Establishment and Validation of Computational Model for MT1-MMP Dependent ECM Degradation and Intervention Strategies

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

Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia

Microtubules and intermediate filaments cooperate with actin and other components of filopodia during invadopodia maturation

5′-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion

Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5′-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2–Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease

Dysregulation of Gene Expression in the Artificial Human Trisomy Cells of Chromosome 8 Associated with Transformed Cell Phenotypes

A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells) by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression