Pan-Cancer Proteomics Analysis Reveals Wiskott-Aldrich Syndrome Protein as a Potential Regulator of Programmed Death-Ligand 1.

The programmed death-ligand 1 (PD-L1) is a key mediator of immunosuppression in the tumor microenvironment. The expression of PD-L1 in cancer cells is useful for the clinical determination of an immune checkpoint blockade (ICB). However, the regulatory mechanism of the PD-L1 abundance remains incompletely understood. Here, we integrated the proteomics of 52 patients with solid tumors and examined immune cell infiltration to reveal PD-L1-related regulatory modules. Wiskott-Aldrich syndrome protein (WASP) was identified as a potential regulator of PD-L1 transcription. In two independent cohorts containing 164 cancer patients, WASP expression was significantly associated with PD-L1. High WASP expression contributed to immunosuppressive cell composition, including cells positive for immune checkpoints (PD1, CTLA4, TIGIT, and TIM3), FoxP3+ Treg cells, and CD163+ tumor-associated macrophages. Overexpression of WASP increased, whereas knockdown of WASP decreased the protein level of PD-L1 in cancer cells without alteration of PD-L1 protein stability. The WASP-mediated cell migration and invasion were markedly attenuated by the silence of PD-L1. Collectively, our data suggest that WASP is a potential regulator of PD-L1 and the WASP/PD-L1 axis is responsible for cell migration and an immunosuppressive microenvironment.

LINC00493-encoded microprotein SMIM26 exerts anti-metastatic activity in renal cell carcinoma.

Human microproteins encoded by long non-coding RNAs (lncRNA) have been increasingly discovered, however, complete functional characterization of these emerging proteins is scattered. Here, we show that LINC00493-encoded SMIM26, an understudied microprotein localized in mitochondria, is tendentiously downregulated in clear cell renal cell carcinoma (ccRCC) and correlated with poor overall survival. LINC00493 is recognized by RNA-binding protein PABPC4 and transferred to ribosomes for translation of a 95-amino-acid protein SMIM26. SMIM26, but not LINC00493, suppresses ccRCC growth and metastatic lung colonization by interacting with acylglycerol kinase (AGK) and glutathione transport regulator SLC25A11 via its N-terminus. This interaction increases the mitochondrial localization of AGK and subsequently inhibits AGK-mediated AKT phosphorylation. Moreover, the formation of the SMIM26-AGK-SCL25A11 complex maintains mitochondrial glutathione import and respiratory efficiency, which is abrogated by AGK overexpression or SLC25A11 knockdown. This study functionally characterizes the LINC00493-encoded microprotein SMIM26 and establishes its anti-metastatic role in ccRCC, and therefore illuminates the importance of hidden proteins in human cancers.

A five-protein prognostic signature with GBP2 functioning in immune cell infiltration of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is of poor clinical outcomes, and currently lacks reliable prognostic biomarkers. By analyzing the datasets of the Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), we established a five-protein prognostic signature containing GBP2, HLA-DRA, ISG15, ISG20 and ITGAX. Our data indicate that this signature was closely correlated with advanced stage, higher pathological grade, and unfavorable survivals in patients with ccRCC. We further functionally characterized GBP2. Overexpression of GBP2 enhanced the phosphorylation of STAT2 and STAT3 to trigger JAK-STAT signaling and promote cell migration and invasion in ccRCC. Treatment of Ruxolitinib, a specific inhibitor of JAK/STAT, attenuated the GBP2-mediated phenotypes. Patients with high GBP2 expression were accompanied with more infiltration of immune cells positively stained with CD3, CD8, CD68, and immune checkpoint markers PD-1 and CTLA4, which was validated by Opal multiplex immunohistochemistry in ccRCC tissues. More CD8 + T cells and CD68 + macrophages were observed in patients expressing high GBP2. Taken together, a five-protein prognostic signature was constructed in our study. GBP2 has an oncogenic role via modulating JAK-STAT signaling and tumor immune infiltration, and thus may serve as a potential therapeutic target in ccRCC.

Solvothermal synthesis of octahedral and magnetic CoFe2O4-reduced graphene oxide hybrids and their photo-Fenton-like behavior under visible-light irradiation.

We report a facile solvothermal synthesis of novel octahedral CoFe2O4-reduced graphene oxide (RGO) hybrid and pure CoFe2O4 that were used as heterogeneous photo-Fenton catalysts for the degradation of organic dyes in water. We investigated the structures, morphologies and catalytic activity of both the CoFe2O4 nanoparticles and CoFe2O4-RGO hybrids. The morphology of CoFe2O4 nanoparticles displays size-dependent shapes changing from granular (or sheet) to octahedral shapes with the introduction of RGO. Compared with bare CoFe2O4, the octahedral CoFe2O4-RGO hybrids serve as novel bifunctional materials displaying higher saturation magnetization values and excellent heterogeneous activation of H2O2 at nearly neutral pH. The high saturation magnetization (41.98 emu g-1) of CoFe2O4-RGO hybrids aids their separation from the reaction mixture. In addition, the remarkable enhancement in the photo-Fenton activity of the CoFe2O4-RGO hybrids under visible light irradiation was attributed to the graphene/CoFe2O4 heterojunction, which aided the separation of excited electrons and holes. Furthermore, the CoFe2O4-RGO hybrids exhibited better removal efficiency for cationic methylene blue (MB) dye than for anionic methyl orange (MO) dye. Meanwhile, the CoFe2O4-RGO hybrids displayed acceptable photocatalytic stability, and we proposed an activation mechanism of H2O2 by the octahedral CoFe2O4-RGO hybrids.

Microsatellite and Single Nucleotide Polymorphisms in the Insulin-Like Growth Factor 1 Promoter with Insulin Sensitivity and Insulin Secretion.

BACKGROUND To investigate associations of the CA microsatellite and rs35767, rs5742612, and rs2288377 polymorphisms and the single nucleotide polymorphism (SNP) haplotypes with and without the CA microsatellite in the IGF1 promoter with insulin sensitivity and secretion. MATERIAL AND METHODS The CA microsatellite and SNPs were genotyped in 389 type 2 diabetes mellitus (T2DM) patients. A 75 g oral glucose tolerance test (OGTT) was given to all the participants. Associations of the genotypes and haplotypes with insulin sensitivity, insulin secretion, glucose tolerance, and insulin-like growth factor 1 (IGF1) were analyzed by ANCOVA (general linear model) and multiple linear regression, after controlling for gender, age, and BMI. RESULTS The CA microsatellite, rs35767 polymorphisms, and SNP haplotypes with or without CA showed no significant association with metabolic parameters. The C allele of rs5742612 was found to be associated with decreased insulin sensitivity (HOMA-S index, ß=-0.131, P=0.008; fasting insulin level, ß=0.022, P=0.006) and increased insulin secretion (HOMA-B index, ß=0.099, P=0.008; insulin AUC, ß=0.112, P=0.012). The linear regression model also indicated that the A allele of rs2288377 was associated with decreased insulin sensitivity (HOMA-S index, ß=-0.159, P=0.001; fasting insulin, ß=0.143, P=0.001) and increased insulin secretion (HOMA-B index, ß=0.114, P=0.017; insulin AUC, ß=0.042, P=0.002). CONCLUSIONS The CA microsatellite and rs35767 have no genotype-related difference in insulin sensitivity or secretion. The rs5742612 and rs2288377 polymorphisms are significantly associated with insulin biology, with the TT genotype exhibiting higher insulin sensitivity and lower insulin secretion compared with carriers of the C allele and A allele, respectively, mostly attributed to the direct functional roles of the two loci.

Influence of type 2 diabetes mellitus on Khorana venous thromboembolism risk in colorectal cancer patients

Background: Many studies have documented the association between venous thromboembolism (VTE) and colorectal cancer (CRC). The Khorana model is a VTE risk assessment model for predicting cancer-associated thrombosis. Type 2 diabetes (T2DM) has also been reported to increase the risk of VTE. Purpose: The aim of this study was to investigate the influence of T2DM on Khorana VTE risk in CRC patients and to explore the relationship between Khorana VTE category and CRC clinicopathological factors. Methods: This analysis included 615 CRC patients (205 with T2DM). Fibrinogen and D-dimer levels were compared within each group. A comparison was made of the proportion of patients in different Khorana VTE risk categories in CRC patients with and without T2DM. The association between Khorana VTE risk category and clinicopathological factors among all the CRC patients was evaluated. Results: Fibrinogen levels of CRC patients with T2DM were significantly higher than those of non-diabetes patients (4.13 ± 1.06 vs 3.94 ± 0.98, p < 0.001). A higher proportion of CRC patients with T2DM were in the Khorana intermediate-to-high risk category (H = 4.749, p = 0.029). Female sex, diabetes, colon location (compared with rectum), larger tumor size, advanced pT stage and pN stage were correlated with the intermediate-to-high Khorana VTE risk category, with odd ratios (95% confidence intervals [CI]) of 1.537 (1.064-2.220), 1.499 (1.027-2.186), 2.313 (1.588-3.370), 2.284 (1.542-3.383), 4.429 (2.088-9.396) and 1.822 (1.230-2.698), respectively. Conclusion: T2DM increases Khorana VTE risk in CRC patients. Female sex, diabetes, colon location, large tumor size and poor stage are associated with the intermediate-to-high Khorana VTE risk category (AU)

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Influence of type 2 diabetes mellitus on Khorana venous thromboembolism risk in colorectal cancer patients.

BACKGROUND: Many studies have documented the association between venous thromboembolism (VTE) and colorectal cancer (CRC). The Khorana model is a VTE risk assessment model for predicting cancer-associated thrombosis. Type 2 diabetes (T2DM) has also been reported to increase the risk of VTE. PURPOSE: The aim of this study was to investigate the influence of T2DM on Khorana VTE risk in CRC patients and to explore the relationship between Khorana VTE category and CRC clinicopathological factors. METHODS: This analysis included 615 CRC patients (205 with T2DM). Fibrinogen and D-dimer levels were compared within each group. A comparison was made of the proportion of patients in different Khorana VTE risk categories in CRC patients with and without T2DM. The association between Khorana VTE risk category and clinicopathological factors among all the CRC patients was evaluated. RESULTS: Fibrinogen levels of CRC patients with T2DM were significantly higher than those of non-diabetes patients (4.13 ± 1.06 vs 3.94 ± 0.98, p < 0.001). A higher proportion of CRC patients with T2DM were in the Khorana intermediate-to-high risk category (H = 4.749, p = 0.029). Female sex, diabetes, colon location (compared with rectum), larger tumor size, advanced pT stage and pN stage were correlated with the intermediate-to-high Khorana VTE risk category, with odd ratios (95% confidence intervals [CI]) of 1.537 (1.064-2.220), 1.499 (1.027-2.186), 2.313 (1.588-3.370), 2.284 (1.542-3.383), 4.429 (2.088-9.396) and 1.822 (1.230-2.698), respectively. CONCLUSION: T2DM increases Khorana VTE risk in CRC patients. Female sex, diabetes, colon location, large tumor size and poor stage are associated with the intermediate-to-high Khorana VTE risk category.

Smilax china L. rhizome extract inhibits nuclear factor-κB and induces apoptosis in ovarian cancer cells.

OBJECTIVE: To study the antitumor effects and associated mechanisms of extract of the Smilax china L. rhizome (SCR) on ovarian cancer cells. METHODS: Ovarian cancer cells A2780 were treated with different concentrations of SCR extract (SCRE), and compared with controls. Effects on cell growth were evaluated by cell counting kit-8 (CCK-8) assay; proliferation effects by EdU incorporation assay; cell cycle by propidium iodide staining; apoptosis by annexin V-fluorescein isothiocyanate/propidium iodide; cellular distribution of nuclear factor-κB (NF-κB) by immunofluorescence; protein levels of NF-κB, caspase-3, poly-adenosine diphosphate (ADP)-ribose polymerase (PARP), Bcl-2-associated X protein (Bax), cellular inhibitor of apoptosis (cIAP)-1, anti-X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-extra large (Bcl-XL), B-cell lymphoma-2 (Bcl-2) and AKT by Western blotting; and effects of SCRE combined with cisplatin or adriamycin on A2780 cells by CCK-8 assay. RESULTS: SCRE suppressed A2780 cell proliferation in a dose-dependent manner (P<0.05,P<0.01), arrested cells in G2/M phase and induced apoptosis by activating caspase-3, PARP and Bax. SCRE treatment also correlated with inhibition of NF-κB and downregulation of Bcl-2, Bcl-XL, cIAP-1, XIAP and AKT. SCRE can promote chemosensitivity to cisplatin and adriamycin in A2780 cells (P<0.01). CONCLUSION: SCR effectively inhibits NF-κB, induces apoptosis and reduces chemoresistance to cisplatin and adriamycin in ovarian cancer cells, which might be its molecular basis for treating ovarian cancer.

[Clinicopathological characteristics of colorectal cancer complicated with type 2 diabetes mellitus: analysis of clinicopathological data from 3, 202 colorectal cancer patients].

OBJECTIVE: the aim of this study was to determine the clinicopathological characteristics of colorectal cancer (CRC) patients complicated with type 2 diabetes mellitus (T2DM ). METHODS: A total of 3, 202 patients with CRC confirmed pathologically in Tianjin Union Medicine Center from January 2005 to December 2009 were included in this study. We analyzed the differences in clinicopathological features between T2DM patients and non-diabetic patients according to age of diagnosis, gender, tumor site, stage, gross type, histological type, and differentiation. RESULTS: From 2005 to 2009, the number of CRC patients increased yearly. The high incidence age of all CRC patients was 51 to 80 years old. The male to female ratio was 1.18:1, showing that the number of female patients with CRC was increased significantly compared with males. The CRC distribution of T2DM patients and non-diabetic patients showed a predominance of rectal cancer (64.4%, 68.7%), followed by sigmoid colon cancer (12.5%, 13.0%), and moderately differentiated ulcer-type adenocarcinoma. Compared with non-diabetic patients, T2DM patients were older (66.2 years versus 62.7 years, P < 0.001) and had more multiple CRCs (3.5% versus 1.6%, P < 0.001). Moreover, the proportion of lymph node or organ metastasis in T2DM patients was higher than that in non-diabetic patients (52.6% versus 45.6%, P < 0.05). No significant differences were observed between both groups in terms of gender, gross type, histological type, and differentiation(P > 0.05 for all). CONCLUSIONS: CRC incidence shows an increasing trend with age. CRC patients with T2DM have an older age of onset, higher proportion of lymph node and distant organ metastasis than in non-diabetic patients.

mRNA expression of IGF-1 and IGF-1R in patients with colorectal adenocarcinoma and type 2 diabetes.

BACKGROUND AND AIMS: Increasing studies show that messenger RNA (mRNA) levels of local IGF-system are overexpressed in cancer tissue of patients with colorectal cancer (CRC). However, the influence of type 2 diabetes (T2DM) on the expression of insulin-like growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R) mRNA in colorectal cancer tissue and adjacent non-cancerous tissue (ANCT) is unknown. The aim of this study was to assess mRNA expression of IGF-1 and IGF-1R in paired samples of cancer tissue and ANCT between colorectal adenocarcinoma (CA) patients with and without T2DM. METHODS: To quantify the levels of IGF-1 and IGF-1R mRNA in CA, we analyzed the expression of IGF-1 and IGF-1R mRNA levels in paired samples of cancer tissue and ANCT in CA patients with and without T2DM using real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: mRNA levels of IGF-1 and IGF-1R were significantly higher in cancer tissue compared with its ANCT in CA patients with and without T2DM. Compared with the CA group, significantly higher levels of IGF-1 and IGF-1R mRNA were observed in cancer tissue in CA with T2DM group. No significant differences were observed in the role of cancer locations, Dukes stages and diabetes duration on mRNA expression of IGF-1. After adjusting for age, gender and Dukes stages, multivariate analysis indicated IGF-1 mRNA level was a risk factor for prognosis (p <0.05). CONCLUSIONS: Our results support the hypothesis that IGF system plays an important role in CRC. Further larger studies are needed.

[The cloning, expression and the binding ability with TRß1 of retinoid X receptor-α gene].

Retinoid X receptor-α (RXR-α), a member of nuclear receptor family, is capable of mediating retinoid signaling pathways and plays a critical role in regulating target gene transcription. To further study the function of RXR-α, abundant of recombinant RXR-α protein in hand is necessary. In this study an intact RXR-α coding sequence was amplified by RT-PCR and subsequently inserted into expression plasmid vector pQE-30Xa to form the recombinant construct of pQE-30Xa/RXR-α. Thereafter, competent bacteria Escherichia coli M15 [PREP4] was transformed and the expression of RXR-α was induced by adding IPTG to the medium. Bacterially expressed recombinant RXR-α was purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blotting analyses. The results showed that a protein, with the molecular mass around 50 kDa, could be selectively recognized by anti-RXR-α antibody. Co-immunoprecipitation assay indicated that this recombinant RXR-α could effectively bind TRß1 to form a heterodimer, which could specifically bind the target DNA fragment. This was confirmed by EMSA. In conclusion, the recombinant human retinoid X receptor-α was prepared successfully, which makes a basic for further study of its function.

Cloning and analysis of rat osteoclast inhibitory lectin gene promoter.

Osteoclast inhibitory lectin (OCIL) is a novel regulator of bone remodeling, however, little is known concerning how OCIL is regulated to date. In this study, approximately 4.4 kb of the 5'-flanking sequence of rat OCIL gene was cloned into the promoter-less reporter vector pGL3-basic and transiently transfected into three different cell lines. The differences in the levels of luciferase activity paralleled well with the levels of OCIL mRNA expression in these cells, suggesting that the regulation of rat OCIL gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments showed that the construct-1819/pGL3 (-1819 to +118) exhibited the highest luciferase activity, suggesting the presence of functional promoter in this region. The region from -4370 to -2805 might contain negative regulatory elements, while the region from -1819 to -1336 might have important positive regulatory elements that enhance OCIL transcription. Sequence analysis of the promoter revealed the absence of both TATA and CAAT boxes. However, in the proximal promoter region (-81 to +118), several potential transcription factor binding sites that may be responsible for the basal transcriptional activity of rat OCIL promoter were observed. The promoter contains several potential Sp1 binding sites, and cotransfection of a shRNA expression plasmid that knockdowns Sp1 significantly reduced OCIL promoter activity and endogenous gene expression and moreover, overexpressing Sp7, a Sp1 family member that also binds to Sp1 binding sequence, increased OCIL promoter activity and gene expression, suggesting a role of Sp1 family proteins in regulation of OCIL transcription.