Abstract P3-01-22: Autocrine motility factor receptor enhances proliferative and migratory capabilities of triple-negative breast cancer

Abstract

Abstract Background: Autocrine motility factor (AMF) is secreted by tumor cells to generate autocrine and paracrine signaling and is received by its receptor, autocrine motility factor receptor (AMFR), to promote aggressive behavior in cancer. The AMF-AMFR interaction has been demonstrated to promote proliferative, anti-apoptotic, and metastatic pathways in many cancers, but its ability to promote these pathways in breast cancer is poorly defined. Our previous work has shown that AMF gene expression is higher in triple-negative breast cancer (TNBC) tumors than in luminal tumors and luminal breast cancers lose a copy of AMFR in their tumor genomes almost twice as frequently as TNBC, suggesting aggressive cancers may need the AMF-AMFR signaling pathway to promote aggressive cancer biology. While AMFR does not presently have a specific inhibitor, erythrose-4-phosphate (E4P) is an established inhibitor of extracellular AMF. Here, we demonstrate that the AMF-AMFR interaction promotes proliferation and migration in TNBC and propose that AMFR may be a potentially potent target for TNBC therapy. Methods: cBioPortal was used to investigate AMFR somatic copy-number alterations (SCNA) and gene expression in METABRIC (n = 1,904) and TCGA Pan-Cancer Atlas (n = 994) patient cohorts. TNBC cell lines MDA-MB-231 and MDA-MB-436 were transduced with lentiviral particles, selected with puromycin, and sorted for high RFP expression using flow cytometry to generate inducible AMFR-shRNA (short hairpin RNA) stable cell lines. To study the ability of AMF to promote migration, MDA-MB-231 and MDA-MB-436 in AMFR-competent and AMFR-knock down conditions were seeded onto Transwell inserts with or without E4P while extracellular AMF was present or absent in the bottom well; after 24 hours, cells were fixed, stained with crystal violet, and counted. To study the ability of AMF to promote proliferation, MDA-MB-231 cells in AMFR-competent and AMFR-knock down conditions were grown in serum-free media with or without AMF for 24 hours. Cells were fixed and incubated in propidium iodide/RNase staining solution, and cell cycle analysis was performed using flow cytometry. Results: Gene expression analysis of AMFR correlates with its copy-number status in the tumor genome. METABRIC patient cohort loss, retention, and gain of AMFR in the tumor genome resulted in median z-score mRNA levels of -0.533, -0.1424, and 0.159, respectively, and TCGA Pan-Cancer Atlas patient cohort loss, retention, and gain of AMFR in the tumor genome resulted in median z-score mRNA levels of -0.5906, -0.274, and -0.09335, respectively. TNBC frequently retains or gains AMFR in their tumor genomes, suggesting TNBC tumors have higher levels of AMFR gene expression. In the TNBC cell line MDA-MB-231, AMF increased migration by 1.4-fold while AMFR knock-down returned migration to baseline and AMF inhibition by E4P decreased migration by 2.7-fold below baseline. In the TNBC cell line MDA-MB-436, AMFR knock-down decreased migration 1.6-fold and AMF inhibition by E4P decreased migration 33-fold below baseline. This data suggest that the signaling of AMF through AMFR promotes migration in TNBC. Cell cycle analysis of MDA-MB-231 cells showed the addition of AMF to culture medium increased actively dividing cells by 8.3%, and AMFR knock-down dropped this effect to 5.9%, suggesting that the AMF-AMFR interaction promotes proliferation of TNBC cells. Conclusion: TNBC has higher AMFR gene expression than luminal tumors. AMF appears to act through AMFR to promote migration and proliferation in TNBC. Targeting AMFR may be a promising therapeutic strategy for patients with TNBC. Citation Format: Cassie Liu, Jeffrey D. Price, Sarah P. Thayer. Autocrine motility factor receptor enhances proliferative and migratory capabilities of triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-22.