Notch3 is involved in the proliferation of renal cancer cells via regulation of cell cycle progression and HIF‑2α

Abstract

Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. Although deregulation of the Notch signaling pathway is common in RCC and is involved in the tumorigenic process, the exact role of Notch3 and its underlying molecular mechanism in RCC, particularly in hypoxia, remain unknown. In the present study, RO4929097, a Notch3 inhibitor, was used to alter NICD3 expression. A Cell Counting Kit-8 assay, EdU incorporation assay, colony formation assay, flow cytometry and western blot analysis were used to investigate the effects of altered NICD3 expression on cell proliferation, cell cycle progression and HIF-2α protein expression. The results of western blot analysis showed that RO4929097 dose-dependently decreased the expression of Notch3 intracellular domain (NICD3) in 786-O and ACHN cells, which originate from clear cell RCC (ccRCC). The results of the Cell Counting Kit-8, EdU incorporation and colony formation assays demonstrated that downregulation of NICD3 significantly suppressed cell proliferation in both normoxia and hypoxia. In addition, flow cytometry and western blot analysis demonstrated that hypoxia (2% O 2) promoted cell cycle progression in ccRCC cells with the increased expression of G 1-S transition-associated proteins, namely cyclin-dependent kinase (CDK)4 and cyclin D1, while downregulation of NICD3 exerted negative effects on cell cycle progression, and the expression levels of CDK4 and cyclin D1. Furthermore, western blot analysis revealed that 2% O 2-induced upregulated hypoxia-inducible factor-2α (HIF-2α) expression decreased following downregulation of NICD3 in 786-O and ACHN cells. Following transfection of the vector containing the NICD3 coding sequence, HIF-2α, CDK4, cyclin D1 and proliferating cell nuclear antigen expression,that were inhibited by RO4929097 in hypoxia, were rescued. Collectively, the results of the present study suggest that Notch3 is closely associated with the cell proliferation of ccRCC cells by regulating the cell cycle and HIF-2α.

Dependence receptors: new targets for cancer therapy

“…Therefore, during this step of tumorigenesis, a prevalent aspect is proliferation of tumor cells, and DR activation has major implications in the promotion of this hallmark. Thus, NOTCH3 was shown to promote the proliferation of renal cancer cells through regulation of cell cycle progression and of hypoxia‐inducible transcription factor 2‐alpha (HIF‐2α) expression (Han et al , 2020). Another example is p75NTR activation, which enhances the clonogenic capabilities of cancer cells by inactivating p53 through activation of MDM2‐mediated ubiquitylation (Zhou et al , 2016).…”

Development and Validation of a Prognostic Gene Signature in Clear Cell Renal Cell Carcinoma

“…Inhibition of the p53 pathway may promote ccRCC cell proliferation and inhibit apoptosis (Noon et al, 2010). Through cell cycle and HIF-2α regulation, Notch3 promotes the proliferation of renal cancer cells (Han et al, 2020). DNA sensing activates innate immune responses not only in immune cells such as dendritic cells (DCs) FIGURE 10 | GSEA analysis of the high-and low-risk groups (A-I) Some cancer-related pathways were prevalent in the high-risk group: "Nod like receptor signaling pathway," "P53 signaling pathway," "cell cycle," "homologous recombination," "base excision repair," "cytosolic dna sensing pathway," "ubiquitin mediated proteolysis," "primary immunodeficiency," "DNA dergradation," NES, Normalized enrichment score.…”

“…but also in non-immune cells such as fibroblasts and tumor cells. Disorders in DNA sensing may lead to cancer (Han et al, 2020;Qiao et al, 2017).…”

Notch3 regulates ferroptosis via ROS‐induced lipid peroxidation in NSCLC cells

“…For specially inhibiting Notch3, the cells were transfected with Notch3 shRNA lentiviral particles (sc-37136-V; Santa Cruz, Dallas, TX, USA) and stable cell lines were established (described previously [12]). For overexpression of NICD3, a pCMV14-Nero vector containing the coding sequence of NICD3 was transfected into cells and stable cell lines were established (described previously [13]). The transfection efficiency was confirmed by western blot analysis.…”