L1-ATP8B1 Exacerbates High-Level Reduction-Oxidation Homeostasis to Promote Carcinogenesis via Affecting PHB1 Sumoylation in LUSC

doi:10.21203/rs.3.rs-1434039/v1

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Research Article

L1-ATP8B1 Exacerbates High-Level Reduction-Oxidation Homeostasis to Promote Carcinogenesis via Affecting PHB1 Sumoylation in LUSC

https://doi.org/10.21203/rs.3.rs-1434039/v1

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posted 21 Mar, 2022

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Background: Long Interspersed Nuclear Element-1 (LINE-1, L1) plays important roles in carcinogenesis. We previously identified 13 LINE-1 insertions in lung squamous cell carcinoma (LUSC), among which L1-ATP8B1 was significantly correlated with poor prognosis of LUSC patients. However, the regulation and function of L1-ATP8B1 in LUSC remain unclear.

Methods: Here we collected 190 LUSC samples from Tianjin Medical University Cancer Institute and Hospital (TJMUCH) and investigated the relationship of L1-ATP8B1 with multiple clinicopathological features. We then constructed L1-ATP8B1-overexpressing LUSC cells and investigated their effects on cell functions and mitochondrial metabolism. We further explored the synergetic therapy effects with an L1 inhibitor and an antioxidant in L1-ATP8B1-induced growth and metastasis of LUSC xenografts in vivo.

Results: We found that L1-ATP8B1 was overexpressed in LUSC patients with short survival and associated with mitochondrial dysfunction in both the TCGA dataset and the TJMUCH cohort. Overexpression of L1-ATP8B1 promoted cell proliferation and invasion in vitro and facilitated LUSC xenograft growth in vivo. L1-ATP8B1 affected mitochondrial complex enzyme I activity via affecting cardiolipin-dependent PHB1 sumoylation and promoting PHB1 ubiquitination, inducing high-level cellular reduction-oxidation (redox) homeostasis. Synergetic therapy with an L1 inhibitor and an antioxidant dramatically interfered with redox homeostasis and restored mitochondrial function, abolishing L1-ATP8B1-induced growth and metastasis of LUSC xenografts in vivo.

Conclusions: We for the first time demonstrated that metabolic disorder plays a vital role in L1-related carcinogenesis and identified L1-ATP8B1 as a promising prognostic biomarker and therapeutic target for LUSC.

LINE-1

ATP8B1

PHB1

redox homeostasis

mitochondrial dysfunction

About half of all cancers have somatic integrations of retrotransposons[1]. Among the retrotransposable elements, Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only known active autonomous transposon in human, accounting for 17% of the entire DNA content[2]. LINE-1 plays an important role in carcinogenesis and many tumor tissues have been found to present high LINE-1 expression, which is caused by an increase in global copy number or hypomethylation in whole chromosomes[3]. Pan-cancer analysis of whole genomes showed that aberrant LINE-1 insertions could lead to the deletion of megabase-scale regions in chromosomes and to complex translocations and large-scale duplications by inducing breakage-fusion-bridge cycles in cancer[1]. In some circumstances, LINE-1 within introns can be transcribed together with adjacent exons to generate L1 chimeric transcripts (LCTs) upon activation of the LINE-1 antisense promoter, which affects the expression and activity of multiple functional genes[4]. Therefore, identification of the core molecular events regulating LINE-1-related carcinogenesis might lead to the discovery of novel predictive biomarkers and therapeutic targets for clinical applications.

LINE-1 contains two open reading frames (ORF1 and ORF2)[5]. ORF1 encodes an RNA-binding protein (ORF1p) with nucleic acid chaperone activity, and ORF2 encodes ORF2p, which has endonuclease and reverse-transcriptase activities but is barely detected in human cancers[6]. LINE-1 ORF1p is an abundant protein in LINE-1-expressing cells, serving as a diagnostic biomarker of LINE-1[7]. Hypomethylation of the LINE-1 promoter in cancer results in high expression of LINE-1 ORF1p and enhanced LINE-1 activity[8]. In our previous study, we used the DeFuse algorithm to identify 13 LCTs in lung squamous cell carcinoma (LUSC) and found that LCTs are correlated with LINE-1 overexpression in tumor tissues[4]. Nevirapine (NVR), an anti-HIV drug, could reduce LCT activity and inhibit LCT-related cell proliferation and invasion[4]. Among 13 LCTs, L1-ATP8B1 was significantly correlated with poor prognosis of LUSC patients, which indicated its significance in LUSC development and progression. But the regulation and function of L1-ATP8B1 in LUSC have not been disclosed.

ATP8B1 encodes a P-type cation transport ATPase. As a tumor suppressor gene, inhibition of ATP8B1 is related to the occurrence of cholangiocarcinoma[9]. Recent studies have shown that ATP8B1 is involved in phospholipid transport in the colorectum, which is crucial for maintaining membrane stability and polarity[10, 11]. The decreased expression of flippases might trigger abnormal distribution of membrane phospholipids, thus enhancing tumor progression and metastasis[12]. ATP8B1 is also involved in the transport of cardiolipin, a major lipid component of mitochondrial intima, and plays an important role in maintaining the electron transport chain (ETC)[13]. Mitochondrial dysfunction caused by ETC damage may promote the progression of tumors into chemoresistance or invasive phenotypes[14]. Thus, we hypothesized that L1-ATP8B1 might promote carcinogenesis by leading to mitochondrial dysfunction and metabolic disorder in LUSC.

To test this hypothesis, we conducted transcriptome and metabolome analysis of LUSC samples from the TCGA dataset and the TJMUCH cohort to investigate L1-ATP8B1-related metabolic disorder. We established L1-ATP8B1-overexpressing LUSC cell lines and generated xenograft-bearing mice to elucidate how L1-ATP8B1 promotes cell proliferation, migration, and invasion in vitro and in vivo. We found that L1-ATP8B1 affects cardiolipin-dependent mitochondrial prohibitin 1 (PHB1) sumoylation and increases the expression of the glutathione (GSH) synthesis transporter protein SLC1A5, which produces excessive reactive oxygen species (ROS) and GSH to induce high-level cellular reduction-oxidation (redox) homeostasis. Interference with redox homeostasis using an antioxidant could inhibit LINE-1 ORF1p expression and reverse L1-ATP8B1-related mitochondrial dysfunction and tumor growth. Therefore, we for the first time demonstrated that metabolic disorder plays a vital role in LCT-related carcinogenesis and proposed that L1-ATP8B1 might serve as a promising predictive biomarker and therapeutic target in LUSC.

Patient information

In total, 190 cases of LUSC patients were obtained from the Cancer Biobank of TJMUCH who were treated with partial lung resection surgery at the Department of Lung Cancer of TJMUCH (Table 1). No prior treatments, including chemotherapy or radiotherapy, were given before lung resection surgery was performed. This project was approved by the Ethics Committee of Tianjin Medical University (Approval No. Ek2021143) and written informed consent was obtained from the patients. All experiments were performed in accordance with the principles of the Declaration of Helsinki.

Cell lines and cell treatment

NCI-H520 and SK-MES-1 cells were purchased from Cellcook Co., Ltd. and verified via the STR multi-amplification method. NCI-H520 cells were cultured in RPMI1640 (Gibco BRL). SK-MES-1 cells were cultured in Eagle’s Minimum Essential Medium. All media contained 10% FBS and 1% penicillin/streptomycin. NVR (TargetMol) was dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich) and NAC (Beyotime) was dissolved in PBS to make stock solutions. The final concentrations of NVR and NAC were 450 µM and 5 mM, respectively. For cell treatment assays, growth media were replaced with media containing NVR, NAC, or both 12 h after cells were seeded. For the MG132 treatment assay, cells were treated with or without 10 µM MG132 (BD Biosciences) for 6 h before cells were harvested. Cell lines were routinely evaluated for Mycoplasma contamination. All experiments were completed less than 2 months after establishing stable cell lines or thawing early-passage cells.

Mouse models

NOD-SCID mice, which were 7 weeks old and weighed about 17–18 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in a specific pathogen-free animal facility. We obtained different H520 cells carrying constructed lentiviruses (OV-NC and OV-L1-ATP8B1) and resuspended 5×10⁴ cells in 100 µL of PBS with Matrigel, followed by subcutaneous injection of the cells into the flanks of NOD-SCID mice. For drug treatment experiments, animals were subjected to treatment with NVR (100 mg/kg/day), NAC (150 mg/kg/d), or both. After model construction, the weights and tumor sizes of each mouse were monitored every 2 days. All animal protocols were approved by the Ethics Committee for Animal Experiments of TJMUCH (Approval No.: NSFC-AE-2021179). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The Wistar IACUC guideline was followed in determining the time for ending the survival experiments (tumor burden exceeds 10% of body weight).

Lentivirus construction and plasmid transfection

For L1-ATP8B1 insertion lentivirus construction, the L1-ATP8B1 fragment was amplified by PCR using cDNA of H520 cells as template. Then the amplified L1-ATP8B1 fragment was inserted into pHBLV-CMV-MCS-EF1-T2A-Puro lentiviral vectors (Hanbio Co., Ltd.) and the constructed positive plasmid was confirmed by DNA sequencing. The recombinant lentivirus with L1-ATP8B1 sequence was generated by co-transfection in cells as previously described[4]. The L1-ATP8B1 and PHB1 overexpression plasmids were purchased from Hanbio Co., Ltd. and transfected into cells as previously described[4].

RNA extraction, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and quantitative Real-time PCR (qPCR) analysis

RNA extraction, cDNA synthesis, RT-PCR and qPCR analysis were performed following the manufacturer’s protocol as previously described[4]. The sequences of primers are shown in Table S1. To confirm the bands detected in the PCR assay were the genes as predicted, we purified and sequenced the PCR products (Invitrogen). High-throughput qPCR analysis was performed on Smartchip (Differential gene technology Co., Ltd.) following the manufacturer’s protocols. All experiments were performed in triplicate. Relative expression level of mRNA was calculated as 2^-^△^Ct (△Ct= Ct _{target gene} – Ct _{reference gene}).

RNA library preparation, sequencing and enrichment analysis

Library preparation and sequencing steps were performed as previously described[4]. Briefly, the libraries were sequenced on Illumina® (NEB) following manufacturer’s recommendations. The RNA sequencing data has been uploaded to GEO database (accession number: GSE181042). Raw sequencing data was aligned to hg38 reference using STAR 2.5.3a[15] and HTseq 0.11.2[16] was used to quantify the gene-sample expression profile. Differentially expressed genes (DEG) was identified with limma voom[17] with FDR adjusted p value<0.01. KEGG and GO function enrichment analysis of the interested gene sets were performed using clusterProfiler package[18].

Gene expression (RNA) profiling: NanoString methodology

Gene expression analysis was also conducted with the NanoString nCounter gene expression platform (NanoString Technologies) as previously described[19]. Briefly, RNA was mixed in a 3′-biotinylated capture probe and 5′-reporter probe tagged with a fluorescent barcode. Probes and target transcripts were hybridized overnight. Hybridized samples were run on the NanoString nCounter preparation station by using a high-sensitivity protocol. The cartridge samples were scanned at maximum resolution by using the nCounter digital analyzer. GEP scores were calculated as a weighted sum of normalized expression values for the genes.

Quantitative methylation-specific PCR analysis for the methylation levels of LINE-1 retrotranspositions

Genomic DNA was obtained and purified from frozen LUSC tissues in quantities sufficient for bisulfite treatment. Bisulfite conversion was carried out on 500 ng genomic DNA using EpiTect Bisulfite Kit (Qiagen), according to the manufacturer’s protocol as described above. The experiments were repeated at least 3 times[4].

Immunohistochemistry

All procedures were performed as described above[4]. The antibodies we used here are in Table S2 and a biotinylated secondary goat anti-mouse IgG antibody (Santa Cruz), labeled with streptavidin-horseradish peroxidase (HRP) using a DAB staining kit (Maixin Biotechnology) according to the manufacturer’s instructions.

RCA-FISH

Tissue samples were treated with xylene and gradient ethanol to be eventually hydrated. The slides were sequentially treated with proteinase K, restriction enzyme, exonuclease reaction solution, probe of target gene, signal amplification reaction solution, and fluorescent probe reaction solution. Quenching-resistant sealing tablets containing DAPI were added to seal the tablets. Fluorescence signals were observed under a fluorescence microscope.

Cell proliferation assay

Cell Counting Kit 8 (CCK8) assay was used to detect proliferation ability. Cells were inoculated in 96-well plates at a density of 2×10³ cells/100μL/well. 10 μl CCK-8 reagent was added to each well. The cells were incubated at 37℃ for 2 h and the absorbance at 450/650 nm was measured by a microplate reader (Synergy HT). The mean values of the three replicates were calculated.

Cell apoptosis assay

Annexin-V-APC Apoptosis Assay Kit was used to detect cell apoptosis. After the cells were collected, they were washed with PBS and resuspended at a concentration of 1×10⁵ cells/mL to prepare single-cell suspension. Subsequently, 5 μL Annexin-V and 5 μL APC were added to the cell suspension and the mixture was incubated in dark for 15 min.

Wound healing assay

Cells were inoculated in 6-well plates. When the density reached 90~100%, draw a line with a 20 μl pipette tip on the monolayer cells to form "scratches". Five fields were selected under the microscope at 0 h and 48 h after scribbling. The scratch distance was measured three times each time to take the average value. Cell migration rate = (0 h scratch distance - 48 h scratch distance) /0 h scratch distance ×100%.

Trans-well invasion assay

The invasion ability was detected using Matrigel gel and Trans-Well plate. The cells were inoculated in Matrigel and 200 μL serum-free RPMI-1640 at a density of 2×10⁴ cells. The number of cells was counted from five fields randomly selected, and the average value of the three complex pores was calculated.

Western blot

The cells were collected in a cell lysate buffer with protease inhibitors. For mitochondria extraction, it was extracted with a mitochondrial extraction kit (Beyotime Biotechnology). The protein was quantified using BCA Protein Assay Kit (Solarbio) and performed western blot as previously described[20].

Immunoprecipitation

Cell lysates were harvested using lysis buffer and rotated at 4°C as previously described[21]. Lysates were clarified by spinning. Protein concentrations were measured using BCA standard curves (Pierce). Flag antibody (for binding to flag-PHB1, Thermo Fisher Scientific) was added to the protein lysate and samples were rotated overnight. Immunoprecipitation was carried out using the Invitrogen Dynabeads Protein G IP Kit. Lysates were next subjected to SDS-PAGE and immunoblot analysis.

Determination of intracellular ROS

ROS were detected using a ROS detection kit (Beyotime Biotechnology) according to the manufacturer’s instructions. The cells were collected, incubated with DCFH-DA at 37°C for 30 min, and then washed with serum-free medium. Flow cytometry was used to detect DCF fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

GSH detection assay

The level of GSH was measured in cell or tumor lysates according to the instructions of the GSH and GSSG Assay Kit (Beyotime Biotechnology).

Measurement of intracellular ATP levels

ATP levels were measured using the Enhanced ATP Assay Kit (Beyotime Biotechnology) according to the manufacturer’s instructions. The substrate was gently mixed with reaction reagent at room temperature. The luminescence was then measured using a Beckman Coulter.

Assessment of mitochondrial membrane potential

JC-1 was used to measure the MMP according to the manufacturer’s instructions (Bioss). Cells in 6-well plates were incubated with JC-1 staining solution at 37°C for 30 min and then washed with JC-1 buffer. Fluorescent signals were obtained using either fluorescence microscopy or flow cytometry.

Mitochondrial complex activity assay

The activities of mitochondrial complex I were determined by the Micro Mitochondrial Respiratory Chain Complex I Activity Assay Kit (Solarbio) according to the manufacturer’s instructions.

TdT-mediated dUTP Nick-End Labeling (TUNEL) assay

Apoptotic DNA fragmentation was examined using the One Step TUNEL Apoptosis Assay kit (Beyotime). Cyanine 3-labeled TUNEL-positive cells were imaged under a fluorescence microscope.

Statistical analysis

Data were analyzed using SPSS 22.0 and GraphPad Prism 8.0 software following the manufacturers’ instructions. Measurement data are presented as median (interquartile range) and compared through the χ² test. Quantitative data are presented as mean ± standard deviation and compared through ANOVA and LSD tests. Spearman’s rank order test and linear regression analysis were performed to assess the correlations between expression levels detected by qPCR. Univariate and multivariate Cox regression analyses were used to identify common genes associated with overall survival. Cumulative survival was determined via the Kaplan–Meier method. Univariate survival analysis between the different LINE-1 insertions and the overall survival of LUSC patients was conducted through the two-sided log-rank test. Statistical significance was set at P < 0.05.

L1-ATP8B1 overexpression predicted poor clinical outcomes in LUSC patients and was correlated with LINE-1 hypomethylation

Previously we identified the 13 most frequently detected LCTs in 448 LUSC samples from the TCGA dataset. Here, we investigated the incidence of these 13 LCTs and their relationships with multiple clinicopathological features in 190 LUSC samples from the Tianjin Medical University Cancer Institute and Hospital (TJMUCH) cohort. L1-ATP8B1 was the most frequently detected LCT in patients with short survival (survival ≤ 40 months), with significantly increased expression compared to that in patients with long survival (survival > 40 months, Fig. 1A–B). Using Kaplan–Meier survival analysis, we confirmed that the L1-ATP8B1 expression level was significantly correlated with poor overall survival (Fig. 1C), large tumor size, lymph node metastasis and distant metastasis (P < 0.05, Fig. 1D) among LUSC patients. Furthermore, L1-ATP8B1 was associated with smoking history and centrally located tumors (Table S3). These data suggest that L1-ATP8B1 might serve as a novel predictive biomarker in LUSC.

Hypomethylation of the LINE-1 promoter was reported to induce the transcriptional activity of LINE-1, which could generate LCTs, leading to tumor progression[22]. We stratified the patients into two groups by the expression level of L1-ATP8B1 by RT-qPCR and then analyzed the methylation level of the LINE-1 promoter in LUSC samples. We found that the average methylation level of the LINE-1 promoter in the LUSC^L1−ATP8B1+ group was significantly lower than that in the LUSC^{L1−ATP8B1−} group (P < 0.0001, Fig. 1E). Since the level of ORF1p is usually applied to assess the transcription and expression of LINE-1 in cells[28, 29], we found that ORF1 expression was negatively correlated with LINE-1 methylation and positively correlated with L1-ATP8B1 expression (Fig. 1F–G). Higher expression of ORF1p was detected in the LUSC^L1−ATP8B1+ group than in the LUSC^{L1−ATP8B1−} group (P = 0.0340, Fig. 1H) by immunohistochemical (IHC) staining.

L1-ATP8B1 promoted LUSC cell proliferation and invasion by directly interfering with ATP8B1 transcription

To explore if L1-ATP8B1 affected ATP8B1 expression, we investigated the correlation between L1-ATP8B1 and ATP8B1 in LUSC samples at the mRNA and protein levels. Spearman rank correlation analysis showed that L1-ATP8B1 was negatively correlated with ATP8B1 at the mRNA level (R = − 0.51, P < 0.0001, Fig. 2A). In addition, the ATP8B1 protein level was dramatically decreased in LUSC^L1−ATP8B1+ compared to LUSC^{L1−ATP8B1−} samples (P < 0.0001, Fig. 2B). L1-ATP8B1 was detected in the LUSC cell line H520, but not in the normal lung epithelium cell line BEAS-2B, using rolling circle amplification–fluorescence in situ hybridization (RCA-FISH). Next, we generated L1-ATP8B1-overexpressing normal lung epithelial cells (BEAS-2B^{OV − L1−ATP8B1}) and LUSC cells NCI-H520 (H520^{OV − L1−ATP8B1}) and confirmed that overexpression of L1-ATP8B1 in cells promoted LINE-1 insertion into exon 3 of the ATP8B1 gene via homologous recombination, thus interfering with the transcription of the wild-type ATP8B1 gene (Figure S1A–C). We confirmed that L1-ATP8B1 reduced ATP8B1 expression in H520^{OV − L1−ATP8B1} cells as effectively as shRNA-mediated knockdown (H520^{SH − ATP8B1}), which could also be reversed by NVR (Fig. 2C).

The increased proliferation and decreased apoptosis in H520^{OV − L1−ATP8B1} cells were also detected in H520^{SH − ATP8B1} cells (Fig. 2D–E). Besides that, enhanced migration and invasion capacities were observed in both H520^{OV − L1−ATP8B1} and H520^{SH − ATP8B1} cells (Fig. 2F–G). The enhanced proliferation, resistance to apoptosis, and aggressiveness could be attenuated by NVR (Fig. 2D–G). Similar results were observed in another LUSC cell line, SK-MES-1 (Figure S2).

Therefore, we knocked down the ORF1 gene in H520^{OV − L1−ATP8B1} to inhibit LINE-1 insertion. The mRNA levels of L1-ATP8B1 decreased while the mRNA levels of ATP8B1 increased accordingly (Fig. 2H). Moreover, after ORF1p expression was suppressed, the proliferation rate and invasion ability of H520^{OV − L1−ATP8B1} cells were dramatically reduced (Fig. 2I–J). These results indicated that L1-ATP8B1 significantly stimulated the proliferation, migration, and invasion of LUSC cells by directly interfering with ATP8B1 transcription through LINE-1-mediated endogenous reverse transcriptase activity.

L1-ATP8B1 affected mitochondrial complex enzyme I activity and induced mitochondrial dysfunction via suppressing PHB1 expression

We divided LUSC samples from the TJMUCH cohort into LUSC^L1−ATP8B1+ and LUSC^{L1−ATP8B1−} groups according to the expression level of L1-ATP8B1. Differentially expressed genes between the two groups were identified for gene function enrichment analysis. Gene Ontology enrichment analysis indicated that multiple mitochondrial-related biological processes (BP) and cellular components (CC) were enriched in the LUSC^L1−ATP8B1+ group, which implied that L1-ATP8B1-mediated protumoral activity might be associated with mitochondrial dysfunction (Fig. 3A). We further compared differentially expressed genes between LUSC^L1−ATP8B1+ and LUSC^{L1−ATP8B1−} samples in the TCGA dataset. Consistently, pathway enrichment analysis indicated that multiple mitochondrial metabolism-related pathways were enriched in LUSC^L1−ATP8B1+ samples, including mitochondrial translational termination, mitochondrial respiratory chain complex I assembly, mitochondrial translational elongation, mitochondrial electron transport, NADH to ubiquinone, and mitochondrial translation, which implied that L1-ATP8B1 might trigger mitochondrial dysfunction (Fig. 3B).

Considering ATP8B1 participates in mitochondrial energy metabolism as a cardiolipin transporter and plays a vital role in maintaining the stability of the mitochondrial membrane[23, 24], we focused on those differentially expressed genes related to mitochondrion organization (GO:0007005, biological process category) and the mitochondrial inner membrane (GO:0005743, cellular component category). The PHB1 and STOML2 genes were selected based on the Venn diagram (Fig. 3C). We compared the mRNA levels of PHB1 and STOML2 in both the TCGA dataset and the TJMUCH cohort, and found that PHB1 rather than STOML2 was significantly downregulated in LUSC^L1−ATP8B1+ samples (P < 0.05, Fig. 3D). IHC results further demonstrated the downregulation of PHB1 in LUSC^L1−ATP8B1+ tissues (P = 0.0027), while STOML2 showed no difference (Fig. 3E). Furthermore, we found that the protein expression of PHB1 was exclusively declined at the mitochondrial level in L1-ATP8B1⁺ cells, which could be restored by NVR treatment (Fig. 3F).

PHB1 is involved in mitochondrial function, including mitochondrial cristae formation, mitochondrial complex I activity, and ROS level homeostasis[25]. We found that the mitochondrial complex enzyme I pathway was downregulated in the LUSC^L1−ATP8B1+ group (Fig. 3B). Furthermore, the activity of mitochondrial complex enzyme I was decreased in L1-ATP8B1⁺ cells (Fig. 3G), while intracellular ROS levels, the mitochondrial membrane potential (MMP, ΔψM), and ATP levels increased simultaneously (Fig. 3H–J). NVR could recover L1-ATP8B1-induced changes of mitochondrial functions. These results indicated that L1-ATP8B1 affected complex enzyme I activity and induced mitochondrial dysfunction via affecting PHB1 expression.

L1-ATP8B1 accelerated PHB1 degradation by affecting cardiolipin-dependent PHB1 sumoylation and promoting PHB1 ubiquitination

We transfected a PHB1 overexpression plasmid into H520^{OV − L1−ATP8B1} cells (H520^{OV − L1−ATP8B1−OV−PHB1}) to evaluate the biological significance of PHB1 in L1-ATP8B1⁺ LUSC. Using the CCK8 proliferation assay, we found that cell viability was suppressed in H520^{OV − L1−ATP8B1−OV−PHB1} cells compared to H520^{OV − L1−ATP8B1} cells (Fig. 4A). The Annexin V apoptosis assay showed that the apoptosis rate of H520^{OV − L1−ATP8B1} cells was increased after PHB1 overexpression (Fig. 4B). Consistently, PHB1 significantly inhibited the migration and invasion capacities of H520^{OV − L1−ATP8B1} cells (Fig. 4C–D). Furthermore, we found that the activity of mitochondrial complex enzyme I in H520^{OV − L1−ATP8B1} cells was restored after PHB1 overexpression (Fig. 4E). In contrast, increased intracellular ROS levels, ΔψM, and ATP levels in H520^{OV − L1−ATP8B1} cells were attenuated after PHB1 overexpression (Fig. 4F–H). These results implied that L1-ATP8B1 induced mitochondrial dysfunction and carcinogenesis in LUSC because of PHB1 deficiency.

We noticed that in H520^{OV − L1−ATP8B1} cells, PHB1 expression decreased significantly at the protein level rather than the mRNA level (Fig. 4I–J), which implied that post-translational modifications might be involved in L1-ATP8B1-induced PHB1 deficiency. Therefore, we treated cells with the protease inhibitor MG132. After MG132 treatment, the protein level of PHB1 was significantly increased, almost reaching control levels, indicating PHB1 is degraded via the ubiquitin–proteasome pathway (Fig. 4K). We found that L1-ATP8B1 significantly increased the ubiquitination level of PHB1 (Fig. 4L). Considering that cardiolipin could mediate protein sumoylation[26], which directly inhibits protein ubiquitination[27, 28], we further explored the sumoylation level of PHB1. PHB1 sumoylation was reduced in H520^{OV − L1−ATP8B1} cells accordingly, which could be recovered by NVR treatment (Fig. 4L). To examine whether cardiolipin regulated PHB1 sumoylation, we added exogenous cardiolipin to H520^{OV − L1−ATP8B1} cells, and found the level of sumoylated PHB1 increased while the level of ubiquitinated PHB1 decreased exclusively in H520^{OV − L1−ATP8B1} cells (Fig. 4M), which indicated that L1-ATP8B1 caused a shortage of cardiolipin in mitochondrial intima and affected sumoylation of PHB1, thus increasing the ubiquitination of PHB1 and accelerating PHB1 degradation.

High-level redox homeostasis was indispensable to promote L1-ATP8B1-mediated LUSC proliferation and invasion

We further profiled 24 cases of primary LUSC tissues to investigate L1-ATP8B1-driven protumoral signaling pathways using the nCounter® PanCancer IO-360™ panel. Consistent with our findings in H520^{OV − L1−ATP8B1} cells, the cell proliferation pathway was upregulated, while the apoptosis and autophagy pathways were downregulated in LUSC^L1−ATP8B1+ tissues compared to LUSC^{L1−ATP8B1−} tissues (Fig. 5A–B). Besides that, we found that the metabolic stress pathway was significantly activated. Therefore, we compared all differentially expressed genes involved in the metabolic stress pathway between LUSC^L1−ATP8B1+ and LUSC^{L1−ATP8B1−} tissues via RNA sequencing, and found that genes related to amino acid metabolism, which encode transporter proteins required for GSH generation and ROS reduction to regulate the redox balance, were highly expressed in LUSC^L1−ATP8B1+ tissues (Fig. 5C–D)[29].

We further examined the mRNA levels of GSH synthesis-related transporter proteins (SLC1A5, SLC2A1, and SLC7A5) in LUSC samples, and found that only SLC1A5 was exclusively positively correlated with L1-ATP8B1 expression (Fig. 5E). The key enzymes regulating intracellular redox homeostasis GCLC, GCLM, and GPX4 were simultaneously upregulated in H520^{OV − L1−ATP8B1} cells (Fig. 5F). Consistently, we found that the intracellular levels of ROS and GSH were dramatically higher than those in H520^{OV − NC} cells (Fig. 5G–H). Then we treated H520^{OV − L1−ATP8B1} cells with NVR and NAC (a precursor of intracellular GSH), and found that NAC significantly increased intracellular GSH and decreased ROS levels, while NVR only decreased ROS levels but had no effect on GSH production (Fig. 5G–H). We also confirmed that the synergetic effect of NVR and NAC suppressed the mRNA level of L1-ATP8B1 (Fig. 5I). Furthermore, synergetic treatment with NAC and NVR fully attenuated the L1-ATP8B1-mediated increases in proliferation, apoptosis resistance, and aggressiveness and additionally disrupted high-level redox homeostasis in H520^{OV − L1−ATP8B1} cells (Fig. 5J–K). Consistently, NVR and NAC synchronously stimulated the protein expression of Bax-2 and LC3II, but reduced the protein expression of Bcl-2 and P62 (Fig. 5L). The above results indicated that high-level redox homeostasis was indispensable to promote L1-ATP8B1-mediated LUSC proliferation and invasion in vitro, which could be efficiently reversed by combined treatment with the LINE-1 inhibitor NVR and the antioxidant NAC.

Synergetic therapy of NVR and NAC significantly abolished L1-ATP8B1-induced growth and metastasis of LUSC xenografts in vivo

We generated LUSC^L1−ATP8B1+ xenograft-bearing mice by subcutaneously implanting H520^{OV − L1−ATP8B1} cells into NOD-SCID mice, which were sequentially treated with NVR (100 mg/kg/d), NAC (150 mg/kg/d), or synergetic therapy. After 17 days of treatment, the mice were sacrificed and tumors were excised. Tumor growth curves showed that both NVR and NAC significantly inhibited the growth of xenografts, and synergetic therapy showed the strongest inhibitory effects (Fig. 6A–B). In addition, synergetic therapy displayed comparable drug safety because no significant weight loss or death occurred during treatment (Fig. 6C, Figure S3). Lung tissues were excised, and the metastatic lesions were detected by hematoxylin-eosin (HE) staining. The number of metastatic nodules was significantly decreased after treatment with NVR, NAC, or both (Fig. 6D).

IHC results showed that NAC or synergetic therapy inhibited L1-ORF1p expression and restored the protein expression of ATP8B1 and PHB1, while NVR only restored ATP8B1 and PHB1 expression, but had no effect on ORF1p expression (Fig. 6E). Consistently, proliferation was inhibited and the number of apoptotic cells was increased in mice treated with NVR, NAC, or both (Fig. 6F–G). As expected, NVR, NAC, or synergetic therapy reduced intracellular ROS levels (Fig. 6H). Compared to NVR, NAC significantly increased the expression of SLC1A5 and promoted GSH production (Fig. 6I). Our results also clearly demonstrated that synergetic therapy had a significantly stronger effect than NVR or NAC alone (6A–6I), implicating that synergetic therapy inhibited tumor growth and metastasis in vivo by suppressing L1-ATP8B1 activity, restraining high-level redox homeostasis, and rectifying mitochondrial dysfunction in the LUSC^L1−ATP8B1+ group.

In LUSC patients, the lack of actionable mutations and frequent immune evasion limit the benefit from target therapy and immune monotherapy. Therefore, investigation of more effective therapeutic targets in LUSC has attracted much attention in recent years. Moreover, the unique metabolic characteristics of LUSC provide clues to find novel prognostic and therapeutic biomarkers.

In our previous work, we screened a novel LCT, L1-ATP8B1, which was highly expressed in LUSC tissues compared to the normal adjacent tissues and significantly correlated with poor prognosis of LUSC patients[4]. In this study, we demonstrated that L1-ATP8B1 could interfere with the transcription of ATP8B1, inhibit the lipid flippase activity of ATP8B1, cause a shortage of cardiolipin in the mitochondrial intima, accelerate ubiquitination-dependent PHB1 degradation, and affect mitochondrial PHB1 expression. Therefore, we propose L1-ATP8B1 promotes carcinogenesis by inducing mitochondrial dysfunction and exacerbating pathogenic high-level redox homeostasis.

ATP8B1 is a tumor suppressor gene and is related to phospholipid homeostasis, which also mediates the transport of cardiolipin on the apical membrane of lung epithelial cells[11]. Deletion of ATP8B1 leads to an abnormal distribution of cardiolipin in mitochondrial intima[30]. Cardiolipin is an important component of the mitochondrial inner membrane and can interact with mitochondrial intima proteins, which is essential for mitochondrial integrity and functions[31]. PHB1 functions as a protein and lipid scaffold to ensure the integrity and functionality of the mitochondrial intima[32]. It has been reported that PHB1, through its role as a scaffold, enables cardiolipin to maintain stability of respiratory chain complexes and regulate apoptosis[32, 33]. PHB1 is also considered to regulate the activity of mitochondrial complex enzyme I of the ETC[34]. In this study, we demonstrated that L1-ATP8B1 impaired ATP8B1 transcription and expression and induced dysfunction of the oxidative phosphorylation (OXPHOS) process of the ETC in mitochondria, which was dependent on PHB1 deficiency and impaired activity of mitochondrial complex enzyme I.

Regarding the regulation of PHB1 expression by L1-ATP8B1, we found that L1-ATP8B1 might regulate PHB1 through post-translational modification. ISince we have demonstrated that L1-ATP8B1 overexpression impaired the transport of cardiolipin into mitochondria, and cardiolipin has been reported to regulate the sumoylation of proteins[25], we proposed that L1-ATP8B1-induced cardiolipin transport disorder might affect the PHB1 sumoylation status. Indeed, we found that PHB1 sumoylation was reduced in H520^{OV − L1−ATP8B1} cells and completely recovered by NVR. Furthermore, we found that the ubiquitination of PHB1 was significantly increased. Sumoylation and ubiquitination are two competitive modification systems with antagonistic actions[26]. Enhanced ubiquitination accelerates PHB1 protein degradation, which finally causes deficiency in PHB1 expression and mitochondrial dysfunction in L1-ATP8B1⁺ cells. Some studies have reported the correlation between PHB1 and cardiolipin[35, 36], but further investigations should be conducted to explore the underlying regulatory mechanism.

Moreover, mitochondrial dysfunction is associated with the development of various types of human cancers by producing ROS[37]. In normal cells, metabolic stress results in the excessive production of ROS, which induce apoptosis. However, tumor cells can adapt to metabolic stress by metabolic reprogramming[38]. In tumor cells, ROS are like a double-edged sword. A certain level of ROS can promote tumor progression, but when ROS levels exceed a certain threshold, they may exert cytotoxic effects, leading to the death of tumor cells[39]. We have shown that L1-ATP8B1⁺ cells possess higher intracellular ROS levels, a higher membrane potential, and higher ATP levels than L1-ATP8B1⁻ cells, which endow it with a higher proliferative capacity and higher aggressiveness. Furthermore, in L1-ATP8B1⁺ cells we detected high intracellular GSH levels and overexpression of the GSH synthesis-related transporter protein SLC1A5, which maintains high-level redox homeostasis to help tumor cells prevent ROS-induced oxidative damage and escape apoptosis[40]. It has been reported that ATP8B1 is involved in maintaining membrane stability[11], but it remains to be determined how ATP8B1 deficiency regulates the expression of SLC1A5 and promotes glutamine transport (Fig. 7). Our research indicated that L1-ATP8B1 induces mitochondrial dysfunction and redox homeostasis maintenance might serve as a novel therapeutic target for LUSC patients, since it has been reported that disrupted redox homeostasis could inhibit tumor progression and promote drug sensitivity[41].

The therapeutic targets of NVR and NAC are different. NVR mainly inhibits the reverse transcriptional activity of LINE-1 ORF2, thus restoring the function of ATP8B1 and fundamentally inhibiting ROS production. NAC indirectly reduces ROS levels by promoting GSH synthesis, and inhibits the protein expression of ORF1p. The combination of the two drugs can directly or indirectly reduce ROS levels and improve redox homeostasis, so as to achieve better therapeutic effects.

In summary, we for the first time proposed a promising predictive LCT biomarker and therapeutic target in LUSC. Furthermore, L1-ATP8B1-induced intracellular ROS overload in turn activated the expression of ORF1p, forming a positive feedback loop to further disrupt ATP8B1 and aggravate metabolic disorder in LUSC. Therefore, the unique mitochondrial metabolic characteristics in LUSC^L1−ATP8B1+ provide a novel and promising therapeutic strategy for this subtype of LUSC patients who might benefit from combined treatment with a LINE-1 inhibitor and an antioxidant, which should be systematically investigated in future clinical trials.

We for the first time proposed a promising predictive LCT biomarker and therapeutic target in LUSC. Furthermore, L1-ATP8B1-induced intracellular ROS overload in turn activated the expression of ORF1p, forming a positive feedback loop to further disrupt ATP8B1 and aggravate metabolic disorder in LUSC. Therefore, the unique mitochondrial metabolic characteristics in LUSC^L1−ATP8B1+ provide a novel and promising therapeutic strategy for this subtype of LUSC patients who might benefit from combined treatment with a LINE-1 inhibitor and an antioxidant, which should be systematically investigated in future clinical trials.

Long Interspersed Nuclear Element-1 (LINE-1, L1)

lung squamous cell carcinoma (LUSC)

reduction-oxidation (redox)

L1 chimeric transcripts (LCT)

open reading frames (ORF)

ORF1 encodes protein (ORF1p)

Nevirapine (NVR)

electron transport chain (ETC)

Tianjin Medical University Cancer Institute and Hospital (TJMUCH)

prohibitin 1 (PHB1)

glutathione (GSH)

reactive oxygen species (ROS)

immunohistochemical (IHC)

rolling circle amplification–fluorescence in situ hybridization (RCA-FISH)

L1-ATP8B1-overexpressing normal lung epithelial cells (BEAS-2B^OV-L1-ATP8B1)

L1-ATP8B1-overexpressing LUSC cells NCI-H520 (H520^OV-L1-ATP8B1)

NCI-H520 cells with ATP8B1 shRNA-mediated knockdown (H520^SH-ATP8B1)

biological processes (BP)

cellular components (CC)

mitochondrial membrane potential (MMP, ΔψM)

H520^OV-L1-ATP8B1 cells with PHB1 overexpression plasmid transfection (H520^{OV-L1-ATP8B1-OV-PHB1})

hematoxylin-eosin (HE)

oxidative phosphorylation (OXPHOS)

Ethics approval and consent to participate: This project was approved by the Ethics Committee of Tianjin Medical University (Approval No. Ek2021143) and written informed consent was obtained from the patients. All animal protocols were approved by the Ethics Committee for Animal Experiments of TJMUCH (Approval No.: NSFC-AE-2021179).

Consent for publication: Not applicable.

Availability of data and materials: The RNA sequencing datasets generated and analyzed during the current study are available in the GEO repository, https://www.ncbi.nlm.nih.gov/geo/ (accession number: GSE181042). And all data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests: The authors declare that they have no competing interests

Funding: This work was supported by the National Natural Science Foundation of China (grant No. 82173198 and 82072588), the National Science and Technology Support Program of China (grant No. 2018ZX09201015), the Project of Science and Technology of Tianjin (grant No. 18JCQNJC82700), Tianjin Research Innovation Project for Postgraduate Students (grant No. 2020YJSB166).

Authors' contributions:

Conceptualization: J Yu, W Zhang

Methodology: X Zhang, R Zhang, W Yu

Investigation: X Zhang, R Zhang, P Liu, G Chen

Data analysis: X Zhang, R Zhang, Z Sun

Supervision: J Yu, W Zhang, X Ren

Writing—original draft: X Zhang, R Zhang

Writing—review & editing: J Yu, W Zhang

All authors read and approved the final manuscript.

Acknowledgements: We thank International Science Editing ( http://www.internationalscienceediting.com) for editing this manuscript.

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Table 1. The basic clinicopathological information of all patients.

Clinicopathological parameters	Number of patients
Total	190
Gender
Male	153
Female	37
Age
<60	82
≥60	108
Stage
Ⅰ-Ⅱ	113
Ⅲ-Ⅳ	77
T stage
1-2	136
3-4	54
N stage
0-1	131
2-3	59
M stage
0	162
1	28
Metastatic site
Negative	162
Lung	2
Bone	5
Brain	7
Others	14
Location
Central	91
Periphery	99
Smoking
Negative	40
Positive	150

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L1-ATP8B1 Exacerbates High-Level Reduction-Oxidation Homeostasis to Promote Carcinogenesis via Affecting PHB1 Sumoylation in LUSC

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