Intradomain cleavage of inhibitory prodomain is essential to protumorigenic function of membrane type-1 matrix metalloproteinase (MT1-MMP) in vivo

Abstract

Invasive cancers use pericellular proteolysis to breach the extracellular matrix and basement membrane barriers and invade the surrounding tissue. Proinvasive membrane type-1 matrix metalloproteinase (MT1-MMP) is the primary mediator of proteolytic events on the cancer cell surface. MT1-MMP is synthesized as a zymogen. The latency of MT1-MMP is maintained by its N-terminal inhibitory prodomain. In the course of MT1-MMP activation, the R(108)RKR(111) ↓ Y(112) prodomain sequence is processed by furin. The intact prodomain released by furin alone, however, is a potent inhibitor of the emerging MT1-MMP enzyme. Evidence suggests that the prodomain undergoes intradomain cleavage at the PGD ↓ L(50) site followed by the release of the degraded prodomain by furin cleavage that finalizes the two-step activation event. These cleavages, only if combined, cause the activation of MT1-MMP. The significance of the intradomain cleavage in the protumorigenic program of MT1-MMP, however, remained unidentified. To identify this important parameter, in our current study, we used the cells that expressed the wild-type prodomain-based fluorescent biosensor and the mutant biosensor with the inactivated PGD↓L(50) cleavage site (L50D mutant) and also the cells with the enforced expression of the wild-type and L50D mutant MT1-MMP. Using cell-based tests, orthotopic breast cancer xenografts in mice, and genome-wide transcriptional profiling of cultured cells and tumor xenografts, we demonstrated that the intradomain cleavage of the PGD ↓ L(50) sequence of the prodomain is essential for the protumorigenic function of MT1-MMP. Our results emphasize the importance of the intradomain cleavages resulting in the inactivation of the respective inhibitory prodomains not only for MT1-MMP but also for other MMP family members.

FIGURE 1.

MT1-MMP cell surface forms. The MCF7-MT1, MCF7-L50D, MCF7-E240A, and original MCF7 cells were surface-biotinylated and then lysed. Biotin-labeled proteins were isolated from the lysate aliquots (1 mg/ml total protein each) using streptavidin beads. Biotin-labeled cell surface proteins were analyzed by Western blotting with the V5 antibody to detect the MT1-MMP-V5 constructs.

FIGURE 2.

RFP-PRO-GFP biosensor is cleaved in cancer cells. A, the prodomain-based RFP-PRO-GFP and mutant RFP-L50D-GFP fluorescent biosensors were expressed in HT1080, MCF7, and MDA-MB-435 cells. The biosensor structure is shown at the top. Red fluorescence alone indicates the intact biosensor. If the biosensor is cleaved, the green fluorescence is merged with the red fluorescence, resulting in an orange-yellow color. Nuclei are DAPI-stained. S, signal peptide; CT, cytoplasmic tail; TM, transmembrane domain. B, the RFP-PRO-GFP biosensor is cleaved in the Golgi compartment in HT1080 cells. The Golgi was stained using a Golgin-97 antibody followed by the secondary Alexa Fluor 350-conjugated antibody.

FIGURE 3.

Tumorigenicity of MCF7-MT1, MCF7-L50D, and MCF7-E240A cells. Xenografts were induced by injection of cells in the mammary fat pad of nude mice. A, mice 10 weeks postinjection. Arrows point to the tumors. B, tumor growth in mice. Tumor size is expressed as mean tumor volume ±S.D. (error bars). C, H&E staining of tumor sections. White arrows, tumor stroma; black arrows, infiltrating immune cells; yellow arrows, mammary epithelial cells; red arrows, blood capillaries. MMTV-PyMT, murine mammary tumor virus-polyoma virus middle T-antigen.

FIGURE 4.

Immunostaining of tumor xenografts. Immunostaining of the MCF7-MT1 and MCF7-L50D frozen tumor sections using the V5 (MT1-MMP), Ki-67, and type I collagen (Col1A) antibodies is shown. Nuclei are DAPI-stained.

FIGURE 5.

Characterization of MCF7-L50D cells and tumors. A, Western blotting of cell and tumor extracts with a Ki-67 antibody. An equal amount of total protein (50 μg) was loaded for each sample we analyzed. B, gelatin zymography of MMP-2 and Western blotting of the tumor extract samples with the V5 antibody (top and bottom panel, respectively). ProMMP-2, proMMP-2 alone; no extract. * indicates the extract of the inoculation site tissue because MCF7-E240 tumors did not emerge in mice. MMTV-PyMT, murine mammary tumor virus-polyoma virus middle T-antigen.

FIGURE 6.

Genome-wide transcriptional profiling of MCF7 xenograft tumors and cultured cells. A, an Ingenuity analysis of the genome-wide transcriptional profiling of the MCF7-L50D cultured cells and tumor xenografts. The line indicates a threshold value. B, cell cycle-related genes, the expression of which was affected in the tumor and cell samples. The number of affected genes is shown above each bar. The compared samples are indicated below the bars. C, RT-PCR of MT1-MMP, Ki-67, and GAPDH (control) in MCF7 cell and tumor xenograft samples. D, Ingenuity network analysis of differentially expressed cell cycle-related genes in MCF7-L50D tumors versus MCF7-L50D cultured cells. Up-regulated and down-regulated genes are shown in red and green, respectively. Genes that are not user-specified but are incorporated into the network through their relationship with other genes are shown in white. Continuous and discontinuous lines represent direct and indirect functional and physical interaction between genes from the literature.