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P6 Promoter methylation profiles and field defect in breast cancer
S14
Poster Session I. Biology
study, we therefore analyze the expression of two ERb isoforms correlates with ERa/PR status. Methods: Sixty samples of primary operable breast carcinomas were analyzed for ERa and PR protein levels and for mRNA expression of two ERb isoforms (ERb1 and ERbD5). ERa and PR proteins were measured by classical biochemical techniques, and ERb mRNAs were measured by real-time RT-PCR. Results: Tumors are divided in three groups according to relative level of mRNA for ERb1 and ERbD5. We found that there is no correlation of ERb1 mRNA expression with ERa and PR protein levels. We confirmed the existence of inverse correlation of ERbD5 with PR and of ERbD5 with ERa in the group of postmenopausal patients. In the subsets of tumors defined by ERa/PR status, we found that percentage of tumors which concomitantly expressed high levels of both transcripts, are parallel with those that do not respond to tamoxifen treatment. Conclusion: Inverse correlation of ERa with ERbD5 and PR with ERbD5 isoforms suggests that ERbD5 may have inhibitory effect on ERa activity in postmenopausal patients. In addition, we point out that determination of expression profiles of ERa and ERb isoforms in the defined groups of patient could be helpful in elucidating its involvement in tamoxifen resistance.
P5 Targeting heparan sulfation in breast cancer therapeutics G. Yip, G. Guo, B.-H. Bay. National University of Singapore, Department of Anatomy, Singapore, Singapore Heparan sulfate is a long, negatively charged glycosaminoglycan made up of repeating disaccharide subunits of glucuronic/iduronic acid and glucosamine. It is ubiquitously distributed on the mammalian cell surface and in the extracellular matrix. In recent years, much interest has been generated in the use of heparan sulfate as a biomarker and prognostic indicator in breast cancer. Furthermore, in vitro and in vivo studies suggest that expression of heparanase, an enzyme that degrades heparan sulfate, leads to increased growth and invasion of breast neoplasm. In this study, we examined the effects of inhibiting heparan sulfation on breast cancer cell behaviour in vitro. MCF-7 human breast cancer cells were maintained in culture and exposed to sodium chlorate, a competitive inhibitor of glycosaminoglycan sulfation. Chlorate treatment reduced growth of the cancer cells without inducing apoptosis. In addition, tumour cell adhesion to fibronectin and collagen was increased, accompanied by upregulation of focal adhesion complex formation. Significant decreases in cancer cell migration and invasion through Matrigel were also observed. These effects of chlorate treatment on MCF-7 cells could be prevented by adding “normally” sulfated heparan sulfate to the culture medium, showing that inhibition of heparan sulfation was the cause of the observed changes in cancer cell behaviour. Taken together, the results suggest that targeting heparan sulfation could be a useful strategy in breast cancer treatment.
P6 Promoter methylation profiles and field defect in breast cancer J.S. Lee1 , J.R. Kim1 , I.S. Yoon1 , T.J. Oh3 , S.W. An3 , K.S. Suh2 , E.S. Chang1 . 1 Chungnam national university collage of medicine, Department of surgery, Daejeon, South Korea, 2 Chungnam national uinversity college of medicine, Department of pathology, Daejeon, South Korea, 3 Genomictree, Inc., Daejeon, South Korea Goal: Aberrant DNA methylation of tumor suppressor genes has been accepted to be a common feature and early event in human cancer. The aim of this study is that analyze methylation profiles of 47 well established methylation-associated genes in accordance with various clinicopathological features in breast cancer and evaluate field cancerization effect with paired tumor adjacent tissues with different distance away from tumor tissues. Methods: Using HpaII-MspI-PCR, we determined methylation status of 47 genes in two breast cancer cell lines MCF7 and MDA-MB231 and 5 genes (APC, CALCA, CDH13, MTHFR, S100A2) were found to be methylated in at least 1 cell line. Methylation of all 5 genes was observed in tumor tissues with different methylation frequency. To examine field cancerization effect of 5 genes in grossly normal appearing tissues, we tested methylation status in paired tumor adjacent tissues with different distance away from tumor tissues in two patients.
Thursday, 15 March 2007 Results: Methylation frequency of five genes is determined as follows: APC (55.2%), MTHFR (75.9%), CALCA (85.7%), CDH13 (93.1%) and S00A2 (96.6%), respectively in breast cancer. The results represent that a panel of these 5 genes will be useful for detection of breast cancer. CALCA gene was methylated in normal appearing tissues even 8 cm away from the tumor in 2 of 2 cases. Meanwhile, methylation of three genes (CDH13, MTHFR and S100A2) was detected 6 cm and 8 cm in 1 and 2 of 2 cases, respectively. However, the methylation status of 5 genes has no statistical significance (chi-square test) in accordance with various clinicopathological parameters (stage, grade, lymph node metastasis status). Conclusions: Methylation of APC, MTHFR, CALCA, CDH13 and S00A2 will be useful for detection of breast cancer and field defect was observed in normal appearing tissues. Detection of this abnormality may be useful in risk assessment for breast cancer.
P7 The CHEK2 1100delC mutation is not present in Korean patients with breast cancer tested for BRCA1 and BRCA2 mutation D.H. Choi1 , H.Y. Cho2 , M.H. Lee3 , H.S. Park4 , S.H. Ahn5 , B.G. Haffty6 . 1 Soonchunhyang University Hospital, Department of Radiation Oncology, Seoul, South Korea, 2 LabGenomics Co., Ltd., Clinical Research Institute, Seoul, South Korea, 3 Soonchunhyang University Hospital, Department of Surgery, Seoul, South Korea, 4 Soonchunhyang University Hospital, Department of Hemato-oncology, Seoul, South Korea, 5 Asan Medical Center, Department of Surgery, Seoul, South Korea, 6 Cancer Institute of New Jersey, Department of Radiation Oncology, New Brunswick, NJ, USA Background: The sequence variant in the cell cycle checkpoint kinase 2 (CHEK2 1100delC) has been associated with a increased risk for breast cancer in women carrying this mutation. It is a low penetrance breast cancer susceptibility allele, frequently observed in patients with family history of breast cancer and/or young age and the frequency varied according to race or ethnicity. In this study, we evaluated the significance of CHEK2 1100delC in predisposition to breast cancer by assessing its frequency in a material of 493 Korean breast cancer patients. Methods: Four hundred and ninty three Korean patients with breast cancer from the two university hospital were selected for this study. All the patients had been screened for BRCA1 and BRCA2 mutations and 42 patients had deleterious mutations. Of the 493 patients entered for this study, 273 were early-onset and/or bilateral, 169 family history of breast cancer, 33 multiple organ cancers, 5 male breast cancer, 13 no risk factors. Mutation detection of CHEK2 1100delC was based upon analysis of primer extension products generated for previously amplified genomic DNA using a chip based MALDI-TOP mass spectrometry platform (Sequenom, Inc., CA). After overall measurement automatically, assays which had bad peaks were checked again manually. Results: None of the 493 Korean patients with breast cancer who were candidate for BRCA1 and BRCA2 test carried the 1100delC mutation. As expected, the mutation confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutation. However, this mutation is not present in Korean patients with family history of breast cancer and early-onset breast cancer, observed in Caucasians with limited frequency. Conclusions: In the previous studies, we observed higher or comparable prevalence of BRCA1 and BRCA2 mutations in Korean patients with breast cancer compared to Caucasian breast cancer population. In our present study, we evaluated role of CHEK2 1100delC as a susceptibility mutation of breast cancer in the Korean population. However our results suggest that this mutation is absent or may be very infrequent in Korean patients with breast cancer who have high risk of BRCA1 and BRCA2 mutation, making its screening irrelevant from the practical point of view.
P8 Potentiation of erythropoietin (EPO) action by membrane steroid receptor agonists V. Pelekanou, M. Kampa, E. Castanas. University of Crete, School of Medicine, Experimental Endocrinology, Heraklion, Greece Background: Recent studies revealed an emerging role of growth factors and their cognitive receptors on breast cancer growth regulation. Among them, erythropoietin (EPO) plays a pivotall role. Additionally, membrane estrogen (mER) and androgen (mAR) receptor agonists, were found to control cell proliferation and apoptosis. As both EPO and mER- and mARagonists share common intracellular signaling pathways and are usually
Poster Session I. Biology
study, we therefore analyze the expression of two ERb isoforms correlates with ERa/PR status. Methods: Sixty samples of primary operable breast carcinomas were analyzed for ERa and PR protein levels and for mRNA expression of two ERb isoforms (ERb1 and ERbD5). ERa and PR proteins were measured by classical biochemical techniques, and ERb mRNAs were measured by real-time RT-PCR. Results: Tumors are divided in three groups according to relative level of mRNA for ERb1 and ERbD5. We found that there is no correlation of ERb1 mRNA expression with ERa and PR protein levels. We confirmed the existence of inverse correlation of ERbD5 with PR and of ERbD5 with ERa in the group of postmenopausal patients. In the subsets of tumors defined by ERa/PR status, we found that percentage of tumors which concomitantly expressed high levels of both transcripts, are parallel with those that do not respond to tamoxifen treatment. Conclusion: Inverse correlation of ERa with ERbD5 and PR with ERbD5 isoforms suggests that ERbD5 may have inhibitory effect on ERa activity in postmenopausal patients. In addition, we point out that determination of expression profiles of ERa and ERb isoforms in the defined groups of patient could be helpful in elucidating its involvement in tamoxifen resistance.
P5 Targeting heparan sulfation in breast cancer therapeutics G. Yip, G. Guo, B.-H. Bay. National University of Singapore, Department of Anatomy, Singapore, Singapore Heparan sulfate is a long, negatively charged glycosaminoglycan made up of repeating disaccharide subunits of glucuronic/iduronic acid and glucosamine. It is ubiquitously distributed on the mammalian cell surface and in the extracellular matrix. In recent years, much interest has been generated in the use of heparan sulfate as a biomarker and prognostic indicator in breast cancer. Furthermore, in vitro and in vivo studies suggest that expression of heparanase, an enzyme that degrades heparan sulfate, leads to increased growth and invasion of breast neoplasm. In this study, we examined the effects of inhibiting heparan sulfation on breast cancer cell behaviour in vitro. MCF-7 human breast cancer cells were maintained in culture and exposed to sodium chlorate, a competitive inhibitor of glycosaminoglycan sulfation. Chlorate treatment reduced growth of the cancer cells without inducing apoptosis. In addition, tumour cell adhesion to fibronectin and collagen was increased, accompanied by upregulation of focal adhesion complex formation. Significant decreases in cancer cell migration and invasion through Matrigel were also observed. These effects of chlorate treatment on MCF-7 cells could be prevented by adding “normally” sulfated heparan sulfate to the culture medium, showing that inhibition of heparan sulfation was the cause of the observed changes in cancer cell behaviour. Taken together, the results suggest that targeting heparan sulfation could be a useful strategy in breast cancer treatment.
P6 Promoter methylation profiles and field defect in breast cancer J.S. Lee1 , J.R. Kim1 , I.S. Yoon1 , T.J. Oh3 , S.W. An3 , K.S. Suh2 , E.S. Chang1 . 1 Chungnam national university collage of medicine, Department of surgery, Daejeon, South Korea, 2 Chungnam national uinversity college of medicine, Department of pathology, Daejeon, South Korea, 3 Genomictree, Inc., Daejeon, South Korea Goal: Aberrant DNA methylation of tumor suppressor genes has been accepted to be a common feature and early event in human cancer. The aim of this study is that analyze methylation profiles of 47 well established methylation-associated genes in accordance with various clinicopathological features in breast cancer and evaluate field cancerization effect with paired tumor adjacent tissues with different distance away from tumor tissues. Methods: Using HpaII-MspI-PCR, we determined methylation status of 47 genes in two breast cancer cell lines MCF7 and MDA-MB231 and 5 genes (APC, CALCA, CDH13, MTHFR, S100A2) were found to be methylated in at least 1 cell line. Methylation of all 5 genes was observed in tumor tissues with different methylation frequency. To examine field cancerization effect of 5 genes in grossly normal appearing tissues, we tested methylation status in paired tumor adjacent tissues with different distance away from tumor tissues in two patients.
Thursday, 15 March 2007 Results: Methylation frequency of five genes is determined as follows: APC (55.2%), MTHFR (75.9%), CALCA (85.7%), CDH13 (93.1%) and S00A2 (96.6%), respectively in breast cancer. The results represent that a panel of these 5 genes will be useful for detection of breast cancer. CALCA gene was methylated in normal appearing tissues even 8 cm away from the tumor in 2 of 2 cases. Meanwhile, methylation of three genes (CDH13, MTHFR and S100A2) was detected 6 cm and 8 cm in 1 and 2 of 2 cases, respectively. However, the methylation status of 5 genes has no statistical significance (chi-square test) in accordance with various clinicopathological parameters (stage, grade, lymph node metastasis status). Conclusions: Methylation of APC, MTHFR, CALCA, CDH13 and S00A2 will be useful for detection of breast cancer and field defect was observed in normal appearing tissues. Detection of this abnormality may be useful in risk assessment for breast cancer.
P7 The CHEK2 1100delC mutation is not present in Korean patients with breast cancer tested for BRCA1 and BRCA2 mutation D.H. Choi1 , H.Y. Cho2 , M.H. Lee3 , H.S. Park4 , S.H. Ahn5 , B.G. Haffty6 . 1 Soonchunhyang University Hospital, Department of Radiation Oncology, Seoul, South Korea, 2 LabGenomics Co., Ltd., Clinical Research Institute, Seoul, South Korea, 3 Soonchunhyang University Hospital, Department of Surgery, Seoul, South Korea, 4 Soonchunhyang University Hospital, Department of Hemato-oncology, Seoul, South Korea, 5 Asan Medical Center, Department of Surgery, Seoul, South Korea, 6 Cancer Institute of New Jersey, Department of Radiation Oncology, New Brunswick, NJ, USA Background: The sequence variant in the cell cycle checkpoint kinase 2 (CHEK2 1100delC) has been associated with a increased risk for breast cancer in women carrying this mutation. It is a low penetrance breast cancer susceptibility allele, frequently observed in patients with family history of breast cancer and/or young age and the frequency varied according to race or ethnicity. In this study, we evaluated the significance of CHEK2 1100delC in predisposition to breast cancer by assessing its frequency in a material of 493 Korean breast cancer patients. Methods: Four hundred and ninty three Korean patients with breast cancer from the two university hospital were selected for this study. All the patients had been screened for BRCA1 and BRCA2 mutations and 42 patients had deleterious mutations. Of the 493 patients entered for this study, 273 were early-onset and/or bilateral, 169 family history of breast cancer, 33 multiple organ cancers, 5 male breast cancer, 13 no risk factors. Mutation detection of CHEK2 1100delC was based upon analysis of primer extension products generated for previously amplified genomic DNA using a chip based MALDI-TOP mass spectrometry platform (Sequenom, Inc., CA). After overall measurement automatically, assays which had bad peaks were checked again manually. Results: None of the 493 Korean patients with breast cancer who were candidate for BRCA1 and BRCA2 test carried the 1100delC mutation. As expected, the mutation confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutation. However, this mutation is not present in Korean patients with family history of breast cancer and early-onset breast cancer, observed in Caucasians with limited frequency. Conclusions: In the previous studies, we observed higher or comparable prevalence of BRCA1 and BRCA2 mutations in Korean patients with breast cancer compared to Caucasian breast cancer population. In our present study, we evaluated role of CHEK2 1100delC as a susceptibility mutation of breast cancer in the Korean population. However our results suggest that this mutation is absent or may be very infrequent in Korean patients with breast cancer who have high risk of BRCA1 and BRCA2 mutation, making its screening irrelevant from the practical point of view.
P8 Potentiation of erythropoietin (EPO) action by membrane steroid receptor agonists V. Pelekanou, M. Kampa, E. Castanas. University of Crete, School of Medicine, Experimental Endocrinology, Heraklion, Greece Background: Recent studies revealed an emerging role of growth factors and their cognitive receptors on breast cancer growth regulation. Among them, erythropoietin (EPO) plays a pivotall role. Additionally, membrane estrogen (mER) and androgen (mAR) receptor agonists, were found to control cell proliferation and apoptosis. As both EPO and mER- and mARagonists share common intracellular signaling pathways and are usually