Establishment and validation of computational model for MT1-MMP dependent ECM degradation and intervention strategies

Abstract

MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion.

Figure 1. MT1-MMP is required for invadopodia-related ECM degradation.

(A) Protein levels of MT1-MMP were assessed by Western blot analysis using anti-MT1-MMP antibody. Actin as a loading control. (B) Representative images show Dylight 633-labeled fibronectin (Green) and actin (Red). (C) Quantification of fibronectin degradation area in Figure 1 B (n = 3). (D) Representative images of MT1-Luo localization (Green), actin staining (Red) and Dylight 633-labeled fibronectin (purple).

Figure 2. MT1-MMP transport to invadopodia via lysosomal secretion.

(A) MT1-MMP-phLuorin-expressing SCC61 cells cultured on Dylight 633-labeled fibronectin were subjected to FRAP- continuous photobleaching experiments. One half of the invadopodia area, indicated by the open box area in region 1, was the FRAP experimental area. Region 2 was a continuous photobleaching area. (B) Quantification of fluorescence recovery in the Figure 2A FRAP region is calculated. (C) Representative images of FRAP experiments at invadopodia without or with bafilomycin. (D) The recovery of FRAP signals are shown in the absence (blue circles) and in the presence (pink circles) of bafilomycin. Reconstructed time courses of fluorescence recovery in the absence (blue line) and in the presence (pink line)of bafilomycin at invadopodia are also shown. The reconstructed FRAP signals show a good agreement with experimental data.

Figure 3. Schematically illustrated model for a rapid turnover.

Two pools of MT1-MMP, pool X (P_X) and pool D (P_D), were assumed. In pool X, insertion was dependent on the surface density of MT1-MMP, and internalization proceeds at a constant rate. While in pool D, insertion proceeds at a constant rate, and internalization was dependent on the surface density.

Figure 4. Outline of the model.

(A) MT-MMPs in the membrane are dimerized, bound with TIMP-2, and activate proMMP-2. Any MT1-MMP, which is TIMP-2-free, was assumed to degrade ECM. MT1-MMP and all of complexes were assumed to be internalized. (B) Complete diagram of interaction between MT1-MMP, TIMP-2 and proMMP-2. Insertion is shown by a thick arrow for MT1-MMP, and internalization of MT1-MMP is shown in broken-lined arrows with internalized species at small squares. MT1-MMP, TIP-2 and proMMP-2 are designated as M14, T2 and M2 for simplicity.

Figure 5. Simulation results of ECM degradation.

(A) Simulated ECM degradation with the initial TIMP-2 concentration at 180 nM is shown as a black line. Degradation rate, which is the rate of degradation in µM/s, is shown in red. (B) The time course of ECM degradation with several turnover intervals. If the turnover interval is increased by the factors shown by small numbers, a longer time is required for the same degree of degradation, reaching the state of no turnover (red curve). τ_h is the time required to reach 50% degradation. (C) τ_h increases concomitantly with an increase in the reduction factor. There is a rapid increase in τ_h when the reduction factor is increased tenfold or more. (D) Concomitantly with the increase in reduction factor, the degradation rate (black) quickly decreases and stays almost unchanged at reduction factor increases larger than tenfold. In contrast, the concentration of inactivated MT1-MMP complexes, which cannot degrade ECM or activate proMMP-2, is increased and reaches a plateau level around a reduction factor of 10.

Figure 6. Simulation results for spatiotemporal model.

(A) Simulated degradation of ECM. With rapid turnover, MT1-MMP causes degradation of ECM (top panel), while in its absence, no appreciable ECM degradation is seen (bottom panel). (B) The time course of ECM degradation in the absence and presence of the turnover of MT1-MMP. The importance of the turnover for the effective degradation of ECM is clearly seen.

Figure 7. Reduction in the ECM-degradation efficacy by a reduced turnover rate and/or reduced concentration of MT1-MMP.

The degradation efficacy is defined as 1/τ_h and plotted as normalized values at the control condition (reduction factor = 1). A larger degradation efficacy indicates a faster degradation of ECM. At the same reduction factor, the reduction in degradation efficacy was higher for a reduced concentration (conc.) than for a reduced turnover rate (turnover). If both were reduced simultaneously, however, an unexpectedly larger reduction in the ECM reduction was observed (turnover+conc.).