NEDD8-conjugating enzyme E2 UBE2F deficiency promotes lung cancer metastasis by stabilizing Snail

doi:10.21203/rs.3.rs-3427704/v1

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NEDD8-conjugating enzyme E2 UBE2F deficiency promotes lung cancer metastasis by stabilizing Snail

https://doi.org/10.21203/rs.3.rs-3427704/v1

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Lung cancer is the most prevalent malignant tumor, resulting in the highest morbidity and mortality rates worldwide. Approximately 30–40% of lung cancer cases progress to distant metastasis, significantly affecting patient quality of life and survival. However, the mechanisms driving this metastasis remain incompletely understood. Our study aims to investigate the impact of the NEDD8-conjugating enzyme UBE2F on lung cancer metastasis. We established a nude mouse model of lung cancer metastasis with stably silencing the UBE2F protein of human lung cancer cells by tail vein injection. In in vivo animal experiments, we observed a significant increase in the number of metastatic micronodules on the lung surface of nude mice following UBE2F knockout compared to the control group. Moreover, the results of cell scratch and transwell chamber assays indicated that UBE2F knockout enhanced the invasion and migration capabilities of lung cancer cells. Western blot analysis further revealed that UBE2F knockout impeded the degradation of Snail protein, a critical factor in epithelial-mesenchymal transition (EMT), resulting in its accumulation. Together, these findings suggested that the downregulation of UBE2F expression inhibits the degradation of Snail protein, thereby promoting the invasion and metastasis of lung cancer cells.

NEDD8-conjugating enzyme E2

UBE2F

Snail

Lung cancer

Metastasis

Lung cancer, the foremost contributor to global morbidity and mortality, presents a formidable threat to human health[1]. Annually, an estimated 1.6 million deaths are attributed to lung cancer, with approximately 85% of cases histologically classified as nonsmall cell lung cancer (NSCLC)[2]. NSCLC often remains asymptomatic in its early stages, leading to delayed diagnosis and missed opportunities for surgical intervention. Consequently, this results in a limited prognosis and overall poor survival rates. While treatment options for NSCLC have expanded in recent years, tumor metastasis continues to be a pivotal factor negatively impacting patient survival and overall outcomes[3, 4]. Thus, it is imperative to explore novel therapeutic strategies to enhance patient survival rates and clinical prognoses in lung cancer. Delving into the intricate molecular mechanisms governing lung cancer invasion and metastasis, followed by targeted interventions, promises to open new therapeutic avenues for a broader spectrum of lung cancer patients.

Neddylation, a critical post-translational protein modification process, involves the successive covalent attachment of the ubiquitin-like small molecule neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to substrate molecules. This modification is catalyzed by NEDD8-activating enzyme E1 (NAE1/UBA3), NEDD8-conjugating enzyme E2 (UBE2M/UBE2F), and NEDD8 ligase E3 (RBX1/2, etc.)[5, 6]. Among the well-established neddylation substrates, the Cullin-RING Ligase (CRL) ubiquitin ligase superfamily stands out[7]. Activation of CRL ubiquitin ligase necessitates neddylation modification of the Cullin subunit, inducing a conformational change in the CRL complex and activating its E3 ubiquitin ligase activity. The CRL superfamily, a multi-subunit ubiquitin ligase family, plays pivotal roles in regulating biological processes, including development and stress responses, with its dysfunction closely linked to tumorigenesis[8–10].

In mammalian cells, there are two NEDD8-conjugating enzyme E2 proteins, UBE2M and UBE2F, each with different biological functions. UBE2F is responsible for mediating the neddylation modification of Cullin5 (CUL5), while UBE2M mediates the same modification for CUL1/2/3/4 [6]. Despite their shared role in the neddylation pathway, UBE2F remains less explored than its counterpart, UBE2M. UBE2F has been implicated as an anti-apoptotic protein, primarily forming a complex with RING-box protein 2 (RBX2), thereby creating the UBE2F/RBX2/CUL5 tricomplex, which activates CRL5. This activation leads to the polyubiquitination and proteasome-mediated degradation of the pro-apoptotic protein NOXA[11]. Intriguingly, a recent study have highlighted that deletion of the CUL5 promotes lung cancer metastasis by stabilizing integrin β1, a crucial protein on cell migration and invasion [12]. However, the specific influence of UBE2F on lung cancer metastasis remains largely unexplored.

This study presents compelling evidence that the downregulation of the NEDD8-cconjuating enzyme UBE2F impedes Snail protein degradation and ultimately promotes lung cancer metastasis, indicating that UBE2F might emerge as a pivotal factor influencing lung cancer metastasis.

2.1 Reagents and Antibodies

High-glucose DMEM medium (containing L-glutamine and pyruvate), fetal bovine serum (FBS), and penicillin-streptomycin (double antibiotic) were procured from Shanghai Shizhi Biotechnology Co., Ltd. The PAGE gel preparation kit, trichrome protein prestaining marker, and Omni-ECL enhanced chemiluminescence detection kit were acquired from Shanghai Yichen Biotechnology Co., Ltd. Antibodies, including Cullin1 (Abcam,75817), Cullin5 (Abcam,184177), UBE2F (Abcam, 12932), Snail(Cell Signaling Technology,3879), E-Cadherin(Cell Signaling Technology,13116), Claudin1(Cell Signaling Technology,13255), ZEB1(Cell Signaling Technology,3396), Vimentin(Cell Signaling Technology,5741), Slug(Cell Signaling Technology,9585), ZO1(Cell Signaling Technology,8193), β-Catenin(Cell Signaling Technology,8480), and β-actin(Cell Signaling Technology,3700), were obtained from Shanghai Yiwei Biotechnology Co., Ltd. HRP-labeled goat anti-mouse secondary antibody and HRP-labeled goat anti-rabbit secondary antibody were sourced from Hangzhou Baoke Biotechnology Co., Ltd. Cycloheximide (CHX) was purchased from Shanghai Solarbio Bioscience & Technology Co., Ltd. Transwell inserts were acquired from Corning Inc., USA.

2.2 Cell Culture

The lung adenocarcinoma cell line A549 was procured from the American Type Culture Collection (ATCC). A549 cells were cultured at 37°C in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution under 5% carbon dioxide in an incubator.

2.3 Generation of stable cell lines by CRISPR/Cas9 system

To produce lentiviral vectors for UBE2F knockout, two guide RNA sequences specific to UBE2F were inserted into the lenti-guide-puro vector, respectively. We co-transfected 293T cells with lentiviral vectors lenti-guide-puro (4 µg) and packaging vectors AGP091 (3.0 µg) and AGP090 (1.2 µg). After 48 hours, the viral supernatants were collected and filtered and were then used to infect A549 cells. Polybrene (Sigma-Aldrich, St. Louis, MO) was added to the viral supernatant at a concentration of 10µg/mL. After a six-hour incubation, the viral supernatant was replaced with normal DMEM containing 10% FBS.

2.4 Protein Expression by Western Blot

A549 cells, cultured individually, were lysed in SDS sample buffer upon collection and denatured by heating at 100°C for 10 minutes. We prepared a polyacrylamide gel using the PAGE gel preparation kit and pre-stained the gel with a trichrome protein marker to indicate protein sizes. Proteins were separated on a 10% Bis-Tris polyacrylamide gel and then transferred to a PVDF membrane. After blocking with milk for 2 hours, the membrane was washed, and the primary antibody was incubated overnight. Following three washes with TBST working solution, the secondary antibody was incubated at room temperature for 2 hours. The membrane was then washed thrice with a TBST working solution, and the developer was prepared according to a 1:1 ratio, as per the instructions. Subsequently, it was applied to the membrane for development using the appropriate machine.

2.5 Animal Experiment

We acquired female nude mice aged eight weeks from Nanjing Junke Bioengineering Co., Ltd. The Institutional Animal Care and Use Committee of Taizhou University approved the mouse experiments conducted in specific pathogen-free facilities. Nude mice were randomly allocated to one of three groups: the control group (CONTROL) injected with wild-type A549 cells and two groups injected with UBE2F knockout A549 cells (UBE2F-KO1 and UBE2F-KO2). Each group received a 100 µL injection of cells at a concentration of 2 × 10⁶/mL via the tail vein and were observed for 40 days. Following euthanasia, mouse lung tissue samples were dissected and collected.

2.6 Cell Scratch Assay

After allowing untreated A549 cells (Control) and A549 stable cell lines (UBE2F-KO1 and UBE2F-KO2) constructed following UBE2F knockout to adhere, the surface was scratched and labeled using a tip. The scratches were assessed 24 hours later, and distances were measured.

2.7 Transwell Migration and Invasion Assays

Migration Assay: Cells were collected, counted, and diluted to 1 × 10⁴/mL with serum-free medium. 100 µl of which was placed in the upper chamber of the Transwell, while 500 µl of serum-containing medium was added to the lower chamber. The cells were cultured for 24 hours, fixed in 4% paraformaldehyde for 30 minutes, stained with crystal violet stain for 30 minutes, washed with PBS, and photographed under a microscope.

Invasion Assay: Matrigel was diluted with serum-free DMEM (dilution ratio 1:3), and the diluted Matrigel was added to the insert. The Transwell plate was placed in a 37°C constant temperature incubator to coagulate for 1 hour. The subsequent steps were similar to those of the migration assay.

2.8 Statistical Methods

We assessed the statistical significance of the variance between groups using GraphPad Prism 6, employing a t-test for between-group comparisons. A p-value of < 0.05 indicated statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).

3.1 UBE2F deficiency promotes lung cancer metastasis

It has been well-established that UBE2F, in partnership with RING-box protein 2 (RBX2), facilitates the neddylation of CUL5[6]. To elucidate the role of UBE2F in CUL5 neddylation modification, stable A549 lung adenocarcinoma cell lines with UBE2F knockout were generated using the CRISPR/Cas9 system. The efficacy of the knockout is depicted in Figure 1A, demonstrating a significant deficiency in UBE2F expression in UBE2F-knockout A549 cells compared to controls. Importantly, the neddylation modification level of CUL1, a classical substrate within the neddylation pathway, remained largely unchanged, while the neddylation modification level of CUL5 exhibited a substantial decrease. These findings highlight that downregulating UBE2F effectively inhibits the neddylation modification of CUL5.

To assess the impact of UBE2F downregulation on lung cancer metastasis, a mouse lung metastasis model was established through a 100 μL injection of A549 cells at a concentration of 2 × 10⁶ per mL via the tail vein. The previously constructed UBE2F knockout A549 cell line was employed in the process. Prior to tail vein injection, successful UBE2F knockout in A549 cells was confirmed (Fig. 1A). Subsequently, following a 40-day observation period, mouse lung tissues were harvested, and metastatic nodules on the lung tissue surface were quantified. We found that the numerous metastatic nodules in the lung of UBE2F knockout group were significantly more than those from control group (Fig. 1B). The results underscore that UBE2F deficiency significantly promotes lung cancer metastasis compared to the control group, indicating that UBE2F plays a crucial role in regulating lung cancer metastasis.

3.2 UBE2F deficiency promotes migration and invasion of lung cancer cells

To examine the impact of UBE2F downregulation on the migration and invasion of lung cancer cells, we conducted in vitro scratch assays and transwell migration assays. We found that UBE2F knockout significantly increased the migration capability of lung cancer cells compared to the control group (Figs. 2A and 2B). Consistently, the transwell invasion and migration analysis revealed a significant increase in the invasion and migration capabilities of lung adenocarcinoma cells upon UBE2F downregulation (Figs. 2C-2F). Together, these results suggest that UBE2F deficiency promotes migration and invasion of lung cancer cells.

3.3 UBE2F deficiency impedes Snail protein degradation

Epithelial-mesenchymal transition (EMT) has been closely associated with malignant phenotypes during cancer progression and metastasis, including increased invasive and metastatic capabilities. EMT involves the transformation of epithelial cells into mesenchymal cells, marked by distinct epithelial and mesenchymal states[13] . Among the EMT transcription factors, Snail has been identified as a factor that represses E-cadherin transcription by binding to the E-box element of its promoter, ultimately leading to the downregulation of E-cadherin expression and the promotion of tumor metastasis[13-15]. In this investigation, we utilized UBE2F-knockout lung cancer cells to analyze the proteins involved in the EMT process. Compared to the control group, UBE2F knockout resulted in an upregulation of the EMT transcription factor Snail protein and a downregulation of E-cadherin protein expression, with minimal changes observed in the expression of other transcription factors such as Slug and Zeb (Fig. 3A). These findings suggest that the crucial role of UBE2F in lung cancer cells invasion and metastasis may be linked to its promotion of E-cadherin via inhibiting Snail expression.

Subsequently, we examined whether UBE2F regulates Snail protein stability. Lung cancer cells were treated with the protein synthesis inhibitor CHX over various time intervals. Notably, UBE2F knockout elevated the basal levels of endogenous Snail and extended the half-life of Snail protein compared to control cells (Fig. 3B). Our results affirm that UBE2F deficiency upregulates Snail protein levels by limiting Snail degradation. In summary, UBE2F deficiency appears to promote lung cancer invasion and metastasis, potentially through its role in inhibiting the degradation of Snail protein.

Here we performed stable human lung adenocarcinoma cell lines A549 with UBE2F knockout using the CRISPR/Cas9 system, and established a lung metastasis mice model by injecting cells into the tail vein. Our results suggested that UBE2F deficiency substantially promoted lung cancer metastasis by stabilizing the EMT transcription factor Snail (Fig. 4). Our study not only uncovers mechanisms underlying lung cancer metastasis but also provides a potential therapeutic strategy for the treatment of metastatic lung cancer.

Neddylation is the reversible covalent conjugation of the ubiquitin-like molecule NEDD to lysine residues on substrate proteins. Recent studies have reported the overexpression of NEDD8 catalytic enzymes (NAE, UBE2M, UBE2F, among others) in various human cancers, implying that key catalytic enzymes participating in neddylation may promote cancer development[11, 19]. In mammalian cells, there are two NEDD8-conjugating enzyme E2 proteins, UBE2M and UBE2F, each with different biological functions. UBE2F is responsible for mediating the neddylation modification of CUL5, while UBE2M mediates the same modification for CUL1/2/3/4 [6]. Interestingly, previous studies have suggested that UBE2F-CUL5 complex may exhibit dual functions in lung cancer. On the one hand, UBE2F is recognized as an anti-apoptotic protein, and its high expression promotes the neddylation modification of CUL5. This activation of CUL5 results in the subsequent polyubiquitylation degradation of the pro-apoptotic protein NOXA, thereby promoting malignant transformation and the development of lung cancer[11, 20]. On the other hand, another study reported that deficiency of either CUL5 or SOCS3, two components of the CRL5-E3 complex, will substantially promote small-cell lung cancer metastasis by stabilizing integrin β1 and stimulating integrin β1 downstream signaling including FAK and SRC[12]. Our experimental results further support the notion that UBE2F within CRL5-E3 complex can inhibit lung cancer metastasis through inducing protein degradation of Snail, a critical factor in EMT. These findings suggest that UBE2F may serve a dual role in lung cancer, promoting tumor growth in the early stages of localized tumors while inhibiting EMT progression as the disease advances. UBE2F suppresses stromal invasion, intravasation, and the survival of circulating tumor cells, thereby inhibiting tumor metastasis. The presence of dual-function proteins in cancer research has been frequently reported and is recognized as a common phenomenon in cancer biology[21-25]. However, the mechanisms and reasons underlying the shift of UBE2F from a pro-cancer factor to an inhibitor of tumor metastasis during later stages of the disease warrant further investigation.

Data Availability Statement

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.

Ethics Statement

The animal study was reviewed and approved by Taizhou University's Institutional Animal Care and Use Committee.

Author Contributions

XZL, DSS, SHY, KC, WJM, LSZ conceived the overall framework for the study, conducted experiments and analyzed the data. XZL, DSS prepared the manuscript. LSZ, YL, BFC reviewed and edited the manuscript. All authors listed have made substantial contributions to this work and have approved its publication.

Funding

This work was supported by the Natural Science Foundation of Zhejiang Province (No. LY21H160008), the National Natural Science Foundation of China (Nos. 81871870, and 82073069), and Students Science and Technology Innovation Activity Plan of Zhejiang Province (known as New Seedling Talent Plan, No. 2023R474013）.

Conflict of Interest

The authors declare that there is no potential conflict of interest in this study.

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Editorial decision: Reject
08 Nov, 2023
Reviewers agreed at journal
13 Oct, 2023
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Editor assigned by journal
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You are reading this latest preprint version

NEDD8-conjugating enzyme E2 UBE2F deficiency promotes lung cancer metastasis by stabilizing Snail

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Reagents and Antibodies

2.2 Cell Culture

2.4 Protein Expression by Western Blot

2.5 Animal Experiment

2.6 Cell Scratch Assay

2.7 Transwell Migration and Invasion Assays

2.8 Statistical Methods

3. Results

4. Discussion

Declarations

References

Status:

Version 1