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Title: Establishment and Validation of Computational Model for MT1-MMP Dependent ECM Degradation and Intervention Strategies}
Article:
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.
Introduction:
Some matrix metalloproteinases( MMPs) are proinvasive and employed by motile and invasive cells to degrade extracellular matrix( ECM). Among the 23 MMPs in mammals, integral membrane type MMPs, especially MT1-MMP, are believed to provide major contributions to cancer cell invasion. In fact, specific inhibition of MT1-MMP activity or knockdown of its expression suppresses not only cancer cell invasion in vitro but also tumor growth in mice[1],[2],[3],[4]. Therefore, MT1-MMP must be an important component of the cellular invasion machinery, and indeed, active ECM degradation is produced at structures called invadopodia[5],[6], which are specialized membrane protrusions extending into the ECM. Thus, invadopodia are hypothesized as machinery to degrade ECM at the initial stage of cancer cell invasion. MT1-MMP was reported to be enriched in invadopodia[7],[8],[9]. In addition, the accumulation of MT1-MMP in invadopodia was followed by the degradation of ECM at the same sites[8]. These observations indicate that MT1-MMP plays a crucial role in ECM degradation at invadopodia[10]. However, the ECM-degrading activity of MT1-MMP will not be persistent, because cell-surface MT1-MMP is inactivated by TIMP-2 which is an endogenous MT1-MMP inhibitor that inhibits the proteolytic degradation of ECM. Therefore, a continuous supply of active MT1-MMP to the surface seems to be critical for maintaining ECM degrading[11]. Several reports have been published that support the importance of a continuous supply of MT1-MMP. It is reported that MT1-MMP is delivered to the invasion front of cells by vesicle transport in a 3D collagen matrix[12]. MT1-MMP is delivered to invadopodia by the exocyst complex, and the process is regulated by cortactin, IQGAP1 and RhoA[8],[13]. VAMP7, a member of the v-snare protein family, also regulates MT1-MMP transport to invadopodia, and MT1-MMP appears to be stored in late endosomal/lysosomal vesicles together with VAMP7[14]. In addition to the supply of MT1-MMP to the surface, the internalization of MT1-MMP was also reported[15],[16]. MT1-MMP was colocated with clathrin, and the internalization proceeded through a clathrin-dependent pathway[16],[17],[18] or in a caveolae-mediated manner[19],[20]. Inhibition of caveolin-1 by siRNA was shown to block the MT1-MMP-dependent ECM degradation[21]. All these previous reports strongly suggest a crucial role of a continuous supply and internalization of surface MT1-MMP( i. e. the turnover of MT1-MMP), particularly by vesicle-mediated mechanisms. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to ECM degradation at invadopodia have not been clarified. In the present study, we attempted to measure experimentally the turnover rate of MT1-MMP at a single invadopodium, and to elucidate the role of the turnover in ECM degradation by computer simulations. First, we analyzed the turnover rate of MT1-MMP at a single invadopodium by FRAP( Fluorescence Recovery after Photobleaching) experiments using a chimeric MT1-MMP with a pH-sensitive GFP protein( pHLuorin). The recovery of fluorescence was plotted in a double-exponential plot, which showed time constants of 26. 0 s and 259 s. Based on these experiments, we constructed a computational model to analyze the role of the rapid MT1-MMP turnover. Simulations showed no appreciable ECM degradation when the rapid turnover was inhibited in our simulation experiments. This was also true in the actual experiments. In addition, simulations have shown a decrease in the ECM degradation concomitantly with an increased reduction of the turnover rate of MT1-MMP. Thus, we have quantified the rate of turnover of MT1-MMP at single invadopodia and shown its critical role in ECM degradation both experimentally and by computer simulations. Furthermore, our simulations have shown a synergetic effect on the inhibition of ECM degradation by simultaneous reductions of the MT1-MMP turnover rate and the vesicular content of it. Our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion.
Discussion:
Here we have revealed a rapid turnover of the surface MT1-MMP with time constants of 26. 0 and 259 s at invadopodia by experiments. In addition, we have demonstrated both by experiments and simulations that ECM degradation was blocked by blocking of the rapid turnover of MT1-MMP. Thus we have revealed a critical role of the rapid turnover for the effective ECM degradation at invadopodia( Figure S9 in Text S1). Furthermore, we have shown in simulations that simultaneous reductions in the turnover rate and the vesicular content of MT1-MMP reduced the ECM degradation synergistically by simulations. It has been reported that invadopodia-associated actin comets represented a biphasic recovery in FRAP signals: the initial fast recovery( 15–20 s) followed by a second slow recovery which reached a plateau level at ∼3 min[25]. It will be of interest to compare these time constants with those for the FRAP signal in the present report. ECM degradation at invadopodia provides room for initial protrusions. Further degradation provides additional space for the elongation and enlargement of invadopodia. These processes are thought to finally lead to the invasion of cancer cells[6],[26],[27]. There is a clear evidence for the involvement of MT1-MMP in these processes[7],[8],[13]. On the other hand, MT1-MMP-containing vesicles trafficking and fusing to the plasma membrane were shown to be important for the ECM degradation[12], and inhibited or decreased ECM degradation by drugs that block vesicle transport has been reported[14],[28],[29]. Our present study presents data supporting the involvement of the rapid turnover of MT1-MMP for the initial stage of invasion. Since rapid delivery of vesicles carrying MT1-MMP to invadopodia is important for the ECM degrading activity, it is of particular interest how this delivery process is regulated. Bafilomycin inhibits acidification of lysosomes. Therefore, MT1-MMP appears to be immediately delivered to invadopodia via an endosome-lysosome pathway rather than by the Golgi apparatus to the plasma membrane, which is preferentially inhibited by brefeldin. Indeed, Steffen et al. reported that VAMP7, which associates with late endosome-lysosome vesicles and plays a role in membrane fusion, is required for MT1-MMP delivery to invadopodia and regulates invadopodia formation by supporting MT1-MMP-mediated ECM degradation. Colocalization of MT1-MMP-mCherry with GFP-VAMP7 at invadopodia accompanying ECM degrading was demonstrated[14]. The internalization of ErbB receptors( ErbBRs) has been extensively studied, and its rate constant was reported to be in the range of 0. 04–0. 2/min depending on the ligand-occupancy[30],[31]. In contrast, the recovery of fluorescence signal from MT1-MMP proceeded with time constants of 26. 0 and 259 s in our study. From these values, we estimated an internalization rate for pools D of 2. 31/min( = 60 s/26. 0 s), which is one-order of magnitude higher than that of ErbBRs. The internalization rate for transferrin receptor was reported to be around 1/min[30],[31]. We have shown a more than twofold higher rate for MT1-MMP. Such a rapid turnover of MT1-MMP at invadopodia is presumably critical for MT1-MMP to degrade ECM because MT1-MMP is inactivated rapidly by TIMP-2 as shown in our simulation results. Endocytosis of receptors has been recognized as a mechanism for their down-regulation. For example, ErbB receptors are known to be internalized upon ligand binding, and this process down-regulated receptor activity[32]. In the present model, however, the internalization of MT1-MMP in pool X increased the ECM-degrading activity by offering free sites for the insertion of new MT1-MMP onto the membrane surface. Thus the internalization of MT1-MMP can provide the opposite effect to that of ErbBR. If the renewal of the surface MT1-MMPs was blocked, the concentration of inactivated MT1-MMP increases by binding of TIMP-2 as shown in Figures 6D and Figure S8B in Text S1. This blocks both binding of MT1-MMP to ECM proteins and activation of proMMP-2. Both of these effects will lead to a decreased ECM degradation. Therefore, we conclude that the rapid turnover and repetitive insertion of surface MT1-MMP is a basis for the effective ECM degradation at invadopodia. In our model we assumed the existence of pools X and D with different insertion and internalization mechanisms for MT1 MMP. However, the observed fluorescence recovery with two time constants can be explained by two pools D or two pools X. Nevertheless, our bafilomycin experiments indicated the existence of different mechanism for the two time-constant processes. Therefore we assumed different insertion and internalization mechanisms for pools X and D. Mathematical models showing cancer cell invasion of tissue have been reported previously[33],[34],[35]. One of the main interests in these models is in the invasive behavior of a population of cells. Our model, as presented in this work, focuses on the invasive behavior of a subcellular structure, invadopodia. These two approaches are different both in spatial and temporal scales. The consolidation of the two approaches will be a challenge that will be approached in the near future. Here we have found a rapid turnover of surface MT1-MMP at invadopodia with a time constant of 26. 0 s. The reduction in the turnover rate according to both fast and slow time constants reduced the rate of ECM degradation as shown in Figures 5 and 6. In addition, with simultaneous reductions in both the turnover rate and the concentration of MT1-MMP, nonlinear and marked reduction in the ECM degradation was seen in our simulations( Figure 7). Thus, the rate of the turnover may be a critical therapeutical parameter in addition to the inhibition of MT1-MMP. The suggested combination treatment derived from our mathematical model may make it possible to inhibit invadopodia-mediated invasion selectively compared to other suggested interventions.
Prevention of invasion is important in cancer therapy . MT1-MMP is a membrane protein involved in degradation of ECM ( extracellular matrix ) that is highly expressed at invadopodia , which are small protrusions of cancer cells . ECM degradation by MT1-MMP at invadopodia is hypothesized as the initial step of cancer cell invasion . However , MT1-MMP is inhibited by the endogenous inhibitor TIMP-2 , so continuous turnover of MT1-MMP at the surface of invadopodia would be required . In agreement , it has been reported that the blockade of vesicle transport , which is one mechanism involved in the turnover , blocked the ECM degradation . However , the turnover rate of MT1-MMP at invadopodia and the extent to which the turnover is critical for the degradation of ECM have not been clarified . In this report we measured the turnover rate of MT1-MMP at a single invadopodium and found rapid turnover rates with time constants of 26 s and 259 s , which primarily depended on the vesicle transport . A computational model was constructed based on the observed kinetics . If we blocked the rapid turnover , the ECM degradation was blocked both experimentally and in simulations . These results established the role of the rapid turnover of MT1-MMP in the ECM degradation at invadopodia .