ALKBH5 in mouse testicular Sertoli cells regulates Cdh2 mRNA translation to maintain blood-testis barrier integrity.

BACKGROUND: RNA N6-methyladenosine (m6A) is involved in mammalian spermatogenesis. In both germ cells and Leydig cells, ALKBH5 regulates spermatogenesis and androgen synthesis in an m6A-dependent manner. However, it is unclear whether ALKBH5 plays a role in testicular Sertoli cells, which constitute the blood-testis barrier (BTB) through cell junctions between adjacent Sertoli cells. METHODS: ALKBH5 expression in the testes of humans and mice was detected by immunohistochemical staining and immunofluorescence staining. BTB integrity was evaluated by BTB assay. m6A-seq was performed to screen for BTB-related molecules regulated by ALKBH5. m6A immunoprecipitation-quantitative real-time polymerase chain reaction (qPCR), RNA immunoprecipitation-qPCR, western blot, coimmunoprecipitation, and polysome fractionation-qPCR analyses were performed to explore the mechanisms of ALKBH5 in BTB. Transmission electron microscopy was applied to observe the BTB ultrastructure. RESULTS: ALKBH5 in Sertoli cells is related to the integrity of the BTB. Subsequently, the m6A level on Cdh2 mRNA, encoding a structural protein N-cadherin in the BTB, was found to be regulated by ALKBH5. IGF2BP1/2/3 complexes and YTHDF1 promoted Cdh2 mRNA translation. In addition, we found that basal endoplasmic specialization, in which N-cadherin is a main structural protein, was severely disordered in the testes of Alkbh5-knockout mice. CONCLUSIONS: Our study revealed that ALKBH5 regulates BTB integrity via basal endoplasmic specialization by affecting Cdh2 mRNA translation.

RNA N6-methyladenosine modification, spermatogenesis, and human male infertility.

RNA N6-methyladenosine (m6A) modification is one of the main forms of posttranscriptional modification, and its dysregulation is involved in a series of pathological processes. RNA m6A regulators, which mediate dynamic RNA m6A modification, are expressed in almost all types of testicular cells, including spermatogenetic cells and somatic cells. Cumulative studies have found that knockout of RNA m6A regulators in the testis leads to abnormal metabolism of the target mRNAs, which eventually causes spermatogenetic disorders and infertility. To date, a role for dysregulated RNA m6A modification in human male infertility remains elusive; however, dysregulated expression of RNA m6A regulators in abnormal human semen samples, including oligospermia, asthenozoospermia and azoospermia, has been found. Therefore, we speculate that abnormal RNA m6A methylation may be an important mechanism of male infertility. In this review, we summarize the recent findings regarding the spatiotemporal expression of RNA m6A regulators in the testes, mechanisms of RNA m6A modification in spermatogenesis and the relation between dysregulated RNA m6A regulators and human male infertility. In addition, we also discuss future directions in studying the molecular mechanism of male infertility and exploring their clinical applications from the viewpoint of RNA m6A modification.

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

The Machado-Joseph Disease Deubiquitinase Ataxin-3 Regulates the Stability and Apoptotic Function of p53.

As a deubiquitinating enzyme (DUB), the physiological substrates of ataxin-3 (ATX-3) remain elusive, which limits our understanding of its normal cellular function and that of pathogenic mechanism of spinocerebellar ataxia type 3 (SCA3). Here, we identify p53 to be a novel substrate of ATX-3. ATX-3 binds to native and polyubiquitinated p53 and deubiquitinates and stabilizes p53 by repressing its degradation through the ubiquitin (Ub)-proteasome pathway. ATX-3 deletion destabilizes p53, resulting in deficiency of p53 activity and functions, whereas ectopic expression of ATX-3 induces selective transcription/expression of p53 target genes and promotes p53-dependent apoptosis in both mammalian cells and the central nervous system of zebrafish. Furthermore, the polyglutamine (polyQ)-expanded ATX-3 retains enhanced interaction and deubiquitination catalytic activity to p53 and causes more severe p53-dependent neurodegeneration in zebrafish brains and in the substantia nigra pars compacta (SNpc) or striatum of a transgenic SCA3 mouse model. Our findings identify a novel molecular link between ATX-3 and p53-mediated cell death and provide an explanation for the direct involvement of p53 in SCA3 disease pathogenesis.

[Long non-coding RNA Gm15577 is involved in mouse cerebellar neurogenesis].

OBJECTIVE: To identify novel lncRNAs involved in cerebellar neurogenesis using neuronal specific Nbs1-deficient (Nbs1(CNS-del)) mouse model. METHODS: Microarray analysis was performed to identify differentially expressed lncRNAs between Nbs1(CNS-ctr) and Nbs1(CNS-del) mice. Expression profiles of lncRNA Gm15577 and coding gene Negr1 in mice, primary cerebellar culture and cell lines were measured using RT-qPCR. Subcellular fractionation was performed to determine the subcellular localization of Gm15577. RESULTS: Gm15577 was specifically expressed in mice cerebellum in a developmentally regulated manner, which could be abolished upon Nbs1-deficiency. Gm15577 was located in the intronic region of Negr1 in a reversed orientation. Gm15577 modulated the RNA expression of Negr1, Shh and ß-catenin. NEGR1 had a distinct expression pattern between normal and medulloblastoma patients. CONCLUSION: Gm15577 may modulate cerebellar granule cell proliferation and differentiation by targeting Negr1, and their dysfunctions or abnormal expression may be related to tumorigenesis of medulloblastoma.

DBC1 maintains skeletal muscle integrity by enhancing myogenesis and preventing myofibre wasting.

BACKGROUND: Skeletal muscle atrophy, particularly ageing-related muscular atrophy such as sarcopenia, is a significant health concern. Despite its prevalence, the underlying mechanisms remain poorly understood, and specific approved medications are currently unavailable. Deleted in breast cancer 1 (DBC1) is a well-known regulator of senescence, metabolism or apoptosis. Recent reports suggest that DBC1 may also potentially regulate muscle function, as mice lacking DBC1 exhibit weakness and limpness. However, the function of DBC1 in skeletal muscle and its associated molecular mechanisms remain unknown, thus prompting the focus of this study. METHODS: Tibialis anterior (TA) muscle-specific DBC1 knockdown C57BL/6J male mice were generated through a single injection of 2.00 E + 11 vg of adeno-associated virus 9 delivering single-guide RNA for DBC1. Grip strength and endurance were assessed 2 months later, followed by skeletal muscle harvest. Muscle atrophy model was generated by cast immobilization of the mouse hindlimb for 2 weeks. Molecular markers of atrophy were probed in muscles upon termination. Cardiotoxin (CTX) was injected in TA muscles of DBC1 knockdown mice, and muscle regeneration was assessed by immunohistochemistry, quantitative PCR and western blotting. DBC1 knockdown C2C12 cells and myotubes were investigated using immunofluorescence staining, Seahorse, immunohistology, fluorescence-activated cell sorting and RNA-sequencing analyses. RESULTS: DBC1 knockdown in skeletal muscle of young mice led to signatures of muscle atrophy, including a 28% reduction in muscle grip force (P = 0.023), a 54.4% reduction in running distance (P = 0.002), a 14.3% reduction in muscle mass (P = 0.007) and significantly smaller myofibre cross-sectional areas (P < 0.0001). DBC1 levels decrease in age-related or limb immobilization-induced atrophic mouse muscles and overexpress DBC1-attenuated atrophic phenotypes in these mice. Muscle regeneration was hampered in mice with CTX-induced muscle injury by DBC1 knockdown, as evidenced by reductions in myofibre cross-sectional areas of regenerating myofibres with centralized nuclei (P < 0.0001), percentages of MyoG+ nuclei (P < 0.0001) and fusion index (P < 0.0001). DBC1 transcriptionally regulated mouse double minute 2 (MDM2), which mediated ubiquitination and degradation of forkhead box O3 (FOXO3). Increased FOXO3 proteins hampered myogenesis in DBC1 knockdown satellite cells by compromising around 50% of mitochondrial functions (P < 0.001) and exacerbated atrophy in DBC1 knockdown myofibres by activating the ubiquitin-proteasome and autophagy-lysosome pathways. CONCLUSIONS: DBC1 is essential in maintaining skeletal muscle integrity by protecting against myofibres wasting and enhancing muscle regeneration via FOXO3. This research highlights the significance of DBC1 for healthy skeletal muscle function and its connection to muscular atrophy.

Protein Phosphatase 2ACα Regulates ATR-Mediated Endogenous DNA Damage Response Against Microcephaly.

Protein phosphatase 2A (PP2A) is an abundant heterotrimeric holoenzyme in eukaryotic cells coordinating with specific kinases to regulate spatial-temporal protein dephosphorylation in various biological processes. However, the function of PP2A in cortical neurogenesis remains largely unknown. Here, we report that neuronal-specific deletion of Pp2acα in mice displayed microcephaly, with significantly smaller brains and defective learning and memory ability. Mechanistically, neuronal Pp2acα deficiency resulted in elevated endogenous DNA damage and activation of ATR/CHK1 signaling. It was further induced by the loss of direct interaction between PP2AC and ATR as well as the function of PP2AC to dephosphorylate ATR. Importantly, ATR/CHK1 signaling dysregulation altered both the expression and activity of several critical downstream factors including P53, P21, Bcl2, and Bax, which led to decreased proliferation of cortical progenitor cells and increased apoptosis in developing cortical neurons. Taken together, our results indicate an essential function of PP2ACα in endogenous DNA damage response-mediated ATR signaling during neurogenesis, and defective PP2ACα in neurons contributes to microcephaly.

Sprouts of RNA epigenetics: the discovery of mammalian RNA demethylases.

More than 100 structurally distinct RNA modifications have been identified in all kingdoms of life. These post-transcriptional modifications are widely present in various RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), etc. We have shown that the methylation of N(6)-methyladenine (m(6)A) can be reversed through the discovery of the first RNA demethylase, the human fat mass and obesity-associated protein, FTO, in 2011. (Most recently, we have identified a new mammalian RNA demethylase, ALKBH5, which is also able to remove the methyl group of m(6)A from RNA both in vitro and in vivo (Fig. 1A). The ALKBH5 protein colocalizes with nuclear speckles where pre-mRNA processing occurs. This protein is actively involved in mRNA export regulation, in which its demethylation activity seems to play an important role, as well as in RNA synthesis. A knockout of the Alkbh5 gene in mice resulted in impaired male fertility due to compromised spermatogenesis. Importantly, increased m(6)A levels were observed in mRNA isolated from the Alkbh5-knockout mouse organs compared to those from wild-type littermates. RNA-Seq results indicate aberrant gene expression in spermatogenic cells of the seminoferous tubulus of testes from Alkbh5-deficient mice, thereby showing that the loss of the m(6)A demethylase influences gene expression, which, in turn, leads to defects in spermatogenesis and increased apoptosis of meiotic cells. Thus, the discovery of FTO and this new RNA demethylase strongly suggests that the methylation of RNA, like DNA and histone modifications, is dynamically regulated and likely to play broad roles in mammalian cells.

Mic60 is essential to maintain mitochondrial integrity and to prevent encephalomyopathy.

Mitochondrial encephalomyopathies (ME) are frequently associated with mutations of mitochondrial DNA, but the pathogenesis of a subset of ME (sME) remains elusive. Here we report that haploinsufficiency of a mitochondrial inner membrane protein, Mic60, causes progressive neurological abnormalities with insulted mitochondrial structure and neuronal loss in mice. In addition, haploinsufficiency of Mic60 reduces mitochondrial membrane potential and cellular ATP production, increases reactive oxygen species, and alters mitochondrial oxidative phosphorylation complexes in neurons in an age-dependent manner. Moreover, haploinsufficiency of Mic60 compromises brain glucose intake and oxygen consumption in mice, resembling human ME syndrome. We further discover that MIC60 protein expression declined significantly in human sME, implying that insufficient MIC60 may contribute for pathogenesis of human ME. Notably, systemic administration of antioxidant N-acetylcysteine largely reverses mitochondrial dysfunctions and metabolic disorders in haplo-insufficient Mic60 mice, also restores neurological abnormal symptom. These results reveal Mic60 is required in the maintenance of mitochondrial integrity and function, and likely a potential therapeutics target for mitochondrial encephalomyopathies.

NF-kappaB protects human papillomavirus type 38 E6/E7-immortalized human keratinocytes against tumor necrosis factor alpha and UV-mediated apoptosis.

Constitutive activation of NF-κB signaling is a key event in virus- and non-virus-induced carcinogenesis. We have previously reported that cutaneous human papillomavirus type 38 (HPV38) displays transforming properties in in vitro and in vivo experimental models. However, the involvement of NF-κB signaling in HPV38-induced cell growth transformation remains to be determined. In this study, we showed that HPV38 E6 and E7 activate NF-κB and that inhibition of the pathway with the IκBα superrepressor sensitizes HPV38E6E7-immortalized human keratinocytes to tumor necrosis factor alpha (TNF-α)- and UVB radiation-mediated apoptosis. Accordingly, inhibition of NF-κB signaling resulted in the downregulation of NF-κB-regulated antiapoptotic genes, including cIAP1, cIAP2, and xIAP genes. These findings demonstrate a critical role of NF-κB activity in the survival of HPV38E6E7-immortalized human keratinocytes exposed to cytokine or UV radiation. Our data provide additional evidence for cooperation between beta HPV infection and UV irradiation in skin carcinogenesis.

Cutaneous human papillomavirus type 38 E7 regulates actin cytoskeleton structure for increasing cell proliferation through CK2 and the eukaryotic elongation factor 1A.

We previously reported that the oncoproteins E6 and E7 from cutaneous human papillomavirus type 38 (HPV38) can immortalize primary human keratinocytes in vitro and sensitize transgenic mice to develop skin cancer in vivo. Immunofluorescence staining revealed that human keratinocytes immortalized by HPV38 E6 and E7 display fewer actin stress fibers than do control primary keratinocyte cells, raising the possibility of a role of the viral oncoproteins in the remodeling of the actin cytoskeleton. In this study, we show that HPV38 E7 induces actin stress fiber disruption and that this phenomenon correlates with its ability to downregulate Rho activity. The downregulation of Rho activity by HPV38 E7 is mediated through the activation of the CK2-MEK-extracellular signal-regulated kinase (ERK) pathway. In addition, HPV38 E7 is able to induce actin fiber disruption by binding directly to eukaryotic elongation factor 1A (eEF1A) and abolishing its effects on actin fiber formation. Finally, we found that the downregulation of Rho activity by HPV38 E7 through the CK2-MEK-ERK pathway facilitates cell growth proliferation. Taken together, our data support the conclusion that HPV38 E7 promotes keratinocyte proliferation in part by negatively regulating actin cytoskeleton fiber formation through the CK2-MEK-ERK-Rho pathway and by binding to eEF1A and inhibiting its effects on actin cytoskeleton remodeling.

m6A demethylase ALKBH5 is required for antibacterial innate defense by intrinsic motivation of neutrophil migration.

Neutrophil migration into the site of infection is necessary for antibacterial innate defense, whereas impaired neutrophil migration may result in excessive inflammation and even sepsis. The neutrophil migration directed by extracellular signals such as chemokines has been extensively studied, yet the intrinsic mechanism for determining neutrophil ability to migrate needs further investigation. N6-methyladenosine (m6A) RNA modification is important in immunity and inflammation, and our preliminary data indicate downregulation of RNA m6A demethylase alkB homolog 5 (ALKBH5) in neutrophils during bacterial infection. Whether m6A modification and ALKBH5 might intrinsically modulate neutrophil innate response remain unknown. Here we report that ALKBH5 is required for antibacterial innate defense by enhancing intrinsic ability of neutrophil migration. We found that deficiency of ALKBH5 increased mortality of mice with polymicrobial sepsis induced by cecal ligation and puncture (CLP), and Alkbh5-deficient CLP mice exhibited higher bacterial burden and massive proinflammatory cytokine production in the peritoneal cavity and blood because of less neutrophil migration. Alkbh5-deficient neutrophils had lower CXCR2 expression, thus exhibiting impaired migration toward chemokine CXCL2. Mechanistically, ALKBH5-mediated m6A demethylation empowered neutrophils with high migration capability through altering the RNA decay, consequently regulating protein expression of its targets, neutrophil migration-related molecules, including increased expression of neutrophil migration-promoting CXCR2 and NLRP12, but decreased expression of neutrophil migration-suppressive PTGER4, TNC, and WNK1. Our findings reveal a previously unknown role of ALKBH5 in imprinting migration-promoting transcriptome signatures in neutrophils and intrinsically promoting neutrophil migration for antibacterial defense, highlighting the potential application of targeting neutrophil m6A modification in controlling bacterial infections.

METTL3-mediated RNA m6A Hypermethylation Promotes Tumorigenesis and GH Secretion of Pituitary Somatotroph Adenomas.

INTRODUCTION: Pituitary growth hormone-secreting (GH) pituitary adenomas (PAs) cause mass effects and dysregulated hypersecretion of GH. However, somatic mutation burden is low in PAs. While progress has been made in identifying the epigenetic changes involved in GH-PA initiation, the precise details of its tumorigenesis in GH-PA patients remains to be elucidated. As N6-methyladenosine (m6A) has been shown to often play a critical role in various tumors, it represents a possible initiation point for the tumorigenesis of pituitary adenomas. However, the role of RNA methylation in GH adenomas remains unclear. METHODS: Protein expression of m6A regulators was measured by immunohistochemistry. Global levels and distribution of m6A methylation were separately analyzed by m6A enzyme-linked immunosorbent assay and m6A sequencing (m6A-seq). RNA interference and lentivirus knockdown system were used to investigate the role of methyltransferase-like 3 (METTL3) and its m6A- dependent regulatory mechanism in tumor progression and GH secretion. RESULTS: We show that both METTL3 messenger RNA and protein expression are elevated in GH-PA samples when compared with both normal pituitary tissue specimens and nonsecreting pituitary adenomas. Levels of m6A modification increased in GH-PAs, and hypermethylated RNAs are involved in hormone secretion and cell development. Knockdown of METTL3 in GH3 cell line resulted in decreased cell growth and GH secretion. Importantly, we found that GNAS and GADD45γ act as the downstream targets in this process. CONCLUSION: Our findings strongly suggest that m6A methyltransferase METTL3 promotes tumor growth and hormone secretion by increasing expression of GNAS and GADD45γ in a m6A-dependent manner. Thus, METTL3 and the methylated RNAs constitute suitable targets for clinical treatment of GH-PAs.

Rare and misincorporated DNA N6-methyladenine is a hallmark of cytotoxic stresses for selectively stimulating the stemness and proliferation of glioblastoma cells.

The entity of DNA N6-methyladenine (6mA) in mammals remains elusive and subsequently its roles in diseases are poorly understood. Here we exploited a bacterial DNA contamination-free and ultrasensitive UHPLC-MS/MS assay to reassess DNA 6mA in human glioblastomas and unveiled that DNA 6mA (~0.08 ppm) is extremely rare. By the use of two independent heavy stable isotope-labeling strategies, we further prove that the observed 6mA is solely generated by DNA polymerase-mediated misinocorporation. In vitro experiments point toward that the generation of misincorporated DNA 6mA is associated with the cellular stresses-caused release of RNA N6-methyladenine (m6A) nucleoside, which is profoundly inhibited by hypoxia milieu. Consistently, compared with normal brain tissues, DNA 6mA decreases in hypoxic human gliomas. Our data also strongly support that rare DNA 6mA rather than relatively abundant DNA 5-methylcytosine and 5-hydroxymethylcytosine is a hallmark of poor prognosis of IDH1/2 mutation-absent glioblastoma patients, reflecting the incidence of cytotoxic stresses and subsequent release of m6A nucleoside. The released m6A nucleoside may selectively preserve a subset of the glioblastoma cells and stimulate their stemness and proliferation. Noteworthily, demethylation-inhibiting IDH1 mutation increases the DNA 6mA content in human gliomas, but the depletion of the demethylase candidate ALKBH1 fails to do so, together suggesting the presence of other unknown 6mA demethylase for erasing misincorporated DNA 6mA. This is the first report on the identification of the misincorporated 6mA together with its origin and roles in diseases.

METTL3 regulates m6A methylation of PTCH1 and GLI2 in Sonic hedgehog signaling to promote tumor progression in SHH-medulloblastoma.

SHH subgroup medulloblastoma (SHH-MB) is one of the most common malignant pediatric tumors that arises in the cerebellum. Previously, we showed that RNA m6A methylation participates in regulation of cerebellar development. Here we investigate whether dysregulated m6A methylation contributes to tumorigenesis of SHH-MB. We show that high expression of m6A methyltransferase METTL3 associates with worse survival in the patients with SHH-MB. A large number of hypermethylated transcripts are identified in SHH-MB tumor cells by m6A-seq. We find that METTL3 promotes tumor progression via activating Sonic hedgehog signaling. Mechanistically, METTL3 methylates PTCH1 and GLI2 RNAs and further regulates their RNA stability and translation. Importantly, targeting METTL3 by depleting METTL3 expression or treatment with its catalytic inhibitor STM2457 restrains tumor progression. Collectively, this study shows a critical function for METTL3 and m6A methylation in SHH-MB, indicative of a potential role of METTL3 as therapeutic target in SHH-MB.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

RNA m6A Demethylase ALKBH5 Protects Against Pancreatic Ductal Adenocarcinoma via Targeting Regulators of Iron Metabolism.

Although RNA m6A regulators have been implicated in the tumorigenesis of several different types of tumors, including pancreatic cancer, their clinical relevance and intrinsic regulatory mechanism remain elusive. This study analyzed eight m6A regulators (METTL3, METTL14, WTAP, FTO, ALKBH5, and YTHDF1-3) in pancreatic ductal adenocarcinoma (PDAC) and found that only RNA m6A demethylase ALKBH5 serves as an independent favorable prognostic marker for this tumor. To better understand the molecular mechanism underlying the protective effect conferred by ALKBH5 against pancreatic tumorigenesis, we performed a transcriptome-wide analysis of m6A methylation, gene expression, and alternative splicing (AS) using the MIA PaCa-2 stable cell line with ALKBH5 overexpression. We demonstrated that ALKBH5 overexpression induced a reduction in RNA m6A levels globally. Furthermore, mRNAs encoding ubiquitin ligase FBXL5, and mitochondrial iron importers SLC25A28 and SLC25A37, were identified as substrates of ALKBH5. Mechanistically, the RNA stabilities of FBXL5 and SLC25A28, and the AS of SLC25A37 were affected, which led to their upregulation in pancreatic cancer cell line. Particularly, we observed that downregulation of FBXL5 in tumor samples correlated with shorter survival time of patients. Owing to FBXL5-mediated degradation, ALKBH5 overexpression incurred a significant reduction in iron-regulatory protein IRP2 and the modulator of epithelial-mesenchymal transition (EMT) SNAI1. Notably, ALKBH5 overexpression led to a significant reduction in intracellular iron levels as well as cell migratory and invasive abilities, which could be rescued by knocking down FBXL5. Overall, our results reveal a previously uncharacterized mechanism of ALKBH5 in protecting against PDAC through modulating regulators of iron metabolism and underscore the multifaceted role of m6A in pancreatic cancer.

Loss of 5-Hydroxymethylcytosine as an Epigenetic Signature That Correlates With Poor Outcomes in Patients With Medulloblastoma.

Medulloblastoma, as the most common malignant brain tumor in children, exhibits highly dysregulated DNA methylation. The novel epigenetic marker-5-hydroxymethylcytosine (5hmC) plays essential role in gene regulation during brain development and in brain tumors. However, the biological and clinical implications of 5hmC in medulloblastoma are still unclear. Here, we detected global 5hmC levels in two independent medulloblastoma patient cohorts (discovery cohort: n = 81; validation cohort: n = 171) using ultra-high performance liquid chromatography-tandem mass spectrometry analysis. Immunohistochemistry was used to identify the cell proliferation and expression of Ten-eleven translocation 1 and 2 (TET1/2). The prognostic impacts of covariates on progression-free survival (PFS) and overall survival (OS) were evaluated using multivariate Cox hazards regression models. We observed that global 5hmC levels were decreased in medulloblastomas compared to normal cerebellums (P < 0.001). Multivariate analysis showed that low global 5hmC levels correlated with poor PFS and OS rates (discovery cohort: PFS: P = 0.003, OS: P = 0.002; validation cohort: PFS: P = 0.0002, OS: P = 0.001). Immunohistochemistry showed an inverse correlation between 5hmC score and Ki-67 index (r = -0.747, P < 0.0001). Moreover, 5hmC score in MB samples was associated with nuclear expression of TET1 (r = -0.419, P = 0.003) and TET2 (r = -0.399, P = 0.005) proteins. Our study demonstrates that loss of 5hmC is an epigenetic biomarker in medulloblastomas. Our results indicate that 5hmC could be a candidate prognostic indicator for improving survival prediction of risk stratification in patients with medulloblastoma.

Genome-wide 5-Hydroxymethylcytosine Profiling Analysis Identifies MAP7D1 as A Novel Regulator of Lymph Node Metastasis in Breast Cancer.

Although DNA 5-hydroxymethylcytosine (5hmC) is recognized as an important epigenetic mark in cancer, its precise role in lymph node metastasis remains elusive. In this study, we investigated how 5hmC associates with lymph node metastasis in breast cancer. Accompanying with high expression of TET1 and TET2 proteins, large numbers of genes in the metastasis-positive primary tumors exhibit higher 5hmC levels than those in the metastasis-negative primary tumors. In contrast, the TET protein expression and DNA 5hmC decrease significantly within the metastatic lesions in the lymph nodes compared to those in their matched primary tumors. Through genome-wide analysis of 8 sets of primary tumors, we identified 100 high-confidence metastasis-associated 5hmC signatures, and it is found that increased levels of DNA 5hmC and gene expression of MAP7D1 associate with high risk of lymph node metastasis. Furthermore, we demonstrate that MAP7D1, regulated by TET1, promotes tumor growth and metastasis. In conclusion, the dynamic 5hmC profiles during lymph node metastasis suggest a link between DNA 5hmC and lymph node metastasis. Meanwhile, the role of MAP7D1 in breast cancer progression suggests that the metastasis-associated 5hmC signatures are potential biomarkers to predict the risk for lymph node metastasis, which may serve as diagnostic and therapeutic targets for metastatic breast cancer.