Non‐cannonical notch signaling pathways regulate breast cancer stem‐like cells function in triple negative breast cancer
Authors: Hossain Fokhrul , Sorrentino Claudia , Bilyeu Ayse , Crabtree Judy , Pannuti Antonio , Golde Todd , Osborne Barbara , Miele Lucio 
Published: November 2023

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive form of breast cancers, characterized by lack of expression of estrogen receptor α (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2).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 Notch signaling in TNBC. Notch1 is highly expressed in Basal‐like 1 (BL1) and especially Mesenchymal‐Stem‐Like (MSL) 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 Notch1 promotes cell survival in MDA‐MB‐231 cells, representative of MSL TNBC, in part by activating NF‐κB. 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 rely on Notch dependent recruitment of NF‐κB subunits, IKKα to the cIAP‐2 promoter. Short term exposure of MDA‐MB‐231 cells [MSL, phosphatase and tensin homolog (PTEN) wild‐type], but not MDA‐MB‐468 cells (BL1, PTEN‐null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by gamma secretase inhibitor (GSI), dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not 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. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF‐κB activation. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1‐activated Notch pathway. We demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA‐MB‐231 cells have increased Notch1, p‐AKT, p‐IKKα, and oxidative metabolism. AKT inhibition or IKKα inhibition alone or in combination with GSI (PF‐03084014) decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by GSI (PF‐03084014) in combination with AKT inhibitor (MK‐2206) or NF‐κB inhibitor (Bay11‐7082) blocks CD90hi or CD44+CD24low (CSCs marker) sorted secondary mammospheres formation. These data suggest that blocking of non‐canonical Notch‐ NF‐κB ‐AKT pathways have potential therapeutic importance in targeting CSCs of TNBC. Our data indicate that therapeutic regimens targeting this pathway deserve further investigation in TNBCs.Support or Funding InformationFunding: NIH 3P01CA166009‐04S1