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Low Expression of Ferritinophagy-Related NCOA4 Gene

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Low Expression of Ferritinophagy-Related NCOA4 Gene Mou et al. BMC Cancer (2021) 21:18 https://doi.org/10.1186/s12885-020-07726-z RESEARCH ARTICLE Open Access Low expression of ferritinophagy-related NCOA4 gene in relation to unfavorable outcome and defective immune cells infiltration in clear cell renal carcinoma Yanhua Mou1,2,3, Jinchun Wu1, Yao Zhang4, Omar Abdihamid1, Chaojun Duan3,5 and Bin Li1,6* Abstract Background: Clear cell renal cell carcinoma is susceptible to ferroptosis, and immunotherapy is recently recommended as a priority for the initial treatment of metastatic clear cell renal carcinoma. Increased ferroptosis and immune activation can synergistically reinforce each other in killing cancer cells. NCOA4 depletion can eliminate iron accumulation and thus weaken ferroptosis. Here, we aim to identify and validate the association between NCOA4 expression, clinicopathologic characteristics, and overall survival in ccRCC by using The Cancer Genome Atlas and Gene Expression Omnibus databases. We further analyze the interacted proteins of NCOA4 and infiltrated immune cells via TIMER and GEPIA databases. Methods: NCOA4 expression in clear cell renal carcinoma (ccRCC) tissues and normal adjacent tissues in The Cancer Genome Atlas (TCGA) data were primarily screened, and further validated in another independent cohort from the gene expression omnibus (GEO) database and human protein atlas. The relationships of NCOA4 expression and clinicopathologic parameters and overall survival (OS) were assessed using multivariate methods and Kaplan-Meier survival curves. And the proteins network with which NCOA4 interacted were also built using the online STRING website. Meanwhile, we use TIMER and GEPIA databases to investigate the relationships between NCOA4 expression and infiltrated immune cells and their corresponding gene marker sets. Results: Contrast to normal tissue, NCOA4 expression was lower in ccRCC tumor tissue(p < 0.05). Lower NCOA4 expression was closely associated with high-grade malignancy and advanced TNM stage. Univariate and multivariate analysis indicated the overall survival of ccRCC cases with low NCOA4 level is shorter than those of patients with high NCOA4 expression (p < 0.05). FTL and FTH1 were the important proteins interacting with NCOA4. ccRCC with NCOA4 deficiency presented the paucity of infiltrated immune cells and their matching marker sets, including CD8+ T cells. Conclusion: Deficient NCOA4 expression was related to disease progression and poor prognosis, as well as impaired infiltration of immune cells in ccRCC. Keywords: Ferritinophagy, NCOA4, Clear cell renal carcinoma, Immune cells, Ferroptosis * Correspondence: [email protected] 1Department of Oncology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China 6National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mou et al. BMC Cancer (2021) 21:18 Page 2 of 12 Background indicator for poor prognosis and was associated with Over the past decades, the global incidence of renal cell deficient infiltration of immune cells in ccRCC. Thus, it carcinoma (RCC) is increasing which ranks first mortality is plausible that NCOA4 defect reduce ferroptosis and amongannualurologiccancers[1]. RCC is a heteroge- thus possibly debilitate antitumor immune effects in neous cancer, most of them is clear cell RCC (ccRCC) ccRCC. Targeting NCOA4 may be a promising thera- which nearly accounts for 75–80% of RCC [2]. Surgery is peutic strategy for ferroptosis-induction or/and with the the mainstay of ccRCC, which still present a high recur- combination of immunotherapy in ccRCC. rence rate of 40% after radical surgery and have high mortality rate once it metastasizes to distant organs [1, 2]. Methods Targeted therapies are currently one of the standard Data source treatments for ccRCC, but almost all patients will ultim- The Cancer Genome Atlas (TCGA) (https://genome- ately develop disease deterioration because drug-induced cancer.ucsc.edu/), a free data portal of largescale cancer cell apoptosis or autophage was escaped by ccRCC cells genome project, provides clinic and pathological informa- [3]. Therefore, as a novel cell death, ferroptosis- tion of 33 types of cancer for scholars and researchers. The induction is becoming an alternative choice for the data of ccRCC patients with the expression of RNA-Seq therapeutic strategy of ccRCC [4–7]. and matching clinical pathologic information were obtained NCOA4, also named as androgen receptor-associated by the TCGA tools cancer browser. The database is protein 70 (ARA70), was originally described as a coacti- publicly open-access and available and therefore there was vator of multiple nuclear hormone receptors. It was no need to get approval from the local ethics committee. closely related to tumorigenesis and progression of ovar- ian cancer, prostate cancer, breast cancer and pancreatic The GEO database and the human protein atlas cancer [8]. Recent studies unravel that NCOA4 is an As one of the biggest collection of gene chips in the autophagosomes component that participates in the world, the GEO database is a comprehensive gene ex- process of ferritinophagy [8, 9]. Surface arginine in pression library in the National Center of Biotechnology ferritin heavy chain 1 (FTH1) can specifically bind the Information (NCBI) (https://www.ncbi.nlm.nih.gov/geo/). C-terminal element of NCOA4 and fused with a The Human Protein Atlas offers a broad amount of prote- lysosome via nascent autophagosomes, thus facilitating omic and transcriptome information of distinct human ferroptotic cell death [10]. NCOA4 was gradually samples, which consists of cell, tissue and pathology Atlas. considered as a key molecule promoting ferroptosis in To date, the online database provides cell-specific location various cancer cells and mounting studies displayed that information for 44 various normal tissues and 20 of the NCOA4 depletion can inhibit ferroptosis by eliminating most typical categories of cancer. Moreover, protein im- the accumulation of intracellular free iron, glutathione munohistochemistry in normal human tissues and tumor production and reactive oxygen species (ROS) [11]. tissues can also be obtained from this online website. Immunotherapy is coming into clinical practice in the treatment of ccRCC due to FDA approval and NCCN Survival and statistical analysis recommendation [12]. Recently, two independent studies According to the median expression of NCOA4 gene, simultaneously reported the same finding that ferropto- patients in test and validation set were split into two sis provoked by T cells in cancer cells is an important groups of high NCOA4 expression and low NCOA4 anti-tumor method of PD-1/PD-L1 antibody and weak expression respectively. To investigate whether NCOA4 effect of PD-L1 antibody was observed in the ferroptosis- expression level affects the clinical outcomes of ccRCC insensitive tumor cells [13, 14]. Increased ferroptosis can patients, we constructed a prognostic classifier using also advance the anti-tumor effect of immunotherapy Kaplan-Meier (KM) survival curves to compare the which pinpointed the positive feedback between ferroptosis survival disparities. and immunotherapy, cooperatively killing cancer cells [13]. In our study, we downloaded and analyzed the rela- Univariate and multivariate logistic regression analysis tionship of NCOA4 expression with clinical information To further determine the effect of NCOA4 expression and overall survival in ccRCC patients utilizing various in ccRCC patients, we use univariate Cox regression databases of TCGA, GEO and human protein atlas. And analysis for calculating the association between the then we used TIMER and GEPIA databases to investi- expression level of NCOA4 and patient’sOSintwo gate the relationships between NCOA4 expression and cohorts. Afterwards, a multivariate analysis was used infiltrated immune cells and their corresponding gene to assess if the NCOA4 is an independent prognostic marker sets. Also, the NCOA4-interacted protein factor for ccRCC patient survival. The NCOA4 has network was explored utilizing the STRING website. statistical significance in Cox regression analysis when The results showed that low NCOA4 level acted as an p value is less than 0.05. Mou et al. BMC Cancer (2021) 21:18 Page 3 of 12 Protein-protein interaction comprehensive analysis was
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