TRIM65 negatively regulates p53 through ubiquitination

doi:10.1016/j.bbrc.2016.03.093

Biochemical and Biophysical Research Communications

Volume 473, Issue 1, 22 April 2016, Pages 278-282

https://doi.org/10.1016/j.bbrc.2016.03.093 Get rights and content

Highlights

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TRIM65 expression is elevated in NSCLC.
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TRIM65 inactivates p53 through mediating p53 ubiquitination and degradation.
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TRIM65 attenuates the response of NSCLC cells to cisplatin.

Abstract

Tripartite-motif protein family member 65 (TRIM65) is an important protein involved in white matter lesion. However, the role of TRIM65 in human cancer remains less understood. Through the Cancer Genome Atlas (TCGA) gene alteration database, we found that TRIM65 is upregulated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Our cell growth assay revealed that TRIM65 overexpression promotes cell proliferation, while knockdown of TRIM65 displays opposite effect. Mechanistically, TRIM65 binds to p53, one of the most critical tumor suppressors, and serves as an E3 ligase toward p53. Consequently, TRIM65 inactivates p53 through facilitating p53 poly-ubiquitination and proteasome-mediated degradation. Notably, chemotherapeutic reagent cisplatin induction of p53 is markedly attenuated in response to ectopic expression of TRIM65. Cell growth inhibition by TRIM65 knockdown is more significant in p53 positive H460 than p53 negative H1299 cells, and knockdown of p53 in H460 cells also shows compromised cell growth inhibition by TRIM65 knockdown, indicating that p53 is required, at least in part, for TRIM65 function. Our findings demonstrate TRIM65 as a potential oncogenic protein, highly likely through p53 inactivation, and provide insight into development of novel approaches targeting TRIM65 for NSCLC treatment, and also overcoming chemotherapy resistance.

Introduction

Tripartite-motif protein family members (TRIM) have been proposed to involve in various human diseases, such as developmental disorders, viral infections and cancer [1], [2], [3], [4]. Interestingly, most of TRIM proteins contain a RING-finger domain, which is a critical functional domain for ubiquitin E3 ligase, and therefore they are defined as a class of E3 ligases [5]. As ubiquitination is an important post-translational modification of proteins, it has drawn increasing attention to characterize the function of TRIM family members and their role in human diseases. For example, as a p53 binding protein identified by mass spectrometry of tandem-affinity-purification (TAP)-purified p53 complex, TRIM24 was reported to negatively regulate p53 through its E3 ligase activity, and therefore is a potential therapeutic target to restore the tumor suppressor role of p53 [6]. However, more information about the TRIM family members still remains largely unknown, especially in human cancers.

In the present study, by analyzing the Cancer Genome Atlas (TCGA) gene alteration database available in cBioportal [7], [8], we found that TRIM65 expression is elevated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Although TRIM65 has been reported as a gene associated with white matter lesions [9], [10], [11], and one of its E3 ligase substrates is trinucleotide repeat containing six (TNRC6) involved in microRNA pathway, little is known about the role of TRIM65 in cancer, particularly in NSCLC. We for the first time showed that TRIM65 could negatively regulate p53 through mediating p53 ubiquitination and proteasomal degradation in NSCLC cell lines, and therefore is a potential oncogenic protein.

Section snippets

Cell lines and reagents

H460, A549 and H1299 cell lines were purchased from American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen), at 37 °C in the presence of 5% CO₂. Cisplatin was purchased from Sigma (St. Louis, MO) and treatment was performed as previously reported [12]. The antibodies used for Western Blotting were from Santa Cruz (anti-p53, DO-1;

TRIM65 is upregulated in non-small cell lung carcinoma (NSCLC)

TRIM65 has been described as a gene associated with white matter lesion [9], [10], [11]. Functional studies have shown that TRIM65 possesses ubiquitin E3 ligase activity with trinucleotide repeat containing six (TNRC6) as its substrate and therefore acts as a cofactor for regulating microRNA pathway [14], [15]. However, little is known about the role of TRIM65 in cancer. We analyzed the Cancer Genome Atlas (TCGA) gene alteration database, particularly in NSCLC, available in cBioportal [7], [8],

Discussion

TP53 has been well accepted as one of the most important tumor suppressor genes, as evidenced by the fact that over 50% of human cancers have p53 mutations that lead to inactivation of this gene [16]. On the other hand, alterations of other cellular events, such as virus infection [17], [18], MDM2 amplification [19], [20], and AKT activation [21] et al., have been demonstrated to suppress wild-type p53 functions. As the research of p53 regulation expands, more proteins have been identified to

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

This study was supported by the following funds: Jilin Province Development and Reform Commission to (2015Y036-3); National Natural Science Foundation of China (81401883).

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TRIM65 knockout inhibits the development of HCC by polarization tumor-associated macrophages towards M1 phenotype via JAK1/STAT1 signaling pathway
2024, International Immunopharmacology
Tumor-associated macrophages (TAMs) are main components of immune cells in tumor microenvironment (TME), and play a crucial role in tumor progression. Tripartite motif-containing protein 65 (TRIM65) has been associated with tumor progression. However, whether TRIM65 regulate the interaction of tumor cell and TAMs in HCC and the underlying mechanisms remain unknown. In this study, we investigated the role of TRIM65 in TME of HCC and explored its underlying mechanisms.
The relation of TRIM65 expression level with tumor grades, TNM stages, and worse prognosis of HCC patients was evaluated by bioinformatics analysis, as well as immune infiltration level of macrophages. TRIM65 shRNA was transfected into HepG2 cells, and TRIM65 overexpression plasmid was transfected into Huh7 cells, and the effect of TRIM65 on cell growth was examined by EdU assay. The mouse subcutaneous Hep1-6 tumor-bearing model with WT and TRIM65^-/- mice was established to study the role of TRIM65 in HCC. Immunohistochemistry staining, Immunofluorescence staining, qRT-PCR and western blot were performed to evaluate the effect of TRIM65 on TAM infiltration, TAM polarization and JAK1/STAT1 signaling pathway.
Bioinformatics analysis revealed that TRIM65 was upregulated in 16 types of cancer especially in HCC, and high level of TRIM65 was strongly correlated with higher tumor grades, TNM stages, and worse prognosis of patients with HCC as well as immune infiltration level of macrophages (M0, M1, and M2). Moreover, we observed that TRIM65 shRNA-mediated TRIM65 knockdown significantly inhibited the HepG2 cells growth while TRIM65 overexpression highly increased the Huh7 cells growth in vitro. TRIM65 knockout significantly inhibited the tumor growth as well as macrophages polarization towards M2 but promoted macrophages polarization towards M1 in vivo. Mechanistically, the results demonstrate that TRIM65 knockout promoted macrophage M1 polarization in conditioned medium-stimulated peritoneal macrophages and in tumor tissues by activating JAK1/STAT1 signaling pathway.
Taken together, our study suggests that tumor cells utilize TRIM65-JAK1/STAT1 axis to inhibit macrophage M1 polarization and promote tumor growth, reveals the role of TRIM65 in TAM-targeting tumor immunotherapy.
Intricate confrontation: Research progress and application potential of TRIM family proteins in tumor immune escape
2023, Journal of Advanced Research
Tripartite motif (TRIM) family proteins have more than 80 members and are widely found in various eukaryotic cells. Most TRIM family proteins participate in the ubiquitin-proteasome degradation system as E3-ubiquitin ligases; therefore, they play pivotal regulatory roles in the occurrence and development of tumors, including tumor immune escape. Due to the diversity of functional domains of TRIM family proteins, they can extensively participate in multiple signaling pathways of tumor immune escape through different substrates. In current research and clinical contexts, immune escape has become an urgent problem. The extensive participation of TRIM family proteins in curing tumors or preventing postoperative recurrence and metastasis makes them promising targets.
The aim of the review is to make up for the gap in the current research on TRIM family proteins and tumor immune escape and propose future development directions according to the current progress and problems.
This up-to-date review summarizes the characteristics and biological functions of TRIM family proteins, discusses the mechanisms of TRIM family proteins involved in tumor immune escape, and highlights the specific mechanism from the level of structure-function-molecule-pathway-phenotype, including mechanisms at the level of protein domains and functions, at the level of molecules and signaling pathways, and at the level of cells and microenvironments. We also discuss the application potential of TRIM family proteins in tumor immunotherapy, such as possible treatment strategies for combination targeting TRIM family protein drugs and checkpoint inhibitors for improving cancer treatment.
Knockdown of TRIM65 suppressed the proliferation and invasiveness of gastric cancer cells by restricting the ubiquitin degradation of PPM1A
2022, Experimental Cell Research
Gastric cancer is a type of serious malignant tumors all around the world. TCGA data showed that the expression of TRIM65 (E3 ubiquitin ligase) was enhanced in the gastric cancer tissues. The role of TRIM65 in the tumorigenesis of gastric cancer remains unclear. In this study, we successfully established TRIM65-knockdown gastric cancer cells. Next, CCK-8, colony formation assays and transwell assays were performed to detect the cell proliferation and invasion. The results showed that suppression of TRIM65 inhibited the proliferation and invasion of gastric cancer cells. Interestingly, the Western blot assay confirmed that downregulation of TRIM65 increased the level of PPM1A and decreased the level of p-TBK1 in gastric cancer cells. Mechanistically, immunoprecipitation assay revealed that knockdown of TRIM65 inhibited the ubiquitin degradation of PPM1A. In rescue experiments, suppression of PPM1A promoted the proliferation and invasion of gastric cancer cells transfected with sh-TRIM65. Therefore, our results suggested that knockdown of TRIM65 inhibited the proliferation and invasion of gastric cancer cells by suppressing the ubiquitin degradation of PPM1A and phosphorylation of TBK1.
The translational values of TRIM family in pan-cancers: From functions and mechanisms to clinics
2021, Pharmacology and Therapeutics
Cancer is the second leading cause of human death across the world. Tripartite motif (TRIM) family, with E3 ubiquitin ligase activities in majority of its members, is reported to be involved in multiple cellular processes and signaling pathways. TRIM proteins have critical effects in the regulation of biological behaviors of cancer cells. Here, we discussed the current understanding of the molecular mechanism of TRIM proteins regulation of cancer cells. We also comprehensively reviewed published studies on TRIM family members as oncogenes or tumor suppressors in the oncogenesis, development, and progression of a variety of types of human cancers. Finally, we highlighted that certain TRIM family members are potential molecular biomarkers for cancer diagnosis and prognosis, and potential therapeutic targets.
Regulation of p53 stability as a therapeutic strategy for cancer
2021, Biochemical Pharmacology
Citation Excerpt :
Studies have shown that TRIM65 promotes the ubiquitination and degradation of p53 by binding to p53 and plays the role of an E3 ligase [100]. Compared with the effect on p53-negative NCI-H460 cells, the effect of TRIM65 knockdown on the viability of p53-positive NCI-H460 cells was significantly greater, which may indicate the internal mechanism of NSCLC development and explain chemotherapy resistance to some extent [100]. LIN-41 (also known as TRIM71), a member of the TRIM family, can facilitate stem cell differentiation.
The tumor suppressor protein p53 participates in the control of key biological functions such as cell death, metabolic homeostasis and immune function, which are closely related to various diseases such as tumors, metabolic disorders, infection and neurodegeneration. The p53 gene is also mutated in approximately 50% of human cancer cells. Mutant p53 proteins escape from the ubiquitination-dependent degradation, gain oncogenic function and promote the carcinogenesis, malignant progression, metastasis and chemoresistance. Therefore, the stability of both wild type and mutant p53 needs to be precisely regulated to maintain normal functions and targeting the p53 stability is one of the therapeutic strategies against cancer. Here, we focus on compound-induced degradation of p53 by both the ubiquitination-dependent proteasome and autophagy-lysosome degradation pathways. We also review other posttranslational modifications which control the stability of p53 and the biological functions involved in these processes. This review provides the current theoretical basis for the regulation of p53 abundance and its possible applications in different diseases.
TRIM proteins in lung cancer: Mechanisms, biomarkers and therapeutic targets
2021, Life Sciences
Citation Excerpt :
TRIM proteins are implicated in extensive cellular processes, and there is increasing evidence that TRIM family proteins participate in the development and progression of multiple tumor types. Many TRIM proteins play an important role in the development of lung cancer via effecting various signal pathways and are regarded as either tumor suppressors or oncogenes [25–55]. The involvement of TRIMs in the progression of lung cancer is summarized in Table 1.
The tripartite motif (TRIM) family is defined by the presence of a Really Interesting New Gene (RING) domain, one or two B-box motifs and a coiled-coil region. TRIM proteins play key roles in many biological processes, including innate immunity, tumorigenesis, cell differentiation and ontogenetic development. Alterations in TRIM gene and protein levels frequently emerge in a wide range of tumors and affect tumor progression. As canonical E3 ubiquitin ligases, TRIM proteins participate in ubiquitin-dependent proteolysis of prominent components of the p53, NF-κB and PI3K/AKT signaling pathways. The occurrence of ubiquitylation events induced by TRIM proteins sustains internal balance between tumor suppressive and tumor promoting genes. In this review, we summarized the diverse mechanism of TRIM proteins responsible for the most common malignancy, lung cancer. Furthermore, we also discussed recent progress in both the diagnosis and therapeutics of tumors contributed by TRIM proteins.

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TRIM65 negatively regulates p53 through ubiquitination

Highlights

Abstract

Introduction

Section snippets

Cell lines and reagents

TRIM65 is upregulated in non-small cell lung carcinoma (NSCLC)

Discussion

Conflicts of interest

Acknowledgments

J. Biol. Chem.

J. Neurol. Sci.

Exp. Gerontol.

Lancet

Cell

Curr. Opin. Genet. Dev.

TRIM family proteins and their emerging roles in innate immunity

Nat. Rev. Immunol.

TRIM proteins and cancer

Nat. Rev. Cancer

TRIM/RBCC, a novel class of 'single protein RING finger' E3 ubiquitin ligases

Bioessays

The tripartite motif family identifies cell compartments

EMBO J.

Trim24 targets endogenous p53 for degradation

Proc. Natl. Acad. Sci. U. S. A.