Ubiquitin-specific peptidase 5 facilitates cancer stem cell-like properties in lung cancer by deubiquitinating ß-catenin.

BACKGROUND: Lung cancer has the highest mortality rate in the world, and mounting evidence suggests that cancer stem cells (CSCs) are associated with poor prognosis, recurrence, and metastasis of lung cancer. It is urgent to identify new biomarkers and therapeutic targets for targeting lung CSCs. METHODS: We computed the single-sample gene set enrichment analysis (ssGSEA) of 1554 Reactome gene sets to identify the mRNA expression-based stemness index (mRNAsi)-associated pathways using the genome-wide RNA sequencing data of 509 patients from The Cancer Genome Atlas (TCGA) cohort of lung adenocarcinoma (LUAD). Phenotypic effects of ubiquitin-specific peptidase 5 (USP5) on the CSC-like properties and metastasis were examined by in vitro sphere formation assay, migration assay, invasion assay, and in vivo xenografted animal models. Cycloheximide chase assay, co-immunoprecipitation assay, and deubiquitination assay were performed to confirm the effect of USP5 on the deubiquitination of ß-catenin. RESULTS: We demonstrated that USP5 expression were positively correlated with the stemness-associated signatures and poor outcomes in lung cancer specimens. Silencing of endogenous USP5 reduced CSC-like characteristics, epithelial-mesenchymal transition (EMT), and metastasis in vitro and in vivo. Furthermore, USP5 interacted with ß-catenin, which resulted in deubiquitination, stabilization of ß-catenin, and activation of Wnt/ß-catenin pathway. Accordingly, expression of USP5 was positively correlated with the enrichment score of the Wnt/TCF pathway signature in human lung cancer. Silencing of ß-catenin expression suppressed USP5-enhancing sphere formation. Targeting USP5 with the small molecule WP1130 promoted the degradation of ß-catenin, and showed great inhibitory effects on sphere formation, migration, and invasion. Finally, we identified a poor-prognosis subset of tumors characterized by high levels of USP5, Wnt signaling score, and Stemness score in both TCGA-LUAD and Rousseaux_2013 datasets. CONCLUSIONS: These findings reveal a clinical evidence for USP5-enhanced Wnt/ß-catenin signaling in promoting lung cancer stemness and metastasis, implying that targeting USP5 could provide beneficial effects to improve lung cancer therapeutics.

Case Report: Low-Frequency Repetitive Transcranial Magnetic Stimulation to Dorsolateral Prefrontal Cortex and Auditory Cortex in a Patient With Tinnitus and Depression.

Repetitive transcranial magnetic stimulation (rTMS) has been widely used as a promising therapy for tinnitus. However, the exact target and stimulation sequence of rTMS that is most effective for treating tinnitus remains unclear. Here, we report a case of a 62-year-old man with treatment-refractory tinnitus and depression whose symptoms markedly improved after undergoing low-frequency rTMS over the right-side dorsolateral prefrontal cortex and left auditory cortex area. Our report indicates that low-frequency rTMS treatment that stimulates multiple brain regions sequentially is feasible and may clinically benefit patients with tinnitus and depression.

Predict Early Recurrence of Resectable Hepatocellular Carcinoma Using Multi-Dimensional Artificial Intelligence Analysis of Liver Fibrosis.

BACKGROUND: Liver fibrosis is thought to be associated with early recurrence of hepatocellular carcinoma (HCC) after resection. To recognize HCC patients with higher risk of early recurrence, we used a second harmonic generation and two-photon excitation fluorescence (SHG/TPEF) microscopy to create a fully quantitative fibrosis score which is able to predict early recurrence. METHODS: The study included 81 HCC patients receiving curative intent hepatectomy. Detailed fibrotic features of resected hepatic tissues were obtained by SHG/TPEF microscopy, and we used multi-dimensional artificial intelligence analysis to create a recurrence prediction model "combined index" according to the morphological collagen features of each patient's non-tumor hepatic tissues. RESULTS: Our results showed that the "combined index" can better predict early recurrence (area under the curve = 0.917, sensitivity = 81.8%, specificity = 90.5%), compared to alpha fetoprotein level (area under the curve = 0.595, sensitivity = 68.2%, specificity = 47.6%). Using a Cox proportional hazards analysis, a higher "combined index" is also a poor prognostic factor of disease-free survival and overall survival. CONCLUSIONS: By integrating multi-dimensional artificial intelligence and SHG/TPEF microscopy, we may locate patients with a higher risk of recurrence, follow these patients more carefully, and conduct further management if needed.

Graphene oxide conjugated with polymers: a study of culture condition to determine whether a bacterial growth stimulant or an antimicrobial agent?

BACKGROUND: The results showed that the deciding factor is the culture medium in which the bacteria and the graphene oxide (GO) are incubated at the initial manipulation step. These findings allow better use of GO and GO-based materials more and be able to clearly apply them in the field of biomedical nanotechnology. RESULTS: To study the use of GO sheets applied in the field of biomedical nanotechnology, this study determines whether GO-based materials [GO, GO-polyoxyalkyleneamine (POAA), and GO-chitosan] stimulate or inhibit bacterial growth in detail. It is found that it depends on whether the bacteria and GO-based materials are incubated with a nutrient at the initial step. This is a critical factor for the fortune of bacteria. GO stimulates bacterial growth and microbial proliferation for Gram-negative and Gram-positive bacteria and might also provide augmented surface attachment for both types of bacteria. When an external barrier that is composed of GO-based materials forms around the surface of the bacteria, it suppresses nutrients that are essential to microbial growth and simultaneously produces oxidative stress, which causes bacteria to die, regardless of whether they have an outer-membrane-Gram-negative-bacteria or lack an outer-membrane-Gram-positive-bacteria, even for high concentrations of biocompatible GO-POAA. The results also show that these GO-based materials are capable of inducing reactive oxygen species (ROS)-dependent oxidative stress on bacteria. Besides, GO-based materials may act as a biofilm, so it is hypothesized that they suppress the toxicity of low-dose chitosan. CONCLUSION: Graphene oxide is not an antimicrobial material but it is a general growth enhancer that can act as a biofilm to enhance bacterial attachment and proliferation. However, GO-based materials are capable of inducing ROS-dependent oxidative stress on bacteria. The applications of GO-based materials can clearly be used in antimicrobial surface coatings, surface-attached stem cells for orthopedics, antifouling for biocides and microbial fuel cells and microbial electro-synthesis.

Efficient two-photon luminescence for cellular imaging using biocompatible nitrogen-doped graphene quantum dots conjugated with polymers.

Nitrogen-doped graphene quantum dot (N-GQD) nanomaterials conjugated with polyethylenimine (PEI)-polystyrene sulfonate (PSS)-anti-epidermal growth factor receptor (AbEGFR) antibody (N-GQD-PEI-PSS-AbEGFR) demonstrated impressive two-photon properties and stability, signifying that they can serve as an effective two-photon contrast agent in two-photon bioimaging. Furthermore, they provided high intensity, brightness, and signal-to-noise ratios at an ultra-low two-photon excitation (TPE) power level in an observation extending to a deep, three-dimensional depth.

Crystal structures of Staphylococcal SaeR reveal possible DNA-binding modes.

Two-component system SaeRS of Staphylococcus regulates virulence factor expression through phosphorylation of the DNA-binding regulator SaeR by the sensor histidine kinase SaeS. Here crystal structures of the DNA-binding domain (DBD) of SaeR from two Staphylococcal species Staphylococcus epidermidis and Staphylococcus aureus were determined and showed similar folds. Analyzing the DNA binding activity of three mutants of SeSaeR, we observed that Thr217 is important in binding to the phosphate group of DNA and Trp219 may interact with the base pairs. Additionally, the tandem arrangement of DBD may represent a possible way for SaeR oligomerization on DNA.

Heterogeneous nuclear ribonucleoproteins A1 and A2 modulate expression of Tid1 isoforms and EGFR signaling in non-small cell lung cancer.

The Tid1 protein is a DnaJ co-chaperone that has two alternative splicing isoforms: Tid1 long form (Tid1-L) and Tid1 short form (Tid1-S). Recent studies have shown that Tid1-L functions as a tumor suppressor by decreasing EGFR signaling in various cancers, including head and neck cancer and non-small cell lung cancer (NSCLC). However, the molecular mechanism responsible for regulating the alternative splicing of Tid1 is not yet known. Two splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and A2, participate in alternative splicing and are known to be overexpressed in lung cancers. In this work, we examined if hnRNP A1 and A2 could regulate the alternative splicing of Tid1 to modulate tumorigenesis in NSCLC. We report that RNAi-mediated depletion of both hnRNP A1/A2 (but not single depletion of either) increased Tid1-L expression, inhibited cell proliferation and attenuated EGFR signaling. Analyses of the expression levels of hnRNP A1, hnRNP A2, EGFR and Tid1-L in NSCLC tissues revealed that hnRNP A1 and A2 are positively correlated with EGFR, but negatively correlated with Tid1-L. NSCLC patients with high-level expression of hnRNP A1, hnRNP A2 and EGFR combined with low-level expression of Tid1-L were associated with poor overall survival. Taken together, our results suggest that hnRNP A1 or A2 are both capable of facilitating the alternative splicing of exon 11 in the Tid1 pre-mRNA, thereby suppressing the expression of Tid1-L and allowing EGFR-related signaling to facilitate NSCLC tumorigenesis.

Small Molecule T315 Promotes Casitas B-Lineage Lymphoma-Dependent Degradation of Epidermal Growth Factor Receptor via Y1045 Autophosphorylation.

RATIONALE: Despite the fact that tyrosine kinase inhibitors (TKIs) have been found effective in treating patients harboring activating mutations of epidermal growth factor receptor (EGFR), an acquired secondary mutation, T790M, which lowers the affinity to TKIs, can lead to EGFR TKI resistance after this standard treatment. OBJECTIVES: To evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo. METHODS: Lung adenocarcinoma cells were treated with T315, and cell proliferation and apoptotic proportion were determined by the CellTiter 96 AQueous MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay and flow cytometry. The effects of T315 on EGFR mRNA and protein levels, autophosphorylation, ubiquitination, and degradation were evaluated by real-time polymerase chain reaction and Western blot, respectively. Direct targeting of T315 to EGFR was confirmed by the in vitro kinase assay and mass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib. MEASUREMENTS AND MAIN RESULTS: We identified T315 as a novel, potent small molecule for suppressing cancer cell proliferation in vitro and in vivo. The therapeutic effect was verified after T315 was combined with a second-generation TKI, afatinib, compared with a single drug administration. We found a new mechanism of action, in that T315 appears to directly bind EGFR and triggers EGFR-Y1045 autophosphorylation, whereby its degradation is triggered through the ubiquitin-proteasome pathway. CONCLUSIONS: Our evidence suggests that T315 is a novel class of anticancer drug that is able to inhibit the growth of EGFR-TKI-resistant lung adenocarcinoma cells by inducing the degradation of EGFR.

MDA-9/Syntenin-Slug transcriptional complex promote epithelial-mesenchymal transition and invasion/metastasis in lung adenocarcinoma.

Melanoma differentiation-associated gene-9 (MDA-9)/Syntenin is a novel therapeutic target because it plays critical roles in cancer progression and exosome biogenesis. Here we show that Slug, a key epithelial-mesenchymal-transition (EMT) regulator, is a MDA-9/Syntenin downstream target. Mitogen EGF stimulation increases Slug expression and MDA-9/Syntenin nuclear translocation. MDA-9/Syntenin uses its PDZ1 domain to bind with Slug, and this interaction further leads to HDAC1 recruitment, up-regulation of Slug transcriptional repressor activity, enhanced Slug-mediated EMT, and promotion of cancer invasion and metastasis. The PDZ domains and nuclear localization of MDA-9/Syntenin are both required for promoting Slug-mediated cancer invasion. Clinically, patients with high MDA-9/Syntenin and high Slug expressions were associated with poor overall survival compared to those with low expression in lung adenocarcinomas. Our findings provide evidence that MDA-9/Syntenin acts as a pivotal adaptor of Slug and it transcriptionally enhances Slug-mediated EMT to promote cancer invasion and metastasis.

Suppression of Aurora-A-FLJ10540 signaling axis prohibits the malignant state of head and neck cancer.

BACKGROUND: Head and neck cancer (HNC) is a highly invasive cancer. Aurora-A has been reported for a number of malignancies. However, the identity of downstream effectors responsible for its aggressive phenotype in HNC remains underinvestigated. METHODS: The mRNA and protein expression levels of Aurora-A and FLJ10540 were assessed in HNC specimens and cell lines using RT-qPCR, western blot, Oncomine, and microarray database analysis. The downstream molecular mechanisms of Aurora-A were confirmed by RT-qPCR, western blot, luciferase reporter, confocal microscopy analyses, immunoprecipitation, colony formation, cell viability, and xenograft model. Cellular functions in response to Aurora-A-modulated downstream targets such as FLJ10540 and MMPs were examined in vitro and in vivo, including cell growth, motility and chemosensitivity. Aurora-A/FLJ10540/MMPs expression was determined in cancer and adjacent normal tissues from HNC patients by immunohistochemistry approach. RESULTS: In the current study, Aurora-A exhibited similar gene expression profiles with FLJ10540 by using accessibly public microarray and Oncomine database analysis, raising the possibility that these molecules might coordinately participate in cancer progression and metastasis of HNC. These two molecules connection were also examined in cell lines and tissues of HNC. Aurora-A overexpression could not only bind to the promoter of FLJ10540 to induce FLJ10540 expression, but also increase both mRNA and protein levels of MMP-7 and MMP-10 in HNC cells. Conversely, depletion of Aurora-A expression by using siRNA or Aurora-A kinase inhibitor, MLN8237, suppressed FLJ10540, MMP-7 and MMP-10 mRNA and protein expressions in vitro and in vivo. In addition, the FLJ10540-PI3K complex was destroyed by inhibition the Aurora-A kinase activity. Forced overexpression of FLJ10540 in Aurora-A-depleted or in MLN8237-treated HNC cells attenuated the effect on cytotoxicity to cisplatin. Elevated Aurora-A expression in HNC cells led to the characteristics of more aggressive malignancy, including enhanced chemoresistance and increased the abilities of proliferation, migration and invasion, which was required for FLJ10540/MMP-7 or FLJ10540/MMP-10 expressions. Finally, immunohistochemical analysis of human HNC specimens showed a significant positively correlation among Aurora-A, FLJ10540, MMP-7 and MMP-10 expressions. CONCLUSION: Together, our findings define a novel mechanism by which Aurora-A promotes cell malignancy, with potential implications for understanding the clinical action of Aurora-A.

Analysis of Protein Stability by the Cycloheximide Chase Assay.

Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a full cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model.

MicroRNA-133a suppresses multiple oncogenic membrane receptors and cell invasion in non-small cell lung carcinoma.

Non-small cell lung cancers (NSCLCs) cause high mortality worldwide, and the cancer progression can be activated by several genetic events causing receptor dysregulation, including mutation or amplification. MicroRNAs are a group of small non-coding RNA molecules that function in gene silencing and have emerged as the fine-tuning regulators during cancer progression. MiR-133a is known as a key regulator in skeletal and cardiac myogenesis, and it acts as a tumor suppressor in various cancers. This study demonstrates that miR-133a expression negatively correlates with cell invasiveness in both transformed normal bronchial epithelial cells and lung cancer cell lines. The oncogenic receptors in lung cancer cells, including insulin-like growth factor 1 receptor (IGF-1R), TGF-beta receptor type-1 (TGFBR1), and epidermal growth factor receptor (EGFR), are direct targets of miR-133a. MiR-133a can inhibit cell invasiveness and cell growth through suppressing the expressions of IGF-1R, TGFBR1 and EGFR, which then influences the downstream signaling in lung cancer cell lines. The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines. In addition, by using the in vivo animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells. Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels. In summary, we identified the tumor suppressor role of miR-133a in lung cancer outcome prognosis, and we demonstrated that it targets several membrane receptors, which generally produce an activating signaling network during the progression of lung cancer.

Tid1-L inhibits EGFR signaling in lung adenocarcinoma by enhancing EGFR Ubiquitinylation and degradation.

Tid1 (DNAJA3), a DnaJ cochaperone, may promote degradation of oncogenic kinases. Tid1 has 2 isoforms, Tid1-L and Tid1-S, that may function differently. In this study, we investigated the role of the Tid1 isoforms in regulating EGF receptor (EGFR) signaling and lung cancer progression. We found that both Tid1-L and Tid1-S expressions were reduced in patients with non-small cell lung cancer compared with normal counterparts. Tid1-L expression correlated inversely with EGFR expression. Low Tid1-L/high EGFR expression predicted poor overall survival in patients with lung adenocarcinoma. Tid1-L overexpression in lung cancer cells attenuated EGFR signaling and inhibited cell proliferation, colony formation, and tumor growth in subcutaneous and orthotropic xenograft models. Conversely, depletion of Tid1 restored EGFR signaling and increased cell proliferation and colony formation. Tid1-L, but not Tid1-S, interacted with EGFR/HSP70/HSP90 through the DnaJ domain, counteracting the EGFR regulatory function of HSP90 by causing EGFR ubiquitinylation and proteasomal degradation. Tid1-L inhibited EGFR signaling even more than the HSP90 inhibitor 17-allylamino-demethoxy geldanamycin. We concluded that Tid1-L acted as a tumor suppressor by inhibiting EGFR signaling through interaction with EGFR/HSP70/HSP90 and enhancing EGFR ubiquitinylation and degradation.

Phosphorylation of LCRMP-1 by GSK3ß promotes filopoda formation, migration and invasion abilities in lung cancer cells.

LCRMP-1, a novel isoform of CRMP-1, can promote cancer cell migration, invasion and associate with poor clinical outcome in patients with non-small-cell lung cancer (NSCLC). However, the underlying regulatory mechanisms of LCRMP-1 in cancer cell invasiveness still remain obscure. Here, we report that GSK3ß can phosphorylate LCRMP-1 at Thr-628 in consensus sequences and this phosphorylation is crucial for function of LCRMP-1 to promote filopodia formation, migration and invasion in cancer cells. Impediment of Thr-628 phosphorylation attenuates the stimulatory effects of LCRMP-1 on filopodia forming, migration and invasion abilities in cancer cells; simultaneously, kinase-dead GSK3ß diminishes regulation of LCRMP-1 on cancer cell invasion. Furthermore, we also found that patients with low-level Ser-9-phosphorylated GSK3ß expression and high-level LCRMP-1 expression have worse overall survival than those with high-level inactive GSK3ß expressions and low-level LCRMP-1 expressions (P<0.0001). Collectively, these results demonstrate that GSK3ß-dependent phosphorylation of LCRMP-1 provides an important mechanism for regulation of LCRMP-1 on cancer cell invasiveness and clinical outcome.

The ability of LCRMP-1 to promote cancer invasion by enhancing filopodia formation is antagonized by CRMP-1.

Metastasis is a predominant cause of death in patients with cancer. It is a complex multistep process that needs to be better understood if we are to develop new approaches to managing tumor metastasis. Tumor cell invasion of the local stroma is suppressed by collapsin response mediator protein-1 (CRMP-1). Recently, we identified a long isoform of CRMP-1 (LCRMP-1), expression of which correlates with cancer cell invasiveness and poor clinical outcome in patients with non-small-cell lung cancer (NSCLC). Here, we report that LCRMP-1 overexpression in noninvasive human cell lines enhanced filopodia formation, cancer cell migration, and invasion via stabilization of actin. This effect required a highly conserved N-terminal region of LCRMP-1 as well as the WASP family verprolin-homologous protein-1/actin nucleation pathway (WAVE-1/actin nucleation pathway). Furthermore, LCRMP-1 appeared to act downstream of Cdc42, a Rho family protein known to be involved in actin rearrangement. In addition, LCRMP-1 associated with CRMP-1, which downregulated cancer cell metastasis by interrupting the association of LCRMP-1 and WAVE-1. Finally, we found that high-level expression of LCRMP-1 and low-level expression of CRMP-1 were associated with lymph node metastasis and poor survival in patients with NSCLC. In sum, we show that LCRMP-1 and CRMP-1 have opposing functions in regulating cancer cell invasion and metastasis and propose that this pathway may serve as a potential anticancer target.

p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug.

The tumour suppressor p53 is known to prevent cancer progression by inhibiting proliferation and inducing apoptosis of tumour cells. Slug, an invasion promoter, exerts its effects by repressing E-cadherin transcription. Here we show that wild-type p53 (wtp53) suppresses cancer invasion by inducing Slug degradation, whereas mutant p53 may stabilize Slug protein. In non-small-cell lung cancer (NSCLC), mutation of p53 correlates with low MDM2, high Slug and low E-cadherin expression. This expression profile is associated with poor overall survival and short metastasis-free survival in patients with NSCLC. wtp53 upregulates MDM2 and forms a wtp53-MDM2-Slug complex that facilitates MDM2-mediated Slug degradation. Downregulation of Slug by wtp53 or MDM2 enhances E-cadherin expression and represses cancer cell invasiveness. In contrast, mutant p53 inactivates Slug degradation and leads to Slug accumulation and increased cancer cell invasiveness. Our findings indicate that wtp53 and p53 mutants may differentially control cancer invasion and metastasis through the p53-MDM2-Slug pathway.

Identification and synthesis of the sex pheromone of the passionvine mealybug, Planococcus minor (Maskell).

The sex pheromone of the mealybug, Planococcus minor was isolated by fractionation of crude pheromone extract obtained by aeration of virgin females. The pheromone was identified as the irregular terpenoid, 2-isopropyl-5-methyl-2,4-hexadienyl acetate, by mass spectrometry, microchemical tests, and (1)H NMR spectroscopy. The stereochemistry of the pheromone was assigned as (E) by comparison with synthetic standards of known geometry. The compound was highly attractive to males in laboratory bioassays, whereas the (Z)-isomer appeared to antagonize attraction.