Reverse Phase Protein Lysate Array (RPPA): Use in Identifying Function for Estrogen-Induced Gene 121 (EIG121)
L Deng, B Hennessy, R Broaddus. M.D. Anderson Cancer Center, Houston, TX
Background: RPPA is a high-throughput dot blot in which dilutions of protein lysate are spotted on nitrocellulose slides. Each slide is probed with a different monospecific antibody. A DAKO-catalyzed signal amplification system is used for signal detection. Each slide holds a maximum of 1,152 spots, thus making the assay amenable to high-throughput analysis. An advantage of RPPA over Western blots is that multiple replicates and dilutions can be incorporated into the experimental design, thus making protein level quantification more accurate. An advantage over immunohistochemistry is that RPPA is quantitative rather than qualitative. We used RPPA to help characterize the function of EIG121, a gene we discovered from a microarray analysis of baseline and post-treatment endometrial biopsies from women taking estrogen-based hormone replacement therapy. EIG121 is over-expressed in estrogen-dependent endometrioid-type endometrial carcinoma, but not non-endometrioid tumors. Other than being a lysosomal/endosomal protein, little is known of EIG121's function.
Design: Protein lysates were prepared from 200 endometrial carcinomas and spotted on RPPA slides. Antibodies against 82 different proteins were applied to the RPPA slides. These antibodies were previously validated by Western. Antibodies were directed against receptor tyrosine kinases, members of the PI3K/AKT, JAK-STAT, and MAPK pathways, and proteins regulating apoptosis and the cell cycle. Correlation coefficients were calculated to identify proteins that were associated with EIG121 expression.
Results: EGFR and HER-2/neu were 2 of the proteins with highest negative correlations with EIG121 expression (i.e., tumors with high EIG121 expression had lowest expression of EGFR and HER-2/neu). Evidence in breast cancer literature suggests that endocrine sensitivity is inversely related to growth factor-mediated signaling. We speculated that lysosomal/endosomal EIG121 binds to and degrades growth factor receptors. In cell-based systems, we have subsequently shown by co-IP that EIG121 binds to EGFR, promotes its degradation, and inhibits downstream signaling via Akt.
Conclusions: RPPA provided critical clues that helped us to identify EIG121 as an important molecular switch that regulates the hormone responsiveness/resistance of tumor cells. We speculate that this switch function of EIG121 may be crucial in dictating an endometrial tumor's histotype (endometrioid vs. non-endometrioid).
Tuesday, March 10, 2009 1:00 PM
Poster Session IV # 198, Tuesday Afternoon