Tissue inhibitor of
metalloproteinase-2
(TIMP2, TIMP-2)

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Gene: TIMP2 maps to 17q25. It spans approximately 83 kb and is composed of five exons and four introns of 54.8, 2.7, 9.1, and 1.7 kb. The 5'-end of the gene contains five Sp1, two AP-2, one AP-1, and three PEA-3 binding sites. Despite the presence of one AP-1 site, the promoter do not respond to the phorbol ester, protein kinase C-activator phorbol 12-myristate 13-acetate (PMA). The TIMP-2 promoter contains a typical CpG island; however, methylation of this island does not seem to influence gene expression (Hammani K. et al., 1996).
mRNA: sizes: 3.5 and 1.0 kb.
Protein: The tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinases, a group of zinc-binding endopeptidases involved in the degradation of the extracellular matrix. TIMP-1 is a 21-kD secreted protein binding to several MMPs. Its amino acid sequence exhibits many similarities to TIMP-1. For example, the positions of the 12 cysteines and 3 of the 4 tryptophan residues are conserved.
See also: TIMPs.

Cell lines:
- TIMP-2 mRNA levels and protein expresssion in MCF-7 and T-47D breast cancer cell (BCC) lines were found to be down-regulated by treatment with the progestin R5020 or R5020 plus estradiol, but not by treatment with estradiol alone. None of these genes were modulated in steroid-independent MDA-MB-231 cells (van den Brûle F.A. et al., 1992).

- In MDA-MB-231 BCC line, MT-MMP-1 (MMP114) secreted into culture media was purified in a complex form with TIMP-2 (Imai K. et al., 1996).

- Calcitonin was able to prevent the indduction of TIMP-1 and -2 mRNAs by the the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (Lacroix M. and Body J.J., 1997).

- Inoculation of MDA-MB-231 BCC into thee left cardiac ventricle of female nude mice causes osteolytic lesions in bone. TIMP-2 was overexpressed in these cells. In mice receiving the TIMP-2-transformed cells, osteolytic lesions were markedly decreased as compared to the animals receiving parent MDA-MB-231 BCC (Yoneda T. et al., 1997).

- Analysis of MMP expression by RT-PCR sshowed expression of MMP-1, MMP-3, and MMP-13 in highly invasive MDA-MB-231 BCC, but not in slightly invasive MCF-7, T-47D, and BT-20 cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516 (Balduyck M et al., 2000).

- Two invasive breast cancer cell lines (MDA-MB-231 and BT-549) were found to be more adherent and have greater migratory capacity on bone marrow fibroblasts than three non-invasive cell lines (MCF-7, T47D and BT-483). TIMP-1 and TIMP-2 were able to attenuate the migration of MDA-MB-231 cells through bone marrow fibroblast monolayers suggesting a role for matrix metalloproteases in the migration of breast cancer cells through bone marrow adherent layers (Saad S. et al., 2000).

- A recombinant adenovirus to deliver huuman TIMP-2 (AdTIMP-2) was generated and its anticancer efficiency was evaluated in a murine model. Overexpression in vitro of TIMP-2 inhibited the invasion of both MDA-MB-231 BCC and endothelial cells without affecting cell proliferation. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of MDA-MB-231 BCC-derived tumor establishment in 100% mice. A single local injection of AdTIMP-2 into preestablished tumors significantly reduced tumor growth rates by 60-80% and tumor-associated angiogenesis index by 25-75% (Li H. et al., 2001).


Tumors:
- TIMP-1 and -2 have been shown to blockk tumor cell invasion both in vitro and in vivo, suggesting that they act as 'metastasis suppressor genes.

- Immunohistological staining of MMP-2 aand MMP-9, basal lamina collagen IV and TIMP-2 were performed on frozen sections of 83 invasive breast carcinomas. MMP-2 and MMP-9 were associated with neoplastic cell plasma membrane in 72% of cases and exhibited inter-tumoral variability of staining intensity. MMP-2 and MMP-9 staining was not correlated with presence of metastases at time of diagnosis or with disease outcome. TIMP-2 was detected in the peri-tumoral stroma and was present in 87% of cases. Residual benign breast tissue was negative for TIMP-2 staining. Neoplasms with diffuse TIMP-2 staining (24%) recurred significantly more frequently (75% recurred) than cases with focal (42% recurred) or absent (27% recurred) TIMP-2. Presence of collagen IV was negatively correlated with gelatinase staining (Visscher D.W. et al., 1994).

- RT-PCR amplicons of MMP-2 and TIMP-2 mmRNA from tissue biopsies of 13 breast carcinomas and one fibrocystic mastopathy were quantitated. In comparison with their normal-tissue counterparts, their expression trends were not uniform: in some cases MMP-2 increased in the tumor without changes in TIMP-2, in others TIMP-2 expression also increased, although to a lesser extent than MMP-2; only in 2 cases was it slightly lower in the tumor tissue (Onisto M. et al., 1995).

- In a series of 55 breast tumours, leveels of both MMP8 and MMP9 were significantly related to concentration of TIMP-1 (for MMP8, P=0.0035; for MMP9, P=0.0021). Levels of MMP8, MMP9, and TIMP-1 showed no significant correlation with either tumor size or axillary node metastasis (Duffy M.J. et al., 1995).

- In a series of 34 breast cancer patiennts, high levels of TIMP-2 mRNA in primary carcinomas correlated with distant metastases (P=0.0055). No correlations were found with patient age, tumor size, grade of anaplasia, steroid receptor status, MMP-2 and MMP-9 mRNA levels (Ree A.H. et al., 1997).

- Allelic deletion at 17q23-q25 is foundd in approximately one-third of breast cancer patients.

- By Western blotting, TIMP-1 and -2 werre measured in paired tumour and normal tissue samples from 43 breast cancer patients. TIMP-1 was found in 82% breast tumours and 50% normal breast samples. For TIMP-2, the corresponding values were 100% and 80%. The amounts of TIMP-1 and TIMP-2 were higher in tumour samples than in normal tissue samples (Garbett E.A. et al., 1999).

- In a series of 79 infiltrating ductal carcinomas (IDCs), 8 tubular carcinomas, and 27 infiltrating lobular carcinomas (ILCs), both tumour cell and stromal staining was observed for TIMP-2, but there was no correlation with metastatic status (Jones J.L. et al., 1999).

- An in situ hybridization study was performed on normal breast tissue (n=6), fibrocystic disease (n=20), five cases of which contained radial scars, lobular carcinoma in situ (CLIS; n=5), ductal carcinoma in situ (DCIS; n=9) and invasive carcinomas (n=24). TIMP-1 and -2 transcript levels were increased in invasive carcinomas correlating with the histological grade (Brummer O. et al., 1999).

- High levels of TIMP-2 were found to coorrelate with both shortened disease-free interval and overall survival. In primary breast cancers, TIMP-2 levels showed no significant relationship with either tumor size or axillary node status but correlated inversely with estrogen receptor levels. TIMP-2 levels also correlated significantly with those for TIMP-1. It was concluded that high levels of endogenous TIMP-2, like other protease inhibitors such as PAI-1 and TIMP-1, correlate with progression of human breast cancer (Remacle A. et al., 2000).

- Thirty one specimens of bone metastasiis from breast carcinoma were stained for MMP1, 2, 9, 14 (MT1-MMP) and TIMP1, and 2 and compared with staining in normal breast tissue, primary breast carcinoma and normal bone. No major differences in the MMP/TIMP staining of tumor cells and fibroblasts were observed between bone metastasis and primary tumor. The number and activity of osteoclasts and osteoblasts was increased dramatically in bone metastases, their MMP/TIMP profiles, however, were not different from normal bone, suggesting that the mechanism of bone degradation by osteoclasts is not different from normal bone remodelling (Lhotak S. et al., 2000).

Balduyck M. et al. (2000) Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro. Clin. Exp. Metastasis 18, 171-178. (PubMed)
Brummer O. et al. (1999) Matrix-metalloproteinases 1, 2, and 3 and their tissue inhibitors 1 and 2 in benign and malignant breast lesions: an in situ hybridization study. Virchows Arch. 435, 566-573. (PubMed)
De Clerck Y. et al. (1992) The gene for tissue inhibitor of metalloproteinases-2 is localized on human chromosome arm 17q25. Genomics 14, 782-784. (PubMed)
Duffy M.J. et al. (1995) Assay of matrix metalloproteases types 8 and 9 by ELISA in human breast cancer. Br. J. Cancer 71, 1025-1028. (PubMed)
Garbett E.A. et al. (1999) Proteolysis in human breast and colorectal cancer. Br. J. Cancer 81, 287-293. (PubMed)
Hajitou A. et al. (2001) Down-regulation of vascular endothelial growth factor by tissue inhibitor of metalloproteinase-2: effect on in vivo mammary tumor growth and angiogenesis. Cancer Res. 61, 3450-3457. (PubMed)
Hammani K. et al. (1996) Structure and characterizationof the human tissue inhibitor of metalloproteinases-2 gene. J. Biol.Chem. 271, 25498-25505. (PubMed)
Imai K. et al. (1996) Membrane-type matrix metalloproteinase 1 is a gelatinolytic enzyme and is secreted in a complex with tissue inhibitor of metalloproteinases 2. Cancer Res. 56, 2707-2710. (PubMed)
Jones J.L. et al. (1999) Expression of MMP-2 and MMP-9, their inhibitors, and the activator MT1-MMP in primary breast carcinomas. J. Pathol. 189, 161-168. (PubMed)
Lacroix M. and Body J.J. (1997) Regulation of c-fos and c-jun expression by calcitonin in human breast cancer cells. Calcif. Tissue Int. 60, 513-519. (PubMed)
Lhotak S. et al. (2000) Immunolocalization of matrix metalloproteinases and their inhibitors in clinical specimens of bone metastasis from breast carcinoma. Clin. Exp. Metastasis 18, 463-470. (PubMed)
Li H. et al. (2001) Adtimp-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice. Hum. Gene Ther. 12, 515-526. (PubMed)
Onisto M. et al. (1995) Gelatinase A/TIMP-2 imbalance in lymph-node-positive breast carcinomas, as measured by RT-PCR. Int. J. Cancer 63, 621-626. (PubMed)
Ree A.H. et al. (1997) High levels of messenger RNAs for tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in primary breast carcinomas are associated with development of distant metastases. Clin. Cancer Res. 3, 1623-1628. (PubMed)
Remacle A. et al. (2000) High levels of TIMP-2 correlate with adverse prognosis in breast cancer. Int. J. Cancer 89, 118-121. (PubMed)
Saad S. et al. (2000) Induction of matrix metalloproteinases MMP-1 and MMP-2 by co-culture of breast cancer cells and bone marrow fibroblasts. Breast Cancer Res. Treat. 63, 105-115. (PubMed)
Stetler-Stevenson W.G. et al. (1989) Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. J. Biol. Chem. 264, 17374-17378. (PubMed)
van den Brûle F.A. et al. (1992) Genes involved in tumor invasion and metastasis are differentially modulated by estradiol and progestin in human breast-cancer cells. Int. J. Cancer 52, 653-657. (PubMed)
Visscher D.W. et al. (1994) Enhanced expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) in the stroma of breast carcinomas correlates with tumor recurrence. Int. J. Cancer 59, 339-344. (PubMed)
Yoneda T. et al. (1997) Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with the bisphosphonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2. J. Clin. Invest. 99, 2509-2517. (PubMed)

Genome Database data (GDB Access Number: 132612)
GeneCard data (TIMP2)
UniGene data (Hs.246948)
OMIM data (ID = 188825)
LocusLink data (LocusID = 7077)
Swiss-Prot (ID = P16035)


MMP1, MMP2, MMP3, MMP7, MMP9, MMP11, MMP13, MMP14, MMP15, MMP16, MMP17, TIMP-1, TIMP-3, TIMP-4