TRIM58 Interacts with ZEB1 to Suppress NSCLC Tumor Malignancy by Promoting ZEB1 Protein Degradation via UPP

Background Currently, how to successfully control refractory and metastatic diseases remains a fundamental goal for clinicians to improve therapeutic effects for patients with non-small cell lung cancer (NSCLC). Several studies have discovered that TRIM58, a member of tripartite motif protein family, shows antitumor effect in multiple types of cancer. In this study, we aimed to further clarify the molecular regulatory network of TRIM58 and corresponding targets for NSCLC patients. Methods TRIM58 expression in clinical tumor tissue samples and cancer cell lines was examined. Functional experiments including cellular invasion, cell metastasis, chemoresistance assay, and ubiquitination evaluation experiments were conducted to investigate the interaction between TRIM58 and ZEB1, which is a prime element of transcription factor network that controls epithelial-to-mesenchymal transition. Results TRIM58 expression was characteristically decreased in NSCLC tumor tissues and cancer cell lines. Functional experiments demonstrated that TRIM58 suppression enhanced malignant biological behaviors including cellular survivability, migration, and invasion, as well as stem-like cellular phenotype of tumor cells. TRIM58 silencing also significantly enhanced the chemoresistance of NSCLC cells to chemoagents. TRIM58-ZEB1 interaction accelerated degradation of ZEB1 protein, thus further leading to the augment of tumor behaviors. Further detailed molecular experiments revealed that the interaction between TRIM58 and ZEB1 was mediated via ubiquitin-proteasome pathway (UPP). Conclusion TRIM58 suppressed NSCLC through interacting with ZEB1 and promoting ZEB1 protein degradation via UPP. The present research sheds light on the interaction between TRIM58 and ZEB1, and TRIM58/ZEB1 axis might be the potential therapeutic targets of NSCLC.


Introduction
Lung cancer remains as one of the most deadly malignant diseases globally [1]. Among all lung cancer types, nonsmall cell lung cancer (NSCLC) occupies a major portion of patient numbers. As compared with other NSCLC subtypes, lung adenocarcinoma usually leads to worse prognosis [2]. Up till now, major progress has been achieved in lung cancer treatment, and novel advances in immunochemotherapy combined with surgical operations have enhanced the general prognosis of lung cancer patients. However, refractory and metastatic diseases are still imminent threats for lung cancer patients. For instance, nearly 20% of advanced NSCLC patients have reduced overall survival due to refractoriness of lung cancer disease [3]. Consequently, research should pay more attention to the underlying mechanism of lung cancer refractoriness and metastasis so as to further improve the efficacy of antitumor treatments.
Epithelial-mesenchymal transition (EMT) is a critical process in the development of tumor cellular invasion that converts polarized immotile epithelial cells to migratory mesenchymal cells. EMT has been intensively involved in tumorigenesis, progression, metastasis, and treatment refractoriness [4][5][6].
Researches utilizing immunodeficient mice model and lineage tracing techniques indicate that EMT is involved in tumor progression, which facilitates the capability of tumor cell self-renewal and differentiation into other types of tumor cells [7][8][9].
E-box binding zinc finger protein 1 (ZEB1) is a member of ZEB homeobox transcription factor family, located on human chromosome 10. ZEB1 is also a key transcription regulator involved in tumor progression [10]. It is abnormally expressed in many cancers and can promote migration, invasion, and metastasis of tumor cells. The enhancement of tumor cell plasticity is an important driving force for the progress of malignant tumors, and ZEB1 is the key factor mediating cell plasticity [11]. ZEB1 could promote tumor cell migration, invasion, and metastasis by changing cell plasticity and mediating EMT mechanism. It was reported that hypoxia-induced ZEB1 promoted cervical cancer progression via CCL8dependent tumor-associated macrophage recruitment [12]. USP18 could promote tumor metastasis in esophageal squamous cell carcinomas via deubiquitinating ZEB1 [13]. The regulatory role of ZEB1 in NSCLC tumor malignancy has not been fully reported.
The ubiquitin-proteasome regulatory system controls protein homeostasis of almost the entire proteome [14][15][16]. Ubiquitin-mediated proteolysis primarily consists E1 activating enzymes, E2 conjugating enzymes, and E3 ubiquitin ligases. Accumulating evidences have found that E3 ligases of both the RING and the HECT family regulate EMT process via ubiquitination, including βTrCP, Skp2, Fbw7, and Cullin 7/Fbxw8 [17][18][19][20][21]. Tripartite motif proteins (TRIMs) belong to the family of E3 ubiquitin ligases. TRIM58, a member of TRIM family, containing a RING motif, a B box type 1 and 2, and a coiled-coil domain in its N-terminal region, has been identified as an E3 ubiquitin ligase and considered as an important regulator in several types of cancers [22]. However, the role of TRIM58 in NSCLC metastasis and progression, especially in cancer cellular EMT process has not been clearly elucidated. Therefore, in this study, we aimed to investigate the role and mechanism of TRIM58 in NSCLC disease progression and invasion. This research might provide novel clues in the management of metastatic disease in NSLCL treatment.

Patient and Sample
Collection. Eligible NSCLC patients diagnosed in our facility from 2019/6/1 to 2020/6/1 were enrolled in this research. Patients with previous surgical treatment, radiotherapy, or chemotherapy were excluded. Tumor tissues and matched normal lung tissues were collected. Samples were stored in liquid nitrogen. Declaration of Helsinki was followed throughout experiments involving humans. The patients' informed consents were obtained. The conduct of this study was approved by the ethical reviewing committee of our institution. We followed the methods of Guo et al. [ 2.4. Vector Construction and Cell Transfection. pLVX lentiviral vector was acquired as vectors for this research (Clontech, USA). Polymerase chain reaction (PCR) was performed using sequence templates of TRIM58 shRNA and TRIM58 and ZEB1 full-length cDNA sequences. Firstly, purification of PCR products were performed by 5% agarose gel, then products were treated by BamHI and EcoRI as well as empty pLVX vectors. And subsequent ligation was conducted using T4DNA ligase. Then, products were used for the E.coli DH5α-competent cells transformation. Afterwards, LB plate with ampicillin was utilized for culture overnight at 37°C. Vectors from positive clones were collected and sequenced (Invitrogen Biotech Co., Ltd). Then, all vectors were packaged and added into each treatment group with multiplicity of infection (MOI) of 15 and vectors titer was set as 1 × 10 8 /mL.
2.6. Cellular Migration and Invasion Assay. Sample cells were inoculated with density of 5 × 10 4 in the upper chamber (Corning, USA). Cells were incubated for 36 h with environmental parameter of 37°C, 5% CO 2 , and lower chamber were then fixed and stained for subsequent observation and quantification. A total of 12 randomly-chose fields per sample were selected for further quantification. The invasion experiment was performed with the same way except that the filters of the transwell chambers were coated with 30 mg matrigel (#4-ME-354262, BD Biosciences, USA). , and then paraformaldehyde was applied to fix cell samples for 20 min. Then, samples were permeabilized using 0.5% NP-40 for 30 min, and were blocked by 10% bovine serum albumin for 20 min. Afterwards, cells were treated with primary antibody for 3 h in room temperature, and were treated with fluorescein conjugated secondary antibody for another 2 h. Finally, DAPI and confocal microscopy were utilized to stain and perform imaging analysis on the slides, respectively. The antibodies applied in this research were listed as follows: anti-TRIM58 antibody (ab254768, Abcam, UK) and anti-ZEB1 antibody (ab203829, Abcam, UK).
2.11. CHX-Chase Assay. CHX-chase assay was performed using CHX (Selleck Chemicals). Cells were treated with 12.5 μg/mL of CHX and the expression of ZEB1 and TRIM58 protein was measured via western blot at 0, 2, 4, and 8 h, respectively. The animals were divided randomly into two groups (n = 5 for each group). The mice in the group TRIM58 were injected with 5 × 10 6 NCI-H441 cells transfected with TRIM58 overexpression vectors, the animals in the group vector were injected with 5 × 10 6 NCI-H441 cells transfected with negative control vectors. Tumor cells were injected into the left armpit of mice. The tumor size was measured with vernier caliper, and the weight of mice was weighed. The drinking water, diet, mental state, and physical condition of the mice were observed. If the mice are found to have limping, extreme malaise, or cachexia, the mice shall be killed by decapitation and recorded. After 21 days, the animals were sacrificed, and the tumor of the mice was weighed.

Ubiquitination
2.14. Statistical Analysis. Statistical analysis was performed using SPSS (Version 21.0, IBM, USA). Results were shown as the mean value + SD with 3 independent experiments. Statistical analysis between two groups was performed by the Student t-test. ANOVA was used to analyze data among more than two groups. Pearson's test was performed to analyze gene correlation. P < 0:05 indicates statistical significance.

Significant TRIM58 Downregulation in NSCLC Tissues
and Cell Lines. Firstly, we examined the protein expression of TRIM58 via IHC method in surgical samples of NSCLC tumor and matched adjacent normal tissues. As shown in Figure 1(a), TRIM58 was notably suppressed in cancer tissue, as compared with adjacent normal lung tissue samples. The average histochemistry score (H-Score) of NSCLC tumors ( Figure 1(b)) was significantly lower than that of normal lung tissue (P < 0:001). Western blot assay in four paired-tumor and normal samples also supported the above finding (Figure 1(c)). Real-time PCR quantification suggested that mRNA expression of TRIM58 was significantly reduced in tumor tissue, compared with normal counterparts (Figures 1(d) and 1(e)).
To investigate the association of TRIM58 expression with the overall survival of NSCLC patients, log-rank test on overall survival differences between TRIM58 low-and high-expression patient groups was analyzed through GEPIA database. As shown in Figure 1(f), patients with high level of TRIM58 expression presented notably better overall survival period in comparison with patients with low TRIM58 expression level (P = 0:00021).
To validate the above findings, we further investigated TRIM58 mRNA and protein levels in NSCLC cell lines and normal human lung epithelial cell line. The results also suggested that NSCLC cell lines exhibited lower mRNA and protein level compared with normal lung epithelial cell line (BEAS-2B) (Figures 1(g) and 1(h)).

TRIM58 Suppression Was Correlated with Boosted
Malignant Biological Behavior of NSCLC Cell Lines. To investigate the role of TRIM58 in NSCLC cells, we performed expression modulation via TRIM58 shRNAs and overexpression vectors utilizing NCI-H1395 and NCI-H441 as cell models, respectively. The effects of shRNAs and overexpres-sion vectors on TRIM58 level were validated via western blot and RT-PCR assay (Figures 2(a) and 2(b)). Next, we performed cellular migration and invasion assays on these two models. Wound-healing assay results suggested that TRIM58 overexpression significantly reduced cellular migration (Figure 2(c)), and TRIM58 shRNA treated tumor cells   (Figure 2(d)). Consistently, transwell assay confirmed that tumor cells with promoted TRIM58 expression demonstrated suppressed cellular mobility and invasiveness (Figure 2(e)), and vice versa (Figure 2(f)). Further molecular study indicated that the proinvasive tendency of tumor cells with TRIM58 suppression was probably related with mesenchymal phenotype transition of tumor cells, as western blot/qRT-PCR study both indicated that mesenchymal cellular markers (N-Cadherin and Vimentin) were significantly increased in TRIM58 silencing cell groups, while epithelial phenotypic marker (E-Cadherin) was considerably suppressed (Figures 2(g) and 2(h)). In addition, transfection with overexpression vector of TRIM58 markedly suppressed tumor growth in vivo (Figure 2(i)).

Association of TRIM58 with NSCLC Chemoresistance
and Stem-Like Cellular Phenotype. We evaluated the tumor cell chemoresistance of NSCLC by examining the survival rates of tumor cells after treated with chemoagents including docetaxel, doxorubicin, and gefitinib. TRIM58 silencing via sh1, sh2 (Figures 3(a)-3(c)), and control vector (Figures 3(d)-3(f)) could significantly enhance the chemoresistance of NSCLC cells to the three chemoagents (P < 0:001 for all comparisons). Western blot and RT-PCR experiments also suggested that TRIM58 silencing tend to increase the stem-like cellular phenotype of these tumor cells, as both protein and mRNA expression of stem-cell marker ALDH1 and CD44 demonstrated notably increase in tumor cells treated by TRIM58 shRNAs (Figures 3(g) and 3(h)).    Figure 4(a), R = −0:683, P < 0:001), while no significant correlation was detected between mRNA expression of TRIM58 and ZEB1 (Figure 4(b), P > 0:05). Subsequently, TRIM58 modulating experiments also suggested that TRIM58 overexpression or silencing showed no impact on ZEB1     (Figure 4(c)). The above evidence suggested that posttranscriptional regulatory effects might be accounted for the impact of TRIM58 on ZEB1. Therefore, autophagy inhibitor (CQ) and proteasome inhibitors (MG132) were applied to investigate autophagy and ubiquitin-proteasome pathway (UPP) in ZEB1 modulation. As shown in Figure 4 10 Disease Markers MG132 treated group. In addition, by transfection of FLAG-TRIM58 vector, we found using HEK-293 T as model that ZEB1 protein expression was inhibited in dose-dependent manner (Figure 4(e)). Subsequent coimmunoprecipitation assay and immunofluorescence colocalization assay also demonstrated clues of TRIM58/ZEB1 protein-protein interaction (Figures 4(f)-4(h)).

Degradation of ZEB1 Was Promoted by TRIM58
Expression. Next, we performed cycloheximide (CHX) chase assay to evaluate protein degradation velocity in tumor cells treated with or without TRIM58 overexpression vectors. As

Discussion
In this article, we demonstrated that TRIM58 suppression was a characteristic in lung cancer cells. And, further detailed experiments depicted novel mechanism that TRIM58 exhibited tumor inhibitory effects via interaction with ZEB1. Through

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Disease Markers binding with ZEB1 protein, TRIM58 enhanced ZEB1 protein degradation via ubiquitin-proteasome pathway so that TRIM58 was capable of minimizing ZEB1-mediated promotion of tumor migrated and survival capability. As reported in previous studies [24,25], TRIM58 downregulation was associated with inferior prognosis and promoted malignancy in several tumors including gastric cancer, colorectal cancer, and lung cancer. As for the functions of TRIM58 in cancer pathogenesis and progression, previous studies have been suggested that TRIM58 takes part in the modulation of tumor microenvironment. As TRIM58 expression was positively correlated with M2-polarized macrophage and mast cells in KRAS-driven lung adenocarcinoma tumor tissues [26], which suggested that TRIM58 might facilitate the infiltration of immune cells in tumor microenvironment and enhance antitumor immune reaction. As a member of tumor suppressor genes (TSGs), previous research indicated that even in early stage of lung adenocarcinoma (LADC), aberrant promoter CpG island methylation resulted in robust expression suppression of TRIM58, which stimulated early carcinogenesis of LADC. Thus, it explained the potential mechanism of suppressed expression of TRIM58 in lung cancer, and such discovery also indicated that targeted aberrant TRIM58 methylation could be novel therapeutic target in future lung cancer treatment development.
ZEB1 protein consists of several protein binding domains including SMAD and p300 protein interaction domains. Researches have demonstrated that ZEB1 tightly control EMT process via transcriptional regulation, a series of genes involved in this process. Detailed study indicated that ZEB1 could inhibit cell polarity factors and basement membrane synthesis, while activate matrix metalloproteases expression including MMP-1, MMP-9, and MMP-14, which could enhance the remodeling of the basement membrane and facilitate tumor invasion into surrounding tissues [27]. And both in vitro and in vivo models have confirmed that overexpression of ZEB1 promoted invasive capability of multiple kinds of tumor cells. Moreover, beyond regulatory role of EMT, ZEB1 also participates in critical cellular func-tions which exerts great impact in tumorigenesis and tumor expansion. For example, Zeb1 knockout resulted in reduction of acinar ductal metaplasia and noninvasive pancreatic lesion [28], which supported the theory that ZEB1 was involved in early steps of pancreatic tumorigenesis. Our study provided novel theory of ZEB1 expression modulation by TRIM58 via UPP (Figure 7), indicating TRIM58triggered ZEB1 depletion via UPP might also serve as a potential treatment strategy in NSCLC patients.
Indeed, the limitation of the in vitro model as well as retrospectively collected clinical samples utilized in our study warrant future consolidation experiments based on TRIM58-ZEB1 axis knockout animal models and clinical evidences based on multicentered perspective clinical trials. However, needless to say, the novel interaction between TRIM58 and ZEB1 may shed new light in NSCLC study and inspire future exploration in the potential significance in NSCLC patient treatment.

Conclusion
In this study, we discovered that TRIM58 was significantly suppressed in NSCLC clinical samples and tumor cell models. And, TRIM58 interacted with ZEB1 to facilitate ZEB1 protein degradation via UPP, which resulted in the exacerbation of NSCLC tumor expansion, invasion, and metastasis. Our study proposed for the first time that TRIM58-ZEB1 interaction might serve as potential target for future NSCLC treatment development.

Data Availability
The data support this study could be requested from corresponding author.

Ethical Approval
All experiment protocols have been approved by the ethics committee of Air Force Medical University.

Consent
Written informed consents have been obtained from the patients for publication of this report and accompanying images. A copy of the written consents is available for review by the Editor-in-Chief of this journal on request.

Conflicts of Interest
The authors declare that there is no conflict of interest.  Figure 7: Schematic diagram of potential molecular mechanism of tumor inhibitory effects of TRIM58. TRIM58 interacted with ZEB1 and promoted ZEB1 protein degradation via UPP. Decreased ZEB1 promoted tumor cellular malignant biological behavior including cellular proliferation, migration, invasion, and chemoresistance.

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Disease Markers