EGFR-T790M Mutation-Derived Interactome Rerouted EGFR Translocation Contributing to Gefitinib Resistance in Non-Small Cell Lung Cancer.

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer.

Transcriptomic Analysis Reveals Intrinsic Abnormalities in Endometrial Polyps.

Endometrial polyps (EPs) are benign overgrowths of the endometrial tissue lining the uterus, often causing abnormal bleeding or infertility. This study analyzed gene expression differences between EPs and adjacent endometrial tissue to elucidate intrinsic abnormalities promoting pathological overgrowth. RNA sequencing of 12 pairs of EPs and the surrounding endometrial tissue from infertile women revealed 322 differentially expressed genes. Protein-protein interaction network analysis revealed significant alterations in specific signaling pathways, notably Wnt signaling and vascular smooth muscle regulation, suggesting these pathways play critical roles in the pathophysiology of EPs. Wnt-related genes DKK1 and DKKL1 were upregulated, while GPC3, GREM1, RSPO3, SFRP5, and WNT10B were downregulated. Relevant genes for vascular smooth muscle contraction were nearly all downregulated in EPs, including ACTA2, ACTG2, KCNMB1, KCNMB2, MYL9, PPP1R12B, and TAGLN. Overall, the results indicate fundamental gene expression changes promote EP formation through unrestrained growth signaling and vascular defects. The intrinsic signaling abnormalities likely contribute to clinical symptoms of abnormal uterine bleeding and infertility common in EP patients. This analysis provides molecular insights into abnormal endometrial overgrowth to guide improved diagnostic and therapeutic approaches for this troublesome women's health condition. Confirmation of expanded cohorts and further investigations into implicated regulatory relationships are warranted.

An experimental model for ovarian cancer: propagation of ovarian cancer initiating cells and generation of ovarian cancer organoids.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynecological cancer due to the recurrence of drug-resistance. Cancer initiating cells (CICs) are proposed to be responsible for the aggressiveness of OC. The rarity and difficulty of in vitro long-term cultivation of CICs challenge the development of CIC-targeting therapeutics. Reprogramming cancer cells into induced cancer initiating cell (iCICs) could be an approach to solve these. Several inducible CICs have been acquired by activating the expression of stemness genes in different cancer cells. However, few reports have demonstrated the feasibility in OC. METHODS: Patients with primary OC receiving surgery were enrolled. Tumor tissue were collected, and OCT4, SOX2, and NANOG expressions were assessed by immunohistochemistry (IHC) staining to investigate the association of stemness markers with overall survival (OS). An high-grade serous ovarian cancer (HGSOC) cell line, OVCAR-3 was reprogrammed by transducing Yamanaka four factors OCT4, SOX2, KLF4 and MYC (OSKM) to establish an iOCIC model, iOVCAR-3-OSKM. CIC characteristics of iOVCAR-3-OSKM were evaluated by RT-PCR, sphere formation assay and animal experiments. Drug-resistance and migration ability were accessed by dye-efflux activity assay, MTT assay and migration assay. Gene profile was presented through RNA-sequencing. Lineage differentiation ability and organoid culture were determined by in vitro differentiation assays. RESULTS: In OC patients, the co-expression of multiple stem-related transcription factors (OCT4, SOX2, and NANOG) was associated with worse OS. iOVCAR-3-OSKM cells generated by reprogramming successfully exhibited stemness characteristics with strong sphere-forming and tumorigenesis ability. iOVCAR-3-OSKM cells also showed malignant potential with higher drug resistance to chemodrug, Paclitaxel (PTX) and migration ability. iOVCAR-3-OSKM was maintainable and expandable on feeder-dependent culture condition, it also preserved ovarian lineage differentiation abilities, which could well differentiate into OC cells with CK-7 and CA125 expressions and develop into an organoid mimic poor prognostic OC histological feature. CONCLUSIONS: The establishment of iOVCAR-3-OSKM not only allows us to fill the gap in the information on induced CICs in OC but also provides a potential strategy to develop personalized CICs and organoid models for treating OC in the near future.

N-glycosylated GPNMB ligand independently activates mutated EGFR signaling and promotes metastasis in NSCLC.

Lung cancer is the leading cause of cancer-related death worldwide. As well as the identified role of epidermal growth factor receptor (EGFR), its association with driver mutations has improved the therapeutics for patients with lung cancer harboring EGFR mutations. These patients usually display shorter overall survival and a higher tendency to develop distant metastasis compared with those carrying the wild-type EGFR. Nevertheless, the way to control mutated EGFR signaling remains unclear. Here, we performed membrane proteomic analysis to determine potential components that may act with EGFR mutations to promote lung cancer malignancy. Expression of transmembrane glycoprotein non-metastatic melanoma protein B (GPNMB) was positively correlated with the status of mutated EGFR in non-small-cell lung cancer (NSCLC). This protein was not only overexpressed but also highly glycosylated in EGFR-mutated, especially EGFR-L858R mutated, NSCLC cells. Further examination showed that GPNMB could activate mutated EGFR without ligand stimulation and could bind to the C-terminus of EGFR, assist phosphorylation at Y845, turn on downstream STAT3 signaling, and promote cancer metastasis. Moreover, we also found that Asn134 (N134) glycosylation of GPNMB played a crucial role in this ligand-independent regulation. Depleting N134-glycosylation on GPNMB could dramatically inhibit binding of GPNMB to mutated EGFR, blocking its downstream signaling, and ultimately inhibiting cancer metastasis in NSCLC. Clarifying the role of N-glycosylated GPNMB in regulating the ligand-independent activation of mutated EGFR may soon give new insight into the development of novel therapeutics for NSCLC.

2-anilino-4-amino-5-aroylthiazole-type compound AS7128 inhibits lung cancer growth through decreased iASPP and p53 interaction.

Lung cancer is the leading cause of cancer-related death worldwide. Thus, developing novel therapeutic agents has become critical for lung cancer treatment. In this study, compound AS7128 was selected from a 2-million entry chemical library screening and identified as a candidate drug against non-small cell lung cancer in vitro and in vivo. Further investigation indicated that AS7128 could induce cell apoptosis and cell cycle arrest, especially in the mitosis stage. In addition, we also found that iASPP, an oncogenic protein that functionally inhibits p53, might be associated with AS7128 through mass identification. Further exploration indicated that AS7128 treatment could restore the transactivation ability of p53 and, thus, increase the expressions of its downstream target genes, which are related to cell cycle arrest and apoptosis. This occurs through disruption of the interactions between p53 and iASPP in cells. Taken together, AS7128 could bind to iASPP, disrupt the interaction between iASPP and p53, and result in cell cycle arrest and apoptosis. These findings may provide new insight for using iASPP as a therapeutic target for non-small cell lung cancer treatment.

Membrane Proteomics of Impaired Energetics and Cytoskeletal Disorganization in Elderly Diet-Induced Diabetic Mice.

Diabetic cardiomyopathy is a well-recognized complication of diabetes, but its pathophysiology is unclear. We aimed to investigate the mechanisms underlying cardiac dysfunction in an elderly type 2 diabetic (T2DM) mouse model, using membrane proteomic analyses. Elderly mice were fed a high fat diet for 12 weeks to induce T2DM, and myocardial structure and function were assessed by echocardiography. Cardiomyocytes were isolated by Langendorff perfusion and subjected to iTRAQ-based quantitative membrane proteomic profiling, immunoblotting, and real-time quantitative reverse-transcriptase polymerase chain reaction. Compared to controls, elderly T2DM mice showed worse systolic function, more myocardial fibrosis and up-regulation of several heart failure markers (all p < 0.05). Cardiomyocyte membrane proteomic profiling revealed that 417 proteins had differential expressions related to perturbations in several biological processes in T2DM mice compared with the control. The most up-regulated proteins were involved in oxidative phosphorylation, whereas many down-regulated proteins were involved in cytoskeletal regulation. Differential protein expression correlated with myocardial systolic velocity by tissue Doppler. In addition, cardiomyocyte immunofluorescence staining showed greater disorganization of thick/parallel F-actin stress fibers and marked reduction in F-to-G-actin ratio in T2DM vs control (p < 0.05), which paralleled worsened myocardial systolic velocity. We concluded that cardiac contractile dysfunction in elderly T2DM mice was associated with impaired energetics and cytoskeletal disorganization.

Shisa3 is associated with prolonged survival through promoting ß-catenin degradation in lung cancer.

RATIONALE: Despite advances in treatment and prognosis of non-small cell lung cancer (NSCLC), patient outcomes are still unsatisfactory. OBJECTIVES: To reduce the morbidity and mortality of patients with NSCLC, a more comprehensive understanding of mechanisms involved in cancer progression is urgently needed. METHODS: By comparison of gene expression profiles in the cell line pair with differential invasion ability, CL1-0 and CL1-5, we found that Shisa3 was highly expressed in the low invasive cells. The effect of Shisa3 on invasion, migration, proliferation, apoptosis, epithelial-mesenchymal transition, and anchorage-independent growth activities in vitro and on tumor growth and metastasis in mice models were examined. The underlying mechanism of Shisa3 was explored by microarray and pathway analysis. Finally, the correlation of Shisa3 expression and clinical outcome was also calculated. MEASUREMENTS AND MAIN RESULTS: We identified Shisa3 as a novel tumor suppressor, which induces ß-catenin degradation resulting in suppression of tumorigenesis and invasion in vitro. Shisa3 decreased the tumor growth in mice with subcutaneous implantation and reduced the number of metastatic nodules in mice with tail vein injection and orthotopic implantation. Shisa3 performs the tumor suppression activity through WNT signaling predicted by microarray analysis. Our data found that Shisa3 accelerates ß-catenin degradation and was positively associated with overall survival and progression-free survival of NSCLC. CONCLUSIONS: Our results reveal that Shisa3 acts as a tumor suppressor by acceleration of ß-catenin degradation and provide new insight for cancer prognosis and therapy.

Dual-targeting compounds possessing enhanced anticancer activity via microtubule disruption and histone deacetylase inhibition.

Dual-targeting anticancer agents 4-29 are designed by combining the structural features of purine-type microtubule-disrupting compounds and HDAC inhibitors. A library of the conjugate compounds connected by appropriate linkers was synthesized and found to possess HDACs inhibitory activity and render microtubule fragmentation by activating katanin, a microtubule-severing protein. Among various zinc-binding groups, hydroxamic acid shows the highest inhibitory activity of Class I HDACs, which was also reconfirmed by three-dimensional quantitative structure-activity relationship (3D-QSAR) pharmacophore prediction. The purine-hydroxamate conjugates exhibit enhanced cytotoxicity against MDA-MB231 breast cancer cells, H1975 lung cancer cells, and various clinical isolated non-small-cell lung cancer cells with different epidermal growth factor receptor (EGFR) status. Pyridyl substituents could be used to replace the C2 and N9 phenyl moieties in the purine-type scaffold, which can help to improve the solubility under physiological conditions, thus increasing cytotoxicity. In mice treated with the purine-hydroxamate conjugates, the tumor growth rate was significantly reduced without causing toxic effects. Our study demonstrates the potential of the dual-targeting purine-hydroxamate compounds for cancer monotherapy.

LCRMP-1 is required for spermatogenesis and stabilises spermatid F-actin organization via the PI3K-Akt pathway.

Long-form collapsin response mediator protein-1 (LCRMP-1) belongs to the CRMP family which comprises brain-enriched proteins responsible for axon guidance. However, its role in spermatogenesis remains unclear. Here we find that LCRMP-1 is abundantly expressed in the testis. To characterize its physiological function, we generate LCRMP-1-deficient mice (Lcrmp-1-/-). These mice exhibit aberrant spermiation with apoptotic spermatids, oligospermia, and accumulation of immature testicular cells, contributing to reduced fertility. In the seminiferous epithelial cycle, LCRMP-1 expression pattern varies in a stage-dependent manner. LCRMP-1 is highly expressed in spermatids during spermatogenesis and especially localized to the spermiation machinery during spermiation. Mechanistically, LCRMP-1 deficiency causes disorganized F-actin due to unbalanced signaling of F-actin dynamics through upregulated PI3K-Akt-mTOR signaling. In conclusion, LCRMP-1 maintains spermatogenesis homeostasis by modulating cytoskeleton remodeling for spermatozoa release.

A Common East Asian aldehyde dehydrogenase 2*2 variant promotes ventricular arrhythmia with chronic light-to-moderate alcohol use in mice.

Chronic heavy alcohol use is associated with lethal arrhythmias. Whether common East Asian-specific aldehyde dehydrogenase deficiency (ALDH2*2) contributes to arrhythmogenesis caused by low level alcohol use remains unclear. Here we show 59 habitual alcohol users carrying ALDH2 rs671 have longer QT interval (corrected) and higher ventricular tachyarrhythmia events compared with 137 ALDH2 wild-type (Wt) habitual alcohol users and 57 alcohol non-users. Notably, we observe QT prolongation and a higher risk of premature ventricular contractions among human ALDH2 variants showing habitual light-to-moderate alcohol consumption. We recapitulate a human electrophysiological QT prolongation phenotype using a mouse ALDH2*2 knock-in (KI) model treated with 4% ethanol, which shows markedly reduced total amount of connexin43 albeit increased lateralization accompanied by markedly downregulated sarcolemmal Nav1.5, Kv1.4 and Kv4.2 expressions compared to EtOH-treated Wt mice. Whole-cell patch-clamps reveal a more pronounced action potential prolongation in EtOH-treated ALDH2*2 KI mice. By programmed electrical stimulation, rotors are only provokable in EtOH-treated ALDH2*2 KI mice along with higher number and duration of ventricular arrhythmia episodes. The present research helps formulate safe alcohol drinking guideline for ALDH2 deficient population and develop novel protective agents for these subjects.

Id2 exerts tumor suppressor properties in lung cancer through its effects on cancer cell invasion and migration.

Background: Despite advances in prognosis and treatment of lung adenocarcinoma (LADC), a notable non-small cell lung cancer subtype, patient outcomes are still unsatisfactory. New insight on novel therapeutic strategies for LADC may be gained from a more comprehensive understanding of cancer progression mechanisms. Such strategies could reduce the mortality and morbidity of patients with LADC. In our previous study, we performed cDNA microarray screening and found an inverse relationship between inhibitor of DNA binding 2 (Id2) expression levels and the invasiveness of LADC cells. Materials and Methods: To identify the functional roles of Id2 and its action mechanisms in LADC progression, we successfully established several Id2-overexpressing and Id2-silenced LADC cell clones. Subsequently, we examined in vitro the effects exerted by Id2 on cell morphology, proliferation, colony formation, invasive, and migratory activities and examined in vivo those exerted by Id2 on cell metastasis. The mechanisms underlying the action of Id2 were investigated using RNA-seq and pathway analyses. Furthermore, the correlations of Id2 with its target gene expression and clinical outcomes were calculated. Results: Our data revealed that Id2 overexpression could inhibit LADC cells' migratory, invasive, proliferation, and colony formation capabilities. Silencing Id2 expression in LADC cells reversed the aforementioned inhibitory effects, and knockdown of Id2 increased LADC cells' metastatic abilities in vivo. Bioinformatics analysis revealed that these effects of Id2 on cancer progression might be regulated by focal adhesion kinase (FAK) signaling and CD44/Twist expression. Furthermore, in online clinical database analysis, patients with LADC whose Id2 expression levels were high and FAK/Twist expression levels were low had superior clinical outcomes.Conclusion: Our data indicate that the Id2 gene may act as a metastasis suppressor and provide new insights into LADC progression and therapy.

Variant Aldehyde Dehydrogenase 2 (ALDH2*2) as a Risk Factor for Mechanical LA Substrate Formation and Atrial Fibrillation with Modest Alcohol Consumption in Ethnic Asians.

Aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism is a common genetic variant in Asians that is responsible for defective toxic aldehyde and lipid peroxidation metabolism after alcohol consumption. The extent to which low alcohol consumption may cause atrial substrates to trigger atrial fibrillation (AF) development in users with ALDH2 variants remains to be determined. We prospectively enrolled 249 ethnic Asians, including 56 non-drinkers and 193 habitual drinkers (135 (70%) as ALDH2 wild-type: GG, rs671; 58 (30%) as ALDH2 variants: G/A or A/A, rs671). Novel left atrial (LA) mechanical substrates with dynamic characteristics were assessed using a speckle-tracking algorithm and correlated to daily alcohol consumption and ALDH2 genotypes. Despite modest and comparable alcohol consumption by the habitual alcohol users (14.3 [8.3~28.6] and 12.3 [6.3~30.7] g/day for those without and with ALDH2 polymorphism, p = 0.31), there was a substantial and graded increase in the 4-HNE adduct and prolonged PR, and a reduction in novel LA mechanical parameters (including peak atrial longitudinal strain (PALS) and phasic strain rates (reservoir, conduit, and booster pump functions), p < 0.05), rather than an LA emptying fraction (LAEF) or LA volume index across non-drinkers, and in habitual drinkers without and with ALDH2 polymorphism (all p < 0.05). The presence of ALDH2 polymorphism worsened the association between increasing daily alcohol dose and LAEF, PALS, and phasic reservoir and booster functions (all Pinteraction: <0.05). Binge drinking superimposed on regular alcohol use exclusively further worsened LA booster pump function compared to regular drinking without binge use (1.66 ± 0.57 vs. 1.97 ± 0.56 1/s, p = 0.001). Impaired LA booster function further independently helped to predict AF after consideration of the CHARGE-AF score (adjusted 1.68 (95% CI: 1.06-2.67), p = 0.028, per 1 z-score increment). Habitual modest alcohol consumption led to mechanical LA substrate formation in an ethnic Asian population, which was more pronounced in subjects harboring ALDH2 variants. Impaired LA booster functions may serve as a useful predictor of AF in such populations.

An informatics-assisted label-free quantitation strategy that depicts phosphoproteomic profiles in lung cancer cell invasion.

Aberrant protein phosphorylation plays important roles in cancer-related cell signaling. With the goal of achieving multiplexed, comprehensive, and fully automated relative quantitation of site-specific phosphorylation, we present a simple label-free strategy combining an automated pH/acid-controlled IMAC procedure and informatics-assisted SEMI (sequence, elution time, mass-to-charge, and internal standard) algorithm. The SEMI strategy effectively increased the number of quantifiable peptides more than 4-fold in replicate experiments (from 262 to 1171, p < 0.05, false discovery rate = 0.46%) by using a fragmental regression algorithm for elution time alignment followed by peptide cross-assignment in all LC-MS/MS runs. In addition, the strategy demonstrated good quantitation accuracy (10-12%) for standard phosphoprotein and variation less than 1.9 fold (within 99% confidence range) in proteome scale and reliable linear quantitation correlation (R(2) = 0.99) with 4000-fold dynamic concentrations, which was attributed to our reproducible experimental procedure and informatics-assisted peptide alignment tool to minimize system variations. In an attempt to explore metastasis-associated phosphoproteomic alterations in lung cancer, this approach was used to delineate differential phosphoproteomic profiles of a lung cancer metastasis model. Without sample fractionation, the SEMI algorithm enabled quantification of 1796 unique phosphopeptides (false discovery rate = 0.56%) corresponding to 854 phosphoproteins from a series of non-small cell lung cancer lines with varying degrees of in vivo invasiveness. Nearly 40% of the phosphopeptides showed >2-fold change in highly invasive cells; validation of phosphoprotein subsets by Western blotting not only demonstrated the consistency of data obtained by our SEMI strategy but also revealed that such dramatic changes in the phosphoproteome result mostly from translational or post-translational regulation. Mapping of these differentially expressed phosphoproteins in multiple cellular pathways related to cancer invasion and metastasis suggests that the site and degree of phosphorylation might have distinct patterns or functions in the complex process of cancer progression.

Galectin-1-mediated tumor invasion and metastasis, up-regulated matrix metalloproteinase expression, and reorganized actin cytoskeletons.

Galectin-1 (Gal-1) is a beta-galactose-binding lectin; its expression level has been reported to correlate with tumor progression. Gal-1 is highly expressed in the invasive front of primary tumors and in the cancer cells of metastatic lesions in the lymph nodes of patients with oral squamous cell carcinoma. However, the molecular mechanism of Gal-1 in tumor metastasis is not completely clear. We found that increased Gal-1 expression is closely associated with its high levels of invasion in lung adenocarcinoma and oral squamous cell carcinoma cell lines. Knocking down Gal-1 with small interfering RNA in highly invasive cancer cells reduced their invasion levels. Moreover, the invasion ability of poorly invasive cancer cells was significantly increased after Gal-1 overexpression of Gal-1. Mechanism studies revealed that Gal-1 promoted tumor invasion mainly by up-regulating matrix metalloproteinase (MMP)-9 and MMP-2 and by reorganizing actin cytoskeleton. Gal-1 enhanced the activation of Cdc42, a small GTPase and member of the Rho family, thus increasing the number and length of filopodia on tumor cells. Furthermore, Gal-1-overexpressing cells had higher metastatic abilities in tail vein metastasis assays in vivo. We conclude that Gal-1 is involved in tumor invasion and metastasis by increasing MMP expression and reorganizing cytoskeletons in oral cancers and lung adenocarcinoma.

Claudin-1 is a metastasis suppressor and correlates with clinical outcome in lung adenocarcinoma.

RATIONALE: Claudin (CLDN)-1, a key component of tight junction complexes, was down-regulated in human lung adenocarcinomas. OBJECTIVES: To investigate the clinical significance of CLDN1 expression in patients with lung adenocarcinoma and its role in cancer invasion and metastasis. METHODS: We examined the CLDN1 mRNA expression in tumor specimens from 64 patients with lung adenocarcinoma and protein expression by immunohistochemistry in an independent cohort of 67 patients with lung adenocarcinoma. CLDN1 functions in cancer cell migration, invasion, and metastatic colonization were studied by overexpression and knockdown of CLDN1. Affymetrix microarrays were performed to identify gene expression changes associated with CLDN1 overexpression. MEASUREMENTS AND MAIN RESULTS: We found that low-CLDN1 mRNA expression had shorter overall survival (P = 0.027, log-rank test) in 64 patients with lung adenocarcinoma, and we confirmed by immunohistochemistry that low CLDN1 expression had shorter overall survival (P = 0.024, log-rank test) in an independent cohort of 67 patients with lung adenocarcinoma. Overexpression of CLDN1 inhibited cancer cell dissociation in time-lapse imaging of wound healing, and suppressed cancer cell migration, invasion, and metastasis. Knockdown CLDN1 expression increased cancer cell invasive and metastatic abilities. Affymetrix microarrays identified a panel of genes altered by CLDN1 overexpression. CLDN1 increased expressions of cancer invasion/metastasis suppressors (e.g., connective tissue growth factor [CTGF], thrombospondin 1 [THBS1], deleted in liver cancer 1 [DLC1], occludin [OCLN], zona occludens 1 [ZO-1]) and suppressed expressions of invasion/metastasis enhancers (e.g., secreted phosphoprotein 1 [SPP1], cut-like homeobox 1 [CUTL1], transforming growth factor alpha [TGF-alpha], solute carrier family 2 [faciliated glucose transporter] member 3 [SLC2A3], placental growth factor [PGF]), supporting a role for CLDN1 as an invasion and metastasis suppressor. CONCLUSIONS: CLDN1 is a cancer invasion/metastasis suppressor. CLDN1 is also a useful prognostic predictor and potential drug treatment target for patients with lung adenocarcinoma.

Suppression of Drug-Resistant Non-Small-Cell Lung Cancer with Inhibitors Targeting Minichromosomal Maintenance Protein.

Drug resistance has been a major threat in cancer therapies that necessitates the development of new strategies to overcome this problem. We report here a cell-based high-throughput screen of a library containing two-million molecules for the compounds that inhibit the proliferation of non-small-cell lung cancer (NSCLC). Through the process of phenotypic screening, target deconvolution, and structure-activity relationship (SAR) analysis, a compound of furanonaphthoquinone-based small molecule, AS4583, was identified that exhibited potent activity in tyrosine kinase inhibitor (TKI)-sensitive and TKI-resistant NSCLC cells (IC50 = 77 nM) and in xenograft mice. The mechanistic studies revealed that AS4583 inhibited cell-cycle progression and reduced DNA replication by disrupting the formation of the minichromosomal maintenance protein (MCM) complex. Subsequent SAR study of AS4583 gave compound RJ-LC-07-48 which exhibited greater potency in drug-resistant NSCLC cells (IC50 = 17 nM) and in mice with H1975 xenograft tumor.

Inhibitor of DNA-Binding Protein 4 Suppresses Cancer Metastasis through the Regulation of Epithelial Mesenchymal Transition in Lung Adenocarcinoma.

Metastasis is a predominant cause of cancer death and the major challenge in treating lung adenocarcinoma (LADC). Therefore, exploring new metastasis-related genes and their action mechanisms may provide new insights for developing a new combative approach to treat lung cancer. Previously, our research team discovered that the expression of the inhibitor of DNA binding 4 (Id4) was inversely related to cell invasiveness in LADC cells by cDNA microarray screening. However, the functional role of Id4 and its mechanism of action in lung cancer metastasis remain unclear. In this study, we report that the expression of Id4 could attenuate cell migration and invasion in vitro and cancer metastasis in vivo. Detailed analyses indicated that Id4 could promote E-cadherin expression through the binding of Slug, cause the occurrence of mesenchymal-epithelial transition (MET), and inhibit cancer metastasis. Moreover, the examination of the gene expression database (GSE31210) also revealed that high-level expression of Id4/E-cadherin and low-level expression of Slug were associated with a better clinical outcome in LADC patients. In summary, Id4 may act as a metastatic suppressor, which could not only be used as an independent predictor but also serve as a potential therapeutic for LADC treatment.

WW Domain-Containing Proteins YAP and TAZ in the Hippo Pathway as Key Regulators in Stemness Maintenance, Tissue Homeostasis, and Tumorigenesis.

The Hippo pathway is a conserved signaling pathway originally defined in Drosophila melanogaster two decades ago. Deregulation of the Hippo pathway leads to significant overgrowth in phenotypes and ultimately initiation of tumorigenesis in various tissues. The major WW domain proteins in the Hippo pathway are YAP and TAZ, which regulate embryonic development, organ growth, tissue regeneration, stem cell pluripotency, and tumorigenesis. Recent reports reveal the novel roles of YAP/TAZ in establishing the precise balance of stem cell niches, promoting the production of induced pluripotent stem cells (iPSCs), and provoking signals for regeneration and cancer initiation. Activation of YAP/TAZ, for example, results in the expansion of progenitor cells, which promotes regeneration after tissue damage. YAP is highly expressed in self-renewing pluripotent stem cells. Overexpression of YAP halts stem cell differentiation and yet maintains the inherent stem cell properties. A success in reprograming iPSCs by the transfection of cells with Oct3/4, Sox2, and Yap expression constructs has recently been shown. In this review, we update the current knowledge and the latest progress in the WW domain proteins of the Hippo pathway in relevance to stem cell biology, and provide a thorough understanding in the tissue homeostasis and identification of potential targets to block tumor development. We also provide the regulatory role of tumor suppressor WWOX in the upstream of TGF-ß, Hyal-2, and Wnt signaling that cross talks with the Hippo pathway.

Structure-guided development of purine amide, hydroxamate, and amidoxime for the inhibition of non-small cell lung cancer.

An 8-oxopurine-6-carboxamide compound (1a) was previously identified as an inhibitor of non-small cell lung cancer (NSCLC). In this study, more than 30 purine-6-carboxamide derivatives with variations at the C2, N7, C8, and N9 positions were synthesized to investigate the structure-activity relationship as a basis for the construction of an advanced pharmacophore model. This model suggests that purine-6-hydroxamate and purine-6-amidoxime analogs could form more hydrogen bonds with a target protein to enhance the inhibitory activities against H1975 cells. Among the series of analogs, hydroxamate 17 and amidoxime 19a exhibited excellent potency against H1975 cells (IC50 < 1.5 µM) and other lung cancer cells with either wild-type or mutated epidermal growth factor receptor (EGFR). Mouse experiments indicated that compounds 17 and 19a were efficient anticancer agents with no appreciable toxicity. The mechanisms of action for the induction of cell apoptosis were determined to involve microtubule fragmentation and p53-mediated signaling pathways.

Hypoxia-induced Slug SUMOylation enhances lung cancer metastasis.

BACKGROUND: The Slug-E-cadherin axis plays a critical role in non-small-cell lung cancers (NSCLCs) where aberrant upregulation of Slug promotes cancer metastasis. Now, the post-translational modifications of Slug and their regulation mechanisms still remain unclear in lung cancer. Hence, exploring the protein linkage map of Slug is of great interest for investigating the scenario of how Slug protein is regulated in lung cancer metastasis. METHODS: The Slug associated proteins, Ubc9 and SUMO-1, were identified using yeast two-hybrid screening; and in vitro SUMOylation assays combined with immunoprecipitation and immunoblotting were performed to explore the detail events and regulations of Slug SUMOylation. The functional effects of SUMOylation on Slug proteins were examined by EMSA, reporter assay, ChIP assay, RT-PCR, migration and invasion assays in vitro, tail vein metastatic analysis in vivo, and also evaluated the association with clinical outcome of NSCLC patients. RESULTS: Slug protein could interact with Ubc9 and SUMO-1 and be SUMOylated in cells. Amino acids 130-212 and 33-129 of Slug are responsible for its binding to Ubc9 and protein inhibitor of activated STAT (PIAS)y, respectively. SUMOylation could enhance the transcriptional repression activity of Slug via recruiting more HDAC1, resulting in reduced expression of downstream Slug target genes and enhanced lung cancer metastasis. In addition, hypoxia could increase Slug SUMOylation through attenuating the interactions of Slug with SENP1 and SENP2. Finally, high expression Slug and Ubc9 levels were associated with poor overall survival among NSCLC patients. CONCLUSIONS: Ubc9/PIASy-mediated Slug SUMOylation and subsequent HDAC1 recruitment may play a crucial role in hypoxia-induced lung cancer progression, and these processes may serve as therapeutic targets for NSCLC.