Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor.

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ T cells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ T cells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ T cell subset by coupled T cell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ T cells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ T cell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ T cell exhaustion and could be targeted to potentiate cancer immunotherapy.

The Ginkgo biloba microRNA160-ERF4 module participates in terpene trilactone biosynthesis.

Terpene trilactones (TTLs) are important secondary metabolites in ginkgo (Ginkgo biloba); however, their biosynthesis gene regulatory network remains unclear. Here, we isolated a G. biloba ethylene response factor 4 (GbERF4) involved in TTL synthesis. Overexpression of GbERF4 in tobacco (Nicotiana tabacum) significantly increased terpenoid content and upregulated the expression of key enzyme genes (3-hydroxy-3-methylglutaryl-CoA reductase [HMGR], 3-hydroxy-3-methylglutaryl-CoA synthase [HMGS], 1-deoxy-D-xylulose-5-phosphate reductoisomerase [DXR], 1-deoxy-D-xylulose-5-phosphate synthase [DXS], acetyl-CoA C-acetyltransferase [AACT], and geranylgeranyl diphosphate synthase [GGPPS]) in the terpenoid pathway in tobacco, suggesting that GbERF4 functions in regulating the synthesis of terpenoids. The expression pattern analysis and previous microRNA (miRNA) sequencing showed that gb-miR160 negatively regulates the biosynthesis of TTLs. Transgenic experiments showed that overexpression of gb-miR160 could significantly inhibit the accumulation of terpenoids in tobacco. Targeted inhibition and dual-luciferase reporter assays confirmed that gb-miR160 targets and negatively regulates GbERF4. Transient overexpression of GbERF4 increased TTL content in G. biloba, and further transcriptome analysis revealed that DXS, HMGS, CYPs, and transcription factor genes were upregulated. In addition, yeast 1-hybrid and dual-luciferase reporter assays showed that GbERF4 could bind to the promoters of the HMGS1, AACT1, DXS1, levopimaradiene synthase (LPS2), and GGPPS2 genes in the TTL biosynthesis pathway and activate their expression. In summary, this study investigated the molecular mechanism of the gb-miR160-GbERF4 regulatory module in regulating the biosynthesis of TTLs. It provides information for enriching the understanding of the regulatory network of TTL biosynthesis and offers important gene resources for the genetic improvement of G. biloba with high contents of TTLs.

Multiomics analysis provides new insights into the regulatory mechanism of carotenoid biosynthesis in yellow peach peel.

Carotenoids, as natural tetraterpenes, play a pivotal role in the yellow coloration of peaches and contribute to human dietary health. Despite a relatively clear understanding of the carotenoid biosynthesis pathway, the regulatory mechanism of miRNAs involved in carotenoid synthesis in yellow peaches remain poorly elucidated. This study investigated a total of 14 carotenoids and 40 xanthophyll lipids, including six differentially accumulated carotenoids: violaxanthin, neoxanthin, lutein, zeaxanthin, cryptoxanthin, and (E/Z)-phytoene. An integrated analysis of RNA-seq, miRNA-seq and degradome sequencing revealed that miRNAs could modulate structural genes such as PSY2, CRTISO, ZDS1, CHYB, VDE, ZEP, NCED1, NCED3 and the transcription factors NAC, ARF, WRKY, MYB, and bZIP, thereby participating in carotenoid biosynthesis and metabolism. The authenticity of miRNAs and target gene was corroborated through quantitative real-time PCR. Moreover, through weighted gene coexpression network analysis and a phylogenetic evolutionary study, coexpressed genes and MYB transcription factors potentially implicated in carotenoid synthesis were identified. The results of transient expression experiments indicated that mdm-miR858 inhibited the expression of PpMYB9 through targeted cleavage. Building upon these findings, a regulatory network governing miRNA-mediated carotenoid synthesis was proposed. In summary, this study comprehensively identified miRNAs engaged in carotenoid biosynthesis and their putative target genes, thus enhancing the understanding of carotenoid accumulation and regulatory mechanism in yellow peach peel and expanding the gene regulatory network of carotenoid synthesis.

SPTAN1/NUMB axis senses cell density to restrain cell growth and oncogenesis through Hippo signaling.

The loss of contact inhibition is a key step during carcinogenesis. The Hippo-Yes-associated protein (Hippo/YAP) pathway is an important regulator of cell growth in a cell density-dependent manner. However, how Hippo signaling senses cell density in this context remains elusive. Here, we report that high cell density induced the phosphorylation of spectrin α chain, nonerythrocytic 1 (SPTAN1), a plasma membrane-stabilizing protein, to recruit NUMB endocytic adaptor protein isoforms 1 and 2 (NUMB1/2), which further sequestered microtubule affinity-regulating kinases (MARKs) in the plasma membrane and rendered them inaccessible for phosphorylation and inhibition of the Hippo kinases sterile 20-like kinases MST1 and MST2 (MST1/2). WW45 interaction with MST1/2 was thereby enhanced, resulting in the activation of Hippo signaling to block YAP activity for cell contact inhibition. Importantly, low cell density led to SPTAN1 dephosphorylation and NUMB cytoplasmic location, along with MST1/2 inhibition and, consequently, YAP activation. Moreover, double KO of NUMB and WW45 in the liver led to appreciable organ enlargement and rapid tumorigenesis. Interestingly, NUMB isoforms 3 and 4, which have a truncated phosphotyrosine-binding (PTB) domain and are thus unable to interact with phosphorylated SPTAN1 and activate MST1/2, were selectively upregulated in liver cancer, which correlated with YAP activation. We have thus revealed a SPTAN1/NUMB1/2 axis that acts as a cell density sensor to restrain cell growth and oncogenesis by coupling external cell-cell contact signals to intracellular Hippo signaling.

[Effects of Xiangqin Jiere Granules on lipid metabolism and chronic inflammation in different obesity model mice].

This paper aims to study the effect of Xiangqin Jiere Granules(XQ) on lipid metabolism and chronic inflammation in different obesity model mice. The monosodium glutamate(MSG) obese mouse model was established by subcutaneous injection of MSG in newborn mice, and the high fat diet(HFD) obese mouse model was established by feeding adult mice with HFD. The normal mice were assigned into the control group; the MSG obese mice were assigned into MSG model group, XQ4.5 group(Xiangqin Jiere Granu-les, 4.5 g·kg~(-1)), XQ22.5 group(Xiangqin Jiere Granules, 22.5 g·kg~(-1)); the HFD obese mice were assigned into HFD model group, XQ4.5 group, and XQ22.5 group. The mice were intragastrically administrated with saline or XQ for 5 weeks. After that, the body weight, visceral fat mass, liver and thymus weight, and the organ indexes in each group were measured. The levels of triglyceride(TG), total cholesterol(TC), and low-density lipoprotein cholesterol(LDL-c) in serum and liver tissue were detected by the kits. The mRNA expression levels of acetyl CoA carboxylase 1(ACC1), fatty acid synthetase(FAS), diacylgycerol acyltransferase 1(DGAT1) and hepatic lipase(HTGL) involved in lipid metabolism in mouse liver tissue were detected by quantitative real-time PCR(qPCR). The protein levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in serum were detected by ELISA, and the mRNA levels of TNF-α and IL-6 in liver tissue were detected by qPCR. Compared with the control group, MSG and HFD mice showed increased body weight, abdominal circumference, Lee index and visceral fat mass as well as elevated levels of TG, TC, and LDL-c in serum. The model mice had up-regulated gene levels of ACC1, FAS and DGAT1 while down-regulated gene level of HTGL in the liver. Furthermore, the mRNA and protein levels of IL-6 increased in the model mice. Compared with the model mice, XQ treatment decreased the body weight, abdominal circumference, Lee index, and visceral fat mass, lowered the levels of TG, TC, and LDL-c in se-rum, down-regulated the gene levels of ACC1, FAS, and DGAT1 in liver tissue, up-regulated the gene level of HTGL, and down-regulated the mRNA and protein levels of IL-6. To sum up, XQ has good therapeutic effect on different obesity model mice. It can improve lipid metabolism and reduce fat accumulation in obese mice by regulating the enzymes involved in lipid metabolism, and alleviate obesity-related chronic low-grade inflammation.

Glycogen accumulation and phase separation drives liver tumor initiation.

Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage in pre-malignant cells. Accumulated glycogen undergoes liquid-liquid phase separation, which results in the assembly of the Laforin-Mst1/2 complex and consequently sequesters Hippo kinases Mst1/2 in glycogen liquid droplets to relieve their inhibition on Yap. Moreover, G6PC or another glycogenolysis enzyme-liver glycogen phosphorylase (PYGL) deficiency in both human and mice results in glycogen storage disease along with liver enlargement and tumorigenesis in a Yap-dependent manner. Consistently, elimination of glycogen accumulation abrogates liver growth and cancer incidence, whereas increasing glycogen storage accelerates tumorigenesis. Thus, we concluded that cancer-initiating cells adapt a glycogen storing mode, which blocks Hippo signaling through glycogen phase separation to augment tumor incidence.

PHF8-promoted TOPBP1 demethylation drives ATR activation and preserves genome stability.

The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.

Bimodal regulation of the PRC2 complex by USP7 underlies tumorigenesis.

Although overexpression of EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is an eminent feature of various cancers, the regulation of its abundance and function remains insufficiently understood. We report here that the PRC2 complex is physically associated with ubiquitin-specific protease USP7 in cancer cells where USP7 acts to deubiquitinate and stabilize EZH2. Interestingly, we found that USP7-catalyzed H2BK120ub1 deubiquitination is a prerequisite for chromatin loading of PRC2 thus H3K27 trimethylation, and this process is not affected by H2AK119 ubiquitination catalyzed by PRC1. Genome-wide analysis of the transcriptional targets of the USP7/PRC2 complex identified a cohort of genes including FOXO1 that are involved in cell growth and proliferation. We demonstrated that the USP7/PRC2 complex drives cancer cell proliferation and tumorigenesis in vitro and in vivo. We showed that the expression of both USP7 and EZH2 elevates during tumor progression, corresponding to a diminished FOXO1 expression, and the level of the expression of USP7 and EZH2 strongly correlates with histological grades and prognosis of tumor patients. These results reveal a dual role for USP7 in the regulation of the abundance and function of EZH2, supporting the pursuit of USP7 as a therapeutic target for cancer intervention.

PHF20L1 as a H3K27me2 reader coordinates with transcriptional repressors to promote breast tumorigenesis.

TUDOR domain-containing proteins (TDRDs) are chiefly responsible for recognizing methyl-lysine/arginine residue. However, how TDRD dysregulation contributes to breast tumorigenesis is poorly understood. Here, we report that TUDOR domain-containing PHF20L1 as a H3K27me2 reader exerts transcriptional repression by recruiting polycomb repressive complex 2 (PRC2) and Mi-2/nucleosome remodeling and deacetylase (NuRD) complex, linking PRC2-mediated methylation and NuRD-mediated deacetylation of H3K27. Furthermore, PHF20L1 was found to serve as a potential MYC and hypoxia-driven oncogene, promoting glycolysis, proliferation, and metastasis of breast cancer cells by directly inhibiting tumor suppressors such as HIC1, KISS1, and BRCA1. PHF20L1 expression was also strongly correlated with higher histologic grades of breast cancer and markedly up-regulated in several cancers. Meanwhile, Phf20l1 deletion not only induces growth retardation and mammary ductal outgrowth delay but also inhibits tumorigenesis in vivo. Our data indicate that PHF20L1 promotes tumorigenesis, supporting the pursuit of PHF20L1 as a target for cancer therapy.

Ubiquitin-specific protease 7 sustains DNA damage response and promotes cervical carcinogenesis.

Central to the recognition, signaling, and repair of DNA double-strand breaks (DSBs) are the MRE11-RAD50-NBS1 (MRN) complex and mediator of DNA damage checkpoint protein 1 (MDC1), the interplay of which is essential for initiation and amplification of the DNA damage response (DDR). The intrinsic rule governing the regulation of the function of this molecular machinery remains to be investigated. We report here that the ubiquitin-specific protease USP7 was physically associated with the MRN-MDC1 complex and that the MRN-MDC1 complex acted as a platform for USP7 to efficiently deubiquitinate and stabilize MDC1, thereby sustaining the DDR. Accordingly, depletion of USP7 impaired the engagement of the MRN-MDC1 complex and the consequent recruitment of the downstream factors p53-binding protein 1 (53BP1) and breast cancer protein 1 (BRCA1) at DNA lesions. Significantly, USP7 was overexpressed in cervical cancer, and the level of its expression positively correlated with that of MDC1 and worse survival rates for patients with cervical cancer. We demonstrate that USP7-mediated MDC1 stabilization promoted cervical cancer cell survival and conferred cellular resistance to genotoxic insults. Together, our study reveals a role for USP7 in regulating the function of the MRN-MDC1 complex and activity of the DDR, supporting the pursuit of USP7 as a potential therapeutic target for MDC1-proficient cancers.

USP9X regulates centrosome duplication and promotes breast carcinogenesis.

Defective centrosome duplication is implicated in microcephaly and primordial dwarfism as well as various ciliopathies and cancers. Yet, how the centrosome biogenesis is regulated remains poorly understood. Here we report that the X-linked deubiquitinase USP9X is physically associated with centriolar satellite protein CEP131, thereby stabilizing CEP131 through its deubiquitinase activity. We demonstrate that USP9X is an integral component of centrosome and is required for centrosome biogenesis. Loss-of-function of USP9X impairs centrosome duplication and gain-of-function of USP9X promotes centrosome amplification and chromosome instability. Significantly, USP9X is overexpressed in breast carcinomas, and its level of expression is correlated with that of CEP131 and higher histologic grades of breast cancer. Indeed, USP9X, through regulation of CEP131 abundance, promotes breast carcinogenesis. Our experiments identify USP9X as an important regulator of centrosome biogenesis and uncover a critical role for USP9X/CEP131 in breast carcinogenesis, supporting the pursuit of USP9X/CEP131 as potential targets for breast cancer intervention.

FOXK2 Elicits Massive Transcription Repression and Suppresses the Hypoxic Response and Breast Cancer Carcinogenesis.

Although clinically associated with severe developmental defects, the biological function of FOXK2 remains poorly explored. Here we report that FOXK2 interacts with transcription corepressor complexes NCoR/SMRT, SIN3A, NuRD, and REST/CoREST to repress a cohort of genes including HIF1ß and EZH2 and to regulate several signaling pathways including the hypoxic response. We show that FOXK2 inhibits the proliferation and invasion of breast cancer cells and suppresses the growth and metastasis of breast cancer. Interestingly, FOXK2 is transactivated by ERα and transrepressed via reciprocal successive feedback by HIF1ß/EZH2. Significantly, the expression of FOXK2 is progressively lost during breast cancer progression, and low FOXK2 expression is strongly correlated with higher histologic grades, positive lymph nodes, and ERα-/PR-/HER2- status, all indicators of poor prognosis.

Ubiquitin ligase RNF20/40 facilitates spindle assembly and promotes breast carcinogenesis through stabilizing motor protein Eg5.

Whether transcriptional regulators are functionally involved in mitosis is a fundamental question in cell biology. Here we report that the RNF20/40 complex, a major ubiquitin ligase catalysing histone H2B monoubiquitination, interacts with the motor protein Eg5 during mitosis and participates in spindle assembly. We show that the RNF20/40 complex monoubiquitinates and stabilizes Eg5. Loss of RNF20/40 results in spindle assembly defects, cell cycle arrest and apoptosis. Consistently, depletion of either RNF20/40 or Eg5 suppresses breast cancer in vivo. Significantly, RNF20/40 and Eg5 are concurrently upregulated in human breast carcinomas and high Eg5 expression is associated with poorer overall survival of patients with luminal A, or B, breast cancer. Our study uncovers an important spindle assembly role of the RNF20/40 complex, and implicates the RNF20/40-Eg5 axis in breast carcinogenesis, supporting the pursuit of these proteins as potential targets for breast cancer therapeutic interventions.

Stabilization of histone demethylase PHF8 by USP7 promotes breast carcinogenesis.

The histone demethylase PHF8 has been implicated in multiple pathological disorders, including X-linked mental retardation and tumorigenesis. However, it is not clear how the abundance and function of PHF8 are regulated. Here, we report that PHF8 physically associates with the deubiquitinase USP7. Specifically, we demonstrated that USP7 promotes deubiquitination and stabilization of PHF8, leading to the upregulation of a group of genes, including cyclin A2, that are critical for cell growth and proliferation. The USP7-encoding gene was also transcriptionally regulated by PHF8, via positive feedback. USP7 was overexpressed in breast carcinomas, and the level of expression positively correlated with expression of PHF8 and cyclin A2 and with the histological grade of breast cancer. We showed that USP7 promotes breast carcinogenesis by stabilizing PHF8 and upregulating cyclin A2 and that the interaction between USP7 and PHF8 is augmented during DNA damage. Moreover, USP7-promoted PHF8 stabilization conferred cellular resistance to genotoxic insults and was required for the recruitment of BLM and KU70, which are both essential for DNA double-strand break repair. Our study mechanistically links USP7 to epigenetic regulation and DNA repair. Moreover, these data support the pursuit of USP7 and PHF8 as potential targets for breast cancer intervention, especially in combination with chemo- or radiotherapies.

Dysfunction of the Reciprocal Feedback Loop between GATA3- and ZEB2-Nucleated Repression Programs Contributes to Breast Cancer Metastasis.

How loss-of-function of GATA3 contributes to the development of breast cancer is poorly understood. Here, we report that GATA3 nucleates a transcription repression program composed of G9A and MTA3-, but not MTA1- or MTA2-, constituted NuRD complex. Genome-wide analysis of the GATA3/G9A/NuRD(MTA3) targets identified a cohort of genes including ZEB2 that are critically involved in epithelial-to-mesenchymal transition and cell invasion. We demonstrate that the GATA3/G9A/NuRD(MTA3) complex inhibits the invasive potential of breast cancer cells in vitro and suppresses breast cancer metastasis in vivo. Strikingly, the expression of GATA3, G9A, and MTA3 is concurrently downregulated during breast cancer progression, leading to an elevated expression of ZEB2, which, in turn, represses the expression of G9A and MTA3 through the recruitment of G9A/NuRD(MTA1).

Hypouricemic effect of the methanol extract from Prunus mume fruit in mice.

CONTEXT: The fruit of the Prunus mume Sieb. et Zucc (Rosaceae) is used as a health food or medicinal material in traditional herb medicine for a long time in Eastern Asian countries. OBJECTIVE: Our present study investigated the hypouricemic effect of the methanol extract from P. mume fruit (MPMF) in mice with potassium oxonate-induced hyperuremia. MATERIALS AND METHODS: Effect of MPMF (35, 70 and 140 mg/kg, p.o.) administrated for 7 days on the serum, liver, urinary uric acid levels and liver xanthine oxidase (XO) activity were assessed in mice. RESULTS: Hyperuricemic mice induced by potassium oxonate demonstrated an elevation in serum and liver uric acid levels (11.0 mg/dL and 0.52 mg/g tissue) and a reduction in urinary uric acid levels (49.9 mg/dL). Oral administration of 140 mg/kg MPMF for 7 days reversed the abnormalities in serum, liver and urinary uric acid levels (7.1 mg/dL, 0.37 mg/g tissue and 69.7 mg/dL, respectively). In addition, 70 and 140 mg/kg MPMF (3.1 and 2.9 nmol/min per mg protein) inhibited liver XO activity compared with hyperuricemic mice (3.9 nmol/min per mg protein). DISCUSSION AND CONCLUSION: The results indicated that the beneficial hypouricaemic effect of MPMF may be mediated, at least in part, by inhibiting XO activity in the liver. Our study suggests that P. mume and its extracts may have a considerable potential for development as an anti-gout agent for clinical application.

Antidepressant-like behavioral, neurochemical and neuroendocrine effects of naringenin in the mouse repeated tail suspension test.

Our previous study demonstrated that the citrus bioflavonoid naringenin ameliorated behavioral alterations via the central serotonergic and noradrenergic systems in the tail suspension test (TST) induced mice. To better understand its pharmacological activity, mice were submitted to three 6min-TSTs one week apart (Day 1: test, Day 7: retest 1, Day 14: retest 2) followed by hippocampal glucocorticoid receptor (GR), monoamine neurotransmitters and serum corticosterone measurement. The results suggested that repeated TST detected the gradual increase in the efficacy of naringenin over time, additionally 1-day (20 mg/kg), 7-day (10, 20 mg/kg) and 14-day (5, 10, 20 mg/kg) naringenin treatment markedly decreased the immobility time. Moreover, administration of naringenin for 14 days (20 mg/kg) increased hippocampal serotonin (5-HT), norepinephrine (NE) and GR levels, and reduced serum corticosterone levels in mice exposed to the repeated TST. Overall, the present study indicated that the re-exposure would facilitate the detection of the anti-immobility effects of antidepressant drugs in the mouse TST, and clearly demonstrated that the antidepressant-like effect of naringenin may be mediated by an interaction with neuroendocrine and neurochemical systems.

[CO2 flux characteristics and their influence on the carbon budget of a larch plantation in Maoershan region of Northeast China].

From January to December 2008, the CO2 flux in a larch plantation (Larix gmeilinii) in Maoershan region of Shangzhi County, Heilongliang Province was measured by eddy covanance method, and the diurnal changes of leaf photosynthetic rate were measured in growth season (from May to October). There existed differences in the net ecosystem exchange (NEE) of the plantation in different time periods under the effects of environmental factors. In the afternoon (12:00-24:00), the NEE changed more slowly with the variation of vapor pressure deficit (VPD) than in the morning (0:00-12:00); and in the morning, tbe light use efficiency was 0.6284 mol x mol(-1), 14% more than that in afternoon. The NEE increased with increasing temperature, and the increment in the morning was 50% higher than that in the afternoon (air temperature > 15 degrees C). These differences in responding to environmental changes led to 88% NEE implemented in the morning, and only 12% NEE implemented in the afternoon. The annual gross ecosystem productivity (GEP) in the morning took a percentage of 60%, and that in afternoon took 40%. These findings were supported by the observation at leaf level, i.e., on average of whole growth season, the leaf photosynthetic capacity in the morning was over 2-fold higher than that in afternoon. Generally, the annual NEE, ecosystem respiration (Re), and GEP of the plantation in 2008 were 263-264 g C x m(-2), 718-725 g C x m(-2), and 981-989 g C x m(-2), respectively.