Abstract P5-05-08: Molecular cross-talk between retinoic acid and NOTCH1 signaling pathways: Role in triple negative breast cancer
Authors: Paroni G , Zanetti A , Bolis M , Vallerga A , Troiani M , Fratelli M , Kurosaki M , Terao M , Garattini E 
Published: May 2019

Triple Negative Breast Cancer (TNBC) represents 10-20% of all breast cancers and it is characterized by poor prognosis and high recurrence rate. The heterogeneity of the disease and the absence of well-defined molecular targets have so far challenged successful therapeutic strategies. NOTCH1 has been found to act as a driver oncogene in a small subset of TNBC characterized by constitutive activation of the protein, acting as a transcription factor. Preclinical studies support an anti-tumor activity of All-Trans Retinoic Acid (ATRA) in specific subsets of breast cancer.

By screening a large panel of breast cancer cell lines recapitulating the heterogeneity of TNBC we identify a specific subset sensitive to the anti-proliferative activity of ATRA. These cell lines (N-TNBC cell lines) are characterized by a Notch1 intragenic fusion transcript conferring gain of function activity to the protein. Indeed, sequence analysis reveals that the cell lines harbor an interstitial deletion in the NOTCH1 gene encompassing the negative regulatory region (NRR) domain. These cell lines depend on Notch active signaling for their proliferation since their cell growth is impaired by Notch inhibitors (γ-secretase inhibitors, e.g. DAPT, PF-3084014). Proliferation assays reveal that ATRA and γ-secretase inhibitors act synergistically in inhibiting cancer cell growth in N-TNBC cell lines suggesting the existence of a cross talk between the molecular pathways engaged by retinoic acid and NOTCH1.

By using retinoic acid receptors (RARs) agonists and antagonists as well as RAR specific silencing experiments we identify RARα as the retinoic acid receptor responsible of the anti-proliferation activity of ATRA in N-TNBC cell lines. In particular N-TNBC cell lines respond to RARα activation by inducing high amounts of the onco-supressor protein RARβ. This feature is unique in ATRA sensitive TNBC cell lines and does not occur in ATRA sensitive luminal cell lines arguing for the existence of a retinoic acid specific mechanism of action in N-TNBC. Since RARs act as transcription factors inside the cells, to gain insights into the molecular pathway at the basis of the observed ATRA/NOTCH1 cross talk, we performed RNAseq analysis of ATRA and/or DAPT treated N-TNBC cells. Gene set enrichment analysis reveal that ATRA is able to directly affect NOTCH1 transduction pathway by modulating the expression of NOTCH1 target genes. In particular, in two out of three N-TNBC cell lines ATRA directly inhibits the NOTCH1 expression at a transcriptional level and its downregulation is increased by ATRA/DAPT combinations. Pathway analysis has allowed the identification of putative molecular hubs responsible for the synergistic effects observed and therefore likely at the basis of the crosstalk between ATRA/NOTCH pathways. These findings are of clinical interest since both the retinoid and NOTCH signaling display crucial physiologic activities and their pleiotropic effects could impinge on the success of therapeutic options based on their pathway modulation.

The newly discovered specificity of ATRA action in the context of NOTCH1 addicted TNBC provides new tools for the identification of patients candidates benefitting from strategies targeting the ATRA/NOTCH axis.

Citation Format: Paroni G, Zanetti A, Bolis M, Vallerga A, Troiani M, Fratelli M, Kurosaki M, Terao M, Garattini E. Molecular cross-talk between retinoic acid and NOTCH1 signaling pathways: Role in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-08.