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0004 DNA hypermethylation in breast cancer and its association with clinicopathological features
The Breast 18 (2009) Supplement 1
S19
Poster Session I
Thursday, 12 March 2009
Biology, experimental 0001
Thymidylate synthase: Tumor promoter and oncogene in sporadic breast tumorigenesis?
B. Barclay1 , D. Murray2 . 1 CEO, Planet Biotech, St Albert, 2 Exptl Oncology, Cross Cancer Inst., Edmonton, Canada Goals: To evaluate the evidence for a potential role for thymidylate synthase (TYMS) as tumor suppressor and oncogene in the etiology of sporadic breast cancer (SBC). Methods: Reviewed over 200 publications cited in Pubmed or Scopus over a 4 1/2 year period and weekly seminars to discuss findings. Results: There is convincing evidence that TYMS activity may serve not only as a useful diagnostic and prognostic indicator in SBC but that altered TYMS expression may be causally related to SBC tumorigenesis in many cases. Conclusion: We suggest here a role for TYMS in the etiology of SBC. The essence of the hypothesis is that thymine nucleotide stress (TNS), caused by interactions between polymorphisms in genes involved in onecarbon metabolism (TYMS, MTHF), nutritional deficiencies (folate, B12) and exposure to environmental toxins (malathion, ethanol) results in the accumulation of gross chromosomal rearrangements (GCRs) throughout the genome of mitotic breast stem cells. GCRs at chromosome band 18p11.32, that include the TYMS gene, create chromosomal (deletions, 18p loss) and extrachromosomal (episomes, large palindromes) cytogenic drivers that alter TYMS activity, stabilizing an endogenous TNS phenotype. TYMS acts as a tumor suppressor in excess by repressing the p53 gene and the network of numerous genes under p53 control. Altered TYMS activity (both limitation and excess) greatly increases rates of mitotic recombination and mutation for both nuclear and mitochondrial genes, giving rise to an early heterogeneous and genetically unstable tumor cell mass. Ongoing TNS causes GCRs and mutations at numerous other tumor suppressor and oncogene loci that produces the genomic variation upon which selection forces act to channel SBC tumor cells into histopathological subtypes and driving later events in disease progression. If supported by critical experiments our hypothesis has implications for SBC diagnosis, treatment and prevention.
0002
Adiponectin differentially affects gene expression in human mammary epithelial cells and breast cancer cells
C.R. Lattrich1 , O. Treeck1 , O. Ortmann1 . 1 Gynaecology, University Hospital Regensburg, Regensburg, Germany Goals: Breast cancer risk is inversely associated with serum levels of adiponectin. Adiponectin is reported to exert direct antitumoral effects by inhibition of cell proliferation and increase of apoptosis of breast cancer cells. We studied the molecular mechanisms underlying the antitumoral action of adiponectin. Methods: We compared the effect of adiponectin on gene expression in a breast cancer cell line and a non-tumorigenic human mammary epithelial cell line. For this purpose, we treated MCF-7 breast cancer cells and MCF-10A immortalized mammary epithelial cells with adiponectin (10 g/ml) for 48h. Adiponectin effect on gene expression was analyzed by means of DNA microarrays and the data was confirmed by real time RT-PCR analysis of expression of estrogen receptor (ER) a, ERb1, ERb2, ERb5, caspase 1, TR2 orphan nuclear hormone receptor, adiponectin receptor 1 and ubiquitin specific peptidase (USP). Results: Cell growth of MCF-10A mammary epithelial cells was reduced after treatment with adiponectin, but not cell growth of MCF-7 breast cancer cells. In MCF-7 breast cancer cells about 6-fold increased ERb2 transcript
levels were observed, whereas no effect in regulation of other analyzed genes was detected. This is in contrast to MCF-10A mammary epithelial cells where adiponectin upregulated mRNA levels of ERb2 and USP more than 5-fold and transcript levels of ERb5, caspase 1 and TR2 were elevated about 2-fold. Conclusion: These findings suggest that adiponectin has different effects on breast cancer and mammary epithelial cells. Upregulation of ERb isoforms and caspase 1, which both are known to affect growth or apoptosis of breast cancer cells, could be at least one molecular mechanism underlying the antitumoral action of adiponectin. This study was funded by an intramural research grant, the ReForM C “Metabolic effects on the development of chronic diseases in sex-hormone sensitive organs” of the University Hospital Regensburg.
0003
Detection of methylation in the CpG islands of the RASSF1A gene promoter in breast cancer (BC)
K.A. Desiris1 , S. Voyatzi2 , P. Stravoravdi2 , A. Kiziridou3 , D. Paikos4 , I. Tremmas1 , E. Stergiou5 , G. Simbilidis1 . 1 2nd Surgical, 2 Research, 3 Pathology, 4 Gastroenterology Oncology, 5 2nd Oncology, Theagenio Cancer Hospital, Thessaloniki, Greece Goals: Epigenetic alterations, such as DNA methylation, are involved in the onset and progression of BC. DNA methylation causes loss or gain of genes expression. Therefore, genome-wide screens are being actively pursued, in order to establish new predictive and prognostic biomarkers in BC. We examined the methylation status of the RASSF1A (Ras-association domain family 1, isoform A) gene involved in the regulation of transcription, angiogenesis, signalling, cell adhesion and apoptosis. Methods: Randomly selected specimens from 37 pts with breast lesions were obtained. The specimens were evaluated for methylation in the CpG islands of the RASSF1A gene. Genomic DNA extracted from archive formalin-fixed paraffin-embedded tumor tissues. DNA methylation was determined by chemical modification of DNA with sodium bisulphate and subsequent double “hot start” Methylation-Specific PCR (MSP), followed by detection on agarose gel. Results on promoter methylation of the gene were correlated with clinicopathological parameters. Results: Out of 37 pts with breast lesions 26 had BC. Promoter methylation of RASSF1A gene was observed in 15/26 pts. The analysis correlating the gene methylation status with clinicopathological features revealed an association with tumor size and ER status. RASSF1A gene was methylated in 12/15 pts with T2 tumors and in 12/15 pts with ER (+) status. However, T2 tumors and ER (+) status together was observed in 9/15 pts. Age, histological types and lymph node metastases were not correlated with methylation status. In benign lesions promoter methylation was present in 5/11 pts. Conclusion: RASSF1A gene is frequently methylated in both benign and malignant breast lesions. The association of methylation of RASSF1A gene with tumor size and ER status is noteworthy. The last observation may be of significance in the evaluation of targeted therapy resistance related to ER status in BC. The small number of pts does not allow us to confirm the exact role of RASSF1A gene methylation in BC yet. Larger studies are required in order to assess if this epigenetic alteration points out a new BC marker, which could be helpful in prognosis and potential biological therapeutic strategies.
0004
DNA hypermethylation in breast cancer and its association with clinicopathological features
F. Chen1 , F. Ou-Yang1 , S. Yang2 , S. Chang3 , M. Hou1 . 1 Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, 2 Department of Pathology, Kaohsiung Medical University Hospital, 3 Department of Public Health, Kaohsiung Medical University, Kaohsiung, Taiwan Goals: Aberrant hypermethylation of gene promoter regions is one of the mechanisms for inactivation of tumor suppressor genes in breast cancer.
S20
Poster Session I. Biology, experimental
We investigated whether hypermethylation identifies breast cancers with distinctive clinical and pathological features. Methods: We evaluated the methylation of RASSF1A, SYK, DAPK, ER-a, BRCA1, CCND2, p16, TMS1, ITIH5 and APC in 95 breast carcinomas using methylation-specific PCR. Results: Methylation frequencies ranged from 9.1% for SYK to 95.8% for RASSF1A. For all age patients, tumors with CCND2 methylation were associated with triple negative (OR = 1.34, CI: 1.165−1.541). However, tumors of patient’s age younger than 50 with BRCA1 methylation were associated with ER (OR = 7, CI: 1.277−38.358) or PR negative (OR = 11.07, CI: 1.245–98.598). And DAPK methylation was associated p53 mutation (OR = 2.714, CI: 1.507−4.890). Nevertheless, for patient’s age older than 50, tumors with ITIH5 and BRCA1 methylation were associated with more advanced t stage (t0−1/t2−4, ITIH5 OR= 0.283, CI= 0.088−0.910; BRCA1 OR= 0.3, CI= 0.095−0.948) and the methylation of APC was associated with PR negative (OR = 4.52, CI= 1.203−16.967). Conclusion: Our data suggests that gene methylation may be linked to various pathological features of breast cancer. Furthermore, the different methylation profile in tumors of different age patient population may imply different carcinogenesis pathway, however, this requires confirmation in larger studies.
0005
Epigallocatechin-3-gallate decreases VASP expression and MCF-7 cells invasion by inhibiting Rac1 activity
Y. Zhang1 , G. Han2 , B. Fan3 , L. Wei4 . 1 Department of Breast and Thyroid Gland Surgery, 2 The First College of Clinical Medicine of Wuhan University, Renmin Hospital of Wuhan University, 3 The Second College of Clinical Medicine of Wuhan University, Zhongnan Hospital of Wuhan University, 4 Department of Pathophysiology, Basic Medical School of Wuhan University, Wuhan, China Goals: To investigate the influence of green tea (−)-epigallocatechin-3gallate (EGCG) on migration and invasion of MCF-7 cells and to identify the altered signalling pathway(s) underlying the response to EGCG exposure. Methods: MCF-7 cells were treated with EGCG (0, 10, 25, 50, 100 mg/ml) for 0 h, 6 h, 12 h, 18 h, 24 h and 30 h, and then expression of vasodilator-stimulated phosphoprotein (VASP) and total Rac1 in cells was detected by RT-PCR and Western Blotting, respectively, at mRNA and protein levels and the activities of Rac1 were examined by GST Pulldown assay, accordingly, cells migration and invasion were analyzed by wound-healing assay and transwell migration/invasion assays. Meanwhile, Rac1 and VASP specific siRNA oligos were applied to knock-down the expression of Rac1 and/or VASP in cells to observe altered signalling pathway(s) underlying the response to EGCG exposure. Results: In a concentration-dependent manner, EGCG decreased the migratory and invasive potential of MCF-7 cells with concomitant downregulated VASP expression and Rac1 activity. By using VASP and Rac1 specific siRNAs to block the expression of VASP and Rac1 in MCF-7 cells which were treated with epidermal growth factor (EGF) previously, we demonstrated that the regulation of cells migration and invasion was associated with Rac1 activity and VASP expression. In addition, siRNA mediated knock-down of Rac1 decreased the amount of VASP expression at mRNA level while VASP specified siRNA revealed no effect on the expression of Rac1 in MCF-7 cells. Conclusion: The inhibitive effect of EGCG on MCF-7 cells migration and invasion may be produced by down-regulated of VASP expression via Rac1 pathway.
0006
Development of novel breast cancer stem cell therapy targeting hedgehog signaling pathway
H. Tanaka1 , M. Nakamura1 , M. Kubo2 , M. Kai1 , M. Katano1 . 1 Cancer Therapy and Research, 2 Surgery and Oncology, Graduate School of Medicine, Kyushu University, Fukuoka, Japan Goals: Hedgehog (Hh) signalling pathway is crucial for growth and patterning during embryonic development. Previously, we reported that this pathway is highly activated in human breast cancer compared with normal tissue, and we can see reports suggesting that the morphogenic signals including Hh signal contribute to the survival and maintenance of cancer stem cells (CSCs) in various types of carcinomas. So, we hypothesized that the Hh signalling pathway might maintain the breast CSCs. Our goal
Thursday, 12 March 2009 is to reveal that the Hh pathway is essential for maintaining breast CSCs and might represent a valuable target for breast cancer stem cell therapy for the first time. Methods: Three human breast cancer cell lines were maintained. Flow Cytometry analysis and sorting were performed to detect the CD24−/low CD44+ population cells and the side population (SP) cells, reported to include much breast CSCs. To confirm the high tumorigenicity and multipotency of them, we performed xenograft tumor experiments in NOD/SCID mice. To assess the expression levels of Hh signalling components, real-time RT-PCR and fluorescent immunostaining were performed. Cyclopamine, which is an inhibitor of Hh pathway, and si-RNA against Gli1, a trans-activator of the Hh signalling pathway were used to inhibit this pathway. Furthermore we created peptides with particular affinity for Patched1 (Ptch1), a transmembrane receptor of Hh ligands. These peptides were used treatment experiment in NOD/SCID mice model. Results: The breast cancer cell lines contained 10−25% of CD24−/low CD44+ population and 1−4% of SP, and these two populations were overlapped with each other. These populations were resistant to anticancer drugs, more tumorigenic than other populations, and had multipotency in xenograft transplantation model. Components of the Hh pathway were more highly expressed in these populations compared with others, and inhibition of Hh signalling activity suppressed the tumorigenicity and the proliferation of both populations. The significance of Hh signalling activity was also confirmed by the effect of si-RNA against Gli1 and Ptch1 affinity peptides in vivo and vitro. Conclusion: The Hh signalling pathway is essential for the survival and maintenance of the breast CSCs, and that this pathway might represent a new candidate for breast cancer therapy targeting cancer stem cells.
0007
Importance of estrogen metabolites (2-OH/16a-OH) in the pathogenesis of breast, ovarian and endometrial cancer in postmenopausal women
I.B. Antonova1 , L.A. Ashrafyan1 , I.O. Basova1 . 1 Oncogynecology, Russian Scientific Center of Radiology, Moscow, Russian Federation Goals: In Russia breast cancer (BC) heads the list of reproductive organs new growth with 45−50 in 100000. The number of endometrial cancer (EC) and ovarian cancer (OC) cases has increased and accounts 12.8 and 10.2 in 10,0000. Conception about nosotropic inhomogeneity of such cancerous process, existence of hormone-dependent and autonomic types has been proved. However numerous examinations confirm that most likely there is no clear-cut line of demarcation between types of cancer. The object of the work was to study endocrinological (metabolite of estrogens, estrogenic receptors) component in process of BC, EC and OC during postmenopause. Methods: The main group was of 40 invasive breast cancer patients, 60 endometrial cancer patients of I−III stages, 60 ovarian cancer patients and a control group of 30 patients without signs of pathology. All patients were postmenopausal women. Results: Most of patients have metabolic syndrome. Also BC, EC and OC is characterized by high level of production of aggressive metabolite 16a-OH (BC: 6.98; EC: 6.66; OC: 8.69 ng/ml) and low expression of 2-OH (BC: 7.12; EC: 6.82, OC: 8.25 ng/ml), ratio of metabolites (2-OH/16a-OH) on the level BC: 1.02 (p < 0.05), EC: 1.16 (p < 0.05), OC: 1.04 (p < 0.05). And OR for control group was 3.5. All EC samples have expression of mRNK of estrogenic receptors. Conclusion: Preponderance of endocrinological aspect in process became visible in BC, EC and OC model. Our analysis using pretreatment urinary samples suggested that a lower 2-OHE/16-OHE was associated with high risk of postmenopausal breast, ovarian and endometrial cancer.
0008
Epigenetic field cancerization of MTHFR gene in breast carcinomas
J. Lee1 , J. Kim1 , K. Suh2 , T. Oh3 , S. An3 , J. Lee4 , B. Choi5 , E. Chang1 . 1 Department of Surgery, 2 Department of pathology, Chungnam National University Hospital, 3 Research and Development Center of Genomic tree, 4 Department of Dermatology, 5 Department of Radiology, Chungnam National University Hospital, Daejeon, South Korea
Goals: Gene promoter hypermethylation has been demonstrated to be a principal mechanism correlating, in cancer cells, to specific gene silencing.
S19
Poster Session I
Thursday, 12 March 2009
Biology, experimental 0001
Thymidylate synthase: Tumor promoter and oncogene in sporadic breast tumorigenesis?
B. Barclay1 , D. Murray2 . 1 CEO, Planet Biotech, St Albert, 2 Exptl Oncology, Cross Cancer Inst., Edmonton, Canada Goals: To evaluate the evidence for a potential role for thymidylate synthase (TYMS) as tumor suppressor and oncogene in the etiology of sporadic breast cancer (SBC). Methods: Reviewed over 200 publications cited in Pubmed or Scopus over a 4 1/2 year period and weekly seminars to discuss findings. Results: There is convincing evidence that TYMS activity may serve not only as a useful diagnostic and prognostic indicator in SBC but that altered TYMS expression may be causally related to SBC tumorigenesis in many cases. Conclusion: We suggest here a role for TYMS in the etiology of SBC. The essence of the hypothesis is that thymine nucleotide stress (TNS), caused by interactions between polymorphisms in genes involved in onecarbon metabolism (TYMS, MTHF), nutritional deficiencies (folate, B12) and exposure to environmental toxins (malathion, ethanol) results in the accumulation of gross chromosomal rearrangements (GCRs) throughout the genome of mitotic breast stem cells. GCRs at chromosome band 18p11.32, that include the TYMS gene, create chromosomal (deletions, 18p loss) and extrachromosomal (episomes, large palindromes) cytogenic drivers that alter TYMS activity, stabilizing an endogenous TNS phenotype. TYMS acts as a tumor suppressor in excess by repressing the p53 gene and the network of numerous genes under p53 control. Altered TYMS activity (both limitation and excess) greatly increases rates of mitotic recombination and mutation for both nuclear and mitochondrial genes, giving rise to an early heterogeneous and genetically unstable tumor cell mass. Ongoing TNS causes GCRs and mutations at numerous other tumor suppressor and oncogene loci that produces the genomic variation upon which selection forces act to channel SBC tumor cells into histopathological subtypes and driving later events in disease progression. If supported by critical experiments our hypothesis has implications for SBC diagnosis, treatment and prevention.
0002
Adiponectin differentially affects gene expression in human mammary epithelial cells and breast cancer cells
C.R. Lattrich1 , O. Treeck1 , O. Ortmann1 . 1 Gynaecology, University Hospital Regensburg, Regensburg, Germany Goals: Breast cancer risk is inversely associated with serum levels of adiponectin. Adiponectin is reported to exert direct antitumoral effects by inhibition of cell proliferation and increase of apoptosis of breast cancer cells. We studied the molecular mechanisms underlying the antitumoral action of adiponectin. Methods: We compared the effect of adiponectin on gene expression in a breast cancer cell line and a non-tumorigenic human mammary epithelial cell line. For this purpose, we treated MCF-7 breast cancer cells and MCF-10A immortalized mammary epithelial cells with adiponectin (10 g/ml) for 48h. Adiponectin effect on gene expression was analyzed by means of DNA microarrays and the data was confirmed by real time RT-PCR analysis of expression of estrogen receptor (ER) a, ERb1, ERb2, ERb5, caspase 1, TR2 orphan nuclear hormone receptor, adiponectin receptor 1 and ubiquitin specific peptidase (USP). Results: Cell growth of MCF-10A mammary epithelial cells was reduced after treatment with adiponectin, but not cell growth of MCF-7 breast cancer cells. In MCF-7 breast cancer cells about 6-fold increased ERb2 transcript
levels were observed, whereas no effect in regulation of other analyzed genes was detected. This is in contrast to MCF-10A mammary epithelial cells where adiponectin upregulated mRNA levels of ERb2 and USP more than 5-fold and transcript levels of ERb5, caspase 1 and TR2 were elevated about 2-fold. Conclusion: These findings suggest that adiponectin has different effects on breast cancer and mammary epithelial cells. Upregulation of ERb isoforms and caspase 1, which both are known to affect growth or apoptosis of breast cancer cells, could be at least one molecular mechanism underlying the antitumoral action of adiponectin. This study was funded by an intramural research grant, the ReForM C “Metabolic effects on the development of chronic diseases in sex-hormone sensitive organs” of the University Hospital Regensburg.
0003
Detection of methylation in the CpG islands of the RASSF1A gene promoter in breast cancer (BC)
K.A. Desiris1 , S. Voyatzi2 , P. Stravoravdi2 , A. Kiziridou3 , D. Paikos4 , I. Tremmas1 , E. Stergiou5 , G. Simbilidis1 . 1 2nd Surgical, 2 Research, 3 Pathology, 4 Gastroenterology Oncology, 5 2nd Oncology, Theagenio Cancer Hospital, Thessaloniki, Greece Goals: Epigenetic alterations, such as DNA methylation, are involved in the onset and progression of BC. DNA methylation causes loss or gain of genes expression. Therefore, genome-wide screens are being actively pursued, in order to establish new predictive and prognostic biomarkers in BC. We examined the methylation status of the RASSF1A (Ras-association domain family 1, isoform A) gene involved in the regulation of transcription, angiogenesis, signalling, cell adhesion and apoptosis. Methods: Randomly selected specimens from 37 pts with breast lesions were obtained. The specimens were evaluated for methylation in the CpG islands of the RASSF1A gene. Genomic DNA extracted from archive formalin-fixed paraffin-embedded tumor tissues. DNA methylation was determined by chemical modification of DNA with sodium bisulphate and subsequent double “hot start” Methylation-Specific PCR (MSP), followed by detection on agarose gel. Results on promoter methylation of the gene were correlated with clinicopathological parameters. Results: Out of 37 pts with breast lesions 26 had BC. Promoter methylation of RASSF1A gene was observed in 15/26 pts. The analysis correlating the gene methylation status with clinicopathological features revealed an association with tumor size and ER status. RASSF1A gene was methylated in 12/15 pts with T2 tumors and in 12/15 pts with ER (+) status. However, T2 tumors and ER (+) status together was observed in 9/15 pts. Age, histological types and lymph node metastases were not correlated with methylation status. In benign lesions promoter methylation was present in 5/11 pts. Conclusion: RASSF1A gene is frequently methylated in both benign and malignant breast lesions. The association of methylation of RASSF1A gene with tumor size and ER status is noteworthy. The last observation may be of significance in the evaluation of targeted therapy resistance related to ER status in BC. The small number of pts does not allow us to confirm the exact role of RASSF1A gene methylation in BC yet. Larger studies are required in order to assess if this epigenetic alteration points out a new BC marker, which could be helpful in prognosis and potential biological therapeutic strategies.
0004
DNA hypermethylation in breast cancer and its association with clinicopathological features
F. Chen1 , F. Ou-Yang1 , S. Yang2 , S. Chang3 , M. Hou1 . 1 Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, 2 Department of Pathology, Kaohsiung Medical University Hospital, 3 Department of Public Health, Kaohsiung Medical University, Kaohsiung, Taiwan Goals: Aberrant hypermethylation of gene promoter regions is one of the mechanisms for inactivation of tumor suppressor genes in breast cancer.
S20
Poster Session I. Biology, experimental
We investigated whether hypermethylation identifies breast cancers with distinctive clinical and pathological features. Methods: We evaluated the methylation of RASSF1A, SYK, DAPK, ER-a, BRCA1, CCND2, p16, TMS1, ITIH5 and APC in 95 breast carcinomas using methylation-specific PCR. Results: Methylation frequencies ranged from 9.1% for SYK to 95.8% for RASSF1A. For all age patients, tumors with CCND2 methylation were associated with triple negative (OR = 1.34, CI: 1.165−1.541). However, tumors of patient’s age younger than 50 with BRCA1 methylation were associated with ER (OR = 7, CI: 1.277−38.358) or PR negative (OR = 11.07, CI: 1.245–98.598). And DAPK methylation was associated p53 mutation (OR = 2.714, CI: 1.507−4.890). Nevertheless, for patient’s age older than 50, tumors with ITIH5 and BRCA1 methylation were associated with more advanced t stage (t0−1/t2−4, ITIH5 OR= 0.283, CI= 0.088−0.910; BRCA1 OR= 0.3, CI= 0.095−0.948) and the methylation of APC was associated with PR negative (OR = 4.52, CI= 1.203−16.967). Conclusion: Our data suggests that gene methylation may be linked to various pathological features of breast cancer. Furthermore, the different methylation profile in tumors of different age patient population may imply different carcinogenesis pathway, however, this requires confirmation in larger studies.
0005
Epigallocatechin-3-gallate decreases VASP expression and MCF-7 cells invasion by inhibiting Rac1 activity
Y. Zhang1 , G. Han2 , B. Fan3 , L. Wei4 . 1 Department of Breast and Thyroid Gland Surgery, 2 The First College of Clinical Medicine of Wuhan University, Renmin Hospital of Wuhan University, 3 The Second College of Clinical Medicine of Wuhan University, Zhongnan Hospital of Wuhan University, 4 Department of Pathophysiology, Basic Medical School of Wuhan University, Wuhan, China Goals: To investigate the influence of green tea (−)-epigallocatechin-3gallate (EGCG) on migration and invasion of MCF-7 cells and to identify the altered signalling pathway(s) underlying the response to EGCG exposure. Methods: MCF-7 cells were treated with EGCG (0, 10, 25, 50, 100 mg/ml) for 0 h, 6 h, 12 h, 18 h, 24 h and 30 h, and then expression of vasodilator-stimulated phosphoprotein (VASP) and total Rac1 in cells was detected by RT-PCR and Western Blotting, respectively, at mRNA and protein levels and the activities of Rac1 were examined by GST Pulldown assay, accordingly, cells migration and invasion were analyzed by wound-healing assay and transwell migration/invasion assays. Meanwhile, Rac1 and VASP specific siRNA oligos were applied to knock-down the expression of Rac1 and/or VASP in cells to observe altered signalling pathway(s) underlying the response to EGCG exposure. Results: In a concentration-dependent manner, EGCG decreased the migratory and invasive potential of MCF-7 cells with concomitant downregulated VASP expression and Rac1 activity. By using VASP and Rac1 specific siRNAs to block the expression of VASP and Rac1 in MCF-7 cells which were treated with epidermal growth factor (EGF) previously, we demonstrated that the regulation of cells migration and invasion was associated with Rac1 activity and VASP expression. In addition, siRNA mediated knock-down of Rac1 decreased the amount of VASP expression at mRNA level while VASP specified siRNA revealed no effect on the expression of Rac1 in MCF-7 cells. Conclusion: The inhibitive effect of EGCG on MCF-7 cells migration and invasion may be produced by down-regulated of VASP expression via Rac1 pathway.
0006
Development of novel breast cancer stem cell therapy targeting hedgehog signaling pathway
H. Tanaka1 , M. Nakamura1 , M. Kubo2 , M. Kai1 , M. Katano1 . 1 Cancer Therapy and Research, 2 Surgery and Oncology, Graduate School of Medicine, Kyushu University, Fukuoka, Japan Goals: Hedgehog (Hh) signalling pathway is crucial for growth and patterning during embryonic development. Previously, we reported that this pathway is highly activated in human breast cancer compared with normal tissue, and we can see reports suggesting that the morphogenic signals including Hh signal contribute to the survival and maintenance of cancer stem cells (CSCs) in various types of carcinomas. So, we hypothesized that the Hh signalling pathway might maintain the breast CSCs. Our goal
Thursday, 12 March 2009 is to reveal that the Hh pathway is essential for maintaining breast CSCs and might represent a valuable target for breast cancer stem cell therapy for the first time. Methods: Three human breast cancer cell lines were maintained. Flow Cytometry analysis and sorting were performed to detect the CD24−/low CD44+ population cells and the side population (SP) cells, reported to include much breast CSCs. To confirm the high tumorigenicity and multipotency of them, we performed xenograft tumor experiments in NOD/SCID mice. To assess the expression levels of Hh signalling components, real-time RT-PCR and fluorescent immunostaining were performed. Cyclopamine, which is an inhibitor of Hh pathway, and si-RNA against Gli1, a trans-activator of the Hh signalling pathway were used to inhibit this pathway. Furthermore we created peptides with particular affinity for Patched1 (Ptch1), a transmembrane receptor of Hh ligands. These peptides were used treatment experiment in NOD/SCID mice model. Results: The breast cancer cell lines contained 10−25% of CD24−/low CD44+ population and 1−4% of SP, and these two populations were overlapped with each other. These populations were resistant to anticancer drugs, more tumorigenic than other populations, and had multipotency in xenograft transplantation model. Components of the Hh pathway were more highly expressed in these populations compared with others, and inhibition of Hh signalling activity suppressed the tumorigenicity and the proliferation of both populations. The significance of Hh signalling activity was also confirmed by the effect of si-RNA against Gli1 and Ptch1 affinity peptides in vivo and vitro. Conclusion: The Hh signalling pathway is essential for the survival and maintenance of the breast CSCs, and that this pathway might represent a new candidate for breast cancer therapy targeting cancer stem cells.
0007
Importance of estrogen metabolites (2-OH/16a-OH) in the pathogenesis of breast, ovarian and endometrial cancer in postmenopausal women
I.B. Antonova1 , L.A. Ashrafyan1 , I.O. Basova1 . 1 Oncogynecology, Russian Scientific Center of Radiology, Moscow, Russian Federation Goals: In Russia breast cancer (BC) heads the list of reproductive organs new growth with 45−50 in 100000. The number of endometrial cancer (EC) and ovarian cancer (OC) cases has increased and accounts 12.8 and 10.2 in 10,0000. Conception about nosotropic inhomogeneity of such cancerous process, existence of hormone-dependent and autonomic types has been proved. However numerous examinations confirm that most likely there is no clear-cut line of demarcation between types of cancer. The object of the work was to study endocrinological (metabolite of estrogens, estrogenic receptors) component in process of BC, EC and OC during postmenopause. Methods: The main group was of 40 invasive breast cancer patients, 60 endometrial cancer patients of I−III stages, 60 ovarian cancer patients and a control group of 30 patients without signs of pathology. All patients were postmenopausal women. Results: Most of patients have metabolic syndrome. Also BC, EC and OC is characterized by high level of production of aggressive metabolite 16a-OH (BC: 6.98; EC: 6.66; OC: 8.69 ng/ml) and low expression of 2-OH (BC: 7.12; EC: 6.82, OC: 8.25 ng/ml), ratio of metabolites (2-OH/16a-OH) on the level BC: 1.02 (p < 0.05), EC: 1.16 (p < 0.05), OC: 1.04 (p < 0.05). And OR for control group was 3.5. All EC samples have expression of mRNK of estrogenic receptors. Conclusion: Preponderance of endocrinological aspect in process became visible in BC, EC and OC model. Our analysis using pretreatment urinary samples suggested that a lower 2-OHE/16-OHE was associated with high risk of postmenopausal breast, ovarian and endometrial cancer.
0008
Epigenetic field cancerization of MTHFR gene in breast carcinomas
J. Lee1 , J. Kim1 , K. Suh2 , T. Oh3 , S. An3 , J. Lee4 , B. Choi5 , E. Chang1 . 1 Department of Surgery, 2 Department of pathology, Chungnam National University Hospital, 3 Research and Development Center of Genomic tree, 4 Department of Dermatology, 5 Department of Radiology, Chungnam National University Hospital, Daejeon, South Korea
Goals: Gene promoter hypermethylation has been demonstrated to be a principal mechanism correlating, in cancer cells, to specific gene silencing.