RNF152 Suppresses Fatty Acid Oxidation and Metastasis of Lung Adenocarcinoma by Inhibiting IRAK1-Mediated AKR1B10 Expression

Lung adenocarcinoma (LUAD) is a common subtype of primary lung cancer. Fatty acid oxidation plays a key role in LUAD development by providing energy for tumor cells. This study aimed to identify the role of ring finger protein 152 (RNF152) in LUAD. RNF152 was down-regulated in LUAD, and low RNF152 expression correlated with a poor prognosis in LUAD patients. RNF152 overexpression inhibited the proliferation and malignant phenotype of LUAD cells, whereas RNF152 knockdown exerted an opposite effect. Tumor cells overexpressing RNF152 showed less fatty acid oxidation compared with control cells, whereas RNF152 knockdown induced fatty acid uptake and oxidation. Further analysis revealed the binding reaction between RNF152 and interleukin-1 receptor-associated kinase 1 (IRAK1). RNF152 reduced the stability of IRAK1 in LUAD cells by promoting its ubiquitination. RNF152-overexpressed tumor cells exhibited a significantly lower level of Aldo-Keto reductase family 1 member 10 (AKR1B10), whereas up-regulation of IRAK1 restored the expression of AKR1B10 in RNF152-overexpressed cells. Furthermore, up-regulation of IRAK1 eliminated the antitumor effect of RNF152 in LUAD cells. Mouse xenograft models confirmed the inhibitory effect of RNF152 on the tumorigenesis and metastasis of LUAD. Taken together, RNF152 played a tumor suppressive role in LUAD by promoting IRAK1 ubiquitination and IRAK1-mediated down-regulation of AKR1B10, thereby reversing the malignant phenotype of LUAD. Lung adenocarcinoma (LUAD) is a common subtype of primary lung cancer. Fatty acid oxidation plays a key role in LUAD development by providing energy for tumor cells. This study aimed to identify the role of ring finger protein 152 (RNF152) in LUAD. RNF152 was down-regulated in LUAD, and low RNF152 expression correlated with a poor prognosis in LUAD patients. RNF152 overexpression inhibited the proliferation and malignant phenotype of LUAD cells, whereas RNF152 knockdown exerted an opposite effect. Tumor cells overexpressing RNF152 showed less fatty acid oxidation compared with control cells, whereas RNF152 knockdown induced fatty acid uptake and oxidation. Further analysis revealed the binding reaction between RNF152 and interleukin-1 receptor-associated kinase 1 (IRAK1). RNF152 reduced the stability of IRAK1 in LUAD cells by promoting its ubiquitination. RNF152-overexpressed tumor cells exhibited a significantly lower level of Aldo-Keto reductase family 1 member 10 (AKR1B10), whereas up-regulation of IRAK1 restored the expression of AKR1B10 in RNF152-overexpressed cells. Furthermore, up-regulation of IRAK1 eliminated the antitumor effect of RNF152 in LUAD cells. Mouse xenograft models confirmed the inhibitory effect of RNF152 on the tumorigenesis and metastasis of LUAD. Taken together, RNF152 played a tumor suppressive role in LUAD by promoting IRAK1 ubiquitination and IRAK1-mediated down-regulation of AKR1B10, thereby reversing the malignant phenotype of LUAD. Lung cancer (LC) is one of the most frequent malignancies that continues to be the leading cause of global cancer-related deaths in both women and men.1Thandra K.C. Barsouk A. Saginala K. Aluru J.S. Barsouk A. Epidemiology of lung cancer.Contemp Oncol (Pozn). 2021; 25: 45-52PubMed Google Scholar Despite knowledge gains in tumor pathophysiology and the development of targeted therapies, mortality from LC remains high in most countries.2Barta J.A. Powell C.A. Wisnivesky J.P. Global epidemiology of lung cancer.Ann Glob Health. 2019; 85: 8Crossref PubMed Scopus (780) Google Scholar In China, LC represents 21% of all cancer-related mortality which is projected to increase by 40% from 2015 to 2030.3Cao M. Chen W. Epidemiology of lung cancer in China.Thorac Cancer. 2019; 10: 3-7Crossref PubMed Scopus (292) Google Scholar Lung adenocarcinoma (LUAD) is a common histologic subtype of primary LC, accounting for 40% of all LC cases.4Myers D.J. Wallen J.M. Lung adenocarcinoma.in: StatPearls [Internet]. StatPearls Publishing, Treasure Island, FL2023https://www.ncbi.nlm.nih.gov/books/NBK519578Google Scholar Although great efforts have been made in smoking prevention, early diagnosis, and clinical management, there are still no significant overall changes in the 5-year survival of LUAD patients in the past decade.5Goldstraw P. Chansky K. Crowley J. Rami-Porta R. Asamura H. Eberhardt W.E. Nicholson A.G. Groome P. Mitchell A. Bolejack V. The IASLC Lung Cancer Staging Project: proposals for revision of the TNM stage groupings in the forthcoming (eighth) edition of the TNM Classification for lung cancer.J Thorac Oncol. 2016; 11: 39-51Abstract Full Text Full Text PDF PubMed Scopus (2752) Google Scholar Therefore, uncovering novel reliable and promising targets for LUAD is of great importance for improving the prognosis of patients with LUAD. Fatty acid oxidation (FAO), also known as ß-oxidation, is a mitochondrial aerobic process of breaking down fatty acids (FAs) to acetyl coenzyme A, which then enters the Krebs cycle to produce energy-carrier molecules, such as adenosine triphosphate.6Bastin J. Regulation of mitochondrial fatty acid β-oxidation in human: what can we learn from inborn fatty acid β-oxidation deficiencies?.Biochimie. 2014; 96: 113-120Crossref PubMed Google Scholar In tumorigenesis, de novo FA biosynthesis and exogenous FA uptake not only allow tumor cells to sustain rapid proliferation, but also provide them an important energy source under metabolic stress conditions.7Koundouros N. Poulogiannis G. Reprogramming of fatty acid metabolism in cancer.Br J Cancer. 2020; 122: 4-22Crossref PubMed Scopus (633) Google Scholar Accumulating evidence has shown that FAO plays a critical role in LC by promoting cancer cell growth, inducing angiogenesis, and providing energy for cancer cell migration and invasion.8Harris F.T. Rahman S.M.J. Hassanein M. Qian J. Hoeksema M.D. Chen H. Eisenberg R. Chaurand P. Caprioli R.M. Shiota M. Massion P.P. Acyl-coenzyme A-binding protein regulates Beta-oxidation required for growth and survival of non-small cell lung cancer.Cancer Prev Res (Phila). 2014; 7: 748-757Crossref PubMed Scopus (34) Google Scholar,9Liu X. Lu Y. Chen Z. Liu X. Hu W. Zheng L. Chen Y. Kurie J.M. Shi M. Mustachio L.M. Adresson T. Fox S. Roszik J. Kawakami M. Freemantle S.J. Dmitrovsky E. The ubiquitin-specific peptidase USP18 promotes lipolysis, fatty acid oxidation, and lung cancer growth.Mol Cancer Res. 2021; 19: 667-677Crossref PubMed Scopus (18) Google Scholar Thus, therapeutically targeting the process of FAO may be an alternative treatment strategy for LUAD. The RING family is a group of ubiquitin E3 ligases responsible for the ubiquitination of essential proteins involved in multiple pathological events, including tumorigenesis and metastasis.10Sinha A. Iyengar P.V. Ten Dijke P. E3 ubiquitin ligases: key regulators of TGFβ signaling in cancer progression.Int J Mol Sci. 2021; 22: 476Crossref PubMed Scopus (15) Google Scholar Ring finger protein 152 (RNF152) is a single-pass transmembrane protein of the RING family, mostly expressed on the surface of lysosomes.11Zhang W. Yang X. Chen L. Liu Y.-Y. Venkatarangan V. Reist L. Hanson P. Xu H. Wang Y. Li M. A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation.PLoS Biol. 2021; 19e3001361Crossref Scopus (14) Google Scholar A recent study reported that RNF152 regulated tumor growth by catalyzing ubiquitination of the Rheb protein and downregulating the mammalian target of rapamycin complex 1 pathway.12Deng L. Chen L. Zhao L. Xu Y. Peng X. Wang X. Ding L. Jin J. Teng H. Wang Y. Pan W. Yu F. Liao L. Li L. Ge X. Wang P. Ubiquitination of Rheb governs growth factor-induced mTORC1 activation.Cell Res. 2019; 29: 136-150Crossref PubMed Scopus (62) Google Scholar In addition, RNF152 suppressed the proliferation and mobility of hepatocellular carcinoma cells via degrading tetraspanin 12.13Wan J. Liu S. Sun W. Yu H. Tang W. Liu W. Ji J. Liu B. Ring finger protein 152-dependent degradation of TSPAN12 suppresses hepatocellular carcinoma progression.Cancer Cell Int. 2021; 21: 122Crossref PubMed Scopus (5) Google Scholar However, RNF152 is rarely reported in LC. Whether RNF152 would exert a regulatory effect on LUAD warrants investigation. The interleukin-1 receptor-associated kinase (IRAK) family is involved in the regulation of inflammatory responses, cancer progression, and therapy resistance.14Singer J.W. Fleischman A. Al-Fayoumi S. Mascarenhas J.O. Yu Q. Agarwal A. Inhibition of interleukin-1 receptor-associated kinase 1 (IRAK1) as a therapeutic strategy.Oncotarget. 2018; 9: 33416-33439Crossref PubMed Scopus (90) Google Scholar IRAK1 is a key member of the IRAK family and its high expression is associated with high malignancy and poor prognosis of many types of cancers, including LC.15Liu Y.-N. Tsai M.-F. Wu S.-G. Chang T.-H. Tsai T.-H. Gow C.-H. Wang H.-Y. Shih J.-Y. miR-146b-5p enhances the sensitivity of NSCLC to EGFR tyrosine kinase inhibitors by regulating the IRAK1/NF-κB Pathway.Mol Ther Nucleic Acids. 2020; 22: 471-483Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Ubiquitous degradation of IRAK1 by S100 calcium-binding protein A14 effectively suppressed the metastasis of nasopharyngeal carcinoma.16Meng D.-F. Sun R. Liu G.-Y. Peng L.-X. Zheng L.-S. Xie P. Lin S.-T. Mei Y. Qiang Y.-Y. Li C.-Z. Xu L. Peng X.-S. Hu H. Lang Y.-H. Liu Z.-J. Wang M.-D. Guo L.-L. Xie D.-H. Shu D.-T. Li H.-F. Luo F.-F. Niu X.-T. Huang B.-J. Qian C.-N. S100A14 suppresses metastasis of nasopharyngeal carcinoma by inhibition of NF-kB signaling through degradation of IRAK1.Oncogene. 2020; 39: 5307-5322Crossref PubMed Scopus (24) Google Scholar Moreover, IRAK1 augments cancer stemness and drug resistance of hepatocellular carcinoma by regulating Aldo-Keto reductase family 1 member 10 (AKR1B10),17Cheng B.Y. Lau E.Y. Leung H.W. Leung C.O. Ho N.P. Gurung S. Cheng L.K. Lin C.H. Lo R.C. Ma S. Ng I.O. Lee T.K. IRAK1 augments cancer stemness and drug resistance via the AP-1/AKR1B10 signaling cascade in hepatocellular carcinoma.Cancer Res. 2018; 78: 2332-2342Crossref PubMed Scopus (68) Google Scholar,18Kim S.Y. Shen Q. Son K. Kim H.S. Yang H.D. Na M.J. Shin E. Yu S. Kang K. You J.S. Yu K.R. Jeong S.M. Lee E.K. Ahn Y.M. Park W.S. Nam S.W. SMARCA4 oncogenic potential via IRAK1 enhancer to activate Gankyrin and AKR1B10 in liver cancer.Oncogene. 2021; 40: 4652-4662Crossref PubMed Scopus (9) Google Scholar a secretory protein that has been recently shown to promote brain metastasis of LC.19Liu W. Song J. Du X. Zhou Y. Li Y. Li R. Lyu L. He Y. Hao J. Ben J. Wang W. Shi H. Wang Q. AKR1B10 (Aldo-keto reductase family 1 B10) promotes brain metastasis of lung cancer cells in a multi-organ microfluidic chip model.Acta Biomater. 2019; 91: 195-208Crossref PubMed Scopus (85) Google Scholar It is worth exploring the potential involvement of IRAK1 and AKR1B10 in the development of LUAD. This study first examined whether RNF152 was aberrantly expressed in LUAD. Cell models with different RNF152 expression levels and mouse xenograft models were then established to elucidate the regulatory mechanisms of RNF152 in LUAD. The relationship of RNF152 with IRAK1 and AKR1B10 was also explored. Gene Expression Profiling Interactive Analysis (GEPIA),20Tang Z. Li C. Kang B. Gao G. Li C. Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses.Nucleic Acids Res. 2017; 45: W98-W102Crossref PubMed Scopus (5841) Google Scholar an interactive web application, was used to compare the expression of RNF152 in LUAD tumors and normal samples based on the Cancer Genome Atlas (TCGA) data.21Tomczak K. Czerwińska P. Wiznerowicz M. The Cancer Genome Atlas (TCGA): an immeasurable source of knowledge.Contemp Oncol (Pozn). 2015; 19: A68-A77PubMed Google Scholar Kaplan-Meier plotter (http://kmplot.com, last accessed November 3, 2021), a web-based survival analysis tool, was used to assess the prognostic significance of RNF152 expression in LUAD. Tumor samples were divided into two groups based on the median expression of RNF152 (low expression, bottom 50%; high expression, top 50%). Kaplan-Meier survival curves of the overall survival (OS) and post-progression survival of the two groups were plotted. LUAD tissue samples (n = 145) and paired paracancerous normal tissues were collected from patients undergoing surgical resection for primary LUAD at the authors’ hospital. The handling of clinical specimens was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University, and performed in accordance with the Declaration of Helsinki.22World Medical AssociationWorld Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects.JAMA. 2013; 310: 2191-2194Crossref PubMed Scopus (14751) Google Scholar Written informed consent was obtained from all patients before enrollment. None of them received preoperative radiotherapy or chemotherapy. The animal study protocol was reviewed and approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University, and all experiments were performed following the Guide for the Care and Use of Laboratory Animals.23Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar Paracancerous normal tissues were at least 2 cm over the tumor and confirmed with hematoxylin and eosin staining as normal controls. The clinicopathological characteristics of all patients are summarized in Table 1. Tissue samples were then prepared for immunohistochemistry (IHC), Western blot, and real-time quantitative PCR (qPCR).Table 1Correlations of RNF152 with Clinical Characteristics in Lung Adenocarcinoma PatientsClinicopathological factorNumber of casesRNF152 expressionPHighLowNumber1454798Sex0.561 Male692445 Female762353Age, years0.524 <60611843 ≥60842955Pathological grading0.809 I582038 II581741 III291019TNM stage0.412 I-II702545 III-IV752253Liver metastasis0.245 Yes812358 No642440 Open table in a new tab Tissue samples were fixed, sectioned, and stained with the following antibodies (Santa Cruz Biotechnology, Dallas, TX) for IHC analysis: anti-RNF152 antibody (5 μg/mL; cat no. sc-398407), anti-IRAK1 (5 μg/mL; cat no. sc-5288), and anti-AKR1B10 (5 μg/mL; cat no. sc-365689). The staining was scored as previously described24Li D. Xing Y. Tian T. Guo Y. Qian J. Overexpression of LRRC59 is associated with poor prognosis and promotes cell proliferation and invasion in lung adenocarcinoma.OncoTargets Ther. 2020; 13: 6453-6463Crossref PubMed Scopus (8) Google Scholar based on the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) and the extensity of positive cells (0, ≤9% cells were stained positive; 1, 10% to 25%; 2, 26% to 50%; 3, 51% to 80%; and 4, ≥81%). The final score was obtained by adding the strongest intensity score and the total extensity score. A sample with a final score of >4 or ≤4 was defined as high or low LRRC59 expression, respectively. The staining was evaluated independently by two experienced pathologists blinded to the clinicopathological data. LUAD cell lines (NCI-H2009, A549, LTEP-A1, HCC827, and NCI-H441), human lung epithelial cell line BEAS-2B, and HEK293T cells were obtained from ATCC (Manassas, VA). NCI-H2009, LTEP-A1, HCC827, and NCI-H441 cells were maintained in RPMI (Sigma-Aldrich, St. Louis, MO), whereas BEAS-2B, A549, and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich). All culture media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA). All cell culture experiments were maintained in a humidified atmosphere of 5% CO2 at 37°C. The lentiviral transduction approach was used to knock down or overexpress target genes in LUAD cells. Lentiviral vectors carrying the full-length sequence of the RNF152 or IRAK1 gene, or packaged with siRNAs against RNF152 (shRNF152-1# and -2#) or IRAK1 (shIRAK1), as well as corresponding control vectors were designed and constructed by GenePharma (Shanghai, China). LUAD cells were transfected with designated vectors using Lipofectamine 2000 (Invitrogen, Waltham, MA) for 48 hours. Infected cells were then selected with fresh culture medium containing 2 μg/mL puromycin (Sigma-Aldrich) for 72 hours. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich). LUAD cells were seeded in a 96-well plate (1 × 103 cells/well). A volume of 10-μL CCK-8 solution (Dojindo Molecular Technologies, Inc., Tokyo, Japan) was added to each well every 24 hours from 0 to 72 hours, followed by another incubation of 3 hours. The absorbance was measured at 450 nm by a plate reader (Thermo Fisher Scientific). Cell proliferation was assessed by EdU incorporation assay. After transfection, LUAD cells were incubated with 100 mL of EdU solution (Ribobio, Guangzhou, China) for 3 hours, and then fixed with 4% paraformaldehyde. After permeabilization, DAPI (Sigma-Aldrich) was used to counterstain cell nuclei. Proliferative cells were detected under a fluorescent microscope (Leica, Wetzlar, Germany). To detect apoptosis, transfected LUAD cells were harvested and stained with Annexin V-FITC/PI Apoptosis Detection Kit (Invitrogen) for 15 minutes in the dark at 4°C. Cells were then detected by an LSR II flow cytometer (BD Biosciences. Franklin Lakes, NJ) and the apoptotic rate was calculated by the FlowJo software version 10.6 (Ashland, OR). Matrigel-coated Transwell chambers (Corning, Corning, NY) were used to evaluate the invasion capacity of LUAD cells, whereas cell migration was assessed using chambers without Matrigel coating. Cells (5 × 103) diluted in serum-free medium were placed into the upper chamber, while culture medium containing 10% fetal bovine serum (complete medium) was added to the lower chamber as the chemoattractant. After 24-hour incubation, cells that were invaded or migrated to the lower chamber were fixed and stained with 1% crystal violet. Cells were then observed under a light microscope (200×). The mean cell number from five randomly selected fields of view was calculated. The relative FAO level in LUAD cells was determined by the FAO Detection Kit (Genmed Scientifics, Shanghai, China) according to the manufacturer's manual. The cellular reactive oxygen species (ROS) level was measured by staining detached LUAD cells with the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Cayman Chemical, Ann Arbor, MI). Cells were harvested and trypsinized to produce single-cell suspension. Cells were then stained with 500 μL of 1 μmol/L DCFH-DA for 30 minutes at 37°C and then washed with phosphate-buffered saline. Flow cytometry (BD Biosciences) was then used to quantify the relative ROS level. The reduced nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) ratio and the glutathione/glutathione disulfide (GSH/GSSG) ratio in LUAD cells were determined by the NADPH/NADP Assay Kit (cat no. ab176724; Abcam, Cambridge, UK) and the GSH/GSSG-Glo assay kit (cat no. V6611; Promega, Madison, WI), respectively, following the manufacturer's manual. To measure FA uptake in LUAD cells, 1 to 3 × 104 cells were diluted in 100 μL of complete medium and seeded onto a 96-well plate. After incubation for 24 hours at 37°C, cells were washed and maintained in serum-free medium for 1 hour. Then, 100 μL of Free Fatty Acid solution from the Free Fatty Acid Uptake Assay Kit (cat no. ab176768; Abcam) was added to each well. After 1-hour incubation at 37°C, the fluorescence signal was measured by a fluorescence microplate reader (excitation 485 nm, emission 515 nm; Thermo Fisher Scientific). A lipid peroxidation sensor BODIPY 581/591 C11 (cat no. D3861; Invitrogen) was used to detect lipid peroxidation in LUAD cells as previously described.25van Weverwijk A. Koundouros N. Iravani M. Ashenden M. Gao Q. Poulogiannis G. Jungwirth U. Isacke C.M. Metabolic adaptability in metastatic breast cancer by AKR1B10-dependent balancing of glycolysis and fatty acid oxidation.Nat Commun. 2019; 10: 2698Crossref PubMed Scopus (63) Google Scholar In brief, cells were cultured in complete medium for 2 days, followed by 30-minute staining with 1 μg/mL BODIPY 581/591 C11 and DAPI (Sigma-Aldrich). Cells were then observed under a confocal microscope (Leica Microsystems) and images were captured: red, nonoxidized cells; green, oxidized cells; blue (DAPI), cell nuclei. RNF152- or IRAK1-encoding sequence was subcloned into pCDH-CMV-MCSEF1-Puro vectors, and tagged with Flag and Myc, respectively, by GenePharma. HEK293T cells were co-transfected with Flag-tagged RNF152 and/or Myc-tagged IRAK1 recombinant plasmids, followed by co-immunoprecipitation analysis of Flag-RNF152 and Myc-IRAK1 overexpression as previously described.26Yu X. Liang C. Zhang Y. Zhang W. Chen H. Inhibitory short peptides targeting EPS8/ABI1/SOS1 tri-complex suppress invasion and metastasis of ovarian cancer cells.BMC Cancer. 2019; 19: 878Crossref PubMed Scopus (11) Google Scholar Briefly, transfected HEK293T cells were lysed in 1 mL of lysis buffer (Cell Signaling Technology, Danvers, MA). Whole-cell lysates were then centrifuged for 10 minutes at 12,000 × g at 4°C. The supernatant was transferred to another tube and incubated with anti-Flag and anti-Myc antibodies (Sigma-Aldrich) overnight at 4°C. Then, 50 μL of magnetic beads (Thermo Fisher Scientific) were added to the mixture and incubated for another hour. The mixture was then centrifuged at 7,800 × g for 5 minutes at 4°C. After the supernatant was removed, the beads were washed, and the interaction between RNF152 and IRAK1 was detected by Western blot. LUAD cells were transfected with vectors overexpressing RNF152 or control vectors as mentioned before, followed by 6-hour incubation with or without 10 μmol/L of MG132 (Sigma-Aldrich). Whole-cell lysates were then prepared by treating cells with lysis buffer. Magnetic beads (Thermo Fisher Scientific) were conjugated with anti-RNF152 (cat no. sc-398407; Santa Cruz Biotechnology) or anti-IRAK1 (cat no. sc-5288; Santa Cruz Biotechnology) antibodies for 15 minutes and then resuspended in cell lysates. After 30 minutes of incubation at room temperature, immunoprecipitates were collected, separated by 10% SDS-PAGE, and stained with poly-ubiquitin antibody (cat no. ab7780; Abcam) to analyze ubiquitination of IRAK1. The cycloheximide (CHX)-chase assay was used to detect the degradation of IRAK1. LUAD cell lines overexpressing RNF152 or with RNF152 knockdown were constructed as aforementioned. After culture in complete medium for 24 hours, cells were treated with 10 μg/mL CHX (Thermo Fisher Scientific) to inhibit de novo synthesis of proteins. The protein level of IRAK1 was detected by Western blot at 0 to 2 hours after treatment to indicate the stability of IRAK1. The expression of E-cadherin, N-cadherin in LUAD cells was detected by immunofluorescence staining. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and permeabilized with 0.4% Triton X-100 (Sigma-Aldrich), followed by staining with anti–E-cadherin (3 μg/mL; cat no. M106; Takara Bio Inc., Shiga, Japan) and anti–N-cadherin antibodies (5 μg/mL; cat no. ab76057; Abcam). Cells were then stained with a goat anti-rabbit IgG (Alexa Fluor 680) secondary antibody (1:8000, cat no. ab175773; Abcam) or a goat anti-rabbit IgG (Alexa Fluor 488) secondary antibody (1:8000, cat no. ab150077; Abcam). After counterstained with DAPI, the fluorescent signals were detected under a fluorescence microscope (Olympus, Tokyo, Japan). Total RNA from LUAD cells was extracted by TRIzol reagent (Life Technologies, Carlsbad, CA). Reverse transcription was then performed using the PrimeScript RT Kit (Takara Bio Inc.). The mRNA level of RNF152 was determined by performing qPCR on the 7300 RT-PCR System (Applied Biosystems, Waltham, MA) using the SYBR Premix Ex Taq Kit (Takara Bio Inc.). The sequences of the primers were as follows: RNF152 F, 5′-GCGCGTATCTAACCCTTCCTG-3′, RNF152 R, 5′-TTCACCCCTTCTGCGTGATG-3′; GAPDH (internal control) F, 5′-ATCACTGCCACCCAGAAGAC-3′, GAPDH R: 5′-TTTCTAGACGGCAGGTCAGG-3′. Total protein from LUAD cells was extracted by RIPA buffer containing protease inhibitor (Pierce Biotechnology, Waltham, MA). The content of protein in each sample was determined by the BCA Kit (Thermo Fisher Scientific). Equal amounts of protein were loaded on 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After blocking, the membranes were stained with designated primary antibodies overnight at 4°C: RNF152 (1:1000; cat no. sc-398407; Santa Cruz Biotechnology), cleaved caspase-3 (1:1000; cat no. ab49822; Abcam), BCL2 (1:2000; cat no. ab182858; Abcam), BAX (1:1000; cat no. ab32503; Abcam), MMP9 (1:1000; cat no. ab76003; Abcam), MMP2 (1:1000; cat no. ab37150; Abcam), acetyl-CoA synthetase 1(ACSL1; 1:1000; cat no. sc-374104; Santa Cruz Biotechnology), acetyl-CoA carboxylase 1 (ACC1; 1:1000; cat no. 4190; Cell Signaling Technology), IRAK1 (1:1000; cat no. sc-5288; Santa Cruz Biotechnology), AKR1B10 (1:1000; cat no. ab96417; Abcam), and β-actin (internal control; 1:5000; cat no. ab32572; Abcam). After 1-hour incubation with a secondary antibody (1:8000; cat no. ab6721; Abcam), the immunoreactive blots were visualized by the ECL Western Blot Kit (Amersham Biosciences, Little Chalfont, UK). An in vivo xenograft model of LUAD was established by injecting male BALB/c nude mice (7-week–old; Charles River Laboratories, Wilmington, MA) with LUAD cells. Mice were housed in a controlled environment with 50% humidity 23 ± 2°C, and 12-hour light-dark cycle. All animals had free access to water and food during the experiment. After 1-week acclimation, mice were randomly divided into two groups (n = 6 per group). A549 cells transfected with vectors carrying the full-length sequence of the RNF152 gene or the control sequence were collected, washed twice with phosphate-buffered saline, and resuspended in normal saline mixed with Matrigel (9:1, v/v). A 200-μL volume of the cell suspension (3 × 106) was injected subcutaneously into the right flank of each mouse. The tumor volume was monitored every 5 days for 30 days. Tumor volume (mm3) = (π × length × width2)/6. On day 30, all mice were euthanized, and the tumors were immediately removed. After weighing, tumor tissues were prepared for IHC staining with anti-RNF152 (5 μg/mL; cat no. sc-398407; Santa Cruz Biotechnology), anti-IRAK1 (5 μg/mL; cat no. sc-5288; Santa Cruz Biotechnology), AKR1B10 (5 μg/mL; cat no. ab96417; Abcam), and KI67 (2 μg/mL; cat no. ab15580; Abcam). A mouse LUAD model of hepatic metastasis was established by injecting LUAD cells into the spleen of male BALB/c nude mice (8-week–old) as previously described with slight modifications.27Soares K.C. Foley K. Olino K. Leubner A. Mayo S.C. Jain A. Jaffee E. Schulick R.D. Yoshimura K. Edil B. Zheng L. A preclinical murine model of hepatic metastases.J Vis Exp. 2014; 51677Crossref PubMed Scopus (105) Google Scholar Mice were obtained and housed as aforementioned. They were randomly divided into two groups (n = 6 per group) and prepared for laparotomy, during which a lateral incision was made to expose the spleen. A549 cells transfected with vectors overexpressing RNF152 or control vectors were collected and resuspended in normal saline. A 10-μL volume of the cell suspension (2 × 105) was injected into the spleen using a 26-gauge × 5/8-inch syringe (BD Biosciences). The injection site was then sealed with Vetbond tissue adhesive (Grovet, Utrecht, the Netherlands) and the peritoneum was closed with a 4-0 running stitch. Three skin clips were used to close the incision. Mice were housed for an additional 3 weeks after implantation. They were then euthanized, and the livers were removed, weighed, and stained for hematoxylin and eosin (Sigma-Aldrich). All experiments were repeated at least three times and performed in triplicate. The normality of the data were tested by the Kolmogorov-Smirnov test. Normally distributed data are shown as means ± SD. One-way analysis of variance was used to compare the results among multiple groups, whereas the t-test was performed for the comparisons between two groups. Pearson's correlation coefficient was used for correlation analysis. All data were analyzed using SPSS software version 24.0 (SPSS, Chicago, IL). P < 0.05 indicated statistical significance. The analysis of the gene expression profiles from TCGA showed that RNF152 was significantly down-regulated in LUAD compared with normal samples (Figure 1A). Next, 145 pairs of LUAD and paired paracancerous normal tissue specimens were collected from patients undergoing surgical resection of LUAD. Consistent with the results of database analysis, the mRNA expression of RNF152 in LUAD samples was significantly lower than that in normal tissues (Figure 1B). Moreover, patients with higher TNM stage and metastasis had significantly lower mRNA expression of RNF152 compared with their counterparts (Figure 1B). The detection of RNF152 protein by Western blot (Figure 1C) and IHC staining (Figure 1D) also revealed markedly lower expression of RNF152 in LUAD tissues, especially those with liver metastasis, in comparison with control. The Kaplan-Meier analysis of LUAD patients with high or low RNF152 expression demonstrated that the group with low RNF152 had significantly poorer OS and post-progression survival compared with the high expression group (Figure 1E), suggesting that low RNF152 level is associated with a poor prognosis in patients with LUAD. The expression of RNF152 was studied in human lung epithelial cell line BEAS-