RBM15 promotes tumorigenic proliferation, suppresses apoptosis, and enhances gefitinib resistance in EGFR-mutant LUAD cells by regulating the EGFR signaling pathway and interacting with DGKE.
Abstract
EGFR is a key oncogenic driver of lung adenocarcinoma, and resistance to its tyrosine kinase inhibitors has become a major clinical challenge in the treatment of EGFR-mutated LUAD patients. Through preliminary experiments including Western blot and immunohistochemistry staining, RBM15 was identified as an oncogene promoting drug resistance in EGFR-mutant LUAD cell lines PC-9 and HCC827. Analysis of the TCGA-LUAD cohort revealed a positive correlation between RBM15 and EGFR expression, whereas RBM15 gain-of-function was negatively associated with LUAD patient survival. RBM15 expression was higher in EGFR-mutant LUAD cell lines PC-9 and HCC827 compared to non-EGFR-mutant cell line A549. Furthermore, RBM15 was downregulated in PC-9 and HCC827 cells following gefitinib treatment. Knockdown of RBM15 reduced proliferation and promoted apoptosis in PC-9 and HCC827 cells in vitro, while also inhibiting the growth of PC-9 xenograft tumors in mice. Notably, RBM15 overexpression rescued these effects and promoted gefitinib resistance in PC-9 and HCC827 cells. Transcriptomic and metabolomic sequencing analyses of RBM15-knockdown PC-9 cells revealed enrichment in multiple cancer signaling pathways, including mitochondrial fatty acid metabolism. Diacylglycerol kinase ε (DGKE) was identified as a novel interacting protein of RBM15, and RBM15 was found to influence fatty acid metabolism by modulating mitochondrial function. In summary, RBM15 promotes tumorigenic proliferation, suppresses apoptosis, and enhances gefitinib resistance in EGFR-mutant LUAD cells by regulating the EGFR signaling pathway and interacting with DGKE. Based on the interplay among RBM15, EGFR, and downstream DGKE, RBM15 may serve as a promising new therapeutic target for EGFR-mutant LUAD.
Gefitinib resistance remains a major obstacle to the effective treatment of non-small cell lung cancer (NSCLC), and the underlying molecular mechanisms have not yet been fully elucidated. Lysosomal-associated transmembrane protein 4B (LAPTM4B) has been shown to be involved in cancer progression, but its specific role in gefitinib resistance and the prognosis of NSCLC patients remains unclear. In this study, we conducted functional experiments including CCK-8, Transwell, and tumor sphere formation assays in LAPTM4B-overexpressing or knockdown HCC827 cells and gefitinib-resistant HCC827-R cells. In vivo validation was performed using a NSCLC xenograft model in nude mice. The results showed that LAPTM4B was upregulated in NSCLC, with a further increase in gefitinib-resistant NSCLC samples. Functional experiments demonstrated that knockdown or overexpression of LAPTM4B regulated gefitinib resistance, cell migration, and stemness in HCC827 cells. Furthermore, RPS3 was identified as a key interacting target of LAPTM4B, and LAPTM4B could enhance the stability of RPS3 protein by inhibiting its ubiquitination. In vivo xenograft experiments showed that knockdown of LAPTM4B significantly suppressed tumor growth and tumor stemness, and this inhibitory effect could be reversed by overexpression of RPS3. Collectively, our findings indicate that LAPTM4B promotes gefitinib resistance and NSCLC progression by stabilizing RPS3 protein through inhibiting its ubiquitination, and the LAPTM4B-RPS3 axis may serve as a potential therapeutic target for overcoming gefitinib resistance in NSCLC.
Yu Jiang, Wen-Jing Xie, Rong-Wei Chen et al.· Biochimica et biophysica act...· 0 citations
Highlights What are the main findings? FOXD3 overexpression suppresses malignant phenotypes in two KRAS G12C-mutant NSCLC models. FOXD3 overexpression is associated with reduced NF-κB transcriptional activity, while TNF-α partially reverses the associated phenotypic changes. What are the implications of the main findings? The FOXD3–NF-κB relationship represents a regulatory feature of tumor-intrinsic behavior in KRAS G12C-mutant NSCLC cells. Direct mechanistic and immune-competent studies are required to establish causality and potential therapeutic relevance. Abstract KRAS G12C mutation is a clinically relevant driver in non-small cell lung cancer (NSCLC), yet the signaling networks that modulate malignant behavior in this context remain incompletely defined. In this study, we examined the functional role of FOXD3 and its relationship with NF-κB signaling in KRAS G12C-mutant NSCLC models. Stable FOXD3 overexpression was established in SW1573 and LU65 cells. FOXD3 reduced cell viability, migration, and invasion while increasing caspase 3/7 activity in both cell lines. Transcriptomic profiling in LU65 cells followed by Hallmark enrichment analysis identified TNFα signaling via NF-κB as a prominently altered pathway associated with FOXD3 overexpression. Consistently, NF-κB dual-luciferase assays showed reduced basal NF-κB transcriptional activity in FOXD3-overexpressing cells. TNFα stimulation partially reversed the inhibitory effects of FOXD3 on proliferation, migration, and invasion and attenuated FOXD3-induced apoptosis. In addition, stable FOXD3 overexpression suppressed xenograft growth in vivo. Collectively, these findings support a functional association between FOXD3 overexpression and reduced NF-κB-related transcriptional activity in KRAS G12C-mutant NSCLC models, although the present data do not establish direct causal mediation by NF-κB.
Peng Xu, Jie Peng, Chao Guo et al.· Cells· 0 citations
The lack of mutation-contextual therapeutic targets remains a major barrier in hepatocellular carcinoma (HCC) management. Immunohistochemical analysis of 341 human HCC specimens identified FK506-binding protein 52 (FKBP52) as a tumor grade-associated biomarker. In vitro, FKBP52 overexpression enhanced proliferation in p53-mutant liver cancer cells but suppressed it in p53-wild-type cells. Using two murine models—subcutaneous xenografts and orthotopic liver implants—genetic manipulation confirmed that FKBP52 overexpression in Huh7 accelerated tumor growth, while shRNA-mediated knockdown attenuated growth and decreased AFP/CD133 expression. Differential p53 status underpinned this disparity. In vitro EdU incorporation assays, colony formation assays and TP53-null xenografts (Hep3B cells) demonstrated that p53 knockdown abrogated the phenotype, confirming that FKBP52 promotes mutant p53-driven tumorigenesis while suppressing wild-type p53 tumors. Mechanistically, co-immunoprecipitation (Co-IP) revealed FKBP52 binds p53 via its FK2 and TPR domains independently of mutation status. This interaction recruited MDM2 into the FKBP52-p53 complex, impairing MDM2-mediated degradation and prolonging stability of both wild-type and mutant p53. Taken together, our findings establish FKBP52 as a novel p53 stabilizer with context-dependent oncogenic effects, critically determined by p53 mutational status. These results highlight the therapeutic potential of targeting the FKBP52-MDM2-p53 axis specifically in TP53-mutant HCC.
Jinfeng Wu, Cheng-Yan Tang, Z. Meng et al.· Journal of Biological Chemis...· 0 citations
Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) represents a major clinical challenge in the management of non-small cell lung cancer (NSCLC). Chromosome 4q12 locus is an important gene locus associated with progression-free survival (PFS) in NSCLC patients receiving EGFR-TKIs therapy. However, it remains poorly characterized how genes at this locus function in NSCLC development and resistance to EGFR-TKIs. Here, we found that CLOCK at this locus is highly expressed in NSCLC tissues and correlates with unfavorable PFS of patients. CLOCK promotes malignant proliferation and metastasis of NSCLC in vitro and in vivo. CLOCK could attenuate treatment efficacies of gefitinib (one of the first-generation EGFR-TKIs) or osimertinib (one of the third-generation EGFR-TKIs). Mechanistically, CLOCK functions as a transcriptional factor to upregulate LAMC2 transcription and expression in NSCLC cells. There was significantly elevated LAMC2 expressed in NSCLC tissues and its high levels were associated with shortened survival of patients. Indeed, CLOCK could activate multiple kinase signaling pathways, such as the PI3K-Akt signaling and the MAPK signaling, by facilitating either the LAMC2-ITGB1 interaction and the LAMC2-EGFR interaction and thereby accelerating NSCLC proliferation and conferring EGFR-TKI resistance. Collectively, these findings for the first time identify CLOCK as a critical mediator of EGFR-TKIs resistance and a promising target to overcome EGFR-TKIs resistance in NSCLC.
Yankang Li, Linyu Han, Huiwen Luan et al.· Archives of Biochemistry and...· 0 citations
The in vitro proliferation of AKP-M4 cells, but not KRAS wild-type SNU-1079 and SSP-25 cells, was reduced by the KRAS G12D mutation inhibitor MRTX-1133, which supports the development of KRAS mutation inhibitor for CCA treatment.
J. Hung, Tsai-Hsien Hung, Chun-Nan Yeh et al.· Journal of Immunology· 0 citations
Lung cancer has the highest mortality rate of all cancers, and lung adenocarcinoma (LUAD) is one of the most common subtypes. TMEM74 expression is upregulated in both lung cancer patients and LUAD cell lines, and its high expression correlates with poor patient survival. Targeted TMEM74 knockdown and overexpression vectors were successfully constructed, and stably transfected LUAD cell lines were established. TMEM74 was found to significantly enhance LUAD cell proliferation and colony formation while suppressing apoptosis. TMEM74 overexpression elevated the LC3II/I ratio, reduced p62 levels, and boosted autolysosome formation, whereas TMEM74 knockdown exerted opposite effects. Bafilomycin A1-mediated autophagy inhibition abrogated the pro-proliferative and anti-apoptotic effects driven by TMEM74 overexpression. Subcutaneous xenograft experiments revealed that TMEM74 knockdown restrained tumor growth, and impaired autophagic activity in tumor. A positive correlation was observed between the expression levels of Runt-related transcription factor 2 (RUNX2) and TMEM74 in LUAD. RUNX2 overexpression in LUAD cells induced upregulation of TMEM74 mRNA levels. Dual luciferase reporter assays demonstrated that RUNX2 significantly enhanced the transcriptional activity of TMEM74 in LUAD cells. TMEM74 knockdown reversed the increased cell proliferation, reduced apoptosis and enhanced autophagosome formation induced by RUNX2 overexpression in LUAD cells. Collectively, our experimental results demonstrate that RUNX2 transcriptionally activates TMEM74, thereby promoting LUAD cell proliferation and autophagy and inhibiting cell apoptosis in vitro; TMEM74 knockdown suppresses tumor growth in vivo.
Ji Zhang, Yuan-Zhu Xia, Bo Huang et al.· Biochemical and Biophysical...· 0 citations
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