Integrin-mediated cell-extracellular matrix (ECM) adhesion is essential for the survival of normal epithelial cells, and loss of this cell-ECM adhesion leads to anoikis. In this dissertation study, we first identify migfilin, a novel focal adhesion protein, as a key sensor of cell-ECM adhesion in epithelial cells. Loss of cell-ECM adhesion significantly reduces migfilin protein levels in untransformed epithelial cells and concomitantly induces anoikis. Migfilin RNAi is sufficient to induce apoptosis in MCF-10A cells while overexpression of FLAG-migfilin partially protects these cells from anoikis, strongly suggesting that migfilin plays a critical role in cell adhesion-mediated cell survival signaling. Cell detachment-induced migfilin reduction is, at least partially, responsible for the induction of anoikis.
Further signaling studies reveal that migfilin regulates cell survival and anoikis by influencing Src activation. Immunoflorescence staining shows that migfilin co-localizes with active Src in focal adhesions, and immunoprecipitation and GST pull-down assays demonstrate that migfilin directly interacts with Src. Moreover, the detailed structural studies show that migfilin strongly binds to the Src SH3 domain via the second PXXP cluster (140-173aa) in its proline-rich region, and weakly binds to the Src SH2 domain via an atypical binding sequence (E6KRVASS12) in its N-terminal. A working model is proposed in which migfilin promotes Src activation via direct interaction, and loss of cell-ECM adhesion triggers the degradation of migfilin protein, thereby causing Src inactivation which contributes to the initiation of anoikis. Interestingly, this migfilin-Src signaling pathway is dysfunctional in some anoikis-resistant cancer cells. During cell detachment, migfilin proteins are stabilized in these cancer cells, and phosph-Y419 Src levels are not reduced concomitantly, representing a novel mechanism for anoikis resistance during tumorigenesis.
In addition, migfilin is found to negatively regulate p27 protein level. Depletion of migfilin significantly increases p27 protein levels in different cell lines. In HCT116 cells, migfilin RNAi increases both cytoplasmic and nuclear p27, and inhibits cell cycle progression. These findings indicate that migfilin provides a linkage between p27 and integrin-mediated cell-ECM adhesion.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12162009-145545 |
| Date | 18 December 2009 |
| Creators | Zhao, Jianping |
| Contributors | Donna Beer Stolz, PhD, Xiao-Ming Yin, MD, PhD, Tianyi Wang, PhD, Chuanyue Wu, PhD, Alan Wells, MD, DMS |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12162009-145545/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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