Focal Adhesion Kinase Induces Matrix Metalloproteinase-2 by Involving α5β1-Mediated Signaling in Breast Cancer Cell, MCF-7

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Acta Medica International,2015,2,1,29-39.
Published:January 2015
Type:Original Article

Focal Adhesion Kinase Induces Matrix Metalloproteinase-2 by Involving α5β1-Mediated Signaling in Breast Cancer Cell, MCF-7

Triparna Sena1, Kirat Kumar Gangulya, Jaydip Biswasb, Amitava Chatterjeea*
aDepartment of Receptor Biology & Tumor Metastasis, Chittaranjan National Cancer Institute, 37, S P Mukherjee Road, Kolkata,
India,

1Present Address: Department of Thoracic Head and Neck Medical Oncology, MD Anderson Cancer Center, Houston, Texas,
USA,

aDepartment of Receptor Biology & Tumor Metastasis, Chittaranjan National Cancer Institute, 37, S P Mukherjee Road, Kolkata,
India,

bDirector and Head, Division of Surgical Oncology, Chittaranjan National Cancer Institute, 37, S P Mukherjee Road, Kolkata, India,
aDepartment of Receptor Biology & Tumor Metastasis, Chittaranjan National Cancer Institute, 37, S P Mukherjee Road, Kolkata, India

Abstract:

Introduction: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays a pivotal role in cell invasion. Matrix metalloproteinases (MMPs) are implicated as the key players in cancer cell invasion. Hence, the role of FAK in MMP regulation is very important in understanding tumor progression. Materials and Methods: Here, we studied the role of FAK, its association with other signaling kinases and involvement in the α5β1 integrin receptor-mediated regulation of MMP-2 activity and expression in human breast cancer cell line MCF-7. Results: Immuno blot analysis revealed that FN treatment causes phosphorylation of FAK and FAK gets localized at the cell attachment focal point of MCF-7 cells. FN treatment did not change the mRNA status of FAK but enhanced mRNA level of MMP-2 and MT1-MMP, also caused down regulation of TIMP-2. Co-imunoprecipitation and inhibitor studies revealed the association of FAK with α5β1, Paxillin, PI3K and ERK. siRNA studies revealed that FAK is critical in regulation of activity and expression of MMP-2 and downstream signaling kinases. Conclusion: Th e interaction of α5β1 integrin with FN initiates a signaling cascade with FAK as its central player. FAK gets phosphorylated and in turn associates with tyrosine kinases like PI3K and ERK. FAK also activates PI3K and ERK that serve as very crucial mediators of the signaling pathway leading to induction of MMP-2 activity and resulting invasion of breast cancer cell, MCF-7.

Effect of FN treatment on the expression of FAK and p-FAK in MCF-7 cells: MCF-7 cells (300,000 cells/ml) were grown in SFCM in absence (C), in presence of 20 μg/ml FN coated for 30 mins (1), 1 hour (2) and 2 hours (3). The cells were collected; extracted and equal protein (100 μg) was subjected to western blot analysis with anti-FAK (upper panel) and anti-p-FAK (lower panel) antibody (1:1000 dilution for 1½ hrs at 37°C). β-tubulin was used as internal control and done in parallel to all the blots. The accompanying graphs represent the comparative densitometric/quantitative analysis of the band intensities using Image J Launcher (version 1.4.3.67). Data are means±SEM of three experiments. (B) Determination of gene expression of MMP-2, FAK, TIMP-2 and MT1-MMP by Quantitative Real-Time RT-PCR: MCF-7 cells (300,000 cells/ml) were grown in absence (C) and in presence 20 μg/ml FN coated for 1 hour and 2 hours in SFCM. Total RNA was extracted from control and experimental MCF-7 cells (1×106 cells). 2 steps RT-PCR wa