Azzam H S, Arand G, Lippman M E, Thompson E W
Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, Washington DC, 20007.
J Natl Cancer Inst. 1993 Nov 3;85(21):1758-64. doi: 10.1093/jnci/85.21.1758.
Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels.
Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism.
Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP-2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator.
MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA-MB-435, MCF-7 ADR) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells.
Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator.
The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.
癌细胞表达的基质金属蛋白酶-2（MMP-2），即72-kd的IV型胶原酶/明胶酶，通过降解IV型胶原介导癌细胞侵袭基底膜，从而与转移相关。然而，这种潜在的酶原在正常组织和体液中的丰度表明，MMP-2酶原的利用受其生理激活的限制，而非仅受表达的限制。我们之前报道过，在I型胶原（维特罗生）凝胶上培养的正常和恶性成纤维样细胞可激活这种酶原。
本研究的目的是1）确定MMP-2激活是否仅限于侵袭性更强的人乳腺癌细胞系，以及2）定位激活机制。
通过检测潜在的MMP-2（72 kd）和较小分子量的成熟形式（59或62 kd），用酶谱法监测MMP-2激活。因此，对在塑料、维特罗生和其他基质上培养的人乳腺癌细胞系进行MMP-2激活筛选。将在I型胶原上培养的细胞暴露于蛋白质合成抑制剂环己酰亚胺或蛋白酶抑制剂，以确定MMP-2激活机制的性质。将来自在I型胶原或塑料上培养的细胞的Triton X-114（TX-114）去污剂提取物与潜在的MMP-2一起孵育，并通过酶谱法分析以定位MMP-2激活剂。
仅在侵袭性更强、雌激素受体阴性、波形蛋白阳性的人乳腺癌细胞系（Hs578T、MDA-MB-436、BT549、MDA-MB-231、MDA-MB-435、MCF-7 ADR）中，I型胶原培养可诱导MMP-2激活，且与MMP-2产生无关。在I型胶原凝胶上培养的细胞中检测到MMP-2激活，但在明胶凝胶、基质胶或I型或IV型胶原、明胶或纤连蛋白薄层上培养的细胞中未检测到。胶原诱导的激活对MMP-2酶具有特异性，因为92-kd的IV型胶原酶/明胶酶MMP-9在类似条件下不可激活。MMP-2激活受环己酰亚胺抑制，对金属蛋白酶抑制剂敏感，但对天冬氨酸、丝氨酸或半胱氨酸蛋白酶抑制剂不敏感。在侵袭性I型胶原培养细胞的富含质膜的疏水性TX-114提取物中检测到MMP-2激活。
I型胶原诱导的MMP-2激活仅限于侵袭性强的雌激素受体阴性、波形蛋白阳性的人乳腺癌细胞系，与MMP-2产生无关，并与转移潜能相关。我们的发现与激活剂的质膜定位一致。
MMP-2激活机制可能代表了人乳腺癌诊断、预后和治疗的新靶点。
