Figure 7. FA-mediated matrix degradation promotes tumor cell invasion. PANC-1 cells were transfected with MT1-MMP WT or mutants (a), and invasive potential was analyzed by the Boyden chamber assay in which filters were coated with 1 mg/ml Matrigel. Constructs that facilitate FA-type degradation (MT1-WT, CP-FAT) significantly promoted invasion com- pared with the GFP vector or the MT1-Y573F mutant that cannot bind p130Cas (d). To test if a reduction of FAK and p130Cas exerts an in- hibitory effect, HT-1080 cells were transfected with siRNA twice, and then rescued with either WT or mutant proteins (b and c). Although knock- down of either FAK or p130Cas significantly impaired invasion (e), this could be reversed by expression of WT proteins. Mutant proteins that cannot reconstitute the FAK–p130Cas–MT1 complex did not provide a functional rescue (e). The graphed data represent results from three independent experiments ± SD, and were nor- malized to the average of control cells (as 1). (f) Illustration depicting the central theme of this study: a FAK–p130Cas complex mediates the docking of MT1-MMP at FAs. MT1-MMP is traf- ficked to the surface via exocytotic vesicles for fusion with the membrane to release MT1 that can be recruited by the FAK–p130Cas complex at FAs. In addition, MT1 is recruited to lamelli- podia by CD44 that may diffuse along the membrane for recruitment to FAs. Src kinase activity at FAs mediates MT1 phosphorylation at Y573 that facilitates an interaction with the FAK–p130Cas complex at FAs. invadopodia (Fig. S5, b and d), exhibited a marked inhibition in the formation of centrally located ECM degradation sites by PP2 treatment (Fig. 6, d and e). Therefore, we tested if a disruption of the FAK–p130Cas–MT1 complex in BxPC-3 cells affects ECM degradation at invadopodia as it did at FAs (Figs. 2 and 4). BxPC3 cells were treated with siRNAs against MT1, FAK, or p130Cas, then plated on gelatin for 16 h, and stained for the invadopodia marker cortactin (Fig. 6, a–c). As expected, the MT1 knockdown affected invadopodia-type degradation due to an inhibition of invadopodia formation (Fig. 6 a). In contrast, FAK or p130Cas knockdown did not affect invadopodia function (Fig. 6, b and c), suggesting that, unlike that observed at FAs, MT1-MMP’s function at inva- dopodia is independent of the FAK–p130Cas complex. Disruption of the FAK–p130Cas–MT1 complex impairs tumor cell invasion in vitro The observations described above have implicated a novel role for a FAK–p130Cas–MT1 complex in the degradation of ECM at FAs. To test if this FA-centric protein complex contributes to tumor cell invasion, an in vitro Boyden chamber invasion assay was implemented. As PANC-1 cells do not express en- dogenous MT1-MMP, cells were transfected to express MT1 WT, Y573F, or MT1CP-FAT (Fig. 7 a). Cells were suspended in serum-free medium and plated in the upper chamber 24 h after transfection, then challenged to invade through a Matrigel- coated filter into the lower chamber containing full serum (10% FBS) medium. After a 36-h period, cells were fixed and the number of invaded cells per field was quantified. Impor- tantly, the protein components that facilitate ECM degradation at FAs (MT1 WT and MT1CP-FAT, Fig. 3 e) also significantly enhanced invasion through the filter compared with MT1 Y573F or GFP vector (Fig. 7 d). As a reciprocal approach, HT-1080 cells were transfected twice with siRNA against FAK or p130Cas, and subsequently rescued with either WT or mutant constructs (Fig. 7, b and c). Interestingly, cells with reduced levels of FAK or p130Cas exhibited significantly reduced invasion compared with siRNA control cells, which is consistent with previous reports (Ueda et al., 2003; Chan et al., 2009; Cunningham-Edmondson and Hanks, 2009). Moreover, reexpression of the respective WT proteins rescued invasion whereas expression of the mutants FAK P2/3A and CasSD that perturb formation of the trimeric complex did not (Fig. 7 e). Taken together, these data suggest that the targeting of MT1-MMP to FAs by the FAK–p130Cas complex is important for cancer cell invasion. Discussion In this study we provide evidence for a novel, unconventional function of focal adhesions that entails the regulated degradation of the surrounding matrix (Fig. 1 and Fig. S1). This process is observed in a wide variety of human tumor cell types such as the fibrosarcoma cell line HT1080 and several different pancreatic tumor cell lines including BxPC3, HuPT3, CFPAC-1, and HPAF. It is mediated by, and dependent upon, MT1-MMP as cells with- out this protease do not degrade matrix at these sites (Fig. 1 and Fig. S2). In addition, we have found that the MT1-MMP is tar- geted to FAs through a novel interaction with a FAK–p130Cas complex. Disruption of these interactions results in a significant reduction of ECM degradation at FAs (Figs. 2–4), but not at 382 JCB • VOLUME 196 • NUMBER 3 • 2012