Lamin B2 contributes to the proliferation of bladder cancer cells via activating the expression of cell division cycle­associated protein 3.

Bladder cancer is the most common malignant tumor of the urinary system, and in China it is first among urogenital system tumors. More therapeutic targets are still urgently required to combat this disease. Lamin B2 (LMNB2) is a type of nuclear lamina filament protein, which is involved in multiple cellular processes, and known as an oncogene affecting the progression of multiple types of cancers. Although the multiple effects of LMNB2 on cancer progression have been elucidated, its possible role in bladder cancer remains unclear. In the present study, it was determined that LMNB2 expression was upregulated in human bladder cancer tissues, and its expression was correlated with the prognosis and the clinical features, including tumor stage (P=0.001) and recurrence (P=0.006) of patients with bladder cancer. In addition, it was further revealed that LMNB2 depletion inhibited bladder cancer cell proliferation, stimulated cell cycle arrest and apoptosis in vitro, and suppressed tumor growth of bladder cancer cells in mice. Furthermore, the present data revealed that LMNB2 promoted the proliferation of bladder cancer cells via transcriptional activation of CDCA3 expression. Therefore, the role of LMNB2 in bladder cancer progression was demonstrated, and may serve as a promising therapeutic target for bladder cancer treatment.

Clinicopathological Significance of AKT1 and PLK1 Expression in Oral Squamous Cell Carcinoma.

Purpose: Oral squamous cell carcinoma (OSCC) is the sixth leading cause of cancer-related death worldwide and is characterized by metastasis and recurrence. We aimed to evaluate the expression of AKT1 and PLK1 in OSCC and identify their correlation with the clinical and histological features and prognosis of patients with OSCC. Methods: Tissue samples were collected from 70 patients with OSCC and 50 patients with normal oral mucosa. The expression levels of AKT1 and PLK1 in OSCC tissues and normal oral mucosa were detected by immunohistochemistry. The chi-square test was used to identify correlations between the expression levels of AKT1 and PLK1 with patients' clinicopathologic characteristics. Survival analysis was assessed by the Kaplan-Meier method. Spearman's rank correlation test was used to determine the relationships between AKT1 and PLK1 expressions. The bioinformatics database GEPIA was used to verify the experimental results. Results: The chi-square test and Fisher's exact test showed that the positive expression rate of AKT1 and PLK1 in OSCC tissue was significantly higher than that in the normal oral mucosa (P < 0.05). PLK1 expression levels were significantly correlated with tumor stage and size (P < 0.05). Kaplan-Meier analysis showed that the survival time of AKT1 and PLK1 with high expression was significantly shorter than that of patients with low expression (P < 0.05). Spearman's rank correlation test showed a strong correlation between AKT1 and PLK1 expression in OSCC tissue (R = 0.53; P < 0.05). GEPIA bioinformatics database analysis results show that the expression and overall survival of AKT1 and PLK1 analysis and the correlation analysis of AKT1 and PLK1 were consistent with experimental results. Conclusion: AKT1 and PLK1 expressions are associated with the occurrence and progression of OSCC and may be used as diagnostic and prognostic indicators of OSCC. There may be a correlation between AKT1 and PLK1 in OSCC tissue.

Targeted inhibition of the expression of both MCM5 and MCM7 by miRNA-214 impedes DNA replication and tumorigenesis in hepatocellular carcinoma cells.

MicroRNAs are noncoding RNAs with a typical length of 22 nucleotides that post-transcriptionally suppress gene expression by inducing target mRNA degradation and/or impairing translation in eukaryotes. Thousands of miRNA genes in the human genome are involved in various physiological and pathological processes. Each miRNA targets many different mRNAs, while each mRNA may be targeted by various miRNAs. Mini-chromosome maintenance (MCM2-7) protein complex functions as essential components of the pre-replicative complex (pre-RC) and forms a helicase together with other proteins to unwind the DNA duplex in S phase. MCM proteins are overexpressed in all cancer cells, while they are strictly regulated in normal cells, with no expression in non-proliferating normal cells. Here we report that miRNA-214-3p (miR-214) targets both MCM5 and MCM7. The level of miR-214 is lower in HepG2 and Hep3B hepatocellular carcinoma cells than the L-02 normal liver cells. Introduction of miRNA-214 mimic into HepG2 and Hep3B cells reduced the mRNA and protein levels of MCM5/7 and inhibited DNA replication, cell cycle progression, cell proliferation and colony formation. Comparatively, miRNA-214 mimic had little effect in L-02 cells. Importantly, miR-214 mimic can also inhibit the growth of HepG2 xenografts in nude mice. Our data suggest that miRNA-214 regulates DNA replication by targeting MCM5/7 and has the potential to be developed into a liver cancer drug. IMPLICATIONS: This study supports the notion that DNA replication-initiation proteins (DRIPs), including MCM2-7 proteins, are attractive anticancer targets. Furthermore, the potential of miR-214 as an anticancer agent, with activity against liver cancer cells but not normal livre cells, may be of high significance.

Myofibroblast YAP/TAZ activation is a key step in organ fibrogenesis.

Fibrotic diseases account for nearly half of all deaths in the developed world. Despite its importance, the pathogenesis of fibrosis remains poorly understood. Recently, the two mechanosensitive transcription cofactors YAP and TAZ have emerged as important profibrotic regulators in multiple murine tissues. Despite this growing recognition, a number of important questions remain unanswered, including which cell types require YAP/TAZ activation for fibrosis to occur and the time course of this activation. Here, we present a detailed analysis of the role that myofibroblast YAP and TAZ play in organ fibrosis and the kinetics of their activation. Using analyses of cells, as well as multiple murine and human tissues, we demonstrated that myofibroblast YAP and TAZ were activated early after organ injury and that this activation was sustained. We further demonstrated the critical importance of myofibroblast YAP/TAZ in driving progressive scarring in the kidney, lung, and liver, using multiple transgenic models in which YAP and TAZ were either deleted or hyperactivated. Taken together, these data establish the importance of early injury-induced myofibroblast YAP and TAZ activation as a key event driving fibrosis in multiple organs. This information should help guide the development of new antifibrotic YAP/TAZ inhibition strategies.

Disuse muscle atrophy-improving effect of ninjin'yoeito in a mouse model.

Disuse syndrome indicates psychosomatic hypofunction caused by excess rest and motionless and muscle atrophy is termed disuse muscle atrophy. Disuse muscle atrophy-induced muscle weakness and hypoactivity further induces muscle atrophy, leading to a vicious cycle, and this is considered a factor causing secondary sarcopenia and subsequently frailty. Since frailty finally leads to a bedridden state requiring nursing, in facing a super-aging society, intervention for a risk factor of frailty, disuse muscle atrophy, is important. However, the main treatment of disuse muscle atrophy is physical therapy and there are fewer effective preventive and therapeutic drugs. The objective of this study was to search for Kampo medicine with a disuse muscle atrophy-improving effect. Ninjin'yoeito is classified as a qi-blood sohozai (dual supplement) in Chinese herbal medicine, and it has an action supplementing the spleen related to muscle. In addition, improvement of muscle mass and muscle weakness by ninjin'yoeito in a clinical study has been reported. In this study, the effect of ninjin'yoeito on disuse muscle atrophy was investigated. A disuse muscle atrophy model was prepared using male ICR mice. After surgery applying a ring for tail suspension, a 1-week recovery period was set. Ninjin'yoeito was administered by mixing it in the diet for 1 week after the recovery period, followed by tail suspension for 14 days. Ninjin'yoeito administration was continued until autopsy including the hindlimb suspension period. The mice were euthanized and autopsied immediately after completion of tail suspension, and the hindlimb muscles were collected. The food and water intakes during the hindlimb unloaded period, wet weight of the collected muscle, and muscle synthesis and muscle degradation-related factors in blood and muscle were evaluated. Ingestion of ninjin'yoeito inhibited tail suspension-induced reduction of the soleus muscle wet weight. In addition, an increase in the blood level of a muscle synthesis-related factor, IGF-1, and promotion of phosphorylation of mTOR and 4E-BP1 in the soleus muscle were observed. It was suggested that ninjin'yoeito has a disuse muscle atrophy-improving action. Promotion of the muscle synthesis pathway was considered the action mechanism of this.

WEE1 promotes endometriosis via the Wnt/ß-catenin signaling pathway.

BACKGROUND: Endometriosis, the presence of active endometrial tissue outside the lining membrane of the uterine cavity, is a common disease in women of childbearing age. The ectopic endometrium has some characteristics of tumor tissue, including invasive and migratory abilities. In addition, endometriosis is associated with inflammation and reduced cellular apoptosis. METHODS: Western blot analysis, qPCR, immunohistochemistry, immunofluorescence microscopy, Transwell assay, wound healing assay, and TUNEL staining. RESULTS: Interleukin-1ß (IL-1ß) induced WEE1 expression in endometrial stromal cells (ESCs), suggesting that WEE1 may be upregulated during the endometriosis-induced inflammatory response. Overexpression of WEE1 in cultured ESCs promoted ESC migration while inhibiting apoptosis, whereas WEE1 knockdown reduced ESC migration while promoting apoptosis. Inhibition of WEE1 attenuates fibrosis in ESCs and female C57BL/6 J mice. This pro-fibrotic effect of WEE1 was significantly decreased by treatment with the Wnt/ß-catenin inhibitor XAV939, suggesting that WEE1 acts via the Wnt/ß-catenin signaling pathway. CONCLUSION: Our study demonstrates that WEE1 promotes ESC migration and fibrosis via the Wnt/ß-catenin signaling pathway. Thus, WEE1 may serve as a potential therapeutic target for the treatment of endometriosis.

Upregulation of miR-29c-3p Hinders Melanoma Progression by Inhibiting CDCA4 Expression.

OBJECTIVE: To investigate the expression and regulation mechanism of miR-29c-3p and cell division cycle associated 4 (CDCA4) in melanoma (MM). Data and Methods. Fifty-nine patients with MM admitted to our hospital were enrolled as the MM group. They were followed up for 3 years to analyze the prognostic factors; meanwhile, 51 healthy subjects were allocated into a normal group. MM cell lines (M21 and C8161) were transfected with miR-29c-3p-mimics, miR-29c-3p-inhibitor, miR-NC, si-CDCA4, and sh-CDCA4. The expression of miR-29c-3p, CDCA4, Bax, Caspase3, Bcl-2, N-cadherin, vimentin, and E-cadherin was quantified, and cell proliferation, migration, invasion, and apoptosis, as well as epithelial-mesenchymal transition (EMT), were determined. RESULTS: Serum miR-29c-3p was lowly expressed and CDCA4 was highly expressed in the MM group. The area under the curve (AUC) of both for diagnosing MM was greater than 0.9. miR-29c-3p and CDCA4 were related to regional lymph node staging (N staging), distant metastasis (M staging), tumor diameter, and pathological differentiation. Low miR-29c-3p and high CDCA4 were associated with poor prognosis of MM. Overexpression of miR-29c-3p and suppression of CDCA4 hindered cell proliferation, migration, invasion, and expression of Bax, Caspase3, N-cadherin, and vimentin, but cell apoptosis and expression of Bcl-2 and E-cadherin were enhanced. Dual-luciferase reporter (DLR) assay confirmed the targeted relationship between miR-29c-3p and CDCA4. After miR-29c-3p-mimics+sh-CDCA4 was transfected into M21 and C8161 cells, the proliferation, invasion, and apoptosis were not different from those in the miR-NC group transfected with unrelated sequences. CONCLUSION: Overexpression of miR-29c-3p suppresses CDCA4 expression and decreases proliferation, migration, invasion, apoptosis, and EMT of MM cells, thus hindering MM progression.

Deficiency of Polη in Saccharomyces cerevisiae reveals the impact of transcription on damage-induced cohesion.

The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication.

The Immunohistochemical Expression of MCM-3, -5, and -7 Proteins in the Uterine Fibroids.

Uterine fibroids are the most common mesenchymal uterine neoplasms; their prevalence is estimated in 40%-60% of women under 35 and in 70%-80% of women over 50 years of age. The current research aims to focus on the etiopathogenesis of uterine fibroids, the factors that affect their growth, and markers with diagnostic and prognostic properties. The MCM (minichromosome maintenance) protein family consists of peptides whose primary function is participation in the molecular mechanism of creating replication forks while regulating DNA synthesis. The aim of this work was to determine the proliferative potential of uterine fibroid cells based on the expression of the Ki-67 antigen and the MCMs-i.e., MCM-3, MCM-5, and MCM-7. In addition, the expression of estrogen (ER) and progesterone (PgR) receptors was evaluated and correlated with the expression of the abovementioned observations. Ultimately, received results were analyzed in terms of clinical and pathological data. MATERIALS AND METHODS: In forty-four cases of uterine fibroids, immunohistochemical reactions were performed. A tissue microarray (TMA) technique was utilized and analyzed cases were assessed in triplicate. Immunohistochemistry was performed using antibodies against Ki-67 antigen, ER, PgR, MCM-3, MCM-5, and MCM-8 on an automated staining platform. Reactions were digitalized by a histologic scanner and quantified utilizing dedicated software for nuclear analysis. Assessment was based on quantification expression of the three histiospots, each representing one case in TMA. RESULTS: In the study group (uterine fibroids), statistically significant stronger expression of all the investigated MCMs was observed, as compared to the control group. In addition, moderate and strong positive correlations were found between all tested proliferative markers. The expression of the MCM-7 protein also correlated positively with ER and PgR. With regard to clinical and pathological data, there was a negative correlation between the expression of MCMs and the number of both pregnancies and births. Significant reductions in MCM-5 and MCM-7 expression were observed in the group of women receiving oral hormonal contraceptives, while smoking women showed an increase in MCM-7, ER, and PgR. CONCLUSIONS: Uterine fibroid cells have greater proliferative potential, as evaluated by expression of the Ki-67 antigen and MCMs, than unaltered myometrial cells of the uterine corpus. The expression of MCM-7 was found to have strong or moderate correlations in all assessed relations. In the context of the clinical data, as well evident proliferative potential of MCMs, further studies are strongly recommended.

miR-181c-5p mediates apoptosis of vascular endothelial cells induced by hyperoxemia via ceRNA crosstalk.

Oxygen therapy has been widely used in clinical practice, especially in anesthesia and emergency medicine. However, the risks of hyperoxemia caused by excessive O2 supply have not been sufficiently appreciated. Because nasal inhalation is mostly used for oxygen therapy, the pulmonary capillaries are often the first to be damaged by hyperoxia, causing many serious consequences. Nevertheless, the molecular mechanism by which hyperoxia injures pulmonary capillary endothelial cells (LMECs) has not been fully elucidated. Therefore, we systematically investigated these issues using next-generation sequencing and functional research techniques by focusing on non-coding RNAs. Our results showed that hyperoxia significantly induced apoptosis and profoundly affected the transcriptome profiles of LMECs. Hyperoxia significantly up-regulated miR-181c-5p expression, while down-regulated the expressions of NCAPG and lncRNA-DLEU2 in LMECs. Moreover, LncRNA-DLEU2 could bind complementarily to miR-181c-5p and acted as a miRNA sponge to block the inhibitory effect of miR-181c-5p on its target gene NCAPG. The down-regulation of lncRNA-DLEU2 induced by hyperoxia abrogated its inhibition of miR-181c-5p function, which together with the hyperoxia-induced upregulation of miR-181c-5p, all these significantly decreased the expression of NCAPG, resulting in apoptosis of LMECs. Our results demonstrated a ceRNA network consisting of lncRNA-DLEU2, miR-181c-5p and NCAPG, which played an important role in hyperoxia-induced apoptosis of vascular endothelial injury. Our findings will contribute to the full understanding of the harmful effects of hyperoxia and to find ways for effectively mitigating its deleterious effects.

CRNDE enhances the expression of MCM5 and proliferation in acute myeloid leukemia KG-1a cells by sponging miR-136-5p.

The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene has been considered to be crucial in tumor malignancy. Although CRNDE is highly expressed in acute myeloid leukemia (AML), its mechanism of action remains unknown. In this study, GEPIA and qRT-PCR were performed to confirm the expression of CRNDE in AML samples and cell lines, respectively. CRNDE shRNA vectors were transfected to explore the biological functions of CRNDE. The cell proliferation was assessed by the CCK8 assay, while apoptosis and cell cycle distribution were measured by flow cytometry and Western blotting. The results showed that CRNDE was overexpressed in both AML samples and cell lines. CRNDE silencing inhibited proliferation and increased apoptotic rate and cell cycle arrest of KG-1a cells. The luciferase reporter assay coupled with RIP assay revealed that CRNDE act as a ceRNA. Rescue assays demonstrated that the effects of CRNDE silencing could be reversed by miR-136-5p inhibitors. In conclusion, our results expound that the CRNDE/miR-136-5p/MCM5 axis modulates cell progression and provide a new regulatory network of CRNDE in KG-1a cells.

Integrated bioinformatics analysis reveals CDK1 and PLK1 as potential therapeutic targets of lung adenocarcinoma.

ABSTRACT: This study is to identify potential biomarkers and therapeutic targets for lung adenocarcinoma (LUAD).GSE6044 and GSE118370 raw data from the Gene Expression Omnibus database were normalized with Robust Multichip Average. After merging these two datasets, the combat function of sva packages was used to eliminate batch effects. Then, limma packages were used to filtrate differentially expressed genes. We constructed protein-protein interaction relationships using STRING database and hub genes were identified based on connectivity degrees. The cBioportal database was used to explore the alterations of the hub genes. The promoter methylation of cyclin dependent kinase 1 (CDK1) and polo-like Kinase 1 (PLK1) and their association with tumor immune infiltration in patients with LUAD were investigated using DiseaseMeth version 2.0 and TIMER databases. The Cancer Genome Atlas-LUAD dataset was used to perform gene set enrichment analysis.We identified 10 hub genes, which were upregulated in LUAD, among which 8 were successfully verified in the Cancer Genome Atlas and Oncomine databases. Kaplan-Meier analysis indicated that the expressions of CDK1 and PLK1 in LUAD patients were associated with overall survival and disease-free survival. The methylation levels in the promoter regions of these 2 genes in LUAD patients were lower than those in normal lung tissues. Their expressions in LUAD were associated with tumor stages and relative abundance of tumor infiltrating immune cells, such as B cells, CD4+ T cells, and macrophages. Moreover, cell cycle, DNA replication, homologous recombination, mismatch repair, P53 signaling pathway, and small cell lung cancer signaling were significantly enriched in CDK1 and PLK1 high expression phenotype.CDK1 and PLK1 may be used as potential biomarkers and therapeutic targets for LUAD.

Overexpressed MPS-1 contributes to endometrioma development through the NF-κB signaling pathway.

BACKGROUND: Endometriosis is a benign gynecological disease that shares some characteristics with malignant tumors and affects approximately 10% of women of reproductive age. Endometrioma refers to endometriosis that appears in the ovary. Metallopanstimulin-1 (MPS-1) is a component of the 40S subunit of ribosomes that has extra-ribosomal functions that contribute to the development of diseases. This study aimed to explore the expression pattern and role of MPS-1 in endometrioma development. METHODS: Quantitative real time polymerase chain reaction, western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay were used to determine the expression of MPS-1 in patients with endometrioma. Following the successful knockdown of MPS-1 by siRNA, CCK-8 assays, flow cytometry, and transwell assays were performed to detect ectopic endometrial stromal cells (EcESCs) proliferation, the rate of apoptosis, and cell cycle, migration, and invasion, respectively. Western blotting was used to explore the effect of MPS-1 knockdown on protein levels in the NF-κB signaling pathway. RESULTS: The expression of MPS-1 was significantly higher in endometrioma and the serum of endometrioma patients than in the patients without endometriosis. In addition, the downregulation of MPS-1 expression inhibited EcESCs proliferation, migration, and invasion. This downregulation led to the arrest of the EcESCs cycle in the G0/G1 phase and apoptosis and depressed the NF-κB signaling pathway. CONCLUSION: MPS-1 can regulate EcESCs proliferation, motility, invasion, apoptosis, and cell cycle via the NF-κB signaling pathway in endometrioma. This may contribute to the formation or development of endometriotic foci. This study suggests the potential role of MPS-1 in the pathogenesis of endometriosis and enabled further research into the use of MPS-1 in the clinical diagnosis of endometrioma.

Cep63 knockout inhibits the malignant phenotypes of papillary thyroid cancer cell line TPC­1.

The present study was designed to observe the expression of the centrosomal protein 63 in papillary thyroid cancer (PTC) tissues and cells and to explore the clinical significance of Cep63 expression in PTC. Primary PTC tissues and matched normal thyroid tissues were collected, and the Cep63 expression level was determined by reverse transcription­quantitative PCR and western blotting. A stable Cep63­knockout cell line was constructed to assess the proliferation, invasion, migration and apoptosis abilities in vitro. A subcutaneous tumorigenesis model was established in nude mice to evaluate the effect of Cep63 on tumor growth and proliferation in vivo. Western blotting was used to explore the relevant signaling pathways. The results revealed that the expression level of Cep63 in PTC tissues was significantly increased. The proliferation, invasion and migration abilities of TPC­1 cells were decreased after Cep63 knockout, and silencing of Cep63 resulted in TPC­1 cell cycle arrest in the S phase. Mechanistically, Cep63 knockout inhibited the activation of the Janus kinase/signal transducer and activator of transcription 3 signaling pathway. In conclusion, Cep63 knockout significantly inhibited biological functions of TPC­1 cells in vitro and in vivo, indicating that Cep63 may be an important oncogene of PTC.

The mechanisms involved in the resistance of estrogen receptor-positive breast cancer cells to palbociclib are multiple and change over time.

PURPOSE: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used for the treatment of advanced estrogen receptor (ER)-positive breast cancer. To develop a treatment strategy for cancers resistant to CDK4/6 inhibitors, here, we established palbociclib-resistant sublines and analyzed their resistance mechanisms. METHODS: Palbociclib-resistant sublines were established from T47D and MCF7 cells. Sensitivity to other drugs was assessed via the WST assay. Altered expression/phosphorylation of proteins related to signal transduction and cell cycle regulation was examined using western blotting. Copy number alterations and mutations in the retinoblastoma (RB1) gene were also analyzed. RESULTS: Although an increase in CDK6 and decrease in retinoblastoma protein (Rb) expression/phosphorylation were commonly observed in the resistant sublines, changes in other cell cycle-related proteins were heterogeneous. Upon extended exposure to palbociclib, the expression/phosphorylation of these proteins became altered, and the long-term removal of palbociclib did not restore the Rb expression/phosphorylation patterns. Consistently a copy number decrease, as well as RB1 mutations were detected. Moreover, although the resistant sublines exhibited cross-resistance to abemaciclib, their response to dinaciclib was the same as that of wild-type cells. Of note, the cell line exhibiting increased mTOR phosphorylation also showed a higher sensitivity to everolimus. However, the sensitivity to chemotherapeutic agents was unchanged in palbociclib-resistant sublines. CONCLUSION: ER-positive breast cancer cells use multiple molecular mechanisms to survive in the presence of palbociclib, suggesting that targeting activated proteins may be an effective strategy to overcome resistance. Additionally, palbociclib monotherapy induces mutations and copy number alterations in the RB1 gene.

NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity.

N-myc downstream regulated gene-1 (NDRG1) has been identified as a putative metastasis suppressor gene and proved to be a key player in cancer spreading and proliferation in our previous work. However, the effects of NDRG1 on tumor invasion and the mechanisms behind it are rarely understood. Here we provided in silico evidence that NDRG1 plays a crucial role in actin reorganization in colorectal cancer (CRC). Through in vitro experiments, we next observed filopodia formation was altered in NDRG1-modified cell lines, while cell division cycle-42 (CDC42) displayed excessive activation in NDRG1-silenced cells. Mechanistically, NDRG1 loss disrupts the binding between RhoGDIα and CDC42 and triggers the activation of CDC42 and the downstream cascades PAK1/Cofilin, thereby promotes the formation of filopodia and invasiveness of CRC. The knockdown of NDRG1 led to enhanced dissemination of CRC cells in vivo and correlates with active CDC42 expression. Using clinical sample analysis, we found an elevated level of active CDC42 in patients with advanced T stage, and it was negatively related to NDRG1 expression. In sum, these results uncover a mechanism utilized by NDRG1 to regulate CDC42 activity in coordinating cytoskeleton reorganization, which was crucial in cancer invasion.

Lappaol F, an anticancer agent, inhibits YAP via transcriptional and post-translational regulation.

CONTEXT: Lappaol F (LAF), a natural lignan from Arctium lappa Linné (Asteraceae), inhibits tumour cell growth by inducing cell cycle arrest. However, its underlying anticancer mechanism remains unclear. OBJECTIVE: The effects of LAF on the Hippo-Yes-associated protein (YAP) signalling pathway, which plays an important role in cancer progression, were explored in this study. MATERIALS AND METHODS: Cervical (HeLa), colorectal (SW480), breast (MDA-MB-231) and prostate (PC3) cancer cell lines were treated with LAF at different concentrations and different durations. BALB/c nude mice bearing colon xenografts were intravenously injected with vehicle, LAF (10 or 20 mg/kg) or paclitaxel (10 mg/kg) for 15 days. The expression and nuclear localisation of YAP were analysed using transcriptome sequencing, quantitative PCR, western blotting and immunofluorescence. RESULTS: LAF suppressed the proliferation of HeLa, MDA-MB-231, SW480 and PC3 cells (IC50 values of 41.5, 26.0, 45.3 and 42.9 µmol/L, respectively, at 72 h), and this was accompanied by significant downregulation in the expression of YAP and its downstream target genes at both the mRNA and protein levels. The expression of 14-3-3σ, a protein that causes YAP cytoplasmic retention and degradation, was remarkably increased, resulting in a decrease in YAP nuclear localisation. Knockdown of 14-3-3σ with small interfering RNA partially blocked LAF-induced YAP inhibition and anti-proliferation effects. In colon xenografts, treatment with LAF led to reduced YAP expression, increased tumour cell apoptosis and tumour growth inhibition. CONCLUSION: LAF was shown to be an inhibitor of YAP. It exerts anticancer activity by inhibiting YAP at the transcriptional and post-translational levels.

Activation of the clock gene TIMELESS by H3k27 acetylation promotes colorectal cancer tumorigenesis by binding to Myosin-9.

BACKGROUND: Colorectal cancer (CRC) is a common tumor characterized by its high mortality. However, the underlying molecular mechanisms that drive CRC tumorigenesis are unclear. Clock genes have important roles in tumor development. In the present study, the expression and functions of clock gene TIMELESS (encoding the Timeless protein) in CRC were investigated. METHODS: Immunohistochemistry, cell proliferation, migration, invasion, EMT and xenograft tumor experiments were used to prove the function of Timeless in the tumorigenesis of CRC. Immunoprecipitation, mass spectrometry, Immunofluorescence and Chromatin immunoprecipitation (ChIP) were utilized to clarify the mechanism of Timeless in regulating CRC tumorigenesis. RESULTS: We found that Timeless was upregulated in CRC tissues compared with corresponding normal tissues and its expression was closely associated with the TNM stages and overall survival of CRC patients. Functional studies demonstrated that Timeless promoted the proliferation, invasion, and EMT of CRC cells in vitro and in vivo. Mechanistic investigations showed that Timeless activated the ß-catenin signal pathway by binding to Myosin-9, which binds to ß-catenin to induce its nuclear translocation. The upregulation of Timeless was attributed to CREB-binding protein (CBP)/p300-mediated H3K27 acetylation of the promoter region of Timeless. CONCLUSION: Timeless regulates the tumorigenesis of CRC by binding to and regulating myosin-9, suggesting Timeless might be a potential prognostic biomarker and therapeutic target for CRC.

Fasting-induced FOXO4 blunts human CD4+ T helper cell responsiveness.

Intermittent fasting blunts inflammation in asthma1 and rheumatoid arthritis2, suggesting that fasting may be exploited as an immune-modulatory intervention. However, the mechanisms underpinning the anti-inflammatory effects of fasting are poorly characterized3-5. Here, we show that fasting in humans is sufficient to blunt CD4+ T helper cell responsiveness. RNA sequencing and flow cytometry immunophenotyping of peripheral blood mononuclear cells from volunteers subjected to overnight or 24-h fasting and 3 h of refeeding suggest that fasting blunts CD4+ T helper cell activation and differentiation. Transcriptomic analysis reveals that longer fasting has a more robust effect on CD4+ T-cell biology. Through bioinformatics analyses, we identify the transcription factor FOXO4 and its canonical target FK506-binding protein 5 (FKBP5) as a potential fasting-responsive regulatory axis. Genetic gain- or loss-of-function of FOXO4 and FKBP5 is sufficient to modulate TH1 and TH17 cytokine production. Moreover, we find that fasting-induced or genetic overexpression of FOXO4 and FKBP5 is sufficient to downregulate mammalian target of rapamycin complex 1 signalling and suppress signal transducer and activator of transcription 1/3 activation. Our results identify FOXO4-FKBP5 as a new fasting-induced, signal transducer and activator of transcription-mediated regulatory pathway to blunt human CD4+ T helper cell responsiveness.