Piperlongumine overcomes osimertinib resistance via governing ubiquitination-modulated Sp1 turnover
Ruirui Wang, Qiang Wang, Jinzhuang Liao, Xinfang Yu, Wei Li
Ruirui Wang, Qiang Wang, Jinzhuang Liao, Xinfang Yu, Wei Li
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Research Article Cell biology Oncology

Piperlongumine overcomes osimertinib resistance via governing ubiquitination-modulated Sp1 turnover

Abstract

Non–small cell lung cancer (NSCLC) is a common cause of cancer-related deaths worldwide, and its incidence has been increasing in recent years. While targeted therapies like osimertinib, an epidermal growth factor receptor tyrosine kinase inhibitor, have brought about notable improvements in patient outcomes for advanced NSCLC, the challenge of acquired drug resistance persists. Here, we found that cellular mesenchymal-epithelial transition factor (c-Met) was highly expressed in osimertinib-resistant cells, and depletion of c-Met markedly inhibited the growth of osimertinib-resistant cells ex vivo and in vivo, suggesting that c-Met is a potential target to address osimertinib resistance. Through a screening process using a natural product compound library, we identified piperlongumine as a potent inhibitor to overcome osimertinib resistance. Furthermore, the combined treatment of piperlongumine and osimertinib exhibited robust antitumor effects in resistant cells, partially restoring their sensitivity to osimertinib. Additionally, we discovered that piperlongumine could enhance the interaction between E3 ligase RNF4 and Sp1, inhibit the phosphorylation of Sp1 at Thr739, facilitate the ubiquitination and degradation of Sp1, lead to c-Met destabilization, and trigger intrinsic apoptosis in resistant cells. In summary, our study sheds light on the potential of piperlongumine in overcoming osimertinib resistance, offering new strategies and perspectives for the clinical management of drug-resistant NSCLC.

Authors

Ruirui Wang, Qiang Wang, Jinzhuang Liao, Xinfang Yu, Wei Li

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Figure 6

Piperlongumine destabilizes Sp1 in a Thr739 phosphorylation–dependent manner.

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Piperlongumine destabilizes Sp1 in a Thr739 phosphorylation–dependent ma...
(A) HCC827OR and H1975OR cells were treated with different concentrations of piperlongumine, and WCE was collected for IB analysis. (B) HCC827OR cells were treated with or without piperlongumine for 24 hours and after coincubation with MG-132 for 8 hours. Immunoprecipitation (IP) assay was performed to detect the interaction between RNF4 and Sp1. (C) Flag-Sp1-WT or -T739D was transfected into HCC827OR cells, followed by piperlongumine treatment for 24 hours. MG-132 was added to the medium and maintained for 8 hours. Cells were collected and IB analysis was performed. (D). The corresponding plasmids were transfected into HCC827OR cells. Piperlongumine treatment was given for 24 hours followed by treatment with CHX (20 μg/mL) for different time points. Cells were collected for IB analysis. (E) Flag-Sp1-T739D plasmid was transfected into HCC827OR and H1975OR cells overnight, followed by various doses of piperlongumine treatment for 24 hours. WCE was collected for IB analysis. (F) Flag-Sp1-WT, or -T739D, was transfected into osimertinib-resistant cells and treated with piperlongumine (4 μM) for 24 hours. MG-132 was added to the medium and maintained for 8 hours. Cells were collected for ubiquitination analysis. (G) Flag-Sp1-WT, or -T739D, was transfected into osimertinib-resistant cells and treated with piperlongumine (4 μM) for 24 hours. Cells were collected for IB analysis. (H and I) MTS (H) and the soft agar assay (I) determined cell viability and colony formation ability, respectively. ***P < 0.001. Comparisons were performed using 1-way ANOVA (H and I, n = 3). Data are presented as the mean ± SD (H and I).