Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by a high rate of metastatic recurrence and poor prognosis. Molecular mechanism underlying the metastatic behavior of TNBC has not been well elucidated, and newer approaches addressing drivers of metastasis are crucial to improving patient outcomes. PEA15 (Phosphoprotein enriched in astrocytes-15) regulates cell proliferation, apoptosis, and autophagy. In breast cancer, PEA15 expression inhibits invasion by binding to ERK and preventing its nuclear translocation. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the effect of PEA15 phosphorylation status on TNBC remains unknown. In this study, we tested the hypothesis that unphosphorylated PEA15 will prevent metastasis in TNBC through inhibition of the epithelial-to-mesenchymal transition (EMT).

Method: We established stable cells overexpressing unphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) PEA15 mutants in MDA-MB-468 cells. To dissect specific Cellular Mechanisms regulated by PEA15 phosphorylation, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to see effects of PEA15 phosphorylation on tumor growth.

Results: The clonogenic growth of PEA15-AA–expressing cells was significantly reduced by 80% compared with empty vector-transfected cells (PEA15-V). Anchorage-independent growth, an indicator of in vivo tumorigenicity, was inhibited in cells expressing PEA15-AA by 60% compared with PEA15-V. PEA15-AA upregulated the expression of E-cadherin and decreased the expression of mesenchymal markers, suggesting that PEA15-AA reverses EMT. Compared with PEA15-V, migration and invasion of cells expressing PEA15-AA were reduced by 65% and 72%, respectively. In contrast, PEA15-DD promoted migration, invasion, and expression of mesenchymal markers. To determine the in vivo effect of PEA15-AA, we injected stable PEA15 transfectants of MDA-MB-468 cells into the mammary fat pad of NOD/SCID mice. The PEA15-DD–injected group showed greater tumor volumes than PEA15-V and PEA15-AA groups, suggesting that PEA15-AA has antitumor effects both in vitro and in vivo. From the immune and metastasis arrays, we found that expression level of IL-8, which is known to induce EMT, was greatly decreased by PEA15-AA, while IL-8 was highly expressed in PEA15-DD cells. Addition of recombinant IL-8 to the cells expressing PEA15-AA partially rescued mesenchymal characteristics, increasing migration and expression of mesenchymal markers. By contrast, IL-8 knockdown in PEA15-DD–expressing cells decreased the mesenchymal phenotype. These findings indicate that IL-8 may play an important role as a mediator of phosphorylation of PEA-15 in breast cancer cell migration and invasion and suggest that PEA15-AA inhibits the expression of IL-8, thereby reversing EMT.

Conclusion: Taken together, our results show that PEA15 phosphorylation serves as an important regulator, having a dual role as an oncogene or tumor suppressor. Further studies are warranted to evaluate the impact of PEA15 phosphorylation status on metastasis in vivo. These findings support the development of PEA15-AA as a potential therapeutic strategy for TNBC.

Citation Format: Park J, Chauhan G, Cohen EN, Ueno NT, Battula VL, Tripathy D, Reuben JM, Bartholomeusz C. PEA15-AA, an unphosphorylatable mutant of PEA15, as a novel therapeutic gene for triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-22.