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TRIM32 overexpression improves chemoresistance through regulation of mitochondrial function in non-small-cell lung cancers.

ABSTRACT: Background:TRIM32 is overexpressed in several human cancers. However, its expression pattern, biological characteristics and mechanisms in human non-small cell lung cancer (NSCLC) have not been reported. Methods:We examined TRIM32 protein in 115 cases of NSCLC specimens. TRIM32 plasmid transfection and siRNA knockdown was carried out in NSCLC cell lines. AnnexinV/PI and JC-1 staining were performed to examine the change of apoptosis and mitochondrial membrane potential. Western blot was used to detect change of downstream proteins. Results:We found that TRIM32 protein was upregulated in 69 cases and positively correlated with advanced TNM stage. TRIM32 overexpression also correlated with poor survival of NSCLC patients. Biological assays demonstrated that TRIM32 overexpression promoted while it depletion inhibited cell growth, colony formation and invasion. In addition, TRIM32 maintained NSCLC cell viability and reduced apoptosis when treated with cisplatin. JC-1 and CellRox staining demonstrated that TRIM32 could maintain mitochondrial membrane potential and reduce Reactive Oxygen Species (ROS) production after cisplatin treatment. Western blot analysis showed that TRIM32 overexpression downregulated caspase 3 cleavage and cytochrome c release. TRIM32 also positively regulated Bcl-2 protein expression and NF-?B signaling. Inhibition of NF-?B abolished the effects of TRIM32 on Bcl-2. Conclusion:Taken together, our results indicated that TRIM32 is overexpressed in NSCLC and regulates cisplatin resistance, possibly through NF-?B and Bcl-2.

SUBMITTER: Du Y

PROVIDER: S-EPMC6225845 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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TRIM32 overexpression improves chemoresistance through regulation of mitochondrial function in non-small-cell lung cancers.

Du Yaming Y Zhang Wei W Du Binghui B Zang Sheng S Wang Xinpeng X Mao Xin X Hu Zhansheng Z

OncoTargets and therapy 20181105

<h4>Background</h4>TRIM32 is overexpressed in several human cancers. However, its expression pattern, biological characteristics and mechanisms in human non-small cell lung cancer (NSCLC) have not been reported.<h4>Methods</h4>We examined TRIM32 protein in 115 cases of NSCLC specimens. TRIM32 plasmid transfection and siRNA knockdown was carried out in NSCLC cell lines. AnnexinV/PI and JC-1 staining were performed to examine the change of apoptosis and mitochondrial membrane potential. Western bl ...[more]

PMID: 30464531

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Project description:BackgroundThe identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures.MethodsGene fitness was determined by bioinformatics analysis from available datasets from genetic screenings. HSPD1 expression was evaluated by immunohistochemistry from formalin-fixed paraffin-embedded tissues from NSCLC patients. Real-time proliferation assays with and without cytotoxicity reagents, colony formation assays and cell cycle analyses were used to monitor growth and drug sensitivity of different NSCLC cells in vitro. In vivo growth was monitored with subcutaneous injections in immune-deficient mice. Cell metabolic activity was analyzed through extracellular metabolic flux analysis. Specific knockouts were introduced by CRISPR/Cas9.ResultsWe show heat shock protein family D member 1 (HSPD1 or HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients' prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on oxidative phosphorylation and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B.ConclusionsThese results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NSCLC.

Project description:The highly refractory nature of non-small cell lung cancer (NSCLC) to chemotherapeutic drugs is an important factor resulting in its poor prognosis. Recent studies have revealed that tumour necrosis factor alpha-induced protein 8 (TNFAIP8) is involved in various biological and pathological processes of cells, but their underlying mechanisms in processes ranging from cancer development to drug resistance have not been fully elucidated.TNFAIP8 expression in clinical NSCLC samples was examined through immunohistochemistry (IHC). After adjusting for patients' characteristics with propensity score matching, Kaplan-Meier analysis and Cox regression analysis were performed for comparison of patients' survival according to the TNFAIP8 level. Lentiviral transfection with TNFAIP8-specific shRNAs was used to establish stable TNFAIP8 knockdown (TNFAIP8 KD) NCI-H460, A549 and cis-diamminedichloroplatinum II resistant A549 (A549/cDDP) cell lines. Cell proliferation and viability were assessed by CCK-8 assay. Cell cycle was examined by flow cytometry. Multiple pathways regulated by TNFAIP8 KD were revealed by microarray analysis.We found that high TNFAIP8 expression was associated with advanced pT stage, advanced pTNM stage, lymph node metastasis and unfavourable survival in NSCLC patients. TNFAIP8 shRNAs reduced in vitro cancer cell proliferation and in vivo tumor growth. Additionally, TNFAIP8 KD increased the sensitivity of NSCLC cells to cisplatin in vitro and in vivo. Conversely, up-regulation of TNFAIP8 promoted the?proliferation and?drug resistance to cisplatin?of NSCLC cells. TNFAIP8 influences cancer progression pathways involving the MDM2/p53 pathway. Indeed, we observed that TNFAIP8 KD mediated the MDM2 downregulation and the p53 ubiquitination, thereby decreasing the degradation of p53 protein. shRNA p53 reversed TNFAIP8 shRNA-mediated regulation of cell proliferation, cell cycle, cisplatin sensitivity, and expression levels of RAD51, a DNA repair gene.Our work uncovers a hitherto unappreciated role of TNFAIP8 in NSCLC proliferation and cisplatin chemoresistance that is mediated through the MDM2/p53 pathway. These findings might offer potential therapeutic targets for reversing cisplatin resistance in NSCLC patients with high TNFAIP8 expression.

Project description:BackgroundNon-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC.MethodsComparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC.ResultsAt DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1 and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944.ConclusionsIntegrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.

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TRIM32 overexpression improves chemoresistance through regulation of mitochondrial function in non-small-cell lung cancers.

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TRIM32 overexpression improves chemoresistance through regulation of mitochondrial function in non-small-cell lung cancers.

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