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Her2 signaling in breast cancer induces dissociation of adherens junction proteins in endothelial cell monolayers
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
and its pathogenesis is incompletely understood. Methods: Potential factors involved in the pathogenesis of large unilateral (ⱖ 50g) and bilateral (ⱖ100g) nodular goiter were investigated including: age, gender, race, body mass index (BMI), family history of thyroid disease, insurance status, cigarette smoking, and alcohol use. Data were obtained from an IRB-approved thyroid database and retrospective chart review of consecutive patients operated on for nodular goiter from 1990-2004. Statistical significance of differences in patients with “large” versus “small” nodular goiter were determined using a Chi-square test and Mann-Whitney U test for nonparametric data and a T-test for parametric data. Results: Of the 460 patients operated on for nodular goiter, 106 (23%) were classified as “large”, 40 with unilateral enlargement ⱖ50g (mean 114 ⫾ 86g) and 66 with bilateral enlargement ⱖ100g (mean 169 ⫾ 88g) and 354 (77%) were classified as “small”, 165 unilateral (18 ⫾ 10g) and 189 bilateral (36 ⫾ 23g). Epidemiologic factors associated with large nodular goiter were: African American race (55% vs. 22%, p⬍0.001), increased BMI (mean 35 ⫾ 11 kg/m 2 vs. 29 ⫾ 8 kg/m 2, p⫽0.003), and no health insurance (32% vs. 19%, p⫽ 0.003). Alcohol use was less common in patients with large nodular goiter (33% vs. 49%, p⫽0.004). No significant differences were observed in age, gender, family history of thyroid disease or cigarette smoking (p⬎0.1). Conclusions: There is a predilection for large nodular goiter among African-American, obese and uninsured patients whereas alcohol use may be protective. Closer scrutiny of “at-risk” patients may help to identify specific causative factors and appropriate interventions for prevention of large nodular goiter and its sequelae.
147. HER2 SIGNALING IN BREAST CANCER INDUCES DISSOCIATION OF ADHERENS JUNCTION PROTEINS IN ENDOTHELIAL CELL MONOLAYERS. B. Carter 1, G. Small 2, M. Ward 3, J. Hahn 2; 1H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, 2University of Maryland School of Medicine, Baltimore, MD, 3Eastern Virginia Medical School, Norfolk, VA. Background: Her2 signaling imparts a metastatic advantage to breast cancer cells. In addition to increased proliferation, improved survival, and robust angiogenesis, early metastases in Her2 overexpressing breast cancer patients are postulated to be driven by angioinvasion. We have shown that Her2 signaling in breast cancer cells induces a paracrine-driven microvessel breakdown and endothelial cell (EC) retraction, critical steps in angioinvasion and transendothelial migration. Because Vascular Endothelial (VE) cadherin complexes of the adherens junctions regulate EC-EC adherence, we postulated that this EC retraction involves dissociation of the adherens junction proteins. Methods: Human iliac vein EC (HIVEC) monolayers were cocultured with estrogen-independent MCF-7 cells, or stable transfectant cells overexpressing Her2 6 fold (HER cells). Using immunoprecipitation with anti-VE cadherin antibody, we tested for complexed ␣, , and ␥ catenins in control or cocultured EC monolayers. We then manipulated Her2 signaling in these cells by stimulating Her2 signaling with Heregulin 1, or blockade of Her2 signaling with trastuzumab, and tested for VE cadherin/catenin dissociation. Results: MCF-7 cells induced a time-dependent dissociation of the catenins from VE cadherin, reaching 80% by 24 hrs (p ⬍ 0.01 vs control). Treatment of the MCF-7 cells with Heregulin 1 (100 ng/ml) enhanced VE-cadherin complex protein dissociation (65% of ␥ catenin dissociated vs 42% in untreated MCF-7 cells at 12 hrs, p ⬍ 0.05). Treatment with trastuzumab (100ng/ml) blocked MCF-7 driven catenin dissociation by 50% at 24 hrs (p ⬍ 0.01 vs untreated MCF-7 cells). HER cells induced greater dissociation of catenins from VE cadherin compared to MCF-7 cells (81% of ␥ catenin dissociated vs 42% of untreated MCF-7 cells at 12 hrs, p ⬍ 0.05). Conclusions: We conclude that Her2 signaling in breast cancer cells induces EC retraction through dissociation of VE cadherin from
catenin proteins in the adherens junctions of EC monolayers. Because vascular destabilization is key to angioinvasion, transendothelial migration, and angiogenesis, stabilization of the adherens junction may limit the metastatic capabilities of breast cancer cells.
148. THE INTRATUMORAL MICROENVIRONMENT PERMITS INSULINOMA DEVELOPMENT DESPITE INITIAL PROTECTION BY AUTOREACTIVE CD8ⴙ T CELLS. Zwicker KA, Gratton K, Santamaria P, Demetrick D, Bathe OF; University of Calgary Because tumors express self-antigens that are shared by their tissue of origin, normal tolerogenic mechanisms preclude the generation of effective anti-tumor immunity. Autoimmunity emerges when immune tolerance fails, causing inappropriate activation of CD8⫹ T cells against autoantigen. We postulated that pathologically primed CD8⫹ T cells in individuals prone to autoimmunity are capable of protecting against tumor expressing the targeted autoantigens. We generated double transgenic 8.3-NOD-RIPTAg mice to test this hypothesis. The RIPTAg transgene confers a propensity to develop spontaneous insulinomas. In individuals with the 8.3-TCR transgene, the vast majority of CD8⫹ T cells recognize the autoantigen IGRP, which is expressed on normal and transformed (insulinoma) -cells. The two transgenes were bred onto the NOD background, which is associated with autoimmune diabetes mellitus (DM). Tumor incidence at 100 days was decreased in 8.3-NODRIPTAg mice (N⫽45; 69%) compared to NOD-RIPTAg mice (N ⫽ 41; 95%)(P⫽0.002), demonstrating that the 8.3-TCR transgene was protective against tumor. However, despite a delay in tumor development (Figure 1), including in individuals with overt autoimmune DM, virtually all mice ultimately developed tumor. Clonal T cell anergy or deletion were not responsible. CD8⫹ T cell priming could be demonstrated in peripancreatic lymph nodes, and this was particularly pronounced in tumor-bearing mice. CD8⫹ T cells were effectively recruited to tumor (Figure 2), although the CD8⫹ T cell infiltrate was generally more pronounced in the periphery of the tumor than in the center. MHC class I downregulation and antigen loss were not observed. The intratumoral inflammatory infiltrate of 8.3-NOD-RIPTAg mice was particularly rich in macrophages and CD4⫹ T cells (including CD4⫹CD25⫹ T cells). Preliminary studies suggest 8.3-TCR CD8⫹ T cell proliferation was impaired directly at the tumor site. Further studies are warranted to define why a vigorous CD8⫹ T cell response, which so effectively kills normal -cells, is inhibited by factors present in the tumor microenvironment.
149. PHASE IB TRIAL OF THE HER2/NEU PEPTIDE (E75) VACCINE FOR OPTIMAL DOSING IN NODE-NEGATIVE BREAST CANCER PATIENTS. E. A. Mittendorf 1, S. Khoo 1, Z. A. Dehqanzada 1, M. T. Hueman 1, C. E. Storrer 1, K. A. Harris 1, C. D. Shriver 2, S. Ponniah 1, G. E. Peoples, Jr. 2; 1 Clinical Breast Care Project, Immunology and Research Center, Uniformed Services University of the Health Sciences,
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
and its pathogenesis is incompletely understood. Methods: Potential factors involved in the pathogenesis of large unilateral (ⱖ 50g) and bilateral (ⱖ100g) nodular goiter were investigated including: age, gender, race, body mass index (BMI), family history of thyroid disease, insurance status, cigarette smoking, and alcohol use. Data were obtained from an IRB-approved thyroid database and retrospective chart review of consecutive patients operated on for nodular goiter from 1990-2004. Statistical significance of differences in patients with “large” versus “small” nodular goiter were determined using a Chi-square test and Mann-Whitney U test for nonparametric data and a T-test for parametric data. Results: Of the 460 patients operated on for nodular goiter, 106 (23%) were classified as “large”, 40 with unilateral enlargement ⱖ50g (mean 114 ⫾ 86g) and 66 with bilateral enlargement ⱖ100g (mean 169 ⫾ 88g) and 354 (77%) were classified as “small”, 165 unilateral (18 ⫾ 10g) and 189 bilateral (36 ⫾ 23g). Epidemiologic factors associated with large nodular goiter were: African American race (55% vs. 22%, p⬍0.001), increased BMI (mean 35 ⫾ 11 kg/m 2 vs. 29 ⫾ 8 kg/m 2, p⫽0.003), and no health insurance (32% vs. 19%, p⫽ 0.003). Alcohol use was less common in patients with large nodular goiter (33% vs. 49%, p⫽0.004). No significant differences were observed in age, gender, family history of thyroid disease or cigarette smoking (p⬎0.1). Conclusions: There is a predilection for large nodular goiter among African-American, obese and uninsured patients whereas alcohol use may be protective. Closer scrutiny of “at-risk” patients may help to identify specific causative factors and appropriate interventions for prevention of large nodular goiter and its sequelae.
147. HER2 SIGNALING IN BREAST CANCER INDUCES DISSOCIATION OF ADHERENS JUNCTION PROTEINS IN ENDOTHELIAL CELL MONOLAYERS. B. Carter 1, G. Small 2, M. Ward 3, J. Hahn 2; 1H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, 2University of Maryland School of Medicine, Baltimore, MD, 3Eastern Virginia Medical School, Norfolk, VA. Background: Her2 signaling imparts a metastatic advantage to breast cancer cells. In addition to increased proliferation, improved survival, and robust angiogenesis, early metastases in Her2 overexpressing breast cancer patients are postulated to be driven by angioinvasion. We have shown that Her2 signaling in breast cancer cells induces a paracrine-driven microvessel breakdown and endothelial cell (EC) retraction, critical steps in angioinvasion and transendothelial migration. Because Vascular Endothelial (VE) cadherin complexes of the adherens junctions regulate EC-EC adherence, we postulated that this EC retraction involves dissociation of the adherens junction proteins. Methods: Human iliac vein EC (HIVEC) monolayers were cocultured with estrogen-independent MCF-7 cells, or stable transfectant cells overexpressing Her2 6 fold (HER cells). Using immunoprecipitation with anti-VE cadherin antibody, we tested for complexed ␣, , and ␥ catenins in control or cocultured EC monolayers. We then manipulated Her2 signaling in these cells by stimulating Her2 signaling with Heregulin 1, or blockade of Her2 signaling with trastuzumab, and tested for VE cadherin/catenin dissociation. Results: MCF-7 cells induced a time-dependent dissociation of the catenins from VE cadherin, reaching 80% by 24 hrs (p ⬍ 0.01 vs control). Treatment of the MCF-7 cells with Heregulin 1 (100 ng/ml) enhanced VE-cadherin complex protein dissociation (65% of ␥ catenin dissociated vs 42% in untreated MCF-7 cells at 12 hrs, p ⬍ 0.05). Treatment with trastuzumab (100ng/ml) blocked MCF-7 driven catenin dissociation by 50% at 24 hrs (p ⬍ 0.01 vs untreated MCF-7 cells). HER cells induced greater dissociation of catenins from VE cadherin compared to MCF-7 cells (81% of ␥ catenin dissociated vs 42% of untreated MCF-7 cells at 12 hrs, p ⬍ 0.05). Conclusions: We conclude that Her2 signaling in breast cancer cells induces EC retraction through dissociation of VE cadherin from
catenin proteins in the adherens junctions of EC monolayers. Because vascular destabilization is key to angioinvasion, transendothelial migration, and angiogenesis, stabilization of the adherens junction may limit the metastatic capabilities of breast cancer cells.
148. THE INTRATUMORAL MICROENVIRONMENT PERMITS INSULINOMA DEVELOPMENT DESPITE INITIAL PROTECTION BY AUTOREACTIVE CD8ⴙ T CELLS. Zwicker KA, Gratton K, Santamaria P, Demetrick D, Bathe OF; University of Calgary Because tumors express self-antigens that are shared by their tissue of origin, normal tolerogenic mechanisms preclude the generation of effective anti-tumor immunity. Autoimmunity emerges when immune tolerance fails, causing inappropriate activation of CD8⫹ T cells against autoantigen. We postulated that pathologically primed CD8⫹ T cells in individuals prone to autoimmunity are capable of protecting against tumor expressing the targeted autoantigens. We generated double transgenic 8.3-NOD-RIPTAg mice to test this hypothesis. The RIPTAg transgene confers a propensity to develop spontaneous insulinomas. In individuals with the 8.3-TCR transgene, the vast majority of CD8⫹ T cells recognize the autoantigen IGRP, which is expressed on normal and transformed (insulinoma) -cells. The two transgenes were bred onto the NOD background, which is associated with autoimmune diabetes mellitus (DM). Tumor incidence at 100 days was decreased in 8.3-NODRIPTAg mice (N⫽45; 69%) compared to NOD-RIPTAg mice (N ⫽ 41; 95%)(P⫽0.002), demonstrating that the 8.3-TCR transgene was protective against tumor. However, despite a delay in tumor development (Figure 1), including in individuals with overt autoimmune DM, virtually all mice ultimately developed tumor. Clonal T cell anergy or deletion were not responsible. CD8⫹ T cell priming could be demonstrated in peripancreatic lymph nodes, and this was particularly pronounced in tumor-bearing mice. CD8⫹ T cells were effectively recruited to tumor (Figure 2), although the CD8⫹ T cell infiltrate was generally more pronounced in the periphery of the tumor than in the center. MHC class I downregulation and antigen loss were not observed. The intratumoral inflammatory infiltrate of 8.3-NOD-RIPTAg mice was particularly rich in macrophages and CD4⫹ T cells (including CD4⫹CD25⫹ T cells). Preliminary studies suggest 8.3-TCR CD8⫹ T cell proliferation was impaired directly at the tumor site. Further studies are warranted to define why a vigorous CD8⫹ T cell response, which so effectively kills normal -cells, is inhibited by factors present in the tumor microenvironment.
149. PHASE IB TRIAL OF THE HER2/NEU PEPTIDE (E75) VACCINE FOR OPTIMAL DOSING IN NODE-NEGATIVE BREAST CANCER PATIENTS. E. A. Mittendorf 1, S. Khoo 1, Z. A. Dehqanzada 1, M. T. Hueman 1, C. E. Storrer 1, K. A. Harris 1, C. D. Shriver 2, S. Ponniah 1, G. E. Peoples, Jr. 2; 1 Clinical Breast Care Project, Immunology and Research Center, Uniformed Services University of the Health Sciences,