Abstract P2-08-16: The cross-talk between nuclear p-ser294-FOXO3a and cytoplasmic MMP-2 in breast cancer survival and treatment response
Authors: Hou Ming-Feng , Yang Sheau-Fang , Huang Yu-Chun , Chen Fang-Ming , Yeh Yao-Tsung 
Published: March 2022

Abstract
Background The forkhead protein FOXO3a, the member of the forkhead box O (FOXO) transcription factors, controls a wide spectrum of carcinogenesis, therapy response and progression in human cancer. Emerging evidence suggest that phosphorylation of FOXO3a by ERK and p38 at Serine 294 induces its nuclear localization or exclusion to the cytosol, therefore regulating FOXO3a transcriptional activity and consequently promoting cell proliferation and tumorigenesis. Several proteins regulated by FOXO3a, such as MMP-2,9 and 13, are involved in metastasis. We aim to explore the potential role of p-ser294-FOXO3a and MMPs, and interplay between both in breast cancer. Materials and Methods Paraffin-embedded tissue microarrays from 350 breast cancer patients were enrolled. Immunohistochemical analysis was performed, and the expression patterns of p-ser294-FOXO3a, MMP-2, MMP-9, MMP-13 were categorized by semiquantitative method and further correlated with the clinicopathological parameters using the x2 test. Survival curves were calculated using the Kaplan-Meier method, while the significance was determined by log rank test. The biological impacts and underlying mechanisms of p-ser294-FOXO3a were analyzed by transwell assay, RT-PCR, western blotting immunofluorescence staining. co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assay in breast cancer cells upon transfection with FOXO3a and FOXO3a mutant (S294A). Results Our results showed that nuclear p-Ser294-FOXO3a index was inversely correlated with tumor grade (p=0.011) and relapse (p=0.053), but positively correlated only with cytoplasmic MMP-2 intensity (p=0.010). It is noted that cytoplasmic MMP-2 intensity was positively correlated with tumor grade (p=0.032) and size (p=0.028). Intriguingly, decreased nuclear p-Ser294-FOXO3a index was significantly associated with an improved overall survival rate (p=0.004) and of those patients bearing high MMP-2 staining (p&lt;0.001) or negative ER status (p=0.006), and those receiving hormone therapy (HT) (p=0.030) or chemotherapy (p=0.001). Ectopic FOXO3a overexpression increased invasion rate which was inhibited by tamoxifen (TAM) in only ER-positive MCF-7 cells. In addition, TAM inhibited the invasion rates of ERα-negative MDA-MB-231 cells when co-expressed FOXO3a and ERα, and Ser294Ala-FOXO3a mutant (S294A) is incompetent for TAM-mediated suppression of invasion. Real time qPCR showed that TAM decreased FOXO3a-induced MMP-2 mRNA expression in MCF-7 cells and in MDA-MB-231 cells co-expressed with ERα. Co-immunoprecipitation and immunofluorescent staining showed that Ser294 phosphorylation of FOXO3a might be essential for its nuclear localization and protein-protein interaction with ERα in response to TAM. Conclusion The present study highlights that the real index of nuclear p-ser294-FOXO3a can predict a better survival rate and treatment response of HT and chemotherapy in breast cancer. Furthermore, the specific interplay among p-ser294-FOXO3a, ERα, and MMP-2 might be involved in breast cancer metastasis and treatment response. A better understanding of the underlying mechanism may improve the treatment strategy of specific breast cancer subtype.
Citation Format: Ming-Feng Hou, Sheau-Fang Yang, Yu-Chun Huang, Fang-Ming Chen, Yao-Tsung Yeh. The cross-talk between nuclear p-ser294-FOXO3a and cytoplasmic MMP-2 in breast cancer survival and treatment response [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-16.