Metabolic-associated signature and hub genes associated with immune microenvironment and prognosis in bladder cancer.

The relationship between metabolism and immune microenvironment remains to be studied in bladder cancer (BCa). We aimed to construct a metabolic-associated signature for prognostic prediction and investigate its relationship with the immune microenvironment in BCa. The RNA expression of metabolism associated genes was obtained from a combined data set including The Cancer Genome Atlas, GSE48075, and GSE13507 to divide BCa patients into different clusters. A metabolic-associated signature was constructed using the differentially expressed genes between clusters in the combined data set and validated in the IMvigor210 trial and our center. The composition of tumor-infiltrating immune cells (TIICs) was evaluated using the single-sample Gene Set Variation Analysis. BCa patients in Cluster A or high-risk level were associated with advanced clinicopathological features and poor survival outcomes. The percentage of high-risk patients was significantly lower in patients responding to anti-PD-L1 treatment. Compared with low-risk patients, the IC50 values of cisplatin and gemcitabine were significantly lower in high-risk patients. Thiosulfate transferase (TST) and S100A16 were significantly associated with clinicopathological features and prognosis. Downregulation of TST promoted BCa cell invasion, migration, and epithelial-to-mesenchymal transition, which are inhibited by downregulation of S100A16. CD8 + T cells, neutrophils, and dendritic cells had higher infiltration in the TST low-level and the S100A16 high-level. Furthermore, loss of function TST and S100A16 significantly affected the expression of PD-L1 and CD47. The metabolic-associated signature can stratify BCa patients into distinct risk levels with different immunotherapeutic susceptibility and survival outcomes. Metabolism disorder promoted the dysregulation of immune microenvironment, thus contributing to immunosuppression.

Reciprocal regulation between ßTrCP and Smurf1 suppresses proliferative capacity of liver cancer cells.

We previously reported that both the ubiquitin E3 ligases ßTrCP (beta-transducin repeat-containing E3 ubiquitin protein ligase) and Smurf1 (SMAD-specific E3 ubiquitin protein ligase 1) play similar antitumorigenic roles in liver cancer cells. However, whether and how they are reciprocally regulated remains elusive. Here, we show that ßTrCP interacts with Smurf1 through the 7 × tryptophan (W) aspartic acid (D)(WD) 40 and the region homologous to the E6-AP carboxyl terminus (HECT) domains, which are the E3 ligase domains of ßTrCP and Smurf1, respectively. The E3 ligase domains of ßTrCP and Smurf1 are also critical for maintaining the protein expressions of Smurf1 and ßTrCP. Moreover, a positive correlation between ßTrCP and Smurf1 was also revealed by tissue microarray analysis, indicating that this relationship might be important in liver cancer. Further, we found that Smurf1 increases the protein stability of ßTrCP, possibly by reducing autoubiquitination of ßTrCP, and vice versa. Interestingly, such effects depended on the presence of E3 ligase domains. Importantly, depletion of Smurf1- or ßTrCP-enhanced proliferative capacity of liver cancer cells could be partially reversed by overexpression of wild-type ßTrCP or Smurf1 but not their E3 ligase-dead mutants. Collectively, a reciprocal post-translational regulation between ßTrCP and Smurf1 has been uncovered in this study. Simultaneous enhancement of ßTrCP and Smurf1 functions might be helpful in the treatment of liver cancer.

The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function.

LPS is an immunostimulatory component of Gram-negative bacteria. Acting on the immune system in a systemic fashion, LPS exposes the body to the hazard of septic shock. In this study we report that cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2/Crispld2; human and mouse/rat versions, respectively), expressed by multitissues and leukocytes, is a novel LPS-binding protein. As a serum protein, median CRISPLD2 concentrations in health volunteers and umbilical cord blood samples are 607 microg/ml and 290 microg/ml, respectively. Human peripheral blood granulocytes and mononuclear cells including monocytes, NK cells, and T cells spontaneously release CRISPLD2 (range, 0.2-0.9 microg/ml) and enhance CRISPLD2 secretion (range, 1.5-4.2 microg/ml) in response to stimulation of both LPS and humanized anti-human TLR4-IgA Ab in vitro. CRISPLD2 exhibits significant LPS binding affinity similar to that of soluble CD14, prevents LPS binding to target cells, reduces LPS-induced TNF-alpha and IL-6 production, and protects mice against endotoxin shock. In in vivo experiments, serum Crispld2 concentrations increased in response to a nontoxic dose of LPS and correlated negatively with LPS lethality, suggesting that CRISPLD2 serum concentrations not only are indicators of the degree of a body's exposure to LPS but also reflect an individual's LPS sensitivity.

Exendin-4 Reversed the PC12 Cell Damage Induced by circRNA CDR1as/miR-671/GSK3ß Signaling Pathway.

The purpose of this paper is to study the effect of circRNA cerebellar degeneration-related protein 1 antisense RNA(CDR1as)/miR-671/GSK3ß signaling pathway on PC12 cell injury and the mechanism of Exendin-4 (Ex-4) in PC12 cell injury protection. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of circular RNA CDR1as and miR-671 in PC12 cells. By overexpressing or knocking out CDR1as, miR-671, and GSK3ß, the role of CDR1as, miR-671, and GSK3ß in PC12 cell injury was analyzed. The binding of CDR1as to miR-671 and GSK3ß to miR-671 was verified by dual luciferase reporter assay. PC12 cells were treated with 1-methyl-4 phenyl-pyridine ion (MPP+) to construct a PC12 cell damage model. PC12 cell transfection experiments were used to confirm the role of CDR1as/miR-671/GSK3ß signal axis in PC12 cell damage, and the role of Ex-4 in the association of circRNA CDR1as/miR-671/GSK3ß signaling axis and PC12 cell damage. PC12 cell damage was detected by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cellular lactate dehydrogenase (LDH) release. Ex-4 reversed the phosphorylation levels of PI3K, AKT, and GSK-3ß in MPP+-treated PC12 cells, and reduced MPP+-induced PC12 cell damage. CircRNA CDR1as upregulated the expression of GSK3ß by sponge miR-671. Ex-4 downregulated CDR1as expression and upregulated miR-671 expression in MPP+-induced PC12 cell. Silencing of CDR1as reduced MPP+-induced PC12 cell damage. CDR1as transfection downregulated the expression of miR-671 in PC12 cells, promoted the expression and phosphorylated of GSK3ß, and induced PC12 cell damage. GSK3ß silencing reversed CDR1as-induced PC12 cell damage. CDR1as promoted the phosphorylation level of GSK3ß in PC12 cells to cause cell damage; Ex-4 reversed the phosphorylation of GSK3ß caused by CDR1as in PC12 cells and reduced the PC12 cell damage caused by CDR1as. Ex-4 reverses the damage of PC12 cells induced by CDR1as/miR-671/GSK3ß signaling pathway.

Knockdown of Nestin inhibits proliferation and migration of colorectal cancer cells.

Nestin, a member of type VI intermediate filament protein family, is widely expressed in mammalian nervous tissue and stem/precursor cells of non-neuronal normal tissues. Nestin has also been investigated to determine possible tumor-promoting functions. However, whether Nestin is involved in colorectal cancer (CRC) cells remains unclear. In this report, Nestin expression was upregulated in stromal cells of human CRC tissues. Endogenous Nestin expression in CRC cell lines SW480 and HCT116 was knocked down by a lentivirus. MTT and colony formation assays revealed that Nestin deletion significantly inhibits the proliferation of CRC cell lines; flow cytometer analysis showed that Nestin deletion causes cell cycle arrest at S phase. Transwell chamber and wound healing scratch assays also revealed that Nestin deletion suppresses cell migration. Our findings indicated that Nestin plays an essential role in CRC progression; thus, Nestin can be applied as a therapeutic target of CRC.

ING4 inhibits the translation of proto-oncogene MYC by interacting with AUF1.

The ING4 tumor suppressor plays a significant role in various cancer-related cellular processes. AUF1 affects the stability and/or translation of multiple mRNAs via binding to an AU-rich element in the 3'-untranslated regions. In this study, we identify AUF1 as a novel and direct binding partner of ING4. mRNP immunoprecipitation assays indicated that ING4, AUF1 and MYC mRNA present in the same mRNP complex. ING4 suppressed MYC protein expression without altering MYC mRNA levels, and abolished the cell proliferation induced by AUF1 in K562 cells. These results suggest that ING4 may regulate MYC translation by its association with AUF1.

Downregulation of inhibitor of growth 3 is correlated with tumorigenesis and progression of hepatocellular carcinoma.

ING3, a member of the inhibitor of growth (ING) family, has been reported to be involved in transcription modulation, cell cycle control and the induction of apoptosis. Previous studies have demonstrated that the expression of ING3 decreased in melanoma and head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the role of ING3 in hepatocellular carcinoma (HCC) tumorigenesis and progression. The correlation between ING3 expression and clinicopathological variables of HCC was analyzed. Using the real-time reverse transcription-polymerase chain reaction (RT-PCR), it was found that ING3 was downregulated in HCC tissues compared with adjacent non-cancerous tissues (p<0.05). The immunohistochemical staining of tissue microarray data indicated a significant reduction of ING3 expression in 57.14% of HCC cases (64/112). In addition, the downregulation of ING3 was associated with the tumor differentiation stage. Most HCC samples of Edmondson-Steiner grades II to III exhibited inhibition of ING3 expression. The overexpression of ING3 in HCC cells was found to suppress cell proliferation, colony formation and cell migration, suggesting that ING3 acts as a tumor suppressor in HCC cells. Taken together, the data revealed that ING3 may serve as a suppression factor during tumorigenesis and progression of HCC.

Insulin receptor tyrosine kinase substrate enhances low levels of MDM2-mediated p53 ubiquitination.

The tumor suppressor p53 controls multiple cellular functions including DNA repair, cell cycle arrest and apoptosis. MDM2-mediated p53 ubiquitination affects both degradation and cytoplasmic localization of p53. Several cofactors are known to modulate MDM2-mediated p53 ubiquitination and proteasomal degradation. Here we show that IRTKS, a novel IRSp53-like protein inhibited p53-induced apoptosis and depressed its transcription activity. IRTKS bound directly to p53 and increased p53 ubiquitination and cytoplasmic localization. Further studies revealed that IRTKS interacted with MDM2 and promoted low levels of MDM2-mediated p53 ubiquitination in vitro and in vivo. In unstressed cells with low levels of MDM2, IRTKS was found to stabilize the interaction of p53 and MDM2. In stressed cells, IRTKS dissociated from p53, and high levels of MDM2 induced by p53 activation mediate IRTKS poly-ubiquitination and subsequent proteasomal degradation. These data suggest that IRTKS is a novel regulator of p53, modulating low level of MDM2-mediated p53 ubiquitination in unstressed cells.