Phosphopeptide mapping of DLC1 in ER+ breast cancer reveals AMOTL2, a key hippo pathway component, as an important target.

Abstract

11592 Background: Metastases suppressor genes are believed to control tumor progression and metastases. Deleted in Liver Cancer 1 (DLC1) acts as a gatekeeper for tumor and metastasis suppression. Low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer. It is essential to understand the impact of DLC1 and its functional network in preventing tumor and metastasis suppression. Methods: T47D cells with stable DLC1-Full-Length (DLC1-FL) were generated using mammalian expression cloning vector, pcDNA3.1+/C-(K)-DYK, and CloneEZ™ technology. Growth rate of control and DLC1-FL knock-in cells was assessed for 2 weeks using clonogenic assay. Proteomic and phosphopeptide enrichment assays (Pierce TiO2 enrichment kit) were performed in triplicates to examine the basis of altered growth phenotype. The PeakJuggler node in Proteome Discoverer was utilized for label-free quantitation of both protein and peptide MS peak areas. In addition, GO term enrichment analysis was performed in DAVID for the significantly changed phosphopeptides. Results: Stable knock-in of T47D-DLC1-FL inhibits cell growth significantly in vitro compared to T47D-control in clonogenic assay. The phosphopeptide enrichment proteomic analyses showed 199 phosphopeptides were identified only in T47D-DLC1-FL and 182 peptides were identified only in T47D-control cells. Pathway analysis using DAVID showed 3 main clusters of significantly differently identified phosphopeptides (p-values ≤ 0.01) involving cadherin binding (p = 4.9e-12), cell-cell adherens junction (p = 3.9e-10), and cell-cell adhesion (p = 9.4e-7). Analysis of specific phosphopeptides showed canonical pathways such as CTNNB1 (Thr552), and BCL2L13 (370-385). More importantly, phosphorylation of HIPPO-pathway component AMOTL2 at S766, critical for promoting YAP signaling and invasion was only identified in T47D-control but not T47D-DLC1-FL (p = 5.78e-5). Conclusions: This data suggest that the absence of DLC1 promotes phosphorylation of AMOTL2, and thereby activating YAP-TAZ signaling leading to invasiveness. This data provides mechanistic basis for targeting this pathway to prevent recurrence in ER+ breast cancer.