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DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma
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Biomedicine & Pharmacotherapy 59 (2005) $363-$365 http://france.elsevier.com/direct/BIOPHA/
DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma E.L.H. Lui, W.T.Y. Loo, L. Zhu, M.N.B. Cheung, L.W.C. Chow* Department of Surgery, Universityof Hong Kong Medical Center, Pokfulam Road, Queen Mary Hospital, Pokfulam, Hong Kong
Abstract B a c k g r o u n d . - Neoplastic cells often display aberrant methylation and silencing of multiple genes, including tumor suppressor genes (TSGs) that regulate critical processes such as cell cycle control, DNA repair and angiogenesis. Tissue inhibitor of metalloproteinase-3 (TIMP3) is an extracellular matrix-bound protein which regulates matrix composition and affects tumor growth, invasion and angiogenesis. It mediates vascular endothelial growth factor (VEGF) by blocking the binding of VEGF to VEGF receptor-2 and inhibits downstream signaling. This study focused on the hypermethylation status of the T I M P 3 gene with clinical parameters in invasive breast ductal carcinoma (IDC) samples. M a t e r i a l s a n d m e t h o d s . - DNA extraction and methylation specific PCR (MSP) was performed on 173 patients with invasive breast carcinoma. Both specific methylated and unmethylated primers for each gene were used for PCR and the products were visualized on agarose gel. The methylation status of T I M P 3 was then compared with corresponding patients' clinicopathologic characteristics. R esults. - Methylation frequencies of T1MP3 in the breast cancer samples were 20.81%. Among the hypermethylated cancers, 50% were tumor grade II-III, 44.44% were positive in lymph node involvement (LN), 36.11% were positive in lymphovascular permeation (LVP), 44.44%, 22.22% and 47.22% for the overexpressions in estrogen receptor (ER), progesterone receptor(PR) and c-erbB2, respectively. C o n c l u s i o n . - The result demonstrated that hypermethylation of T I M P 3 in IDC might be associated with high tumor grading and lymph nodes metastasis, and overexpression of ER, PR and c-erbB2, respectively. © 2005 Elsevier SAS. All rights reserved.
Keywords: TIMP3; Invasive ductal carcinoma; Methylation
1. Introduction
DNA methylation is a common epigenetic process controlling gene expression in mammalian cells and often required for proper development [1]. It usually takes place in the "CpG islands" in the promoter region of genes, with the exception of genes on the inactive X chromosome in females and imprinted genes [2]. The enzymes responsible for this chemical reaction are DNA methyl transferase I, IIIB and I l i a [2,3]. The DNA methyl transferase I is mainly involved in the maintenance of methylation status of genomes through DNA replication, whereas DNA methyl transferase IIIA and IIIB act principally for the de novo DNA methylation in the early stages of development [1,4].
* Corresponding author. E-mail address: [email protected] (L.W.C. Chow). © 2005 Elsevier SAS. All rights reserved.
Neoplastic cells often display aberrant methylation and silencing of multiple genes, including genes that regulate critical processes such as cell cycle control, DNA repair, and angiogenesis [5]. The cause of aberrant promoter methylation in neoplastic cells remains to be elucidated. In the case of cancer development, aberrant hypermethylation of "housekeeping" genes, as in this study, T I M P 3 gene, is a common mechanism for tumor suppressor inactivation in human cancer [5,6]. Such alteration in gene expression patterns causes the change in many involved signaling pathways, and the patterns are different for different tumor types [7-13]. Methylation-specific PCR (MSP) was used to quantitatively assess the methylation status of CpG islands located in the regulatory regions of T I M P 3 gene in a series of tumors representative of invasive breast ductal carcinoma (IDC). T I M P 3 , an extracellular matrix-bound protein that is expressed ubiquitously, is a potent inhibitor of angiogenesis
$364
E.L.H. Lui et al. / Biomedicine & Pharmacotherapy59 (2005) $363-$365
and tumor growth [14-16]. It contributes to the regulation of VEGF-mediated angiogenesis [ 17]. Our study focused on the hypermethylation pattern of TIMP3 in IDC and its relationships with histopathological features of the corresponding IDC patients. 2. Materials and methods 2.1. Patients and samples collection
One hundred and seventy-three patients with invasive ductal carcinoma were recruited for the MSP investigation on TIMP3. Written consent was obtained from all patients. The breast cancer tissues were collected in sterilized bottle containing 0.9% normal saline from the Department of Surgery, Queen Mary Hospital, The University of Hong Kong. 2.2. DNA extraction and methylation-specific PCR (MSP)
DNA was extracted by QIAamp® DNA Mini Kit (Qiagen, Canada). The tumors' DNA was isolated by filter column and washed with provided buffer as to elute the genomic DNA into sterile !.5 ml tube. The extracted DNA was then modified by CpG DNA Modification Kit (Chemicon International, USA), under the action of bisulfide. Both the specific methylated and unmethylated primers for TIMP3 were used for PCR. The products The PCR products generated by MSP were electrophoresed, using 2% agarose gel stained with ethidium bromide, against a 50 bp DNA ladder (Invitrogen, USA) and the images were captured under ultra-violet light. 3. Results 3.1. Methylation-specific PCR
Both methylated (M) and unmethylated (U) TIMP3 products were shown in Fig. 1. Methylation frequencies of TIMP3 in the breast cancer samples were 20.81%. 3.2. Pathological results o f patients
Patients' tissues obtained from the surgery were stained for estrogen receptor (ER), progesterone receptor (PR) and c-erbB2, the lymphovascular permeation (LVP) and histological grading of tumor differentiation were identified Methylation-specific PCR of TIMP3 gene Marker M U U U
(data not shown). The relationships between hypermethylated TIMP3 cases and tumor grade, LN, LVP, ER, PR and c-erbB2 are detailed in Table 1. 4. Discussion The growing number of tumor suppressor and other cancer genes reported to be hypermethylated with associated transcriptional silencing provides an opportunity for the examination of the pattern of epigenetic alteration in breast cancer [1--6]. TIMP3 may show activity to inhibit tumor growth, angiogenesis and metastasis [18], though the inhibition of angiogenesis by it are through the blockage of VEGF receptor-binding [17] and inhibition of matrix metalloproteinases (MMPs) [14] instead of the COX-2 pathway. MMPs would promote invasion and entry into and out of blood or lymphatic vessels (intravasation, extravasation) [14-16]. Primary and metastatic tumors need a certain environment to support and maintain their growth [18]. Such environment is provided by the presence of MMPs which is believed to initiate angiogenesis, working in combination with serine proteases, by proteolytically degrade the capillary basement membrane [19]. From our observation, 44.44% of the hypermethylated cases were associated with lymph node involvement (LN). In the hypermethylated cases, in which TIMP3 protein was not expressed, and so MMPs would not be inhibited resulting for the high possibility for lymphatic vessels invasion [1416]. Among the TIMP3 hypermethylated group, 50% were of tumor grades II-III. TIMP3 plays a great role in angiogenesis inhibition and it may be associated with tumor grade shown by a former research [20]. This further supports our investigation here with TIMP3 and tumor grade. Apart from the above two parameters, hypermethylated TIMP3 cases are also likely to be associated with estrogen receptor and c-erbB2 receptor overexpression, though the reasons behind require further investigation with larger scale of study which can possibly generate statistical significant figures. At present, studies about the relationship between hypermethylation status of TIMP3 gene and the histopathological records of breast carcinoma patients are very few; our investigation can provide a general view of the above relationship and as a possible guideline for later investigations. To conclude the likely associations of the examined TIMP3 hypermethylation with high tumor grade, lymph node positive, and estrogen receptor and c-erbB2 Table 1 Comparisonof hypermethylatedT1MP3with clinical status Hypermethylated Grade cases (%) (>or = 2) (%)
Fig. 1. M indicatespositivein methylatedprimersequence,and U indicates positivein unmethylatedprimersequence.
TIMP3(20.81)
50.00
LN (%)
LVP ER PR c-erbB2 (%) (%) (%) (%)
44.44 36.11 44.44 22.22 47.22
E.L.H. Lui et al. / Biomedicine & Pharmacotherapy 59 (2005) $363-$365
overexpressions may enable it to be a prediction agent of the tumor characteristics of patients. References [1]
Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science (Wash. DC) 1999;286:481-6.
[2]
Panlsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development and disease. J Patho12001; 195:97-110.
[3]
Jones PA, Takai DT. The role of DNA methylation in mammalian epigenetics, Science 2001 ;293:1068-70.
[4]
Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000; 16:168-74.
[5]
Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999;21:163-7.
[6]
Baylin SB, Herman JG, Graft JR, Vertino PM, Issa J-PJ. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 1998;72:141-96.
[7]
Melki JR, Vincent PC, Clark SJ. Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. Cancer Res 1999;59:3730-40.
[8]
Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res 2001 ;61:3225-9.
[9]
Ueki T, Toyota M, Sohn T, Yeo CJ, Issa JP, Hruban RH, et al. Hypermethylation of multiple genes in pancreatic adenocarcinoma. Cancer Res 2000;60:1835-9.
[10] Zochbauer-Muller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. Aberrant promoter methylation of multiple genes in nonsmall cell lung cancers. Cancer Res 2001 ;61:249-55.
$365
[11] Dong SM, Kim HS, Rha SH, Sidransky D. Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix. Clin Cancer Res 2001 ;7:1982~6. [12] Maruyama R, Toyooka S, Toyooka KO, Harada K, Virmani AK, Zochbauer-Muller S, et al. Aberrant promoter methylation profile of bladder cancer and its relationship to histopathological features. Cancer Res 2001 ;61:8659-63. [13] Kwong J, Lo KW, To KF, Teo PM, Johnson PJ, Huang DP. Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res 2002;8:131-7. [14] Anand-Apte B, Bao L, Smith R, Iwata K, Olsen BR, Zetter B, et al. A review of tissue inhibitor of metalloproteinases-3 (TIMP-3) and experimental analysis of its effect on primary tumor growth. Biochem Cell Biol 1996;74(6):853-62. [15] Anand-Apte B, Pepper MS, Voest E, Montesano R, Olsen B, Murphy G, et al. Inhibition of angiogenesis by tissue inhibitor of metalloproteinase-3. Invest Ophthalmol Vis Sci 1997;38:817-23. [16] Apte SS, Mattei MG, Olsen BR. Cloning of the cDNA encoding human tissue inhibitor of metalloproteinase-3 (TIMP-3) and mapping of the TIMP-3 gene to chromosome 22. Genomics 1994; 19:86-90. [17] Qi JH, Ebrahem Q, Moore N, Murphy G, Claesson-Welsh L, Bond M, et al. A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2. Nat Med 2003;9:407-15. [ 18] Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 1997;89:1260-70. [19] Pepper MS, Montesano R, Mandriota SJ, Orci L, Vassalli JD. Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis. Enzyme Protein 1996;49:138-62. [20] Olewniczak S, Chosia M, Kwas A, Kram A, Domagala W. Angiogenesis and some prognostic parameters of invasive ductal breast carcinoma in women. Pol J Patho12002;53:183-8.
8CIENCIE ~IDIRBCT
ELSEVIER
e
& BIOMEDICINE PHARMACOTHERAPY
Biomedicine & Pharmacotherapy 59 (2005) $363-$365 http://france.elsevier.com/direct/BIOPHA/
DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma E.L.H. Lui, W.T.Y. Loo, L. Zhu, M.N.B. Cheung, L.W.C. Chow* Department of Surgery, Universityof Hong Kong Medical Center, Pokfulam Road, Queen Mary Hospital, Pokfulam, Hong Kong
Abstract B a c k g r o u n d . - Neoplastic cells often display aberrant methylation and silencing of multiple genes, including tumor suppressor genes (TSGs) that regulate critical processes such as cell cycle control, DNA repair and angiogenesis. Tissue inhibitor of metalloproteinase-3 (TIMP3) is an extracellular matrix-bound protein which regulates matrix composition and affects tumor growth, invasion and angiogenesis. It mediates vascular endothelial growth factor (VEGF) by blocking the binding of VEGF to VEGF receptor-2 and inhibits downstream signaling. This study focused on the hypermethylation status of the T I M P 3 gene with clinical parameters in invasive breast ductal carcinoma (IDC) samples. M a t e r i a l s a n d m e t h o d s . - DNA extraction and methylation specific PCR (MSP) was performed on 173 patients with invasive breast carcinoma. Both specific methylated and unmethylated primers for each gene were used for PCR and the products were visualized on agarose gel. The methylation status of T I M P 3 was then compared with corresponding patients' clinicopathologic characteristics. R esults. - Methylation frequencies of T1MP3 in the breast cancer samples were 20.81%. Among the hypermethylated cancers, 50% were tumor grade II-III, 44.44% were positive in lymph node involvement (LN), 36.11% were positive in lymphovascular permeation (LVP), 44.44%, 22.22% and 47.22% for the overexpressions in estrogen receptor (ER), progesterone receptor(PR) and c-erbB2, respectively. C o n c l u s i o n . - The result demonstrated that hypermethylation of T I M P 3 in IDC might be associated with high tumor grading and lymph nodes metastasis, and overexpression of ER, PR and c-erbB2, respectively. © 2005 Elsevier SAS. All rights reserved.
Keywords: TIMP3; Invasive ductal carcinoma; Methylation
1. Introduction
DNA methylation is a common epigenetic process controlling gene expression in mammalian cells and often required for proper development [1]. It usually takes place in the "CpG islands" in the promoter region of genes, with the exception of genes on the inactive X chromosome in females and imprinted genes [2]. The enzymes responsible for this chemical reaction are DNA methyl transferase I, IIIB and I l i a [2,3]. The DNA methyl transferase I is mainly involved in the maintenance of methylation status of genomes through DNA replication, whereas DNA methyl transferase IIIA and IIIB act principally for the de novo DNA methylation in the early stages of development [1,4].
* Corresponding author. E-mail address: [email protected] (L.W.C. Chow). © 2005 Elsevier SAS. All rights reserved.
Neoplastic cells often display aberrant methylation and silencing of multiple genes, including genes that regulate critical processes such as cell cycle control, DNA repair, and angiogenesis [5]. The cause of aberrant promoter methylation in neoplastic cells remains to be elucidated. In the case of cancer development, aberrant hypermethylation of "housekeeping" genes, as in this study, T I M P 3 gene, is a common mechanism for tumor suppressor inactivation in human cancer [5,6]. Such alteration in gene expression patterns causes the change in many involved signaling pathways, and the patterns are different for different tumor types [7-13]. Methylation-specific PCR (MSP) was used to quantitatively assess the methylation status of CpG islands located in the regulatory regions of T I M P 3 gene in a series of tumors representative of invasive breast ductal carcinoma (IDC). T I M P 3 , an extracellular matrix-bound protein that is expressed ubiquitously, is a potent inhibitor of angiogenesis
$364
E.L.H. Lui et al. / Biomedicine & Pharmacotherapy59 (2005) $363-$365
and tumor growth [14-16]. It contributes to the regulation of VEGF-mediated angiogenesis [ 17]. Our study focused on the hypermethylation pattern of TIMP3 in IDC and its relationships with histopathological features of the corresponding IDC patients. 2. Materials and methods 2.1. Patients and samples collection
One hundred and seventy-three patients with invasive ductal carcinoma were recruited for the MSP investigation on TIMP3. Written consent was obtained from all patients. The breast cancer tissues were collected in sterilized bottle containing 0.9% normal saline from the Department of Surgery, Queen Mary Hospital, The University of Hong Kong. 2.2. DNA extraction and methylation-specific PCR (MSP)
DNA was extracted by QIAamp® DNA Mini Kit (Qiagen, Canada). The tumors' DNA was isolated by filter column and washed with provided buffer as to elute the genomic DNA into sterile !.5 ml tube. The extracted DNA was then modified by CpG DNA Modification Kit (Chemicon International, USA), under the action of bisulfide. Both the specific methylated and unmethylated primers for TIMP3 were used for PCR. The products The PCR products generated by MSP were electrophoresed, using 2% agarose gel stained with ethidium bromide, against a 50 bp DNA ladder (Invitrogen, USA) and the images were captured under ultra-violet light. 3. Results 3.1. Methylation-specific PCR
Both methylated (M) and unmethylated (U) TIMP3 products were shown in Fig. 1. Methylation frequencies of TIMP3 in the breast cancer samples were 20.81%. 3.2. Pathological results o f patients
Patients' tissues obtained from the surgery were stained for estrogen receptor (ER), progesterone receptor (PR) and c-erbB2, the lymphovascular permeation (LVP) and histological grading of tumor differentiation were identified Methylation-specific PCR of TIMP3 gene Marker M U U U
(data not shown). The relationships between hypermethylated TIMP3 cases and tumor grade, LN, LVP, ER, PR and c-erbB2 are detailed in Table 1. 4. Discussion The growing number of tumor suppressor and other cancer genes reported to be hypermethylated with associated transcriptional silencing provides an opportunity for the examination of the pattern of epigenetic alteration in breast cancer [1--6]. TIMP3 may show activity to inhibit tumor growth, angiogenesis and metastasis [18], though the inhibition of angiogenesis by it are through the blockage of VEGF receptor-binding [17] and inhibition of matrix metalloproteinases (MMPs) [14] instead of the COX-2 pathway. MMPs would promote invasion and entry into and out of blood or lymphatic vessels (intravasation, extravasation) [14-16]. Primary and metastatic tumors need a certain environment to support and maintain their growth [18]. Such environment is provided by the presence of MMPs which is believed to initiate angiogenesis, working in combination with serine proteases, by proteolytically degrade the capillary basement membrane [19]. From our observation, 44.44% of the hypermethylated cases were associated with lymph node involvement (LN). In the hypermethylated cases, in which TIMP3 protein was not expressed, and so MMPs would not be inhibited resulting for the high possibility for lymphatic vessels invasion [1416]. Among the TIMP3 hypermethylated group, 50% were of tumor grades II-III. TIMP3 plays a great role in angiogenesis inhibition and it may be associated with tumor grade shown by a former research [20]. This further supports our investigation here with TIMP3 and tumor grade. Apart from the above two parameters, hypermethylated TIMP3 cases are also likely to be associated with estrogen receptor and c-erbB2 receptor overexpression, though the reasons behind require further investigation with larger scale of study which can possibly generate statistical significant figures. At present, studies about the relationship between hypermethylation status of TIMP3 gene and the histopathological records of breast carcinoma patients are very few; our investigation can provide a general view of the above relationship and as a possible guideline for later investigations. To conclude the likely associations of the examined TIMP3 hypermethylation with high tumor grade, lymph node positive, and estrogen receptor and c-erbB2 Table 1 Comparisonof hypermethylatedT1MP3with clinical status Hypermethylated Grade cases (%) (>or = 2) (%)
Fig. 1. M indicatespositivein methylatedprimersequence,and U indicates positivein unmethylatedprimersequence.
TIMP3(20.81)
50.00
LN (%)
LVP ER PR c-erbB2 (%) (%) (%) (%)
44.44 36.11 44.44 22.22 47.22
E.L.H. Lui et al. / Biomedicine & Pharmacotherapy 59 (2005) $363-$365
overexpressions may enable it to be a prediction agent of the tumor characteristics of patients. References [1]
Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science (Wash. DC) 1999;286:481-6.
[2]
Panlsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development and disease. J Patho12001; 195:97-110.
[3]
Jones PA, Takai DT. The role of DNA methylation in mammalian epigenetics, Science 2001 ;293:1068-70.
[4]
Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000; 16:168-74.
[5]
Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999;21:163-7.
[6]
Baylin SB, Herman JG, Graft JR, Vertino PM, Issa J-PJ. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 1998;72:141-96.
[7]
Melki JR, Vincent PC, Clark SJ. Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. Cancer Res 1999;59:3730-40.
[8]
Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res 2001 ;61:3225-9.
[9]
Ueki T, Toyota M, Sohn T, Yeo CJ, Issa JP, Hruban RH, et al. Hypermethylation of multiple genes in pancreatic adenocarcinoma. Cancer Res 2000;60:1835-9.
[10] Zochbauer-Muller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. Aberrant promoter methylation of multiple genes in nonsmall cell lung cancers. Cancer Res 2001 ;61:249-55.
$365
[11] Dong SM, Kim HS, Rha SH, Sidransky D. Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix. Clin Cancer Res 2001 ;7:1982~6. [12] Maruyama R, Toyooka S, Toyooka KO, Harada K, Virmani AK, Zochbauer-Muller S, et al. Aberrant promoter methylation profile of bladder cancer and its relationship to histopathological features. Cancer Res 2001 ;61:8659-63. [13] Kwong J, Lo KW, To KF, Teo PM, Johnson PJ, Huang DP. Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res 2002;8:131-7. [14] Anand-Apte B, Bao L, Smith R, Iwata K, Olsen BR, Zetter B, et al. A review of tissue inhibitor of metalloproteinases-3 (TIMP-3) and experimental analysis of its effect on primary tumor growth. Biochem Cell Biol 1996;74(6):853-62. [15] Anand-Apte B, Pepper MS, Voest E, Montesano R, Olsen B, Murphy G, et al. Inhibition of angiogenesis by tissue inhibitor of metalloproteinase-3. Invest Ophthalmol Vis Sci 1997;38:817-23. [16] Apte SS, Mattei MG, Olsen BR. Cloning of the cDNA encoding human tissue inhibitor of metalloproteinase-3 (TIMP-3) and mapping of the TIMP-3 gene to chromosome 22. Genomics 1994; 19:86-90. [17] Qi JH, Ebrahem Q, Moore N, Murphy G, Claesson-Welsh L, Bond M, et al. A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2. Nat Med 2003;9:407-15. [ 18] Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 1997;89:1260-70. [19] Pepper MS, Montesano R, Mandriota SJ, Orci L, Vassalli JD. Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis. Enzyme Protein 1996;49:138-62. [20] Olewniczak S, Chosia M, Kwas A, Kram A, Domagala W. Angiogenesis and some prognostic parameters of invasive ductal breast carcinoma in women. Pol J Patho12002;53:183-8.