Comparative study of lung cancer between smokers and nonsmokers: A real-world study based on the whole population from Tianjin City, China.

INTRODUCTION: The purpose of this study was to examine the prevalence, clinical characteristics, and changing trends of non-smokers with lung cancer (LC) based on data from a population-wide cancer registry in northern China. METHODS: The study used LC incidence and follow-up data from 2010 to 2019 from the Cancer Registry System of Tianjin city, which included 82769 cases. Trends in the incidence and proportion of non-smokers with LC were examined by joinpoint regression analysis. Life table and Cox survival analyses were used to calculate the survival rates and compare the death hazard ratios (HRs) in different groups, respectively. RESULTS: Among the 82769 new diagnosis cases of LC during 2010 to 2019, there were 34589 (41.8%) current smokers, 14913 (18.0%) ex-smokers, 28123 (34.0%) non-smokers, and 5144 (6.2%) unknowns. The proportion of non-smokers changed slightly from 2010 (35.36%) to 2019 (36.87%) (annual percentage change, APC= -0.01%, p>0.05). This proportion declined in men (2010 vs 2019; 22.06% vs 20.66%) and increased in women (2010 vs 2019; 53.02% vs 62.35%), and in the 0-44 years age group it showed an upward trend from 2015 to 2019 (APC=4.82%, 95% CI: 1.8-7.9). Compared with smokers with LC, non-smokers with LC were predominantly females (64.15% vs 27.26%), had a predominantly adenocarcinoma histological subtypes (76.71% vs 42.22%), and had a 20% lower risk of death than smokers (HR=0.80; 95% CI: 0.78-0.81). CONCLUSIONS: The proportion of non-smokers with LC was relatively high in northern China, with an increasing trend in the proportion of females and younger age groups. Non-smokers with LC had different epidemiological and clinical characteristics compared with smokers with LC.

The prognostic role of circulating tumor DNA across breast cancer molecular subtypes: A systematic review and meta-analysis.

Objective: Circulating tumor DNA (ctDNA) is increasingly being used as a potential prognostic biomarker in cancer patients. We aimed to assess the prognostic value of ctDNA in different subtypes of breast cancer patients throughout the whole treatment cycle. Materials and methods: PubMed, Web of Science, Embase, Cochrane Library, Scopus, and clinical trials.gov databases were searched from January 2016 to May 2022. The following search terms were used: ctDNA OR circulating tumor DNA AND breast cancer OR breast carcinoma. Only studies written in English were included. The following pre-specified criteria should be met for inclusion: (i) original articles, conference abstracts, etc.; (ii) patients with breast cancer; (iii) ctDNA measurement; and (iv) clinical outcome data such as recurrence-free survival (RFS) and overall survival (OS). The random-effects model was preferred considering the potential heterogeneity across studies. The main outcomes are ctDNA detection rate and postoperative long-term outcomes (RFS and OS). Results: A total of 24 studies were screened. At every measurement time, the ctDNA detection rate of the HR+ subgroup was similar to that of the HR- subgroup (P = 0.075; P = 0.458; P = 0.744; and P = 0.578), and the ctDNA detection rate of the HER2+ subgroup was similar to that of the HER2- subgroup (P = 0.805; P = 0.271; P = 0.807; and P = 0.703). In the HR+ subgroup, RFS and OS of ctDNA positive patients were similar to those of ctDNA negative patients (P = 0.589 and P = 0.110), while RFS and OS of the ctDNA positive group was significantly shorter than those of the ctDNA negative patients in the HR- subgroup (HR = 4.03, P < 0.001; HR = 3.21, P < 0.001). According to HER grouping, the results were the same as above. In the triple negative breast cancer (TNBC) subgroup, the RFS and OS of ctDNA-positive patients was significantly shorter than of the ctDNA negative patients before and after surgery. Conclusions: ctDNA was more predictive of recurrence-free survival and overall survival in the HR- subgroup than in the HR+ subgroup, and the same result was showed in the HER2- subgroup vs. HER2+ subgroup. The prognosis of the TNBC subtype is closely related to ctDNA before and after surgery.

[Association between serum uric acid and airflow obstruction based on the health-checkup population].

OBJECTIVE: To investigate the association between serum uric acid, pulmonary function and airflow obstruction in Chinese Taiwan healthy subjects. METHODS: All the cross-sectional analysis was performed in the population over 40 years old using the physical examination data of Chinese Taiwan MJ Health Resource Center between 1996 and 2016 stratification by gender. The correlation analyses between serum uric acid were done and multivariate Logistic regression analysis was used to explore the effect of serum uric acid on airflow obstruction. RESULTS: A total of 35 465 people were included in the study, including 16 411 men and 19 054 women. Among them, the serum uric acid concentration of men was higher than that of women, and the serum uric acid concentration of the people with airflow obstruction was higher than that of the people without airflow obstruction. There was a negative correlation between serum uric acid level and the forced expiratory volume in one second (FEV1) and the force vital capacity (FVC) in women (P < 0.05), but in men the correlation didn' t exist (P>0.05). After adjusting for age, education, smoking status, drinking status, work strength, body mass index, history of cough, history of hypertension, history of diabetes, history of dyslipidemia, white blood cells and blood albumin, the airflow obstruction in women was more likely to exist with the serum uric acid elevated (OR=1. 12, 95%CI: 1.02-1.22, P < 0.05). The results showed that women with hyperuricemia were more likely to have airflow obstruction than those without hyperuricemia (OR=1.36, 95%CI: 1.06-1.75, P < 0.05). There was no correlation between serum uric acid concentration and airflow obstruction in men (OR=1.04, 95%CI: 0.96-1.13, P>0.05), also the hyperuricemia and airflow obstruction (OR=1.12, 95%CI: 0.89-1.39, P>0.05). CONCLUSION: There is a negative correlation between serum uric acid and FEV1 and FVC in relatively healthy women, and there is an association between elevated serum uric acid and airflow obstruction in women, but not in men. Further prospective studies are needed to explore whether high serum uric acid level can increase the risk of airflow obstruction.

Circulating tumor DNA as prognostic markers of relapsed breast cancer: a systematic review and meta-analysis.

Objective: Circulating tumor DNA (ctDNA) is increasingly being used as a potential prognosis biomarker in patients of breast cancer. This review aims to assess the clinical value of ctDNA in outcome prediction in breast cancer patients throughout the whole treatment cycle. Methods: PubMed, Web of Science, Embase, Cochrane Library, Scopus, and clinical trials.gov were searched from January 2016 to May 2022. Conference abstracts published in last three years were also included. The following search terms were used: ctDNA OR circulating tumor DNA AND breast cancer OR breast carcinoma. Only studies written in English languages were included. The following pre-specified criteria should be met for inclusion: (1) observational studies (prospective or retrospective), randomized control trials, case-control studies and case series studies; (2) patients with breast cancer; (3) ctDNA measurement; (4) clinical outcome data such as objective response rate (ORR), pathological complete response (pCR), relapse-free survival (RFS), overall survival (OS), and so on. The random-effect model was preferred considering the potential heterogeneity across studies. The primary outcomes included postoperative short-term outcomes (ORR and pCR) and postoperative long-term outcomes (RFS, OS, and relapse). Secondary outcomes focused on ctDNA detection rate. Results: A total of 30 studies, comprising of 19 cohort studies, 2 case-control studies and 9 case series studies were included. The baseline ctDNA was significantly negatively associated with ORR outcome (Relative Risk [RR] = 0.65, 95% confidence interval [CI]: 0.50-0.83), with lower ORR in the ctDNA-positive group than ctDNA-negative group. ctDNA during neoadjuvant therapy (NAT) treatment was significantly associated with pCR outcomes (Odds Ratio [OR] = 0.15, 95% CI: 0.04-0.54). The strong association between ctDNA and RFS or relapse outcome was significant across the whole treatment period, especially after the surgery (RFS: Hazard Ratio [HR] = 6.74, 95% CI: 3.73-12.17; relapse outcome: RR = 7.11, 95% CI: 3.05-16.53), although there was heterogeneity in these results. Pre-operative and post-operative ctDNA measurements were significantly associated with OS outcomes (pre-operative: HR = 2.03, 95% CI: 1.12-3.70; post-operative: HR = 6.03, 95% CI: 1.31-27.78). Conclusions: In this review, ctDNA measurements at different timepoints are correlated with evaluation indexes at different periods after treatment. The ctDNA can be used as an early potential postoperative prognosis biomarker in breast cancer, and also as a reference index to evaluate the therapeutic effect at different stages.

An innovative prognostic auxiliary for colon adenocarcinoma based on zinc finger protein genes.

Background: The carcinogenesis and progression of colon adenocarcinoma (COAD) are intensively related to the abnormal expression of the zinc finger (ZNF) protein genes. We aimed to employ these genes to provide a reliable prognosis and treatment stratification tool for COAD patients. Methods: Cox and the least absolute shrinkage and selection operator (LASSO) regression analysis were applied, utilizing The Cancer Genome Atlas (TCGA) metadata, to build a ZNF protein gene-based prognostic model. Using this model, patients in the training cohort and testing cohort (GSE17537) were labelled as either high or low risk. Kaplan-Meier (KM) survival analysis and time-dependent receiver operating characteristic (ROC) curve analysis were performed in the patients with opposite risk status to assess the predictive ability in each cohort. The potentiality of the mechanism was explored by the estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE), single-sample gene set enrichment analysis (ssGSEA), gene set enrichment analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, the degrees of expression of model genes were validated by immunohistochemistry (IHC). Results: The prognostic model consisting of INSM1, PHF21B, RNF138, SYTL4, WRNIP1, ZNF585B, and ZNF514, classified patients into opposite risk statuses. Patients in the high-risk subset had a considerably lower chance of surviving compared to those in the low-risk subset. There is a high probability that these model genes were attached to immune-related biological processes, which can be confirmed by the results of the above mechanistic methods. Moreover, patients in the low-risk subset also significantly outperformed the patients in the high-risk subset when calculating immune cells and function scores. Drug sensitivity and tumor immune dysfunction and exclusion (TIDE) analyses showed a clear difference in the immunological and chemotherapeutic efficacy predictions within the two risk groups. Additionally, the degrees of expression of model genes in high-risk and low-risk subsets presented great discrepancies. Conclusions: The signature may be applied as a predictive classifier to shepherd special medication for COAD patients.

Molecular mechanisms of the chemical constituents from anti-inflammatory and antioxidant active fractions of Ganoderma neo-japonicum Imazeki.

Ganoderma neo-japonicum Imazeki is a rare medicinal mushroom that has been reported to play a role in scavenging free radicals, protecting the liver, and inhibiting tumor cell activity. In this study, crude extracts were prepared, and 47 triterpenoids were identified by Ultra-high-performance liquid chromatography coupled with triple quadrupole time-of flight mass spectrometry (UHPLC-Triple TOF-MS/MS). Then, the crude extracts were subjected to column chromatography for the first time to obtain six fractions (Fr. (a), (b), (c), (d), (e) and (f)). Antioxidant and anti-inflammatory active tracking assays of all fractions found that Fr. (c) exhibited the strongest bioactivity. Subsequently, the chemical composition of Fr. (c) was clarified, and eight triterpenoids were determined in combination with the standard substances. In addition, this study demonstrated that Fr. (c) reduced the levels of inflammatory cytokines and reactive oxygen species (ROS) in LPS-stimulated RAW264.7 macrophages. Further studies showed that Fr. (c) could down-regulate the expression level of proteins associated of NF-κB signaling pathway, and upregulated Nrf2 and HO-1 protein level. In conclusion, our study showed that Fr. (c) inhibited LPS-mediated inflammatory response and oxidative stress by activating the Nrf2/HO-1 pathway and inactivating the NF-κB pathway. In the future, with the clearing of its composition and activity mechanism, Fr. (c) of G. neo-japonicum are expected to become a functional food for health and longevity.

Green walnut husk extracts Proliferation and Migration in Gastric Cancer.

Background: In the past few decades, natural products have become an increasingly important source of potential anti-cancer agents. The green walnut husk(GWH) extracts have been reported to inhibit multiple tumor cells and might be a promising chemopreventive agent in human neoplasia. However, it is not clear whether GWH extracts inhibit gastric cancer. Methods: Proliferation, invasion, and migration of gastric cancer cells were assessed by the CCK-8, wound-healing, and Transwell assay. The apoptotic rate was detected by flow cytometry(FCM). The expressions of Bcl-2, Bax, and Caspase-3 proteins were examined by Western blot. Moreover, the growth of gastric cancer cells was assessed using orthotopic xenograft models, and related proteins expressions were evaluated using immunohistochemistry. Finally, the Gene expression profile of gastric cancer treated with GWH extracts was evaluated by using High-throughput RNA sequencing(RNA-seq). Results: GWH extracts effectively inhibited gastric cancer cell growth in vitro and in vivo. In vivo, GWH extracts inhibited the survival of gastric cancer cells in a dose and time-dependent manner, inhibited the migration and invasion of gastric cancer cells, regulated the expressions of apoptosis-related proteins, and induced apoptosis of gastric cancer cells. In vitro, GWH extracts inhibited the growth of mouse xenografted tumors. A total of differentially expressed genes, of which 41 genes were up-regulated, and 610 genes were down-regulated, were identified by RNA-seq. GO, and KEGG analysis showed that these differentially expressed genes might be related to the mechanism of the anti-gastric cancer effect of GWH extracts. Conclusion: GWH extracts played an anti-gastric cancer effect by inducing apoptosis and inhibiting invasion. Secondly, the differential expression of many genes, multiple signal pathways, and metabolic pathways in gastric cancer play an essential role in the anti-gastric cancer effect of GWH extracts. The results suggested that GWH extracts are expected to be a low toxic drug for the treatment of gastric cancer in the future.

Methionine oxidation of CLK4 promotes the metabolic switch and redox homeostasis in esophageal carcinoma via inhibiting MITF selective autophagy.

BACKGROUND: Metabolic reprogramming and redox homeostasis contribute to esophageal squamous cell carcinoma (ESCC). CDC-like kinase 4 (CLK4) is a dual-specificity kinase that can phosphorylate substrates' tyrosine or serine/threonine residue. However, the role and mechanism of CLK4 in ESCC remain unknown. METHODS: CLK4 expression was analysed using publicly available datasets and confirmed in ESCC tissues and cell lines. The biological roles of CLK4 were studied with gain and loss-of-function experiments. Mass spectrometry was employed to examine the effects of CLK4 on metabolic profiling. In vitro kinase assay, co-immunoprecipitation, glutathione S-transferase pulldown, chromatin immunoprecipitation and luciferase reporter were used to elucidate the relationship among CLK4, microphthalmia-associated transcription factor (MITF), COP1 and ZRANB1. RESULTS: CLK4 down-regulation was observed in ESCC cell lines and clinical samples and associated with the methylation of its promoter. Low levels of CLK4 promoted ESCC development by affecting the purine synthesis pathway and nicotinamide adenine dinucleotide phosphate (NADPH)/nicotinamide adenine dinucleotide phosphate (NADP+ ) ratio. Interestingly, CLK4 inhibited ESCC development by blocking MITF-enhanced de novo purine synthesis and redox balance. Mechanistically, wild type CLK4 (WT-CLK4) but not kinase-dead CLK4-K189R mutant phosphorylated MITF at Y360. This modification promoted its interaction with E3 ligase COP1 and its K63-linked ubiquitination at K308/K372, leading to sequestosome 1 recognition and autophagic degradation. However, the deubiquitinase ZRANB1 rescued MITF ubiquitination and degradation. In turn, MITF bound to E- rather than M-boxes in CLK4 promoter and transcriptionally down-regulated its expression in ESCC. Clinically, the negative correlations were observed between CLK4, MITF, and purine metabolic markers, which predicts a poor clinical outcome of ESCC patients. Notably, CLK4 itself was a redox-sensitive kinase, and its methionine oxidation at M307 impaired kinase activity, enhanced mitochondria length and inhibited lipid peroxidation, contributing to ESCC. CONCLUSIONS: Our data highlight the potential role of CLK4 in modulating redox status and nucleotide metabolism, suggesting potential therapeutic targets in ESCC treatment.

Breast cancer screening using synthesized two-dimensional mammography: A systematic review and meta-analysis.

PURPOSE: We conducted a systematic review and meta-analysis to compare the screening performance of synthesized mammography (SM) plus digital breast tomosynthesis (DBT) with digital mammography (DM) plus DBT or DM alone. METHODS: Medline, Embase, Web of Science, and the Cochrane Library databases were searched from January 2010 to January 2021. Eligible population-based studies on breast cancer screening comparing SM/DBT with DM/DBT or DM in asymptomatic women were included. A random-effect model was used in this meta-analysis. Data were summarized as risk differences (RDs), with 95 % confidence intervals (CIs). RESULTS: Thirteen studies involving 1,370,670 participants were included. Compared with DM/DBT, screening using SM/DBT had similar breast cancer detection rate (CDR) (RD = -0.1/1000 screens, 95 % CI = -0.4 to 0.2, p = 0.557, I2 = 0 %), but lower recall rate (RD = -0.56 %, 95 % CI = -1.03 to -0.08, p = 0.022, I2 = 90 %) and lower biopsy rate (RD = -0.33 %, 95 % CI = -0.56 to -0.10, p = 0.005, I2 = 78 %). Compared with DM, SM/DBT improved CDR (RD = 2.0/1000 screens, 95 % CI = 1.4 to 2.6, p < 0.001, I2 = 63 %) and reduced recall rate (RD = -0.95 %, 95 % CI = -1.91 to -0.002, p = 0.049, I2 = 99 %). However, SM/DBT and DM had similar interval cancer rate (ICR) (RD = 0.1/1000 screens, 95 % CI = -0.6 to 0.8, p = 0.836, I2 = 71 %) and biopsy rate (RD = -0.05 %, 95 % CI = -0.35 to 0.24, p = 0.727, I2 = 93 %). CONCLUSIONS: Screening using SM/DBT has similar breast cancer detection but reduces recall and biopsy when compared with DM/DBT. SM/DBT improves CDR when compared with DM, but they have little difference in ICR. SM/DBT could replace DM/DBT in breast cancer screening to reduce radiation dose.

Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5.

Although the treatment of brain tumors by targeting kinase-regulated macroautophagy/autophagy, is under investigation, the precise mechanism underlying autophagy initiation and its significance in glioblastoma (GBM) remains to be defined. Here, we report that PAK1 (p21 [RAC1] activated kinase 1) is significantly upregulated and promotes GBM development. The Cancer Genome Atlas analysis suggests that the oncogenic role of PAK1 in GBM is mainly associated with autophagy. Subsequent experiments demonstrate that PAK1 indeed serves as a positive modulator for hypoxia-induced autophagy in GBM. Mechanistically, hypoxia induces ELP3-mediated PAK1 acetylation at K420, which suppresses the dimerization of PAK1 and enhances its activity, thereby leading to subsequent PAK1-mediated ATG5 (autophagy related 5) phosphorylation at the T101 residue. This event not only protects ATG5 from ubiquitination-dependent degradation but also increases the affinity between the ATG12-ATG5 complex and ATG16L1 (autophagy related 16 like 1). Consequently, ELP3-dependent PAK1 (K420) acetylation and PAK1-mediated ATG5 (T101) phosphorylation are required for hypoxia-induced autophagy and brain tumorigenesis by promoting autophagosome formation. Silencing PAK1 with shRNA or small molecule inhibitor FRAX597 potentially blocks autophagy and GBM growth. Furthermore, SIRT1-mediated PAK1-deacetylation at K420 hinders autophagy and GBM growth. Clinically, the levels of PAK1 (K420) acetylation significantly correlate with the expression of ATG5 (T101) phosphorylation in GBM patients. Together, this report uncovers that the acetylation modification and kinase activity of PAK1 plays an instrumental role in hypoxia-induced autophagy initiation and maintaining GBM growth. Therefore, PAK1 and its regulator in the autophagy pathway might represent potential therapeutic targets for GBM treatment.Abbreviations: 3-MA: 3-methyladenine; Ac-CoA: acetyl coenzyme A; ATG5: autophagy related 5; ATG16L1, autophagy related 16 like 1; BafA1: bafilomycin A1; CDC42: cell division cycle 42; CGGA: Chinese Glioma Genome Atlas; CHX, cycloheximide; ELP3: elongator acetyltransferase complex subunit 3; GBM, glioblastoma; HBSS: Hanks balanced salts solution; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K1: mitogen-activated protein kinase kinase 1; MAPK14, mitogen-activated protein kinase 14; PAK1: p21 (RAC1) activated kinase 1; PDK1: pyruvate dehydrogenase kinase 1; PGK1, phosphoglycerate kinase 1; PTMs: post-translational modifications; RAC1: Rac family small GTPase 1; SQSTM1: sequestosome 1; TCGA, The Cancer Genome Atlas.

Targeting CLK3 inhibits the progression of cholangiocarcinoma by reprogramming nucleotide metabolism.

CDC-like kinase 3 (CLK3) is a dual specificity kinase that functions on substrates containing serine/threonine and tyrosine. But its role in human cancer remains unknown. Herein, we demonstrated that CLK3 was significantly up-regulated in cholangiocarcinoma (CCA) and identified a recurrent Q607R somatic substitution that represented a gain-of-function mutation in the CLK3 kinase domain. Gene ontology term enrichment suggested that high CLK3 expression in CCA patients mainly was associated with nucleotide metabolism reprogramming, which was further confirmed by comparing metabolic profiling of CCA cells. CLK3 directly phosphorylated USP13 at Y708, which promoted its binding to c-Myc, thereby preventing Fbxl14-mediated c-Myc ubiquitination and activating the transcription of purine metabolic genes. Notably, the CCA-associated CLK3-Q607R mutant induced USP13-Y708 phosphorylation and enhanced the activity of c-Myc. In turn, c-Myc transcriptionally up-regulated CLK3. Finally, we identified tacrine hydrochloride as a potential drug to inhibit aberrant CLK3-induced CCA. These findings demonstrate that CLK3 plays a crucial role in CCA purine metabolism, suggesting a potential therapeutic utility.

Long non-coding RNA TPT1-AS1 promotes angiogenesis and metastasis of colorectal cancer through TPT1-AS1/NF90/VEGFA signaling pathway.

LncRNAs have been proven closely correlated to tumor progression. A recent study identified LncRNA TPT1-AS1 (TPT1-AS1) as one of the liver-metastasis associated LncRNAs in colorectal cancer (CRC). In this study, we report that TPT1-AS1 is upregulated in CRC tissues, which is associated with poor prognosis. Functional assays unravel a pro-angiogenesis and metastasis role of TPT1-AS1. Mechanistically, Flexmap 3D assays reveal that TPT1-AS1 upregulates the VEGFA secretion in CRC cells. RNA immunoprecipitation and mRNA stability assays further show that TPT1-AS1 interacts with nuclear factor 90 (NF90) and subsequently promotes the association between NF90 and VEGFA mRNA, which leads to the upregulation of VEGFA mRNA stability. Therefore, we elucidate a new regulatory mechanism of TPT1-AS1 in CRC angiogenesis and targeting the TPT1-AS1/NF90/VEGFA axis may provide a useful strategy for diagnosis and treatment for colorectal cancer patients.

Application of antagonist Bacillus amyloliquefaciens NCPSJ7 against Botrytis cinerea in postharvest Red Globe grapes.

We investigated the effects and possible mechanisms of Bacillus amyloliquefaciens NCPSJ7 against the gray mold caused by Botrytis cinerea in the postharvest Red Globe grapes. The disease incidence, lesion diameter, decay index, and some resistance-related enzymes were evaluated. The antioxidant capacity of grape treated with 1 × 104 CFU/ml B. cinerea alone and combined with 1 × 107 CFU/ml NCPSJ7 was also determined. The results showed that NCPSJ7 + B. cinerea reduced the disease incidence, lesion diameter, and decay index of postharvest grapes and enhanced the activities of polyphenol oxidase, peroxidase, chitinase, and ß-1,3-glucanase during different storage periods. Furthermore, the oxidative resistance, demonstrated by an escalating trend in the total phenolic content, DPPH free radical clearance rate, reducing power, and superoxide anion clearance rate after lesion presence, was improved. However, NCPSJ7 showed an inhibitory effect on gray mold, but resulted in the reduced antioxidant capacity in the grapes.

UHMK1 promotes gastric cancer progression through reprogramming nucleotide metabolism.

UHMK1 is a nuclear serine/threonine kinase recently implicated in carcinogenesis. However, the functions and action mechanisms of UHMK1 in the pathogenesis of human gastric cancer (GC) are unclear. Here, we observed that UHMK1 was markedly upregulated in GC. UHMK1 silencing strongly inhibited GC aggressiveness. Interestingly, UHMK1-induced GC progression was mediated primarily via enhancing de novo purine synthesis because inhibiting purine synthesis reversed the effects of UHMK1 overexpression. Mechanistically, UHMK1 activated ATF4, an important transcription factor in nucleotide synthesis, by phosphorylating NCOA3 at Ser (S) 1062 and Thr (T) 1067. This event significantly enhanced the binding of NCOA3 to ATF4 and the expression of purine metabolism-associated target genes. Conversely, deficient phosphorylation of NCOA3 at S1062/T1067 significantly abrogated the function of UHMK1 in GC development. Clinically, Helicobacter pylori and GC-associated UHMK1 mutation induced NCOA3-S1062/T1067 phosphorylation and enhanced the activity of ATF4 and UHMK1. Importantly, the level of UHMK1 was significantly correlated with the level of phospho-NCOA3 (S1062/T1067) in human GC specimens. Collectively, these results show that the UHMK1-activated de novo purine synthesis pathway significantly promotes GC development.

Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 3 Loss Activates Purine Metabolism and Promotes Hepatocellular Carcinoma Progression.

Cancer cells metabolize different energy sources to generate biomass rapidly. The purine biosynthetic pathway was recently identified as an important source of metabolic intermediates for these processes. However, very little was known about the regulatory mechanisms of purine metabolism in hepatocellular carcinoma (HCC). We explored the role of dual-specificity tyrosine (Y) phosphorylation-regulated kinase 3 (Dyrk3) in HCC metabolism. Dyrk3 was significantly down-regulated in HCC compared with normal controls. Its introduction in HCC cells markedly suppressed tumor growth and metastasis in xenograft tumor models. Mass spectrometric analysis of metabolites suggests that the effect of Dyrk3 on HCC occurred at least partially through down-regulating purine metabolism, as evidenced by the fact that inhibiting purine synthesis reverted the HCC progression mediated by the loss of Dyrk3. We further provide evidence that this action of Dyrk3 knockdown requires nuclear receptor coactivator 3 (NCOA3), which has been shown to be a coactivator of activating transcription factor 4 (ATF4) to target purine pathway genes for transcriptional activation. Mechanistically, Dyrk3 directly phosphorylated NCOA3 at Ser-1330, disrupting its binding to ATF4 and thereby causing the inhibition of ATF4 transcriptional activity. However, the phosphorylation-resistant NCOA3-S1330A mutant has the opposite effect. Interestingly, the promoter activity of Dyrk3 was negatively regulated by ATF4, indicating a double-negative feedback loop. Importantly, levels of Dyrk3 and phospho-NCOA3-S1330 inversely correlate with the expression of ATF4 in human HCC specimens. Conclusion: Our findings not only illustrate a function of Dyrk3 in reprograming HCC metabolism by negatively regulating NCOA3/ATF4 transcription factor complex but also identify NCOA3 as a phosphorylation substrate of Dyrk3, suggesting the Dyrk3/NCOA3/ATF4 axis as a potential candidate for HCC therapy.

Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth.

UVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide in vitro and in vivo evidence that UVRAG ubiquitination at lysine residues 517 and 559 or prevention of Ser522 phosphorylation by D4476, a CSNK1A1 inhibitor, enhances the lysosomal degradation of EGFR, which significantly inhibits hepatocellular carcinoma (HCC) growth. Furthermore, UVRAG S522 phosphorylation levels correlate with ZRANB1 T35/S209 phosphorylation levels and poor prognosis in HCC patients. These findings identify a novel molecular mechanism by which ubiquitination and phosphorylation of UVRAG regulate its function in autophagosome maturation and HCC growth, encouraging further study of their potential therapeutic implications. Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; BECN1: beclin 1; CHX: cycloheximide; CSNK1A1/CK1α: casein kinase 1 alpha 1; CQ: chloroquine; DUB: deubiquitinase; EBSS: Earle's balanced salt solution; EGF: epidermal growth factor; GFP: green fluorescent protein; GST: glutathione S-transferase; HBSS: Hanks balanced salts solution; HCC: hepatocellular carcinoma; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryo fibroblasts; mRFP: monomeric red fluorescent protein; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PTMs: post-translational modifications; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; Ub-AMC: ubiquitin-7-amido-4-methylcoumarin: a fluorogenic substrate; UVRAG: UV radiation resistance associated; ZRANB1/TRABID: zinc finger RANBP2-type containing 1.

Synthesis, stability and bioavailability of astaxanthin succinate diester.

BACKGROUND: We synthesized astaxanthin succinate diester (ASD), a novel astaxanthin (AST) derivate, with succinic anhydride and free AST. ASD was purified and characterized using silica gel column chromatography and spectrometry, respectively. RESULTS: The ASD final synthesis rate was 82.63%. A stability test revealed a high AST and ASD retention rate at pH 5.0-7.0. ASD showed better stability than did AST under acidic conditions. Both sample ions showed lower retention rates under Fe2+ and Fe3+ states. The ASD metabolic curve showed serum and liver area under the curve from 0 h to time t (AUC0-t ) values of 45.05 ± 4.58 and 120.38 ± 23.66 µg h-1  mL-1 , respectively. The long-term accumulation was significantly higher in the ASD group than in the AST group, which showed higher accumulation in the heart, muscle and spleen than in other tissues in vivo. CONCLUSION: The thermal stability and bioavailability of ASD were higher than that of the non-esterified free AST and common free AST, respectively. Additionally, AST accumulation in different tissues of the ASD group was multifold higher than that of free AST. These results prove that ASD may serve as a better source of AST for human nutrition than does free AST. © 2017 Society of Chemical Industry.

GSK-3ß phosphorylation-dependent degradation of ZNF281 by ß-TrCP2 suppresses colorectal cancer progression.

Zinc finger protein 281 (ZNF281) has been recently shown to be critical for CRC progression. However, the immediate upstream regulators of ZNF281 remain unclear. Here we reported that the E3 ligase the ß-transducin repeat-containing protein 2 (ß-TrCP2) governs the ubiquitination and degradation of ZNF281. In human CRC specimens, endogenous ß-TrCP2 were inversely correlated with ZNF281. Beta-TrCP2 reversed the phenotype of CRC cell with overexpressed ZNF281. Moreover, we found that glycogen synthase kinase 3ß (GSK-3ß), not GSK-α, could bind to and phosphorylate ZNF281 at one consensus motif (TSGEHS; phosphorylation site is shown in italics), which promotes the interaction of ZNF281 with ß-TrCP2, not ß-TrCP1, and leads to the subsequent ubiquitination and degradation of phosphorylated ZNF281. A mutant of ZNF281 (ZNF281-S638A) is much more stable than wild-type ZNF281 because ZNF281-S638A mutant abolishes the phosphorylation by GSK-3ß and can not be ubiquitinated and degraded by ß-TrCP2. Conversely, ZNF281 transcriptionally repressed the expression of ß-TrCP2, indicating a negative feedback loop between ZNF281 and ß-TrCP2 in CRC cells. These findings suggest that the turnover of ZNF281 by ß-TrCP2 might provide a potentially novel treatment for patients with CRC.

siRNA-mediated inactivation of HER3 improves the antitumour activity and sensitivity of gefitinib in gastric cancer cells.

The human EGFR family consists of four type-1 transmembrane tyrosine kinase receptors: HER1 (EGFR, ErbB1), HER2 (Neu, ErbB2), HER3 (ErbB3), and HER4 (ErbB4). HER3 can dimerize with EGFR, HER2 and even c-Met and likely plays a central role in the response to EGFR-targeted therapy. Because HER3 lacks significant kinase activity and cannot be inhibited by tyrosine kinase inhibitors, neutralizing antibodies and alternative inhibitors of HER3 have been sought as cancer therapeutics. Here, we describe the stable suppression of HER3 mRNA and protein using siRNA. The inhibition of HER3 expression decreased cell proliferation, suppressed cell cycle progression, induced apoptosis and inhibited cell motility, migration, invasiveness, and soft agar growth. In addition, we found that gefitinib treatment increased the HER3 and HER2 mRNA levels. The administration of various concentrations of gefitinib to HER3-knockdown cells enhanced antitumour activity and sensitivity due to the downregulation of protein phosphorylation via PI3K/AKT and ERK signalling. Our results support the use of combined treatments targeting multiple EGFR receptors, particularly the use of HER3 inhibitors combined with EGFR inhibitors, such as gefitinib.

HnRNPK/miR-223/FBXW7 feedback cascade promotes pancreatic cancer cell growth and invasion.

Several studies have identified miR-223 critically involved in various types of cancer, including pancreatic ductal adenocarcinoma (PDAC). However, its action and regulatory mechanisms in PDAC remains largely unclear. In this study, we found that the expression levels of miR-223 were increased in clinical samples with PDAC (81.6%). The upregulation of miR-223 increases the proliferation, migration, and invasive abilities of PDAC cells in vitro and in vivo. Mechanistically, miR-223 directly targeted FBXW7 and overexpression of FBXW7 reverted miR-223- induced drastic proliferation in PDAC cells. Interestingly, miR-223 promoter was found to form a coprecipitable complex with hnRNPK, and siRNA knockdown of hnRNPK in PDAC cells reduced the levels of miR-223. These results show that hnRNPK is a cellular protein that binds and affects the accumulation of miR-223 in PDAC. Furthermore, FBXW7 interacts with hnRNPK and promotes its degradation, which requires phosphorylation of hnRNPK at threonine 1695 by GSK3. Consistently, we observed an inverse expression pattern between FBXW7 and miR-223, whereas a positive expression pattern between miR-223 and hnRNPK was found in human PDAC tissues. These data unveiled an important new miR-223/FBXW7/HnRNPK feedback cascade in human PDAC.