The Notch inhibitor, FLI-06, increases the chemosensitivity of head and neck Squamous cell carcinoma cells to taxanes-based treatment.

Aberration of Notch signaling is one of the key events involved in the development and progression of head and neck squamous cell carcinoma (HNSCC). The Notch pathway controls the tissue-specific differentiation of normal squamous epithelial cells and is frequently altered in squamous carcinomas, thus affecting their proliferation, growth, survival, and chemosensitivity or resistance against anti-cancer agents. In this study, we show that the use of novel, small-molecule inhibitors of Notch signaling, such as FLI-06, can have a beneficial effect on increasing the chemosensitivity of HNSCC to taxane-based chemotherapy. Inhibition of Notch signaling by FLI-06 alone virtually blocks the proliferation and growth of HNSCC cells in both 2D and 3D cultures and the zebrafish model, which is accompanied by down-regulation of key Notch target genes and proteins. Mechanistically, FLI-06 treatment causes cell cycle arrest in the G1-phase and induction of apoptosis in HNSCC, which is accompanied by increased c-JunS63 phosphorylation. Combining FLI-06 with Docetaxel shows a synergistic effect and partially blocks the cell growth of aggressive HNSCC cells via enhanced apoptosis and modification of c-JunS243 phosphorylation via GSK-3ß inhibition. In conclusion, inhibition of Notch signaling in HNSCC cells that retain active Notch signaling significantly supports taxane-based anticancer activities via modulation of both the GSK-3ß and the c-Jun.

Optimization of a Three-Dimensional Culturing Method for Assessing the Impact of Cisplatin on Notch Signaling in Head and Neck Squamous Cell Carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, with cisplatin being a primary treatment approach. However, drug resistance and therapy failure pose a significant challenge, affecting nearly 50% of patients over time. This research had two aims: (1) to optimize a 3D cell-culture method for assessing the interplay between tumor cells and cancer-associated fibroblasts (CAFs) in vitro; and (2) to study how cisplatin impacts the Notch pathway, particularly considering the role of CAFs. Using our optimized "3D sheet model" approach, we tested two HNSCC cell lines with different cisplatin sensitivities and moderate, non-mutated NOTCH1 and -3 expressions. Combining cisplatin with a γ-secretase inhibitor (crenigacestat) increased sensitivity and induced cell death in the less sensitive cell line, while cisplatin alone was more effective in the moderately sensitive line and sensitivity decreased with the Notch inhibitor. Cisplatin boosted the expression of core Notch signaling proteins in 3D monocultures of both lines, which was counteracted by crenigacestat. In contrast, the presence of patient-derived CAFs mitigated effects and protected both cell lines from cisplatin toxicity. Elevated NOTCH1 and NOTCH3 protein levels were consistently correlated with reduced cisplatin sensitivity and increased cell survival. Additionally, the Notch ligand JAG2 had additional, protective effects reducing cell death from cisplatin exposure. In summary, we observed an inverse relationship between NOTCH1 and NOTCH3 levels and cisplatin responsiveness, overall protective effects by CAFs, and a potential link between JAG2 expression with tumor cell survival.

Evaluation of the anticancer activity of RIN-1, a Notch signaling modulator, in head and neck squamous cell carcinoma.

Notch signalling is one of the key molecular pathways involved in cell-to-cell signal transduction. Although the mechanisms of action of the NOTCH receptors are already relatively well known, their biological implications remain unclear, especially during the initiation and progression of head and neck squamous cell carcinoma (HNSCC). Here, we present the growth- and differentiation-modulating effects of various "next generation" small molecule Notch modulators represented by RIN-1, and CB-103, on HNSCC, compared to gamma secretase inhibitors as "conventional" NOTCH interfering compounds, like DAPT. These molecules were tested in different cell- and tissue culture conditions represented by 2D monolayer, non-adherent or spheroid culture, 3D organoid cultures, and zebrafish in vivo model. The most pronounced, pleiotropic effects were observed for the NOTCH modulator RIN-1. At the molecular level, RIN-1-dependent activation of Notch signalling led to characteristic changes in the expression of NOTCH-regulated targets, i.e., the transcriptional suppressors HES1 and HEY1, p21 (CDKN1A) cell cycle inhibitor, and pro-apoptotic BAX markers. These changes led to restriction of proliferation, growth, and reduced motility of HNSCC cells in 2D cultures. Consequently, cell cycle arrest in the G2-M phase and induction of apoptosis were observed. Similar anticancer effects were observed in 3D cultures and in the zebrafish model. In contrast, RIN-1 treatment resulted in inhibition of Notch signalling and the growth of HNSCC spheroids under non-adherent cell culture conditions. Our results suggest that modulation of Notch signalling could be used as a chemotherapeutic agent in selected patients with intact NOTCH signaling.

Shooting at Moving and Hidden Targets-Tumour Cell Plasticity and the Notch Signalling Pathway in Head and Neck Squamous Cell Carcinomas.

Head and Neck Squamous Cell Carcinoma (HNSCC) is often aggressive, with poor response to current therapies in approximately 40-50% of the patients. Current therapies are restricted to operation and irradiation, often combined with a small number of standard-of-care chemotherapeutic drugs, preferentially for advanced tumour patients. Only very recently, newer targeted therapies have entered the clinics, including Cetuximab, which targets the EGF receptor (EGFR), and several immune checkpoint inhibitors targeting the immune receptor PD-1 and its ligand PD-L1. HNSCC tumour tissues are characterized by a high degree of intra-tumour heterogeneity (ITH), and non-genetic alterations that may affect both non-transformed cells, such as cancer-associated fibroblasts (CAFs), and transformed carcinoma cells. This very high degree of heterogeneity likely contributes to acquired drug resistance, tumour dormancy, relapse, and distant or lymph node metastasis. ITH, in turn, is likely promoted by pronounced tumour cell plasticity, which manifests in highly dynamic and reversible phenomena such as of partial or hybrid forms of epithelial-to-mesenchymal transition (EMT), and enhanced tumour stemness. Stemness and tumour cell plasticity are strongly promoted by Notch signalling, which remains poorly understood especially in HNSCC. Here, we aim to elucidate how Notch signal may act both as a tumour suppressor and proto-oncogenic, probably during different stages of tumour cell initiation and progression. Notch signalling also interacts with numerous other signalling pathways, that may also have a decisive impact on tumour cell plasticity, acquired radio/chemoresistance, and metastatic progression of HNSCC. We outline the current stage of research related to Notch signalling, and how this pathway may be intricately interconnected with other, druggable targets and signalling mechanisms in HNSCC.