RFWD2 Knockdown as a Blocker to Reverse the Oncogenic Role of TRIB2 in Lung Adenocarcinoma

Abstract

RFWD2, an E3 ubiquitin ligase, is overexpressed in numerous human cancers, including leukemia, lung cancer, breast cancer, renal cell carcinoma, and colorectal cancer. The roles of RFWD2 in cancer are related to the targeting of its substrates for ubiquitination and degradation. This study aimed to investigate the role of TRIB2 in relation to the regulation of protein degradation through RFWD2. inBio Discover™ results demonstrated that TRIB2 can perform its functions by interacting with RFWD2 or other factors. TRIB2 can interact with and regulate RFWD2, which further attends the proteasome-mediated degradation of the RFWD2 substrate p-IκB-α. TRIB2 colocalizes with RFWD2-related IκB-α to form a ternary complex and further affects the IκB-α degradation by regulating its phosphorylation. Specific domain analysis showed that TRIB2 may bind to RFWD2 via its C-terminus, whereas it binds to IκB via its pseudokinase domain. TRIB2 acts as an oncogene and promotes cancer cell proliferation and migration, whereas RFWD2 knockdown reversed the role of TRIB2 in promoting cancer cell growth and colony formation in vitro and in vivo. In summary, this study reveals that TRIB2 promotes the progression of cancer by affecting the proteasome-mediated degradation of proteins through the interaction with RFWD2.

WFDC21P promotes triple-negative breast cancer proliferation and migration through WFDC21P/miR-628/SMAD3 axis

“…Immunoblotting was performed as described previously ( 17 – 19 ). Protein was extracted from SUM159 cells using 1× RIPA buffer (P0013B, Beyotime, Shanghai, China).…”

IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8+ T, and CD4+ T Cells

“…3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay Cells proliferation was analyzed by MTT assay following the previous reports. 24,25 In brief, cells (1 × 10 4 ) were cultured in each well of a 96-well flat bottom microtiter plates. Four hours before the end of incubation, each well was added with 10 μL MTT (Sigma, St Louis, MO, USA, 5 mg/mL).…”

“…Western blot analysis was performed as in previous reports. 24,25 Membranes were incubated with rabbit anti-mouse IL-12 (1:500, LSBio, Seattle WA, USA) and β-actin (1:3000, Bioworld Technology, Ltd., Nanjing, China) in tris buffered saline with Tween-20 (TBST) at 4°C overnight. HRP-labeled goat anti-rabbit IgG (1:6000, Zhong Shan-Golden Bridge Technology Co., Ltd., Beijing, China) was added for 1 h. Immunoblotting was incubated with enhanced chemiluminescence (ECL) (Boster Immunoleader, Wuhan, China).…”

“…Immunofluorescence analysis of calreticulin levels was performed as described in a previous study. 25 Cells were prepared and incubated with rabbit anti-calreticulin antibody (1:200, Abcam, San Francisco, CA, USA) overnight at 4 °C, and then with Alexa Fluor 488 donkey anti-rabbit IgG (H+L, Molecular Probes, Eugene, OR, USA) at 37 °C for 1 h. The results were observed under a confocal microscope (LEICA TCS SPE, Leica, Dresden, Germany).…”

Advances of E3 ligases in lung cancer

“…COP1, also called RFWD2, decreases expression of c-Jun via ubiquitination, thus increasing histone H3 acetylation of PD-L1 promoter, which improves proliferation of T cells in cisplatin-resistant NSCLC [ 89 ]. In contrast, COP1 ubiquitinates and induces the degradation of p -IκBα, p53, signal transducer and activator of transcription 3 (STAT3), β-catenin, p27 and C/EBPα in the proteasome-mediated pathway, which promotes proliferation and migration but inhibits apoptosis in NSCLC [ 90 ]. SCF-Skp2 is an E3 ligase complex belonging to CRLs and can induce the degradation of p27 and E-cadherin through ubiquitination, which will upregulate proliferation and migration in NSCLC cells.…”