The MQRG score: a novel prognostic tool for adrenocortical carcinoma patients based on mitochondrial quality.

Objectives: Adrenal tumors are common, but adrenocortical carcinomas (ACCs) are a rare and challenging form of cancer to diagnose and manage.This study aimed to explore the critical role of mitochondrial quality in maintaining cellular function and the implications of the abnormal expression of mitochondrial metabolism-related proteins observed in ACC patients. We focused on identifying the connection between mitochondrial quality and the development of ACC at molecular and genomic levels. Methods: We compared mitochondrial quality-related genes (MQRGs) across ACC subtypes using overall survival (OS) and disease-free survival (DFS) as evaluation indicators. Furthermore, a novel MQRG score was developed to predict clinical prognosis and guide immunotherapy responses accurately. Results: The majority of MQRGs were upregulated in the ACC samples, correlating to poor prognosis. The MQRG score was confirmed as an independent prognostic factor for ACC, with the high-risk MQRG score group showing a significantly shorter overall survival period. Conclusions: Multilayer alterations in MQRGs are associated with patient prognosis and immune cell infiltration characteristics. This comprehensive analysis of MQRGs can contribute to a deeper understanding of potential differences in ACC patients' tumor microenvironment. This can influence clinical decision-making and advanced prognosis prediction, thereby offering new insights into personalized treatments in ACC.

BET proteins inhibitor JQ1 impairs GM-CSF-promoted peritoneal macrophage self-renewal and IL-4-induced alternative polarization.

Peritoneal macrophages (PMs), which resided in peritoneal cavity, are crucial to maintain tissue homeostasis and immunity. Macrophage self-renewal and polarization states are critical for PM population homeostasis and function. However, the underlying molecular mechanism that regulates self-renewal and polarization of PMs is still unclear and needs to be explored. Here, we demonstrated that PMs self-renewal was stimulated by granulocyte macrophage colony-stimulating factor (GM-CSF), but not by macrophage colony-stimulating factor (M-CSF). Pharmacological inhibition of Bromodomain & Extraterminal (BET) Proteins by either JQ1 or ARV-825 significantly reduced GM-CSF-dependent peritoneal macrophage self-renewal by abrogating cell proliferation and decreasing self-renewal-related gene expression, such as MYC and Klf4, at transcriptional and protein levels. In addition, transcriptomic analysis showed that JQ1 blocked alternative PMs polarization by downregulating key transcriptional factor IRF4 expression, but not the activation of AKT or STAT6 in PMs. These findings illustrated that the significance of BET family proteins in GM-CSF-induced PMs self-renewal and IL-4-induced alternative polarization.

The prognostic value of tumor mutation burden (TMB) and its relationship with immune infiltration in breast cancer patients.

OBJECTIVE: Although the tumor mutation burden (TMB) was reported as a biomarker for immunotherapy of various cancers, whether it can effectively predict the survival prognosis in breast cancer patients remains unclear. In this study, the prognostic value of TMB and its correlation with immune infiltration were explored by using multigroup studies. METHODS: The somatic mutation data of 986 breast cancer patients were obtained from TCGA database. Breast cancer patients were divided into a low-TMB group and a high-TMB group according to the quartile of TMB scores. The differentially expressed genes (DEGs) were identified by the "limma" R program. The CIBERSORT algorithm was utilized to estimate the immune cell fraction of each sample. The TIMER database was utilized to evaluate the association between CNVs of immune genes and tumor immune cell infiltration and the prognostic value of the immune cells in breast cancer. RESULTS: In breast cancer, TP53, PIK3CA, TTN, CDH1 and other genes were the most important mutated genes. Higher survival rate of patients was found in the low-TMB group. Among the top 10 DEGs, three of them belong to the KRT gene family. GSEA enrichment analysis showed that MAPK, Hedgehog, mTOR, TGF-bate and GnRH signaling pathways were enriched in the low-TMB group. The infiltration levels of the most of immune cells were higher in the low-TMB group (P < 0.01). Higher expression of CCL18 and TRGC1 was correlated with poor prognosis. Breast cancer patients with CCL18 copy number variations, especially arm-level gains, showed significantly decreased immune cell infiltration. In the low B cell infiltration group, the survival prognosis of breast cancer patients was poor. CONCLUSIONS: TMB is a potential prognosis marker in breast cancer. Immune-related gene CCL18 and TRGC1 are biomarkers of poor prognosis while immune (B cell) infiltration is a biomarker of good prognosis.

A novel ferroptosis-related gene prognostic index for prognosis and response to immunotherapy in patients with prostate cancer.

Background: Prostate cancer (PCa) is among the leading causes of cancer death worldwide. Ferroptosis refers to an iron-dependent form of regulated cell death and is involved in prostate tumorigenesis. A few ferroptosis-related gene signatures have been developed to predict the prognosis for PCa patients. However, previous signatures were typically established based on biochemical recurrence-free survival, which has proven not to be a good surrogate for overall survival (OS). This study aimed to construct a novel ferroptosis-related gene prognostic index (FRGPI) to predict disease-free survival (DFS) and response to immunotherapy for PCa patients after radical prostatectomy. Methods: Gene expression and clinicopathological data on PCa patients were obtained from the TCGA database. Ferroptosis-related hub genes associated with DFS of PCa patients were identified by an in-depth bioinformatics analysis using a novel and comprehensive algorithm based on functional enrichment, consensus clustering, weighted gene co-expression network analysis (WGCNA), and protein-protein interaction (PPI) network construction. The FRGPI was established on the basis of the genes selected using multivariate cox regression analysis and further validated in two additional PCa cohorts. Next, the clinicopathological, molecular, and immune profiles were characterized and compared between FRGPI-high and FRGPI-low subgroups. Finally, the predictive role of the FRGPI in response to immunotherapy was estimated using a metastatic urothelial cancer cohort treated with an anti-PD-L1 agent. Results: The FRGPI was constructed based on four genes (E2F1, CDC20, TYMS, and NUP85), and FRGPI-high patients had worse DFS than FRGPI-low patients. Multivariate cox regression analysis revealed that FRGPI could act as an independent prognostic factor for PCa patients after radical prostatectomy. A prognostic nomogram comprising the FRGPI and other clinicopathological parameters was established to predict the DFS for PCa patients quantitatively. In addition, comprehensive results demonstrated that high FRGPI scores showed a significantly positive correlation with worse clinicopathological features, higher mutation counts, increased frequency of copy number variations (CNVs), higher homologous recombination deficiency (HRD) and immune scores, higher mRNAsi, and more importantly, enhanced sensitivity to immunotherapy. Conclusions: FRGPI is not only a promising and robust prognostic biomarker, but also a potential indicator of immunotherapeutic outcomes for PCa patients after radical prostatectomy.

High mRNA Expression of CENPL and Its Significance in Prognosis of Hepatocellular Carcinoma Patients.

Centromere proteins (CENPs) are the main constituent proteins of kinetochore, which are essential for cell division. In recent years, several studies have revealed that several CENPs were aberrantly expressed in hepatocellular carcinoma (HCC). However, numerous centromere proteins have not been studied in HCC. In this study, we used databases of Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), the Kaplan-Meier Plotter, cBioPortal, the Human Protein Atlas (HPA), and TIMER (Tumor Immune Estimation Resource) and immunohistochemical staining of clinical specimens to investigate the expression of 15 major centromere proteins in HCC to evaluate their potential prognostic value. We found that the mRNA levels of 4 out of 15 centromere proteins (CENPL, CENPQ, CENPR, and CENPU) were significantly higher in HCC than in normal tissues, and their mRNA levels were associated with the tumor stages (p values < 0.01). Patients with higher mRNA levels of CENPL had poorer overall survival, progression-free survival, relapse-free survival, and disease-specific survival (p values < 0.05). Furthermore, the higher levels of CENPL mRNA were associated with worse overall survival in males without hepatitis virus infection (p values < 0.05). The protein expression level of CENPL in human HCC tissue was higher than that in normal liver tissue. In addition, the expression of CENPL was positively correlated with the levels of the tumor-infiltrating lymphocytes. The results suggest that the high mRNA expression of CENPL may be a potential predictor of prognosis in HCC patients.

Exploring the effects of formula feeding on infant immune development in China: A prospective cohort study.

BACKGROUND AND OBJECTIVES: The worldwide exclusive breastfeeding rate is suboptimal and this study aims to evaluate effects on infant immune development of formula feeding. METHODS AND STUDY DESIGN: A prospective study including 221 infants fed with breast milk or formula was conducted. At 3-month and 9-month, the concentrations of total immunoglobulin (Ig)G, IgM, IgA, IgG1, IgG2, interleukin (IL)-4, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were measured by using enzyme-linked immunosorbent assay (ELISA). Natural killer (NK) cell activity and lymphocyte transformation testing were conducted. Furthermore, the occurrence of infantile diarrhea, respiratory infections and allergic diseases were questioned. RESULTS: The levels of total IgG (Z=-3.21, p=0.001), IgG1 (Z=-2.12, p=0.034), IFN-γ (t=-2.09, p=0.039) and NK cell activity (t=-2.14, p=0.034) were significant higher in formula-fed infants compared to breast-fed after 3 months. At 9-month, the levels of total IgG (Z=-4.34, p<0.001), IgA (Z=-2.05, p=0.041) and TNF-α (t=-2.10, p=0.037) of formula-fed infants were higher, but the lymphocyte stimulation index (t=2.76, p=0.007) was lower than breast-fed infants. While, no significant differences were found in the incidences of diarrhea and respiratory tract infection (p>0.05). CONCLUSIONS: This investigation suggested that formula- and breast-feeding have different contributions to infant immune development, but the formula feeding would not cause significantly increase of diarrhea and respiratory infections.

The Predictive Value of Red Cell Distribution Width and Red Cell Distribution Width to Erythrocyte Count Ratio for Adverse Cardiovascular Events During the Hospitalization of Patients of ST-segment Elevation Myocardial Infarction.

BACKGROUND: High red cell distribution width (RDW) is correlated with poor prognosis in acute coronary syndromes (ACS), including ST-segment elevation myocardial infarction (STEMI). However, the association of red cell distribution width to erythrocyte count ratio (RER) with STEMI undergoing percutaneous coronary intervention (PCI) during hospitalization has not been investigated. Therefore, we performed a retrospective study to investigate whether RER is associated with STEMI patients after PCI during hospitalization. METHODS: A total of 331 patients, who were hospitalized for STEMI and underwent PCI, were enrolled. Receiver operating characteristic curve (ROC) analyses were used to find the cutoff value of RER and classify the patients into two groups including higher RER group and lower RER group by cutoff value. Differences between measured parameters in higher RER and lower RER groups were analyzed by the Mann-Whitney U test. The evaluation correlation of RDW, red blood cell, and RER with major adverse cardiovascular events was determined by bivariate regression analysis. Univariate logistic regression analysis was used to determine the factors associated with adverse cardiovascular events during the hospitalization of STEMI patients. Multivariate logistic regression analysis was performed to evaluate the potential independent predictors of STEMI. RESULTS: According to ROC analysis, the cutoff value of RER and RDW is 3.10 and 13.9, the sensitivity is 51% and 35%, the specificity is 76% and 80%, respectively. RER showed improved diagnostic capacity compared to RDW in correlation with adverse cardiovascular events during hospitalization in STEMI patients (p < 0.001). Compared with the lower RER group, the incidence of adverse cardiovascular events in STEMI patients is elevated in the higher RER group (75% vs. 64.5%, p < 0.05). Bivariate regression analysis indicated that RER and RDW showed a good correlation with adverse cardiovascular events, and the difference was statistically significant (R = 0.10 p < 0.05 vs. R = 0.05 p < 0.05). Univariate logistic regression analysis showed that age, heart rate, left ventricular ejection fraction, hyperlipidemia, RDW, mean platelet volume, total cholesterol, and RER were correlated with the occurrence of adverse cardiovascular events during the hospitalization of STEMI patients (p < 0.05). Multivariate logistic regression analysis demonstrated that RER could be an independent predictor of adverse cardiovascular events in STEMI patients (B: 0.574, OR: 1.776, 95% CI: 1.043 ~ 3.023, p < 0.05). CONCLUSIONS: RER and RDW demonstrated good correlation with adverse cardiovascular events during hospitalization in STEMI patients. RER is a potential independent predictor of adverse cardiovascular events during hospitalization in STEMI patients.

A Potential Serum Biomarker, Albumin-to-Glutamyltransferase Ratio, Suggests the Severity of Liver Disease.

BACKGROUND: We aimed to investigate the utility of albumin-to-glutamyltransferase ratio (AGR) as a new biomarker to distinguish hepatic carcinoma from hepatitis, as early disease diagnosis, prognosis or monitoring could improve patient management and outcomes. METHODS: Clinical characteristics of 34 hepatitis (women 19), 88 cirrhosis (women 22) and 52 hepatic carcinoma (women 9) cases were retrospectively reviewed. Patients diagnosed with cirrhosis were classified by Child-Pugh score and the presence of ascites. The differences among groups were evaluated by the Kruskal-Wallis test and Mann-Whitney U. The linear correlation between variables was assessed by Spearman's correlation analysis. The diagnostic value of albumin-to-glutamyltransferase (AGR) was considered using receiver operating characteristic (ROC) curves. Multiple logistic regression analysis and univariate logistic regression analysis were used to identify AGR as an independent predictor in liver disease progression. RESULTS: The significant differences among the hepatitis vs. cirrhosis vs. and hepatic carcinoma were AST (108.50 ± 184.00 vs. 38.00 ± 21.50 vs. 47.00 ± 71.00, p < 0.01), TP/AST (TAR, 0.67 ± 0.69 vs. 1.77 ± 0.87 vs. 1.36 ± 0.95, p < 0.01), and ALB/GGT (AGR, 0.32 ± 0.27 vs. 0.67 ± 0.43 vs. 0.20 ± 0.26, p < 0.05). At the same time, AST (32.00 ± 13.50 vs. 53.00 ± 23.00 vs. 114.50 ± 42.50, p < 0.05) and TAR (2.15 ± 0.72 vs. 1.28 ± 0.74 vs. 0.64 ± 0.39, p < 0.05) were higher but AGR (0.86 ± 0.54 vs. 0.46 ± 0.32 vs. 0.26 ± 0.22, p < 0.05) was lower in Child-Pugh class C group compared with group B and C. TAR (1.92 ± 0.73 vs. 0.98 ± 0.89, p < 0.01) and AGR (0.79 ± 0.52 vs. 0.46 ± 0.28, p < 0.05) were significantly elevated in the serum of cirrhosis with no ascites compared with the cirrhosis patients suffered from ascites, while AST (35.00 ± 14.50 vs. 63.00 ± 44.50, p < 0.01) was reduced in cirrhosis patients with no ascites. Furthermore, AST (r = 0.4490, p<0.01) was positively correlated with AFP, TAR (r = -0.4393, p < 0.01) and AGR (r = -0.4395, p < 0.01) were negatively correlated with AFP. The ROC curve analysis for AST had an area under the curve (AUC) ranging from 0.66 to 0.82, TAR ranged from 0.64 to 0.80 and AGR ranged from 0.54 to 0.72. Multiple logistic regression analysis revealed AGR as an independent parameter to distinguish liver can¬cer to hepatitis, and AGR was associated with the presence of ascites and the progression in cirrhosis patients. CONCLUSIONS: AGR is a potential biomarker for diagnosis of liver disease progression.

The Relationship between Erythrocyte Sedimentation Rate and Erythrocyte Sedimentation/Red Blood Cell Ratio and Disease Activity in Systemic Lupus Erythematosus.

BACKGROUND: The treatment options of systemic lupus erythematosus (SLE) patients in active and inactive phases are very different clinically, and the prognosis of patients with active SLE is much worse than inactive patients. However, the present indicators for diagnosis of SLE in activity are limited and inefficient. METHODS: Three hundred thirty patients with SLE were included. All patients are classified as SLEDAI (systemic lupus erythematosus disease activity index) > 4 as active and SLEDAI ≤ 4 as inactive. The linear correlation between variables was assessed by Pearson's correlation analysis. The difference between parameters in active and inactive patients was evaluated by the Mann-Whitney U test. The evaluation capacity of erythrocyte sedimenta-tion/red blood cell (ERR) and red blood cell/albumin ratio (RAR) on SLE activity was determined by bivariate regression analysis. Sensitivity and specificity are assessed by receiver operating characteristic curve (ROC). RESULTS: Compared with the inactive SLE, ESR (52.97 ± 35.66 vs. 32.38 ± 29.16 p < 0.001), ERR (15.40 ± 12.41 vs. 8.19 ± 8.10 p < 0.001) and RAR (0.13 ± 0.10 vs. 0.11 ± 0.20 p = 0.038) are all elevated in active SLE (52.97 ± 35.66 vs. 32.38 ±2 9.16 p < 0.001). ERR shows better correlation than RAR with ESR (p < 0.001 vs. p = 0.911). Patients with active SLE exhibited higher SLEDAI than those with inactive SLE (8.67 ± 2.67 vs. 3.27 ± 1.36, p < 0.001). According to ROC analysis, when ESR levels > 58.5 and ERR levels > 13.18, the sensitivity is 37.6% and 45.2%, the specificity is 83.0% and 83.2%. CONCLUSIONS: ESR and ERR are potential indicators for diagnosis of active and inactive SLE.

LRH-1 drives hepatocellular carcinoma partially through induction of c-myc and cyclin E1, and suppression of p21.

BACKGROUND: To explore potential therapeutic target is one of the areas of great interest in both clinical and basic hepatocellular carcinoma (HCC) studies. Nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) is proved to play a positive role in several cancers including breast cancer, pancreatic cancer and intestinal cancer in recent years. However, the exact role of LRH-1 in the development and progression of HCC is not fully elucidated. METHODS: The LRH-1 expression level in HCC clinical samples was examined by immunohis-tochemistry (IHC). Stable LRH-1-suppressed HepG2 clones (HepG2LRH-1/-) were generated by transcription activator-like effector nucleases (TALENs) and both in vitro and in vivo experiments were conducted. RESULTS: We confirmed that LRH-1 showed an increased expression pattern in HCC clinical samples. Our in vitro and in vivo results indicated that suppression of LRH-1 in HepG2 significantly attenuated its proliferation rate and tumorigenic capacity. Gene expression microarray analysis indicated that LRH-1mostly regulated gene expression involved in cell cycle. In addition, our gain-of-function experiments indicated that ectopic expression of LRH-1 dramatically induced the mRNA and protein levels of c-myc and cyclin E1, while attenuating the expression of p21. CONCLUSION: Our results suggest that LRH-1 might be a potential therapeutic target for clinical HCC treatment.

A Tumor-Specific Tissue-Penetrating Peptide Enhances the Efficacy of Chemotherapy Drugs in Gastric Cancer.

PURPOSE: C-end rule (CendR) peptides are found to enhance the penetration of chemotherapeutic agents into tumor cells, while GX1 is a peptide that homes to gastric cancer (GC) vasculature. This study aimed to synthesize a novel peptide GX1-RPAKPAR (GXC) and to explore the effect of GXC on sensitizing GC cells to chemotherapeutic agents. MATERIALS AND METHODS: Intracellular Adriamycin concentration analysis was applied to conform whether GXC peptide increases the penetration of chemotherapeutic agents into GC cells in vitro. The effect of GXC peptide on sensitizing GC cells to chemotherapeutics was validated by apoptosis assay and in vitro/vivo drug sensitivity assay. The specificity of GXC to GC tissue was validated by ex vivo fluorescence imaging. RESULTS: In vitro, administration of GXC significantly increased Adriamycin concentrations inside SGC-7901 cells, and enhanced the efficacy of chemotherapeutic agents by decreasing the IC50 value. In vivo, FITC-GXC specifically accumulated in GC tissue. Moreover, systemic co-injection with GXC peptide and Adriamycin statistically improved the therapeutic efficacy in SGC-7901 xenograft models, surprisingly, without obviously increasing side effects. CONCLUSION: These results demonstrated that co-administration of the novel peptide GXC with chemotherapeutic agents may be a potential way to enhance the efficacy of anticancer drugs in GC treatment.

Orphan nuclear receptor TLX contributes to androgen insensitivity in castration-resistant prostate cancer via its repression of androgen receptor transcription.

The metastatic castration-resistant prostate cancer (CRPC) is a lethal form of prostate cancer, in which the expression of androgen receptor (AR) is highly heterogeneous. Indeed, lower AR expression and attenuated AR signature activity is shown in CRPC tissues, especially in the subset of neuroendocrine prostate cancer (NEPC) and prostate cancer stem-like cells (PCSCs). However, the significance of AR downregulation in androgen insensitivity and de-differentiation of tumor cells in CRPC is poorly understood and much neglected. Our previous study shows that the orphan nuclear receptor TLX (NR2E1), which is upregulated in prostate cancer, plays an oncogenic role in prostate carcinogenesis by suppressing oncogene-induced senescence. In the present study, we further established that TLX exhibited an increased expression in metastatic CRPC. Further analyses showed that overexpression of TLX could confer resistance to androgen deprivation and anti-androgen in androgen-dependent prostate cancer cells in vitro and in vivo, whereas knockdown of endogenous TLX could potentiate the sensitivity to androgen deprivation and anti-androgen in prostate cancer cells. Our study revealed that the TLX-induced resistance to androgen deprivation and anti-androgen was mediated through its direct suppression of AR gene transcription and signaling in both androgen-stimulated and -unstimulated prostate cancer cells. We also characterized that TLX could bind directly to AR promoter and repress AR transcription by recruitment of histone modifiers, including HDAC1, HDAC3, and LSD1. Together, our present study shows, for the first time, that TLX can contribute to androgen insensitivity in CRPC via repression of AR gene transcription and signaling, and also implicates that targeting the druggable TLX may have a potential therapeutic significance in CRPC management, particularly in NEPC and PCSCs.

Nuclear Receptor LRH-1 Functions to Promote Castration-Resistant Growth of Prostate Cancer via Its Promotion of Intratumoral Androgen Biosynthesis.

Targeting of steroidogenic enzymes (e.g., abiraterone acetate targeting CYP17A1) has been developed as a novel therapeutic strategy against metastatic castration-resistant prostate cancer (CRPC). However, resistance to steroidal inhibitors inevitably develops in patients, the mechanisms of which remain largely unknown. Liver receptor homolog-1 (LRH-1, NR5A2) is a nuclear receptor, originally characterized as an important regulator of some liver-specific metabolic genes. Here, we report that LRH-1, which exhibited an increased expression pattern in high-grade prostate cancer and CRPC xenograft models, functions to promote de novo androgen biosynthesis via its direct transactivation of several key steroidogenic enzyme genes, elevating intratumoral androgen levels and reactivating AR signaling in CRPC xenografts as well as abiraterone-treated CRPC tumors. Pharmacologic inhibition of LRH-1 activity attenuated LRH-1-mediated androgen deprivation and anti-androgen resistance of prostate cancer cells. Our findings not only demonstrate the significant role of LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management.Significance: These findings not only demonstrate the significant role of the nuclear receptor LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management. Cancer Res; 78(9); 2205-18. ©2018 AACR.

STAT3-mediated TLR2/4 pathway upregulation in an IFN-gamma-induced Chlamydia trachomatis persistent infection model.

Inflammatory pathological injury caused by Chlamydia trachomatis persistent infection could lead to a variety of urogenital tract diseases. By comparing the cytokine production and PRR (pattern recognition receptor) expression between cell models with acute or persistent C. trachomatis infection, our data supported that persistent infection of C. trachomatis led to abnormal activation of toll-like receptor (TLR)2/4 signaling pathway and elevated IL-1α and IL-6 production, which was mediated by signal transducer and activator of transcription3 (STAT3). Studying the effects of abnormal activation of TLR signaling pathway in the cells with C. trachomatis persistent infection could provide new hints for chronic infection treatment and an important experimental basis for understanding the pathogenesis of C. trachomatis persistent infection.

TGF-ß2 induces epithelial-mesenchymal transition in cultured human lens epithelial cells through activation of the PI3K/Akt/mTOR signaling pathway.

The present study aimed to investigate whether the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is involved in the transforming growth factor ß2 (TGF-ß2)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial (HLE) cells. HLEB-3 cells were cultured and stimulated with 10 ng/ml TGF-ß2 for 24 h. Western blotting was then performed to analyze the expression levels of connexin 43 and fibronectin, and the activities of Akt and mTOR. Confocal cell immunofluorescence was used to observe the expression of phosphorylated (p)-Akt. The toxicity of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) was assessed using a Cell Counting Kit-8 assay, and inhibition investigations were performed using a PI3K inhibitor. The expression of connexin 43 was suppressed and the expression of fibronectin was increased when the cells were stimulated with 10 ng/ml TGF-ß2 for 24 h. In addition, Akt and mTOR were activated during TGF-ß2-induced EMT. Treatment of with LY294002 (20 µM) inhibited the activation of Akt and mTOR and effectively prevented TGF-ß2-induced EMT in the HLECs. Therefore, the results of the present study indicated that TGF-ß2 induces EMT by activating the PI3K/Akt/mTOR signaling pathway in cultured HLECs.

The lncRNA H19 promotes epithelial to mesenchymal transition by functioning as miRNA sponges in colorectal cancer.

Recently, the long non-coding RNA (lncRNA) H19 has been identified as an oncogenic gene in multiple cancer types and elevated expression of H19 was tightly linked to tumorigenesis and cancer progression. However, the molecular basis for this observation has not been characterized in colorectal cancer (CRC) especially during epithelial to mesenchymal transition (EMT) progression. In our studies, H19 was characterized as a novel regulator of EMT in CRC. We found that H19 was highly expressed in mesenchymal-like cancer cells and primary CRC tissues. Stable expression of H19 significantly promotes EMT progression and accelerates in vivo and in vitro tumor growth. Furthermore, by using bioinformatics study and RNA immunoprecipitation combined with luciferase reporter assays, we demonstrated that H19 functioned as a competing endogenous RNA (ceRNA) for miR-138 and miR-200a, antagonized their functions and led to the de-repression of their endogenous targets Vimentin, ZEB1, and ZEB2, all of which were core marker genes for mesenchymal cells. Taken together, these observations imply that the lncRNA H19 modulated the expression of multiple genes involved in EMT by acting as a competing endogenous RNA, which may build up the missing link between the regulatory miRNA network and EMT progression.

Endostar attenuates melanoma tumor growth via its interruption of b-FGF mediated angiogenesis.

To develop optimal therapeutics is one of the hotspots in both clinical and basic melanoma studies. Previous studies indicate that fibroblast growth factors (b-FGF/FGF-2), an angiogenesis inducer beyond VEGF, might be a potential drug target in melanoma. As a novel anti-angiogenesis peptide drug, Endostar has shown promising therapeutic efficacy in non-small cell lung cancer. However, the effect of Endostar on b-FGF-induced angiogenesis in melanoma is unraveled. To this end, both in vivo and in vitro experiments were conducted and it was found that treatment of Endostar could inhibit tumor growth, which was accompanied by decreased micro-vessel density and serum b-FGF levels in a mouse melanoma model. In addition, treatment with Endostar in blood vessel endothelial cells could reduce their proliferation, cell migration and tube formation capacity in a dosage-dependent manner. Moreover, treatment of Endostar could also attenuate b-FGF-activated phosphorylation of p38 and ERK1/2 in HUVECs. These findings indicate that Endostar might exert its anti-tumor effect via suppressing b-FGF-induced angiogenesis and b-FGF-activated MAPK signaling pathway, suggesting that Endostar might be a potential choice for clinical melanoma treatment.

Orphan nuclear receptor TLX functions as a potent suppressor of oncogene-induced senescence in prostate cancer via its transcriptional co-regulation of the CDKN1A (p21(WAF1) (/) (CIP1) ) and SIRT1 genes.

Oncogene-induced senescence is an important tumour-suppressing mechanism to prevent both premalignant transformation and cancer progression. Overcoming this process is a critical step in early cancer development. The druggable orphan nuclear receptor TLX (NR2E1) is characterized as an important regulator of neural stem cells and is also implicated in the development of some brain tumours. However, its exact functional roles in cancer growth regulation still remain unclear. Here we report that TLX can act as a promoter of tumourigenesis in prostate cancer by suppressing oncogene-induced senescence. We determined that TLX exhibited an increased expression in high-grade prostate cancer tissues and many prostate cancer cell lines. Functional studies revealed that TLX could perform an oncogenic function in prostate cancer cells, as its knockdown triggered cellular senescence and cell growth arrest in vitro and in vivo, whereas its over-expression promoted the malignant growth of prostate cancer cells. Furthermore, enhancement of TLX activity, by either ectopic expression or ligand stimulation, could potently prevent doxorubicin-induced senescence in prostate cancer cells and also allow prostatic epithelial cells to escape oncogene-induced senescence induced either by activated oncogene H-Ras(G12V) or knockdown of tumour suppressor PTEN, via a mechanism of direct but differential transcriptional regulation of two senescence-associated genes, repression of CDKN1A and transactivation of SIRT1. Together, our present study shows, for the first time, that TLX may play an important role in prostate carcinogenesis through its suppression of oncogene-induced senescence, and also suggests that targeting the senescence-regulatory TLX is of potential therapeutic significance in prostate cancer.

Identification of miRNAs that specifically target tumor suppressive KLF6-FL rather than oncogenic KLF6-SV1 isoform.

The Krüppel like factor 6 (KLF6) gene encodes multiple protein isoforms derived from alternative mRNA splicing, most of which are intimately involved in hepatocarcinogenesis and tumor progression. Recent bioinformatics analysis shows that alternative mRNA splicing of the KLF6 gene produces around 16 alternatively spliced variants with divergent or even opposing functions. Intriguingly, the full-length KLF6 (KLF6-FL) is a tumor suppressor gene frequently inactivated in liver cancer, whereas KLF6 splice variant 1 (KLF6-SV1) is an oncogenic isoform with antagonistic function against KLF6-FL. Compelling evidence indicates that miRNA, the small endogenous non-coding RNA (ncRNA), acts as a vital player in modulating a variety of cellular biological processes through targeting different mRNA regions of protein-coding genes. To identify the potential miRNAs specifically targeting KLF6-FL, we utilized bioinformatics analysis in combination with the luciferase reporter assays and screened out two miRNAs, namely miR-210 and miR-1301, specifically targeted the tumor suppressive KLF6-FL rather than the oncogenic KLF6-SV1. Our in vitro experiments demonstrated that stable expression of KLF6-FL inhibited cell proliferation, migration and angiogenesis while overexpression of miR-1301 promoted cell migration and angiogenesis. Further experiments demonstrated that miR-1301 was highly expressed in liver cancer cell lines as well as clinical specimens and we also identified the potential methylation and histone acetylation for miR-1301 gene. To sum up, our findings unveiled a novel molecular mechanism that specific miRNAs promoted tumorigenesis by targeting the tumor suppressive isoform KLF6-FL rather than its oncogenic isoform KLF6-SV1.

mTOR regulates TGF-ß2-induced epithelial-mesenchymal transition in cultured human lens epithelial cells.

BACKGROUND: Post-cataract surgery fibrosis in the lens capsule is caused by epithelial to mesenchymal transition (EMT) of the lens epithelium. Mammalian target of rapamycin (mTOR) has been demonstrated to be a key regulator of EMT. The aim of this study was to investigate the role of mTOR in transforming growth factor ß2 (TGF-ß2)-induced EMT in human lens epithelial cells (HLECs). METHODS: Human lens epithelial B-3 (HLEB-3) cells were cultured with 10 ng/ml TGF-ß2 for different periods of time. The expression of E-cadherin, connexin 43, fibronectin and α-smooth muscle actin (α-SMA), and activation of mTOR were determined by Western blots. Cell migration was assessed by wound healing assay. An inhibition test was performed using two kinds of mTOR inhibitors. RESULTS: E-cadherin and connexin 43 expressions were suppressed, whereas fibronectin and α-SMA expressions were increased in HLEB-3 cells after treatment with TGF-ß2. mTOR was activated during the TGF-ß2-induced EMT in a time-dependent manner. Rapamycin or Ku-0063794 with 100 nM was able to inhibit the phosphorylation of mTOR and impaired EMT induced by TGF-ß2. Cell motility enhanced by TGF-ß2 for 24 h was attenuated by both rapamycin and Ku-0063794. CONCLUSIONS: mTOR is activated during TGF-ß2-induced EMT in HLECs, suggesting that it is involved in the regulation of TGF-ß2-induced EMT and may contribute to the development of posterior capsule opacification.