Abstract #2050

Cell migration is a tightly regulated event critical for normal development and adult wound healing, but it is also a key step in the tumor metastasis cascade. Growth factors initiate intra-cellular signaling pathways that stimulate cell movement by virtue of remodeling the actin cytoskeleton. Fine regulation of this process renders a degree of polarity to the cells, altering their cytoskeleton and providing the requisite mechanical torsion to move forward. Breast cancer cells extravasate from their primary site and travel to other organs forming metastatic colonies.Various signaling molecules including members of the Mitogen Activated Protein Kinase (MAPK) such as c-Jun N-Terminal Kinase (JNK) have been implicated in regulating cancer cell migration. How growth factors transmit signals to form dorsal ruffles and filopodia for cell migration is not precisely understood. Additionally, JNK is encoded by three genes, resulting in 10 isoforms, hence its role in metastasis is complicated. Here, we used the Polyoma Virus Middle T Antigen (PyVMT) transgenic mouse model where wildtype jnk2 was expressed or knocked out. In tumors we found that PyVMT jnk2-/- tumors express higher Epidermal Growth Factor Receptor Substrate 8 (EPS8) mRNA and protein. EPS8 was originally identified as a substrate for Epidermal Growth Factor Receptor (EGFR), and was recently reported to have an actin capping function. Hence, EPS8 is a prime candidate for connecting the EGFR signaling to actin cytoskeletion remodeling, and therefore mediate cell migration. In vitro using Boyden Chamber based migration assays, we found that the PyV MT jnk2+/+ cells migrated fivefold more than the PyV MT/jnk2-/- cells. Pan inhibition of JNK using a small peptide inhibitor TAT-JIP VII significantly reduced migration of the PyV MT/jnk2+/+ cells but was ineffective at further reducing PyVMT jnk2-/- cell migration, suggesting that the JNK2 is the major isoform to convey this function. Furthermore, shRNA mediated knock down of EPS8 in the PYVMT jnk2-/- cells, increased migration of these cells to levels comparable with the wild type counterparts. We subsequently undertook immunocytochemistry studies to evaluate EPS8 localization and cytoskeletal changes upon serum stimulation. Cells expressing JNK2 showed more dorsal ruffles upon serum stimulation, and EPS8 localized to the plasma membrane and in dorsal ruffles. In contrast, EPS8 was predominantly localized in focal adhesions in the PyVMT jnk2-/- cells, which may explain their reduced migration potential. Together our model supports an integral role for JNK2 regulation of EPS8 expression and localization to facilitate cell migration. Our current studies are aimed at further deciphering the JNK/EPS8 pathway to understand the exact molecular mechanism by which these proteins regulate a critical step in mammary cancer cell metastasis.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2050.

Thirty-first San Antonio Breast Cancer Symposium Dec 10-14, 2008; San Antonio, TX