Abstract
Ubiquitin ligases have been shown to regulate drug sensitivity. This study aimed to explore the role of the ubiquitin ligase WD repeat and HMG-box DNA binding protein 1 (WDHD1) in regulating cisplatin sensitivity in lung adenocarcinoma (LUAD). A quantitative analysis of the global proteome identified differential protein expression between LUAD A549 cells and the cisplatin-resistant strain A549/DDP. Public databases revealed the relationship between ubiquitin ligase expression and the prognosis of patients with LUAD. Quantitative real-time polymerase chain reaction and Western blotting were used to estimate the WDHD1 expression levels. Analysis of public databases predicted the substrate of WDHD1. Western blotting detected the effect of WDHD1 on microtubule-associated protein RP/EB family member 2 (MAPRE2) and DSTN. Functional analysis of MAPRE2 verified the interaction between WDHD1 and MAPRE2, as well as the interacting sites by methyl-thiazolyl-tetrazolium assay and flow cytometry, immunoprecipitation, protein stability, and immunofluorescence. Cell and animal experiments confirmed the effect of WDHD1 and MAPRE2 on cisplatin sensitivity in LUAD. Clinical data evaluated the impact of WDHD1 expression level on cisplatin sensitivity. Quantitative analysis of the global proteome revealed ubiquitin-dependent protein catabolism to be more active in A549/DDP cells than in A549 cells. WDHD1 expression was higher in A549/DDP cells than in A549 cells, and knocking out WDHD1 increased the sensitivity of A549/DDP cells to cisplatin. WDHD1 overexpression negatively correlated with the overall survival of LUAD patients. We observed that MAPRE2 was upregulated when WDHD1 was knocked out. A MAPRE2 knockout in A549 cells resulted in increased cell viability while decreasing apoptosis when the A549 cells exposed to cisplatin. WDHD1 and MAPRE2 were found to interact in the nucleus, and WDHD1 promoted the ubiquitination of MAPRE2. Following cisplatin exposure, the WDHD1 and MAPRE2 knockout groups facilitated cell proliferation and migration, inhibited apoptosis in A549/DDP cells, decreased apoptosis, and increased tumor size and growth rate in animal experiments. Immunohistochemistry showed that Ki67 levels increased, and levels of apoptotic indicators significantly decreased in the WDHD1 and MAPRE2 knockout groups. Clinical data confirmed that WDHD1 overexpression negatively correlated with cisplatin sensitivity. Thus, the ubiquitin ligase WDHD1 induces cisplatin resistance in LUAD by promoting MAPRE2 ubiquitination.
Highlights
Lung cancer is the most common type of malignant tumor
Ubiquitin-Dependent Protein Catabolism Was More Active in A549/DDP Cells Than That in A549 Cells Cisplatin resistance in patients with Lung adenocarcinoma (LUAD) is one of the main reasons for poor tumor prognosis
Among the quantified proteins in A549/DDP cells, we found that 312 proteins were upregulated, and 345 proteins were downregulated as compared to A549 cells (Figure 1A)
Summary
Lung cancer is the most common type of malignant tumor. It ranks first in morbidity and mortality globally, as well as in China, and is the leading cause of cancer-related deaths [1, 2]. The treatment of lung cancer is achieved by a combination of various therapies, including surgery, chemotherapy, radiotherapy, molecular targeted therapy, and immunotherapy. Chemotherapy continues to be an important treatment strategy for lung cancer. The chemotherapy regimen for lung cancer is based on platinum combined with other chemotherapeutic drugs, the most commonly used platinum drug being cisplatin (DDP). The failure of lung cancer treatment has typically occurred due to cisplatin resistance. Lung adenocarcinoma (LUAD) accounts for the highest proportion of lung cancer, ranging from 40 to 55%.
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