Abstract 1499: MT1-MMP two-step activation in tumorigenesis

Abstract

Abstract Elevated activity of membrane type-1 matrix metalloproteinase (MT1-MMP) promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intracellular intradomain cleavage of the PGD-L50 site initiates the MT1-MMP activation (first step), whereas the 108RRKR111-Y112 cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP (second step). Here we used the prodomain-based fluorescent biosensors to demonstrate that the proteolytic steps in MT1-MMP activation are differentially regulated in cancer cells versus normal cells. We determined that the intracellular cleavage of the PGD-L50 site represents an “on” and “off” mechanism that discriminates cancer cells from normal cells. To evaluate the significance of the PGD-L50 site cleavage in tumorigenic potential of MT1-MMP, the MCF7 cells transfected with the wild type MT1-MMP (MCF7-MT1) and the uncleavable L50D mutant (MCF7-L50D) were inoculated in the breast fat pads of nude mice. In 4 weeks palpable tumors were evident in all of the mice that received MCF7-MT1 and MCF7-L50D cells. The MCF7-L50D cells formed small tumors that reached ∼50 mm3 volume and remained unchanged from week 4 till week 10. On the contrary, the MCF7-MT1 tumors grew rapidly and reached ∼700 mm3 volume in 10 weeks. These data suggest that the intracellular cleavage of PGD-L50 site is critical for MT1-MMP two-step activation and tumorigenic activity. This intracellular cleavage does not occur in normal cells and the enzyme remained inhibited by it's non-covalently associated prodomain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1499. doi:10.1158/1538-7445.AM2011-1499