RHBDD1 induces cell cycle arrest in TP53-mutant NSCLC cells by promoting endoplasmic reticulum-associated degradation of p53 and DNA-PKcs

Abstract

Background

RHBDD1 is a tumor-promoting protein that enhances endoplasmic reticulum-associated degradation (ERAD). This study aimed to explore its effects on non-small cell lung cancer (NSCLC) cells.

Methods

The potential roles of RHBDD1 in NSCLC were predicted through bioinformatics analysis. The predicted regulation of p53/DNA-PKcs signaling by RHBDD1-associated ERAD was further validated in TP53 wild-type and mutant NSCLC cells.

Results

Bioinformatics analysis revealed that in the RHBDD1-highly expressed NSCLC samples, cell cycle was significantly downregulated, protein processing in endoplasmic reticulum (ER) was significantly upregulated, and p53 signaling pathway was inhibited. TP53 and PRKDC were identified as hub genes. Inhibition of cell cycle and activation of protein processing in ER were specifically enriched in TP53-mutant, RHBDD1-highly expressing samples, but not in TP53 wild-type ones. The protein levels of p53 and DNA-PKcs (encoded by the PRKDC gene) were more significantly altered by the DNA-PKcs inhibitor STL127705 and sh-RHBDD1 in NCI-H596 cells compared to A549 cells. In NCI-H596 cells transfected with pcDNA-RHBDD1, the reduced levels of DNA-PKcs and p53 were restored by co-treatment with p53 activators HBX41108 or Nutlin-3a. Cell cycle progression in NCI-H596 cells was significantly arrested at the S phase in both sh-RHBDD1 and pcDNA-RHBDD1 transfected groups. Both interventions led to decreased cell viability, which was reversed by co-treatment with the DNA-PKcs inhibitor STL127705 and further enhanced by Nutlin-3a. Increased levels of DNA-PKcs and p53, along with a modest reduction in ER stress markers, were observed in the sh-RHBDD1 group, whereas opposite trends were seen in the pcDNA-RHBDD1 group. Apoptosis was obviously inhibited in pcDNA-RHBDD1 transfected NCI-H596 cells, but not in transfected A549 cells. Co-localization of p53 with RHBDD1 and DNA-PKcs was prominently observed in TP53-mutant NSCLC cells, but not in wild-type cells.

Conclusion

RHBDD1 arrests the cell cycle in TP53-mutant NSCLC cells through ERAD-dependent downregulation of p53 and DNA-PKcs. However, cell survival may be enhanced due to the inhibition of DNA-PKcs-regulated apoptosis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-03116-y.

Highlights

RHDBB1 could arrest cell cycle in the TP53 mutant NCI-H596 cells;

RHDBB1 promotes NSCLS by reducing DNA-PKcs dependent cells apoptosis;

Targeting both TP53 and RHDBB1 may have beneficial effects on NSCLC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-03116-y.

Introduction

Lung cancer, particularly non-small cell lung cancer (NSCLC), is one of the most prevalent and deadly cancers worldwide [1]. In 2022, there were approximately 2.5 million new cases of NSCLC, accounting for 12.4% of all cancer diagnoses globally, with particularly high incidence rates in China [2]. NSCLC consists of two main subtypes: lung adenocarcinoma (LUAD), which accounts for 50–60% of cases, and lung squamous cell carcinoma (LUSC), which represents 20–30%. These subtypes share similar pathological features and are generally treated with comparable strategies [3]. However, treating NSCLC presents numerous challenges, including late-stage diagnosis, evolving tumor microenvironments, tumor heterogeneity, and ongoing tumor evolution [4]. Investigating common mutations is essential for understanding its tumorigenic signaling and improving treatment efficacy and patient prognosis [5].

Rhomboid domain-containing 1 (RHBDD1), also known as protease rhomboid-like protein 4 (RHBDL4), is primarily localized in the endoplasmic reticulum (ER) and has been shown to be upregulated in various cancers, promoting tumor progression [6]. Studies have demonstrated that RHBDD1 enhances proliferation, cell cycle progression, metastasis, and inhibits apoptosis in tumors such as pancreatic cancer, glioma, and renal cancer [7–9]. In lung cancer, high expression of RHBDD1 is associated with poor prognosis [10]. Our previous research demonstrated that RHBDD1 promotes the proliferation and invasion of NSCLC by modulating the ZEB1/PI3K/AKT signaling pathway [11]. Mechanistically, RHBDD1 recruits valine-containing protein (VCP, also known as p97 or Cdc48) via its C-terminal binding motif [12], facilitating the recognition and binding of ubiquitinated substrate proteins with atypical positively charged transmembrane domains. This RHBDD1-VCP complex then promotes their cleavage and translocation [13]. This process is crucial for the distribution, transport to the Golgi apparatus, and secretion of certain proteins [14], and may also contribute to the non-canonical secretion of endogenous soluble ER-resident molecular chaperones [15]. ER-associated degradation (ERAD) mediated by RHBDD1 and VCP is a novel protein quality control mechanism, though it remains underexplored [16]. Some studies suggest that RHBDD1 may regulate the expression and distribution of key tumor-related proteins, such as TLR4, via this mechanism [17]. Such a quality control mechanism may have important implications for gene mutations, which are frequently observed across tumors. Mutations can lead to protein misfolding, initiating ER retention and subsequent ERAD, including through the RHBDD1/VCP-dependent pathway [18]. These suggest potential associations of RHBDD1-regulated ERAD and mutant proteins in NSCLC.

The tumor suppressor protein p53, encoded by the TP53 gene on chromosome 17p13.1, has been identified as the most frequently mutated gene across human tumors [19]. It is also recognized as a critical substrate for both ERAD and the unfolded protein response (UPR) [20]. The stability, expression, and nuclear localization of p53 can be enhanced in various cell lines under conditions such as glucose deprivation, or ER stress induced by UPR activators like thapsigargin, tunicamycin, and brefeldin A [21]. This phenomenon has also been observed in lung endothelial cells, where UPR activation increased p53 expression [22]. Structurally, the C-terminal domain of p53 is a disordered lysine-rich region with positive charges [23], making it a potential target for RHBDD1 regulation. However, it remains unreported whether RHBDD1 regulates mutant p53 or if this regulation can be targeted to alleviate NSCLC. In this study, we explore the role of RHBDD1-associated ERAD in NSCLC using bioinformatics analysis of both wild-type (wt) and mutant (mut) samples, and further investigate the RHBDD1-ERAD-dependent degradation of p53 and DNA-PKcs in TP53-mutant NSCLC cells.

Materials and methods

Bioinformatics analysis

Proteomics data from 141 NSCLC patients, including relative protein abundance (both relative intensity and normalized rates under Log2), as well as associated clinicopathological characteristics, were directly obtained from the report by Lehtiö et al. [24]. The data were cataloged in the PXD database as PXD025560, PXD020548, and PXD020191. LUAD and LUSC datasets were downloaded from The Cancer Genome Atlas (TCGA) on 14/06/2024 (https://portal.gdc.cancer.gov/). For both proteome and transcriptome data, all samples with RHBDD1 expression were included. The samples were divided into RHBDD1-high and RHBDD1-low expression groups. Sequencing data were analyzed using the limma package, with Log₂ fold change (FC, for RHBDD1-high vs. -low) > 1 and p < 0.05 considered as differentially expressed proteins (DEPs) or genes (DEGs). Enrichment analysis was performed using the clusterProfiler or GSEA packages based on human KEGG gene sets. Protein–protein interactions (PPIs) were analyzed via the STRING database (https://string-db.org/). Enrichment plots for the most significantly enriched pathways were drawn using online bioinformatics tools (https://www.bioinformatics.com.cn/) and modified with Adobe Illustrator CC to improve presentation. The correlation between hub proteins and RHBDD1 in NSCLC was predicted using GEPIA (http://gepia.cancer-pku.cn/), and Pearson correlations for proteomics data were analyzed using GraphPad Prism 9.5.

Cell culture, transfection, and treatments

TP53 wild-type A549 (CL-0016, Pricella, Wuhan, China) and NCI-H838 (JY350, Jinyuan Biotechnology, Shanghai, China), as well as TP53-mutant NCI-H596 (CL-0404, Pricella) and NCI-H1155 (JY1055, Jinyuan Biotechnology) cells, were cultured in RPMI-1640 medium (#GNM31800, GENOM Bio, Zhejiang, China), supplemented with 10% FBS (DX-1002, DAXI, Inner Mongolia, China) and 1% penicillin–streptomycin solution (GNM15140-1, GENOM Bio). The cells were maintained in a 5% CO₂ incubator at 37 °C.

For transfection, sh-RHBDD1, sh-NC, pcDNA-NC, and pcDNA-RHBDD1 vectors were purchased from GenePharma (Suzhou, China) and cloned into the pcDNA3.1 vector (E0648, Sigma-Aldrich) by GENOM Bio. Cells at 70% confluence were transfected with the vectors in Opti-MEM^™ medium (31985070, Invitrogen, USA) using PEI reagent (40815ES03, Yeason, Shanghai, China) for 48 h. After transfection, cells were treated with other reagents and collected within 72 h. The shRNAs used were:

shRNA 1: 5′-GATCGCCTATGTTATCACCGCATTTCTCGAGAAATGCGGTGATAACATAGGCTTTTTG-3′;

shRNA 2: 5′-GATCGCTGGGATTCTTGTTGGACTATCGAGTAGTCCAACAAGAATCCCAGCTTTTTG-3′.

The DNA-PKcs inhibitor STL127705 (3.5 µM, HY-122727, MCE, Shanghai, China), USP7 inhibitor HBX 41108 (424 nM, HY-101666, MCE), MDM2 inhibitor Nutlin-3a (90 nM, N129972, Aladdin, Shanghai, China), proteasome inhibitor PYR-41 (10 μM, HY-13296, MCE), and cisplatin (13 μM, HY-17394, MCE) were dissolved in DMSO and added to the medium for 48-h treatments.

Real-time quantitative PCR (qPCR)

Total RNA was extracted from treated cells using 0.5 mL of Trizol (15596026, Invitrogen) per well of a 6-well plate. cDNA was synthesized using the HiScript III All-in-One RT SuperMix Perfect for qPCR (R333-01, Vazyme). qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). The cycling conditions were: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Gene expression was calculated using the 2^−∆∆Ct method, with GAPDH as the reference gene. The primers used were:

RHBDD1: Forward (F) 5′-ACGCAGGCGGGTCGTA-3′, Reverse (R) 5′-GGGCCAAAGTTGCTAGGGT-3′;

DNA-PKcs: F 5′-CCAAGTCCAACACCAAGTAGCCACCCA-3′, R 5′-CCGCCATGCCGCCGAGTCCC-3′;

p53: F 5′-CTGGATTGGCAGCCAGACT-3′, R 5′-TGGGCATCCTTGAGTTCC-3′;

GAPDH: F 5′-GGTGAAGGTCGGAGTCAACG-3′, R 5′-GCATCGCCCCACTTGATTTT-3′.

Western blotting

Proteins were extracted from treated cells by adding 50 µL of protein lysis buffer (P0013, Beyotime, Shanghai, China) per well on ice. After centrifugation at 12,000 rpm for 10 min at 4 °C, the supernatant was quantified using a BCA protein assay kit (P0010, Beyotime). Equal amounts of protein were mixed with loading buffer (P0015, Beyotime) and heated at 100 °C for 10 min. Proteins were separated by 8–12% SDS-PAGE and transferred to a nitrocellulose membrane (BS-NC-22, Biosharp, Anhui, China), then blocked with 5% non-fat powdered milk in TBST. Membranes were incubated overnight with primary antibodies at 4 °C, followed by incubation with HRP-labeled secondary antibodies (Goat Anti-Mouse IgG [A0216, Beyotime] or Goat Anti-Rabbit IgG [A0208, Beyotime]) at room temperature for 1 h. Protein bands were visualized using ECL reagent (P0018M, Beyotime). ImageJ (v1.53t, NIH) was used to analyze band intensities, normalized to GAPDH levels.

The primary antibodies involved were: BIP (ab108615, Abcam), Caspase-3 (DF6879, Affinity), Caspase-7 (AF5118, Affinity), CHOP (66,741-1-Ig, Proteintech), Cleaved Caspase-3 (AF7022, Affinity), Cleaved Caspase-7 (AF4023, Affinity), Cleaved PARP (AF4023, Affinity), DNA-PKcs (19,983-1-AP, Proteintech), GAPDH (P04406, Abways), IRE1 (27,528-1-AP, Proteintech), p-IRE1 (ab124945, Abcam), p53 (CY5047, Abways, Shanghai, China), PARP (13,371-1-AP, Proteintech), PERK (24,390-1-AP, Proteintech), RHBDD1 (20,869-1-AP, Proteintech, Rosemont, USA), and VCP (10,736-1-AP, Proteintech).

Cell viability

Cell viability was assessed using the CCK-8 assay. Approximately 3000 cells were seeded per well in a 96-well plate. Transfection was performed before seeding, and treatments were initiated 12 h post-seeding. After 46 h of culture, 10 µL of CCK-8 reagent (C0038, Beyotime) was added to each well, and optical density at 450 nm was measured after 2 h.

Flow cytometry

For testing cell cycle, cells were harvested by centrifugation at 500 rpm for 5 min and washed twice with cold PBS. The cell pellets were fixed in cold 70% ethanol at 4 °C overnight, then resuspended in propidium iodide/RNase/PBS and incubated in the dark for 30 min. After filtration through a 400-mesh membrane, cell cycle analysis was performed using a flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo 10.8.1 software.

Apoptosis induced by cisplatin (3.90 μg/mL) treatment was investigated in A549 and NCI-H596 cells transfected with pcDNA-NC or pcDNA-RHBDD1, using an Annexin V-FITC/PI kit (abs50001, Absin, Shanghai, China). Briefly, cells were digested with 0.25% EDTA-free trypsin, resuspended in Binding Buffer, stained with 5 μL of Annexin V-FITC for 15 min in the dark, 5 μL of PI for 5 min, and 200 μL of Binding Buffer before testing on the flow cytometer.

Immunoprecipitation

To validate the interaction of DNA-PKcs, RHBDD1, and p53 in NSCLC cells, A549, NCI-H838, NCI-H596, and NCI-H1155 cells were treated with 10 μM PYR-41 to inhibit proteolysis for 48 h. Then immunoprecipitation was performed with an Immunoprecipitation Kit with Protein G Magnetic Beads (P2177M, Beyotime). Briefly, cells were lysed with 100 μL lysis buffer supplemented with 1% Protease Inhibitor Cocktail. After lysis on ice for 30 min, the lysates were incubated with anti-p53 or anti-IgG-coated beads. Proteins were eluted from the beads using 100 μL Acid Elution Buffer and 10 μL Neutralization Buffer, respectively. Harvested proteins, including raw lysate and immunoprecipitation products, were analyzed by western blotting.

Statistical analysis

R v3.2 was used for bioinformatics analysis, including data processing and enrichment of both proteomics and transcriptomics data. All other quantitative and semi-quantitative data were analyzed using GraphPad Prism 9.5. Relative protein expression levels were assessed with ImageJ. Data are presented as mean ± SD in bar graphs. N = 6 for the CCK-8 assay and N = 3 for other cell-based tests. Statistical comparisons were performed using Student’s t-test for comparisons between two groups, and one-way ANOVA for multiple comparisons. A p-value of < 0.05 was considered statistically significant.

Results

RHBDD1 negatively regulates the cell cycle in NSCLC

Proteomic and transcriptomic analyses of RHBDD1 high- and low-expression samples revealed significant changes in several pathways. In the proteomic data, cell cycle, DNA replication, and spliceosome pathways were downregulated, while hematopoietic cell lineage, cytokine-cytokine receptor interaction, and focal adhesion pathways were upregulated (Fig. 1a). Transcriptomic data showed upregulation of viral protein interactions with cytokines, Th17 cell differentiation, and hematopoietic cell lineage, while cell cycle and carbon metabolism were downregulated (Fig. 1b). Cell cycle regulation emerged as the most significantly enriched pathway in both datasets, highlighting its importance in RHBDD1-mediated effects in NSCLC. Additionally, protein processing in the ER was upregulated, while the p53 signaling pathway was downregulated in the proteomic analysis, further supporting the role of RHBDD1 in regulating cancer cell cycle progression. As shown in Fig. 1c, interaction analysis revealed that the two typically enriched pathways, protein processing in ER and cell cycle, were closely linked by TP53 (p53). Other key proteins involved in both pathways included the upregulated HSPA5 (BiP), HSP90B1, SYVN1, MAP3K5, WFS1, and SEC31A, while CRYAB, PRKDC (DNA-PKcs), and the downregulated CDC25C.

Fig. 1.

Role of RHBDD1 in NSCLC Predicted by Bioinformatics Analysis. Proteomic data were analyzed using the PXD025560, PXD020548, and PXD020191 datasets from the PXD database, while transcriptomic data were obtained from the TCGA-LUAD and TCGA-LUSC datasets. A Gene set enrichment analysis (GSEA) of the proteomic data based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. B GSEA of the transcriptomic data based on the KEGG database. C Protein–protein interaction (PPI) network analysis using differentially expressed proteins (DEPs) from the protein processing in the ER and cell cycle pathways. For GSEA plots, the top 25 most significant pathways and five additional NSCLC or ERAD-associated pathways are shown. For the PPI network, the proteins were clustered into six groups using k-means

RHBDD1 may regulate the cell cycle via the DNA-PKcs–p53 axis in TP53-mutant NSCLC Cells

Correlations between RHBDD1 and key hub proteins in LUAD and LUSC datasets were examined using GEPIA. RHBDD1 showed a strong correlation with TP53 in healthy tissues, but this correlation was less significant in NSCLC tissues (Fig. 2a). Among the hub proteins regulating the cell cycle, PRKDC levels were most significantly correlated with RHBDD1 in both healthy and NSCLC tissues (Fig. 2b–d). At the protein level, p53, DNA-PKcs, and PPP1R60 were negatively correlated with RHBDD1, while CRYA2 was positively correlated (Fig. 2e). p53 levels were higher in TP53-mutant samples (Fig. 2f) and negatively correlated with RHBDD1 in both TP53 wild-type and mutant samples (Fig. 2g). Interestingly, DNA-PKcs was positively correlated with p53 only in TP53-mutant samples (Fig. 2h). Furthermore, patients with low RHBDD1 expression and wild-type TP53 showed significantly higher overall survival rates compared to other groups (Fig. 2i). Reanalysis of proteomic data revealed that protein processing in the ER was upregulated for RHBDD1 -high versus -low expression samples in both TP53 wild-type and mutant samples, while cell cycle regulation was downregulated only in TP53-mutant samples with high RHBDD1 expression (Fig. 2j, k). These suggest RHBDD1’s role in cell cycle regulation through the DNA-PKcs–p53 axis, particularly in TP53-mutant NSCLC.

Fig. 2.

Correlations of Predicted Hub Proteins with RHBDD1. The correlations of A TP53, B PRKDC, C CDC25C, and D CRYAB with RHBDD1 were predicted using GEPIA with LUAD and LUSC datasets, comparing normal and NSCLC tissues. E The correlations of TP53, PRKDC, CDC25C, and CRYAB with RHBDD1 were analyzed using proteomic data from PXD025560, PXD020548, and PXD020191, which were also used in panels F-K. F p53 protein levels in TP53 wild-type (TP53_wt) and TP53-mutant (TP53_mut) samples. G Correlation between TP53 and RHBDD1 in TP53_wt and TP53_mut samples. H Correlation between TP53 and PRKDC (DNA-PKcs) in TP53_wt and TP53_mut samples. I Overall survival rates of patients grouped by TP53 mutation status and RHBDD1 protein levels. J GSEA of the proteomic data by KEGG database for p53_wt samples, comparing RHBDD1 high vs low. K GSEA of the proteome data using the KEGG database in TP53_mut samples, comparing RHBDD1 high versus low. The top 23 most significant pathways and two additional NSCLC- or ERAD-associated pathways are shown

Expression of p53 protein in NSCLC is sensitive to rhbdd1 levels

To verify RHBDD1’s regulation of the cell cycle via the DNA-PKcs–p53 axis, A549 (wild-type TP53) and NCI-H596 (mutant TP53) cells were transfected with sh-RHBDD1 or pcDNA-RHBDD1 to induce RHBDD1 silencing or overexpression. Successful silencing and overexpression of RHBDD1 in A549 cells were confirmed via qPCR (Fig. 3a) and Western blotting (Fig. 3b). In A549 cells, DNA-PKcs and p53 levels modestly increased in the sh-RHBDD1 group, with no significant changes upon co-treatment with STL127705 (Fig. 3c, d). Similarly, successful silencing and overexpression of RHBDD1 in NCI-H596 cells were confirmed via qPCR (Fig. 3e) and Western blotting (Fig. 3f). Both DNA-PKcs and p53 levels increased significantly in the sh-RHBDD1 group, and p53 levels were further elevated with STL127705 treatment (Figs. 3g, h). Conversely, DNA-PKcs and p53 levels were significantly reduced by pcDNA-RHBDD1 transfection, with HBX41108 or Nutlin-3a co-treatment reversing these effects (Fig. 3i, j). These results suggest that RHBDD1 negatively regulates DNA-PKcs and p53, particularly in TP53-mutant NSCLC cells.

Fig. 3.

Effect of RHBDD1 on DNA-PKcs/p53 Signaling. A Validation of transfection in A549 cells by qPCR. B Validation of transfection in A549 cells by western blotting. C Levels of RHBDD1, DNA-PKcs, and p53 proteins in sh-RHBDD1-transfected A549 cells treated with the DNA-PKcs inhibitor STL127705. D Protein levels analyzed using ImageJ. E Validation of transfection in NCI-H596 cells by qPCR. F Validation of transfection in in NCI-H596 cells by western blotting. G Levels of RHBDD1, DNA-PKcs and p53 in sh-RHBDD1 transfected NCI-H596 cells treated with STL127705. H The protein levels were analyzed by ImageJ. I Levels of RHBDD1, DNA-PKcs, and p53 in pcDNA-RHBDD1 transfected NCI-H596 cells co-treated with HBX41108 or Nutlin-3a. J Protein levels were analyzed using ImageJ. Data are shown as mean ± SD. N = 3 for all tests. *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons test

RHBDD1 reduces DNA-PKcs and p53 to promote the cell cycle in TP53-mutant NSCLC cells

Cell cycle analysis of NCI-H596 cells revealed that the G1 phase was reduced, while the S phase increased in both sh-RHBDD1 and pcDNA-RHBDD1 transfected group cells (Fig. 4a, b). STL127705 treatment modestly arrested the cell cycle in the S phase in sh-RHBDD1 cells, while Nutlin-3a had no effect in the pcDNA-RHBDD1 group. Cell viability was measured using the CCK-8 assay; both sh-RHBDD1 and pcDNA-RHBDD1 transfected cells showed modest reductions in viability, which were reversed by STL127705 co-treatment and enhanced by Nutlin-3a co-treatment (Fig. 4c). These results suggest that RHBDD1 regulates the cell cycle in TP53-mutant NSCLC cells in a DNA-PKcs and p53-dependent manner.

Fig. 4.

RHBDD1 Regulates the Cell Cycle through DNA-PKcs/p53 Signaling. A Cell cycle analysis of transfected NCI-H596 cells co-treated with STL127705 or Nutlin-3a for 48 h by flow cytometry. B Cell cycle distribution. C Viability of transfected NCI-H596 cells co-treated with STL127705 or Nutlin-3a for 48 h. Data are shown as mean ± SD. N = 3 for flow cytometry and N = 6 for CCK-8 assays. *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons test

RHBDD1 reduces DNA-PKcs and p53 via an ERAD-dependent mechanism

To investigate the mechanism by which RHBDD1 reduces DNA-PKcs and p53 levels, we examined the presence of ER-associated degradation (ERAD) markers in NCI-H596 cells (Fig. 5a, b). In the sh-RHBDD1 group, DNA-PKcs and p53 were elevated. DNA-PKcs was reduced, while p53 was modestly increased, by co-treatment with STL127705. ERAD markers, such as VCP, PERK, BIP, and CHOP were reduced by STL127705 in the sh-RHBDD1 group. In contrast, in the pcDNA-RHBDD1-transfected cells, DNA-PKcs and p53 levels were significantly reduced, while VCP and ERAD markers modestly increased, and were modestly reduced upon Nutlin-3a co-treatment. The relative levels of cleaved-PARP./PARP, cleaved Caspase-7/Caspase-7, and cleaved Caspase-3/Caspase-3 were not significantly altered by these treatments, implying that apoptosis was not prominently induced. These results suggest that RHBDD1 mediates the reduction of DNA-PKcs and p53, possibly through an ERAD-dependent mechanism in TP53-mutant NSCLC cells.

Fig. 5.

RHBDD1 Regulates DNA-PKcs and p53 Levels in an ERAD-Dependent Manner. A ERAD marker analysis in sh-RHBDD1- or pcDNA-RHBDD1-transfected cells treated with STL127705 or Nutlin-3a, assessed by western blotting. B Relative protein levels in sh-RHBDD1 transfected cells. C Apoptosis induced by cisplatin in pcDNA-RHBDD1-transfected A549 and NCI-H596 cells. Data are shown as mean ± SD. N = 3 for western blot analysis. *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons test

Next, apoptosis under cisplatin treatment was assessed in both wild-type and mutant NSCLC cells transfected with pcDNA-RHBDD1. Apoptosis was inhibited by pcDNA-RHBDD1 in both A549 and NCI-H596 cells, with a more pronounced apoptosis observed in A549 cells (Fig. 5c). These results indicate that RHBDD1 inhibits apoptosis in lung cancer cells, regardless of p53 mutation.

To investigate the interaction between RHBDD1, DNA-PKcs, and p53, immunoprecipitation assays were conducted using TP53 wild-type cells (A549 and NCI-H838) and TP53-mutant cells (NCI-H596 and NCI-H1155), all pre-treated with PYR-41 to prevent ERAD-mediated proteolysis. Both RHBDD1 and p53 were co-precipitated with the DNA-PKcs antibody in TP53 wild-type and mutant cells (Fig. 6a, b), with noticeably stronger interactions observed in the TP53-mutant cells. These findings suggest that the interaction among RHBDD1, DNA-PKcs, and p53 is enhanced in TP53-mutant NSCLC cells.

Fig. 6.

Immunoprecipitation of p53 with DNA-PKcs and RHBDD1. Cells were treated with 10 μM PYR-41 for 48 h to prevent ERAD-mediated proteolysis, and immunoprecipitation was performed in A TP53 wild-type cells (A549 and NCI-H838), and B TP53-mutant cells (NCI-H596 and NCI-H1155)

Discussion

The importance of ERAD, ER stress, and UPR in lung cancer has been well supported by numerous studies. The ER is a critical organelle that regulates essential biological processes, particularly protein folding and trafficking. When misfolded proteins accumulate, ER stress is often triggered within the tumor microenvironment, leading to processes such as epithelial-mesenchymal transition, increased cell invasion, resistance to therapies, and poor prognosis in lung cancer [25]. To mitigate ER stress-induced UPR and apoptosis, tumor cells can activate ERAD as an adaptive mechanism to restore normal ER function [26]. RHBDD1, a key regulator of ERAD, has been shown to protect against ER stress by modulating the morphology and distribution of ER sheets [27]. Notably, other ERAD-regulating proteins, including RHBDD2 and VCP, along with four additional ER stress-related genes, have been identified as promising prognostic markers in LUAD [28]. Our bioinformatics analysis revealed that cell cycle was the most significantly enriched and inhibited pathway in RHBDD1-high versus -low expressing NSCLC samples. The predicted inhibition of cell cycle by RHBDD1 contrasts with previous reports. For example, downregulation of RHBDD1 inactivated AKT and CDK2 in response to the proteasome pathway, thereby inhibiting cell cycle progression in breast cancer cells [29]. Our previous reports also supported the regulation of CDK2 by RHBDD1 in NSCLC [30]. To further explore the potential regulation of cell cycle by RHBDD1 in NSCLC, we investigated protein processing in ER, the enriched ERAD-associated pathway, and its involvement in protein interactions in cell cycle. Interestingly, p53 and DNA-PKcs were identified as key hub proteins in this interaction network, and cell cycle was further enriched in TP53-mutant cases, rather than wild-type ones. The bioinformatics analysis suggests that RHBDD1-mediated inhibition of cell cycle may occur in TP53-mutant NSCLC cases, which does not conflict with our previous observations in TP53 wild-type cells [30].

RHBDD1 has gained significant attention due to its membrane-embedded active sites, involvement in protein maturation and allocation, and its association with ERAD [31]. Unfolded proteins, particularly those with hydrophobic residues, can form deposits through phase separation when present in sufficient concentrations [32]. RHBDD1 interacts with the erlin ERAD complex to prevent the aggregation of peptides and proteins [33], and plays a critical role in processing specific proteins, such as the amyloid precursor protein [34]. Elevated RHBDD1 levels have been observed to promote tumor progression in breast, gastric, and colorectal cancers [6]. Previous studies suggest that RHBDD1 may drive NSCLC progression by activating the ZEB1/PI3K/AKT pathway [11], and its expression can be increased by EGFR or MEK inhibitors, further promoting NSCLC progression [30]. Recently, the Wnt/β-catenin signaling pathway was found to be activated by RHBDD1 across various tumors, supporting tumor growth, metastasis, and stemness [9, 35]. Additionally, RHBDD1 downregulation by miR-924 inhibits cell proliferation, migration, and invasion in NSCLC cells in a Wnt/β-catenin-dependent manner [36]. These studies collectively support the tumor promoting effects of RHBDD1. In the current study, we found that inhibition of the cell cycle was the most significantly enriched signaling pathway in NSCLC samples with high RHBDD1 expression and TP53 mutation. In vitro tests revealed that RHBDD1 more strongly regulated DNA-PKcs and p53 in TP53-mutant cells compared to wild-type cells. Highly expressed RHBDD1 significantly reduced DNA-PKcs and p53 levels in NCI-H596 cells, which could be reversed by co-treatment with p53 degradation inhibitors HBX 41108 or Nutlin-3a. By using the two proteolysis inhibitors for p53, we aimed to minimize potential off-target effects. Both silencing and overexpression of RHBDD1 resulted in S-phase cell cycle arrest and reduced cell viability in NCI-H596 cells. Our results also support the benefit of inhibiting RHBDD1 in TP53-mutant NSCLC cells, showing that highly expressed RHBDD1 can arrest the cell cycle as predicted. Furthermore, STL127705 and Nutlin-3a increased cell viability in NCI-H596 cells, with the greatest reduction observed with pcDNA-RHBDD1 transfection combined with Nutlin-3a treatment. These results suggest that p53 is a central player in RHBDD1-regulated cell cycle arrest, and that DNA-PKcs is regulated alongside p53.

TP53 is one of the most well-studied tumor suppressor genes, primarily functioning as a transcription factor for a wide range of tumor-suppressive genes. TP53 mutations are especially frequent across cancers, leading to the disruption of normal p53 functions and the acquisition of oncogenic properties [19]. In lung injury, p53 plays a crucial role in alveolar regeneration by promoting the self-renewal of alveolar type 2 (AT2) cells and their transition into alveolar type 1 (AT1) cells [37]. TP53 mutations are highly prevalent in lung cancers, contributing to genomic instability. Approximately 86% of small cell lung cancer, 47% of LUAD, and 81% of LUSC patients harbor TP53 mutations [19]. These mutations can initiate the development and progression of NSCLC, enhance therapy resistance, and result in poor prognosis and resistance to EGFR-TKIs and immunotherapy [38, 39]. In this study, we demonstrated that RHBDD1 regulates p53, and the effects of RHBDD1 and p53 on NSCLC may depend on DNA-PKcs protein levels. DNA-PKcs is a critical component of DNA damage repair. It acts as a DNA damage sensor and is recruited to damaged DNA ends by the Ku70/80 complex. Within this complex, DNA-PKcs serves as a scaffold to recruit DNA repair machinery, initiating non-homologous end joining (NHEJ), a process that contributes to genomic instability [40]. Given its multifunctional role, DNA-PKcs has been widely recognized as a tumor-promoting gene. It also inhibits p53 function, as its inhibition leads to ATM overactivation and enhanced p53 activity [41]. Moreover, EGFR-mediated increased binding of DNA-PKcs and p53 suppresses p53 function [42]. Despite these interactions, DNA-PKcs and p53 can work together to suppress p21 transcription [43]. We found that both DNA-PKcs and p53 were elevated in the sh-RHBDD1 group and reduced in the pcDNA-RHBDD1 group in TP53-mutant NCI-H596 cells. The loss of p53–DNA-PKcs interaction has previously been observed in p53-mutant cells [41]. We further showed that the cell cycle was significantly arrested at the S phase in pcDNA-RHBDD1-transfected NCI-H596 cells, a process likely dependent on p53 and DNA-PKcs. To further explore the implication of RHBDD1-regulated cell cycle arrest in TP53-mutant cells, we investigated apoptosis in transfected NCI-H596 cells. ERAD markers were modestly reduced with shRHBDD1 but significantly increased with STL127705. In contrast, ERAD markers were reduced with pcDNA-RHBDD1, which were abolished with Nutlin-3a. Cisplatin-induced apoptosis was more significant in pcDNA-RHBDD1 transfected NCI-H596 cells than in transfected A549 cells. Finally, the direct interaction of RHBDD1 with DNA-PKcs and p53 was validated through immunoprecipitation. These findings support the regulation of p53 and DNA-PKcs signaling by RHBDD1-associated ERAD, and suggest that although RHBDD1 can arrest the cell cycle in TP53-mutant NSCLC cells, it may inhibit apoptosis, thereby enhancing tumor cell survival and promoting NSCLC progression.

As summarized in Fig. 7, p53 is captured by MDM2 in the absence of a DNA damage response, leading to its ubiquitination and proteasomal degradation. In response to DNA damage, ATM and ATR are activated; ATM competes with MDM2 to release and activate p53, promoting transcriptional activation, cell cycle arrest, DNA repair, and apoptosis. Both DNA-PKcs and p53 can individually promote cell cycle arrest and DNA repair individually, but apoptosis requires the nuclear translocation of both proteins [43]. Therefore, RHBDD1 may reduce mutant p53 and DNA-PKcs in NSCLC cells to arrest the cell cycle and facilitate DNA repair. However, the absence of apoptosis may allow the survival of cells with severe DNA damage, ultimately contributing to NSCLC malignancy.

Fig. 7.

Role of RHBDD1-Regulated ERAD-Dependent p53 and DNA-PKcs Degradation in NSCLC. Apoptosis in NSCLC cells potentially requires co-localization of p53 and DNA-PKcs to initiate transcription, while cell cycle arrest can occur in response to either p53 or DNA-PKcs alone. Mutant p53 enhances ERAD and promotes the degradation of DNA-PKcs, thereby facilitating cell survival in NSCLC cells

The regulation of DNA-PKcs and p53 by RHBDD1 in NSCLC may have important therapeutic implications. Despite extensive characterization in biological functions, p53 lacks easily druggable target sites, encompasses a wide variety of mutant forms, and is regulated by complex networks [19], making it historically considered undruggable. Nevertheless, p53 has regained attention in recent years, especially in context of immunotherapy [44]. Our results indicate that loss of DNA-PKcs may be a key tumor promoting mechanism of mutant p53, and restoring DNA-PKcs expression could counteract the tumor-promoting effects of mutant p53. Considering that NSCLC patients with low RHBDD1 expression and wild-type p53 have a much better prognosis than other patients, targeting RHBDD1 and p53 may offer promising anticancer strategies.

There were limitations in this work. We validated only one aspect of the predicted roles of RHBDD1 and p53 in NSCLC. Other predicted changes, such as the widely enriched immune responses, remain unexplored. Many potentially correlated pathways within RHBDD1-regulated p53/DNA-PKcs signaling, such as DNA damage response and ER stress were not investigated. The use of chemical inhibitors may introduce off-target effects, and RHBDD1 regulation of to p53 and DNA-PKcs was not fully captured in the few cell lines and limited clinical samples used. Importantly, our findings were validated only in cell-based experiments; in vivo validation is required in future studies. Nevertheless, this study improves our understanding of RHBDD1 and p53 in tumor pathology and provides potential therapeutic targets for NSCLC treatment.

Conclusion

RHBDD1 arrests the cell cycle in TP53-mutant NSCLC cells through ERAD-dependent downregulation of p53 and DNA-PKcs. However, cell survival may be enhanced due to the inhibition of DNA-PKcs-regulated apoptosis. Our results suggest that restoring DNA-PKcs could suppress the tumor promotive role of mutant p53, and that targeting RHBDD1 and p53 may represent a promising therapeutic strategy for NSCLC treatment.

Author contributions

YC and YW participated in data collection and analysis, and contributed to writing the paper. SS, LZ, JL, and XY were responsible for literature search and data visualization. HP and ZX conceived and designed the study, and revised the manuscript critically for important intellectual content. All authors give final approval of the version to be published. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.

Funding

This study was supported by Yunnan Provincial First People’s Hospital Clinical Medical Center for Thoracic Diseases (Grant No.: 2022LCZXKF-XB01), the Joint Foundation of Science and Technology Department of Yunnan Province and Kunming Medical University (Grant Nos.: 202301AY070001-231, 202401AY070001-257), Yunnan Provincial Clinical Research Center for Respiratory System Diseases (Grant No.: 2023YJZX-HX01), and Yunnan Provincial Basic Research Program (Grant No.: 202501AT070123).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.