The Roles of Akt1 and Akt2 Downstream of Src Family Kinases in the Migration and Invasion of Breast Cancer Cells.

摘要

The leading cause of death in breast cancer patients is metastasis. Cancer metastasis involves a complex series of events, including cell detachment from the primary tumor, invasion into the surrounding tissue, intravasation into the circulatory system, extravasation, and growth in a new organ. Src Family Kinases (SFKs) comprise a group of non-receptor tyrosine kinases that regulate a variety of pathways that promote cell survival, growth, proliferation, motility, and invasion. Previous studies have found a correlation between the activity of SFKs and the progression of breast cancer into metastasis. Since the pathways regulated by SFKs are often activated in metastasis, I investigated whether SFKs play a role in the metastasis of breast cancer cells.;To determine whether SFKs regulate breast cancer metastasis, I analyzed the role of SFKs in the migration and invasion of a metastatic breast cancer cell line, MDA-MB-231 cells. SFKs were inhibited with expression of dominant negative Src or by treatment with the pharmacological inhibitors PP2 and SU6656. The inhibition of SFKs in MDA-MB-231 cells led to decreased cell migration and invasion through transwell migration and invasion chambers. To explore the mechanism by which SFKs regulate cellular migration and invasion, I investigated whether SFKs regulate the Akt1 and Akt2 isoforms in MDA-MB-231 breast cancer cells. Akt isoforms are serine/threonine kinases that are often activated in cancer cells. The activity of Akt isoforms were decreased in SFK-inhibited MDA-MB-231 cells as determined by immunoblot detection of the phosphorylation of Threonine 308 and Serine 473 residues, in vitro kinase assays, and immunoblot detection of the phosphorylation of an Akt substrate. To determine whether the Akt isoforms play a role in the migration and/or invasion of breast cancer cells, small interfering RNAs were used to knockdown the expression of each Akt isoform. Akt2 knockdown specifically led to decreased MDA-MB-231 cell migration and invasion by mechanisms that did not involve the attachment of cells onto extracellular matrix or the regulation of Pak1. Co-immunoprecipitation and mass spectrometry assays were used in an effort to identify an Akt2-specific binding partner that could mediate its role in breast cancer cell migration and invasion. The Arf6-GAP ACAP2 was identified as a protein that co-immunoprecipitated with Akt2-Flag, but additional analysis found that ACAP2 was immunoprecipitated non-specifically by the mouse anti-Flag antibody and does not interact with Akt2.;These results point to a role of SFKs in breast cancer metastasis, specifically in promoting cell migration and invasion. Although SFKs regulate the activation of both Akt1 and Akt2 isoforms in MDA-MB-231 cells, only Akt2 is required for the migration and invasion of MDA-MB-231 cells.