- Email: [email protected]
103
194
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
dependent kinase 2, is an important regulator of the G1 phase transition and its overexpression has been shown to shorten the length of the G1 phase. HER2 amplification and cyclin E overexpression have each been shown to be important prognostic indicators in breast cancer. We hypothesized therefore that HER2 overexpression may modulate cyclin E regulation in breast cancer. Methods: HER2 expression was downregulated in the HER2-overexpressing breast cancer cell lines MCF-7-HER-18 (HER18) and SKBr3 using two different mechanisms; treatment with the monoclonal antibody trastuzumab dosed at 10 and 20 g/ml and transfection with 2 different HER2 siRNA sequences. The low-HER2 expressing cell line MCF-7 was used as a negative control. Western blot analysis was performed to determine levels of HER2 and cyclin E expression. Flow cytometry was performed to determine cell cycle profiles and kinase assays were used to evaluate cyclin E activity. To determine in vivo effects of trastuzumab on cyclin E levels, a HER2-overexpressing breast xenograft model was used. Results: HER2 inactivation by trastuzumab resulted in a dose dependent decrease in cyclin E expression in both HER18 and SKBr3 cells. This was confirmed in vivo using immunohistochemistry analysis of cyclin E expression in tumors from mice treated with trastuzumab. HER2 downregulation by two different siRNA sequences to HER2 also caused a decrease in HER2 and cyclin E expression compared to transfection with random sequence siRNA controls. Cell cycle analysis of trastuzumab treated cells demonstrated an increase in the percentage of cells in the G1 phase (figure). Kinase assays demonstrated decreased cyclin E activity in both SKBr3 and HER18 cell lines (60% decrease for SKBr3 and 25% decrease for HER18 cells after treatment with trastuzumab 20 g/ml). Conclusions: HER2 downregulation results in decreased expression of cyclin E with a resulting increase in the percentage of cells in G1 arrest and decrease in cyclin E kinase activity. Further study into the mechanism of interaction between these two may identify a role for cyclin E expression in predicting response to HER2-directed therapy with trastuzumab.
ONCOLOGY IV: IMMUNOLOGY 103. MESOTHELIN: A MALIGNANT FACTOR AND A NOVEL THERAPEUTIC VACCINE TARGET FOR PANCREATIC CANCER. M. Li, U. Bharadwaj, R. Zhang, S. Zhang, H. Mu, C. Chen, Q. Yao; Baylor College of Medicine, Houston, TX Background: Mesotheline (MSLN) is a GPI-linked glycoprotein, which is overexpressed in several cancer types including mesothelioma, ovarian cancer, and pancreatic cancer, but very little or no expression of MSLN is seen in normal tissues and other cancer types. In this study, we investigated the expression and functions of MSLN in human pancreatic cancer cells, and further studied the effect and mechanism of chimeric virus-like particle (VLP)-hMSLN as a potent vaccine candidate for immunotherapy in pancreatic cancer. Methods: The expression of MSLN in human pancreatic cancer cell lines, HPDE cells, clinical specimens of human pancreatic adenocarcinoma were determined by real-time RT-PCR and western blot. MSLN stable overexpression and silencing cells were selected in MIA PaCa-2, and BxPC-3 cells using retrovirus vectors. In vitro cell proliferation, migration, and cell cycle were performed by MTS, modified Boyden chamber assay, and flow cytometry analysis, re-
spectively. In vivo tumor growth was performed in subcutaneous and orthotopic nude mice model. Chimeric VLP-hMSLN was constructed, and C57BL/6J mice were immunized by VLP-hMSLN to treat preexisting pancreatic tumor. MSLN-specific T cells and antibodies, and regulatory T cells were assessed by ELISPOT and ELISA, and CD4 ⫹CD25 ⫹Foxp3 ⫹ staining, respectively. Results: The MSLN expression levels were significantly increased in all four pancreatic cancer cell lines tested as compared with that in HPDE cells (p⬍0.05). In 10 clinical pancreatic carcinoma samples, MSLN was found to be substantially overexpressed as compared with the surrounding normal tissues (p⬍0.05). Forced expression of MSLN boosts tumor cell proliferation and migration in vitro by 90% and 300% when compared with the vector control cells (p⬍0.05). MSLN overexpression cells lines significantly promote tumor progression in both subcutaneous and orthotopic pancreatic cancer xenograft mouse models. Silencing of MSLN decreased cell proliferation and migration in pancreatic cancer cells by 50% and 70% compared with the vector control cells (p⬍0.05), and most significantly ablated tumor growth in an in vivo mouse pancreatic cancer model. To evaluate the efficacy of immunotherapy with chimeric VLP which contains MSLN incorporated at the surface of the particle (VLP-hMSLN), C57BL/6J mice were orthotopically planted with pancreatic tumors and VLP-hMSLN immunized group showed significantly regressed pre-existing tumor progression as compared with VLP control immunized group. Significant higher amount of MSLN-specific IgG antibody production, the elevated levels of MSLN specific CD8 ⫹ T cell responses and down regulated T regulatory cells were found in the group that immunized with VLP-hMSLN but not in VLP control immunized group. The MSLN-specific humoral and cellular immune responses maybe responsible for controlling tumor growth and correlated with prolonged survival. Conclusions: These data demonstrate that MSLN is overexpressed, and is a malignant factor in pancreatic cancer. VLP-hMSLN immunization in C57BL/6J mice significantly regressed the pre-existing pancreatic tumor and prolonged the survival, and therefore, VLP-hMSLN can be a novel therapeutic vaccine. This study may provide a new treatment strategy for therapeutic intervention in pancreatic cancer. 104. GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) TRANSFECTED VACCINE FOR ESOPHAGEAL CANCER CAUSES REGRESSION OF SUBCUTANEOUS IMPLANTS IN RATS. T. Miyashita, T. D. Armstrong, J. Wang, M. K. Gibson, D. S. Chen, K. Yoshimura, P. Mohebi, G. Marti, E. A. Montgomery, M. Duncan, E. M. Jaffee, J. W. Harmon; Johns Hopkins University School of Medicine, Baltimore, MD Introduction: We aim to protect individuals with Barrett’s esophageal dysplasia from proceeding to esophageal cancer by using a tumor vaccine. As an initial assessment of this approach, we have developed a whole-cell esophageal cancer vaccine and have tested its ability to impede the growth of esophageal cancer cells implanted subcutaneously in rats. Methods: We established three adenosquamous cancer cell lines from reflux-induced esophageal cancers in a rat model. While the cells had a squamous phenotype, microarray and karyotyping revealed that these cell lines shared molecular characteristics of human esophageal adenocarcinoma cells (J Thorac Cardiovasc Surg in press). We transfected the JA cell line to express GM-CSF at the rate of 55 ng/24 hours/1⫻10 6 cells as measured by ELISA. The growth of subcutaneous tumor implants in Sprague Dawley rats was then assessed. Rats were vaccinated on day minus 7 with 3 ⫻10 7 irradiated vaccine cells secreting GM-CSF. PBS was injected in the control group animals. All animals were implanted subcutaneously with 1.5 ⫻ 10 7 cultured tumor cells along with Matrigel(tm) on day 0, and implant sites were assessed histologically at 11 and 14 days. Tumor size was assessed at 0, 4, 7, 11 and 14 days. Results: In the vaccinated group, tumors on four of six rats disappeared after 4 days. By day 14, tumors in all six vaccinated rats had
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
dependent kinase 2, is an important regulator of the G1 phase transition and its overexpression has been shown to shorten the length of the G1 phase. HER2 amplification and cyclin E overexpression have each been shown to be important prognostic indicators in breast cancer. We hypothesized therefore that HER2 overexpression may modulate cyclin E regulation in breast cancer. Methods: HER2 expression was downregulated in the HER2-overexpressing breast cancer cell lines MCF-7-HER-18 (HER18) and SKBr3 using two different mechanisms; treatment with the monoclonal antibody trastuzumab dosed at 10 and 20 g/ml and transfection with 2 different HER2 siRNA sequences. The low-HER2 expressing cell line MCF-7 was used as a negative control. Western blot analysis was performed to determine levels of HER2 and cyclin E expression. Flow cytometry was performed to determine cell cycle profiles and kinase assays were used to evaluate cyclin E activity. To determine in vivo effects of trastuzumab on cyclin E levels, a HER2-overexpressing breast xenograft model was used. Results: HER2 inactivation by trastuzumab resulted in a dose dependent decrease in cyclin E expression in both HER18 and SKBr3 cells. This was confirmed in vivo using immunohistochemistry analysis of cyclin E expression in tumors from mice treated with trastuzumab. HER2 downregulation by two different siRNA sequences to HER2 also caused a decrease in HER2 and cyclin E expression compared to transfection with random sequence siRNA controls. Cell cycle analysis of trastuzumab treated cells demonstrated an increase in the percentage of cells in the G1 phase (figure). Kinase assays demonstrated decreased cyclin E activity in both SKBr3 and HER18 cell lines (60% decrease for SKBr3 and 25% decrease for HER18 cells after treatment with trastuzumab 20 g/ml). Conclusions: HER2 downregulation results in decreased expression of cyclin E with a resulting increase in the percentage of cells in G1 arrest and decrease in cyclin E kinase activity. Further study into the mechanism of interaction between these two may identify a role for cyclin E expression in predicting response to HER2-directed therapy with trastuzumab.
ONCOLOGY IV: IMMUNOLOGY 103. MESOTHELIN: A MALIGNANT FACTOR AND A NOVEL THERAPEUTIC VACCINE TARGET FOR PANCREATIC CANCER. M. Li, U. Bharadwaj, R. Zhang, S. Zhang, H. Mu, C. Chen, Q. Yao; Baylor College of Medicine, Houston, TX Background: Mesotheline (MSLN) is a GPI-linked glycoprotein, which is overexpressed in several cancer types including mesothelioma, ovarian cancer, and pancreatic cancer, but very little or no expression of MSLN is seen in normal tissues and other cancer types. In this study, we investigated the expression and functions of MSLN in human pancreatic cancer cells, and further studied the effect and mechanism of chimeric virus-like particle (VLP)-hMSLN as a potent vaccine candidate for immunotherapy in pancreatic cancer. Methods: The expression of MSLN in human pancreatic cancer cell lines, HPDE cells, clinical specimens of human pancreatic adenocarcinoma were determined by real-time RT-PCR and western blot. MSLN stable overexpression and silencing cells were selected in MIA PaCa-2, and BxPC-3 cells using retrovirus vectors. In vitro cell proliferation, migration, and cell cycle were performed by MTS, modified Boyden chamber assay, and flow cytometry analysis, re-
spectively. In vivo tumor growth was performed in subcutaneous and orthotopic nude mice model. Chimeric VLP-hMSLN was constructed, and C57BL/6J mice were immunized by VLP-hMSLN to treat preexisting pancreatic tumor. MSLN-specific T cells and antibodies, and regulatory T cells were assessed by ELISPOT and ELISA, and CD4 ⫹CD25 ⫹Foxp3 ⫹ staining, respectively. Results: The MSLN expression levels were significantly increased in all four pancreatic cancer cell lines tested as compared with that in HPDE cells (p⬍0.05). In 10 clinical pancreatic carcinoma samples, MSLN was found to be substantially overexpressed as compared with the surrounding normal tissues (p⬍0.05). Forced expression of MSLN boosts tumor cell proliferation and migration in vitro by 90% and 300% when compared with the vector control cells (p⬍0.05). MSLN overexpression cells lines significantly promote tumor progression in both subcutaneous and orthotopic pancreatic cancer xenograft mouse models. Silencing of MSLN decreased cell proliferation and migration in pancreatic cancer cells by 50% and 70% compared with the vector control cells (p⬍0.05), and most significantly ablated tumor growth in an in vivo mouse pancreatic cancer model. To evaluate the efficacy of immunotherapy with chimeric VLP which contains MSLN incorporated at the surface of the particle (VLP-hMSLN), C57BL/6J mice were orthotopically planted with pancreatic tumors and VLP-hMSLN immunized group showed significantly regressed pre-existing tumor progression as compared with VLP control immunized group. Significant higher amount of MSLN-specific IgG antibody production, the elevated levels of MSLN specific CD8 ⫹ T cell responses and down regulated T regulatory cells were found in the group that immunized with VLP-hMSLN but not in VLP control immunized group. The MSLN-specific humoral and cellular immune responses maybe responsible for controlling tumor growth and correlated with prolonged survival. Conclusions: These data demonstrate that MSLN is overexpressed, and is a malignant factor in pancreatic cancer. VLP-hMSLN immunization in C57BL/6J mice significantly regressed the pre-existing pancreatic tumor and prolonged the survival, and therefore, VLP-hMSLN can be a novel therapeutic vaccine. This study may provide a new treatment strategy for therapeutic intervention in pancreatic cancer. 104. GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) TRANSFECTED VACCINE FOR ESOPHAGEAL CANCER CAUSES REGRESSION OF SUBCUTANEOUS IMPLANTS IN RATS. T. Miyashita, T. D. Armstrong, J. Wang, M. K. Gibson, D. S. Chen, K. Yoshimura, P. Mohebi, G. Marti, E. A. Montgomery, M. Duncan, E. M. Jaffee, J. W. Harmon; Johns Hopkins University School of Medicine, Baltimore, MD Introduction: We aim to protect individuals with Barrett’s esophageal dysplasia from proceeding to esophageal cancer by using a tumor vaccine. As an initial assessment of this approach, we have developed a whole-cell esophageal cancer vaccine and have tested its ability to impede the growth of esophageal cancer cells implanted subcutaneously in rats. Methods: We established three adenosquamous cancer cell lines from reflux-induced esophageal cancers in a rat model. While the cells had a squamous phenotype, microarray and karyotyping revealed that these cell lines shared molecular characteristics of human esophageal adenocarcinoma cells (J Thorac Cardiovasc Surg in press). We transfected the JA cell line to express GM-CSF at the rate of 55 ng/24 hours/1⫻10 6 cells as measured by ELISA. The growth of subcutaneous tumor implants in Sprague Dawley rats was then assessed. Rats were vaccinated on day minus 7 with 3 ⫻10 7 irradiated vaccine cells secreting GM-CSF. PBS was injected in the control group animals. All animals were implanted subcutaneously with 1.5 ⫻ 10 7 cultured tumor cells along with Matrigel(tm) on day 0, and implant sites were assessed histologically at 11 and 14 days. Tumor size was assessed at 0, 4, 7, 11 and 14 days. Results: In the vaccinated group, tumors on four of six rats disappeared after 4 days. By day 14, tumors in all six vaccinated rats had