PTEN decreases NR2F1 expression to inhibit ciliogenesis during EGFRL858R-induced lung cancer progression.

Lung cancer is the major cause of death worldwide. Activation of oncogenes or inhibition of tumor suppressors causes cancer formation. Previous studies have indicated that PTEN, as a tumor suppressor, inhibits cancer formation. In this study, we studied the role of PTEN in EGFRL858R-induced lung cancer in vivo. Interestingly, loss of PTEN increased bronchial cell hyperplasia but decreased alveolar cell hyperplasia in EGFRL858R*PTEN-/--induced lung cancer. Systematic analysis of gene expression by RNA-seq showed that several genes related to ciliogenesis were upregulated in EGFRL858R*PTEN-/--induced lung cancer and subsequently showed that bronchial ciliated cells were hyperplastic. Several critical ciliogenesis-related genes, such as Mucin5A, DNAI2, and DNAI3, were found to be regulated by NR2F1. Next, NR2F1 was found to be inhibited by overexpression of PTEN, indicating that PTEN negatively regulates NR2F1, thereby inhibiting the expression of ciliogenesis-related genes and leading to the inhibition of bronchial cell hyperplasia during EGFRL858R-induced lung cancer progression. In addition, we also found that PTEN decreased AKT phosphorylation in A549, KRAS mutant, and H1299 cells but increased AKT phosphorylation in PC9, EGFRL858R, and H1299L858R cells, suggesting that PTEN may function as a tumor suppressor and an oncogene in lung cancers with KRAS mutation and EGFR mutation, respectively. PTEN acts as a double-edged sword that differentially regulates EGFRL858R-induced lung cancer progression in different genomic backgrounds. Understanding the PTEN in lung cancer with different genetic backgrounds will be beneficial for therapy in the future.

USP24-i-101 targeting of USP24 activates autophagy to inhibit drug resistance acquired during cancer therapy.

Drug resistance in cancer therapy is the major reason for poor prognosis. Addressing this clinically unmet issue is important and urgent. In this study, we found that targeting USP24 by the specific USP24 inhibitors, USP24-i and its analogues, dramatically activated autophagy in the interphase and mitotic periods of lung cancer cells by inhibiting E2F4 and TRAF6, respectively. USP24 functional knockout, USP24C1695A, or targeting USP24 by USP24-i-101 inhibited drug resistance and activated autophagy in gefitinib-induced drug-resistant mice with doxycycline-induced EGFRL858R lung cancer, but this effect was abolished after inhibition of autophagy, indicating that targeting USP24-mediated induction of autophagy is required for inhibition of drug resistance. Genomic instability and PD-L1 levels were increased in drug resistant lung cancer cells and were inhibited by USP24-i-101 treatment or knockdown of USP24. In addition, inhibition of autophagy by bafilomycin-A1 significantly abolished the effect of USP24-i-101 on maintaining genomic integrity, decreasing PD-L1 and inhibiting drug resistance acquired in chemotherapy or targeted therapy. In summary, an increase in the expression of USP24 in cancer cells is beneficial for the induction of drug resistance and targeting USP24 by USP24-i-101 optimized from USP24-i inhibits drug resistance acquired during cancer therapy by increasing PD-L1 protein degradation and genomic stability in an autophagy induction-dependent manner.

NCI677397 targeting USP24-mediated induction of lipid peroxidation induces ferroptosis in drug-resistant cancer cells.

Cancer represents a profound challenge to healthcare systems and individuals worldwide. The development of multiple drug resistance is a major problem in cancer therapy and can result in progression of the disease. In our previous studies, we developed small-molecule inhibitors targeting ubiquitin-specific peptidase 24 (USP24) to combat drug-resistant lung cancer. Recently, we found that the USP24 inhibitor NCI677397 induced ferroptosis, a type of programmed cell death, in drug-resistant cancer cells by increasing lipid reactive oxygen species (ROS) levels. In the present study, we investigated the molecular mechanisms and found that the targeting of USP24 by NCI677397 increased gene expression of most lipogenesis-related genes, such as acyl-CoA synthetase long-chain family member 4 (ACSL4), and activated autophagy. In addition, the activity of several antioxidant enzymes, such as glutathione peroxidase 4 (GPX4) and dihydrofolate reductase (DHFR), was inhibited by NCI677397 treatment via an increase in protein degradation, thereby inducing lipid ROS production and lipid peroxidation. In summary, we demonstrated that NCI677397 induced a marked increase in lipid ROS levels, subsequently causing lipid peroxidation and leading to the ferroptotic death of drug-resistant cancer cells. Our study provides new insights into the clinical use of USP24 inhibitors as ferroptosis inducers (FINs) to block drug resistance during chemotherapy.