Abstract A59: A tumor supressor role for nSMase2 in triple negative breast cancer

Abstract PURPOSEIn this study, we assess a putative tumor suppressor role for neutral sphingomyelinase-2 (nSMase2) in triple negative breast cancer (TNBC) and determine if nSMase2 effects are through cell intrinsic or extrinsic mechanisms. METHODSLentivirus was used to stably overexpress wild-type nSMase2 (N2) and a catalytically inactive variant (HA) in 4T1(mouse basal BC), EMT6 (mouse basal BC), and MDA-MB-231 (human basal BC) cells that harbor epigenetic nSMase2 suppression. In vitro tumorigenicity were assessed by soft-agar colony formation assay and anchorage-independent survival was analyzed by trypan blue exclusion assay following 48h of suspension culture. Monolayer growth was assessed by MTT assay. For in vivo studies, cells were orthotopically implanted in the mammary fat pad of Balb/c (4T1, EMT6) or NSG (MDA-MB-231) mice. Time to palpable tumor formation was assessed and once detected, tumor size was measured with calipers (twice a week for 3 weeks) for a maximum of 40 days post injection. For end point analysis, tumors were dissected and measured for tumor size and weight. RESULTSValidation experiments confirmed expression of lentiviral constructs with increased nSMase activity and ceramide levels observed in N2 but not HA cells. Biological assays revealed that nSMase2 expression resulted in decreased colony formation compared to HA controls, but this was not due to alterations in cell viability (either in monolayer or suspension). There were also no differences in monolayer growth between the two cell lines. In vivo studies in immunocompetent mice showed a drastic reduction in tumor growth of N2 cells compared to HA controls. Intriguingly, the effects of N2 overexpression on tumor growth were significantly milder in immunocompromised mice. CONCLUSIONThese studies reveal that restoration of nSMase2 expression has tumor suppressive effects in TNBC and suggest this occurs through both cell intrinsic and extrinsic mechanisms. In vitro studies demonstrate that nSMase2 suppression of tumorigenicity (as assessed by colony formation) is through effects on anchorage-independent growth (AIG) and independent of cell viability. This is consistent with reduced tumor growth in immunocompromised mice. However, the larger suppressive effects seen in immunocompetent models suggest that tumor cell nSMase2 can also interact with the host immune system to exert anti-cancer roles. We are currently exploring the mechanisms by which nSMase2 regulates AIG and defining if and how nSMase2 interacts with the tumor immune environment to promote anti-tumor immunity. Citation Format: Andrew E Resnick, Fabiola N Valazquez, Samia Mohammed, Joseph Bonica, Danielle Guida, Victoria Alvarado, Deanna M Peperno, Yusuf A Hannun, Christopher J Clarke. A tumor supressor role for nSMase2 in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A59.