Combined physicochemical and transcriptomic analyses reveal the effect of the OsGA20ox1 gene on the starch properties of germinated brown rice.

Genes play a pivotal role in regulating the germination of cereal grains; however, there is limited research on the impact of germination genes on the physicochemical properties of germinated cereal starch. We investigated the effects of the OsGA20ox1 gene on the multiscale structural features and adhesion behavior of germinated brown rice starch. Compared to the knockout lines group, the wild type exhibited a decrease in double-helix content (62.74 %), relative crystallinity (47.39 %), and short-range molecular ordering (2.47 %), accompanied by enhanced erosion on the surface of starch granules. The damage to glycosidic bonds at the double-helix level and the heightened structural amorphization (90.95 %) led to reduced entanglement and interaction among starch molecules, ultimately resulting in reduced characteristic viscosity. Further transcriptomic analysis revealed that OsGA20ox1 could regulate the expression of starch-related enzyme genes in the starch metabolism pathway during germination of brown rice. This study contributes to understanding the role of germination genes in promoting the physicochemical properties of starch in germinated grains, thereby opening up new avenues for the improvement of plant-based starch, and paving the way for further research in this field.

Paternal Preconception Hepatitis B Virus Infection and Risk of Congenital Heart Disease in Offspring.

Importance: Previous evidence suggests that maternal hepatitis B virus (HBV) infection during prepregnancy or pregnancy is associated with congenital heart diseases (CHDs) in offspring. However, the association of paternal HBV infection with CHDs is not well examined. Objective: To explore the association of paternal preconception HBV infection with CHDs in offspring. Design, Setting, and Participants: This retrospective cohort study used propensity score matching of data from the Chinese National Free Preconception Checkup Project (NFPCP) from January 1, 2010, to December 31, 2018. Male participants whose wives were aged 20 to 49 years, were uninfected with HBV, and successfully conceived within 1 year after prepregnancy examination were enrolled. Data were analyzed from March 2023 to February 2024. Exposures: The primary exposure was paternal preconception HBV infection status, including uninfected, previous infection (both serum hepatitis B surface antigen and hepatitis B envelope antigen negative), and new infection (serum hepatitis B surface antigen positive). Maternal HBV immune status was further classified as immune or susceptible. Main Outcomes and Measures: The main outcome was CHDs, which were collected from the birth defect registration card of the NFPCP. Logistic regression with robust error variances was used to estimate the association between paternal preconception HBV infection and CHDs in offspring. Results: A total of 6 675 540 couples participated in the NFPCP service. After matching husbands with and without preconception HBV infection in a 1:4 ratio, 3 047 924 couples (median age of husbands, 27 years [IQR, 25-30 years]) were included in this study. Of these couples, 0.025% had offspring with CHDs. Previous paternal HBV infection was independently associated with CHDs in offspring (adjusted relative risk [ARR], 1.40; 95% CI, 1.11-1.76) compared with no infection. Similar results were obtained in subgroup analyses according to maternal HBV immune status. Compared with couples with uninfected husbands and susceptible wives, the risk of CHDs in offspring among couples with previously HBV-infected husbands was similar in couples with wives with susceptible immune status (ARR, 1.49; 95% CI, 1.10-2.03) and in those with wives with immunity (ARR, 1.49; 95%CI, 1.07-2.09). A significantly higher CHD risk in offspring was found among couples with newly infected husbands and immune wives (ARR, 1.38; 95% CI, 1.05-1.82), but there was no difference in risk among those with newly infected husbands and susceptible wives (ARR, 0.99; 95% CI, 0.72-1.36). No interactions were found between maternal immune status and paternal HBV infection. Conclusions and Relevance: In this cohort study using propensity score matching, previous paternal preconception HBV infection was associated with CHD risk in offspring. The findings suggest that personalized reproductive guidance regarding HBV screening and staying free of HBV infection should be provided for both wives and husbands.

Husband smoking is associated with Wife's thyrotropin abnormality: A population-based cohort study among Chinese reproductive-aged women.

OBJECTIVES: To comprehensively assess the association of husband smoking with wives' thyrotropin abnormality. METHODS: This population-based retrospective cohort study included 2 406 090 Chinese reproductive-aged women who had participated twice in the National Free Pre-pregnancy Checkups Project between 2010 and 2020. Multivariate-adjusted odds ratios and 95% confidence intervals for subnormal and supranormal thyrotropin were estimated according to the husband's smoking status. RESULTS: Husband smoking at the first visit was associated with a 17% (15%-20%) and 26% (24%-28%) increased odds of subnormal thyrotropin and supranormal thyrotropin respectively compared to participants in neither-smoker group. In non-smoking participants with normal thyrotropin levels at the first visit, the corresponding increased risk of subnormal thyrotropin and supranormal thyrotropin at the second visit were 15% (12%-18%) and 19% (16%-21%) in contrast to participants without husband-smoking exposure. In non-smoking participants with abnormal thyrotropin levels at their first visit, husband smoking cessation was associated with 27% (17%-35%) and 36% (31%-40%) reduced odds of subnormal thyrotropin and supranormal thyrotropin at the second visit compared with the participants whose husband still smoking at the second visit. CONCLUSION: Husband smoking was associated with wives' subnormal thyrotropin and supranormal thyrotropin, and cessation of husband smoking could reduce the odds of thyrotropin abnormality. Couple-focused smoking intervention should be developed to reduce the burden of asymptomatic thyroid disease in females.

An immune window of opportunity to prevent spontaneous abortion: prepregnancy peripheral leukocytes and subsets were associated with a decreased risk of spontaneous abortion.

STUDY QUESTION: Do prepregnancy peripheral leukocytes (PPLs) and their subsets influence the risk of spontaneous abortion (SAB)? SUMMARY ANSWER: PPLs and their subsets are associated with the risk of SAB. WHAT IS KNOWN ALREADY: Compelling studies have revealed the crucial role of maternal peripheral leukocytes in embryo implantation and pregnancy maintenance. Adaptive changes are made by PPLs and their subsets after conception. STUDY DESIGN, SIZE, DURATION: This population-based retrospective cohort study was based on data from the National Free Pre-pregnancy Check-up Project (NFPCP) in mainland China. Couples preparing for pregnancy within the next six months were provided with free prepregnancy health examinations and counseling services for reproductive health. The current study was based on 1 310 494 female NFPCP participants aged 20-49 who became pregnant in 2016. After sequentially excluding 235 456 participants lost to follow-up, with multiple births, and who failed to complete blood tests, a total of 1 075 038 participants were included in the primary analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: PPLs and their subset counts and ratios were measured. The main outcome was SAB. A multivariable logistic regression model was used to estimate the odds ratio (OR) and 95% CI of SAB associated with PPLs and their subsets, and restricted cubic spline (RCS) was used to estimate the nonlinear exposure-response relationship. MAIN RESULTS AND ROLE OF CHANCE: Of the included pregnant participants, a total of 35 529 SAB events (3.30%) were recorded. Compared to participants with reference values of PPLs, the ORs (95% CIs) of leukopenia and leukocytosis for SAB were 1.14 (1.09-1.20) and 0.74 (0.69-0.79), respectively. The RCS result revealed a monotonous decreasing trend (Pnonlinear < 0.05). Similar relationships were observed for the neutrophil count and ratio, monocyte count, and middle-sized cell count and ratio. The lymphocyte ratio showed a positive and nonlinear relationship with the risk of SAB (Pnonlinear < 0.05). Both eosinophils and basophils showed positive relationships with the risk of SAB (eosinophil Pnonlinear > 0.05 and basophil Pnonlinear < 0.05). LIMITATIONS, REASONS FOR CAUTION: Chemical abortion events and the cause of SAB were not collected at follow-up. Whether women with abnormal PPLs had recovered during periconception was not determined. WIDER IMPLICATIONS OF THE FINDINGS: PPLs and their subsets are associated with the risk of SAB. Leukopenia and neutropenia screening in women preparing for pregnancy and developing a feasible PPL stimulation approach should be emphasized to utilize the immune window of opportunity to prevent SAB. STUDY FUNDING/COMPETING INTEREST(S): This study was approved by the Institutional Research Review Board of the National Health and Family Planning Commission. This study was supported by the National Key Research and Development Program of China (grants 2021YFC2700705 [Y.Y.] and 2016YFC100307 [X.M.]) and the National Natural Science Foundation of China (grant no. 82003472 [L.W.]). The funding source was not involved in the study design, data collection, analysis and interpretation of the data, writing the report, or the decision to submit this article for publication. No competing interests. TRIAL REGISTRATION NUMBER: N/A.

Pulsed light treatment enhances starch hydrolysis and improves starch physicochemical properties of germinated brown rice.

BACKGROUND: Recently, germinated brown rice (GBR) has gained substantial attention as a functional food because of its nutritional attributes. Notably, pulsed light technology (PLT) has emerged as a promising tool for enhancing rice germination and, consequently, has improved the nutritional and functional qualities of GBR-derived products. However, further research is required to comprehensively understand the impact of PLT on GBR physicochemical properties. The present study aimed to investigate the stimulating effects of PLT on starch hydrolysis, starch structure and functional properties of GBR. RESULTS: The PLT substantially boosted α-amylase activity during brown rice germination, leading to a 10.9% reduction in total starch content and a 17.3% increase in reducing sugar content, accompanied by elevated free water levels. Structural analysis indicated no changes in starch crystalline types, whereas gelatinization temperature slightly increased. Pasting properties exhibited a significant drop in peak viscosity. Scanning electron microscopy showed surface erosion of starch granules with microstructural changes. Furthermore, correlation analysis established positive links between α-amylase activity, reducing sugar accumulation, starch structure and functional properties in GBR. CONCLUSION: The present study demonstrates that PLT enhanced the physicochemical properties of GBR starch, significantly improving the stability of GBR products, thereby contributing to expanded applicability of rice starch in the food industry. © 2023 Society of Chemical Industry.

PLK1 maintains DNA methylation and cell viability by regulating phosphorylation-dependent UHRF1 protein stability.

PLK1 is a key serine/threonine kinase as well as a master mitotic regulator, but it has never been reported that PLK1 regulates DNA methylation. In the present study, we for the first time found that PLK1 inhibition disrupted global DNA methylation and elevated the expression level of tumor suppressor genes. Mechanistically, we found that PLK1 interacts UHRF1 protein to induce its phosphorylation at serine 265. Phosphorylation is required for the maintenance of UHRF1 protein stability by recruiting a deubiquitinase USP7. Conversely, PLK1 inhibition decreases UHRF1 protein interaction with USP7 and activates the ubiquitin-proteasome pathway, thereby accelerating UHRF1 protein degradation. UHRF1 degradation decreases the recruitment of DNMT1 to chromatin, and decreases the level of genome-wide DNA methylation, thereby elevating the expression of tumor suppressor genes and decreasing cell viability. We here presented the first report on the novel role of PLK1 in DNA methylation maintenance through UHRF1-DNMT1 pathway, and revealed a novel anticancer mechanism of PLK1 inhibitors.

Declined prevalence, improved awareness and control of hypertension are associated with spousal educational attainment: A mega-data study.

Cardiovascular diseases, including hypertension, have posed a serious threat to human health in recent decades. Family-centered health promotion and disease control for the management of hypertension is gaining attention. In this study, we assessed the association between spousal educational attainment (SEA) and hypertension prevalence, awareness, and control, intending to provide new directions for family health care. A total of 71 211 191 reproductive-aged participants from the National Free NFPCP during 2013-2019 were included in the current study. Inverse probability weighting (IPW) via propensity models were used to adjust for the imbalance by SEA. Both multivariable-adjusted ORs and inverse-probability-weighted ORs were used to assess the association between SEA and the prevalence, awareness, and control of hypertension. ORs of prevalence, awareness, and control of hypertension with SEA stratified by sex, age, and residency type were also reported. Compared with participants with SEA of compulsory education, the inverse-probability-weighted ORs for hypertension were 0.97 (0.96-0.97), 0.99 (0.98-1.00), and 0.91 (0.88-0.93) for participants with SEA of senior high, college, and postgraduate, respectively. The corresponding ORs for hypertension awareness were 1.12 (1.10-1.13), 1.15 (1.13-1.16), and 1.38 (1.34-1.41). The increment of hypertension control associated with SEA was only identified in urban areas. Modification analyses revealed that urban participants were observed to have more healthy benefits associated with SEA; additional decreased prevalent hypertension and increased hypertension awareness associated with SEA were observed in wives and husbands respectively. Thus, SEA was associated with decreased prevalent hypertension and increased awareness and control of hypertension. Our findings call for increased participation of spouses in family-centered healthcare, with consideration of modified effects by gender, age, and residency type, to improve chronic disease prevention and control including hypertension.

UHRF1 promotes spindle assembly and chromosome congression by catalyzing EG5 polyubiquitination.

UHRF1 is an epigenetic coordinator bridging DNA methylation and histone modifications. Additionally, UHRF1 regulates DNA replication and cell cycle, and its deletion induces G1/S or G2/M cell cycle arrest. The roles of UHRF1 in the regulation of G2/M transition remain poorly understood. UHRF1 depletion caused chromosome misalignment, thereby inducing cell cycle arrest at mitotic metaphase, and these cells exhibited the defects of spindle geometry, prominently manifested as shorter spindles. Mechanistically, UHRF1 protein directly interacts with EG5, a kinesin motor protein, during mitosis. Furthermore, UHRF1 induced EG5 polyubiquitination at the site of K1034 and further promoted the interaction of EG5 with spindle assembly factor TPX2, thereby ensuring accurate EG5 distribution to the spindles during metaphase. Our study clarifies a novel UHRF1 function as a nuclear protein catalyzing EG5 polyubiquitination for proper spindle architecture and faithful genomic transmission, which is independent of its roles in epigenetic regulation and DNA damage repair inside the nucleus. These findings revealed a previously unknown mechanism of UHRF1 in controlling mitotic spindle architecture and chromosome behavior and provided mechanistic evidence for UHRF1 deletion-mediated G2/M arrest.

AR antagonists develop drug resistance through TOMM20 autophagic degradation-promoted transformation to neuroendocrine prostate cancer.

BACKGROUND: Prostate cancer(PCa) is the most commonly occurring male cancer in the USA. Abiraterone or Enzalutamide have been approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). However, the treatment-emergent neuroendocrine PCa (t-NEPC) may develop, resulting in drug resistance in about 10-17% CRPC patients. The detailed mechanisms remain unclear.. METHODS: The expression correlation of TOMM20 and AR in PCa was determined by analyzing publicly available datasets, or by IHC staining in tumor specimens. The protein interaction of TOMM20 and AR was validated by co-immunoprecipitation or GST pull-down assay. The impact of TOMM20 depletion on drug sensitivity were elucidated by assays of cell proliferation, invasion, sphere formation, xenograft growth and intravenous metastasis. The intracellular ROS level was measured by flow cytometry, and the NEPC transdifferentiation and characteristics of cancer stem-like cells were validated by RNA-seq, RT-PCR and western blotting. RESULTS: The protein level of TOMM20 is positively correlated with AR in PCa cells and specimens. TOMM20 protein physically interacts with AR. AR antagonists induced the protein degradation of TOMM20 through autophagy-lysosomal pathway, thereby elevating the intracellular ROS level and activating PI3K/AKT signaling pathway. When TOMM20 was depleted, PCa cells underwent EMT, acquired the characteristics of cancer stem-like cells, and developed resistance to AR antagonists. The stable depletion of TOMM20 promoted the transdifferentiation of PCa adenocarcinoma into NEPC and metastasis. Conversely, the rescue of TOMM20 re-sensitized the resistant PCa cells to AR antagonists. CONCLUSIONS: TOMM20 protein degradation induced by AR antagonists promoted the transdifferentiation of PCa to NEPC, thereby revealing a novel molecular mechanism by which AR antagonists develop drug resistance through mitochondrial outer membrane-mediated signaling pathway. These findings suggested that the decreasing or loss of TOMM20 expression in PCa tissues might become a useful predictor of PCa resistance to AR antagonists.

Isolation of a Novel QTL, qSCM4, Associated with Strong Culm Affects Lodging Resistance and Panicle Branch Number in Rice.

Rice breeders are now developing new varieties with semi-high or even high plant height to further increase the grain yield, and the problem of lodging has re-appeared. We identified a major quantitative trait locus (QTL), qSCM4, for resistance to lodging by using an F2 segregant population and a recombinant self-incompatible line population from the cross between Shennong265 (SN265) and Lijiangxintuanheigu (LTH) after multiple years and multiple environments. Then, the residual heterozygous derived segregant population which consisted of 1781 individual plants, and the BC3F2 segregant population which consisted of 3216 individual plants, were used to shorten the physical interval of qSCM4 to 58.5 kb including 11 genes. DNA sequencing revealed the most likely candidate gene for qSCM4 was Os04g0615000, which encoded a functional protein with structural domains of serine and cysteine. There were 13 DNA sequence changes in LTH compared to SN265 in this gene, including a fragment deletion, two base changes in the 3' UTR region, six base changes in the exons, and four base changes in the introns. A near-isogenic line carrying qSCM4 showed that it improved the lodging resistance through increasing stem thickness by 25.3% and increasing stem folding resistance by 20.3%. Furthermore, it was also discovered that qSCM4 enhanced the primary branch per panicle by 16.7%, secondary branch by per panicle 9.9%, and grain number per panicle by 14.7%. All the above results will give us a valuable genetic resource for concurrently boosting culm strength and lodging resistance, and they will also provide a basis for further research on the lodging resistance mechanism of rice.

Diosgenin inhibits prostate cancer progression by inducing UHRF1 protein degradation.

Prostate cancer (PCa) represents the second cause of cancer death in adult men. Aberrant overexpression of UHRF1 has been reported in several cancer types, and is regarded as a novel drug target for cancer therapy. Nevertheless, no UHRF1-targeted small molecule inhibitor has been testing in clinical trials. Traditional Chinese medicine (TCM) prescriptions have a long history for the treatment of PCa in China, and Chinese herbal extracts are important resources for new drug discovery. In the present study, we first screened the potentially effective components from the commonly used TCMs for PCa treatment in clinic by using network pharmacology together with molecular docking. We identified diosgenin (DSG) as a small molecule natural compound specifically targeting UHRF1 protein. Furthermore, we validated the results by using the wet lab experiments. DSG, by directly binding UHRF1 protein, induced UHRF1 protein degradation through the ubiquitin-proteasome pathway. Importantly, DSG induced UHRF1 protein degradation by reducing the protein interaction with a deubiquitinase USP7. DSG reduced the level of genomic DNA methylation, and elevated the expression of such tumor suppressor genes as p21, p16 and LXN, thereby resulting in cell cycle arrest, cellular senescence and the inhibition of xenograft tumor growth. We here presented the first report that DSG specifically induced UHRF1 protein degradation, thereby revealing a novel anticancer mechanism of DSG. Altogether, this present study provided a promising strategy to discover new molecule-targeted drugs from small-molecule natural products.

AKT1 regulates UHRF1 protein stability and promotes the resistance to abiraterone in prostate cancer.

Oncogenic activation of PI3K/AKT signaling pathway, together with epigenetic aberrations are the characters of castration-resistant prostate cancer (CRPC). UHRF1 as a key epigenetic regulator, plays a critical role in prostate cancer (PCa) development, and its expression is positively correlated with the degree of malignancy. In this present study we investigated the potential regulatory mechanism of AKT1 on UHRF1, and further validated the in vitro and in vivo anticancer efficacy of AKT phosphorylation inhibitor MK2206 in combination with abiraterone. Both UHRF1 and p-AKT aberrantly overexpressed in the abiraterone-resistant PCa cells. Further studies revealed that AKT1 protein interacts with UHRF1, and AKT1 directly phosphorylates UHRF1 via the site Thr-210. MK2206 induced UHRF1 protein degradation by inhibiting AKT1-induced UHRF1 phosphorylation, and then reduced the interaction between UHRF1 and deubiquitinase USP7, while promoted the interaction between UHRF1 and E3 ubiquitin protein ligase BTRC. MK2206 significantly promoted the sensitivity of abiraterone-refractory PCa cells and xenografts to abiraterone by decreasing UHRF1 protein level, and reversed the phenotype of NEPC, evently induced cellular senescence and cell apoptosis. Altogether, our present study for the first time revealed a novel molecular mechanism of abiraterone resistance through PI3K/AKT-UHRF1 pathway, and provided a novel therapeutic modality by targeting PI3K/AKT1 to promote the drug sensitivity of abiraterone in PCa patients.

Combined transcriptome and metabolome analyses reveal the mechanisms of ultrasonication improvement of brown rice germination.

This study investigated the effects of ultrasonication treatment on the germination rate of brown rice. Brown rice grains were subjected to ultrasound (40 kHz/30 min) and then incubated for 36 h at 37 °C to germinate the seeds. Ultrasonic treatment increased the germination rate of brown rice by up to ∼28 % at 30 h. Transcriptomic and metabolomic analyses were performed to explore the mechanisms underlying the effect of ultrasonic treatment on the brown rice germination rate. Comparing the treated and control check samples, 867 differentially expressed genes (DEGs) were identified, including 638 upregulated and 229 downregulated), as well as 498 differentially accumulated metabolites (DAMs), including 422 up accumulated and 76 down accumulated. Multi-omics analysis revealed that the germination rate of brown rice was promoted by increased concentrations of low-molecular metabolites (carbohydrates and carbohydrate conjugates, fatty acids, amino acids, peptides, and analogues), and transcription factors (ARR-B, NAC, bHLH and AP2/EREBP families) as well as increased carbon metabolism. These findings provide new insights into the mechanisms of action of ultrasound in improving the brown rice germination rate and candidate DEGs and DAMs responsible for germination have been identified.

Norcantharidin promotes cancer radiosensitization through Cullin1 neddylation-mediated CDC6 protein degradation.

Radiotherapy (RT) is a conventional cancer therapeutic modality. However, cancer cells tend to develop radioresistance after a period of treatment. Diagnostic markers and therapeutic targets for radiosensitivity are severely lacking. Our recently published studies demonstrated that the cell division cycle (CDC6) is a critical molecule contributing to radioresistance, and maybe a potential therapeutic target to overcome radioresistance. In the present study, we for the first time reported that Norcantharidin (NCTD), a demethylated form of cantharidin, re-sensitized radioresistant cancer cells to overcome radioresistance, and synergistically promoted irradiation (IR)-induced cell killing and apoptosis by inducing CDC6 protein degradation. Mechanistically, NCTD induced CDC6 protein degradation through the ubiquitin-proteasome pathways. By using small interfering RNA (siRNA) interference or small compound inhibitors, we further determined that NCTD induced CDC6 protein degradation through a neddylation-dependent pathway, but not through Huwe1, Cyclin F, and APC/C-mediated ubiquitin-proteasome pathways. We screened the six most relevant Cullin subunits (CUL1, 2, 3, 4A, 4B, and 5) using siRNAs. The knockdown of Cullin1 but not the other five cullins remarkably elevated CDC6 protein levels. NCTD promoted the binding of Cullin1 to CDC6, thereby promoting CDC6 protein degradation through a Cullin1 neddylation-mediated ubiquitin-proteasome pathway. NCTD can be used in combination with radiotherapy to achieve better anticancer efficacy, or work as a radiosensitizer to overcome cancer radioresistance.

Comparative multi-omics analysis of hypoxic germination tolerance in weedy rice embryos and coleoptiles.

Hypoxic germination tolerance is an important trait for seedling establishment of direct-seeded rice. Our comparative metabolomics analysis revealed that weedy rice accumulated more sugar and amino acids than cultivated rice accumulated in the embryo and coleoptile tissues under hypoxic stress. At the transcriptional level, oxidative phosphorylation activity in weedy rice was higher than in cultivated rice that likely led to more efficient energy metabolism during hypoxic stress. Based on our comparative proteomics analysis, enriched proteins related to cell wall implied that the advantages in energy metabolism of weedy rice were ultimately reflected in the formation of tissue structures. In this study, we found that most of key hypoxic germination tolerance (HGT) genes shared the same genetic backgrounds with Oryza japonica, however, several of them genetically similar to other Oryza plant also play important roles. Our findings suggest weedy rice can serve as genetic resources for the improvement of direct-seeding rice.

UHRF1 promotes androgen receptor-regulated CDC6 transcription and anti-androgen receptor drug resistance in prostate cancer through KDM4C-Mediated chromatin modifications.

The UHRF1 and CDC6, oncogenes play critical roles in therapeutic resistance. In the present study, we found that UHRF1 mediates androgen receptor (AR)-regulated CDC6 transcription in prostate cancer cells. In prostate cancer tissues and cell lines, levels of UHRF1 and CDC6 were simultaneously upregulated, and this was associated with worse survival. UHRF1 silencing significantly promoted the cytotoxicity and anti-prostate cancer efficacy of bicalutamide in mouse xenografts by inhibiting CDC6 gene expression. UHRF1 promoted AR-regulated CDC6 transcription by binding to the CCAAT motif near the androgen response element (ARE) in the CDC6 promoter. We further found that UHRF1 promoted androgen-dependent chromatin occupancy of AR protein by recruiting the H3K9me2/3-specific demethyltransferase KDM4C and modifying the intense heterochromatin status. Altogether, we found for the first time that UHRF1 promotes AR-regulated CDC6 transcription through a novel chromatin modification mechanism and contributes to anti-AR drug resistance in prostate cancer. Targeting AR and UHRF1 simultaneously may be a novel and promising therapeutic modality for prostate cancer.

USP7 is a novel Deubiquitinase sustaining PLK1 protein stability and regulating chromosome alignment in mitosis.

BACKGROUND: The deubiquitinase USP7 has been identified as an oncogene with key roles in tumorigenesis and therapeutic resistance for a series of cancer types. Recently small molecular inhibitors have been developed to target USP7. However, the anticancer mechanism of USP7 inhibitors is still elusive. METHODS: Cell viability or clonogenicity was tested by violet crystal assay. Cell apoptosis or cell cycle was analyzed by flow cytometry, and chromosome misalignment was observed by a fluorescent microscopy. The protein interaction of PLK1 and USP7 was detected by tandem affinity purification and high throughput proteomics, and further confirmed by co-immunoprecipitation, GST pull-down and protein co-localization. The correlation between USP7 level of tumor tissues and taxane-resistance was evaluated. RESULTS: Pharmacological USP7 inhibition by P5091 retarded cell proliferation and induced cell apoptosis. Further studies showed that P5091 induced cell cycle arrest at G2/M phase, and particularly induced chromosome misalignment, indicating the key roles of USP7 in mitosis. USP7 protein was detected in the PLK1-interacted protein complex. USP7 interacts with PLK1 protein through its PBD domain by catalytic activity. USP7 as a deubiquitinase sustained PLK1 protein stability via the C223 site, and inversely, USP7 inhibition by P5091 promoted the protein degradation of PLK1 through the ubiquitination-proteasome pathway. By overexpressing PLK1, USP7 that had been depleted by RNAi ceased to induce chromosome misalignment in mitosis and again supported cell proliferation and cell survival. Both USP7 and PLK1 were overexpressed in taxane-resistant cancer cells, and negatively correlated with the MP scores in tumor tissues. Either USP7 or PLK1 knockdown by RNAi significantly sensitized taxane-resistant cells to taxane cell killing. CONCLUSION: This is the first report that PLK1 is a novel substrate of USP7 deubiquitinase, and that USP7 sustained the protein stability of PLK1. USP7 inhibition induces cell apoptosis and cell cycle G2/M arrest, and overcomes taxane resistance by inducing the protein degradation of PLK1, resulting in chromosome misalignment in mitosis.

Radiation-promoted CDC6 protein stability contributes to radioresistance by regulating senescence and epithelial to mesenchymal transition.

Ionizing radiation (IR) is a conventional cancer therapeutic, to which cancer cells develop radioresistance with exposure. The residual cancer cells after radiation treatment also have increased metastatic potential. The mechanisms by which cancer cells develop radioresistance and gain metastatic potential are still unknown. In this study acute IR exposure induced cancer cell senescence and apoptosis, but after long-term IR exposure, cancer cells exhibited radioresistance. The proliferation of radioresistant cells was retarded, and most cells were arrested in G0/G1 phase. The radioresistant cells simultaneously showed resistance to further IR-induced apoptosis, premature senescence, and epithelial to mesenchymal transformation (EMT). Acute IR exposure steadily elevated CDC6 protein levels due to the attenuation of ubiquitination, while CDC6 overexpression was observed in the radioresistant cells because the insufficiency of CDC6 phosphorylation blocked protein translocation from nucleus to cytoplasm, resulting in subcellular protein accumulation when the cells were arrested in G0/G1 phase. CDC6 ectopic overexpression in CNE2 cells resulted in apoptosis resistance, G0/G1 cell cycle arrest, premature senescence, and EMT, similar to the characteristics of radioresistant CNE2-R cells. Targeting CDC6 with siRNA promoted IR-induced senescence, sensitized cancer cells to IR-induced apoptosis, and reversed EMT. Furthermore, CDC6 depletion synergistically repressed the growth of CNE2-R xenografts when combined with IR. The study describes for the first time cell models for IR-induced senescence, apoptosis resistance, and EMT, three major mechanisms by which radioresistance develops. CDC6 is a novel radioresistance switch regulating senescence, apoptosis, and EMT. These studies suggest that CDC6highKI67low represents a new diagnostic marker of radiosensitivity, and CDC6 represents a new therapeutic target for cancer radiosensitization.