PTEN Deficiency Facilitates Exosome Secretion and Metastasis in Cholangiocarcinoma by Impairing TFEB-mediated Lysosome Biogenesis.

BACKGROUND & AIMS: In eukaryotes, the ubiquitin-proteasome system and the autophagy-lysosome pathway are essential for maintaining cellular proteostasis and associated with cancer progression. Our previous studies have demonstrated that phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, limits proteasome abundance and determines chemosensitivity to proteasome inhibitors in cholangiocarcinoma (CCA). However, whether PTEN regulates the lysosome pathway remains unclear. METHODS: We tested the effects of PTEN on lysosome biogenesis and exosome secretion using loss- and gain-of-function strategies in CCA cell lines. Using in vitro dephosphorylation assays, we explored the regulatory mechanism between PTEN and the key regulator of lysosome biogenesis, transcription factor EB (TFEB). Using the migration assays, invasion assays, and trans-splenic liver metastasis mouse models, we evaluated the function of PTEN deficiency, TFEB-mediated lysosome biogenesis, and exosome secretion on tumor metastasis. Moreover, we investigated the clinical significance of PTEN expression and exosome secretion by retrospective analysis. RESULTS: PTEN facilitated lysosome biogenesis and acidification through its protein phosphatase activity to dephosphorylate TFEB at Ser211. Notably, PTEN deficiency increased exosome secretion by reducing lysosome-mediated degradation of multi-vesicular bodies, which further facilitated the proliferation and invasion of CCA. TFEB agonist curcumin analog C1 restrained the metastatic phenotype caused by PTEN deficiency in mouse models, and we highlighted the correlation between PTEN deficiency and exosome secretion in clinical cohorts. CONCLUSIONS: In CCA, PTEN deficiency impairs lysosome biogenesis to facilitate exosome secretion and cancer metastasis in a TFEB phosphorylation-dependent manner.

Purification and characterization of a chitinase from Aeromonas media CZW001 as a biocatalyst for producing chitinpentaose and chitinhexaose.

Developing chitinase suitable for the bioconversion of chitin to chitin oligosaccharides has attracted significant attention due to its benefits in environmental protection. In this study, chitinase from Aeromonas media CZW001 (AmChi) was purified and characterized. The molecular weight of AmChi was approximately 40 kDa. AmChi exhibited maximum catalytic activity at pH 8.0 with an optimum temperature of 55°C and showed broad stability between 15 and 65°C and between pH 5.0 and 9.0. AmChi was activated by Mg2+ , Na+ , and K+ and inhibited by Hg+ , Co2+ , Fe2+ , Ca2+ , Ag+ , Zn2+ , and EDTA. The main products of AmChi on colloidal chitin were chitinhexaose and chitinpentaose. AmChi had better substrate specificity for powdered chitin than colloidal chitin and had a higher catalytic efficiency toward (GlcNAc)5 than colloidal chitin. AmChi inhibited fungal growth in a dose-dependent manner. These results suggest that AmChi could be used for the enzymatic degradation of chitin to produce chitinhexaose and chitinpentaose, which have several industrial applications.

Bioinformatic analysis and validation of cardiac hypertrophy-related genes.

In this study, we have screened genes involved in myocardial hypertrophy (MH) using a mice model for compensatory stress overload (transverse aortic constriction, TAC) and bioinformatics. Microarrays were downloaded, and according to the Venn diagram, three groups of data intersections were obtained. Gene function was analyzed by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), whereas protein-protein interactions (PPI) were analyzed using the STRING database. A mouse aortic arch ligation model was established to verify and screen the expression of hub genes. A total of 53 (DEGs) and 32 PPI genes were screened out. GO analysis showed DEGs mainly involved in cytokine and peptide inhibitor activity. KEGG analysis focused on ECM receptor interaction and osteoclast differentiation. Expedia co-expression gene network analysis showed that Serpina3n, Cdkn1a, Fos, Col5a2, Fn1 and Timp1 participated in the occurrence and development of MH. RT-qPCR verified that all the other 9 hub genes except Lox were highly expressed in TAC mice. This study lays a foundation for further study on the molecular mechanism of MH and for screening of molecular markers.

Insights into peptide profiling of sturgeon myofibrillar proteins with low temperature vacuum heating.

BACKGROUND: Protein oxidation during food processing causes changes in the balance of protein-molecular interactions and protein-water interactions, ultimately leading to protein denaturation, which results in the loss of a range of functional properties. Therefore, how to control the oxidative modification of proteins during processing has been the focus of research. RESULTS: In the present study, the intrinsic fluorescence value of the myofibrillar proteins (MP) decreased and the surface hydrophobicity value increased, indicating that the heat treatment caused a significant change in the conformation of the MP. With an increase in heating temperature, protein carbonyl content increased, total sulfhydryl content decreased, and protein secondary structure changed from α-helix to ß-sheet, indicating that protein oxidation and aggregation occurred. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that heat treatment can lead to the degradation of proteins, especially myosin heavy chain, although actin had a certain thermal stability. In total, 733 proteins were identified by proteomics, and the protein oxidation caused by low temperature vacuum heating (LTVH) was determined to be mild oxidation dominated by malondialdehyde and 4-hydroxynonenal by oxidation site division. CONCLUSION: The present study has revealed the effect of LTVH treatment on the protein oxidation modification behavior of sturgeon meat, and explored the effect mechanism of LTVH treatment on the processing quality of sturgeon meat from the perspective of protein oxidation. The results may provide a theoretical basis for the precise processing of aquatic products. © 2023 Society of Chemical Industry.

Gankyrin and TIGAR cooperatively accelerate glucose metabolism toward the PPP and TCA cycle in hepatocellular carcinoma.

Oncogene-derived metabolic reprogramming is important for anabolic growth of cancer cells, which is now considered to be not simply rely on glycolysis. Pentose phosphate pathway and tricarboxylic acid cycle also play pivotal roles in helping cancer cells to meet their anabolic and energy demands. The present work focused on gankyrin, a relatively specific oncogene in hepatocellular carcinoma (HCC), and its impact on glycolysis and mitochondrial homeostasis. Metabolomics, RNA-seq analysis, and subsequent conjoint analysis illustrated that gankyrin regulated the pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and mitochondrial function and homeostasis, which play pivotal roles in tumor development. Mechanistically, gankyrin was found to modulate HCC metabolic reprogramming via TIGAR. Gankyrin positively regulated the transcription of TIGAR through Nrf2, which bound to the antioxidant response elements (AREs) in the promoter of TIGAR. Interestingly, TIGAR feedback regulated the transcription of Nrf2 and subsequently gankyrin by promoting nuclear importation of PGC1α. The loop between gankyrin, Nrf2, and TIGAR accelerated glucose metabolism toward the PPP and TCA cycle, which provided vital building blocks, such as NADPH, ATP, and ribose of tumor and further facilitated the progression of HCC.

Monitoring the lipid oxidation and flavor of Russian sturgeon fillets treated with low temperature vacuum heating: formation and relationship.

BACKGROUND: Sturgeon is one of the most precious fish resources worldwide. Low temperature vacuum heating (LTVH) has been confirmed as a good way of maintaining food quality. However, there is a lack of in-depth studies assessing the impact of LTVH on lipid oxidation and flavor formation. RESULTS: The present study compared the effect of LTVH and traditional cooking on lipid oxidation and flavor of sturgeon fillets. In total, 13 fatty acids were detected, of which polyunsaturated fatty acids content was the highest (P < 0.05). LTVH prevented the formation of conjugated diene and thiobarbituric acid reactive substances (P < 0.05), as manifested by an increased signal intensity of free radicals of electron spin resonance. The characteristic peaks intensity of lipid by Raman at 970 cm-1 , 1080 cm-1 and 1655 cm-1 were reduced, whereas peaks at 1068 cm-1 and 1125 cm-1 displayed the opposite trend. Confocal fluorescence microscopy showed that the lipids particles were reduced and distributed more evenly with an increase in heating temperature. Principal component analysis of electronic nose cannot effectively separate all groups; however, gas chromatography-ion migration spectrometry showed that the volatile flavor compounds were relatively stable during LTVH. Correlation analysis of all the above lipid oxidation indices and characteristic flavor substances showed that each treatment group was located in different quadrants and demonstrated great differentiation. CONCLUSION: Overall, the results of the present study support the view that LTVH is a healthier way of cooking. © 2022 Society of Chemical Industry.

RPB5-Mediating Protein Promotes Cholangiocarcinoma Tumorigenesis and Drug Resistance by Competing With NRF2 for KEAP1 Binding.

BACKGROUND AND AIMS: Cancer cell survival depends on the balance between reactive oxygen species production and scavenging, which is regulated primarily by NRF2 during tumorigenesis. Here, we demonstrate that deletion of RBP5-mediating protein (RMP) in an autonomous mouse model of intrahepatic cholangiocarcinoma (ICC) delays tumor progression. APPROACH AND RESULTS: RMP-overexpressing tumor cells exhibited enhanced tolerance to oxidative stress and apoptosis. Mechanistically, RMP competes with NRF2 for binding to the Kelch domain of KEAP1 (Kelch-like ECH-associated protein 1) through the E**E motif, leading to decreased NRF2 degradation via ubiquitination, thus increasing NRF2 nuclear translocation and downstream transactivation of antioxidant genes. This RMP-KEAP1-NRF2 axis promotes ICC tumorigenesis, metastasis, and drug resistance. Consistent with these findings, the RMP level in human ICC is positively correlated with the protein level of NRF2 and is associated with poor prognosis. CONCLUSION: These findings reveal that RMP is involved in the oxidative stress defense program and could be exploited for targeted cancer therapies.

A predictive and prognostic model for hepatocellular carcinoma with microvascular invasion based TCGA database genomics.

BACKGROUND: Microvascular invasion (MVI) adversely affects postoperative long-term survival outcomes in patients with hepatocellular carcinoma (HCC). There is no study addressing genetic changes in HCC patients with MVI. We first screened differentially expressed genes (DEGs) in patients with and without MVI based on TCGA data, established a prediction model and explored the prognostic value of DEGs for HCC patients with MVI. METHODS: In this paper, gene expression and clinical data of liver cancer patients were downloaded from the TCGA database. The DEG analysis was conducted using DESeq2. Using the least absolute shrinkage and selection operator, MVI-status-related genes were identified. A Kaplan-Meier survival analysis was performed using these genes. Finally, we validated two genes, HOXD9 and HOXD10, using two sets of HCC tissue microarrays from 260 patients. RESULTS: Twenty-three MVI-status-related key genes were identified. Based on the key genes, we built a classification model using random forest and time-dependent receiver operating characteristic (ROC), which reached 0.814. Then, we performed a survival analysis and found ten genes had a significant difference in survival time. Simultaneously, using two sets of 260 patients' HCC tissue microarrays, we validated two key genes, HOXD9 and HOXD10. Our study indicated that HOXD9 and HOXD10 were overexpressed in HCC patients with MVI compared with patients without MVI, and patients with MVI with HOXD9 and 10 overexpression had a poorer prognosis than patients with MVI with low expression of HOXD9 and 10. CONCLUSION: We established an accurate TCGA database-based genomics prediction model for preoperative MVI risk and studied the prognostic value of DEGs for HCC patients with MVI. These DEGs that are related to MVI warrant further study regarding the occurrence and development of MVI.

The CD68+ macrophages to CD8+ T-cell ratio is associated with clinical outcomes in hepatitis B virus (HBV)-related hepatocellular carcinoma.

BACKGROUND: Tumor microenvironment plays an essential role during the progression of hepatocellular carcinoma (HCC). Tumor infiltrating immune cells (TILs) was an important component of tumor microenvironment. However, whether TIL features are correlated with the prognosis of HCC patients remains unclear. METHODS: Cancer tissue and paired paracancerous tissues from 220 stage II∼III HBV-related HCC patients were collected. TILs were analyzed using a tyramide signal amplification system combined with immunohistochemistry. Kaplan-Meier survival analysis was conducted to investigate the associations between the prognosis and the infiltrating pattern of TILs. RESULTS: The patients were classified into three distinct subgroups (Clusters (C)1-3) with different overall survival (OS) and disease-free survival (DFS) according to the distribution pattern of TILs. The CD68/CD8 ratio in the cancer SA was correlated with the prognosis. Patients with a higher CD68/CD8 ratio exhibited poorer OS and DFS than those with a lower ratio. The CD68/CD8 ratio in the cancer SA was an independent factor for OS prediction but not DFS. CONCLUSION: CD68+ macrophages and CD8+ T-cells are essential immunological determinants for HBV-related HCC prognosis, and the CD68/CD8 ratio in cancer SA is a novel, prognostic factor for OS prediction in HBV-related HCC patients.

Ultrasound biomicroscopy validation of a murine model of cardiac hypertrophic preconditioning: comparison with a hemodynamic assessment.

In mice, myocardial hypertrophic preconditioning (HP), which is produced by the removal of short-term transverse aortic constriction (TAC), was recently reported to render the heart resistant to hypertrophic responses induced by subsequent reconstriction (Re-TAC). However, there is no efficient noninvasive method for ensuring that the repeated aortic manipulations were successfully performed. We previously demonstrated that ultrasound biomicroscopy (UBM) is a noninvasive and effective approach for predicting TAC success. Here, we investigated the value of UBM for serial predictions of load conditions in establishing a murine HP model. C57BL/6J mice were subjected to a sham operation, TAC, or Re-TAC, and the peak flow velocity at the aortic banding site (PVb) was measured by UBM. Left ventricular end-systolic pressure (LVESP) was examined by micromanometric catheterization. The PVb was positively associated with LVESP (R2 = 0.8204, P < 0.001, for TAC at 3 days and R2 = 0.7746, P < 0.001, for Re-TAC at 4 wk). PVb and LVESP values were markedly elevated after aortic banding, became attenuated to the sham-operated level after debanding, and increased after aortic rebanding. The cardiac hypertrophic responses were examined by UBM, histology, RT-PCR, and Western blot analysis. Four weeks after the last operation, with PVb ≥ 3.5 m/s as an indicator of successful aortic constriction, Re-TAC mice showed less cardiac hypertrophy, fetal gene expression, and ERK1/2 activation than TAC mice. Therefore, we successfully established a UBM protocol for the serial assessment of aortic flow and the prediction of LVESP during repeated aortic manipulations in mice, which might be useful for noninvasive evaluations of the murine HP model.NEW & NOTEWORTHY We successfully developed an ultrasound biomicroscopy protocol for the serial assessment of aortic bandings and the relevant left ventricular pressure in a murine model of cardiac hypertrophic preconditioning. The protocol may be of great importance in the successful establishment of the hypertrophic preconditioning model for further mechanistic and pharmacological studies.

ABORTED MICROSPORES Acts as a Master Regulator of Pollen Wall Formation in Arabidopsis.

Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning.

Evolution of Vertebrate Ryanodine Receptors Family in Relation to Functional Divergence and Conservation.

Ryanodine receptors (RyRs), the large homotetrameric protein complexes, regulate the release of calcium from intracellular stores into the cytosol and play vital roles in the excitation-contraction coupling of cells. However, the evolutionary relationship of RyRs in vertebrates has yet to be elucidated. We identified 22 RyRs from Homo sapiens, Mus musculus, Rattus norvegicus, Gallus gallus, Anolis carolinensis, Rana catesbeiana, and Danio rerio. The phylogenetic relationship, motifs analysis and reconstruction of ancestral RyRs showed that the members of RyR family in vertebrates were grouped into three clades: the RyR1 clade, the RyR2 clade, and the RyR3 clade. Positive selection existed in RyR gene evolution, which is consistent in three site models, and gene ontology (GO) analysis showed that the evolution of RyR family in vertebrates promotes RyRs function differentiation. At last, we predicted 140 mutation sites which may be involved in diseases and 57 phosphorylation sites among RyR1 sequence in human, as well as 61 mutation sites and 70 phosphorylation sites in human RyR2 sequences. Most of these potential sites are arranged in clusters. Our work provides insight into the origin and evolutionary process of RyRs in vertebrates, facilitating their functional investigations in the future.

Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFß-1.

Ryanodine receptor type 2 (RyR-2), the main Ca2+ release channel from sarcoplasmic reticulum in cardiomyocytes, plays a vital role in the regulation ofmyocardial contractile function and cardiac hypertrophy. However, the role of RyR-2 in cardiac fibrosis during the development of cardiac hypertrophy remains unclear.In this study, we examined whether RyR-2 regulates TGFß1, which is secreted from cardiomyocytes and exerts on cardiac fibrosis using cultured cardiomyocytes and cardiac fibroblasts of neonatal rats. The expression of RyR-2 was found only in cardiomyocytesbut not in cardiac fibroblasts. Mechanical stretch induced upregulation of TGFß1 in cardiomyocytes and RyR-2 knockdown significantly suppressed the upregulation of TGFß1 expression. The transcript levels of collagen genes were also decreased in fibroblasts compare with wild type, although the expression of both two kinds was higher than those in stationary cardiomyocytes (non-stretch). With the inhibition of the TGFß1-neutralizing antibody, the expression of collagen genes has no significant difference between the mechanically stretched cardiomyocytes and non-stretchedones. These results indicate that RyR-2 regulated TGFß1 expression in mechanically stretched cardiomyocytes and TGFß1 promoted collagen formation of cardiac fibroblasts by a paracrine mechanism.RyR-2 in mechanical stretch could promote the development of cardiac fibrosis involving TGFß1-dependent paracrine mechanism. Our findings provided more insight into comprehensively understanding the molecular role of RyR-2 in regulating cardiac fibrosis.

The effects of different angiotensin II type 1 receptor blockers on the regulation of the ACE-AngII-AT1 and ACE2-Ang(1-7)-Mas axes in pressure overload-induced cardiac remodeling in male mice.

Angiotensin II (AngII) type 1 receptor blockers (ARBs) have been effectively used in hypertension and cardiac remodeling. However, the differences among them are still unclear. We designed this study to examine and compare the effects of several ARBs widely used in clinics, including Olmesartan, Candesartan, Telmisartan, Losartan, Valsartan and Irbesartan, on the ACE-AngII-AT1 axis and the ACE2-Ang(1-7)-Mas axis during the development of cardiac remodeling after pressure overload. Although all of the six ARBs, attenuated the development of cardiac hypertrophy and heart failure induced by transverse aortic constriction (TAC) for 2 or 4weeks in the wild-type mice evaluated by echocardiography and hemodynamic measurements, the degree of attenuation by Olmesartan, Candesartan and Losartan tended to be larger than that of the other three drugs tested. Additionally, the degree of downregulation of the ACE-AngII-AT1 axis and upregulation of the ACE2-Ang(1-7)-Mas axis was higher in response to Olmesartan, Candesartan and Losartan administration in vivo and in vitro. Moreover, in angiotensinogen-knockdown mice, TAC-induced cardiac hypertrophy and heart failure were inhibited by Olmesartan, Candesartan and Losartan but not by Telmisartan, Valsartan and Irbesartan administration. Furthermore, only Olmesartan and Candesartan could downregulate the ACE-AngII-AT1 axis and upregulate the ACE2-Ang(1-7)-Mas axis in vitro. Our data suggest that Olmesartan, Candesartan and Losartan could effectively inhibit pressure overload-induced cardiac remodeling even when with knockdown of Ang II, possibly through upregulation of the expression of the ACE2-Ang(1-7)-Mas axis and downregulation of the expression of the ACE-AngII-AT1 axis. In contrast, Telmisartan, Valsartan and Irbesartan only played a role in the presence of AngII, and Losartan had no effect in the presence of AngII in vitro.

Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis.

Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis.

Combination Treatment With Antihypertensive Agents Enhances the Effect of Qiliqiangxin on Chronic Pressure Overload-induced Cardiac Hypertrophy and Remodeling in Male Mice.

We previously showed that Qiliqiangxin (QL) capsules could ameliorate cardiac hypertrophy and remodeling in a mouse model of pressure overload. Here, we compared the effects of QL alone with those of QL combined with the following 3 types of antihypertensive drugs on cardiac remodeling and dysfunction induced by pressure overload for 4 weeks in mice: an angiotensin II type 1 receptor (AT1-R) blocker (ARB), an angiotensin-converting enzyme inhibitor (ACEI), and a ß-adrenergic receptor (ß-AR) blocker (BB). Adult male mice (C57B/L6) were subjected to either transverse aortic constriction or sham operation for 4 weeks, and the drugs (or saline) were orally administered through gastric tubes. Cardiac function and remodeling were evaluated through echocardiography, catheterization, histology, and analysis of hypertrophic gene expression. Cardiomyocyte apoptosis and autophagy, AT1-R and ß1-AR expression, and cell proliferation-related molecules were also examined. Although pressure overload-induced cardiac remodeling and dysfunction, hypertrophic gene reprogramming, AT1-R and ß1-AR expression, and ERK phosphorylation were significantly attenuated by QL alone, QL + ARB, QL + ACEI, and QL + BB, the attenuation was stronger in the combination treatment groups. Moreover, apoptosis was reduced to a larger extent by each combination treatment than by QL alone, whereas autophagy was more strongly attenuated by either QL + ARB or QL + ACEI. None of the treatments significantly upregulated ErbB2 or ErbB4 phosphorylation, and none significantly downregulated C/EBPß expression. Therefore, the effects of QL on chronic pressure overload-induced cardiac remodeling may be significantly increased when QL is combined with an ARB, an ACEI, or a BB.

Insulin-like growth factor binding protein 4 enhances cardiomyocytes induction in murine-induced pluripotent stem cells.

Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes cardiomyocytes induction of iPSCs is unclear. In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. We observed that IGFBP4 treatment at late stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac-specific transcription factor), α-MHC, α-actinin, and Troponin I (cardiac-specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that α-MHC, TNNT2 and connexin 43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs, but it significantly promoted the proliferation of cardiomyocytes from iPSCs characterized by higher ratio EdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibited ß-catenin expression in cytosol of EBs at late stage during differentiation of iPSCs. Knockdown of ß-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhanced cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocytes differentiation of iPSCs has relationship with ß-catenin signaling pathway. In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through inhibiting ß-catenin signaling.

Prognostic significance of cytoskeleton-associated membrane protein 4 and its palmitoyl acyltransferase DHHC2 in hepatocellular carcinoma.

BACKGROUND: The functions of cytoskeleton-associated membrane protein 4 (CKAP4), one kind of type II transmembrane protein, are associated with the palmitoyl acyltransferase DHHC2. The objective of the current study was to investigate CKAP4/DHHC2 expression and its prognostic significance in patients with hepatocellular carcinoma (HCC). METHODS: Two independent cohorts of 416 patients with HCC were enrolled. All the patients included had defined clinicopathologic and follow-up data. Using real-time polymerase chain reaction and immunohistochemical assay, CKAP4 and DHHC2 expression were evaluated. The association between CKAP4/DHHC2 expression and HCC-specific disease-free survival and overall survival was analyzed by Kaplan-Meier curves, the log-rank test, and Multivariate Cox regression analyses. RESULTS: The data documented that CKAP4 expression was much higher in HCC tumor tissues compared with adjacent normal tissues and its expression was significantly correlated with tumor size, intrahepatic metastases, portal venous invasion, and Barcelona Clinic Liver Cancer stage of disease in 2 cohorts of patients. On survival analysis, patients with high CKAP4 expression appeared to have a favorable overall survival and a longer disease-free survival compared with those with low expression. DHHC2 expression was also examined in tissue microarray analysis by immunohistochemistry and the results demonstrated that 87.6% of the cases had low expression of DHHC2. Kaplan-Meier analysis indicated that a high level of DHHC2 expression predicted favorable overall survival and disease-free survival rates in both the training cohort and validation set. Furthermore, the combination of CKAP4 and DHHC2 was found to have a more powerful efficiency in prognosis prediction than either one alone. CONCLUSIONS: To the best of our knowledge, the current study is the first to demonstrate that the expression of CKAP4 and its palmitoyl acyltransferase DHHC2 correlates with disease progression and metastasis in patients with HCC and may provide prognostic and therapeutic value.

Signal regulatory protein α is associated with tumor-polarized macrophages phenotype switch and plays a pivotal role in tumor progression.

UNLABELLED: Macrophages (Mψ) are the major component of infiltrating leukocytes in tumors and exhibit distinct phenotypes according to the microenvironment. We have recently found that signal regulatory protein α (SIRPα), the inhibitory molecule expressed on myeloid cells, plays a critical role in controlling innate immune activation. Here, we identify that SIRPα is down-regulated on monocytes/Mψ isolated from peritumoral areas of hepatocellular carcinoma (HCC) samples, while its level is moderately recovered in intratumor Mψ. In vitro assays demonstrate that SIRPα expression is significantly reduced on Mψ when cocultured with hepatoma cells. This reduction is partly due to the soluble factors in the tumor microenvironment. Knockdown (KD) of SIRPα prolongs activation of nuclear factor kappa B (NF-κB) and PI3K-Akt pathways as Mψ encounter tumor cells, leading to an increased capacity of Mψ for migration, survival, and proinflammatory cytokine production. Enhanced Stat3 and impaired Stat1 phosphorylation are also observed in tumor-exposed SIRPα-KD Mψ. Adoptive transfer with SIRPα-KD Mψ accelerates mouse hepatoma cells growth in vivo by remolding the inflammatory microenvironment and promoting angiogenesis. SIRPα accomplishes this partly through its sequestration of the signal transducer Src homology 2-containing phosphotyrosine phosphatase (SHP2) from IκB kinase ß (IKKß) and PI3K regulatory subunit p85 (PI3Kp85). CONCLUSION: These findings suggest that SIRPα functions as an important modulator of tumor-polarized Mψ in hepatoma, and the reduction of SIRPα is a novel strategy used by tumor cells to benefit their behavior. Therefore, SIRPα could be utilized as a potential target for HCC therapy.

CKAP4 inhibited growth and metastasis of hepatocellular carcinoma through regulating EGFR signaling.

CKAP4, one kind of type II trans-membrane protein, plays an important role to maintain endoplasmic reticulum structure and inhibits the proliferation of bladder cancer cells by combining its ligand anti-proliferative factor (APF). However, the biological function of CKAP4 in the progression of liver cancer has not been clearly demonstrated. In the present study, we knocked down or overexpressed CKAP4 in hepatocellular carcinoma (HCC) cells and cell proliferation, invasion, and migration capacities were investigated by CCK-8 and transwell assays. In vivo tumor model in mice was used to evaluate the role of CKAP4 on growth and metastasis of HCC. The data documented that HCC cells with high CKAP4 levels were featured by low proliferation capability as well as low invasion potential. Interestingly, we found that CKAP4 suppressed the activation of epithelial growth factor receptor (EGFR) signaling, which may partly explain the role of CKAP4 in cell biological behavior of HCC. Further study revealed that CKAP4 could associate with EGFR at basal status and the complex was reduced upon EGF stimulation, leading to release EGFR into cytoplasm. Thus, we demonstrate the novel mechanism, for the first time, expression of CKAP4 regulates progression and metastasis of HCC and it may provide therapeutic values in this tumor.