A homozygous loss-of-function mutation in FBXO43 causes human non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) represents one of the most serious forms of male infertility caused by spermatogenic failure. Despite multiple genes found to be associated with human NOA, the genetic basis of this idiopathic disease remains largely unknown. FBXO43 is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) E3 ligase and crucially important in mouse spermatogenesis. In this study, for the first time, we identified a homozygous nonsense mutation in FBXO43 c.1747C > T:p.Gln583X in two NOA brothers from a Chinese consanguineous family via whole-exome sequencing. FBXO43 was absent from testicular tissue of the proband, and FBXO43-immunostaining signals were invisible in the affected seminiferous tubules. Furthermore, in humans, FBXO43 defects cause meiotic arrest within early diplotene of prophase I. The results here demonstrate the pathogenicity of this loss-of-function mutation and confirmed that spermatocytes were unable to complete meiotic divisions without FBXO43 in humans. In mouse testicular protein extracts, three subunits of the APC/C, including ANAPC2, ANAPC8 and ANAPC10, were validated to interact directly with FBXO43, whereas no interactions were detected for FBXO43 and SKP1. This study furthers our understanding of the genetic basis of human NOA and provides insights into FBXO43 and male infertility.

Downregulation of the FBXO43 gene inhibits tumor growth in human breast cancer by limiting its interaction with PCNA.

BACKGROUND: The function and regulatory mechanism of FBXO43 in breast cancer (BC) are still unclear. Here, we intended to determine the role and mechanism of FBXO43 in BC. METHODS: FBXO43 expression in BC was evaluated by analysis of The Cancer Genome Atlas (TCGA). RT-qPCR and western blotting were utilized to detect FBXO43 expression in BC cell lines. Lentivirus was applied to downregulate FBXO43 in human BC cells. Proliferation assays were performed to evaluate the proliferative ability of BC cells. The apoptosis and cell cycle analysis of BC cells were analyzed by flow cytometry. Cell migration and invasion were investigated via Transwell assays. The function of FBXO43 in vivo was evaluated by constructing a xenograft mouse model. The proteins that might interact with FBXO43 in BC were identified by mass spectrometry, bioinformatics analysis, and co-immunoprecipitation (Co-IP) assays. Finally, rescue experiments were conducted to validate the recovery effects of the proteins interacting with FBXO43. RESULTS: FBXO43 was highly expressed in BC and was significantly downregulated after FBXO43 knockdown. The proliferation, migration, and invasion of BC cells were inhibited, and cell apoptosis was induced by FBXO43 knockdown. In addition, an in vivo experiment indicated that FBXO43 knockdown could inhibit the cell growth of BC. The results of the Co-IP assay showed that FBXO43 interacted with PCNA. Further rescue experiments confirmed that overexpression of PCNA significantly reversed the effects of FBXO43 knockdown on BC cells. CONCLUSION: Downregulation of FBXO43 inhibits the tumor growth of BC by limiting its interaction with PCNA. FBXO43 might be a new potential oncogene and a therapeutic target for BC.

FBXO43 variants in patients with female infertility characterized by early embryonic arrest.

STUDY QUESTION: Can any new genetic factors responsible for early embryonic arrest in infertile patients be identified, together with the mechanism of pathogenic variants? SUMMARY ANSWER: We identified three homozygous variants in the F-box protein 43 gene (FBXO43) in infertile patients and studies on the effects of the variants in HEK293T cells and mouse oocytes provided evidence for a causal relation between FBXO43 and female infertility. WHAT IS KNOWN ALREADY: FBXO43, an inhibitor of the anaphase-promoting complex/cyclosome, mediates Metaphase II arrest as a component of the cytostatic factor in oocytes. Both male and female Fbxo43 knockout mice are viable but sterile. FBXO43, therefore, appears to be an essential component of the mammalian cell-cycle machinery that regulates both male and female meiosis. Until now, only one article has reported a homozygous FBXO43 variant associated with teratozoospermia, but the causal relationship was not established with functional evidence. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing (WES) and homozygosity mapping were performed in 24 probands from consanguineous families who suffered from early embryonic arrest, and two different homozygous variants in FBXO43 were identified in two independent families. WES data from a further 950 infertile women with early embryonic arrest were screened for homozygous and compound heterozygous variants in FBXO43, and a third individual with an additional homozygous variant in FBXO43 was identified. The infertile patients presenting with early embryonic arrest were recruited from August 2016 to May 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: The women diagnosed with primary infertility were recruited from the reproduction centers of local hospitals. Genomic DNA samples from the affected individuals, their family members, and healthy controls were extracted from peripheral blood. The FBXO43 variants were identified using WES, homozygosity mapping, in silico analysis, and variant screening. All of the variants were confirmed by Sanger sequencing, and the effects of the variants were investigated in human embryonic kidney (HEK) 293T cells by western blotting and in mouse oocytes by complementary RNA injection. MAIN RESULTS AND THE ROLE OF CHANCE: We identified three homozygous variants in FBXO43 (NM_001029860.4)-namely, c.1490_1497dup (p.(Glu500Serfs*2)), c.1747C>T (p.(Gln583*)), and c.154delG (p.(Asp52Thrfs*30))-in three independent families. All of the homozygous variants reduced the protein level of FBXO43 and reduced the level of its downstream target Cyclin B1 in HEK293T cells. In addition, the variants reduced the ability of exogenous human FBXO43 to rescue the parthenogenetic activation phenotype in Fbxo43 knockdown mouse oocytes. LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from the oocytes of patients, the exact molecular mechanism remains unknown and should be further investigated using knock out or knock in mice. WIDER IMPLICATIONS OF THE FINDINGS: Our study has identified three pathogenic variants in FBXO43 that are involved in human early embryonic arrest. These findings contribute to our understanding of the role of FBXO43 in human early embryonic development and provide a new genetic marker for female infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2018YFC1003800, 2017YFC1001500, and 2016YFC1000600), the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, 81971382, and 82001552), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Foundation of the Shanghai Health and Family Planning Commission (20154Y0162), the Capacity Building Planning Program for Shanghai Women and Children's Health Service, and the collaborative innovation center project construction for Shanghai Women and Children's Health. None of the authors have any competing interests. TRIAL REGISTRATION NUMBER: N/A.

FBXO43 promotes cell cycle progression in cancer cells through stabilizing SKP2.

FBXO43 is a member of the FBXO subfamily of F-box proteins, known to be a regulatory hub during meiosis. A body of data showed that FBXO43 is overexpressed in a number of human cancers. However, whether and how FBXO43 affects cell cycle progression and growth of cancer cells remain elusive. In this study, we provide first piece of evidence, showing a pivotal role of FBXO43 in cell cycle progression and growth of cancer cells. Specifically, FBXO43 acts as a positive cell cycle regulator with an oncogenic activity in variety types of human cancer, including non-small cell lung cancer, hepatocellular carcinoma and sarcoma. Mechanistically, FBXO43 interacts with phosphorylated SKP2 induced by AKT1, leading to reduced SKP2 auto-ubiquitylation and subsequent proteasome degradation. Taken together, our study demonstrates that FBXO43 promotes cell cycle progression by stabilizing SKP2, and FBXO43 could serve as a potential anti-cancer target.

High Expression of F-Box Protein 43 Is Associated With Poor Prognosis and Adjuvant Chemotherapy Resistance in Colorectal Cancer.

Background: The F-box protein 43 (FBXO43), also referred to as endogenous meiotic inhibitor 2 (EMI2), has been linked to the advancement of various types of cancer, such as hepatocellular carcinoma, breast cancer, cholangiocarcinoma, and gastric cancer. Nevertheless, the precise function of FBXO43 in colorectal cancer (CRC) remains unclear. This study employed data from The Cancer Genome Atlas (TCGA) and clinical specimens to analyze the expression, prognostic value, and chemotherapeutic advantages of FBXO43 in CRC. Methods: Level 3 RNA sequencing data pertaining to 631 cases of colon and rectal adenocarcinomas (COAD-READ) were downloaded from TCGA. The data were utilized to analyze the expression, prognosis, and related signal pathways of FBXO43. The expression of FBXO43 in clinical samples was subsequently confirmed through the use of real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Lastly, a tissue microarray (TMA) consisting of 120 cases of CRC and corresponding normal tissues was established to investigate the relationship between FBXO43 and survival outcomes. Results: Results from both the TCGA analysis and clinical samples indicated that FBXO43 was significantly upregulated in CRC tissues in comparison to normal tissues. Moreover, high level of FBXO43 was found to be relevant to malignant clinical features, such as differentiation, lymph node metastasis, and pathological stage, as well as unfavorable prognosis in CRC patients. Subgroup analysis further demonstrated that FBXO43 could be an effective parameter for stratifying low-risk CRC patients. Notably, survival analysis showed that patients with high level of FBXO43 had worse overall survival (OS) and disease-free survival (DFS) following adjuvant chemotherapy, and FBXO43 was distinctly upregulated in chemotherapy-resistant patients' primary CRC tissues. Conclusions: FBXO43 was upregulated and associated with poor prognosis of CRC; patients with high expression of FBXO43 may not be benefit from adjuvant chemotherapy.

Association between F-box-only protein 43 overexpression and hepatocellular carcinoma pathogenesis and prognosis.

BACKGROUND: Despite great advances in the prevention, diagnosis, treatment, and management regarding hepatocellular carcinoma (HCC), the overall prognosis of HCC remains unfavorable. The expression profile, prognostic role, and biological functions of F-box-only protein 43 (FBXO43) in HCC remain unclear. Here, we determine the expression profile and prognostic value of FBXO43 in patients with HCC. MATERIALS AND METHODS: A total of 467 HCC patients and their clinicopathological data were collected from the Second Affiliated Hospital of Jiaxing University, the Cancer Genome Atlas (TCGA), and Genotype-Tissue Expression (GTEx) databases. The expression profile, prognostic value, biological functions, and underlying mechanism of its involvement of FBXO43 were explored based on TCGA, Gene Expression Omnibus (GEO), LinkedOmics, and Cancer Dependency Map (DepMap). The expression of FBXO43 in 93 paired liver tissues was investigated via immunohistochemical staining, tissue microarray analysis, and Western blot. The prognostic value was assessed using survival analysis. RESULTS: FBXO43 RNA was upregulated in HCC liver tissues and was associated with an unfavorable prognosis (p < 0.05). Furthermore, FBXO43 protein was overexpressed in HCC liver tissues compared with that in paired normal liver tissues. Overexpression of FBXO43 protein was significantly associated with advanced TNM stage, large tumor size, lymphatic invasion, distant metastasis, earlier cancer recurrence, and decreased overall survival after radical surgery (p < 0.05). Cox regression analysis showed that FBXO43 had significant prognostic value in HCC. Importantly, FBXO43 and its co-expressed genes were mainly involved in cell cycle regulation, DNA replication, metabolic regulation, and so on. FBXO43 knockdown could significantly affect the HCC cell lines growth and proliferation. CONCLUSIONS: We first revealed that FBXO43 was overexpressed in liver HCC tissues at the RNA and protein levels and served as an independent prognostic factor for HCC patients. Therefore, FBXO43 is worth investigating as a potential HCC treatment target.

Clinical significance of FBXO43 in hepatocellular carcinoma and its impact on tumor cell proliferation, migration and invasion.

Background: The effects of FBXO43 on hepatocellular carcinoma (HCC) and its clinical significance have not yet been determined. This study aims to determine the clinical significance of FBXO43 in HCC and its impact on the biological functions of HCC cells. Methods: Data from TCGA database were downloaded to investigate the expression of FBXO43 in HCC and its correlation with prognosis and immune infiltration. Immunohistochemical staining images of FBXO43 in HCC were acquired from the HPA website. HCC cells (BEL-7404 and SMMC-7721) were transfected with the lentivirus targeting FBXO43 to decrease FBXO43 expression in HCC cells. Western blotting assay was conducted to evaluate the expression level of FBXO43 protein. MTT assay was used to detect the proliferation of HCC cells. The migration and invasion of HCC cells were investigated by performing scratch wound-healing and Transwell invasion assays, respectively. Results: In comparison to normal tissues, FBXO43 is overexpressed in HCC tissue, and high FBXO43 expression is linked to late T stage, TNM stage and tumor grade. Elevated FBXO43 expression is a risk factor for HCC. In patients with high FBXO43 expression, the overall survival, disease-specific survival, progression-free survival and disease-free survival are poorer. The proliferation, migration and invasion of HCC cells are significantly attenuated in FBXO43 knockdown cells. Also, TCGA data analysis reveals that FBXO43 exhibits a positive correlation with immunosuppression of HCC. Conclusion: FBXO43 is overexpressed in HCC, and is linked to late tumor stage, worse prognosis and tumor immunosuppression. FBXO43 knockdown restrains the proliferation, migration and invasion of HCC.

FBXO43 increases CCND1 stability to promote hepatocellular carcinoma cell proliferation and migration.

Background and Aims: Abnormal expression of E3 ubiquitin ligase plays an important role in the development and progression of hepatocellular carcinoma (HCC), although the mechanism has remained elusive. This study aimed to investigate the biological function and potential mechanism of FBXO43 in HCC. Methods: FBXO43 expression in tissues and cells were detected by quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry (IHC). The Kaplan-Meier method and Cox regression analysis were used to explore the correlation between the expression level of FBXO43 and the clinical survival. MTT assay, EdU incorporation, colony formation, Transwell, and wound healing assays were performed to evaluate the function of FBXO43 in cell proliferation and migration in vitro. The interaction between FBXO43 and cyclin D1 (CCND1) was assessed by co-immunoprecipitation (Co-IP) assay and in vivo ubiquitination assay. Results: We found that FBXO43 was upregulated in HCC patient tissues and positively associated with poor clinicopathological features. Meanwhile, HCC patients with high expression of FBXO43 had shorter overall survival (OS) and disease-free survival (DFS). Furthermore, knockdown of FBXO43 inhibited HCC cell proliferation, migration and epithelial-mesenchymal transition (EMT) in HCC cells. Mechanistically, FBXO43 interacted with CCND1 and promoted its stability by polyubiquitination, leading to HCC cell proliferation, migration and EMT. Functional rescue experiments demonstrated that knockdown of CCND1 blocks FBXO43-mediated cell proliferation and metastasis. Conclusions: FBXO43, as an independent prognostic biomarker, promotes HCC cell proliferation, metastasis and EMT by stability of CCND1, which provides a new potential strategy for HCC treatment by targeting FBXO43-CCND1 axis.

F-box protein 43 promoter methylation as a novel biomarker for hepatitis B virus-associated hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) has a high prevalence and poor prognosis worldwide. Therefore, it is urgent to find effective and timely diagnostic markers. The objective of this study was to evaluate the diagnostic value of F-box protein 43 promoter methylation in peripheral blood mononuclear cells (PBMCs) for HCC. Method: A total of 247 participants were included in this study, comprising individuals with 123 hepatitis B virus-associated HCC, 79 chronic hepatitis B, and 45 healthy controls. F-box protein 43 methylation and mRNA levels in PBMCs were detected by MethyLight and quantitative real-time PCR. Result: F-box protein 43 promoter methylation levels were significantly lower in HCC PBMCs than the chronic hepatitis B (P < 0.001) and healthy control PBMCs (P < 0.001). Relative mRNA expression levels of F-box protein 43 in HCC PBMCs were significantly higher than those in chronic hepatitis B (P < 0.001) and healthy control PBMCs (P < 0.001). Receiver operating characteristic analysis of F-box protein 43 promoter methylation levels yielded an area under curve (AUC) of 0.793 with 76.42% sensitivity and 68.35% specificity when differentiating HCC from chronic hepatitis. These values for the F-box protein 43 promoter methylation level were superior to those of the alpha-fetoprotein serum (AFP) level (AUC: 0.780, sensitivity: 47.97%, and specificity: 96.20%), with increments in values for the combination of F-box protein 43 promoter methylation AFP levels (AUC: 0.888, sensitivity: 76.42%, and specificity: 86.08%). Conclusion: Hypomethylation of the F-box protein 43 promoter in PBMCs is a promising biochemical marker for HBV-associated HCC.

F-Box Protein 43, Stabilized by N6-Methyladenosine Methylation, Enhances Hepatocellular Carcinoma Cell Growth and Invasion via Promoting p53 Degradation in a Ubiquitin Conjugating Enzyme E2 C-Dependent Manner.

The roles of F-box protein 43 (FBXO43) in carcinogenesis have been rarely revealed. The present study investigates the expression, function, and underlying mechanism of FBXO43 in hepatocellular carcinoma (HCC). Firstly, the expression and clinical significance of FBXO43 in HCC were investigated bioinformatically and experimentally using online omics data and local tissue samples. The role of N6-methyladenosine modification (m6A) of mRNA in regulating FBXO43 expression and the effects of m6A/FBXO43 axis alteration on cell proliferation and invasion were investigated further. Moreover, the underlying mechanism of the oncogenic FBXO43 was also explored. The results demonstrated that FBXO43 was significantly upregulated in HCC and was positively correlated with advanced progression and poor prognosis in patients. METTL3 and IGF2BP2 expressions were positively correlated with FBXO43 expression and served as the writer and reader of FBXO43 m6A, respectively, which stabilized and upregulated FBXO43 mRNA in HCC. FBXO43 silencing significantly reduced cell proliferation and invasion, and ectopic expression of FBXO43 could significantly restore the inhibitory effects caused by METTL3 and IGF2BP2 depletion in HCC cells. Mechanistically, FBXO43 depletion reduced the expression of UBE2C, a p53 ubiquitin-conjugating enzyme, suppressed proteasomal degradation of p53, and thus inhibited cell proliferation and invasion in HCC. In summary, the present study revealed that METTL3/IGF2BP2 mediated m6A contributed to the upregulation of FBXO43 that promoted the malignant progression of HCC by stimulating p53 degradation in a UBE2C-dependent manner, highlighting the promising application of FBXO43 as a target in HCC treatment.

EMI2 expression as a poor prognostic factor in patients with breast cancer.

Early mitotic inhibitor 2 (EMI2, gene symbol FBXO43), an APC/C inhibitor regulated by Plx1, is essential for cytostatic factor (CSF) activity. It belongs to subclass FBXO of the F-box proteins family. The aim of this study is to examine the clinicopathological significance of EMI2 in breast cancer. In this study, immunohistochemistry analysis was used to evaluate EMI2 expression in breast cancer tissues and then the association between EMI2 expression and clinicopathological factors was examined. Correlation of EMI2 with patient survival was analyzed by Kaplan-Meier survival curves. Among 192 patients analyzed, 105 (54.7%) had high expression of EMI2, and this was significantly associated with shortened disease free survival and overall survival in breast cancer patients. EMI2 expression was significantly associated with tumor grade (P = .006), tumor size (P < .001), and lymph node metastasis (P = .008). However, there was no significant correlation between EMI2 status and other biomarkers including ER, PR and Her2 status. Our results revealed that elevated EMI2 expression is a risk factor (hazard ratio = 3.93) for breast cancer and overexpression of EMI2 in breast cancer predicts higher risk of metastasis and worse survival. Therefore, EMI2 may be a potential therapeutic target for breast cancer.

A novel homozygous FBXO43 mutation associated with male infertility and teratozoospermia in a consanguineous Chinese family.

OBJECTIVE: To identify the genetic causes of male infertility characterized by teratozoospermia. DESIGN: Genetic studies. SETTING: Medical university. PATIENT(S): Two infertile brothers with teratozoospermia in a consanguineous Chinese family, another 124 sporadic infertile male patients presenting with teratozoospermia, and 200 male controls with normal fertility. INVENTION(S): None. MAIN OUTCOME MEASURE(S): Whole exome sequencing and genotype analysis to identify the potential pathogenic mutation, Sanger sequencing to validate the mutation in family members, in silico structural modeling to predict the functional consequences of mutation, and targeted next-generation sequencing to validate the mutation in sporadic cases. RESULT(S): A novel homozygous nonsynonymous mutation (C1991T, p.G664D) in FBXO43 (F-box only protein 43) was observed in two brothers from a consanguineous Chinese family. The mutation was segregated with the disease phenotype and was predicted to be a disease causing protein by SIFT, PolyPhen-2, and Mutation Taster. An in silico mutant FBXO43 model predicts that the mutation p.G664D causes shortening of two ß-sheets, an additional α-helix, and change in loops, which may result in loss of function of the protein. The homozygous mutation of FBXO43 was absent in the 1000 Genomes Project (1000 G) and the Exome Aggregation Consortium (ExAC) databases. Subsequent mutation screening of FBXO43 in a cohort of 124 cases identified four additional cases with heterozygous FBXO43 mutations. No mutations were found in FBXO43 in 200 fertile controls. CONCLUSION(S): The mutation in FBXO43 is a causative factor of male infertility and teratozoospermia.