Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, accounting for 15-25% of all breast cancer cases. It is characterised by marked molecular heterogeneity, high metastatic potential, and the absence of effective targeted therapies due to the lack of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression. Tumour progression is sustained by a dynamic bidirectional crosstalk between cancer cells and the tumour microenvironment (TME), while the scaffold protein RACK1 coordinates signalling pathways involved in tumour cell survival, migration, and invasion. The anti-tumour activity of the plant-derived extract A1 was evaluated in the mesenchymal-like TNBC cell lines MDA-MB-231 and SUM-159PT, using complementary 2D and 3D in vitro models. Cell viability was assessed by MTT, CellTiter-Glo® and LIVE/DEAD assays, while wound healing, Transwell migration and Matrigel invasion assays were performed to evaluate migratory and invasive capacities. RACK1 protein expression was analysed by Western blotting. Tumour-immune crosstalk was investigated through Transwell assays measuring THP-1 monocytes recruitment toward tumour conditioned media. A1 reduced TNBC cell viability in a dose-dependent manner and markedly impaired migration and invasion in both cell lines. These effects were associated with a significant downregulation of RACK1 expression. Moreover, conditioned media from A1-treated TNBC cells significantly reduced THP-1 monocytes recruitment, suggesting that A1 impairs tumour-derived paracrine signalling involved in TME communication. In 3D spheroids models, A1 effectively suppressed spheroids viability and markedly inhibited invasive outgrowth and branching structures, confirming its anti-invasive activity in a more physiologically relevant setting. These findings demonstrate that A1 simultaneously targets multiple hallmarks of TNBC progression by reducing tumour cells survival, invasion, and TME crosstalk. The associated downregulation of RACK1 further supports its potential role as a molecular mediator of A1 activity. Overall, A1 emerges as a promising candidate for the development of novel adjuvant therapeutic strategies against TNBC, warranting further mechanistic studies and in vivo validation.
Il carcinoma mammario triplo-negativo (TNBC) rappresenta il sottotipo più aggressivo di carcinoma mammario, costituendo il 15-25% di tutti i casi di tumore al seno. È caratterizzato da una marcata eterogeneità molecolare, un elevato potenziale metastatico e dall’assenza di terapie mirate efficaci, a causa della mancata espressione del recettore per gli estrogeni (ER), del recettore per il progesterone (PR) e di HER2. La progressione tumorale è sostenuta da un dinamico crosstalk bidirezionale tra le cellule tumorali e il microambiente tumorale (TME), mentre la proteina scaffold RACK1 coordina diversi pathway di segnalazione coinvolti nella sopravvivenza, migrazione e invasione delle cellule tumorali. L’attività antitumorale dell’estratto vegetale A1 è stata valutata nelle linee cellulari di TNBC a fenotipo mesenchimale MDA-MB-231 e SUM-159PT, utilizzando modelli in vitro 2D e 3D complementari. La vitalità cellulare è stata valutata mediante saggi MTT, CellTiter-Glo® e LIVE/DEAD, mentre i saggi di wound healing, migrazione in Transwell e invasione con Matrigel sono stati impiegati per valutare la capacità migratoria e invasiva. L’espressione della proteina RACK1 è stata analizzata mediante Western blot. La comunicazione tra tumore e sistema immunitario è stata indagata attraverso saggi Transwell per valutare il reclutamento di monociti THP-1 verso il terreno condizionato tumorale. A1 ha dimostrato di ridurre la vitalità delle cellule TNBC in maniera dose-dipendente e ha marcatamente compromesso migrazione e invasione in entrambe le linee cellulari. Questi effetti sono stati associati ad una significativa downregulation dell’espressione di RACK1. Inoltre, il terreno condizionato proveniente da cellule di TNBC trattate con A1 ha ridotto in maniera significativa il reclutamento dei monociti THP-1, suggerendo che A1 alteri la segnalazione paracrina tumore-derivata coinvolta nella comunicazione con il TME. Nei modelli di sferoidi 3D, A1 ha efficacemente soppresso la vitalità degli sferoidi e ha marcatamente inibito l’invasività e la formazione di strutture ramificate, confermando la sua attività anti-invasiva in un contesto più fisiologicamente rilevante. Questi risultati dimostrano che A1 agisce simultaneamente su molteplici hallmark della progressione del TNBC, riducendo la sopravvivenza, l’invasione e il crosstalk con il TME delle cellule tumorali. La concomitante downregulation di RACK1 supporta ulteriormente il suo possibile ruolo come mediatore molecolare dell’attività di A1. Nel complesso, A1 emerge come un candidato promettente per lo sviluppo di nuove strategie terapeutiche adiuvanti contro il TNBC, meritando ulteriori studi meccanicistici e di validazione in vivo.
Valutazione dell’efficacia funzionale dell’estratto vegetale A1 nel contrastare l’aggressività del TNBC.
GIUGNETTI, VERONICA
2025/2026
Abstract
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, accounting for 15-25% of all breast cancer cases. It is characterised by marked molecular heterogeneity, high metastatic potential, and the absence of effective targeted therapies due to the lack of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression. Tumour progression is sustained by a dynamic bidirectional crosstalk between cancer cells and the tumour microenvironment (TME), while the scaffold protein RACK1 coordinates signalling pathways involved in tumour cell survival, migration, and invasion. The anti-tumour activity of the plant-derived extract A1 was evaluated in the mesenchymal-like TNBC cell lines MDA-MB-231 and SUM-159PT, using complementary 2D and 3D in vitro models. Cell viability was assessed by MTT, CellTiter-Glo® and LIVE/DEAD assays, while wound healing, Transwell migration and Matrigel invasion assays were performed to evaluate migratory and invasive capacities. RACK1 protein expression was analysed by Western blotting. Tumour-immune crosstalk was investigated through Transwell assays measuring THP-1 monocytes recruitment toward tumour conditioned media. A1 reduced TNBC cell viability in a dose-dependent manner and markedly impaired migration and invasion in both cell lines. These effects were associated with a significant downregulation of RACK1 expression. Moreover, conditioned media from A1-treated TNBC cells significantly reduced THP-1 monocytes recruitment, suggesting that A1 impairs tumour-derived paracrine signalling involved in TME communication. In 3D spheroids models, A1 effectively suppressed spheroids viability and markedly inhibited invasive outgrowth and branching structures, confirming its anti-invasive activity in a more physiologically relevant setting. These findings demonstrate that A1 simultaneously targets multiple hallmarks of TNBC progression by reducing tumour cells survival, invasion, and TME crosstalk. The associated downregulation of RACK1 further supports its potential role as a molecular mediator of A1 activity. Overall, A1 emerges as a promising candidate for the development of novel adjuvant therapeutic strategies against TNBC, warranting further mechanistic studies and in vivo validation.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/36050