Citation: Ilari, A.; Cogliati, V.; Sherif, N.; Grassilli, E.; Ramazzotti, D.; Cordani, N.; Cazzaniga, G.; Di Bella, C.; Lavitrano, M.; Cazzaniga, M.E.; et al. Differential Expression of NOTCH-1 and Its Molecular Targets in Response to Metronomic Followed by Conventional Therapy in a Patient with Advanced Triple-Negative Breast Cancer. Biomedicines 2024, 12, 272. https://doi.org/10.3390/ biomedicines12020272 Academic Editors: Daniela Carlisi and Anna De Blasio Received: 8 December 2023 Revised: 19 January 2024 Accepted: 23 January 2024 Published: 25 January 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomedicines Article Differential Expression of NOTCH-1 and Its Molecular Targets in Response to Metronomic Followed by Conventional Therapy in a Patient with Advanced Triple-Negative Breast Cancer Alice Ilari 1 , Viola Cogliati 2 , Noorhan Sherif 1 , Emanuela Grassilli 1 , Daniele Ramazzotti 1 , Nicoletta Cordani 1 , Giorgio Cazzaniga 3 , Camillo Di Bella 3 , Marialuisa Lavitrano 1 , Marina Elena Cazzaniga 1,2 and Maria Grazia Cerrito 1, * 1 School of Medicine and Surgery, Milano-Bicocca University, 20900 Monza, Italy; a.ilari@campus.unimib.it (A.I.); n.sherif@campus.unimib.it (N.S.); emanuela.grassilli@unimib.it (E.G.); daniele.ramazzotti@unimib.it (D.R.); nicoletta.cordani@unimib.it (N.C.); marialuisa.lavitrano@unimib.it (M.L.); marina.cazzaniga@unimib.it (M.E.C.) 2 Phase 1 Research Centre, Fondazione IRCCS San Gerardo dei Tintori, Via Pergolesi 33, 20900 Monza, Italy; viola.cogliati@irccs-sangerardo.it 3 Department of Pathology, Fondazione IRCCS San Gerardo dei Tintori, Via Pergolesi 33, 20900 Monza, Italy; giorgio9cazzaniga@gmail.com (G.C.); camillo.dibella@irccs-sangerardo.it (C.D.B.) * Correspondence: mariagrazia.cerrito@unimib.it; Tel.: +39-0264488339 Abstract: A group of 27 patients diagnosed with metastatic triple-negative breast cancer (mTNBC) was randomly distributed into two groups and underwent different lines of metronomic treatment (mCHT). The former group (N 14) received first-line mCHT and showed a higher overall survival rate than the second group (N 13), which underwent second-line mCHT. Analysis of one patient still alive from the first group, diagnosed with mTNBC in 2019, showed a complete metabolic response (CMR) after a composite approach implicating first-line mCHT followed by second-line epirubicin and third-line nab-paclitaxel, and was chosen for subsequent molecular characterization. We found altered expression in the cancer stemness-associated gene NOTCH-1 and its corresponding protein. Additionally, we found changes in the expression of oncogenes, such as MYC and AKT, along with their respective proteins. Overall, our data suggest that a first-line treatment with mCHT followed by MTD might be effective by negatively regulating stemness traits usually associated with the emergence of drug resistance. Keywords: metastatic triple-negative breast cancer (mTNBC); metronomic therapy (mCHT); cancer stem cells (CSCs); NOTCH-1; c-MYC; pAKT 1. Introduction Triple-negative breast cancer (TNBC) represents 10–20% of all breast cancers and is characterized by the absence of the expression of estrogen receptors (ER), progesterone receptors (PR), and the absence of amplification/over-expression of the human epidermal growth factor receptor 2 (HER2) [1]. These receptors are common targets for breast cancer therapies, and their absence in TNBC makes it more challenging to treat compared to other types of breast cancer. Chemotherapy and surgery have represented for years the only possible treatment approach for these patients [2], who very often relapse or have a spread of the tumor to other organs [3]. Currently, immune checkpoint inhibitors [4] and the new class of antibody–drug conjugates have provided valuable options for the treatment of TNBC [5,6]. Nevertheless, better therapeutic options are needed, both in terms of new drugs and methods of administering chemotherapy treatments. Many efforts are underway to identify new therapeutic targets and treatment strategies for TNBC. Metronomic chemotherapy (mCHT), consisting of continuous administration of chemotherapy drugs at reduced doses, has been explored in selected patients as an alterna- Biomedicines 2024, 12, 272. https://doi.org/10.3390/biomedicines12020272 https://www.mdpi.com/journal/biomedicines