Erianin inhibits the growth and metastasis through autophagy-dependent ferroptosis in KRASG13D colorectal cancer.

Colorectal cancer (CRC) is the third most common cause of cancer mortality worldwide. Approximately 40% of CRC patients are KRAS sequence variation, including KRAS G13D mutation (KRASG13D) CRC patients, accounting for approximately 8% of all KRAS mutations in CRC patients and showing little benefit from anti-EGFR therapy. Therefore, there is an urgent need for new and effective anticancer agents in patients with KRASG13D CRC. Here, we identified a natural product, erianin, that directly interacted with purified recombinant human KRASG13D with a Kd of 1.1163 µM, which also significantly improve the thermal stability of KRASG13D. The cell viability assay showed that KRASG13D cells were more sensitive to erianin than KRASWT or KRASG12V cells. In vitro, results showed that erianin suppressed the migration, invasion and epithelial-mesenchymal transition (EMT) of KRASG13D CRC cells. Furthermore, erianin induced ferroptosis, as evidenced by the accumulation of Fe2+ and reactive oxygen species (ROS), lipid peroxidation, and changes in the mitochondrial morphology of KRASG13D CRC cells. Interestingly, we also found that erianin-induced ferroptosis was accompanied by autophagy. Moreover, the occurrence of erianin-induced ferroptosis is reversed by autophagy inhibitors (NH4Cl and Bafilomycin A1) and ATG5 knockdown, suggesting that erianin-induced ferroptosis is autophagy-dependent. In addition, we evaluated the inhibition of tumor growth and metastasis by erianin in vivo using a subcutaneous tumor model and a spleen-liver metastasis model, respectively. Collectively, these data provide novel insights into the anticancer activity of erianin, which is valuable for the further discussion and investigation of the use of erianin in clinical anticancer chemotherapy for KRASG13D CRC.

Periplocin inhibits hepatocellular carcinoma progression and reduces the recruitment of MDSCs through AKT/NF-κB pathway.

AIMS: We aimed to evaluate the effect of periplocin on inhibiting hepatocellular carcinoma (HCC) and further determine its mechanisms. MAIN METHODS: Cytotoxic activity of periplocin against HCC cells was tested by CCK-8 and colony formation assays. The antitumor effects of periplocin were evaluated in human HCC SK-HEP-1 xenograft and murine HCC Hepa 1-6 allograft mouse models. Flow cytometry was used to measure cell cycle distribution, apopotosis, and the number of myeloid-derived suppressor cells (MDSCs). Hoechst 33258 dye was applied to observe the nuclear morphology. Network pharmacology was performed to predict possible signaling pathways. Drug affinity responsive target stability assay (DARTS) was used to evaluate AKT binding of periplocin. Western blotting, immunohistochemistry, and immunofluorescence were used to examine the protein expression levels. KEY FINDING: Periplocin inhibited cell viability with IC50 values from 50 nM to 300 nM in human HCC cells. Periplocin disrupted cell cycle distribution and promoted cell apoptosis. Moreover, AKT was predicted as the target of periplocin by network pharmacology, which was confirmed by that AKT/NF-κB signaling was inhibited in periplocin-treated HCC cells. Periplocin also inhibited the expression of CXCL1 and CXCL3, leading to decreased accumulation of MDSCs in HCC tumors. SIGNIFICANCE: These findings reveal the function of periplocin in inhibiting HCC progression by G2/M arrest, apoptosis and suppression of MDSCs accumulation through blockade of the AKT/NF-κB pathway. Our study further suggests that periplocin has the potential to be developed as an effective therapeutic agent for HCC.

Single-cell RNA sequencing reveals the immune microenvironment and signaling networks in cystitis glandularis.

Introduction: Cystitis glandularis (CG) is a rare chronic bladder hyperplastic disease that mainly manifests by recurrent frequent urination, dysuria and gross hematuria. The current lack of unified diagnosis and treatment criteria makes it essential to comprehensively describe the inflammatory immune environment in CG research. Methods: Here, we performed scRNA-sequencing in CG patients for the first time, in which four inflamed tissues as well as three surrounding normal bladder mucosa tissues were included. Specifically, we isolated 18,869 cells to conduct bioinformatic analysis and performed immunofluorescence experiments. Results: Our genetic results demonstrate that CG does not have the classic chromosomal variation observed in bladder tumors, reveal the specific effects of TNF in KRT15 epithelial cells, and identify a new population of PIGR epithelial cells with high immunogenicity. In addition, we confirmed the activation difference of various kinds of T cells during chronic bladder inflammation and discovered a new group of CD27-Switch memory B cells expressing a variety of immunoglobulins. Discussion: CG was regarded as a rare disease and its basic study is still weak.Our study reveals, for the first time, the different kinds of cell subgroups in CG and provides the necessary basis for the clinical treatment of cystitis glandularis. Besides, our study significantly advances the research on cystitis glandularis at the cellular level and provides a theoretical basis for the future treatment of cystitis glandularis.

Optimization of prostate cancer patient lymph node staging via the integration of neutrophil-lymphocyte ratios, platelet-lymphocyte ratios, and 68 Ga-PSMA-PET-derived SUVmax values.

BACKGROUND: At present, standardized parameters for quantitatively evaluating 68 Ga-PSMA-PET/CT outcomes when diagnosing lymph node metastasis in prostate cancer patients are lacking. Inflammatory hematological biomarkers offer value as robust predictors of certain cancer-related outcomes. The present study was thus developed to explore approaches to improving the utility of 68 Ga-PSMA-PET/CT for diagnosing lymph node metastasis through the combined evaluation of inflammatory hematological markers in prostate cancer patients. METHODS: Pretreatment patient details including age, initial TPSA levels, hematological findings, biopsy pathology results (Gleason score and ISUP grouping), radical pathology results, and imaging details were collected. Optimal cutoff values for each predictor then being determined based upon Youden's index, with univariate and multivariate analyses were then used to identify independent predictors of lymph node metastasis and used to construct a nomogram. RESULT: Independent predictors of lymph node metastasis in this patient cohort included SUVmax (odds ratio [OR]: 30.549, 95% confidence interval [CI]: 10.855-85.973, p < 0.001), neutrophil-lymphocyte ratio (OR:8.221, 95%CI: 1.335-50.614, p = 0.023), platelet-lymphocyte ratio (OR:8.221, 95% CI: 1.335-50.614, p = 0.023), initial TPSA (OR:2.761, 95% CI: 1.132-6.733, p = 0.026), and clinical T-stage (T3 vs. T2, OR:11.332, 95% CI:3.929-32.681, p < 0.001; T4 vs. T2, OR:9.101, 95% CI:1.962-42.213, p = 0.005), with corresponding optimal cutoff values of 2.3 (area under the curve [AUC]: 0.873, sensitivity: 0.736, specificity: 0.902), 1.72 (AUC: 0.558, sensitivity: 0.529, specificity: 0.643), 83.305 (AUC: 0.651, sensitivity: 0.299, specificity: 0.979), and 21.875 (AUC: 0.672, sensitivity: 0.736, specificity: 0.601). Subsequent nomogram construction was associated with good predictive ability, with a C-index of 0.887(95% CI: 0.793-0.981) and an AUC of 0.924 (95% CI: 0.882-0.965). CONCLUSION: SUVmax, the neutrophil-lymphocyte ratio, the platelet-lymphocyte ratio, initial TPSA, and clinical T-stage represent valuable independent predictors of lymph node metastasis in prostate cancer patients, offering an opportunity to further optimize lymph node staging for these patients.

Renal phenotypes correlate with genotypes in unrelated individuals with tuberous sclerosis complex in China.

PURPOSE: To explore the relationship between the genotype and renal phenotype in a Chinese cohort and guide clinical decision-making for treating tuberous sclerosis complex (TSC). MATERIALS AND METHODS: We reviewed 173 patients with definite TSC at three centers in China from September 2014 to September 2020. All the patients underwent TSC1 and TSC2 genetic testing as well as renal phenotypic evaluation. All analyses were performed using the SPSS software, version 19.0, with a cut-off P value of 0.05 considered statistically significant. RESULTS: We identified variants in 93% (161/173) cases, including 16% TSC1 and 77% TSC2 variants. Analysis of the relationship between the genotype and renal phenotype, revealed that those with TSC2 variants were more likely to develop severe renal AML (> 4) (P = 0.044). In terms of treatment, TSC2 variants were more likely to undergo nephrectomy/partial nephrectomy (P = 0.036) and receive mTOR medication such as everolimus (P < 0.001). However, there was no significant difference between the two groups in terms of their response to the everolimus treatment. CONCLUSION: Patients with TSC2 variants exhibit more severe renal phenotypes, especially those associated with renal angiomyolipomas (AML), and they often require nephrectomy/partial nephrectomy or mTOR medication. Detection of the genotype is helpful in TSC management.

Synthesis and biological evaluation of marine natural product, Cryptoechinuline D derivatives as novel antiangiogenic agents.

Tumor angiogenesis is an important biological process involved in the proliferation and migration of endothelial cells, regulated by Ang/Tie-2 signaling pathways, which is essential for tumor growth and metastasis. Therefore, blocking Ang/Tie-2 signaling pathways is a promising anti-angiogenic strategy for tumor treatment. 2,5-Diketopiperazines (DKPs) are a kind of bioactive compounds derived from marine fungi and they present a wide spectrum of pharmacological properties, particularly in the field of cancer treatment. Herein, a DKP marine natural product, Cryptoechinuline D (Cry D) was applied to structural modification and twelve derivatives were synthesized. Among which, compound 5 showed significant inhibitory activity against HUVECs with an IC50 value of 12.6 µmol/L, which weakened the proliferation, migration and invasion of HUVECs by inhibiting the Ang2/Tie-2 signaling pathway. The results of these evaluations indicated that compound 5 might be a promising anti-angiogeneic agent and worth further optimization and development for cancer therapy.

Perfect match: mTOR inhibitors and tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome that presents with diverse and complex clinical features and involves multiple human systems. TSC-related neurological abnormalities and organ dysfunction greatly affect the quality of life and can even result in death in patients with TSC. It is widely accepted that most TSC-related clinical manifestations are associated with hyperactivation of the mammalian target of rapamycin (mTOR) pathway caused by loss­of­function mutations in TSC1 or TSC2. Remarkable progress in basic and translational research has led to encouraging clinical advances. Although mTOR inhibitors (rapamycin/everolimus) demonstrate great potential in TSC management, two major concerns hamper their generalized application. One is the frequent manifestation of adverse events, such as stomatitis, infections, and menstrual disorders; and the other is the poor response in certain patients. Thus, indicators are required to effectively predict the efficacy of mTOR inhibitors. Herein, we have summarized the current utilization of mTOR inhibitors in the treatment of TSC and focused on their efficacy and safety, in an attempt to provide a reference to guide the treatment of TSC.

Angiogenesis-Inhibitory Piperidine Alkaloids from the Leaves of Microcos paniculata.

Eight new 2,6-disubstituted piperidin-3-ol alkaloids (1-8), featuring a C10 unsaturated alkyl side chain, together with three previously reported analogues (9-11) were isolated from the leaves of medicinal plant Microcos paniculata. Their structures and absolute configurations were elucidated unambiguously by means of 1D and 2D NMR spectroscopic data analysis, modified Mosher's method, Snatzke's method, and quantum chemical electronic circular dichroism (ECD) calculations, as well as single-crystal X-ray crystallography. The isolates were evaluated for their antiangiogenic effects on human umbilical vein endothelial cells (HUVECs). Compound 2 displayed an inhibitory effect on tube formation of HUVECs in a concentration-dependent manner.

m6A modification: recent advances, anticancer targeted drug discovery and beyond.

Abnormal N6-methyladenosine (m6A) modification is closely associated with the occurrence, development, progression and prognosis of cancer, and aberrant m6A regulators have been identified as novel anticancer drug targets. Both traditional medicine-related approaches and modern drug discovery platforms have been used in an attempt to develop m6A-targeted drugs. Here, we provide an update of the latest findings on m6A modification and the critical roles of m6A modification in cancer progression, and we summarize rational sources for the discovery of m6A-targeted anticancer agents from traditional medicines and computer-based chemosynthetic compounds. This review highlights the potential agents targeting m6A modification for cancer treatment and proposes the advantage of artificial intelligence (AI) in the discovery of m6A-targeting anticancer drugs. Three stages of m6A-targeting anticancer drug discovery: traditional medicine-based natural products, modern chemical modification or synthesis, and artificial intelligence (AI)-assisted approaches for the future.

Probing Indole Diketopiperazine-Based Hybrids as Environmental-Induced Products from Aspergillus sp. EGF 15-0-3.

Comprehensive analyses of the metabolite spectra of Aspergillus sp. EGF 15-0-3 under different culture conditions revealed the presence of unique environmental-induced metabolites exclusively from the rice medium. Subsequent target isolation afforded four unprecedented indole diketopiperazine-based hybrids with a pyrano[3',2':7,8]isochromeno[4,3-b]pyrazino[2,1-i]indole core (1 and 2) or a spiro[piperazine-2,2'-pyrano[3,4,5-de]chromene] scaffold (3 and 4). Putative biosynthetic pathways for 1-4, with Diels-Alder cycloadditions as key steps, were proposed. 1-4 exhibited selective cytotoxicities among several human cancer cells.

Dual-tracer PET/CT-targeted, mpMRI-targeted, systematic biopsy, and combined biopsy for the diagnosis of prostate cancer: a pilot study.

PURPOSE: Growing evidence proved the efficacy of multi-parametric MRI (mpMRI) and prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT)-guided targeted biopsy (TB) in prostate cancer (PCa) diagnosis, but there is no direct comparison between mpMRI-TB and PSMA PET/CT-TB. Gastrin-releasing peptide receptor (GRPR) is highly expressed in PCa, which can compensate for the unstable expression of PSMA in PCa. Therefore, we designed a study to compare the efficiency of mpMRI-TB, dual-tracer (GRPR and PSMA) PET/CT-TB, systematic biopsy, and combined biopsy for the diagnosis of prostate cancer. METHODS: One hundred twelve suspicious PCa patients were enrolled from September 2020 to June 2021. Patients with anyone of positive dual-tracer PET/CT or mpMRI underwent TB, and all enrolled patients underwent systematic biopsy (SB) after TB. The primary outcome was the detection rates of PCa in different biopsy strategies. Secondary outcomes were the performance of three imaging methods, omission diagnostic rates, and upgrading and downgrading of biopsy samples relative to those of prostatectomy specimens in different biopsy strategies. McNemar's tests and Bonferroni correction in multiple comparisons were used to compare the primary and secondary outcomes. RESULTS: In 112 men, clinically significant PCa (grade group[GG] ≥ 2) accounted for 34.82% (39/112), and nonclinically significant PCa (GG = 1) accounted for 4.46% (5/112). 68 Ga-PSMA PET/CT-TB achieved higher PCa detection rate (69.77%) and positive ratio of biopsy cores (0.44) compared with SB (39.29% and 0.12) and mpMRI-TB (36.14% and 0.23), respectively (P < 0.005). Dual-tracer PET/CT screen out patients for avoiding 52.67% (59/112) unnecessary biopsy, whereas dual-tracer PET/CT-TB plus SB achieved high detection rate (77.36%) without misdiagnosis of csPCa. CONCLUSION: Dual-tracer PET/CT might screen patients for avoiding unnecessary biopsy. Dual-tracer PET/CT-TB plus SB might be a more effective and promising strategy for the definite diagnosis of clinically significant PCa than mpMRI-TB.

Low-Dose Everolimus Maintenance Therapy for Renal Angiomyolipoma Associated With Tuberous Sclerosis Complex.

Objective: To assess the safety and efficacy of low-dose everolimus maintenance therapy for tuberous sclerosis complex-related renal angiomyolipoma (TSC-RAML) patients that had previously undergone standard-dose treatment for a minimum of 6 months. Materials and Methods: In total, 24 patients with a definitive TSC diagnosis were enrolled from April 2018 - April 2019 at Xiangya Hospital, Central South University. All patients underwent low-dose everolimus maintenance therapy following standard-dose everolimus induction therapy for a minimum of 6 months. Patients additionally underwent TSC1/TSC2 genetic testing, And they were followed-up at 3, 6, 12, 18, and 24 months. The Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1) criteria were used to monitor patient RAML responses, while adverse events (AEs) were assessed as per the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, version 4.0). P < 0.05 was the significance level for all analyses, which were performed using SPSS 19.0. Results: TSC1/TSC2 gene mutations were present in all 24 patients, all of whom achieved a significant reduction in TSC-RAML volume within the initial 6-month induction therapy period, and exhibited volume stabilization during the low-dose maintenance therapy treatment period without any instances of TSC-RAML regrowth. Adverse events (AEs) were significantly less severe and less frequent over the course of maintenance therapy relative to standard therapy. Conclusions: Low-dose everolimus maintenance therapy represents an effective approach to achieving TSC-RAML control following a minimum of 6 months of full-dose induction therapy, and may be associated with decreases in everolimus-related AE frequency and severity.

Everolimus versus sirolimus for angiomyolipoma associated with tuberous sclerosis complex: a multi-institutional retrospective study in China.

PURPOSE: To evaluate the efficacy and safety of everolimus and sirolimus in patients with tuberous sclerosis complex-associated angiomyolipomas (TSC-AML). MATERIALS AND METHODS: We performed a multi-institutional retrospective study of TSC-AML patients treated with oral everolimus 10 mg or sirolimus 2 mg per day for at least 3 months. Angiomyolipoma volume was estimated using orthogonal measurements by MRI or CT. Adverse events (AEs) were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events. All analyses were performed using SPSS 19.0 software. RESULTS: Response rates were high in both groups. With the prolonged medication durations, the therapeutic efficacy of both agents became more significant. The TSC-AML volume reduction after 6 and 12 months was more pronounced in patients with everolimus than those with sirolimus. More than half of the patients treated with everolimus had ≥ 50% reduction, and approximately 80% of them had ≥ 30% reduction, which was higher than that in patients treated with sirolimus. Regarding safety, there was no significant difference in the incidence of AEs between the two groups. CONCLUSIONS: Both everolimus and sirolimus are excellent therapeutic options for TSC-AML. However, everolimus has a better therapeutic efficacy than sirolimus, particularly in reducing TSC-AML volume. Everolimus is therefore recommended as the first choice of therapy for TSC-AML.

Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth.

Epidermal growth factor receptor (EGFR) activation plays a pivotal role in EGFR-driven non-small cell lung cancer (NSCLC) and is considered as a key target of molecular targeted therapy. EGFR tyrosine kinase inhibitors (TKIs) have been canonically used in NSCLC treatment. However, prevalent innate and acquired resistances and EGFR kinase-independent pro-survival properties limit the clinical efficacy of EGFR TKIs. Therefore, the discovery of novel EGFR degraders is a promising approach towards improving therapeutic efficacy and overcoming drug resistance. Here, we identified a 23-hydroxybetulinic acid derivative, namely DPBA, as a novel EGFR small-molecule ligand. It exerted potent in vitro and in vivo anticancer activity in both EGFR wild type and mutant NSCLC by degrading EGFR. Mechanistic studies disclosed that DPBA binds to the EGFR extracellular domain at sites differing from those of EGF and EGFR. DPBA did not induce EGFR dimerization, phosphorylation, and ubiquitination, but it significantly promoted EGFR degradation and repressed downstream survival pathways. Further analyses showed that DPBA induced clathrin-independent EGFR endocytosis mediated by flotillin-dependent lipid rafts and unaffected by EGFR TKIs. Activation of the early and late endosome markers rab5 and rab7 but not the recycling endosome marker rab11 was involved in DPBA-induced EGFR lysosomal degradation. The present study offers a new EGFR ligand for EGFR pharmacological degradation and proposes it as a potential treatment for EGFR-positive NSCLC, particularly NSCLC with innate or acquired EGFR TKI resistance. DPBA can also serve as a chemical probe in the studies on EGFR trafficking and degradation.

Hunzeylanines A-E, Five Bisindole Alkaloids Tethered with a Methylene Group from the Roots of Hunteria zeylanica.

Five novel bisindole alkaloids, hunzeylanines A-E (1-5), with an unprecedented skeleton were isolated from the roots of Hunteria zeylanica. Compounds 1-5 represent the first examples of akuammine-pleioarpamine-type bisindole alkaloids fused with a dihydropyran unit. Their structures including absolute configurations were established through comprehensive spectroscopic data analyses and computational calculation methods. The plausible biogenetic pathway of 1 was also proposed. Alkaloids 1 and 2 displayed moderate cytotoxicity toward three human cancer cell lines (MDA-MB-231, AV3, and Huh7).

ISYNA1 is overexpressed in bladder carcinoma and regulates cell proliferation and apoptosis.

BACKGROUND: Bladder cancer (BCa) is one of the most common urological malignancies. While Inositol-3-phosphate synthase 1 (ISYNA1) expression and function were largely unknown in BCa. We aimed to study the expression and role of ISYNA1 in bladder cancer and investigate its potential mechanisms via ingenuity pathway analysis (IPA). METHODS: ISYNA1 expression was quantified by qRT-PCR in bladder cancer cell lines as well as normal urothelial cell line. Knocking down ISYNA1 gene in BCa T24 cells was achieved by shRNA lentivirus transfection. MTT and Celigo assay were used to assess cell proliferation. Flow cytometry was applied to test cell cycle and apoptosis. In addition, IPA was performed using PrimeView™ Human Gene Expression Array. Imunohistochemistry (IHC) was performed in BCa patient tissue microarray to verify the association between ISYNA1 expression and patients' clinicopathological features. RESULTS: ISYNA1 was significantly upregulated in BCa samples vs. para-tumor tissues. Higher expression were significantly associated with tumor T stage and lymph node metastasis of bladder cancer patients. Similarly, it was elevated in BCa cell lines (5637 and T24) compared with SVHUC cells. Knocking down ISYNA1 significantly decreased proliferation, induced apoptosis and cell cycle arrest in T24 cells. Furthermore, IPA indicated that ISYNA1 was an important regulatory factors and related networks were involved in multiple functional processes. CONCLUSION: Taken together, current study suggest ISYNA1 promotes proliferation and inhibit apoptosis in bladder cancer cells, and its expression correlated with BCa patients' clinicopathological features. Thus, ISYNA1 may serve as a potential biomarker and therapeutic target for BCa patients.

Microarray expression profiles analysis revealed lncRNA OXCT1-AS1 promoted bladder cancer cell aggressiveness via miR-455-5p/JAK1 signaling.

Bladder cancer (BCa) is one of the most prevalent cancers of the urinary system worldwide. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) perform a vital function in the pathogenesis and progression of BCa. In the current study, we identified a novel lncRNA OXCT1-AS1 and investigated its role and potential mechanisms in BCa. The microarray results showed the expression of lncRNAs, microRNAs, and messenger RNAs between BCa primary tumor tissues and metastatic lymph nodes were significantly different. The quantitative polymerase chain reaction verification was performed to ensure the reliability of the screening results. The Cell Counting Kit 8 and transwell assay were used to assess the tumor cell proliferation and invasion abilities in vitro, respectively. The dual-luciferase activity assay was performed to investigate the potential mechanism of competing endogenous RNA network. lncRNA OXCT1-AS1, which elevated in metastasis lymph node, was significantly upregulated in BCa cell lines compared with SVHUC-1. We demonstrated OXCT1-AS1 inhibited miR-455-5p to decrease its binding to the JAK1 3'-untranslated region, which could upregulate the expression of JAK1 at the protein level, thus promoting BCa proliferation and invasion. Therefore, lncRNA OXCT1-AS1 could act as a potential biomarker and therapeutic target for patients with BCa.

Arenobufagin induces MCF-7 cell apoptosis by promoting JNK-mediated multisite phosphorylation of Yes-associated protein.

BACKGROUND: It has been demonstrated that bufadienolides exert potent anti-cancer activity in various tumor types. However, the mechanisms that underlie their anti-cancer properties remain unclear. Yes-associated protein, a key effector of Hippo signaling, functions as a transcription coactivator, plays oncogenic and tumor suppressor roles under different conditions. Here, we report that arenobufagin (ABF), a representative bufadienolide, induced breast cancer MCF-7 cells to undergo apoptosis, which occurred through the JNK-mediated multisite phosphorylation of YAP. METHODS: Cytotoxicity was examined using an MTT assay. ABF-induced apoptosis was measured with a TUNEL assay and Annexin V-FITC/PI double staining assay. Western blotting, immunofluorescence, qRT-PCR and coimmunoprecipitation were employed to assess the expression levels of the indicated molecules. Lose-of-function experiments were carried out with siRNA transfection and pharmacological inhibitors. ABF-induced phosphopeptides were enriched with Ti4+-IMAC chromatography and further subjected to reverse-phase nano-LC-MS/MS analysis. RESULTS: ABF significantly reduced the viability of MCF-7 cells and increased the percentage of early and late apoptotic cells in a concentration- and time-dependent manner. Following ABF treatment, YAP accumulated in the nucleus and bound to p73, which enhanced the transcription of the pro-apoptotic genes Bax and p53AIP1. YAP knock-down significantly attenuated ABF-induced apoptotic cell death. Importantly, we found that the mobility shift of YAP was derived from its phosphorylation at multiple sites, including Tyr357. Moreover, mass spectrometry analysis identified 19 potential phosphorylation sites in YAP, with a distribution of 14 phosphoserine and 5 phosphothreonine residues. Furthermore, we found that the JNK inhibitor SP600125 completely diminished the mobility shift of YAP and its phosphorylation at Tyr357, the binding of YAP and p73, the transcription of Bax and p53AIP1 as well as the apoptosis induced by ABF. These data indicate that ABF induced YAP multisite phosphorylation, which was associated with p73 binding, and that apoptosis was mediated by the JNK signaling pathway. CONCLUSIONS: Our data demonstrate that ABF suppresses MCF-7 breast cancer proliferation by triggering the pro-apoptotic activity of YAP, which is mediated by JNK signaling-induced YAP multisite phosphorylation as well as its association with p73. The present work not only provides additional information on the use of ABF as an anti-breast cancer drug, but also offers evidence that the induction of the tumor suppressor role of YAP may be a therapeutic strategy.

Glucocorticoid-Inducible Kinase 2 Promotes Bladder Cancer Cell Proliferation, Migration and Invasion by Enhancing ß-catenin/c-Myc Signaling Pathway.

Background: Bladder cancer is one of the most common malignancies in urologic system. The glucocorticoid-inducible kinase 2 (SGK2) expression and function were largely unknown in cancers. Current study was aimed to investigate the role of SGK2 in bladder cancer and its potential mechanisms. Methods: SGK2 expression was quantified by western blot (WB) in multiple bladder cancer cell lines (T24, 5637, J82 and UMUC3) compared with normal urothelial cell line (SVHUC). SGK2 knocking down and overexpression model were established by lentivirus transfection. MTT, colony formation, wound healing and transwell assay were used to assess the tumor cell proliferation, migration and invasion abilities, respectively. In addition, molecular function analysis was performed using FunRich software V3. Immunoprecipitation (IP) assay was applied to investigate the interaction between SGK2 and ß-catenin at protein level. TCGA database was retrieved to verify the association between these genes and clinical tumor stage as well as prognosis among bladder cancer patients. Results: SGK2 expression was significantly upregulated in multiple bladder cancer cell lines compared with SVHUC at protein level. Cell proliferation, migration and invasion abilities were significantly decreased after knocking down SGK2 in J82 and UMUC3 cell lines. Inversely, cell aggressive phenotypes were significantly increased after overexpressing SGK2 in T24 cell line. Furthermore, functional analyses of SGK2 based on TCGA database showed that SGK2 related genes were involved in receptor activity, ATP binding, DNA repair protein, trans-membrane receptor activity and lipid binding. In addition, protein interaction analysis identified c-Myc was significantly enriched in SGK2 positively associated genes. The prediction was validated by WB and IP assay that SGK2 could directly bind with ß-catenin at protein level to regulate their downstream gene c-Myc expression in bladder cancer to influence tumor progression. And clinical data generated from TCGA database also identified these downstream genes were significantly associated with tumor stage and survival status of bladder cancer patients. Conclusion: Taken together, our findings suggest SGK2 promotes bladder cancer progression via mediating ß-catenin/c-Myc signaling pathway, which may serve as a potential therapeutic target for bladder cancer patients.

Association Between 12 Polymorphisms of VEGF/Hypoxia/Angiogenesis Pathway Genes and Risk of Urogenital Carcinomas: A Meta-Analysis Based on Case-Control Studies.

Objective: Previous studies indicated potential associations between polymorphisms in genes of VEGF/hypoxia/angiogenesis pathway and risk of urogenital carcinomas However, the results were controversial and inconclusive. Here, we conducted an in-depth meta-analysis to investigate the precise associations between polymorphisms in VEGF/hypoxia/angiogenesis related genes and risk of urogenital carcinomas. Methods: We searched PubMed, Web of Science, EMBASE, and Cochrane Library to identify all eligible publications. Pooled odds ratios (ORs) corresponding with the 95% confidence intervals (CIs) were calculated to evaluate their associations. Subgroup analysis was conducted to further ascertain such relationship and investigate sources of heterogeneity. Results: In the end, a total of 96 case-control studies fulfilled the inclusion criteria were enrolled for 12 polymorphisms in 4 VEGF/hypoxia/angiogenesis related genes. The pooled results showed eNOS-rs2070744 polymorphism conferred a significantly increased overall risk of urogenital carcinomas in allele, homozygote, and recessive models, respectively. In addition, eNOS-Intron 4a/b VNTR polymorphism was identified related to an increased risk of urogenital carcinomas in recessive model. And VEGF-rs699947 polymorphism was also identified an increased risk of renal cell carcinoma (RCC) in allelic, heterozygote, dominant, homozygote, and recessive models. Conclusion: To conclude, eNOS-rs2070744 and eNOS-Intron 4a/b VNTR polymorphisms are risk factors for urogenital carcinomas. VEGF-rs699947 polymorphism was also identified as an increased risk factor for renal carcinoma.