Abstract
To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early gene Cyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61 was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61 mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development.
Highlights
To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library
Northern analysis revealed that Cyr61 was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A
Purified Cyr61 protein has been reported to mediate cell adhesion, stimulate chemotaxis, augment growth factor-induced DNA synthesis, enhance cell survival, and induce angiogenesis in vivo (10 –12). Because these characteristics may foster the progression of breast cancer, we studied this gene in detail and found it to be highly expressed in some invasive breast cancer cell lines and 36% of primary breast tumors
Summary
To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early gene Cyr, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Stable expression of Cyr cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Purified Cyr protein has been reported to mediate cell adhesion, stimulate chemotaxis, augment growth factor-induced DNA synthesis, enhance cell survival, and induce angiogenesis in vivo (10 –12) Because these characteristics may foster the progression of breast cancer, we studied this gene in detail and found it to be highly expressed in some invasive breast cancer cell lines and 36% of primary breast tumors. Characterization of the oncogenic activity of Cyr demonstrated that forced expression of Cyr enhanced MCF-7 cell growth in soft agar and promoted tumor growth in both normal breast and breast cancer cells in nude mice
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