Arsenic promotes ROS-mediated malignant transformation of bronchial epithelial cells by specifically downregulating TXNL1 expression

Zhao, Lingling; Jiao, Xueli; Li, Hongyan; Chang, Yuanyuan; Li, Minghao; Liu, Yu; Xie, Qipeng; Huang, Chuanshu; Huang, Haishan

doi:10.1038/s42003-025-09216-z

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Published: 22 November 2025

Arsenic promotes ROS-mediated malignant transformation of bronchial epithelial cells by specifically downregulating TXNL1 expression

Lingling Zhao ORCID: orcid.org/0000-0002-8907-6495¹^na1,
Xueli Jiao^1,2^na1,
Hongyan Li¹^na1,
Yuanyuan Chang³,
Minghao Li¹,
Yu Liu³,
Qipeng Xie⁴,
Chuanshu Huang¹ &
…
Haishan Huang ORCID: orcid.org/0000-0002-7658-2188¹

Communications Biology volume 8, Article number: 1827 (2025) Cite this article

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Abstract

Arsenic exposure is a known cause of lung cancer, primarily mediated through reactive oxygen species (ROS) generation and oxidative DNA damage. However, the precise mechanism by which arsenic modulates ROS levels remains unclear. This study reveals that, contrary to the upregulation of various key ROS scavenging genes, arsenic specifically downregulates the expression of the redox-active protein thioredoxin-like 1 (TXNL1) both in vitro and in vivo. Enhancing TXNL1 expression significantly suppresses arsenic-induced ROS production, DNA oxidative damage, and malignant transformation. Mechanistic investigations indicate that arsenic downregulates TXNL1 expression through the downregulation of the deubiquitinase USP10, which leads to increased ubiquitination and degradation of TXNL1. Additionally, arsenic promotes hypermethylation of the USP10 promoter region by upregulating the expression of DNA methyltransferase 1 (DNMT1), resulting in transcriptional repression of USP10. In summary, our results reveal that arsenic disrupts redox homeostasis via the DNMT1–USP10–TXNL1 axis, identifying a potential target for preventing arsenic-induced lung carcinogenesis.

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Introduction

Lung cancer accounts for approximately 2.2 million new cases worldwide and has the second highest incidence rate among all cancers. Each year, it leads to 1.8 million fatalities, making it the cancer with the highest mortality rate¹. Many lung cancer patients have a history of environmental and occupational exposures². Both the International Agency for Research on Cancer (IARC) and the U.S. Environmental Protection Agency (EPA) identify arsenic as a significant environmental carcinogen^3,4. Arsenic is commonly found in nature and is often listed as the highest priority substance by the Agency for Toxic Substances and Disease Registry (ATSDR). It can enter and accumulate in the human body through various routes, including skin contact, consumption of tainted water, and inhalation. Long-term exposure to a high-arsenic environment can induce lung cancer⁵. Therefore, comprehending the mechanisms behind arsenic-induced lung cancer holds substantial importance for the prevention and management of this illness; nonetheless, the way in which arsenic triggers lung cancer is still not fully understood.

Multiple mechanisms have been identified in arsenic carcinogenesis, including epigenetic alterations, abnormal activation of signaling pathways, and imbalances in cellular oxidative stress⁶. The generation of reactive oxygen species (ROS) and the resulting oxidative damage to DNA are essential factors in the development of cancer caused by arsenic^7,8,9. Arsenic induces the overproduction of ROS through intracellular metabolism and mitochondrial dysfunction. However, mammals also have an antioxidant system to combat excessive ROS¹⁰. Elevated levels of ROS induced by arsenic stimulate antioxidant pathways, specifically the Nrf2-ARE signaling cascade^11,12. The transcription factor known as nuclear factor erythroid-derived factor 2–related factor 2 (NRF2) is vital for countering oxidative stress and protecting cells by promoting the synthesis of antioxidant enzymes like heme oxygenase-1 (HO-1), thioredoxin reductase (TrxR), and NAD(P)H quinone oxidoreductase-1 (NQO1)^13,14. However, prolonged accumulation of ROS can disrupt the oxidation-antioxidant balance, leading to abnormal cell signaling and damage to DNA, proteins, lipids, and other cellular components, ultimately contributing to tumor development. Therefore, elucidating the regulatory mechanism of ROS under arsenic exposure is essential for comprehending the carcinogenic process of arsenic.

In order to explore the onset of lung cancer induced by arsenic, we subjected the human bronchial epithelial cell line BEAS-2B to arsenic for 24 h. Through iTRAQ proteomic analysis, thioredoxin-like protein-1 (TXNL1) was identified as one of the most significantly down-regulated proteins. TXNL1, referred to as thioredoxin-related 32 kDa protein (TRP32), functions as a thioredoxin that plays a crucial role in managing oxidative stress, thereby safeguarding cells from damage via the maintenance of redox equilibrium^15,16,17. TXNL1 has multiple functions during human disease progression¹⁸. Notably, downregulation of TXNL1 is linked to unfavorable outcomes in colorectal cancer¹⁹ and is connected to cisplatin-induced apoptosis²⁰, highlighting its relevance in cancer treatment and oxidative stress-related diseases. However, the regulatory mechanisms governing TXNL1 expression and its specific function during arsenic-induced carcinogenesis remain unclear.

In this study, we found that, contrary to the upregulation of most antioxidant proteins, arsenic specifically downregulated the expression of the antioxidant protein TXNL1, leading to the accumulation of ROS and promoting arsenic-induced bronchial epithelial cell malignant transformation. Further investigation into how arsenic downregulates TXNL1 expression revealed that arsenic can upregulate the expression level of DNA methylase DNMT1, which promotes hypermethylation of the USP10 promoter and decreases its transcriptional activity, thereby downregulating USP10 expression and ultimately facilitating the malignant transformation of bronchial epithelial cells. These findings provide new insights into the mechanism by which arsenic induce lung cancer, suggesting valuable strategies for diagnosing and preventing the disease.

Results

Expression of TXNL1 is downregulated by arsenic both in vitro and in vivo

To enhance the detection of arsenic-induced signaling alterations and identify key mechanisms underlying the malignant transformation of bronchial epithelial cells, we exposed BEAS-2B cells to 2.5 and 5.0 μM arsenic—concentrations that are both environmentally relevant and sufficient to amplify molecular responses²¹. After 24 h of acute exposure, we performed iTRAQ-based proteomic analysis to identify differentially expressed proteins. The results indicated a general trend of downregulation following arsenic treatment (Fig. 1a, b). Further analysis focused on proteins with a fold change (FC) ≤ 0.5 that were consistently downregulated in both treatment groups (Supplementary Data 1). Among the 28 commonly suppressed proteins, TXNL1 emerged as the most significantly downregulated protein overall. This observation suggests that decreased TXNL1 expression may represent a critical molecular event in arsenic-associated lung carcinogenesis (Fig. 1c).

**Fig. 1: TXNL1 expression is downregulated in response to arsenic exposure in vitro, in vivo, and in human lung cancer tissues.**

To validate the iTRAQ proteomics findings, we assessed TXNL1 expression under varying arsenic concentrations using western blotting, which revealed a dose-dependent reduction in TXNL1 levels (Fig. 1d). Additionally, TXNL1 expression in BEAS-2B cells exposed to arsenic over an extended period (0.5 μM, 5 months) showed a consistent downregulation (Fig. 1e), suggesting its potential role throughout the arsenic-induced malignant transformation process. To evaluate the effect of arsenic on TXNL1 expression in vivo, Wistar rats were administered arsenic (NaAsO₂, 100 mg/L) via drinking water. After 8 months of exposure, lung tissues were collected and TXNL1 expression was measured. Western blotting and immunohistochemical (IHC) analyses both showed a marked decrease in TXNL1 expression in the lung tissues of arsenic-exposed rats compared to controls (Figs. 1f, S1a, b). To further explore the relevance of TXNL1 downregulation in human lung cancer progression, we analyzed TXNL1 expression in 50 paired clinical samples of lung cancer and adjacent normal tissues using IHC. The results demonstrated that TXNL1 expression was significantly lower in tumor tissues compared to matched normal counterparts (Fig. 1g). Collectively, these findings demonstrate that TXNL1 showed a significant decrease in both human and rat lung epithelial cells upon arsenic exposure, consistent with the expression pattern observed in human lung cancer tissues. We therefore hypothesize that TXNL1 may play an important role in the pathogenesis of arsenic-induced lung cancer.

TXNL1 downregulation plays an important role in arsenic-induced malignant transformation of human bronchial epithelial cells

To investigate the potential role of TXNL1 downregulation in arsenic-induced malignant transformation, we first established a stable TXNL1-overexpression BEAS-2B cell line. Even under arsenic treatment, TXNL1 expression remained at functionally significant levels, as the overexpressed protein exceeded the cellular degradation capacity (Fig. 2a). We then exposed both TXNL1-overexpressing and vector control cells to 0.5 μM arsenic for five months. Following this long-term exposure, the cells were assessed for anchorage-independent growth—a key hallmark of malignant transformation—using a soft agar assay. The results demonstrated that TXNL1 overexpression significantly suppressed arsenic-induced colony formation (Fig. 2b). To further validate these findings, we knocked down endogenous TXNL1 using shRNA, generating two stable knockdown lines (shTXNL1-#1 and shTXNL1-#4; Fig. 2c). Soft agar assays showed that TXNL1 knockdown significantly promoted the arsenic-induced malignant transformation (Fig. 2d). Together, these gain- and loss-of-function experiments provide compelling evidence that TXNL1 downregulation plays a critical role in arsenic-driven malignant transformation of lung epithelial cells.

We next employed a nude mouse tumorigenicity model—a widely accepted standard for assessing malignant transformation²²—to evaluate the effects of TXNL1 in vivo. BEAS-2B vector control cells, arsenic-exposed vector cells (0.5 μM, 5 months), and arsenic-exposed TXNL1-overexpressing cells were subcutaneously injected into nude mice and allowed to form tumors for 5 weeks. The size, volume, and weight of the subcutaneous tumors were recorded. No tumors developed in mice injected with unexposed vector control cells, whereas large tumors formed in those injected with arsenic-exposed vector cells. In contrast, tumors derived from TXNL1-overexpressing cells exposed to arsenic were significantly smaller in size, volume, and weight (Fig. 2e–h). IHC staining confirmed sustained TXNL1 overexpression in the xenograft tissues (Fig. S2a, b). These in vitro and in vivo results consistently demonstrate that TXNL1 acts as an inhibitor of arsenic-induced malignant transformation in human bronchial epithelial cells.

TXNL1 downregulation enhances arsenic-induced DNA damage by promoting ROS accumulation in BEAS-2B cells

ROS are pivotal in arsenic-induced carcinogenesis^8,9. In response, intracellular antioxidant mechanisms are activated by these species to mitigate the damage caused by arsenic¹⁰. The Nrf2-ARE signaling pathway serves as a crucial antioxidant response mechanism that is activated in response to ROS^11,12, leading to the increased transcription of several key antioxidant enzymes, including heme oxygenase-1 (HO-1), peroxiredoxin 1 (Prx1), and NAD(P)H:quinone oxidoreductase 1 (NQO1). Additionally, the thioredoxin (Trx) system has been shown to be upregulated under arsenic stress, further contributing to ROS suppression^23,24. TXNL1, a thioredoxin-like protein, is also involved in ROS clearance. To explore the specific function of TXNL1 in arsenic-induced ROS generation, we evaluated the expression of major antioxidant genes—including Trx1, Nrf2, and HO-1—after both acute and chronic arsenic exposure. We observed that arsenic treatment increased the expression of Nrf2, HO-1, and Trx1 (Fig. 3a, b), which contrasted with the consistent downregulation of TXNL1. Since arsenic-induced antioxidant gene expression is ROS-dependent, we also treated cells with H₂O₂ to simulate ROS stimulation. Interestingly, H₂O₂ not only up-regulated Nrf2, HO-1, and Trx1, but also did not reduce TXNL1 expression (Fig. S3a), suggesting that the arsenic-mediated downregulation of TXNL1 is ROS-independent. Together, compared with the upregulation of other ROS scavenging genes, the specific downregulation of TXNL1 suggested that TXNL1 may have a significant role in arsenic-induced ROS accumulation and subsequent carcinogenesis.

**Fig. 3: TXNL1 downregulation promotes arsenic-induced DNA damage by increasing arsenic-induced ROS production in BEAS-2B cells.**

To assess whether TXNL1 modulates arsenic-induced ROS activity, we measured intracellular ROS levels using flow cytometry. The findings demonstrated that TXNL1 overexpression significantly attenuated arsenic-triggered ROS accumulation in BEAS-2B cells (Fig. 3c; gating strategy shown in Fig. S4). Given that ROS-induced DNA damage is a pivotal event in arsenic-related lung carcinogenesis, we also evaluated oxidative DNA damage by immunocytochemistry (ICC) for 8-hydroxy-2′-deoxyguanosine (8-OHdG). TXNL1 overexpression effectively reduced arsenic-induced 8-OHdG formation (Fig. 3d). Consistent with these in vitro findings, decreased 8-OHdG levels were also observed in subcutaneous tumors formed by TXNL1-overexpressing cells following chronic arsenic exposure (Fig. 3e). Furthermore, in lung tissues of arsenic-exposed Wistar rats, elevated 8-OHdG levels were accompanied by reduced TXNL1 expression (Fig. 3f). Taken together, these findings suggest that overexpression of TXNL1 can mitigate arsenic-induced oxidative DNA damage by suppressing ROS production.

Arsenic attenuates TXNL1 protein stability by downregulating USP10

To investigate how arsenic downregulates TXNL1, BEAS-2B cells were treated with 1 μM arsenic for 0, 12, or 24 h, and TXNL1 expression was assessed at both mRNA and protein levels. The findings indicated that arsenic treatment led to a decrease in TXNL1 protein (Fig. 4a), but an increase in its mRNA levels (Fig. 4b), indicating that post-transcriptional regulation is primarily responsible for the reduction in TXNL1. Since H₂O₂ was previously shown to not downregulate TXNL1 protein expression (Fig. S3a), we also measured its effect on TXNL1 mRNA and observed significant upregulation (Fig. S3b). Collectively, these findings suggest that arsenic-induced ROS may enhance TXNL1 transcription, while the inhibitory effect of arsenic on TXNL1 protein expression is more pronounced, ultimately overriding the ROS-mediated transcriptional upregulation.

**Fig. 4: Arsenic destabilizes TXNL1 protein by downregulating USP10 in human bronchial epithelial cells.**

We next examined the stability of the TXNL1 protein using cycloheximide (CHX) to inhibit new protein synthesis. TXNL1 degradation was markedly accelerated in cells co-treated with arsenic and CHX compared to CHX alone (Fig. 4c), suggesting arsenic enhances TXNL1 turnover. Since ubiquitin-mediated proteasomal degradation is a major route for protein regulation in eukaryotes, we applied the proteasome inhibitor MG132. Treatment with MG132 effectively prevented arsenic-induced TXNL1 degradation (Fig. 4d). Furthermore, ubiquitylation immunoprecipitation assays confirmed that arsenic promotes ubiquitin conjugation to TXNL1 (Fig. 4e), demonstrating that arsenic stimulates TXNL1 degradation via the ubiquitin–proteasome pathway.

To identify upstream regulators affecting TXNL1 ubiquitination, we performed co-immunoprecipitation coupled with mass spectrometry (Co-IP-MS) to isolate TXNL1-interacting proteins. A protein interaction network generated using the STRING database (https://string-db.org/), followed by Gene Ontology (GO) analysis, indicated that TXNL1 may have roles beyond antioxidation (Figure. S5, Supplementary Data 2). Cross-referencing these interactors with proteins altered by arsenic exposure revealed USP10—a deubiquitinating enzyme—as a potential regulatory candidate (Fig. 4f). Although the E3 ligases RBBP7 and WDR82 were downregulated in our proteomic data, their reduced expression makes them unlikely to mediate arsenic-induced TXNL1 degradation, as diminished E3 ligase activity would typically lead to substrate stabilization. We further validated the interaction between TXNL1 and USP10, as well as the effect of arsenic on USP10 expression, using Co-IP and western blotting. The results confirmed that USP10 binds to TXNL1 and that arsenic exposure downregulates USP10 as well as TXNL1 (Fig. 4g, h). As a deubiquitinating enzyme, USP10 stabilizes its target proteins by reducing their ubiquitination. To investigate whether USP10 is involved in arsenic-induced TXNL1 downregulation, we established a USP10-overexpressing BEAS-2B cell line and evaluated TXNL1 expression under both normal and arsenic-treated conditions. USP10 overexpression not only elevated basal TXNL1 protein levels (Fig. 4i) but also counteracted the arsenic-induced decrease in TXNL1 expression (Fig. 4j). A protein degradation assay further confirmed that USP10 overexpression enhanced TXNL1 stability (Fig. 4k). Additionally, in vitro ubiquitination IP experiments showed that USP10 significantly reduced ubiquitin modification of TXNL1 in HEK293 cells (Fig. 4l). Together, these results demonstrate that TXNL1 is a substrate of USP10 and that arsenic facilitates TXNL1 degradation by suppressing USP10 expression.

The downregulation of USP10 is critical in the malignant transformation of human bronchial epithelial cells caused by arsenic

The abnormal expression of USP10 has been reported in multiple human cancers^25,26, particularly in non-small cell lung cancer²⁷. However, the potential role of USP10 in lung tumorigenesis triggered by environmental carcinogens, such as arsenic, remains unexplored. To address this question, we stably overexpressed USP10 in BEAS-2B cells and exposed both USP10-overexpressing and vector control cells to 0.5 μM arsenic for five months. Subsequent soft agar assays demonstrated that USP10 overexpression significantly suppressed arsenic-induced malignant transformation (Fig. 5a, b). To validate these findings in vivo, we performed subcutaneous tumor formation assays in nude mice. Tumors derived from USP10-overexpressing cells exposed to arsenic exhibited markedly reduced size, volume, and weight compared to those from control cells (Fig. 5c–e). IHC analysis of the xenograft tissues confirmed elevated expression of both USP10 and TXNL1 (Fig. 5f–h), and their expression levels showed a significant positive correlation (Fig. 5i). We further investigated whether USP10 modulates arsenic-induced ROS production and DNA damage. Flow cytometry analysis demonstrated that USP10 overexpression significantly attenuated ROS accumulation under both basal and arsenic-treated conditions (Fig. 5j, k). Furthermore, ICC and IHC analyses consistently revealed that USP10 overexpression markedly reduced arsenic-induced 8-OHdG formation in both cellular models and mouse subcutaneous tumor tissues (Fig. 5l–o). Collectively, these observations suggest that USP10 can inhibit the accumulation of ROS and DNA damage, thus contributing to the suppression of arsenic-induced malignant transformation in bronchial epithelial cells.

DNMT1-mediated promoter hypermethylation contributes to USP10 downregulation under arsenic exposure

To investigate the mechanism underlying arsenic-induced downregulation of USP10, we first assessed its mRNA expression and observed a significant reduction after arsenic treatment (Fig. 6a). A dual-luciferase reporter assay further revealed markedly reduced promoter activity of USP10 under arsenic treatment (Fig. 6b), suggesting transcriptional repression. To identify the specific promoter region mediating this effect, we generated a series of truncated USP10 promoter luciferase constructs (Fig. 6c). Transfection of these constructs into BEAS-2B cells, along with pRL-TK for normalization, followed by arsenic treatment, revealed that the repression primarily occurred within the region spanning −487 to +25 (Pro-Usp10-3) (Fig. 6d). Bioinformatic analysis using the PROMO database (https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) predicted potential transcription factor (TF) binding sites within this region (Fig. 6e). We therefore evaluated the expression of the main candidate TFs, Sp1 and ETS-1, but observed no significant changes following arsenic exposure (Fig. 6f). Based on this finding, we speculate that arsenic may not primarily influence the expression of USP10 by directly impacting on the expression of TFs.

**Fig. 6: DNMT1-mediated promoter hypermethylation contributes to USP10 downregulation and TXNL1 protein downregulation following arsenic exposure.**

DNA methylation also serves as a crucial regulatory mechanism for gene transcription. Hypermethylation of promoter regions can result in the silencing of specific tumor suppressor genes, thereby facilitating carcinogenesis. To examine whether DNA methylation contributes to USP10 regulation, we utilized MethPrimer (http://www.urogene.org/methprimer/)²⁸ to predict CpG islands within the USP10 promoter region and identified three CpG islands (Fig. 6g). We then designed methylation-specific primers and performed methylation-specific PCR (MS-PCR) to evaluate arsenic’s effect on USP10 promoter methylation. The results showed that arsenic treatment time-dependently increased methylated USP10 promoter levels (M: indicated by a 219 bp band), while unmethylated DNA (U: 245 bp) decreased (Fig. 6h). Moreover, treatment with the DNMT inhibitor 5-aza-2′-deoxycytidine (5-Aza) suppressed arsenic-induced promoter methylation (Fig. 6i) and rescued both mRNA and protein expression of USP10 (Fig. 6j, k), indicating that arsenic suppresses USP10 through DNA hypermethylation.

The process of DNA methylation is mainly driven by DNA methyltransferases (DNMTs), including DNMT1, DNMT3a, and DNMT3b. Therefore, we examined their expression following arsenic exposure. After 12 hours, arsenic specifically upregulated DNMT1 protein levels, while DNMT3a and DNMT3b remained unchanged (Fig. 6l). To determine whether DNMT1 mediates arsenic-induced USP10 downregulation, we stably knocked down DNMT1 using shRNA in BEAS-2B cells (Fig. 6m). DNMT1 knockout reduced basal USP10 promoter methylation and elevated USP10 expression. Furthermore, it significantly attenuated arsenic-induced hypermethylation and restored USP10 mRNA and protein levels (Fig. 6n–p). Although arsenic still partially reduced USP10 expression in DNMT1-knockdown cells, both USP10 and TXNL1 levels remained substantially higher than in control cells. Together, these findings indicate that DNMT1-mediated promoter hypermethylation represents a major mechanism by which arsenic regulates USP10-TXNL1 expression, while transcription factors may also contribute to the downregulation of USP10 in response to arsenic.

To make the DNMT1-USP10-TXNL1 axis clearer, we further evaluated the protein levels of DNMT1, USP10 and TXNL1 in cells with TXNL1 knockdown, TXNL1 overexpression, and USP10 overexpression (Fig. S6). The results indicate that DNMT1 downregulates USP10, which subsequently lead to a decrease in TXNL1 expression. In contrast, TXNL1 does not significantly influence the expression of either USP10 or DNMT1, and USP10 also exerts minimal effects on DNMT1 expression.

DNMT1-USP10-TXNL1 cascade caused by arsenic was determined in lung tissues in vivo

To investigate the effect of arsenic exposure on the expression of the DNMT1-USP10-TXNL1 cascade in vivo, we assessed the protein levels of DNMT1, USP10, and TXNL1 in lung tissues of Wistar rats following 8 months of arsenic exposure using IHC. The results revealed a marked increase in DNMT1 expression in the arsenic-exposed group compared to the ddH₂O-treated control group, whereas USP10 and TXNL1 levels were significantly reduced (Fig. 7a–c). Further analysis indicated a positive correlation between USP10 and TXNL1 expression, and a negative correlation between TXNL1 and DNMT1 levels (Fig. 7d, e). A schematic diagram summarizing the proposed mechanism is presented in Fig. 7f. Collectively, these findings suggest that arsenic exposure upregulates DNMT1 expression, which promotes methylation of the USP10 promoter and subsequently downregulates USP10 expression. The reduction in USP10 leads to enhanced ubiquitin-mediated degradation of TXNL1, further decreasing its expression. Suppression of TXNL1 results in the accumulation of ROS and DNA damage in bronchial epithelial cells, ultimately promoting their malignant transformation.

**Fig. 7: Correlation between DNMT1, USP10, and TXNL1 expression in arsenic exposed rat lung tissues.**

Discussion

Despite numerous studies, the mechanisms underlying arsenic-induced carcinogenesis remain unclear. Environmental carcinogens, including arsenicals, can drive malignant transformation of normal cells via epigenetic alterations, abnormal protein modifications, and disruptions in cellular oxidative balance^29,30,31. However, the intrinsic connections between these key biological phenomena and their effects on cell malignant transformation remain poorly defined. Here, we demonstrate that arsenic exposure specifically downregulates TXNL1 through the DNMT1–USP10–TXNL1 axis, resulting in elevated ROS and subsequent malignant transformation of bronchial epithelial cells.

Under normal conditions, ROS play beneficial physiological roles. However, under metabolic disturbance or external stress, the cellular redox balance becomes disrupted, leading to a sharp increase in ROS and subsequent oxidative stress³². Excess ROS can cause genomic damage, increase instability, dysregulate gene expression, and facilitate cancer development³³. Key antioxidant factors such as TRX1, Nrf2, and HO-1 normally maintain redox homeostasis by clearing excess ROS^34,35,36. Arsenic exposure promotes carcinogenesis partly through ROS generation³⁷. Our study reveals that arsenic continuously downregulates the expression of the antioxidant protein TXNL1, which consequently impairs cellular antioxidant capacity and leads to the accumulation of intracellular ROS.

TXNL1 is a multifunctional protein implicated in cell cycle regulation, protein synthesis, modification, degradation, vesicular trafficking, transcription, apoptosis, viral replication, and oxidative stress management. Growing evidence underscores its importance in the proliferation, apoptosis, and drug resistance of gastric, colorectal, and pancreatic cancers¹⁸, highlighting its potential as a therapeutic target. However, TXNL1 expression and function appear context-dependent, varying across cancer types—likely due to tissue-specific regulatory networks and the functional versatility of TXNL1 itself¹⁸. Although TXNL1 is involved in diverse processes, its regulatory mechanisms remain elusive, particularly in the context of arsenic exposure. Our research indicates that arsenic exposure upregulates the mRNA levels of TXNL1 while simultaneously inhibiting its protein expression. However, arsenic appears to promote TXNL1 protein degradation with remarkable efficiency. According to the central dogma of molecular biology, newly synthesized TXNL1 protein may be rapidly targeted for degradation. Whether the transiently generated TXNL1 retains any functional role under these conditions remains to be elucidated.

Interestingly, we also observed that, in contrast to the consistent upregulation of ROS scavenging proteins TRX1, Nrf2, and HO-1 under both H₂O₂ and arsenic treatment conditions, TXNL1 exhibited a distinct regulatory pattern. Specifically, TXNL1 protein levels were not downregulated by H₂O₂, but were significantly reduced upon arsenic exposure, suggesting that the downregulation of TXNL1 by arsenic may be specific. The suppression of TXNL1 protein induced by arsenic disrupts the cellular redox balance, resulting in the accumulation of ROS, oxidative stress, and genomic instability. These findings establish TXNL1 as a critical regulator of arsenic-induced oxidative stress and a potential key mediator in arsenic-driven bronchial epithelial cell transformation. Our results provide novel insights into TXNL1’s biological function and reveal a unique molecular mechanism underlying arsenic carcinogenesis.

Of note, when investigating the role of TXNL1 in arsenic-induced malignant transformation, its arsenic-triggered downregulation must be considered. Although the overexpressed TXNL1 construct remains susceptible to degradation, quantitative analysis confirms that ectopic expression exceeds the cellular degradation capacity, maintaining functional TXNL1 levels. In knockdown experiments, 0.5 μM arsenic effectively revealed the impact of TXNL1 loss on transformation. Nonetheless, lower doses may better preserve residual TXNL1, potentially offering clearer functional insights in future knockdown studies.

The ubiquitin–proteasome pathway plays a pivotal role in regulating intracellular protein levels and functions^38,39,40. In this process, ubiquitinating enzymes (UBs) and deubiquitinating enzymes (DUBs) critically determine protein stability and activity, thereby influencing cellular protein fate^41,42. While ubiquitinases have been extensively studied in human diseases, research on DUBs remains relatively limited. To date, approximately 100 DUBs have been identified, with the ubiquitin-specific peptidases (USPs) constituting the largest subfamily^41,43. Among these, USP10 is an evolutionarily conserved and widely expressed enzyme that targets key regulators such as TP53, Beclin1, AMP-activated protein kinase alpha (AMPKα), sirtuin6 (SIRT6), and nuclear factor kappa B (NF-κB)-essential modulator (NEMO), underscoring its importance in diverse cellular processes and disease progression^{44,45,46,47,48,49,50}. Interestingly, USP10 exhibits tissue-specific expression patterns in cancer—elevated in breast cancer and glioblastoma but reduced in gastric, colon, and lung cancers⁴⁴. However, its role in arsenic-induced lung cancer remains unexplored. Our study reveals for the first time that TXNL1 is a key substrate of USP10. During arsenic-induced malignant transformation of BEAS-2B cells, USP10 expression was significantly downregulated, leading to enhanced TXNL1 ubiquitination and degradation. Given the significant role of the ubiquitin–proteasome pathway in the occurrence and progression of human tumors, inhibitors of this pathway and PROTACs therapy have emerged as cutting-edge clinical treatments for cancer^51,52. This research offers an important theoretical foundation for the clinical development of drugs and treatments targeting arsenic-induced lung cancer. Intriguingly, a recent preprint by Gao, Nardone, Yip et al. (biorxiv, 2024.11.08.622741) revealed that in HEK293 cells, TXNL1 associates with the proteasome 19S regulatory particle through interactions with PSMD1/Rpn2, PSMD4/Rpn10, and PSMD14/Rpn11, leading to ubiquitin-independent degradation of TXNL1 upon arsenate exposure. This finding suggests that there may also be ubiquitin-independent degradation pathways in Beas-2B cells exposed to arsenic. The role of this pathway in the TXNL1-dependent arsenic-induced malignant transformation of bronchial epithelial cells warrants further investigation.

A comparison of the roles of USP10 and TXNL1 in inhibiting ROS accumulation, 8-OHdG formation, and arsenic-induced malignant transformation revealed that USP10 exerts a stronger inhibitory effect than TXNL1 on ROS accumulation and 8-OHdG formation. However, soft agar colony formation assays and nude mouse xenograft experiments showed that TXNL1 overexpression suppressed arsenic-induced malignant transformation to an extent comparable to USP10 overexpression, suggesting that TXNL1 acts as the primary downstream effector of USP10 in this process. The stronger inhibitory effect of USP10 may be attributed to its more efficient upregulation of endogenous TXNL1 compared to exogenous TXNL1 overexpression, observed under both basal and arsenic-treated conditions. Notably, USP10 is a key deubiquitinating enzyme in tumor progression, regulating multiple established substrates (such as p53⁵³, PTEN⁵⁴, KLF4⁵⁵) that impact tumorigenesis and development. Given this, other USP10 substrates may also exert synergistic effects. Whether these molecules are involved in arsenic-induced malignant transformation remains to be further investigated. Proteomic analysis of arsenic-treated BEAS-2B cells showed downregulation of multiple USP10-regulated targets, such as p53, DDX21, FASN, and RFC2, with TXNL1 exhibiting the most pronounced reduction. Moreover, the regulatory relationship between USP10 and its substrates under arsenic exposure remain to be fully elucidated. Future studies should focus on comprehensively delineating the functional hierarchy and mechanistic interplay between USP10 and its substrate network in arsenic-associated carcinogenesis.

USP10 is regulated at the post-transcriptional level by miRNAs as well as by genes such as ATM and BECN1^45,50,56, but the mechanisms governing its transcriptional regulation remain uncertain. While our dual-luciferase assays demonstrate arsenic’s transcriptional repression of USP10 via the Pro-USP10-3 region, the specific TFs responsible remain elusive. Future studies should investigate more candidate TFs and their activation states (e.g., phosphorylation). Epigenetic alterations, particularly alterations in promoter DNA methylation, are critically implicated in both the occurrence and progression of cancer⁵⁷. Abnormal DNA methylation may result in the excessive activation of oncogenes or the reduced expression and silencing of tumor suppressor genes, which together facilitate the advancement of cancer^57,58. However, the impact of arsenic exposure on the methylation of genomic DNA remains a subject of debate. Numerous researches have indicated that arsenic exposure can cause a reduction in the expression of DNA methyltransferases (DNMTs) and result in global DNA hypomethylation^59,60. On the other hand, alternative studies have found that arsenic exposure might also trigger DNA hypermethylation in specific tumor suppressor genes, including p53, p16, and Foxp3^29,61,62. Both hypomethylation and hypermethylation of DNA seem to play a role in the initiation and advancement of cancer. However, the mechanisms by which arsenic induces hypermethylation of specific genes are not yet fully understood. In this research, we observed that arsenic enhances DNA methylation in the promoter region of USP10, decreasing its expression during the malignant transformation of BEAS-2B cells induced by arsenic. The addition of methyl groups to DNA is facilitated by three members of the DNMT family: DNMT1, DNMT3a, and DNMT3b⁵⁸. Our findings indicate that the main cause of changes in DNA modification of the USP10 promoter is a change in DNMT1 levels, offering a new theoretical basis for understanding the role of arsenic in DNA methylation. Research on the regulation of DNMT1 following arsenic exposure also remains controversial. Some studies report that DNMT1 expression is down regulated in human keratinocyte cells (HaCaT) after acute arsenic exposure⁵⁹, while others demonstrate an upregulation of DNMT1 expression in regulatory T cells (Tregs) following the same exposure⁶². This discrepancy suggests that different cell types and treatment regimens may contribute to the uncertainty regarding the effect of arsenic on DNMT expression. Furthermore, the specific mechanisms by which arsenic regulates DNMT1, as well as how DNMT1 specifically regulates USP10, necessitate further in-depth investigation.

In this study, we conducted iTRAQ proteomics experiments with acute exposure of BEAS-2B cells to high concentrations of arsenic (2.5 and 5.0 μM) to enhance the detection of arsenic-induced alterations in signaling pathways and to facilitate the identification of proteins that play a critical role in arsenic-induced lung cancer. Additionally, we performed validation experiments using moderate concentrations (0.1–1 μM) aimed at elucidating the mechanisms of arsenic carcinogenesis. Notably, the arsenic concentrations of 0.1–1 μM are more representative of those found in the bodies of patients with chronic exposure and have been widely applied in experiments investigating arsenic-induced malignant transformation of BEAS-2B cells to study the mechanisms of arsenic-induced lung cancer^63,64,65. Therefore, the results of the proteomics experiments are intended to provide clues, while the actual mechanisms still require validation through biological experiments. Further analysis of the proteomics data revealed a significant upregulation of HMOX1 consistency within both groups, along with numerous significant downregulated proteins. In addition to TXNL1 exhibiting the most significant downregulation, several ribosomal proteins, including RL7, RL15, RL34, RL31, RPL10, and RL23A, also underwent extensive downregulation. Previous studies have confirmed that the reduction in ribosome numbers following arsenic exposure is a rapid, reversible, and effective protein toxicity stress response, through which cells cope with the protein misfolding toxicity induced by arsenic⁶⁶. Furthermore, the study found that several proteins involved in DNA replication and repair, such as MCM2, MCM6, and WDHD1, also exhibited downregulation after arsenic exposure. Among these, MCM2 and MCM6 are typically overexpressed in lung cancer tissues, and their expression levels are closely correlated with patient prognosis. However, the specific mechanisms by which these proteins contribute to arsenic-induced lung cancer development remain to be further investigated⁶⁷. Further proteomic analysis identified significant downregulation of MSH2^68,69, MBD4⁷⁰, NAMPT⁷¹, BUB1⁷², and MCM7⁷³, a phenomenon previously associated with arsenic exposure. Notably, MSH2 and MBD4 have been shown to undergo promoter hypermethylation under arsenic treatment, suggesting that their suppression may stem from DNMT1 upregulation. These observations raise the possibility that DNMT1-mediated epigenetic silencing contributes to the downregulation of these and other proteins in our dataset.

Collectively, our findings revealed that arsenic can specifically downregulate TXNL1 expression in human bronchial epithelial cells and induce ROS accumulation, ultimately promoting malignant transformation. Mechanistic studies revealed that arsenic specifically down regulates TXNL1 expression through the upregulation of DNMT1, which hypermethylates the promoter region of USP10, thereby inhibiting its transcriptional activity; ultimately, this increases the ubiquitination level of TXNL1 and promotes its degradation. Together, these results indicated that targeting the DNMT1–USP10–TXNL1 pathway could serve as an effective strategy for preventing and treating arsenic-induced lung cancer.

Methods

Reagents and plasmids

Cycloheximide (CHX, 66-81-9) was obtained from Santa Cruz Biotechnology (Shanghai, China). Sodium arsenite (NaAsO₂, S7400) and 5-aza-2’-deoxycytidine (5-Aza, A3656) were acquired from Sigma-Aldrich (Shanghai, China). Puromycin (P8230) was sourced from Solarbio (Beijing, China), while MG132 (S2619) was obtained from Selleck Chemicals (Shanghai, China). G418 (108321-42-2) was acquired from Shanghai GoldBio Tech. The TXNL1 overexpression plasmid, myc-Ubiquitin plasmid, and their corresponding control plasmids were purchased from Miaoling Company (Wuhan, China). Additionally, the USP10 overexpression plasmid, TXNL1 shRNA plasmids, DNMT1 shRNA plasmids, and the control plasmids were obtained from Public Protein/Plasmid Library (Nanjing, China). Luciferase reporters driven by USP10 promoter-1 (from −1604 to +25), USP10 promoter-2 (from −1021 to +25), and USP10 promoter-3 (from −487 to +25) were generated using genomic DNA extracted from BEAS-2B cells, utilizing data obtained from the NCBI database. Nested PCR was performed using the forward outside primers (F1) 5′-CCC AAT AAT TCA CGA AGA TCA CCC AT-3′, (F2) 5′-CCT CCTCTG CTC AGT GTA CGG TCT-3′, and (F3) 5′-CGG CAG GAC TTG GGG AGT GAA T-3′; and the reverse primers (R1,2,3) 5′-CCG GAA TGC CAA GCT TTC TTC TCG CCC GCA CAT ACA-3′. The resulting PCR products underwent digestion, were cloned into the pGL3-Basic vector (E1751; Promega, Madison, WI, USA), and were subsequently confirmed through DNA sequencing.

Human clinical samples and cell lines

A total of 50 pairs of human lung cancer tissue specimens alongside adjacent normal tissues were obtained from the Affiliated Hospital of Wenzhou Medical University (Supplemental Table 1). The ethics committee of Wenzhou Medical University approved all experiments involving clinical specimens. BEAS-2B cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA; ATCC® CRL-9609), which originate from normal human bronchial epithelial cells, typically do not exhibit tumorigenic properties. The cell line was authenticated by Genetic Testing Biotechnology Corporation (Suzhou, China) using short tandem repeat (STR) profiling. The cell line was found to be negative for mycoplasma contamination. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma), enriched with 10% fetal bovine serum (FBS; Gibco), and supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 2 mM GlutaMAX (Gibco).

Cell transfection and generation of stable cell lines

Cells were transfected using Polyjet^TM DNA in Vitro Transfection Reagent (SignaGen Laboratories) to introduce plasmids. Stable cell lines for TXNL1-HA, TXNL1 knockdown, DNMT1 knockdown and vector control were stablished through lentiviral infection and screened by puromycin (0.5 μg/mL). The lentivirus packaging and infection processes were carried out according to previously outlined methods⁷⁴. Additionally, stable cells for USP10 expression and the corresponding vector control were generated through direct plasmid transfection and selected using G418 (2000 μg/mL).

Cell arsenic exposure

For acute arsenic exposure, we selected a concentration range of 0–5.0 μM and a treatment duration of 12–24 h. When the cell density reaches 70–80%, the cells were first treated with DMEM medium supplemented with 0.1% FBS for a duration of 12 h. Subsequently, 0–5.0 μM NaAsO₂ was introduced into the medium, and the cells were then cultured for the specified duration. For long-term arsenic exposure, following each cell passage and attachment, the medium was replaced with DMEM complete medium containing 0.5 μM NaAsO₂, and the exposure period was extended for 5 months. One micromolar of arsenic is equivalent to 75 ppb, which is similar to levels found in the environment, and these concentrations are widely recognized doses in arsenic toxicity research⁷⁵.

Animal arsenic exposure

Animal experiments were conducted at the animal institute of Wenzhou Medical University, following the protocols authorized by both the Laboratory Animal Center and the Laboratory Animal Ethics Committee of the same university, in compliance with the ARRIVE guidelines. The Wistar rats (4–5 weeks old) to be poisoned were purchased from Zhejiang Weitonglihua Animal Technology Co., Ltd. (Jiaxing, China) and housed in the SPF-class animal facility at Wenzhou Medical University. To ensure age consistency, age-matched rats (4 females and 8 males) were randomly assigned to the control and experimental groups, with each group containing six animals and an equivalent sex distribution. During the experiment, the drug was administered orally. NaAsO₂ powder was dissolved into ddH₂O at a final concentration of 100 mg/L. This concentration has been well-established in previous arsenic exposure studies using rat models⁷⁶. The control group received no drug treatment, while the experimental group underwent long-term drug treatment. After 8 months of administration, the rats were euthanized, and their lungs were collected for subsequent experimentation. The lung tissue was divided into two portions: one portion was preserved by freezing at −80 °C for the extraction of RNA or protein, whereas the other portion was fixed in formalin for 24 h before being paraffin-embedded.

iTRAQ proteomic analysis

BEAS-2B cells were seeded in 10 cm dishes and serum-starved for 12 hours in DMEM containing 0.1% FBS to synchronize growth. Following this, the cells were treated with NaAsO₂ at concentrations of 0, 2.5, or 5.0 μM for 24 h. After harvesting, the cells underwent iTRAQ-based quantitative proteomic analysis (Lianchuang Biotech, Project ID: 2017D25pmA81). In brief, proteins were extracted, reduced, and alkylated to cleave disulfide bonds. The protein concentration was quantified using the Bradford assay and verified through SDS-PAGE. Equal amounts of protein (100 µg) were trypsin-digested, labeled with iTRAQ reagents, pooled, and pre-fractionated using strong cation exchange chromatography (SCX) before LC-MS/MS analysis. Differentially expressed proteins (DEPs) were identified with a threshold of fold change ≥1.2 or ≤0.83, and a p-value < 0.05, with statistical significance assessed using the Significance A/B test.

Western blot analysis

Following the rupture of the cells in the lysis buffer containing 10% SDS, 100 mM Na₃VO₄, and 1 M Tris-HCl, the resulting proteins were extracted and subjected to SDS-PAGE, subsequently transferred onto a PVDF membrane. The membranes were subsequently treated with the specified primary antibodies, followed by a reaction with a secondary antibody conjugated to alkaline phosphatase (AP). Antibodies against TXNL1 (Ab188328, 1:1000) and USP10 (Ab72486, 1:1000) were purchased from Abcam (Shanghai, China). The following antibodies were used for protein detection: anti-Thioredoxin 1 (Trx1, 2429S, CST, 1:1000), anti-Nrf2 (12721S, CST, 1:1000), anti-HO-1 (70081S, CST, 1:1000), anti-HA (3724S, CST, 1:1000), anti-Myc-Tag (2276S, CST, 1:1000), anti-Ubiquitin (3933S, CST, 1:1000), anti-Sp1 (9389S, CST, 1:1000), anti-β-Actin (Ab0011, Abways Technology, 1:10000), anti-ETS-1 (sc-350, Santa Cruz, 1:500), anti-8-OHdG (sc-66036, Santa Cruz, 1:500), anti-DNMT1 (24206-1-AP, Proteintech, 1:1000), anti-DNMT3a (20954-1-AP, Proteintech, 1:10000), and anti-DNMT3b (NB300-516, Novus Biologicals, 1:500). To visualize the signals, an enhanced ECF chemifluorescence system was employed, and the images were captured using a phosphorimager (Typhoon FLA 7000; GE Healthcare, MA, USA).

Immunohistochemistry (IHC)

Fresh lung tissues and subcutaneous tumor tissues were fixed in formalin solution or 4% paraformaldehyde (PFA) for 24 h. Following a gradient dehydration, the tissue specimens were embedded in paraffin, and 4 μm sections were prepared. After dewaxing with xylene and rehydrating through a series of alcohol concentrations (100%, 95%, 90%, 80%, 70%, and 50%), microwave antigen retrieval was carried out in citrate buffer. Once the samples returned to room temperature, they were treated with 3% H₂O₂ for 10 min and then incubated with 3% FBS for 30 min at room temperature. The tissue specimens were allowed to incubate overnight at 4 °C with antibodies targeting TXNL1 (1:250), USP10 (1:250), DNMT1 (1:300), and 8-OHdG (1:200). Staining was conducted using the Ready-to-Use SABC-POD Kit (SA1022; BOSTER, Wuhan, China). Images of the immunostained samples were captured using a Nikon Eclipse Ni microscope (DS-Ri2). The analysis of protein expression levels was performed using Image-Pro Plus version 6.0 (Media Cybernetics, MD, USA).

Soft agar assay

Soft agar assays were conducted to evaluate the capacity of arsenic-treated BEAS-2B cells to undergo anchorage-independent growth. In these experiments, 1 × 10⁴ BEAS-2B cells were evenly mixed with basal medium eagle (BME) supplemented with 1% FBS and containing 0.33% soft agar, then layered over a base of 0.5% agar in BME with 1% FBS. The plates were cultured for 2–3 weeks, and the colonies were observed using a microscope (DMi1; Leica, Germany). Colonies containing over 32 cells were included in the count.

Nude mouse subcutaneous tumorigenesis experiments

Female BALB/c athymic nude mice (3–4 weeks old) were procured from GemPharmatech (Nanjing, Jiangsu, China). Following a week of acclimatization, the mice were randomly sorted into three groups (n = 5 per group) and received subcutaneous injections of 8.0 × 10⁶ cells each: BEAS-2B (Vector), BEAS-2B (Vector-As, 5 m), and BEAS-2B (TXNL1-As, 5 m) with the cell suspensions delivered in 100 μL of PBS on the right dorsal side. During the culture, tumor volumes were recorded every 5 days. The modified ellipsoid formula (length × width² × 0.5) was used to calculate tumor volume. All procedures involving animals were sanctioned by the Ethics Committee of Wenzhou Medical University.

Intracellular ROS determination

Intracellular ROS were assessed utilizing a cellular ROS assay kit (CA1420, Solarbio). Initially, cells were seeded in a 6-well plate and subjected to arsenic treatment for 24 h. Following this, the cells were cultured in complete DMEM for 12 h before being collected through centrifugation. The harvested cells were then incubated for 30 min with dihydroethidium (DHE) red fluorescent dye at a final concentration of 10 μM. After centrifugation, the cells were rinsed three times with PBS. The detection of intracellular ROS was carried out via CytoFLEX flow cytometry (BECKMAN, USA), and the results were analyzed with CytExpert software.

Immunocytochemistry

After 24 h of exposure to 1 μM arsenic, cells were subjected to fixation with 4% paraformaldehyde for 20 min at room temperature. This was followed by a permeabilization step using 0.5% Triton X-100 for 20 min, after which a treatment with 3% H₂O₂ was conducted for 10 min. Blocking was then performed with 3% BSA for 30 min at room temperature. Next, the 8-ohDG antibody (sc-66036, 1:200) was introduced and incubated overnight at 4 °C. The SABC-POD kit (SA1022; BOSTER Biological Technology) was utilized for staining, and the results were observed under a light microscope. Five images were captured for each cell slide and subjected to statistical analysis.

RNA isolation and real-time PCR

Total RNA was extracted from cultured cells by utilizing the TRIzol reagent (254708, Invitrogen). Following this, the RNA underwent reverse transcription to produce total cDNA with the assistance of the PrimeScript^TM RT reagent kit (RR037B, Takara), and was subsequently analyzed using RT-PCR. The quantification of mRNA expression levels was performed through qPCR on a Q6 real-time PCR System, using SYBR Green Master Mix (4309155, Applied Biosystems), with GAPDH serving as an internal loading control. The primers utilized in this investigation were as follows: human TXNL1 (forward: 5′-CCA CAG GCT GTT TTC TTG-3′ and reverse: 5′-ATT GCT TCC AGG GTC ATT-3′), human USP10 (forward: 5′-CCA TAC AGT GGA ACA GTT CTG T-3′ and reverse: 5′-GGG TTC AGT GTG CTT GAA ATA C-3′), and human GAPDH (forward: 5′-GAC TCA TGA CCA CAG TCC ATG C-3′ and reverse: 5′-CAG GTC AGG TCC ACC ACT GA-3′).

Co-immunoprecipitation assay

BEAS-2B cells were grown in 10 cm dishes until confluence reached 70%, and then transfected with TXNL1 overexpression plasmid or vector control plasmid. Following transfection, the cells were harvested and lysed using 1× Cell Lysis Buffer (9803S, CST) supplemented with protease inhibitors (Roche, Branchburg, NJ, USA), followed by a brief sonication step. The cell extracts were then incubated overnight at 4 °C with HA Magnetic Beads (M180-11, MBL). A magnetic stand was employed to adsorb the magnetic beads, after which the supernatant was discarded. The beads underwent washing 5–6 times with 1× Cell Lysis Buffer, and the proteins bound to the beads were eluted using 2× SDS sample buffer before being analyzed through western blotting.

Ubiquitylation immunoprecipitation

BEAS-2B or 293 T cells were utilized in this assay. Briefly, the cells underwent co-transfection with the Myc-Ub plasmid alongside the specified plasmids for a duration of 36 h. Prior to harvesting, the cells received treatment with MG132 at a concentration of 2.5 μM for 12 h. Cell extracts were then prepared using 1× cell lysis buffer and immunoprecipitated with HA Magnetic Beads. A Western blotting assay was conducted to identify the ubiquitination status of TXNL1.

Dual luciferase reporter assay

Luciferase reporters driven by the USP10 promoter (P-USP10-1, P-USP10-2, and P-USP10-3) and their control plasmid, were co-transfected with pRL-TK into BEAS-2B cells for 24 h. Following this, the cells underwent starvation in 0.1% FBS DMEM for 12 h, after which 1 μM arsenic was added for either 12 h or 24 h. Subsequently, the cells were lysed to assess luciferase and Renilla luciferase activity using the Dual-Luciferase Reporter Assay System (E1500, Promega). Measurements were obtained with a luminometer (Centro LB 960; Berthold, Germany). The relative promoter activity is presented as normalized luciferase activity compared to Renilla luciferase activity.

DNA extraction, bisulfite DNA modification, and methylation-specific PCR

Intracellular DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). Human peripheral blood leukocyte genomic DNA served as a negative control, while methylated DNA was prepared as a positive control by methylating Buffy coat DNA with M.SssI CpG methyltransferase (M0226V, NEB). Following the instructions of the EZ DNA Methylation-Gold™ Kit (ZymoResearch, Irvine, CA, USA), the sample DNA was treated with bisulfite, and the final product was collected. Methylation-specific PCR (MSP) primers were designed based on the DNA methylation sites predicted by MethPrimer, and optimized MSP was performed on the bisulfite-treated genomic DNA. The primers used were as follows: MF: 5′-GGT GAG GAG TCG GGT TCG TC-3′, MR:5′-GCC TAC GCG ACC GAC AAA TAA AC-3′, UF:5′-TTT AGT AGG TGA GGA GTT GGG-3′, UR: 5′-AAA ACC AAC CCA CTA CTA C-3′. The PCR products were subsequently subjected to agarose gel electrophoresis.

Statistics and reproducibility

Experimental results are presented as means ± SD from at least three biologically independent samples, and the data were analyzed and visualized using GraphPad Prism 6.0 statistical software for statistical analysis. Student’s t-test was used to evaluate differences between two groups. Two-way ANOVA was employed to examine statistical differences involving two different independent variables. The Pearson correlation coefficient was calculated to evaluate gene expression correlation. P < 0.05 was considered to indicate a significant difference relative to the control.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Ethical approval

All experiments related to clinical specimens were approved by the ethics committee of Wenzhou Medical University (LCKY2018-48) and signed the informed consent with all patients before the research started. All animal experiments were performed in accordance with the management regulations of the Experimental Animal Ethics Committee of Wenzhou Medical University (xmsq 2021-0083).

Data availability

The graphical abstract representing the overview of this research is shown in Fig. 7f. All data are available from the corresponding author on reasonable request. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD067927. Uncropped blot images were shown in Supplemental Fig. S7. The Numerical source data for graphs and charts can be found in Supplementary Data 3.

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Acknowledgements

This work was supported by Zhejiang Provincial Natural Science Foundation of China (LQ22H260006), Key Discipline of Zhejiang Province in Medical Technology (First Class, Category A), and Key Project of Science and Technology Innovation Team of Zhejiang Province (2013TD10).

Author information

These authors contributed equally: Lingling Zhao, Xueli Jiao, Hongyan Li.

Authors and Affiliations

Zhejiang Provincial Key Laboratory of Medical Genetics, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China
Lingling Zhao, Xueli Jiao, Hongyan Li, Minghao Li, Chuanshu Huang & Haishan Huang
Department of Microbiology, Nantong Center for Disease Control and Prevention, Nantong City, Jiangsu, 226007, China
Xueli Jiao
The First Affiliated Hospital of Wenzhou Medical University, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, China
Yuanyuan Chang & Yu Liu
The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325027, China
Qipeng Xie

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Lingling Zhao
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Xueli Jiao
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Hongyan Li
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Yuanyuan Chang
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Minghao Li
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Yu Liu
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Qipeng Xie
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Chuanshu Huang
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Haishan Huang
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Contributions

Conception and design: H.S.H., L.L.Z. and C.S.H. Perform experiments and acquisition of data: X.L.J., H.Y.L., Y.Y.C., M.H.L., Y.L., Q.P.X. and L.L.Z. Analysis and interpretation of data: X.L.J., H.Y.L. and L.L.Z. Writing, review, and/or revision of the paper: X.L.J., H.Y.L. and L.L.Z. and H.S.H. The supplemental experiments and textual modifications in response to the reviewers’ comments were performed by L.L.Z. Administrative, technical, or material support: H.S.H. and C.S.H. Study supervision: H.S.H. and L.L.Z. All authors reviewed and approved the final version.

Corresponding authors

Correspondence to Lingling Zhao or Haishan Huang.

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Communications Biology thanks Matteo Trovato and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christina Karlsson Rosenthal. A peer review file is available.

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Zhao, L., Jiao, X., Li, H. et al. Arsenic promotes ROS-mediated malignant transformation of bronchial epithelial cells by specifically downregulating TXNL1 expression. Commun Biol 8, 1827 (2025). https://doi.org/10.1038/s42003-025-09216-z

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Received: 07 February 2025
Accepted: 10 November 2025
Published: 22 November 2025
Version of record: 29 December 2025
DOI: https://doi.org/10.1038/s42003-025-09216-z