shZEBl cells, B16F10/GPI-IL21 cells and B16F10/miR200c, respectively. The capabilities of colony formation and migration in the different cells were tested by Wound-Healing assay and Colony forming assay, respectively.(2) The C57BL/6 mice were initially immunized subcutaneous (s.c.) in a mouse’s groin with 1×107 B16F10 cells inactivated with the 100μg/ml-1 mitomycin C; this was performed three times at a two-week interval. The blood was collected from the ophthalmic vein at week 1, week 2, week 3, and week 4, respectively. The serum levels of IFN-y, IL-4 and TGF-β was detected by enzyme linked immunosorbent assay (ELISA) of double antibody sandwich with the Kits. The splenocyte cytotoxicity was analyzed by Flow Cytometry (FCM). About 7 days after the final immunization, the mice were randomly divided into four groups:the B16F10/WT group, the B16F10/shZEB1 group, the B16F10/miR200 group, and the B16F10/GPI-IL-21 group. All mice were challenged s.c. with 1×105 above-mentioned different B16F10 cells. Six mice/group were used in the study. Tumor growth and life span was monitored, and then the counts of lung metastases were also examined by hematoxylin and eosin (HE) stain after mice was sacrificed. The expressions of EMT relevant molecules in tumor tissues were analyzed with Immunohistochemistry and Western-blot.2) Study of the strengthening antitumor efficacy and mechanisms of B16F10/GPI-IL-21 vaccine by combining injection of recombinant shZEB1 and miR200c in mice(1) The method of mouse immunization is the same as before, and the mouse’s right flank was s.c. challenged with 1×105 B16F10 cells after the third immunization. The mice were randomly divided into three groups:the PBS group, the shZEB1 group, and the miR200 agomir group 3 days after challenge. The mice were s.c. treated with above-mentioned different reagents, once every four day and in total six times. Twelve mice/group were used in the study.(2) The blood was collected from the ophthalmic vein at finish of treatment, and the serum levels of IFN-y and TGF-β was detected by double antibody sandwich ELISA Six mice/group were sacrifice, and the cytotoxicities of natural killer (NK) cells and cytotoxic T lymphocyte (CTL) as well as the proportion of CD4^CD25+Treg cells in the tumor-draining lymph nodes and the splenocytes were respectively analyzed by FCM. Tumor growth, survival, and distant metastases were observed. The lung metastatic focuses were stained by HE; the expression of EMT relevant molecules in tumor tissues were simultaneously analyzed with Immunohistochemistry and Western-blot.3) Study of the reinforcing antimelanoma efficacy and mechanisms of B16F10/GPI-IL-21 Vaccine by combining downregulation of TGF-β1 and miR200c in mice.(1) The recombinant pSUPER-EGFP1-TGF-β1 (shTGF-(31) was constructed and transfected into B16F10 cells by Lipo-fectamine 2000, and then the stable transfected clones were selected with G418, and labeled "B16F10/shTGF-β1". The interfere effectiveness was identified by RT-PCR. The expression of EMT relevant molecules in the B16F10/shTGF-β1 cells was analyzed with Western blot. The capabilities of colony formation and migration in the B16F10/shTGF-β1 cells were detected by Wound-Healing assay and Colony forming assay, respectively.(2) The mice were immunized according to above-mentioned method, and were randomly divided into the B16F10/GPI-IL-21+B16F10/shTGF-β1 group (mice were challenged s.c. with 1×105 B16F10/shTGF-β1cells), the B16F10/GPI-IL-21+B16F10/ shTGF-β1+miR200c agomir group (mice were challenged s.c. with 1×105 B16F10/ shTGF-β1 cells and treated with miR200c agomir), and the B16F10/GPI-IL-21+B16F10/Scrambled+miR200c/Scrambled group (mice were challenged s.c. with 1×105 B16F10/Scramble, and treated with miR200c/Scramble). Meanwhile, two group mice were serves as a control, which are of the B16F10/shTGF-β1 group (mice were challenged S.C. with 1×105 B16F10/shTGF-β1cells) and the B16F10/shTGF-β1miR200c agomir group (mice were challenged S.C. with 1×105 B16F10/shTGF-β1 cells, and treated with miR200c agomir). The time of tumor growth and survival, and distant metastases were observed. The lung metastatic focus was stained by HE, and the expression of EMT relevant molecules in tumor tissues were simultaneously analyzed with Immunohistochemistry and Western-blot.3. Results:1)) The serum cytokine levels of IFN-y, TNF-a and IL-4, and the splenocyte cytotoxicity were significantly increased, whereas the serum TGF-β level was markedly decreased compared with the control vaccine after the mice were immunized with the tumor vaccine B16F10/GPI-IL-21 in combination with either overexpression of miR200c or knockdown of ZEB1 expression in B16F10 cells. In addition, the immunized mice significantly reduced melanoma growth and lung metastasis. The EMT relevant molecules in tumor tissues detected by Immunohistochemistry and Western blot showed that the expression of TGF-P, ZEB1 and N-Cadherin was remarkablely decreased; in contrast, the expression of E-Cadherin and Smad7 was obviously increased, which was statistically significant compared with the control group.3) The mice was challenged with 1x10 B16F10 cells after the third immunization of the tumor vaccine B16F10/GPI-IL-21, and treated with shZEBlplasmid and miR200c agomir again. The results indicated that the cytotoxicities of NK cells and CTL were significantly enhanced with the reduction of CD4+CD25+Treg cells in the tumor-draining lymph nodes and the splenocytes. The rate of tumor formation, growth speed and lung metastases were markedly decreased compared with the control group. Immunohistochemistry and Western-blot results showed that the expression of epithelial phenotypic relevant molecules in tumor tissues was significantly increased with the decrease of mesenchymal phenotypic relevant molecules in contrast to the control group.3) The mice was challenged with 1×105 B16F10/shTGF-β1cells after the third immunization of the tumor vaccine B16F10/GPI-IL-21, and treated with miR200c agomir. The results of post-treatment showed that the mice generated stronger immune responses than that of control mice, such as the enhanced cytotoxicities of NK cells and CTL, repressed expression of FoxP3, a specific mark of CD4+CD25+Treg cells, changed inhibitory action of Treg cells to CD8T cells, promoted level of IFN-y secreted by NK cells and CD8+T cells, and reduced level of serum TGF-β. Importantly, the rate of tumor formation, growth speed and lung metastases were significantly repressed compared with the results that were shown in previous two sections. Additionally, the survival time of tumor bearing mice was longer than that of control mice.4. Conclusions:Regulation expression of miR200c, ZEB1 and TGF-β1 in B16F10 cells and tumor microenvironment positively enhances anti-tumor effect of tumor vaccine B16F10/GPI-IL-21, inhibiting or inversing the melanoma’EMT and metastasis. These findings may open up a new visual angle for immunotherapy of melanoma in clinical trials.