LncRNA WT-AS inhibits metastatic ability of non-small cell lung cancer by regulating KLK13.

OBJECTIVE: The aim of this study was to investigate the effect of long non-coding RNA (lncRNA) WT-AS on the invasiveness and migration of non-small cell lung cancer (NSCLC) cells, and to explore the underlying molecular mechanism of lncRNA WT-AS in the pathogenesis of NSCLC. PATIENTS AND METHODS: LncRNA WT-AS expression in 50 pairs of NSCLC tissues and adjacent ones was studied by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and the correlations of WT-AS with clinicopathological indicators and prognosis of NSCLC patients were analyzed. Meanwhile, NSCLC expression levels in NSCLC cell lines were also evaluated by qPCR assay. In addition, WT-AS overexpression and knockdown models were constructed using lentivirus in NSCLC cell lines A549 and H1299, respectively. Thereafter, transwell and cell wound healing assays were carried out to assess the implication of WT-AS in biological functions of NSCLC cells. Furthermore, the interaction between WT-AS and KLK13 was determined via Luciferase assay. RESULTS: The results showed that WT-AS expression in NSCLC was remarkably lower than that in normal tissues adjacent to the cancer. Univariate analysis suggested that compared with patients with high expression of WT-AS, patients in low expression group showed higher incidence of metastasis and lower survival rates. Overexpression of WT-AS suppressed cell invasion and metastasis capacity, while the opposite result was observed in WT-AS knockdown group. KLK13 expression showed an increase in NSCLC cell lines and tissues, which was negatively correlated with WT-AS level. Meanwhile, Luciferase assay confirmed the binding between WT-AS and KLK13. Western blotting revealed that KLK13 expression was remarkably elevated in EC tissues and was positively correlated with TRIM62. In addition, it was also found that WT-AS and KLK13 had a mutual regulatory effect, which together affect the malignant progress of NSCLC. CONCLUSIONS: This study shows for the first time that LncRNA WT-AS interacts with KLK13 to serve as a negative regulator of NSCLC progression.

Data-Driven Discovery of Extravasation Pathway in Circulating Tumor Cells.

Circulating tumor cells (CTCs) play a crucial role in cancer dissemination and provide a promising source of blood-based markers. Understanding the spectrum of transcriptional profiles of CTCs and their corresponding regulatory mechanisms will allow for a more robust analysis of CTC phenotypes. The current challenge in CTC research is the acquisition of useful clinical information from the multitude of high-throughput studies. To gain a deeper understanding of CTC heterogeneity and identify genes, pathways and processes that are consistently affected across tumors, we mined the literature for gene expression profiles in CTCs. Through in silico analysis and the integration of CTC-specific genes, we found highly significant biological mechanisms and regulatory processes acting in CTCs across various cancers, with a particular enrichment of the leukocyte extravasation pathway. This pathway appears to play a pivotal role in the migration of CTCs to distant metastatic sites. We find that CTCs from multiple cancers express both epithelial and mesenchymal markers in varying amounts, which is suggestive of dynamic and hybrid states along the epithelial-mesenchymal transition (EMT) spectrum. Targeting the specific molecular nodes to monitor disease and therapeutic control of CTCs in real time will likely improve the clinical management of cancer progression and metastases.

An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers.

Epithelial-mesenchymal transition (EMT), a crucial mechanism in development, mediates aggressiveness during carcinoma progression and therapeutic refractoriness. The reversibility of EMT makes it an attractive strategy in designing novel therapeutic approaches. Therefore, drug discovery pipelines for EMT reversal are in need to discover emerging classes of compounds. Here, we outline a pre-clinical drug screening platform for EMT reversal that consists of three phases of drug discovery and validation. From the Phase 1 epithelial marker promoter induction (EpI) screen on a library consisting of compounds being approved by Food and Drug Administration (FDA), Vorinostat (SAHA), a histone deacetylase inhibitor (HDACi), is identified to exert EMT reversal effects by restoring the expression of an epithelial marker, E-cadherin. An expanded screen on 41 HDACi further identifies 28 compounds, such as class I-specific HDACi Mocetinosat, Entinostat and CI994, to restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells. Mocetinostat is the most potent HDACi to restore epithelial differentiation with the lowest concentration required for 50% induction of epithelial promoter activity (EpIC-50).The HDACi exerts paradoxical effects on EMT transcriptional factors such as SNAI and ZEB family and the effects are context-dependent in epithelial- and mesenchymal-like cells. In vitro functional studies further show that HDACi induced significant increase in anoikis and decrease in spheroid formation in ovarian and bladder carcinoma cells with mesenchymal features. This study demonstrates a robust drug screening pipeline for the discovery of compounds capable of restoring epithelial differentiation that lead to significant functional lethality.

Epithelial-mesenchymal status renders differential responses to cisplatin in ovarian cancer.

Chemoresistance to platinums, such as cisplatin, is of critical concern in the treatment of ovarian cancer. Recent evidence has linked epithelial-mesenchymal transition (EMT) as a contributing mechanism. The current study explored the connection between cellular responses to cisplatin and EMT in ovarian cancer. Expression microarrays were utilized to estimate the EMT status as a binary phenotype, and the transcriptional responses of 46 ovarian cancer cell lines to cisplatin were measured at dosages equivalent to 50% growth inhibition. Phenotypic responses to cisplatin were quantified with respect to cell number, proliferation rate and apoptosis, and then compared with the epithelial or mesenchymal status. Ovarian cancer cell lines with an epithelial status exhibited higher resistance to cisplatin treatment in the MTS assay than those with a mesenchymal status. Pathway analyses revealed the induction of G1/S- and S-phase genes (P=0.001) and the activation of multiple NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) downstream genes (P=0.0016) by cisplatin selectively in epithelial-like cell lines. BrdU incorporation and Caspase-3/7 release assays confirmed impaired apoptosis in epithelial-like ovarian cancer cells. In clinical samples, we observed resistance to single platinum treatment and the selective activation of the NF-κB pathway by platinum in ovarian cancers with an epithelial status. Overall, our results suggest that, in epithelial-like ovarian cancer cells, NF-κB activation by cisplatin may lead to defective apoptosis, preferential proliferation arrest and a consequential decreased sensitivity to cisplatin.

Targeting WNT1-inducible signaling pathway protein 2 alters human breast cancer cell susceptibility to specific lysis through regulation of KLF-4 and miR-7 expression.

The molecular basis for the resistance of tumor cells to cell-mediated cytotoxicity remains poorly understood and thus poses a major challenge for cancer immunotherapy. The present study was designed to determine whether the WNT1-inducible signaling pathway protein 2 (WISP2, also referred to as CCN5), a key regulator of tumor cell plasticity, interferes with tumor susceptibility to cytotoxic T-lymphocyte (CTL)-mediated lysis. We found that silencing WISP2 signaling in human breast adenocarcinoma MCF7 cells impairs CTL-mediated cell killing by a mechanism involving stem cell marker Kruppel-like factor-4 (KLF-4) induction and microRNA-7 (miR-7) downregulation. Inhibition of transforming growth factor beta (TGF-ß) signaling using the A83-01 inhibitor in MCF7-shWISP2 cells resulted in a significant reversal of the epithelial-to-mesenchymal-transitioned (EMT) phenotype, the expression of KLF-4 and a partial recovery of target susceptibility to CTLs. More importantly, we showed that silencing KLF-4 was accompanied by a reduction in MCF7-shWISP2 resistance to CTLs. Using human breast cancer tissues, we demonstrated the coexpression of KLF-4 with EMT markers and TGF-ß pathway signaling components. More importantly, we found that KLF-4 expression was accompanied by miR-7 inhibition, which is partly responsible for impairing CTL-mediated lysis. Thus, our data indicate that WISP2 has a role in regulating tumor cell susceptibility through EMT by inducing the TGF-ß signaling pathway, KLF-4 expression and miR-7 inhibition. These studies indicate for the first time that WISP2 acts as an activator of CTL-induced killing and suggests that the loss of its function promotes evasion of immunosurveillance and the ensuing progression of the tumor.

FZD7 drives in vitro aggressiveness in Stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway.

Ovarian cancer (OC) can be classified into five biologically distinct molecular subgroups: epithelial-A (Epi-A), Epi-B, mesenchymal (Mes), Stem-A and Stem-B. Among them, Stem-A expresses genes relating to stemness and is correlated with poor clinical prognosis. In this study, we show that frizzled family receptor 7 (FZD7), a receptor for Wnt signalling, is overexpressed in the Stem-A subgroup. To elucidate the functional roles of FZD7, we used an RNA interference gene knockdown approach in three Stem-A cell lines: CH1, PA1 and OV-17R. Si-FZD7 OC cells showed reduced cell proliferation with an increase in the G0/G1 sub-population, with no effect on apoptosis. The cells also displayed a distinctive morphologic change by colony compaction to become more epithelial-like and polarised with smaller internuclear distances and increased z-axis height. Immunofluorescence (IF) staining patterns of pan-cadherin and ß-catenin suggested an increase in cadherin-based cell-cell adhesion in si-FZD7 cells. We also observed a significant rearrangement in the actin cytoskeleton and an increase in tensile contractility in si-FZD7 OC cells, as evident by the loss of stress fibres and the redistribution of phospho-myosin light chain (pMLC) from the sites of cell-cell contacts to the periphery of cell colonies. Furthermore, there was reciprocal regulation of RhoA (Ras homolog family member A) and Rac1 (Ras-related C3 botulinum toxin substrate 1 (Rho family, small GTP-binding protein Rac1)) activities upon FZD7 knockdown, with a significant reduction in RhoA activity and a concomitant upregulation in Rac1 activity. These changes in pMLC and RhoA, as well as the increased TopFlash reporter activities in si-FZD7 cells, suggested involvement of the non-canonical Wnt/planar cell polarity (PCP) pathway. Selected PCP pathway genes (cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), prickle homolog 4 (Drosophila) (PRICKLE4), dishevelled-associated activator of morphogenesis 1 (DAAM1), profilin 2 (PFN2), protocadherin 9 (PCDH9), protocadherin α1 (PCDHA1), protocadherin ß17 pseudogene (PCDHB17), protocadherin ß3 (PCDHB3), sprouty homolog 1 (SPRY1) and protein tyrosine kinase 7 (PTK7)) were found to be more highly expressed in Stem-A than non Stem-A subgroup of OC. Taken together, our results suggest that FZD7 might drive aggressiveness in Stem-A OC by regulating cell proliferation, cell cycle progression, maintenance of the Mes phenotype and cell migration via casein kinase 1ɛ-mediated non-canonical Wnt/PCP pathway.

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor, saracatinib (AZD0530).

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial-mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial-mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.

A novel cognitive interpretation of breast cancer thermography with complementary learning fuzzy neural memory structure.

Early detection of breast cancer is the key to improve survival rate. Thermogram is a promising front-line screening tool as it is able to warn women of breast cancer up to 10 years in advance. However, analysis and interpretation of thermogram are heavily dependent on the analysts, which may be inconsistent and error-prone. In order to boost the accuracy of preliminary screening using thermogram without incurring additional financial burden, Complementary Learning Fuzzy Neural Network (CLFNN), FALCON-AART is proposed as the Computer-Assisted Intervention (CAI) tool for thermogram analysis. CLFNN is a neuroscience-inspired technique that provides intuitive fuzzy rules, human-like reasoning, and good classification performance. Confluence of thermogram and CLFNN offers a promising tool for fighting breast cancer.

Ovarian cancer diagnosis by hippocampus and neocortex-inspired learning memory structures.

Early detection and accurate staging of ovarian cancer are the keys to improving survival rate. However, at present there is no single diagnosis modality that is sufficiently sensitive. DNA microarray analysis is an emerging technique that has potential for ameliorating the hardship in early detection and staging of ovarian disease. However, microarray data is ultra-huge and difficult to analyze. Hence, computational intelligence methods are often utilized to assist in the diagnosis and analysis process. Fuzzy Neural Networks (FNN) are more suitable for this task as FNN provides not only the accuracy, but also the interpretability of its reasoning process. Hippocampus-inspired Complementary Learning FNN (CLFNN) is able to rapidly derive fuzzy sets and formulate fuzzy rules. CLFNN uses positive and negative learning, and hence it reduces the effect of the curse of dimensionality and is capable of modeling the dynamics of the problem space with relatively good classification performance. One of its successors, a hybrid of complementary hippocampal learning and associative neocortical learning called Pseudo Associative Complementary Learning (PACL), is a structure that seeks to functionally model the memory consolidation process. Both PACL and CLFNN have human-like reasoning that allows physicians to examine their computation using familiar terms. They can construct intuitive fuzzy rules autonomously to justify their reasoning, which is important to generate trust among the users. Hence, CLFNN and PACL are applied as a diagnostic decision support system in ovarian cancer diagnosis. The experimental results are encouraging.

Review of patients with infective endocarditis of native valves over a five-year period.

The clinical characteristics, echocardiographic features, bacteriologic data, morbidity and mortality of patients who were admitted to our hospital with infective endocarditis of their native valves over a five-year period were reviewed. There were 32 patients with a mean age of 38.2 +/- 16.2 years (range: 17 to 71 years) in our study population; 24 patients had underlying valvular abnormalities, six patients had congenital heart disease and two patients had no structural cardiac abnormality. Echocardiography was performed for all patients. Vegetations were absent in three (9.4%) patients, single in 19 (59.4%) patients and multiple in ten (31.3%) patients. Of the 24 (75%) patients who had left-sided endocarditis, mitral valve disease was the commonest valvular abnormality (16 patients). Ventricular septal defect was the commonest underlying abnormality in patients with right-sided endocarditis. Blood cultures were positive in 26 (81.3%) patients; the commonest organism was streptococcal (16 or 50% patients). Complications were present in 13 (40.6%) patients, of which eight patients had evidence of embolism, four patients had cardiac failure and one patient had a paravalvular abscess. Four (12.5%) patients died, two as a result of refractory heart failure and two as a consequence of septic embolism. Advances in medicine have resulted in a better outcome for patients with infective endocarditis, however, it remains an important disease with significant morbidity and mortality.

Anticancer gelatin microspheres with multiple functions.

Biodegradable, hydrophilic gelatin microspheres (GM) with an average diameter of 70 microns were prepared by cross-linking gelatin with glutaraldehyde for hepatic intra-arterial infusion. An anticancer agent, mitomycin C (MMC), together with a radioisotope, 131I, were bound to the GM for chemotherapy and local internal radiotherapy. The 131I-labelled MMC-GM (131I-MMC-GM) could accumulate in the specific site and embolize the hepatic arteries after the hepatic intra-arterial infusion, while it caused various effects to the liver cells. The 131I-MMC-GM remained within the hepatic arteries for at least one month. In vitro release of drugs from the GM was also quantified using a dynamic dialysis method.

[The pharmacokinetic study of 3H-alpha-2-butylhydroxybenzyl alcohol in rabbits].

A derivative of Gastrodigenin, alpha-2-butylhydroxybenzyl alcohol, has shown a high distribution in the mice brain and stronger pharmacologic effects than its parent compounds. With 3H-alpha-2-butylhydroxybenzyl alcohol as radioactive tracer, pharmacokinetic data were obtained after the intravenous administration of a single dose alpha-2-butylhydroxybenzyl alcohol to rabbits, according to a cross-over design. Then, serial plasma samples were taken from 1 min to 480 min and measured by liquid scintillation counter. The data were analyzed by IBMPC computer for the estimation of pharmacokinetic parameters and the judgement of compartment model with a program recently developed by ourselves. The results suggested that the pharmacokinetics of alpha-2-butylhydroxybenzyl alcohol accords with the two compartment open model based on either the comparison of the calculated theoretic value with measured concentration or F test for the model judgement, and that the process of distribution was quite rapid, and the elimination half-life (T1/2 beta) was 12 h, indicating a slow elimination process.

[Autoradiographic studies on the metabolism of gingival glycosaminoglycans in experimental periodontitis].

The purpose of this study was to investigate the changes in glycosaminoglycans (GAGs) of the gingiva during a period of experimental periodontitis induced by placing a silk ligature below the gingival margin of dog molars. Incorporation of 3H-glucosamine into the gingiva was determined autoradiographically. The gingiva was collected at 0, 7, 21, 60 and 90 days and cultured in vitro in the presence of 3H-glucosamine. The autoradiographs showed a predominantly epithelial location of the silver grains in all gingival epithelia. The location was intercellular in all epithelia. The results suggest greater 3H-glucosamine incorporation by the epithelium compared with the connective tissue and markedly more rapid metabolic turnover of epithelial GAGs.

[The distribution of 3H-alpha-sec-butyl-p hydroxybenzyl alcohol in mice].

alpha-sec-butyl-p-hydroxybenzyl alcohol is a derivative of gastrodigenin. It dissolves in lipid and passes through the blood-brain barrier more easily than gastrodin does. The distribution experiment has demonstrated that 3H-alpha-sec-butyl-p-hydroxybenzyl alcohol in the brain of mice is higher than in other organs, such as the liver, kidney and intestines. Radioactivity of brain tissue rises to the highest peak in about 1/2-2 min after intravenous injection. Each gram of the brain tissue takes up 21.89% radioactivity of the total dose administered. The sedative effect of alpha-sec-butyl-p-hydroxybenzyl alcohol on the center is rapid and short. It has been proved by the reactions in mice, results indicated by the electrocorticogram obtained from the mice after giving alpha-sec-butyl-p-hydroxybenzyl alcohol, and the correspondence with the distribution of alpha-sec-butyl-p-hydroxybenzyl alcohol in the mouse brain.