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.

MicroRNA-320 suppresses the stem cell-like characteristics of prostate cancer cells by downregulating the Wnt/beta-catenin signaling pathway.

Prostate cancer (PCa) is a leading cause of mortality and morbidity in men worldwide, and emerging evidence suggests that the CD44(high) prostate tumor-initiating cells (TICs) are associated with its poor prognosis. Although microRNAs are frequently dysregulated in human cancers, the influence of microRNAs on PCa malignancy and whether targeting TIC-associated microRNAs inhibit PCa progression remain unclear. In this study, we found that miR-320 is significantly downregulated in PCa. Overexpression of miR-320 in PCa cells decreases PCa tumorigenesis in vitro and in vivo. Global gene expression profiling of miR-320-overexpressing PCa cells reveals that downstream target genes of Wnt/ß-catenin pathway and cancer stem cell markers are significantly decreased. MicroRNA-320 inhibits ß-catenin expression by targeting the 3'-untranslated region of ß-catenin mRNA. The reduction of miR-320 associated with increased ß-catenin was also found in CD44(high) subpopulation of prostate cancer cells and clinical PCa specimens. Interestingly, knockdown of miR-320 significantly increases the cancer stem-like properties, such as tumorsphere formation, chemoresistance and tumorigenic abilities, although enriching the population of stem-like TICs among PCa cells. Furthermore, increased miR-320 expression in prostate stem-like TICs significantly suppresses stem cell-like properties of PCa cells. These results support that miR-320 is a key negative regulator in prostate TICs, and suggest developing miR-320 as a novel therapeutic agent may offer benefits for PCa treatment.

Galectin-1 orchestrates an inflammatory tumor-stroma crosstalk in hepatoma by enhancing TNFR1 protein stability and signaling in carcinoma-associated fibroblasts.

Most cases of hepatocellular carcinoma (HCC) arise with the fibrotic microenvironment where hepatic stellate cells (HSCs) and carcinoma-associated fibroblasts (CAFs) are critical components in HCC progression. Therefore, CAF normalization could be a feasible therapy for HCC. Galectin-1 (Gal-1), a ß-galactoside-binding lectin, is critical for HSC activation and liver fibrosis. However, few studies has evaluated the pathological role of Gal-1 in HCC stroma and its role in hepatic CAF is unclear. Here we showed that Gal-1 mainly expressed in HCC stroma, but not cancer cells. High expression of Gal-1 is correlated with CAF markers and poor prognoses of HCC patients. In co-culture systems, targeting Gal-1 in CAFs or HSCs, using small hairpin (sh)RNAs or an therapeutic inhibitor (LLS30), downregulated plasminogen activator inhibitor-2 (PAI-2) production which suppressed cancer stem-like cell properties and invasion ability of HCC in a paracrine manner. The Gal-1-targeting effect was mediated by increased a disintegrin and metalloprotease 17 (ADAM17)-dependent TNF-receptor 1 (TNFR1) shedding/cleavage which inhibited the TNF-α → JNK → c-Jun/ATF2 signaling axis of pro-inflammatory gene transcription. Silencing Gal-1 in CAFs inhibited CAF-augmented HCC progression and reprogrammed the CAF-mediated inflammatory responses in a co-injection xenograft model. Taken together, the findings uncover a crucial role of Gal-1 in CAFs that orchestrates an inflammatory CSC niche supporting HCC progression and demonstrate that targeting Gal-1 could be a potential therapy for fibrosis-related HCC.

Raised Source/Drain (RSD) and Vertical Lightly Doped Drain (LDD) Poly-Si Thin-Film Transistor.

The raised source/drain (RSD) structure is one of thin film transistor designs that is often used to improve device characteristics. Many studies have mentioned that the high impact ionization rate occurring at a drain side can be reduced, owing to a raised source/drain area that can disperse the drain electric field. In this study, we will discuss how the electric field at the drain side of an RSD device is reduced by a vertical lightly doped drain (LDD) scheme rather than a RSD structure. We used different raised source/drain forms to simulate the drain side electric field for each device, as well as their output characteristics, using Integrated Systems Engineering (ISE-TCAD) simulators. Different source and drain thicknesses and doping profiles were applied to verify the RSD mechanism. We found that the electric fields of a traditional device and uniform doping RSD structures are almost the same (~2.9 × 105 V/cm). The maximum drain electric field could be reduced to ~2 × 105 V/cm if a vertical lightly doped drain RSD scheme was adopted. A pure raised source/drain structure did not benefit the device characteristics if a vertical lightly doped drain design was not included in the raised source/drain areas.

DNA methylation maintains the CLDN1-EPHB6-SLUG axis to enhance chemotherapeutic efficacy and inhibit lung cancer progression.

The loss of cancer-cell junctions and escape from the primary-tumor microenvironment are hallmarks of metastasis. A tight-junction protein, Claudin 1 (CLDN1), is a metastasis suppressor in lung adenocarcinoma. However, as a metastasis suppressor, the underlying molecular mechanisms of CLDN1 has not been well studied. Methods: The signaling pathway regulated by CLDN1 was analyzed by Metacore software and validated by immunoblots. The effect of the CLDN1-EPHB6-ERK-SLUG axis on the formation of cancer stem-like cells, drug resistance and metastasis were evaluated by sphere assay, aldefluor assay, flow cytometry, migration assay, cytotoxicity, soft agar assay, immunoprecipitation assay and xenograft experiments. Furthermore, the methylation-specific PCR, pyrosequencing assay, chromatin immunoprecipitation and reporter assay were used to study the epigenetic and RUNX3-mediated CLDN1 transcription. Finally, the molecular signatures of RUNX3/CLDN1/SLUG were used to evaluate the correlation with overall survival by using gene expression omnibus (GEO) data. Results: We demonstrated that CLDN1 repressed cancer progression via a feedback loop of the CLDN1-EPHB6-ERK1/2-SLUG axis, which repressed metastasis, drug resistance, and cancer stemness, indicating that CLDN1 acts as a metastasis suppressor. CLDN1 upregulated the cellular level of EPHB6 and enhanced its activation, resulting in suppression of ERK1/2 signaling. Interestingly, DNA hypermethylation of the CLDN1 promoter abrogated SLUG-mediated suppression of CLDN1 in low-metastatic cancer cells. In contrast, the histone deacetylase inhibitor trichostatin A or vorinostat facilitated CLDN1 expression in high-metastatic cancer cells and thus increased the efficacy of chemotherapy. Combined treatment with cisplatin and trichostatin A or vorinostat had a synergistic effect on cancer-cell death. Conclusions: This study revealed that DNA methylation maintains CLDN1 expression and then represses lung cancer progression via the CLDN1-EPHB6-ERK1/2-SLUG axis. Because CLDN1 enhances the efficacy of chemotherapy, CLDN1 is not only a prognostic marker but a predictive marker for lung adenocarcinoma patients who are good candidates for chemotherapy. Forced CLDN1 expression in low CLDN1-expressing lung adenocarcinoma will increase the chemotherapy response, providing a novel therapeutic strategy.

MicroRNA-150-5p promotes cell motility by inhibiting c-Myb-mediated Slug suppression and is a prognostic biomarker for recurrent ovarian cancer.

Treatment of ovarian cancer (OvCa) remains challenging owing to its high recurrence rates. Detachment of cancer cells into the peritoneal fluid plays a key role in OvCa relapse, but how this occurs remains incompletely understood. Here we examined global miRNA expression profiles of paired primary/recurrent OvCa specimens and identified a novel biomarker, microRNA-150-5p (miR-150-5p), that was significantly upregulated in 16 recurrent OvCa tissues compared with their matched primary specimens. Analyses of cohorts from two other groups confirmed that expression of miR-150-5p was associated with early relapse and poor survival of OvCa patients. Inhibition of miR-150-5p significantly inhibited the migration and invasion of OvCa cells and induced a mesenchymal-epithelial transition (MET) phenotype. We demonstrated that the proto-oncogene, MYB, is an miR-150-5p target in OvCa cells and that the miR-150-5p/c-Myb/Slug axis plays important roles in regulating epithelial-mesenchymal transition (EMT) in OvCa cells. Expression of MYB was significantly correlated with good clinical outcome in OvCa and was negatively correlated with Slug expression in late-stage clinical specimens. These results suggest that miR-150-5p upregulation mediates the progression of recurrent OvCa by targeting the c-Myb/Slug pathway. Inhibition of miR-150-5p may serve as a new therapeutic strategy for preventing recurrence of OvCa.

Association of increased pain threshold by noise with central opioid neurons.

Several studies indicated that stress would induce analgesia. Noise, one of the stressors, was assumed to be one of the elements to enhance the threshold of pain tolerance. Since noise might affect human's daily life, it is important to know the mechanism underlying this phenomenon. The objective of this study was to explore the possible mechanism which was trying to explain how the noise affects central nervous system and the possible relationship between this effect and the involvement of opioid neurons. In the preliminary study, the analgesic effect was corroborated in ICR mice in a formalin study. The results are as follows: [1] Naloxone (a micro-opioid receptor antagonist; 1 mg/kg, i.p.), beta-FNA (a delta-opioid receptor antagonist; 5, 10 mg, i.c.v.) and naltrindole (a delta-opioid receptor antagonist; 1, 5 mg/kg, i.p.) were found to reduce antinociceptive effect. [2] nor-BNI (a kappa-antagonist; 1 microg, i.c.v.) had much effect on noise induced analgesic. In conclusion, this study suggests that noise stress enhanced the threshold of analgesia, which might be related to micro- and delta-opioid receptors in the central nervous system.

A DNA Aptamer Targeting Galectin-1 as a Novel Immunotherapeutic Strategy for Lung Cancer.

Galectin-1 (Gal-1) is a pleiotropic homodimeric ß-galactoside-binding protein with a single carbohydrate recognition domain. It has been implicated in several biological processes that are important during tumor progression. Several lines of evidence have indicated that Gal-1 is involved in cancer immune escape and induces T cell apoptosis. These observations all emphasized Gal-1 as a novel target for cancer immunotherapy. Here, we developed a novel Gal-1-targeting DNA aptamer (AP-74 M-545) and demonstrated its antitumor effect by restoring immune function. AP-74 M-545 binds to Gal-1 with high affinity. AP-74 M-545 targets tumors in murine tumor models but suppresses tumor growth only in immunocompetent C57BL/6 mice, not in immunocompromised non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. Immunohistochemistry revealed increased CD4+ and CD8+ T cells in AP-74 M-545-treated tumor tissues. AP-74 M-545 suppresses T cell apoptosis by blocking the binding of Gal-1 to CD45, the main receptor and apoptosis mediator of Gal-1 on T cells. Collectively, our data suggest that the Gal-1 aptamer suppresses tumor growth by blocking the interaction between Gal-1 and CD45 to rescue T cells from apoptosis and restores T cell-mediated immunity. These results indicate that AP-74 M-545 may be a potential strategy for cancer immunotherapy.

Targeting FXYD2 by cardiac glycosides potently blocks tumor growth in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is an aggressive neoplasm with a high recurrence rate that frequently develops resistance to platinum-based chemotherapy. There are few prognostic biomarkers or targeted therapies exist for patients with OCCC. Here, we identified that FXYD2, the modulating subunit of Na+/K+-ATPases, was highly and specifically expressed in clinical OCCC tissues. The expression levels of FXYD2 were significantly higher in advanced-stage of OCCC and positively correlated with patients' prognoses. Silencing of FXYD2 expression in OCCC cells inhibited Na+/K+-ATPase enzyme activity and suppressed tumor growth via induction of autophagy-mediated cell death. We found that high FXYD2 expression in OCCC was transcriptionally regulated by the transcriptional factor HNF1B. Furthermore, up-regulation of FXYD2 expression significantly increased the sensitivity of OCCC cells to cardiac glycosides, the Na+/K+-ATPase inhibitors. Two cardiac glycosides, digoxin and digitoxin, had a great therapeutic efficacy in OCCC cells in vitro and in vivo. Taken together, our results demonstrate that FXYD2 is functionally upregulated in OCCC and may serve as a promising prognostic biomarker and therapeutic target of cardiac glycosides in OCCC.

α-Catulin drives metastasis by activating ILK and driving an αvß3 integrin signaling axis.

α-Catulin is an oncoprotein that helps sustain proliferation by preventing cellular senescence. Here, we report that α-catulin also drives malignant invasion and metastasis. α-Catulin was upregulated in highly invasive non-small cell lung cancer (NSCLC) cell lines, where its ectopic expression or short-hairpin RNA-mediated attenuation enhanced or limited invasion or metastasis, respectively. α-Catulin interacted with integrin-linked kinase (ILK), a serine/threonine protein kinase implicated in cancer cell proliferation, antiapoptosis, invasion, and angiogenesis. Attenuation of ILK or α-catulin reciprocally blocked cell migration and invasion induced by the other protein. Mechanistic investigations revealed that α-catulin activated Akt-NF-κB signaling downstream of ILK, which in turn led to increased expression of fibronectin and integrin αvß3. Pharmacologic or antibody-mediated blockade of NF-κB or αvß3 was sufficient to inhibit α-catulin-induced cell migration and invasion. Clinically, high levels of expression of α-catulin and ILK were associated with poor overall survival in patients with NSCLC. Taken together, our study shows that α-catulin plays a critical role in cancer metastasis by activating the ILK-mediated Akt-NF-κB-αvß3 signaling axis.