Abstract 3335: Characterizing the MAXH28R secretome in endometrial cancer

Abstract

Abstract We have previously shown that the recurrent endometrial cancer MAX p.His28Arg mutation (MAXH28R) is associated with a proangiogenesis phenotype in both in vitro and xenograft models. The purpose of this study is to characterize the MAXH28R secretome to identify the proangiogenic and other paracrine factors contributing to MAXH28R associated tumorigenesis. Bottom-up high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of secreted proteins coupled with microarray analysis of global gene expression was performed in AN3CA endometrial cancer cell lines that stably express the long isoform of MAX-wild type (MAXWT) or MAXH28R. Orthogonal validation of differentially expressed proteins was performed by Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) using MAXWT- or MAXH28R-expressing AN3CA cell lines. Bioinformatic analysis of the proteins secreted into conditioned media (secretome) identified 114 proteins differentially expressed by MAXH28R compared to MAXWT-expressing AN3CA cells (P < 0.05). Differential expression of proteins was highly correlated with mRNA expression (Pearson r = 0.75, P < 0.001), indicating that a subset of the differentially expressed proteins are potential direct transcriptional targets of MAX. Secretome analysis revealed that Laminin-β1 expression in MAXH28R was reduced more than 100-fold compared to MAXWT (P < 0.001), while ALCAM (CD166) expression was increased 3.5-fold (P < 0.001). Decreased expression of TGFβ1 (5.9-fold decrease, P = 0.023) and TGFβ2 (3-fold decrease, P = 0.009) was seen for MAXH28R cells compared to MAXWT. Differential expression of Laminin-β1 and ALCAM was confirmed by WB analysis and TGFβ1 and TGFβ2 by ELISA. Our analyses revealed extensive changes in the MAXH28R secretome, as exemplified above. Reduced Laminin-β1 highlights alteration in the extracellular matrix with implications for cell adhesion and integrin signaling. Increased ALCAM expression indicates changes to cell-cell adhesion. Elevated levels of ALCAM in conditioned media are consistent with increased ALCAM shedding, which is a marker of more aggressive tumors in a variety of malignancies. Reduced TGFβ ligand levels suggest potential alterations in paracrine and autocrine TGFβ signaling. However, the impact on canonical and noncanonical TGFβ signaling in MAX mutant endometrial cancers remains unknown. ChIP-qPCR experiments are ongoing to assess MAXH28R binding at the promoters of differentially expressed genes. Biologic validation of MAXH28R effects on differential expression of secreted proteins is being performed in the Ishikawa and RL95-2 endometrial cancer cell lines that stably express MAXH28R and MAXWT. The effects of reduced Laminin-β1 on integrin signaling and anchorage independent growth will be reported, as will effects of altered TGFβ expression on canonical and noncanonical TGFβ signaling. Citation Format: Craig M. Rush, Miranda L. Gardner, Caroline E. Sapp, Michael A. Freitas, Paul J. Goodfellow. Characterizing the MAXH28R secretome in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3335.