Abstract PD3-05: The paradoxical role of RalA and RalB in triple negative breast cancer
Authors: Spehar Jonathan M. , Thies Katie A. , Cole Matthew W. , Schafer Rachel E. , Richardson Dillon S. , Steck Sarah A. , Das Manjusri , Lian Arthur W. , Ray Alo , Knoblaugh Sue E. , Trimmers Cynthia D. , Sizemore Gina M. , Sizemore Steven T. 
Published: March 2022

Abstract
Background: Breast Cancer (BC) is the most common cancer and leading cause of cancer associated mortality in women worldwide. TNBC patients have the highest mortality mainly due to lack of receptors for targeted therapies. RalA and RalB are small GTPases that are known to regulate growth and metastasis in several cancers. However, roles for these GTPases in BC is poorly understood. The goal of this study was to investigate the contributions of RalA and RalB in TNBC. Methods: Control, RalA or RalB CRISPR knockout (KO) MDA-MB-231 cells were injected into the mammary gland of nod-skid-gamma (NSG) mice. Tumor growth was monitored and groups were taken as they met early removal criteria. Tumors and lungs were formalin-fixed and paraffin embedded. Tumors underwent immunohistochemical staining for Ki-67 and Cleaved caspase-3 and lungs were stained for hematoxylin and eosin and imaged on a Leica Aperio ScanScope XT to calculate lung metastasis. RalA or RalB were also depleted in MDA-MB-231 and MVT1 cells by shRNA. In addition, RalA depleted MDA-MB-231 cells were labeled with luciferase and injected into the tail vein of NSG mice and imaged on an IVIS spectrum to test seeding and lung colonization. Immunohistochemistry of patient TMAs was preformed on a Bond RX autostainer using RalA (Abcam, ab126627, 1:2000). Immunohistochemical stains were imaged on a PerkinElmer’s Vectra® Automatic Imaging System and quantified using inForm® Advanced Image Analysis software. Statistical significance of Kaplan-Meier survival curves were determined by log rank. Results: RalA knockout and depletion slowed primary orthotopic tumor growth in MDA-MB-231 and MVT1 cells. RalB KO had the opposite effect and increased growth rate compared to controls and RalA KO cells. Ki67 and cleaved caspase 3 IHC staining of tumors indicate KO of RalA decreased proliferation, whereas KO RalB increased proliferation with no change in apoptosis. RalA KO decreased the number and area of lung metastasis in both spontaneous and experimental metastasis assays. RalB KO or depletion caused an increase in the area and number of metastasis. Utilizing data from the METABRIC and TCGA BC datasets, elevated RALA, but not RALB, was prognostic of worse outcome in the overall BC populations and the TNBC populations specifically. RALA was shown to be more highly expressed in BC, particularly TNBC, relative to normal mammary tissue whereas RalB was decreased in BC and TNBC. IHC staining of a TMA comprised of all BC subtypes and a TMA of only TNBC samples confirmed RalA as a prognostic marker of patient outcome. Conclusions: RalA and RalB have important but paradoxical roles in TNBC.
Citation Format: Jonathan M. Spehar, Katie A. Thies, Matthew W. Cole, Rachel E. Schafer, Dillon S. Richardson, Sarah A. Steck, Manjusri Das, Arthur W. Lian, Alo Ray, Sue E. Knoblaugh, Cynthia D. Trimmers, Gina M. Sizemore, Steven T. Sizemore. The paradoxical role of RalA and RalB in triple negative breast cancer [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 PD3-05.