A novel pathway of Stat3 interaction with the Rho GTPases

297 Unlike cultured cells, cells in a tumor have extensive opportunities for adhesion to their neighbors in a three-dimensional structure, therefore in the study of signal transduction it is important to take into account the effect of surrounding cells. Stat3 has emerged as an important signal transducer with a role in the etiology of many cancers. In resting cells, Stat3 is inactive in the cytoplasm but following receptor activation it is phosphorylated at tyr-705, dimerizes and migrates to the nucleus where it activates transcription from a number of genes involved in cell division and survival. Our lab has demonstrated that cell-to-cell adhesion, as observed in confluent cultured cells, can lead to a dramatic increase in Stat3 activity, which peaks at 2-4 days post-confluence. Most importantly, this Stat3 activation required Calcium but was found to be resistant to inhibition of a number of tyrosine kinases, including the Src family, IGF1-R, EGFR and Fer individually, often activated in a variety of cancers (Oncogene 23:2600, Mol.Biol.Cell 16:3832). It has recently emerged that engagement of cadherins, surface receptors responsible for the formation of cell-to-cell adhesion junctions, results in the rapid activation of the Rac1 and Cdc42, Rho family GTPases. Furthermore, expression of activated forms of the Rho GTPases leads to Stat3, ptyr705 and pser727 phosphorylation and activation. In an attempt to investigate whether the Rho GTPases might be mediating the Stat3 activation we observe at post-confluence, we examined their activity in extracts from sparse vs confluent mouse normal breast epithelial HC11 cells and NIH3T3 fibroblasts. In agreement with previous results, a modest increase in Rac1 activity was noted as cells approached confluence. However, examination of the levels of Rac1, as well as Cdc42 and RhoA by Western blotting revealed a dramatic increase in total levels of these proteins, starting at confluence, which could be due to an increase in protein stability. This increase preceded the increase in Stat3-ptyr705 by approximately 24 hours. Moreover, inhibition of Rac1, RhoA and cdc42 activity with dominant-negative mutants or pharmacological inhibitors prevented the cell-to-cell adhesion-mediated, Stat3 activation, indicating that these GTPases could be responsible for the dramatic increase observed at post-confluence. Most importantly, inhibition of Rac1, RhoA and cdc42, or Stat3 at post-confluence induced apoptosis, while inhibition in sparsely growing cultures caused merely a growth retardation. These findings reveal demonstrate the existence of a novel mechanism of upregulation of the Rho GTPases which is engaged following extensive cell-to-cell adhesion. This pathway leads to Stat3 upregulation and cellular survival, and is present both in epithelial cells as well as fibroblasts.

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