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The extracellular matrix plays a critical role in modulating the growth, motility and differentiation of cells. The response of cells to matrix proteins is mediated primarily through the integrin family of adhesion receptors. The functional mechanisms of bidirectional signaling initiated by integrins are poorly understood. Our experiments aimed at determining the mechanism by which human colon cancer GEO cells activate c-Src by “outside-in signaling” on attachment to collagen type IV (CNIV; integrin α2-mediated) which in turn controls cell adhesion and motility (inside-out signaling). The relationship of c-Src with cancer metastasis is not clear. GEO cells on attachment to CNIV (0-2h) showed, in a temporal fashion, distinct activation kinetics of c-Src (Y418). Immunoblotting showed c-Src (60 kDa) and its cleaved form (55 kDa) which seems to be cell type specific. Activation of c-Src was inhibited by antimetastatic drug PP2, a selective and potent c-Src inhibitor. Similarly, cell adhesion to CNIV was inhibited (48.3 and 71.3%) in the presence of 10 and 20 μM concentrations of PP2. Interference of signals from inside the cells by PP2 decreased the integrin α2 affinity for its ligand CNIV. To determine the role of c-Src activation in cell locomotion, we used a sensitive, real-time, and quantitative Electric Cell-Impedance Sensing (ECIS) technique. Cells were plated on gold electrodes precoated with CNIV, subconfluent cultures were treated with DMSO or with 10 and 20 μM concentrations of PP2 and cell motility (micromotion) was recorded. We observed a concentration-dependent inhibitory effect of PP2 (35.5 and 61% respectively) on cell micromotion. Blocking antibodies (P1E6) to integrin α2 inhibited cell micromotion by 60%. We further sought to identify c-Src downstream targets regulating cell motility. Treatment of cells with PP2 and MEK inhibitor U0126 inhibited ERK activation and cell micromotion in parallel fashion indicating that c-Src regulates cell micromotion via ERK/MAPK signaling. Preliminary coimmunoprecipitation experiments indicate FAK and PLCγ as partners of c-Src activation. To further study the mechanism of limited c-Src cleavage and its relation to cell locomotion, we identified the role of specific intracellular protease mu- and m- calpains. Currently, the mechanism by which calpain is activated during integrin-mediated signaling is speculative. Calpain antisense nucleotides, both anti-mu and anti-m showed inhibition in micromotion of 52 and 12 % respectively, demonstrating the major role of mu isoform in integrin α2-mediated cell motility. In conclusion, our results suggest integrin α2-mediated c-Src and calpain activation critically regulate motility, the former via ERK/MAPK pathway. Further, our data support the notion that c-Src and mu-calpain are potential target molecules for designing novel therapeutic interventions, especially for cancer metastasis.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]