PRMT1 promotes Warburg effect by regulating the PKM2/PKM1 ratio in non-small cell lung cancer.

Abnormal epigenetic modifications are involved in the regulation of Warburg effect in tumor cells. Protein arginine methyltransferases (PRMTs) mediate arginine methylation and have critical functions in cellular responses. PRMTs are deregulated in a variety of cancers, but their precise roles in Warburg effect in cancer is largely unknown. Experiments from the current study showed that PRMT1 was highly expressed under conditions of glucose sufficiency. PRMT1 induced an increase in the PKM2/PKM1 ratio through upregulation of PTBP1, in turn, promoting aerobic glycolysis in non-small cell lung cancer (NSCLC). The PRMT1 level in p53-deficient and p53-mutated NSCLC remained relatively unchanged while the expression was reduced in p53 wild-type NSCLC under conditions of glucose insufficiency. Notably, p53 activation under glucose-deficient conditions could suppress USP7 and further accelerate the polyubiquitin-dependent degradation of PRMT1. Melatonin, a hormone that inhibits glucose intake, markedly suppressed cell proliferation of p53 wild-type NSCLC, while a combination of melatonin and the USP7 inhibitor P5091 enhanced the anticancer activity in p53-deficient NSCLC. Our collective findings support a role of PRMT1 in the regulation of Warburg effect in NSCLC. Moreover, combination treatment with melatonin and the USP7 inhibitor showed good efficacy, providing a rationale for the development of PRMT1-based therapy to improve p53-deficient NSCLC outcomes.

Unique progerin C-terminal peptide ameliorates Hutchinson-Gilford progeria syndrome phenotype by rescuing BUBR1.

An accumulating body of evidence indicates an association between mitotic defects and the aging process in Hutchinson-Gilford progeria syndrome (HGPS), which is a premature aging disease caused by progerin accumulation. Here, we found that BUBR1, a core component of the spindle assembly checkpoint, was downregulated during HGPS cellular senescence. The remaining BUBR1 was anchored to the nuclear membrane by binding with the C terminus of progerin, thus further limiting the function of BUBR1. Based on this, we established a unique progerin C-terminal peptide (UPCP) that effectively blocked the binding of progerin and BUBR1 and enhanced the expression of BUBR1 by interfering with the interaction between PTBP1 and progerin. Finally, UPCP significantly inhibited HGPS cellular senescence and ameliorated progeroid phenotypes, extending the lifespan of LmnaG609G/G609G mice. Our findings reveal an essential role for the progerin-PTBP1-BUBR1 axis in HGPS. Therapeutics designed around UPCP may be a beneficial strategy for HGPS treatment.

Anti-hsa-miR-59 alleviates premature senescence associated with Hutchinson-Gilford progeria syndrome in mice.

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature aging disorder without an effective therapeutic regimen. Because of their targetability and influence on gene expression, microRNAs (miRNAs) are attractive therapeutic tools to treat diseases. Here we identified that hsa-miR-59 (miR-59) was markedly upregulated in HGPS patient cells and in multiple tissues of an HGPS mouse model (LmnaG609G/G609G ), which disturbed the interaction between RNAPII and TFIIH, resulting in abnormal expression of cell cycle genes by targeting high-mobility group A family HMGA1 and HMGA2. Functional inhibition of miR-59 alleviated the cellular senescence phenotype of HGPS cells. Treatment with AAV9-mediated anti-miR-59 reduced fibrosis in the quadriceps muscle, heart, and aorta, suppressed epidermal thinning and dermal fat loss, and yielded a 25.5% increase in longevity of LmnaG609G/G609G mice. These results identify a new strategy for the treatment of HGPS and provide insight into the etiology of HGPS disease.

Dual Inhibition of H3K9me2 and H3K27me3 Promotes Tumor Cell Senescence without Triggering the Secretion of SASP.

Chemotherapy remains the most common cancer treatment. Although chemotherapeutic drugs induce tumor cell senescence, they are often associated with post-therapy tumor recurrence by inducing the senescence-associated secretory phenotype (SASP). Therefore, it is important to identify effective strategies to induce tumor cell senescence without triggering SASP. In this study, we used the small molecule inhibitors, UNC0642 (G9a inhibitor) and UNC1999 (EZH2 inhibitor) alone or in combination, to inhibit H3K9 and H3K27 methylation in different cancer cells. Dual inhibition of H3K9me2 and H3K27me3 in highly metastatic tumor cells had a stronger pro-senescence effect than either inhibitor alone and did not trigger SASP in tumor cells. Dual inhibition of H3K9me2 and H3K27me3 suppressed the formation of cytosolic chromatin fragments, which inhibited the cGAS-STING-SASP pathway. Collectively, these data suggested that dual inhibition of H3K9 and H3K27 methylation induced senescence of highly metastatic tumor cells without triggering SASP by inhibiting the cGAS-STING-SASP pathway, providing a new mechanism for the epigenetics-based therapy targeting H3K9 and H3K27 methylation.

EZH2-CCF-cGAS Axis Promotes Breast Cancer Metastasis.

Cytoplasmic chromatin fragments (CCF) are recognized by the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS), which activates the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway and promotes the production of inflammatory factors and breast cancer metastasis. However, the mechanisms by which CCF are formed in tumor cells and CCF activation cGAS promotes breast cancer metastasis remain unclear. Here, we report that the enhancer of zeste homolog 2 (EZH2) can promote the formation of CCF and activate the cGAS-STING pathway to promote breast cancer metastasis. Further research found that the EZH2-mediated CCF formation depended on high mobility group A1 (HMGA1), while the stability of EZH2 required ubiquitin-specific peptidase 7 (USP7), indicating that the EZH2-HMGA1-USP7 complex regulated CCF formation. Moreover, EZH2 can activate cGAS through CCF, requiring USP7 to deubiquitinate cGAS and stabilize cGAS. In vivo experimental results showed that EZH2 could promote breast cancer metastasis through CCF. Our findings highlight a new target for breast cancer metastasis. Targeting the EZH2-CCF-cGAS axis may be a potential therapeutic strategy for inhibiting breast cancer metastasis.

Arginine and lysine methylation of MRPS23 promotes breast cancer metastasis through regulating OXPHOS.

Mitochondrial oxidative phosphorylation (OXPHOS) is a vital regulator of tumor metastasis. However, the mechanisms governing OXPHOS to facilitate tumor metastasis remain unclear. In this study, we discovered that arginine 21(R21) and lysine 108 (K108) of mitochondrial ribosomal protein S23 (MRPS23) was methylated by the protein arginine methyltransferase 7 (PRMT7) and SET-domain-containing protein 6 (SETD6), respectively. R21 methylation accelerated the poly-ubiquitin-dependent degradation of MRPS23 to a low level. The MRPS23 degradation inhibited OXPHOS with elevated mtROS level, which consequently increased breast cancer cell invasion and metastasis. In contrast, K108 methylation increased MRPS23 stability, and K108 methylation coordinated with R21 methylation to maintain a low level of MRPS23, which was in favor of supporting breast cancer cell survival through regulating OXPHOS. Consistently, R21 and K108 methylation was correlated with malignant breast carcinoma. Significantly, our findings unveil a unique mechanism of controlling OXPHOS by arginine and lysine methylation and point to the impact of the PRMT7-SETD6-MRPS23 axis during breast cancer metastasis.

The Interaction of the Senescent and Adjacent Breast Cancer Cells Promotes the Metastasis of Heterogeneous Breast Cancer Cells through Notch Signaling.

Chemotherapy is one of the most common strategies for tumor treatment but often associated with post-therapy tumor recurrence. While chemotherapeutic drugs are known to induce tumor cell senescence, the roles and mechanisms of senescence in tumor recurrence remain unclear. In this study, we used doxorubicin to induce senescence in breast cancer cells, followed by culture of breast cancer cells with conditional media of senescent breast cancer cells (indirect co-culture) or directly with senescent breast cancer cells (direct co-culture). We showed that breast cancer cells underwent the epithelial-mesenchymal transition (EMT) to a greater extent and had stronger migration and invasion ability in the direct co-culture compared with that in the indirect co-culture model. Moreover, in the direct co-culture model, non-senescent breast cancer cells facilitated senescent breast cancer cells to escape and re-enter into the cell cycle. Meanwhile, senescent breast cancer cells regained tumor cell characteristics and underwent EMT after direct co-culture. We found that the Notch signaling was activated in both senescent and non-senescent breast cancer cells in the direct co-culture group. Notably, the EMT process of senescent and adjacent breast cancer cells was blocked upon inhibition of Notch signaling with N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) in the direct co-cultures. In addition, DAPT inhibited the lung metastasis of the co-cultured breast cancer cells in vivo. Collectively, data arising from this study suggest that both senescent and adjacent non-senescent breast cancer cells developed EMT through activating Notch signaling under conditions of intratumoral heterogeneity caused by chemotherapy, which infer the possibility that Notch inhibitors used in combination with chemotherapeutic agents may become an effective treatment strategy.

Methylation of EZH2 by PRMT1 regulates its stability and promotes breast cancer metastasis.

Enhancer of zeste homolog 2 (EZH2), a key histone methyltransferase and EMT inducer, is overexpressed in diverse carcinomas, including breast cancer. However, the molecular mechanisms of EZH2 dysregulation in cancers are still largely unknown. Here, we discover that EZH2 is asymmetrically dimethylated at R342 (meR342-EZH2) by PRMT1. meR342-EZH2 was found to inhibit the CDK1-mediated phosphorylation of EZH2 at T345 and T487, thereby attenuating EZH2 ubiquitylation mediated by the E3 ligase TRAF6. We also demonstrate that meR342-EZH2 resulted in a decrease in EZH2 target gene expression, but an increase in breast cancer cell EMT, invasion and metastasis. Moreover, we confirm the positive correlations among PRMT1, meR342-EZH2 and EZH2 expression in the breast cancer tissues. Finally, we report that high expression levels of meR342-EZH2 predict a poor clinical outcome in breast cancer patients. Our findings may provide a novel diagnostic target and promising therapeutic target for breast cancer metastasis.

Arginine methylation of SHANK2 by PRMT7 promotes human breast cancer metastasis through activating endosomal FAK signalling.

Arginine methyltransferase PRMT7 is associated with human breast cancer metastasis. Endosomal FAK signalling is critical for cancer cell migration. Here we identified the pivotal roles of PRMT7 in promoting endosomal FAK signalling activation during breast cancer metastasis. PRMT7 exerted its functions through binding to scaffold protein SHANK2 and catalyzing di-methylation of SHANK2 at R240. SHANK2 R240 methylation exposed ANK domain by disrupting its SPN-ANK domain blockade, promoting in co-accumulation of dynamin2, talin, FAK, cortactin with SHANK2 on endosomes. In addition, SHANK2 R240 methylation activated endosomal FAK/cortactin signals in vitro and in vivo. Consistently, all the levels of PRMT7, methylated SHANK2, FAK Y397 phosphorylation and cortactin Y421 phosphorylation were correlated with aggressive clinical breast cancer tissues. These findings characterize the PRMT7-dependent SHANK2 methylation as a key player in mediating endosomal FAK signals activation, also point to the value of SHANK2 R240 methylation as a target for breast cancer metastasis.

ELT-2 promotes O-GlcNAc transferase OGT-1 expression to modulate Caenorhabditis elegans lifespan.

O-GlcNAc transferase (OGT) is the enzyme catalyzing protein O-GlcNAcylation by addition of a single O-linked-ß-N-acetylglucosamine molecule (O-GlcNAc) to nuclear and cytoplasmic targets, and it uses uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a donor. As UDP-GlcNAc is the final product of the nutrient-sensing hexosamine signaling pathway, overexpression or knockout of ogt in mammals or invertebrate models influences cellular nutrient-response signals and increases susceptibility to chronic diseases of aging. Evidence shows that OGT expression levels decrease in tissues of older mice and rats. However, how OGT expression is modulated in the aging process remains poorly understood. In Caenorhabditis elegans, the exclusive mammalian OGT ortholog OGT-1 is crucial for lifespan control. Here, we observe that worm OGT-1 expression gradually reduces during aging. By combining prediction via the "MATCH" algorithm and luciferase reporter assays, GATA factor ELT-2, the homolog of human GATA4, is identified as a transcriptional factor driving OGT-1 expression. Chromatin immunoprecipitation-quantitative polymerase chain reaction and electrophoretic mobility shift assays show ELT-2 directly binds to and activates the ogt-1 promoter. Knockdown of elt-2 decreases the global O-GlcNAc modification level and reduces the lifespan of wild-type worms. The reduction in lifespan caused by elt-2 RNA interference is abrogated by the loss of ogt-1. These results imply that GATA factors are able to activate OGT expression, which could be beneficial for longevity and the development of therapeutic treatment for aging-related diseases.

Arginine hypomethylation-mediated proteasomal degradation of histone H4-an early biomarker of cellular senescence.

Senescence is accompanied with histones level alteration; however, the roles and the mechanisms of histone reduction in cellular senescence are largely unknown. Protein arginine methyltransferase 1 (PRMT1) is the major enzyme that generates monomethyl and asymmetrical dimethyl arginine. Here we showed that abrogation of PRMT1-mediated senescence was accompanied with decreasing histone H4 level. Consistently, under multiple classic senescence models, H4 decreasing was also been found prior to the other 3 core histones. Noticeably, asymmetric demethylation of histone H4 at arginine 3 (H4R3me2as), catalyzed by PRMT1, was decreased prior to histone H4. In addition, we showed that the PRMT1-mediated H4R3me2as maintained H4 stability. Reduction of H4R3me2as level increased the interaction between proteasome activator PA200 and histone H4, which catalyzes the poly-ubiquitin-independent degradation of H4. Moreover, H4 degradation promoted nucleosome decomposition, resulting in increased senescence-associated genes transcription. Significantly, H4 was restored by 3 well-informed anti-aging drugs (metformin, rapamycin, and resveratrol) much earlier than other senescence markers detected under H2O2-induced senescence. Thus, we uncovered a novel function of H4R3me2as in modulation of cellular senescence via regulating H4 stability. This finding also points to the value of histone H4 as a senescence indicator and a potential anti-aging drug screening marker.

Arginine methylation-dependent LSD1 stability promotes invasion and metastasis of breast cancer.

Histone lysine demethylase 1 (LSD1), the first identified histone demethylase, is overexpressed in multiple tumor types, including breast cancer. However, the mechanisms that cause LSD1 dysregulation in breast cancer remain largely unclear. Here, we report that protein arginine methyltransferase 4 (PRMT4 or CARM1) dimethylates LSD1 at R838, which promotes the binding of the deubiquitinase USP7, resulting in the deubiquitination and stabilization of LSD1. Moreover, CARM1- and USP7-dependent LSD1 stabilization plays a key role in repressing E-cadherin and activating vimentin transcription through promoter H3K4me2 and H3K9me2 demethylation, respectively, which promotes invasion and metastasis of breast cancer cells. Consistently, LSD1 arginine methylation levels correlate with tumor grade in human malignant breast carcinoma samples. Our findings unveil a unique mechanism controlling LSD1 stability by arginine methylation, also highlighting the role of the CARM1-USP7-LSD1 axis in breast cancer progression.

Autism candidate gene DIP2A regulates spine morphogenesis via acetylation of cortactin.

Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing.

SHON expression predicts response and relapse risk of breast cancer patients after anthracycline-based combination chemotherapy or tamoxifen treatment.

BACKGROUND: SHON nuclear expression (SHON-Nuc+) was previously reported to predict clinical outcomes to tamoxifen therapy in ERα+ breast cancer (BC). Herein we determined if SHON expression detected by specific monoclonal antibodies could provide a more accurate prediction and serve as a biomarker for anthracycline-based combination chemotherapy (ACT). METHODS: SHON expression was determined by immunohistochemistry in the Nottingham early-stage-BC cohort (n = 1,650) who, if eligible, received adjuvant tamoxifen; the Nottingham ERα- early-stage-BC (n = 697) patients who received adjuvant ACT; and the Nottingham locally advanced-BC cohort who received pre-operative ACT with/without taxanes (Neo-ACT, n = 120) and if eligible, 5-year adjuvant tamoxifen treatment. Prognostic significance of SHON and its relationship with the clinical outcome of treatments were analysed. RESULTS: As previously reported, SHON-Nuc+ in high risk/ERα+ patients was significantly associated with a 48% death risk reduction after exclusive adjuvant tamoxifen treatment compared with SHON-Nuc- [HR (95% CI) = 0.52 (0.34-0.78), p = 0.002]. Meanwhile, in ERα- patients treated with adjuvant ACT, SHON cytoplasmic expression (SHON-Cyto+) was significantly associated with a 50% death risk reduction compared with SHON-Cyto- [HR (95% CI) = 0.50 (0.34-0.73), p = 0.0003]. Moreover, in patients received Neo-ACT, SHON-Nuc- or SHON-Cyto+ was associated with an increased pathological complete response (pCR) compared with SHON-Nuc+ [21 vs 4%; OR (95% CI) = 5.88 (1.28-27.03), p = 0.012], or SHON-Cyto- [20.5 vs. 4.5%; OR (95% CI) = 5.43 (1.18-25.03), p = 0.017], respectively. After receiving Neo-ACT, patients with SHON-Nuc+ had a significantly lower distant relapse risk compared to those with SHON-Nuc- [HR (95% CI) = 0.41 (0.19-0.87), p = 0.038], whereas SHON-Cyto+ patients had a significantly higher distant relapse risk compared to SHON-Cyto- patients [HR (95% CI) = 4.63 (1.05-20.39), p = 0.043]. Furthermore, multivariate Cox regression analyses revealed that SHON-Cyto+ was independently associated with a higher risk of distant relapse after Neo-ACT and 5-year tamoxifen treatment [HR (95% CI) = 5.08 (1.13-44.52), p = 0.037]. The interaction term between ERα status and SHON-Nuc+ (p = 0.005), and between SHON-Nuc+ and tamoxifen therapy (p = 0.007), were both statistically significant. CONCLUSION: SHON-Nuce+ in tumours predicts response to tamoxifen in ERα+ BC while SHON-Cyto+ predicts response to ACT.

Arginine methylation of SKN-1 promotes oxidative stress resistance in Caenorhabditis elegans.

Caenorhabditis elegans NRF (NF-E2-related factor)/CNC (Cap'n'collar) transcription factor, Skinhead-1 (SKN-1), is conservatively critical for promoting phase II detoxification gene expressions in response to oxidative stress. SKN-1 activity is controlled by well-known phosphorylation and recently-reported O-GlcNAcylation. Whether other kinds of posttanslational modifications of SKN-1 occur and influence its function remains elusive. Here, we found arginines 484 and 516 (R484/R516) of SKN-1 were asymmetrically dimethylated by PRMT-1. Oxidative stress enhanced the binding of PRMT-1 to SKN-1. Consequently, asymmetrical dimethylation of arginines on SKN-1 was elevated. Loss of prmt-1 or disruption of R484/R516 dimethylation decreased the enrichment of SKN-1 on the promoters of SKN-1-driven phase II detoxification genes, including gamma-glutamine cysteine synthetase gcs-1, glutathione S-transferases gst-7 and gst-4, which resulted in reduced ability of worms to defense against oxidative stress. These findings have important implications for investigating the physiological and pathological functions of arginine methylation on conserved NRF/CNC transcription factors in human diseases related to oxidative stress response.

Muscle-Specific Histone H3K36 Dimethyltransferase SET-18 Shortens Lifespan of Caenorhabditis elegans by Repressing daf-16a Expression.

Mounting evidence shows that histone methylation, a typical epigenetic mark, is crucial for gene expression regulation during aging. Decreased trimethylation of Lys 36 on histone H3 (H3K36me3) in worms and yeast is reported to shorten lifespan. The function of H3K36me2 in aging remains unclear. In this study, we identified Caenorhabditis elegans SET-18 as a histone H3K36 dimethyltransferase. SET-18 deletion extended lifespan and increased oxidative stress resistance, dependent on daf-16 activity in the insulin/IGF pathway. In set-18 mutants, transcription of daf-16 isoform a (daf-16a) was specifically upregulated. Accordingly, a decrease in H3K36me2 on daf-16a promoter was observed. Muscle-specific expression of SET-18 increased in aged worms (day 7 and day 11), attributable to elevation of global H3K36me2 and inhibition of daf-16a expression. Consequently, longevity was shortened. These findings suggested that chromatic repression mediated by tissue-specific H3K36 dimethyltransferase might be detrimental to lifespan and may have implications in human age-related diseases.

miR-51 regulates GABAergic synapses by targeting Rab GEF GLO-4 and lysosomal trafficking-related GLO/AP-3 pathway in Caenorhabditis elegans.

A deficit of GABA (γ-aminobutyric acid) transmission will lead to epilepsy and other cognitive disorders. Recent evidence has shown that neuronal miRNAs affect various synapses, including GABAergic synapses. However, the miRNAs that control GABAergic synapses remain not fully understood. Here, we identified miR-51, a member of Caenorhabditis elegans miR-99/100 family, as a key regulator of GABAergic synapses. Loss of mir-51 increased PTZ (Pentylenetetrazole) and aldicarb hypersensitivities, and decreased the number of GABAergic synapses and abundance of GABAA receptors. A Rab guaninenucleotide exchange factor (GEF) GLO-4, a well-known component in lysosomal trafficking-related GLO-4/GLO-1/AP-3 (GLO/AP-3) pathway, was discovered to be the direct target of miR-51. Rescue experiments showed that GLO-4 expressed in GABAergic motor neurons functioned as a suppressor of miR-51. Disruption of glo-1 or AP-3 gene apm-3 attenuated the defects of GABAergic synapse in mir-51 mutants, suggesting miR-51 regulated GABAergic synapses through GLO/AP-3 pathway. The present study implies the essential roles of miRNAs on the nervous pathologies characterized by mis-regulated GABA signaling, such as epilepsy.

HDAC inhibitor PAC-320 induces G2/M cell cycle arrest and apoptosis in human prostate cancer.

HDAC inhibitors (HDACis) have been demonstrated with profound antiproliferative activities in various tumor types. Previously, we screened several polyoxometalate HDACis based on our p21 luciferase promoter system and demonstrated that such HDACis have antitumor activity. Here, we further investigate the antitumor mechanism of PAC-320, a compound among the polyoxometalates, in human prostate cancer. We demonstrate that PAC-320 is a broad-spectrum HDACi and could inhibit growth of prostate cancer cells in vitro and in vivo. Furthermore, we find that PAC-320 induces cell cycle arrest at G2/M phase and apoptosis. Mechanically, PAC-320 induced cell cycle arrest is associated with an increase of p21 and decrease of cyclin A and cyclin B1, while PAC-320 induced apoptosis is mediated through mitochondria apoptotic pathway and is closely associated with increase of BH3-only proteins Noxa and Hrk. Meanwhile, we demonstrate that p38 MAPK pathway is involved in PAC-320 induced antiproliferative activities in prostate cancer. Taken together, our data indicates that PAC-320 has potent prostate cancer inhibitory activity in vitro and in vivo, which is mediated by G2/M cell cycle arrest and apoptosis.