Autocrine Motility Factor Signaling Pathway Promotes Aggressive Behavior and Migration in Breast Cancer Cassie Liu1,2, Jeffrey D. Price1,2 , Dulce Maroni1,2, Jadd M. Stevens3, Amaris Z. VanDyke3, Pamela A. Althof3, Pinaki Mondal1,2, Jennifer N. Sanmann3, and Sarah P. Thayer1,2 Division of Surgical Oncology1, Fred & Pamela Buffett Cancer Center2, and the Human Genetics Laboratory at the Munroe-Meyer Institute for Genetics and Rehabilitation3, University of Nebraska Medical Center, Omaha, NE, United States 68198

INTRODUCTION RESULTS AMF Expression in Human Breast AMF-AMFR Interaction Promotes Migration in Autocrine motility factor (AMF) is a secreted by cancer cells that acts in an Cancer Positively Correlates with Molecular Aggressive TNBC Cancer Cell Line autocrine or paracrine fashion to bind to its receptor, AMFR, at the surface of tumor Loss of AMFR in Tumor Genome Correlates cells. The AMF-AMFR pathway has been implicated in promoting proliferation, anti- Subtype Aggression apoptosis, motility and migration, invasion, and metastasis pathways in various with Good Prognosis Tumors A B cancers[1-8]. A

AMFR appears upregulated in breast cancer cells[9]. Studies in breast cancer have shown that the upregulation of AMF causes enhanced migration, epithelial-to- mesenchymal transition (EMT), cell viability, and proliferation in aggressive breast cancer molecular subtypes[4,10,11]. However, AMF functions intracellularly as glucose- 6-phosphate isomerase (GPI), a key enzyme that regulates the second step of glycolysis, and therefore the mechanism through which AMF acts in these studies is not certain.

We propose that the extracellular signaling of AMF through AMFR is the key mediator for breast cancer tumor aggression, and thus, AMFR may be a potential therapeutic target.

Figure 2. Patient breast cancer tissue from the METABRIC (n = 1,667) (A) and Pan- Cancer Atlas (n = 981) (B) datasets show a progressive trend of increasing AMF as tumor molecular subtypes become more aggressive. Figure 4. Exogenous AMF increases metastatic TNBC cell migration by 1.6-fold. Knock down of AMFR eliminates cell migration towards exogenous AMF. Aggressive TNBC Cell Line Expresses Higher (n=3; *p<0.05, ***p<0.001) B AMFR Gene and AMFR Protein Expression Our Patient Cohort Than Luminal A Cell Line SUMMARY • Loss of AMFR in tumor genomes is associated with good prognosis breast cancers. • High AMF gene expression in human breast cancer tissue correlates with A B aggressive molecular subtypes. METABRIC • High AMFR gene expression and AMFR protein expression correlates with Patient Cohort aggressive cell behavior in breast cancer cell lines. • Downregulating AMFR leads to decreased breast cancer cell migration in METHODOLOGY aggressive TBNC cell line. Somatic Copy-Number Alterations (SCNAs) of AMFR in Breast Cancer Patient Samples Tumor tissue obtained from consented breast cancer patients (n = 34) were analyzed by the genome-wide array platform Affymetrix OncoscanTM FFPE Assay (Thermo Fisher Scientific, Waltham, MA, USA) and examined for SCNA of AMFR. cBioPortal was used to investigate SCNA of AMFR in primary breast cancer tumors from METABRIC (n = 1,784) and TCGA Pan-Cancer Atlas (n = 945) datasets[12,13]. CONCLUSION Gene Expression for AMF in Breast Cancer Patient Samples Pan-Cancer Atlas cBioPortal was used to investigate gene expression of AMF in primary breast cancer tumors from METABRIC (n = 1,667) and TCGA AMF may depend on its signaling through AMFR to promote an Pan-Cancer Atlas (n = 981) datasets[12,13]. Patient Cohort Gene Expression of AMF and AMFR in Breast Cancer Cell Lines aggressive phenotype in breast cancer. Therefore, AMFR may be RNA was isolated from luminal A (MCF-7) and metastatic TNBC (MDA-MB-231) cell lines and converted to cDNA. AMF and AMFR Taqman probes were used for qPCR and the results were normalized against GAPDH. an attractive therapeutic target for patients with aggressive breast Protein Expression of AMFR in Breast Cancer Cell Lines Figure 1. A. Single-copy loss is shown in green. Patient breast cancer tissue Whole cell protein lysates were isolated from luminal A (MCF-7) and metastatic TNBC (MDA-MB-231) cell lines. Western blot cancer. analysis was performed with AMFR rabbit polyclonal (ProteinTech, Rosemont, IL, USA) and β-actin (13E5) rabbit shows frequent single-copy loss of the AMFR gene in good prognosis tumors monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA). Densitometry was used for band quantification with the help of while poor prognosis tumors frequently retain both copies of the AMFR gene. References ImageJ. Figure 3. A. Metastatic TNBC cell line MDA-MB-231 has 2.1-fold increased gene [1]Funasaka et al. Cancer Res, 2007, 67(9): 4236-43. [8]Tsutsumi et al. Clin Cancer Res, 2004, 10(22): 7775-84. Migration of Metastatic TNBC Cell Line Towards AMF *Patient 25 only had analyzable lymph node tissue. B. Our patient cohort (n = 34), METABRIC [2]Haga et al. Int J Cancer, 2003, 107: 707-14. [9]Jiang et al. J Histochem Cytochem, 2006, 54(2): 231-41. [3]Watanabe et al. Int J Cancer, 1993, 53: 689-95. [10]Funasaka et al. Cancer Res, 2009, 69(13): 5349-56. Non-targeting (Scramble) and AMFR (AMFR KD) siRNA were transfected into MDA-MB-231 cells. After 48 hours, the cells were expression of AMFR compared to luminal A cell line MCF-7. (n = 3; **p<0.01) (n = 1764), and Pan-Cancer Atlas (n = 945) datasets show AMFR is lost over 50% of [4]Jung et al. Oncol Lett, 2017, 13: 4925-32. [11] Gallardo-Pérez et al. Biochim Biophys Acta, 2017, 1864(10): 1679-90. added to transwell migration inserts. The lower chamber contained either only DMEM media with 10% FBS or 10 µg/mL AMF in B. MDA-MB-231 has 1.85-fold increased protein expression of AMFR compared [5]Araki et al. J Biol Chem, 2009, 284(47): 32305-11. [12]Gao et al. Sci Signal, 2013, 6(269): pl1. DMEM media with 10% FBS. Cells were given 24 hours to migrate before they were fixed with 4% paraformaldehyde (PFA) and the time in less aggressive molecular subtypes while disproportionately retained [6]Timar et al. Cancer Res, 1996, 56(8): 1902-8. [13]Cerami et al. Cancer Discov, 2012, 2(50: 401-4. stained with crystal violet. and even gained in more aggressive molecular subtypes. to luminal A cell line MCF-7. (n = 1) [7]Torimura et al. Hepatology, 2001, 34(1): 62-71.