Genetic dissection of proteolytic and non-proteolytic contributions of MT1-MMP to macrophage invasion

MT1-MMP/MMP-14 is a major invasion-promoting membrane protease expressed in macrophages. In addition to its proteolytic activity that degrades the extracellular matrix, MT1-MMP also boosts ATP production in cells in a manner independent of its proteolytic activity. It remains unclear to what extent the proteolytic and energy-boosting activities of MT1-MMP contribute to macrophage invasion. Recently, we demonstrated that the cytoplasmic tail of MT1-MMP makes use of APBA3/Mint3 to activate HIF-1 and thereby boosts glycolysis for ATP production. Here, we used Apba3^−/− macrophages to dissect the contribution of the proteolytic and the energy-boosting activities of MT1-MMP. The proteolytic activity of MT1-MMP was not affected by the lack of APBA3 in macrophages. Apba3^−/− and Mmp14^−/− macrophages exhibited a 55% reduction of ATP levels compared to wild-type (WT) cells and the rate of motility of the mutant cells was accordingly reduced. In contrast, matrigel invasion by Mmp14^−/− and Apba3^−/− macrophages was reduced to 24% and 55.4%, respectively, of the level observed in WT cells. These results represent the first attempt to dissect the contribution of the two invasion-promoting activities of MT1-MMP to macrophage invasion.     

Hypoxia-Inducible Factor 1 Regulation through Cross Talk between mTOR and MT1-MMP

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the cellular adaptation to hypoxia. Although HIF-1 is usually strongly suppressed by posttranslational mechanisms during normoxia, HIF-1 is active and enhances tumorigenicity in malignant tumor cells that express the membrane protease MT1-MMP. The cytoplasmic tail of MT1-MMP, which can bind a HIF-1 suppressor protein called factor inhibiting HIF-1 (FIH-1), promotes inhibition of FIH-1 by Mint3 during normoxia. To explore possible links between HIF-1 activation by MT1-MMP/Mint3 and tumor growth signals, we surveyed a panel of 252 signaling inhibitors. The mTOR inhibitor rapamycin was identified as a possible modulator, and it inhibited the mTOR-dependent phosphorylation of Mint3 that is required for FIH-1 inhibition. A mutant Mint3 protein that cannot be phosphorylated exhibited a reduced ability to inhibit FIH-1 and promoted tumor formation in mice. These data suggest a novel molecular link between the important hub proteins MT1-MMP and mTOR that contributes to tumor malignancy.     

Cleavage of amyloid-beta precursor protein (APP) by membrane-type matrix metalloproteinases

Amyloid-beta precursor protein (APP) was identified on expression cloning from a human placenta cDNA library as a gene product that modulates the activity of membrane-type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with APP in HEK293T cells induced cleavage and shedding of the APP ectodomain when co-expressed with APP adaptor protein Fe65. Among the MT-MMPs tested, MT3-MMP and MT5-MMP also caused efficient APP shedding. The recombinant APP protein was cleaved by MT3-MMP in vitro at the A463-M464, N579-M580, H622-S623, and H685-Q686 peptide bonds, which included a cleavage site within the amyloid beta peptide region known to produce a C-terminal fragment. The Swedish-type mutant of APP, which produces a high level of amyloid beta peptide, was more effectively cleaved by MT3-MMP than wild-type APP in both the presence and absence of Fe65; however, amyloid beta peptide production was not affected by MT3-MMP expression. Expression of MT3-MMP enhanced Fe65-dependent transactivation by APP fused to the Gal4 DNA-binding and transactivation domains. These results suggest that MT1-MMP, MT3-MMP and MT5-MMP should play an important role in the regulation of APP functions in tissues including the central nervous system.     

Novel processing of beta-amyloid precursor protein catalyzed by membrane type 1 matrix metalloproteinase releases a fragment lacking the inhibitor domain against gelatinase A

In various mammalian cell lines, beta-amyloid precursor protein (APP) is proteolytically processed to release its NH(2)-terminal extracellular domain as a soluble APP (sAPP) that contains the inhibitor domain against gelatinase A. To investigate roles of sAPP in the regulation of gelatinase A activity, we examined the correlation between the activation of progelatinase A and processing of APP. We found that stimulation of HT1080 fibrosarcoma cells with concanavalin A led to an activation of endogenous progelatinase A and to a novel processing of APP, which releases a COOH-terminally truncated form of sAPP (sAPPtrc) into the culture medium. Reverse zymographic analysis showed that sAPPtrc lacked an inhibitory activity against gelatinase A. Analyses of production of sAPPtrc in the presence of various metalloproteinase inhibitors showed that membrane type 1 matrix metalloproteinase (MT1-MMP), an activator of progelatinase A, is most likely responsible for the production of sAPPtrc. When the concanavalin A-stimulated HT1080 cells were cultured in the condition that inhibited MT1-MMP activity, sAPP and APP were associated with the extracellular matrix deposited by the cells, whereas these gelatinase A inhibitors in the matrix were displaced by sAPPtrc after exertion of MT1-MMP activity. Taken together, these data support a model in which MT1-MMP-catalyzed release of sAPPtrc leads to reduction of the extracellular matrix-associated gelatinase A inhibitor, sAPP, thus making it feasible for gelatinase A to exert proteolytic activity only near its activator, MT1-MMP.     

ECM regulates MT1-MMP localization with beta1 or alphavbeta3 integrins at distinct cell compartments modulating its internalization and activity on human endothelial cells

Regulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) by different extracellular matrices (ECMs) on human endothelial cells (ECs) has been investigated. First, MT1-MMP is found at the intercellular contacts of confluent ECs grown on beta1 integrin-dependent matrix such as type 1 collagen (COL I), fibronectin (FN), or fibrinogen (FG), but not on gelatin (GEL) or vitronectin (VN). The novel localization of MT1-MMP at cell-cell contacts is assessed by confocal videomicroscopy of MT1-MMP-GFP-transfected ECs. Moreover, MT1-MMP colocalizes with beta1 integrins at the intercellular contacts, whereas it is preferentially found with alphavbeta3 integrin at motility-associated structures on migrating ECs. In addition, clustered integrins recruit MT1-MMP and neutralizing anti-beta1 or anti-alphav integrin mAb displace MT1-MMP from its specific sites, pointing to a biochemical association that is finally demonstrated by coimmunoprecipitation assays. On the other hand, COL I, FN, or FG up-regulate cell surface MT1-MMP on confluent ECs by an impairment of its internalization, whereas expression and internalization are not modified on GEL or VN. In addition, MT1-MMP activity is diminished in confluent ECs on COL I, FN, or FG. Finally, MT1-MMP participates and cooperates with beta1 and alphavbeta3 integrins in the migration of ECs on different ECM. These data show a novel mechanism by which ECM regulates MT1-MMP association with beta1 or alphavbeta3 integrins at distinct cellular compartments, thus modulating its internalization, activity, and function on human ECs.     

Prointegrin maturation follows rapid trafficking and processing of MT1-MMP in Furin-Negative Colon Carcinoma LoVo Cells

Understanding the function of invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP) is of paramount importance for understanding cancer biology. MT1-MMP is synthesized in cells as a latent zymogen that requires the cleavage of its prodomain to exert the proteolytic activity. The mature alphav integrin subunit is also generated by endoproteolytic cleavage of the alphav subunit precursor (pro-alphav). Cleavage by furin is considered to be a principal event in the activation of both MT1-MMP and pro-alphav. To elucidate the alternative activation pathway of MT1-MMP and pro-alphav, we employed furin-negative LoVo cells, which co-express MT1-MMP with integrin alphavbeta3. In these cells the MT1-MMP proenzyme was rapidly trafficked to the plasma membrane via an unconventional Brefeldin A-resistant pathway and, then, autocatalytically processed on the cell surface. Next, the MT1-MMP activity converted the cell surface-associated pro-alphav into the mature alphav integrin, represented by the disulfide-bonded heavy and light chains, and promoted the formation of the functional integrin alphavbeta3 heterodimer. These events stimulated cell motility in vitro, and malignant invasion and tumor growth in vivo. Our data suggest that in furin-negative colon carcinoma cells MT1-MMP is autocatalytically processed and the active protease then operates as a prointegrin convertase. Our findings argue strongly that the processing by furin is not a prerequisite for the activation of MT1-MMP.     

Membrane-type matrix metalloproteinase-1 and -3 activity in primate smooth muscle cells.

Membrane-type matrix metalloproteinases-1 and -3 (MT1- and MT3-MMPs) are expressed by activated smooth muscle cells (SMCs) both in vitro and in vivo (19). To define their functions in SMCs, we transduced MT1- and MT3-MMP cDNAs into baboon SMCs by using adenoviral vectors. Overexpression of MT1-MMP increased the conversion of proMMP-2 to the intermediate and active forms. In contrast, in MT3-MMP-overexpressing cells, MMP-2 was activated partially. Immunoblot analyses revealed that MT1-MMP protein was present in the SMCs and accumulated in the presence of the synthetic MMP inhibitor, BB94, or tissue inhibitor of metalloproteinase-2 (TIMP-2). However, MT3-MMP protein was detectable only when BB94, but not TIMP-2, was present. Zymographic analyses showed that MT3-MMP had much stronger casein- and gelatin-degrading activities than did MT1-MMP. Furthermore, when MT3-MMP and MT1-MMP were coexpressed, MT1-MMP degradation was enhanced; this result supports the possibility that MT3-MMP can degrade MT1-MMP. SMCs overexpressing either MT1- or MT3-MMP exhibited altered morphology, without changing their proliferation. This alteration was prevented by BB94 addition. The cells, which underwent this change, showed reduced adhesion to both collagen and fibronectin and increased migration in a Boyden chamber. The present study demonstrates that MT1- and MT3-MMPs have different enzymatic activities but may nevertheless affect SMC function in the same way.     

Direct regulation of membrane type 1 matrix metalloproteinase following myocardial infarction causes changes in survival, cardiac function, and remodeling

The membrane type 1 matrix metalloproteinase (MT1-MMP) is increased in left ventricular (LV) failure. However, the direct effects of altered MT1-MMP levels on survival, LV function, and geometry following myocardial infarction (MI) and the proteolytic substrates involved in this process remain unclear. MI was induced in mice with cardiac-restricted overexpression of MT1-MMP (MT1-MMPexp; full length human), reduced MT1-MMP expression (heterozygous; MT1-MMP(+/-)), and wild type. Post-MI survival was reduced with MT1-MMPexp and increased with MT1-MMP(+/-) compared with WT. LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT post-MI and was higher in the MT1-MMP(+/-) mice. In vivo localization of MT1-MMP using antibody-conjugated microbubbles revealed higher MT1-MMP levels post-MI, which were the highest in the MT1-MMPexp group and the lowest in the MT1-MMP(+/-) group. LV collagen content within the MI region was higher in the MT1-MMPexp vs. WT post-MI and reduced in the MT1-MMP(+/-) group. Furthermore, it was demonstrated that MT1-MMP proteolytically processed the profibrotic molecule, latency-associated transforming growth factor-1-binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by over fourfold in the post-MI MT1-MMPexp group and reduced in the MT1-MMP(+/-) group, which was directionally paralleled by phospho-Smad-3 levels, a critical signaling component of the profibrotic transforming growth factor pathway. We conclude that modulating myocardial MT1-MMP levels affected LV function and matrix structure, and a contributory mechanism for these effects is through processing of profibrotic signaling molecules. These findings underscore the diversity of biological effects of certain MMP types on the LV remodeling process.     

Membrane type 1-matrix metalloproteinase is regulated by chemokines monocyte-chemoattractant protein-1/ccl2 and interleukin-8/CXCL8 in endothelial cells during angiogenesis

We have investigated the putative role and regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in angiogenesis induced by inflammatory factors of the chemokine family. The absence of MT1-MMP from null mice or derived mouse lung endothelial cells or the blockade of its activity with inhibitory antibodies resulted in the specific decrease of in vivo and in vitro angiogenesis induced by CCL2 but not CXCL12. Similarly, CCL2- and CXCL8-induced tube formation by human endothelial cells (ECs) was highly dependent on MT1-MMP activity. CCL2 and CXCL8 significantly increased MT1-MMP surface expression, clustering, activity, and function in human ECs. Investigation of the signaling pathways involved in chemokine-induced MT1-MMP activity in ECs revealed that CCL2 and CXCL8 induced cortical actin polymerization and sustained activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. Inhibition of PI3K or actin polymerization impaired CCL2-induced MT1-MMP activity. Finally, dimerization of MT1-MMP was found to be enhanced by CCL2 in ECs in a PI3K- and actin polymerization-dependent manner. In summary, we identify MT1-MMP as a molecular target preferentially involved in angiogenesis mediated by CCL2 and CXCL8, but not CXCL12, and suggest that MT1-MMP dimerization might be an important mechanism of its regulation during angiogenesis.     

Oxidative stress and prostate cancer progression are elicited by membrane-type 1 matrix metalloproteinase

Oxidative stress caused by high levels of reactive oxygen species (ROS) has been correlated with prostate cancer aggressiveness. Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP), which has been implicated in cancer invasion and metastasis, is associated with advanced prostate cancer. We show here that MT1-MMP plays a key role in eliciting oxidative stress in prostate cancer cells. Stable MT1-MMP expression in less invasive LNCaP prostate cancer cells with low endogenous MT1-MMP increased activity of ROS, whereas MT1-MMP knockdown in DU145 cells with high endogenous MT1-MMP decreased activity of ROS. Expression of MT1-MMP increased oxidative DNA damage in LNCaP and in DU145 cells, indicating that MT1-MMP-mediated induction of ROS caused oxidative stress. MT1-MMP expression promoted a more aggressive phenotype in LNCaP cells that was dependent on elaboration of ROS. Blocking ROS activity using the ROS scavenger N-acetylcysteine abrogated MT1-MMP-mediated increase in cell migration and invasion. MT1-MMP-expressing LNCaP cells displayed an enhanced ability to grow in soft agar that required increased ROS. Using cells expressing MT1-MMP mutant cDNAs, we showed that ROS activation entails cell surface MT1-MMP proteolytic activity. Induction of ROS in prostate cancer cells expressing MT1-MMP required adhesion to extracellular matrix proteins and was impeded by anti-β1 integrin antibodies. These results highlight a novel mechanism of malignant progression in prostate cancer cells that involves β1 integrin-mediated adhesion, in concert with MT1-MMP proteolytic activity, to elicit oxidative stress and induction of a more invasive phenotype.     

The non-redundant role of N-WASP in podosome-mediated matrix degradation in macrophages

Wiskott-Aldrich Syndrome Protein (WASP) is a hematopoietic cell-specific regulator of Arp2/3-dependent actin polymerization. Despite the presence of the highly homologous N-WASP (neural-WASP), macrophages from WAS patients are devoid of podosomes, adhesion structures in cells of the monocytic lineage capable of matrix degradation via matrix metalloproteases (MMPs), suggesting that WASP and N-WASP play unique roles in macrophages. To determine whether N-WASP also plays a unique role in macrophage function, N-WASP expression was reduced using silencing RNA in a sub-line of RAW 264.7 macrophages (RAW/LR5). Similar to reduction in WASP levels, cells with reduced N-WASP levels were rounder and less polarized. Interestingly, podosomes still formed when N-WASP was reduced but they were unable to perform matrix degradation. This defect was rescued by re-expression of N-WASP, but not by over-expression of WASP, indicating that these proteins play distinct roles in podosome function. Additionally, reducing N-WASP levels mistargets the metalloprotease MT1-MMP and it no longer localizes to podosomes. However, N-WASP was only found to co-localize with MT1-MMP positive vesicles at podosomes, suggesting that N-WASP may play a role on the targeting or fusion of MMP-containing vesicles to podosomes in macrophage-like cells.     

Rho-ROCK-Myosin Signaling Meditates Membrane Type 1 Matrix Metalloproteinase-induced Cellular Aggregation of Keratinocytes

Membrane type 1-matrix metalloproteinase (MT1-MMP, MMP14), which is associated with extracellular matrix (ECM) breakdown in squamous cell carcinoma (SCC), promotes tumor formation and epithelial-mesenchymal transition. However, in this report we demonstrate that MT1-MMP, by cleaving the underlying ECM, causes cellular aggregation of keratinocytes and SCC cells. Treatment with an MMP inhibitor abrogated MT1-MMP-induced phenotypic changes, but decreasing E-cadherin expression did not affect MT1-MMP-induced cellular aggregation. As ROCK1/2 can regulate cell-cell and cell-ECM interaction, we examined its role in mediating MT1-MMP-induced phenotypic changes. Blocking ROCK1/2 expression or activity abrogated the cellular aggregation resulting from MT1-MMP expression. Additionally, blocking Rho and non-muscle myosin attenuated MT1-MMP-induced phenotypic changes. Moreover, SCC cells expressing only the catalytically active MT1-MMP protein demonstrated increased cellular aggregation and increased myosin II activity in vivo when injected subcutaneously into nude mice. Together, these results demonstrate that expression of MT1-MMP may be anti-tumorigenic in keratinocytes by promoting cellular aggregation.     

Identification and Characterization of Lutheran Blood Group Glycoprotein as a New Substrate of Membrane-type 1 Matrix Metalloproteinase 1 (MT1-MMP): A SYSTEMIC WHOLE CELL ANALYSIS OF MT1-MMP-ASSOCIATING PROTEINS IN A431 CELLS*

Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates.Cells in tissues are surrounded by an extracellular cellular matrix that interacts with cells to regulate their activity (1, 2). Matrix metalloproteinases (MMPs)³ are endopeptidases responsible for extracellular matrix degradation and thereby regulate turnover of the extracellular matrix. However, recent studies have demonstrated that substrates of MMPs are expanded to a variety of pericellular proteins.MT1-MMP/MMP14 is an integral membrane proteinase that cleaves multiple proteins in the pericellular milieu and thereby regulates various cell functions. Substrates of MT1-MMP identified to date include extracellular matrix proteins (type I collagen, fibronectin, vitronectin, laminin-1 and -5, and others), cell adhesion molecules (CD44, syndecan-1, and αv integrin), cytokines (SDF-1 and transforming growth factor-β and others), and latent forms of pro-MMPs (pro-MMP-2 and pro-MMP13) (3–5). Processing of these proteins by MT1-MMP alters their activities and thereby regulates a variety of cellular functions, such as motility, invasion, growth, differentiation, and apoptosis. Consistent with these functions, forced expression of MT1-MMP in tumor cells enhances behavior consistent with increased malignancy, such as rapid tumor growth, invasion, and metastasis (6). However, MT1-MMP is normally expressed in various types of cell and mice deficient in MT1-MMP expression (MT1^−/−) display pleiotropic defects (7–10). However, we as yet have only limited knowledge of the physiological substrates of MT1-MMP that could explain such pleiotropic effects.Proteases interact with their substrates at least transiently, but in some cases such interaction is more stable. For instance, type I collagen binds MT1-MMP via a hemopexin-like domain and is cleaved (11, 12). Cleavage of collagen by MT1-MMP regulates cell growth and invasion in a collagen-rich environment (13). CD44, a hyaluronic acid receptor, also binds to the hemopexin of MT1-MMP and is cleaved (14). Expression of CD44 and MT1-MMP in tumor cells promotes cell migration, accompanied by the shedding of CD44 by MT1-MMP (14, 15). pro-MMP-2, which is cleaved by MT1-MMP for activation, forms a tri-molecular complex with MT1-MMP and TIMP-2 (3, 16). Therefore, screening of proteins that associate with MT1-MMP may provide a systematic method to identify potential substrates of MT1-MMP in cells. In addition, these proteins may also be regulatory proteins of MT1-MMP.To identify proteins associating with MT1-MMP in different types of tumor cells, we first studied conditions for cell lysis using malignant melanoma A375 cells and following purification method of the proteins as reported recently (17). Proteins purified in this manner were analyzed by high-throughput proteomic analysis (18–21). Interestingly, approximately one-half of the membrane proteins identified in our previous study could be cleaved by MT1-MMP at least in vitro. Here, we applied this approach to human carcinoma cells (A431) that originate from epidermoid cells and further validated the systemic whole cell analysis method. To evaluate whether the MT1-MMP-associated membrane proteins so identified include physiological targets of MT1-MMP activity, we select one of them, Lutheran blood group glycoprotein (Lu), and evaluate its processing in A431 cells.     

Matrix-dependent proteolysis of surface transglutaminase by membrane-type metalloproteinase regulates cancer cell adhesion and locomotion

Cell invasion requires cooperation between adhesion receptors and matrix metalloproteinases (MMPs). Membrane type (MT)-MMPs have been thought to be primarily involved in the breakdown of the extracellular matrix. Our report presents evidence that MT-MMPs in addition to the breakdown of the extracellular matrix may be engaged in proteolysis of adhesion receptors on tumor cell surfaces. Overexpression of MT1-MMP by glioma and fibrosarcoma cells led to proteolytic degradation of cell surface tissue transglutaminase (tTG) at the leading edge of motile cancer cells. In agreement, structurally related MT1-MMP, MT2-MMP, and MT3-MMP but not evolutionary distant MT4-MMP efficiently degraded purified tTG in vitro. Because cell surface tTG represents a ubiquitously expressed, potent integrin-binding adhesion coreceptor involved in the binding of cells to fibronectin (Fn), the proteolytic degradation of tTG by MT1-MMP specifically suppressed cell adhesion and migration on Fn. Reciprocally, Fn in vitro and in cultured cells protected its surface receptor, tTG, from proteolysis by MT1-MMP, thereby supporting cell adhesion and locomotion. In contrast, the proteolytic degradation of tTG stimulated migration of cells on collagen matrices. Together, our observations suggest both an important coreceptor role for cell surface tTG and a novel regulatory function of membrane-anchored MMPs in cancer cell adhesion and locomotion. Proteolysis of adhesion proteins colocalized with MT-MMPs at discrete regions on the surface of migrating tumor cells might be controlled by composition of the surrounding ECM.     

Induction of autophagy biomarker BNIP3 requires a JAK2/STAT3 and MT1-MMP signaling interplay in Concanavalin-A-activated U87 glioblastoma cells

Plant lectins have been considered as possible anti-tumor drugs because of their property to induce autophagic cell death. Given that expression of membrane type-1 matrix metalloproteinase (MT1-MMP) has been found to regulate expression of the autophagy biomarker Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), we sought to investigate possible signaling interplay mechanisms between MT1-MMP and BNIP3 in Concanavalin-A (ConA) lectin-activated U87 glioblastoma cells. ConA induced acidic vacuole organelle formation as well as BNIP3 and MT1-MMP gene and protein expressions, whereas only BNIP3 expression was dose-dependently inhibited by the JAK2 tyrosine kinase inhibitor AG490 suggesting a requirement for some STAT-mediated signaling. Gene silencing of MT1-MMP and of STAT3 abrogated ConA-induced STAT3 phosphorylation and BNIP3 expression. Correlative analysis shows that STAT3 signaling events occur downstream from MT1-MMP induction. Overexpression of a full length MT1-MMP recombinant protein led to increased BNIP3 gene and protein expressions. The cytoplasmic domain of MT1-MMP was also found necessary for transducing STAT3 phosphorylation. Among JAK1, JAK2, JAK3, and TYK2, only JAK2 gene silencing abrogated ConA's effects on MT1-MMP and BNIP3 gene and protein expressions. Our study elucidates how MT1-MMP signals autophagy, a process which could contribute to the chemoresistance phenotype in brain cancer cells.