Characterization and Targeting Cancer Stem‐like Cells in Triple Negative Breast Cancer
Authors: Hossain Fokhrul , Sorrentino Claudia , Bilyeu Ayse , Matossian Margarite , Crabtree Judy , Pannuti Antonio , Burow Matthew , Xu Keli , Golde Todd , Osborne Barbara , Miele Lucio 
Published: June 2021

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem‐like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore, CSC‐targeted therapies are keenly sought. There is strong evidence for the involvement of canonical‐Notch signaling in TNBC, however the role of non‐canonical Notch signaling in TNBC remains unknown. Notch1 is highly expressed in Basal‐like 1 (BL1) and especially Mesenchymal (M) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch activation by Jagged1‐expressing stromal cells enhances transcription of the anti‐apoptotic gene cIAP‐2 (BIRC3), a known NF‐κB target. This event is dependent on recruitment to the cIAP‐2 promoter of NF‐κB subunits, IKKα and Notch1. Short term exposure of MDA‐MB‐231 cells (M, PTEN wild‐type), but not MDA‐MB‐468 cells (BL1, PTEN‐null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by AKT inhibitors, IKK inhibitors, and dual mTORC1/2 inhibitors but not an mTORC1‐selective inhibitor. These observations support a model where canonical and non‐canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF‐κB activation in PTEN wild type TNBC cells. Importantly, we find co‐localization of Notch1 with Mitochondria in MDA‐MB‐231 cells by confocal microscopy and Western blot of isolated mitochondrial fractions. We demonstrate that recombinant Jagged1 increases the metabolism of TNBC cells. Knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. CSCs derived from MDA‐MB‐231 cells have increased Notch1, p‐AKT, and oxidative metabolism compared to non‐stem cells. AKT inhibition or IKK inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF‐03084014) in combination with AKT inhibitor (MK‐2206) or IKK inhibitor (Bay11‐7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. Notably, we find similar results in TNBC patient derived xenograft (PDX) models. However, Notch inhibition may have adverse effects on tumor immunity, due to the role of Notch in CD4 and CD8 T‐cell activation. Thus, we explored an immunocompetent tumor mouse model to study the effects of these pharmacological interventions in that setting. We developed an immunocompetent TNBC mouse model by injecting TNBC mouse cells (C0321) into mammary fat pads of isogenic FVB female mice. Combination treatments with GSI (PF‐03084014) and AKT inhibitor (MK‐2206) significantly delayed tumor growth as well as decreased CSCs number in that model. These data suggest that combination treatments affecting the intersection of Notch and AKT pathways have potential therapeutic applications in targeting CSCs in TNBC cases with high Notch1 expression. The possible immunological effects of such combination treatments require additional investigation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.