ERCC6L drives lung adenocarcinoma metastasis: a PJA2/p53 ubiquitination-dependent mechanism

Abstract

Lung adenocarcinoma (LUAD) is prone to metastasis and exhibits a poor prognosis. The DNA repair-related protein ERCC6L is implicated in tumorigenesis, but its role in LUAD invasion and metastasis remains unclear. The expression, function, and underlying mechanisms of ERCC6L were investigated using bioinformatics analysis, cellular assays, and animal experiments. ERCC6L was significantly upregulated in LUAD tissues and cell lines, and its high expression was associated with poor overall and recurrence-free survival. Functional experiments demonstrated that ERCC6L overexpression promoted the migration, invasion, and epithelial-mesenchymal transition (EMT) of LUAD cells. Mechanistically, ERCC6L activated the E3 ubiquitin ligase PJA2, which mediated K48-linked polyubiquitination and subsequent degradation of p53, thereby attenuating its tumor-suppressive function. In vivo studies confirmed that ERCC6L knockout suppressed tumor growth, metastasis, and EMT progression by regulating the PJA2/p53 signaling axis. ERCC6L promotes migration, invasion, and EMT in LUAD by facilitating PJA2-mediated ubiquitination and degradation of p53. The ERCC6L/PJA2/p53 axis represents a potential novel therapeutic target for inhibiting metastasis in LUAD.

Introduction

Lung cancer (LC) remains the leading cause of cancer-related death worldwide, accounting for 12.4% of all newly diagnosed malignancies and 18.7% of cancer-related fatalities, thereby constituting a threat to global public health [1]. Non-small cell lung cancer constitutes the majority of all LC cases, with lung adenocarcinoma (LUAD) being the most prevalent subtype [2]. Although comprehensive approaches including surgical resection, radiotherapy, chemotherapy, and targeted therapy have substantially improved LUAD treatment outcomes [3, 4], the 5-year survival rate for patients with locally advanced LUAD remains below 20% [5], primarily due to the markedly enhanced invasiveness and high metastatic propensity of advanced LUAD [6, 7]. Therefore, elucidating the molecular regulatory mechanisms underlying LUAD invasion and metastasis is crucial for identifying new therapeutic avenues and improving patient prognosis.

Post-translational modifications (PTMs) are vital regulatory processes for cellular proteins, extensively involved in physiological and pathological processes such as cell death and innate immunity [8]. Ubiquitination, one of the most common PTMs, involves the specific attachment of ubiquitin molecules to target proteins via an enzymatic cascade, thereby regulating protein stability, localization, and function [9]. Numerous studies indicate that ubiquitination can influence tumor progression by mediating the degradation or stabilization of tumor-associated proteins, thereby regulating cell cycle, apoptosis, and related signaling pathway activation [10–12]. E3 ubiquitin ligase-mediated protein ubiquitination in LUAD can remarkably promote tumor metastasis and malignant progression by regulating key factors, highlighting the critical role of ubiquitination in the progression [13, 14]. However, the upstream regulators and detailed molecular mechanisms of ubiquitination in LUAD remain incompletely understood and require further investigation.

Excision repair protein ERCC6-like (ERCC6L), also known as PICH, is a DNA helicase belonging to the SNF2 family [15]. It is primarily involved in mitotic spindle stability and chromosome segregation, assuming key roles in cell proliferation and chromosomal stability [16]. High ERCC6L expression is linked with poor prognosis in various tumors, including gastric cancer [17], laryngeal squamous cell carcinoma [18], and hepatocellular carcinoma [19]. Notably, evidence suggests that ERCC6L is highly expressed in LUAD cells, significantly accelerating cell proliferation, invasion, and the epithelial-mesenchymal transition (EMT) process, indicating its strong potential to drive tumor invasion and metastasis [20]. However, the core mechanism by which ERCC6L acts within the molecular network to regulate LUAD metastasis and EMT warrants in-depth investigation.

This study demonstrates that ERCC6L expression is pronouncedly upregulated in LUAD tissues and cells, and its high expression is closely linked with increased tumor malignancy and poor prognosis. Mechanistically, ERCC6L recruits the E3 ubiquitin ligase Praja2 (PJA2) and enhances its interaction with the P53 protein, inducing K48-linked ubiquitination and degradation of P53. This subsequently facilitates EMT pathway activity, ultimately driving LUAD metastasis. Overall, this research reveals a novel mechanism by which ERCC6L promotes LUAD invasion and metastasis via the PJA2/P53 axis, suggesting that targeting ERCC6L represents a potential therapeutic strategy for LUAD.

Materials and methods

Bioinformatics analysis

Transcriptomic data for LUAD tissue samples (n = 515) and adjacent normal tissue samples (n = 59) were sourced from the TCGA database [21]. Differential analysis was performed utilizing the “edgeR” package in R software (v 4.2.3) (P < 0.05, |log2FC| > 1). Violin plots visualizing ERCC6L expression differences between LUAD and normal tissues were generated using “ggplot2”. By implementing the Kaplan-Meier Plotter [22], the correlation between ERCC6L expression levels and overall survival (OS) and relapse-free survival (RFS) in LUAD patients was explored. The GSEA revealed potential signaling pathways related to ERCC6L. Furthermore, according to the UbiBrowser platform [23], we predicted potential substrate proteins of PJA2.

Cell cultivation

All cells used were purchased from BNCC (China), including human bronchial epithelial cells BEAS-2B (BNCC359274), human LUAD cell lines A549 (BNCC337696), Calu-3 (BNCC359757), NCI-H1975 (BNCC340345) and NCI-H1299 (BNCC100268), and human embryonic kidney cells HEK-293 (BNCC100449). DMEM-H medium was used for BEAS-2B and HEK-293 cells; F-12 K complete medium for A549 cells; EMEM complete medium for Calu-3 cells; and RPMI-1640 medium for NCI-H1975 cells and NCI-H1299 cells. All reagents were provided by BNCC (China). All media were containing 10% FBS (BNCC, China) and 1% P/S solution (MCE, USA). Cells were maintained in a constant temperature incubator (37℃, 5% CO₂) (Thermo Fisher Scientific, USA).

Plasmids, shRNA, lentivirus, and transfection

Specific overexpression plasmids (oe-ERCC6L), shRNA interference vectors (sh-ERCC6L), and negative controls were sourced from RiboBio (China). Various plasmids were also ordered and synthesized from Addgene (USA), including: pcDNA3.1-3×Flag-ERCC6L, pcDNA3.1-Myc-P53, pcDNA3.1-HA-PJA2, and pcDNA3.1-HA-Ub K48 (all lysine residues except K48 mutated to arginine); Myc-tagged wild-type P53 and its lysine residue mutants (#101, #164, #351, #357, #386); and the HA-tagged single-lysine ubiquitin series (Ub-K6, Ub-K11, Ub-K27, Ub-K29, Ub-K33, Ub-K48, Ub-K63). HEK-293 and LUAD cells were transfected at 70–90% confluency using Lipofectamine™ 3000 (Thermo Fisher Scientific, USA), and the target plasmids or shRNA were diluted separately in Opti-MEM™ medium (Gibco, USA). The mixtures were combined at a 1:1 volume ratio and incubated at room temperature (RT) for 5 min. The resulting complexes were then added to the wells, followed by an incubation for 1–3 days at 37℃. Cells were prepared for subsequent experiments after confirming transfection efficiency.

Lentiviral vectors for stable transfection were derived from Addgene (USA), including pLKO.1-sh-ERCC6L targeting ERCC6L, pLVX-EF1α-IRES-Puro-PJA2 driving PJA2 overexpression, and their respective controls. pLKO.1-sh-ERCC6L or pLVX-EF1α-IRES-Puro-PJA2 were co-transfected with packaging plasmids pMD2G and pSPAX2 into HEK-293 cells. After 48 h, cell culture supernatant was collected and filtered through a 0.45 μm membrane. The resulting viral supernatant was applied to infect specified cells in the presence of 10 µg/mL Polybrene (Sigma-Aldrich, USA) for 8 h. Cells were then rinsed twice with medium and selected with 1.5 µg/mL puromycin (Sigma-Aldrich, USA) for 5 days to obtain stable cell lines for subsequent experiments.

qRT-PCR

Following extraction with TRIzol™ reagent (Thermo Fisher Scientific, USA), total RNA concentration/purity was measured employing a spectrophotometer (Agilent, USA). RNA was reverse-transcribed into cDNA by implementing the PrimeScript RT reagent kit (Takara, Japan). Subsequently, qRT-PCR was conducted in a 20 µL reaction system using an ABI 7500 PCR detection system (Thermo Fisher Scientific, USA) and specific primers (Supplemental Table 1). Each experiment was performed in triplicate. Relative mRNA expression was quantified by the 2^−ΔΔCT method, normalizing to GAPDH as an internal control.

Western Blot (WB)

Total protein was extracted from differently treated cell or tissue samples using RIPA lysis buffer (Yeasen, China). After measuring protein concentration, equal amounts of protein were loaded for SDS-PAGE separation and blotted to PVDF membranes (Thermo Fisher Scientific, USA) via wet transfer. After transfer, membranes underwent blocking with 5% skim milk at RT for 45 min. Membranes were then incubated with primary antibodies (Supplemental Table 2) overnight at 4℃ and washed three times with PBST (0.1% Tween-20) the next day, followed by incubation with HRP-conjugated secondary antibodies (Supplemental Table 2) for 1 h at RT and subsequent PBST washes. Protein signals were detected using the BeyoECL Plus kit (Beyotime, China) and captured/analyzed via an iBright imaging system (Thermo Fisher Scientific, USA).

Colony formation assay

Treated LUAD cells were cultured in 12-well plates (Corning, USA) (1000 cells/well) for 7–14 days in a 37℃, 5% CO₂ environment until visible cell colonies formed. Following 30-min fixation with 4% PFA (Beyotime, China) at 4℃, colonies were stained with 0.5% crystal violet solution (Beyotime, China) at RT for 30 min. After staining, excess dye was gently washed off with PBS (Beyotime, China). Finally, colonies were photographed and counted.

Transwell assay

For the migration assay, after culturing, digestion, and collection, the cells were resuspended in serum-free medium (2 × 10⁴ cells/mL). 200 µL of cell suspension was inoculated into the apical chamber of a transparent Transwell insert (Corning, USA). The basal one contained RPMI-1640 medium (BNCC, China) with 10% FBS as a chemoattractant. For the invasion assay, 200 µL of cell suspension (2 × 10⁵ cells/mL) was seeded in the apical chamber of Transwell inserts coated with100 µL of Matrigel (BD Biosciences, USA). After solidifying at 37℃ for 2 h, 500 µL of RPMI-1640 medium with 10% FBS was inoculated into the basal chamber. Following a 24-h incubation, cells on the membrane were fixed with 75% ethanol and stained with 0.5% crystal violet solution (Beyotime, China), and photographed under a microscope (Carl Zeiss, Germany). Five random fields were selected for counting to quantify cell migration and invasion.

Immunofluorescence (IF)

Cells were inoculated onto sterile coverslips placed in 6-well plates (Corning, USA). After fixation with 75% ethanol (Nanjing Reagent, China) at RT for 15 min, cells were permeabilized with PBS (Beyotime, China) containing 0.2% Triton X-100 (Beyotime, China) for 5 min, followed by blocking with 1% BSA (Beyotime, China). Cells were then maintained with primary antibodies (Supplemental Table 3) overnight at 4℃. After three PBS washes the next day, cells were incubated with fluorescently labeled secondary antibodies (Supplemental Table 3) for 1 h at RT, and nuclei were counterstained with DAPI (Sigma-Aldrich, USA) for 5 min. Processed coverslips were mounted with anti-fade mounting medium (Beyotime, China) and observed/imaged under a microscope (Carl Zeiss, Germany).

Cycloheximide (CHX) assay

Cells subjected to distinct treatments were inoculated into 12-well plates (Corning, USA) (3 × 10⁵ cells/well). Subsequently, cells were cultivated in 2 µM CHX (Sigma-Aldrich, USA), and samples were collected at designed time points (0, 2, 4, and 6 h). After lysis, P53 protein expression was determined by WB. Antibody information is provided in Supplemental Table 2.

Co-immunoprecipitation (Co-IP) and immunoprecipitation (IP)

After lysis and centrifugation, the collected supernatant was applied for subsequent Co-IP experiments. For endogenous Co-IP, cell lysates were incubated overnight at 4 ℃ under constant rotation with the indicated primary antibodies, anti-ERCC6L (H00054821-PW1, Abnova, China), anti-p53 (ab32049, Abcam, UK), or anti-PJA2 (#40180, Cell Signaling Technology, USA), or with an isotype-matched IgG control (ab172730, Abcam, UK). Protein A/G agarose beads (Santa Cruz Biotechnology, USA) were added simultaneously to capture immune complexes. After incubation, immune complexes were washed three times with IP buffer, followed by two washes with PBS (Beyotime, China). Then, 1× loading buffer was introduced, and bound proteins were eluted by heating at 95℃ for 5 min for WB. For exogenous Co-IP, cell lysates were incubated with anti-Flag M2 magnetic beads (Sigma-Aldrich, USA) or anti-Myc magnetic beads (Dyean Biotech, China) overnight at 4℃. After thorough washing, competitive elution was conducted by employing PBS buffer (Beyotime, China) containing 200 ng of 3×Flag peptide (Sigma-Aldrich, USA) or Myc tag peptide (Dyean Biotech, China), and the resulting products were attained for subsequent WB. Antibody involved is detailed in Supplemental Table 2.

In Situ proximity ligation assay (PLA)

The assay was conducted using the Duolink In Situ Red Starter Kit (DUO92101, Sigma-Aldrich, USA) following the manufacturer’s protocol. Cells were seeded onto coverslips placed in 24-well plates. After fixation with 4% paraformaldehyde for 15 min, the cells were permeabilized with 0.2% Triton X-100. Following blocking with Duolink blocking solution for 1 h at 37 °C, the cells were incubated overnight at 4 °C with anti-ERCC6L (PA5-50405, Thermo Fisher, USA) and anti-PJA2 (H00009867-M01, Thermo Fisher, USA). After three washes with Buffer A, PLA probe solution was added, and the cells were incubated for 1 h at 37 °C. Subsequently, the cells were incubated in ligation buffer for 30 min, followed by incubation with amplification solution for 100 min at 37 °C. The coverslips were then mounted using Duolink In Situ mounting medium containing DAPI and allowed to stand for 15 min. Finally, images were captured under a fluorescence microscope.

Ubiquitination assay

Flag-, Myc-, and HA-tagged plasmids underwent 24-h co-transfection into HEK-293 cells and culture. The proteasome inhibitor MG132 (20 µM/L, MCE, USA) was added 4 h before cell collection. Cells were subjected to lysis, followed by boiling with loading buffer at 95℃ for 5 min to prepare lysates. To detect ubiquitination levels, IP was performed using an anti-P53 antibody (ab32049, Abcam, UK), and the immunoprecipitates were analyzed by WB using anti-Ub antibody (ab134953, Abcam, UK) or anti-K48 Ub antibody (F0527, Selleck, USA). Antibody information is listed in Supplemental Table 2.

LC-MS/MS

HEK-293T cells transfected with Flag-ERCC6L were subjected to IP using anti-Flag antibody (ab205606, Abcam, UK) or IgG negative control antibody (ab172730, Abcam, UK). Immunocomplexes were then incubated with Flag M2 magnetic beads (Sigma-Aldrich, USA). After four washes, immunoprecipitates were boiled in 1× loading buffer and isolated by SDS-PAGE. LC-MS/MS was performed by High Precision (Hangzhou) Life Technology Co., LTD (China).

Animal studies

Six-week-old female BALB/c nude mice used, sourced commercially from BioRigino (China) (License No.: SCXK (Zhe) 2024-0011), were randomly assigned to three groups (n = 6 per group). To construct the subcutaneous xenograft model, NCI-H1975 cells stably transfected with sh-ERCC6L + oe-NC, sh-ERCC6L + oe-PJA2, or sh-NC + oe-NC (1 × 10⁶ cells) were inoculated into nude mice. Tumor dimensions were monitored using a vernier caliper at 7-day intervals, and volume was calculated using the formula V = 1/2 × length × (width²). Following a 35-day inoculation period, mice were euthanized. Tumors were dissected, and their volume and weight were measured. Tumors were then fixed in 4% PFA (Beyotime, China) for subsequent studies. For the metastasis experiment, NCI-H1975 cells stably transfected with sh-ERCC6L + oe-NC, sh-ERCC6L + oe-PJA2, or sh-NC + oe-NC (1 × 10⁷ cells) were injected into the tail vein. Animals were euthanized 30 days post-injection, and the number of surface lung nodules was counted.

Immunohistochemistry (IHC)

Freshly collected tissue blocks were immersed in 4% PFA (Beyotime, China) and fixed at 4℃ for 48 h. Fixed tissues were subjected to paraffin embedding to obtain tissue blocks, followed by cutting into Sect.  (4 μm thickness). Before staining, dried sections were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 95%, 85%, 75%). Antigen retrieval was performed using Tris-EDTA buffer (Beyotime, China). Subsequently, receiving the treatment of 3% H₂O₂ solution for 10 min to block endogenous peroxidase activity, sections were blocked with 10% goat serum (Beyotime, China) at RT for 30 min and incubated with primary antibodies (Supplemental Table 4) overnight at 4℃. The next day, they were mixed with secondary antibodies (Supplemental Table 4) for 1 h at RT. For color development, the mixture was treated with DAB (Yeasen, China) for 5 min, and nuclei were counterstained with hematoxylin (Beyotime, China) for 1 min. After staining, sections were dehydrated through a graded ethanol series, cleared in xylene, and mounted with neutral balsam. Finally, five randomly selected fields per section were observed and imaged under a microscope (Carl Zeiss, Germany).

Hematoxylin and Eosin (H&E) staining

Paraffin-embedded tissues fixed in 4% PFA (Beyotime, China) were sectioned at 4 μm thickness and baked at 65℃ for 2 h to ensure firm attachment to slides. Sections were sequentially deparaffinized in xylene and rehydrated through a graded ethanol series. Following rehydration, nuclei were visualized by an 8-min incubation in hematoxylin (Beyotime, China). Slides were briefly washed with PBS (Beyotime, China) and subsequently counter-stained with eosin (Beyotime, China) for 3 min. The specimens were then taken through a graded ethanol dehydration sequence, cleared in xylene, and allowed to air-dry. Finally, coverslips were affixed with neutral balsam, and representative micrographs were acquired on a microscope (Carl Zeiss, Germany).

Data processing

All experiments were conducted with a minimum of three independent replicates (n ≥ 3). Data are presented as mean ± SD. Intergroup comparisons employed independent Student’s t-test (two groups) and one-way ANOVA (≥ 3 groups). Statistical analysis was performed using SPSS v21.0 software (IBM, USA), and graphs were processed by GraphPad Prism 8 (GraphPad, USA). A P-value < 0.05 was considered to denote statistical significance.

Results

Upregulated ERCC6L expression promotes LUAD invasion, metastasis, and EMT

To elucidate the potential role of ERCC6L in LUAD, we first analyzed its mRNA levels based on RNA sequencing data from the TCGA database. The expression was noticeably upregulated in LUAD tissues (n = 515) compared with adjacent normal tissues (n = 59) (Fig. 1A). We then evaluated the correlation between ERCC6L expression and clinical prognosis in LUAD patients. Survival curve analysis indicated that the high ERCC6L expression group had significantly shorter OS and RFS than the low expression group (Fig. 1B-C). Subsequently, we also detected ERCC6L expression levels in human BEAS-2B cells and LUAD cell lines A549, Calu-3, and NCI-H1975 to corroborate this finding. As shown in qRT-PCR, ERCC6L was generally highly expressed in LUAD cells, with the highest expression in NCI-H1975 cells and relatively lower expression in A549 cells (Fig. 1D). Subsequently, an ERCC6L-overexpressing A549 cell model was generated to assess its role in LUAD progression. The efficacy of overexpression was then validated by qRT-PCR (Fig. 1E). Colony formation assays revealed that ERCC6L upregulation markedly enhanced the proliferative potential of LUAD cells (Fig. 1F). Transwell assays demonstrated that cells with upregulated ERCC6L exhibited significantly strengthened migration and invasion compared with controls (Fig. 1G). To further analyze the underlying mechanism, the expression of EMT-related markers was examined. In WB, ERCC6L overexpression led to decreased E-Cadherin levels and markedly increased levels of N-Cadherin, Vimentin, and the matrix metalloproteinases MMP2 and MMP9 (Fig. 1H). These results suggest that ERCC6L may enhance the invasion and metastasis in LUAD cells by accelerating the EMT process.

Fig. 1.

Upregulated ERCC6L expression promotes LUAD invasion, metastasis, and EMT process. A Violin plot of ERCC6L expression differences between LUAD tissues (n = 515) and adjacent normal tissues (n = 59) from the TCGA database. B Kaplan-Meier curves of OS based on high/low ERCC6L expression groups. C Kaplan-Meier curves of RFS based on high/low ERCC6L expression groups. D qRT-PCR of ERCC6L mRNA levels in BEAS-2B and LUAD cells (A549, Calu-3, NCI-H1975). A549 cells were transfected with oe-NC or oe-ERCC6L, groups: oe-NC, oe-ERCC6L. E qRT-PCR validation of ERCC6L mRNA expression in different groups. F Colony formation assay of cell proliferation capacity; left panel: representative colony images, right panel: quantitative results. G Transwell assay detecting cell migration and invasion capacities; left panel: typical images, right panel: quantitative analysis. H WB detection of EMT-related protein (E-Cadherin, N-Cadherin, Vimentin, MMP2, MMP9) expression levels. *, P < 0.05

ERCC6L modulates ubiquitination of the P53 protein

To elucidate the molecular mechanism by which ERCC6L mediates LUAD metastasis and invasion, we performed GSEA, which revealed that ERCC6L was significantly enriched in the P53 signaling pathway in LUAD (Fig. 2A). Thus, the regulatory effect of ERCC6L on P53 was investigated. NCI-H1975 cells transfected with sh-NC or sh-ERCC6L, and A549 cells transfected with oe-NC or oe-ERCC6L, were employed to construct ERCC6L knockdown and overexpression models, respectively. In qRT-PCR, no significant changes in P53 mRNA levels regardless of the expression status of ERCC6L (Fig. 2B). WB results demonstrated that ERCC6L knockdown significantly increased P53 protein levels, while its overexpression led to a marked downregulation of P53 (Fig. 2C). IF co-localization experiments further revealed that ERCC6L, primarily localized in the nucleus, was significantly co-localized with P53 (Fig. 2D). These results suggest that ERCC6L may function by regulating P53 protein stability. To further test this hypothesis, we conducted CHX-induced protein degradation assays. ERCC6L knockdown significantly prolonged the half-life of the P53 protein compared with controls (Fig. 2E). Next, we treated NCI-H1975 cells with the proteasome inhibitor MG132 (MCE, USA) and the lysosomal pathway inhibitor chloroquine (CQ, MCE, USA) to clarify the degradation pathway of P53. MG132 effectively blocked ERCC6L-mediated P53 protein degradation, while CQ treatment had no significant effect (Fig. 2F), suggesting that ERCC6L likely modulates P53 protein levels via the ubiquitin-proteasome pathway. Furthermore, Co-IP results indicated that ERCC6L interacted with the P53 protein under both endogenous and exogenous conditions (Fig. 2G-H). Notably, after IP pull-down of P53 protein, ERCC6L knockdown markedly reduced the ubiquitination level of P53 (Fig. 2I), further clarifying that ERCC6L mediates P53 protein ubiquitination.

Fig. 2.

ERCC6L mediates ubiquitination of the P53 protein. A GSEA of enrichment pathways for ERCC6L. NCI-H1975 or A549 cell groups: sh-NC, sh-ERCC6L, oe-NC, oe-ERCC6L. B qRT-PCR detection of P53 mRNA expression in cells. C WB detection of ERCC6L and P53 protein expression in NCI-H1975 and A549 cells. D Immunofluorescence staining of co-localization of ERCC6L and P53 proteins in NCI-H1975 cells. E WB of P53 protein expression levels in NCI-H1975 cells transfected with sh-NC or sh-ERCC6L using CHX at indicated time points (0, 2, 4, 6 h) and line graph for quantitative analysis. F WB of ERCC6L and P53 protein expression levels in NCI-H1975 cells transfected with sh-NC or sh-ERCC6L under the treatment of MG132 or CQ. G-H Endogenous and exogenous Co-IP validating the interaction between ERCC6L and P53 protein. I Co-IP experiment pulling down P53 protein using NCI-H1975 cells transfected with sh-NC or sh-ERCC6L; WB detection of ubiquitination levels. J-K Different ubiquitin molecules J and ubiquitin mutant Ub K48 R K were co-transfected with Flag-ERCC6L and Myc-P53 tagged plasmids into HEK-293 cells. Cell lysates were subjected to Co-IP using P53 antibody, followed by WB detection with specified antibodies. L HA-K48 was co-transfected with Flag-ERCC6L and wild-type Myc-P53 or K-to-R mutant Myc-P53 tagged plasmids into HEK-293 cells. Cell lysates were subjected to Co-IP using P53 antibody, followed by WB detection with specified antibodies. *, P < 0.05; ns, no significant difference

Subsequently, we constructed Ub mutants retaining only a single lysine site, including HA-Ub K6, HA-Ub K11, HA-Ub K27, HA-Ub K29, HA-Ub K33, HA-Ub K48, and HA-Ub K63 (all lysine residues except K6 mutated to arginine) in order to identify the specific type of ubiquitination modification associated with ERCC6L. These different Ub mutants were co-transfected with ERCC6L and P53 tagged plasmids (Flag-ERCC6L, Myc-P53) into HEK-293 cells and detected by Co-IP. ERCC6L overexpression primarily promoted K48-linked ubiquitination of the P53 protein compared with cells transfected with other ubiquitin mutants (Fig. 2J). However, this effect was effectively abolished by the K48R ubiquitin plasmid (Fig. 2K). Through a combination of bioinformatics prediction and Co-IP analysis of K-R mutants for potential ubiquitination sites on the P53 protein (K101R, K164R, K351R, K357R, K386R), we identified the K357 site as a major ubiquitination site. The K357R mutation markedly decreased the P53 ubiquitination levels (Fig. 2L). These results indicate that ERCC6L negatively regulates P53 stability by enhancing its K48-linked ubiquitination modification of P53 protein and accelerating its degradation via the proteasome pathway.

ERCC6L promotes LUAD invasion, metastasis, and EMT via P53

To further verify whether ERCC6L affects the malignant behavior of LUAD cells by regulating P53, we treated NCI-H1975 cells transfected with sh-NC or sh-ERCC6L with DMSO or the P53-specific inhibitor Pifithrin-α (PFT, MCE, USA). As shown in colony formation assays, ERCC6L knockdown significantly inhibited NCI-H1975 cell proliferation, and this effect was markedly reversed by PFT addition (Fig. 3A). Transwell assays further demonstrated that ERCC6L knockdown noticeably suppressed NCI-H1975 cell migration and invasion, while PFT treatment partially restored their migration and invasion potential (Fig. 3B). Additionally, WB results showed that ERCC6L knockdown significantly upregulated P53 and E-Cadherin protein expression while downregulating N-Cadherin, Vimentin, MMP2, and MMP9 levels. Moreover, these expression changes were effectively reversed upon PFT treatment (Fig. 3C), suggesting that ERCC6L boosts the EMT process and expression of matrix degradation-related molecules via P53 suppression. Notably, to determine whether the effects mediated by ERCC6L are dependent on p53, we further overexpressed ERCC6L (oe-ERCC6L) in the p53 deficient NCI H1299 cell line to investigate the impact of its expression on the malignant behavior of cancer cells. The results revealed that, compared with the control group, ERCC6L overexpression alone in NCI H1299 cells did not significantly affect cancer cell proliferation, migration, or the EMT process (oe-NC group vs. oe-ERCC6L group) (Fig. 3D-F). However, upon transfection with p53 in NCI H1299 cells, ERCC6L overexpression markedly attenuated the inhibitory effect of p53 on cancer cells (oe-P53 group vs. oe-ERCC6L + oe-P53 group). Taken together, ERCC6L promotes LUAD cell proliferation, migration, invasion, and EMT by inhibiting P53 activity.

Fig. 3.

ERCC6L Promotes LUAD Invasion, Metastasis, and EMT Process via P53NCI-H1975 cells transfected with sh-NC, sh-ERCC6L were treated with DMSO or P53 inhibitor (PFT). Groups: sh-NC+DMSO, sh-ERCC6L+DMSO, sh-ERCC6L+PFT. A Colony formation assay detecting cell proliferation capacity; left panel: representative experimental images, right panel: quantitative analysis results. B Transwell assay detecting cell migration and invasion capacities; left panel: representative experimental images, right panel: quantitative analysis results. C WB detection of ERCC6L, P53, and EMT pathway-related protein (E-Cadherin, N-Cadherin, Vimentin, MMP2, MMP9) expression. In NCI H1299 cells, oe-NC (control), oe-ERCC6L, or oe-P53 were transfected, and the following experimental groups were established: oe-NC, oe-ERCC6L, oe-P53, and oe-ERCC6L+oe-P53. D Colony formation assay for assessing cell proliferation capacity; representative images are shown on the left, with corresponding quantitative results on the right. E Transwell assay for evaluating cell migration and invasion capacity; representative images are presented on the left, and quantitative analyses on the right. F WB detecting the protein expression levels of ERCC6L, P53, and EMT related markers (E Cadherin, N Cadherin, Vimentin, MMP2, and MMP9). *, P < 0.05.

ERCC6L promotes P53 ubiquitination and degradation by activating PJA2

Ubiquitination modification relies on E3 ubiquitin ligases. To further explore the E3 ligase involved in ERCC6L-promoted P53 ubiquitination and degradation, we identified the E3 ubiquitin ligase PJA2 (Fig. 4A) through LC-MS/MS analysis on ERCC6L immunoprecipitates. Combined with UbiBrowser database prediction, a potential interaction between PJA2 and P53 was discovered (Fig. 4B). Therefore, we further validated using Co-IP and PLA experiments that ERCC6L had a significant interaction with PJA2 in LUAD cells (Fig. 4C-D). Further Co-IP assays revealed that PJA2 robustly precipitated P53, and this association was markedly intensified upon ERCC6L overexpression. Reciprocally, P53 efficiently retrieved PJA2, with the interaction strength similarly potentiated by ERCC6L upregulation (Fig. 4E). Furthermore, PJA2 overexpression decreased P53 protein levels, which was further enhanced upon ERCC6L overexpression (Fig. 4F), suggesting that ERCC6L may mediate P53 degradation via PJA2. The enhancement of P53 ubiquitination by ERCC6L was significantly attenuated by PJA2 knockdown, as demonstrated by ubiquitination assays (Fig. 4G). To clarify the linkage type of ubiquitination, we co-transfected P53, ERCC6L, PJA2 plasmids with the HA-Ub K48 mutant and performed Co-IP. Results showed that ERCC6L cooperated with PJA2 to promote K48-linked ubiquitination of P53 (Fig. 4H), thereby accelerating its degradation. In summary, ERCC6L enhances K48-linked ubiquitination of P53 by interacting with PJA2.

Fig. 4.

ERCC6L promotes P53 ubiquitination and degradation by activating PJA2. A Protein components displayed by SDS-PAGE and silver staining after ERCC6L immunoprecipitation from NCI-H1975 cells; arrow indicates the target protein band. B UbiBrowser database shows PJA2 as a potential E3 ubiquitin ligase for P53. C NCI-H1975 cells were collected and lysed; Co-IP was performed on lysates using ERCC6L or PJA2 antibody, followed by WB detection with specified antibodies; IgG served as negative control. D The interaction between ERCC6L and PJA2 was assessed using PLA. PLA signals indicate the strength of the interaction between ERCC6L and PJA2. Blue: DAPI; red: PLA signal. E-F Endogenous and exogenous Co-IP validating the interaction among ERCC6L, PJA2, and P53 proteins. G HEK-293 cells were co-transfected with oe-ERCC6L or sh-PJA2 and corresponding plasmids; cell lysates were subjected to Co-IP using P53 antibody and analyzed by WB with specified antibodies. H HEK-293 cells were co-transfected with oe-NC or oe-ERCC6L and corresponding plasmids; cell lysates were subjected to Co-IP using P53 antibody and analyzed by WB with specified antibodies

ERCC6L regulates LUAD invasion, metastasis, and EMT via the PJA2/P53 axis confirmed by In Vivo validation

To further validate the impact of ERCC6L on LUAD progression in vivo, we generated subcutaneous xenograft models in mice (n = 6 per group): sh-NC + oe-NC, sh-ERCC6L + oe-NC, and sh-ERCC6L + oe-PJA2 (Fig. 5A). Compared with control tumors, ERCC6L silencing markedly blunted both tumor growth speed and volume, and tumor weight was also significantly reduced. This growth suppression was substantially reversed when PJA2 was concurrently overexpressed (Fig. 5B-C). IHC results showed that ERCC6L knockdown markedly reduced intra-tumoral Ki67, ERCC6L and PJA2 abundance while concurrently elevating p53 levels; these alterations were partially rescued when PJA2 was re-expressed in the sh-ERCC6L + oe-PJA2 cohort (Fig. 5D). WB of EMT-related proteins in tumor tissues revealed that ERCC6L knockdown cuased upregulated E-Cadherin expression in vivo, while levels of N-Cadherin, Vimentin, MMP2, and MMP9 decreased. However, this expression trend was remarkably reversed when PJA2 was simultaneously overexpressed (Fig. 5E). To further study the effect of ERCC6L on LUAD cell metastasis, indicated NCI-H1975 cells were implanted into nude mice via tail vein injection. The number of lung metastatic nodules formed by ERCC6L-knockdown NCI-H1975 cells in nude mice was substantially reduced, and PJA2 overexpression reversed this inhibitory effect (Fig. 5F). For H&E staining, compared with controls, lungs from the ERCC6L-depleted cohort retained an essentially intact tissue structure and exhibited markedly fewer pink-stained metastatic nodules. However, upon concurrent PJA2 overexpression, these nodular areas were further expanded (Fig. 5G). The above data indicate that ERCC6L drives LUAD proliferation, invasion, and distant metastasis by activating PJA2 to promote P53 ubiquitination and degradation.

Fig. 5.

ERCC6L regulates LUAD invasion, metastasis, and EMT via the PJA2/P53 Axis confirmed by In Vivo validation. Animal groups: sh-NC + oe-NC, sh-ERCC6L + oe-NC, sh-ERCC6L + oe-PJA2. A Photographs of dissected tumors from each group of mice. B Changes in tumor volume in different groups. C Comparison of tumor weight among groups. D Representative IHC staining images of expression levels of KI67, ERCC6L, P53, and PJA2 in tumor tissues. E WB results of EMT-related protein (E-Cadherin, N-Cadherin, Vimentin, MMP2, MMP9) expressions in tumor tissues of each group. NCI-H1975 cells transfected with sh-ERCC6L or oe-PJA2 and their controls were injected into mice via the tail vein to establish lung metastasis models. Groups: sh-NC + oe-NC, sh-ERCC6L + oe-NC, and sh-ERCC6L + oe-PJA2. F Representative images and quantitative statistics of lung nodules in each group. G H&E staining results of lung tissue nodules. *, P < 0.05

Discussion

Despite recent therapeutic advances, the clinical outcome of LUAD remains largely unsatisfactory, primarily owing to the high propensity for metastatic dissemination [24]. Consequently, elucidating the molecular underpinnings that drive LUAD metastasis continues to represent a critical and unmet challenge in current oncological research. In this study, we found that ERCC6L is highly expressed in LUAD, and its upregulation drives tumor cell metastasis, invasion, and EMT by activating the E3 ubiquitin ligase PJA2 to promote K48-linked ubiquitination and degradation of P53. This finding suggests that ERCC6L may be an important intervention point and opens new avenues for related strategy research.

As an important member of the chromosome segregation-associated helicase family, ERCC6L has been confirmed to have tumor-promoting effects in multiple cancers [25, 26]. ERCC6L accelerates hepatocellular carcinoma cell proliferation by activating PI3K/AKT signaling [27]. Furthermore, Zhang et al.. reported that upregulated ERCC6L in renal cell carcinoma boosts in vivo tumor progression by regulating the MAPK signaling pathway [28]. Notably, ERCC6L is not only a biomarker and therapeutic target for LUAD [29] but also facilitates LUAD malignant progression through HIF-1α-induced glycolysis and stemness [30]. Consistently, our data revealed that ERCC6L is markedly upregulated in both LUAD tissues and cell lines, and its elevated expression strongly correlates with unfavorable clinical outcomes. Further overexpression of ERCC6L significantly promoted the proliferation, migration, invasion, and activity of the EMT pathway in LUAD cells. Therefore, elucidating the molecular regulatory network by which ERCC6L orchestrates EMT in LUAD is expected to furnish a theoretical framework for the discovery of next-generation biomarkers.

Although ERCC6L has been confirmed to be associated with LUAD risk, the molecular mechanisms by which ERCC6L regulates LUAD metastasis and EMT remain poorly understood. Preliminary GSEA demonstrated that high ERCC6L expression in LUAD was significantly related to the P53 pathway. The canonical tumor suppressor p53 is central to safeguarding genomic integrity and triggering apoptosis; loss of its function constitutes a critical prerequisite for both tumorigenesis and malignant progression [31, 32]. DCAF13, as a novel negative regulator of P53, accelerates LUAD progression by facilitating its ubiquitination and degradation [33]. Additionally, the novel colorectal cancer-associated gene SHARPIN can promote tumor growth by inhibiting P53 expression [34]. Consequently, we hypothesized that ERCC6L might exert its protumor effect by inhibiting P53. Consistent with our hypothesis, ERCC6L interacts with P53 and catalyzes its K48-linked ubiquitylation and proteasomal degradation, and inhibiting P53 could counteract the suppressive effects of ERCC6L knockdown on the malignant phenotype of LUAD cells. These results confirm ERCC6L drives LUAD initiation and EMT by orchestrating ubiquitin-dependent destruction of the tumor suppressor P53.

E3 ubiquitin ligases are essential for proteasomal degradation via ubiquitination [35, 36]. Subsequently, we further screened for E3 ligases interacting with ERCC6L using LC-MS/MS and identified PJA2, which in silico algorithms predict to be a putative E3 ubiquitin ligase for P53. PJA2, as an E3 ubiquitin ligase, may hold dual roles as either a tumor promoter or suppressor in distinct tumor types by mediating substrate ubiquitination and degradation. As shown by Lignitto L et al.., PJA2 accelerates glioblastoma growth by hydrolyzing MOB1 protein and suppressing the Hippo signaling pathway [37]. In contrast, Zhao et al.. demonstrated that PJA2-mediated KSR1 ubiquitination and degradation inhibits the MEK-ERK signaling pathway, ultimately preventing gastric cancer progression [38]. Given that the biological function and upstream regulatory mechanisms of PJA2 in LUAD remain unclear, this study investigated the potential role of PJA2 as a key downstream effector of ERCC6L in LUAD progression. Through in vitro and in vivo experiments, we found that ERCC6L upregulation activates PJA2, which in turn promotes K48-linked ubiquitination and subsequent degradation of P53, a mechanism significantly driving LUAD progression and metastasis. In conclusion, our findings uncover an ERCC6L/PJA2/P53 regulatory axis that governs the invasive and metastatic capacity of lung adenocarcinoma cells, thereby providing a conceptual framework for understanding the molecular underpinnings of LUAD dissemination.

Overall, this study reveals that ERCC6L drives LUAD cell proliferation, invasion, and EMT by activating PJA2 to promote K48-linked ubiquitination and degradation of P53, expanding the understanding of LUAD molecular mechanisms and providing a theoretical basis for exploring intervention strategies targeting ERCC6L.Despite advances, there are several limitations. First, the potential for ERCC6L to cooperate with other E3 ligases in governing P53 stability remains an unexplored area. Second, the conclusions of this study are primarily derived from limited cell and animal models, lacking systematic validation in large-scale clinical samples. To address these issues, future research will combine multi-omics technologies to further screen potential cooperative regulatory molecules and validate the clinical relevance of the ERCC6L/PJA2/P53 axis in large-scale LUAD cohorts, with the ultimate goal of accelerating the translation toward precision therapeutics.

Authors' contributions

Lingyu Jiang: Writing – original draft, Supervision, Software, Resources, Formal analysis, Conceptualization. Huaihai Zhou, Yifan Zhou: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Data curation, Conceptualization. Zhaoke Wen, Junqi Qin, Zhenniu Lei, Jianwei Huang: Resources, Data curation. Rongling Li, Yunzhi Ma, Shucong Peng, Shuping Huang, Xiaoyan Huang: Formal analysis. Lingyu Jiang, Yonglong Zhong: Writing – review & editing, Funding acquisition. Lingyu Jiang, Shengjing Liang, Yonglong Zhong: Writing – review & editing, Writing – original draft, Software, Resources, Methodology, Investigation, Funding acquisition, Conceptualization.

Funding

This work was supported by National Natural Science Foundation of China (82560725), Guangxi Natural Science Foundation (2023GXNSFAA026129).

Data availability

The RNA-sequencing data for the lung adenocarcinoma (LUAD) cohort utilized in this study are available in The Cancer Genome Atlas (TCGA) repository, accessible at http://portal.gdc.cancer.gov/.

Declarations

Ethics approval and consent to participate

This study and all animal experiments were in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Guangxi Zhuang Autonomous Region People’s Hospital (KY-KJT-2023-62).

Not applicable.

Competing interests

The authors declare no competing interests.