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Background: Extracellular vesicles (EVs) are derived from internal cellular compartments, and have potential as a diagnostic and therapeutic tool in degenerative disease associated with aging. Mesenchymal stem cells (MSCs) have become a promising tool for functional EVs production. This study investigated the efficacy of EVs and its effect on differentiation capacity. Methods: The characteristics of MSCs were evaluated by flow cytometry and stem cell differentiation analysis, and a production mode of functional EVs was scaled from MSCs. The concentration and size of EVs were quantitated by Nanoparticle Tracking Analysis (NTA). Western blot analysis was used to assess the protein expression of exosome-specific markers. The effects of MSC-derived EVs were assessed by chondrogenic and adipogenic differentiation analyses and histological observation. Results: The range of the particle size of adipose-derived stem cells (ADSCs)- and Wharton’s jelly -MSCs-derived EVs were from 130 to 150 nm as measured by NTA, which showed positive expression of exosomal markers. The chondrogenic induction ability was weakened in the absence of EVs in vitro. Interestingly, after EV administration, type II collagen, a major component in the cartilage extracellular matrix, was upregulated compared to the EV-free condition. Moreover, EVs decreased the lipid accumulation rate during adipogenic induction. Conclusion: The results indicated that the production model could facilitate production of effective EVs and further demonstrated the role of MSC-derived EVs in cell differentiation. MSC-derived EVs could be successfully used in cell-free therapy to guide chondrogenic differentiation of ADSC for future clinical applications in cartilage regeneration.

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of β-catenin was reversed by proteasome inhibitor via the β-catenin/ GSK3β signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the β-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.

Cancer stem cells (CSCs) play an important role in cancer treatment resistance and disease progression. Identifying an effective anti‐CSC agent may lead to improved disease control. We used CSC‐associated gene signatures to identify drug candidates that may inhibit CSC growth by reversing the CSC gene signature. Thiostrepton, a natural cyclic oligopeptide antibiotic, was the top‐ranked candidate. In non–small‐cell lung cancer (NSCLC) cells, thiostrepton inhibited CSC growth in vitro and reduced protein expression of cancer stemness markers, including CD133, Nanog and Oct4A. In addition, metastasis‐associated Src tyrosine kinase signalling, cell migration and epithelial‐to‐mesenchymal transition (EMT) were all inhibited by thiostrepton. Mechanistically, thiostrepton treatment led to elevated levels of tumour suppressor miR‐98. Thiostrepton combined with gemcitabine synergistically suppressed NSCLC cell growth and induced apoptosis. The inhibition of NSCLC tumours and CSC growth by thiostrepton was also demonstrated in vivo. Our findings indicate that thiostrepton, an established drug identified in silico, is an inhibitor of CSC growth and a potential enhancer of chemotherapy in NSCLC.

Dysregulation of pericellular proteolysis is often required for tumor invasion and cancer progression. It has been shown that down-regulation of hepatocyte growth factor activator inhibitor-2 (HAI-2) results in activation of matriptase (a membrane-anchored serine protease), human prostate cancer cell motility and tumor growth. In this study, we further characterized if HAI-2 was a cognate inhibitor for matriptase and identified which Kunitz domain of HAI-2 was required for inhibiting matriptase and human prostate cancer cell motility. Our results show that HAI-2 overexpression suppressed matriptase-induced prostate cancer cell motility. We demonstrate that HAI-2 interacts with matriptase on cell surface and inhibits matriptase proteolytic activity. Moreover, cellular HAI-2 harnesses its Kunitz domain 1 (KD1) to inhibit matriptase activation and prostate cancer cell motility although recombinant KD1 and KD2 of HAI-2 both show an inhibitory activity and interaction with matriptase protease domain. The results together indicate that HAI-2 is a cognate inhibitor of matriptase, and KD1 of HAI-2 plays a major role in the inhibition of cellular matritptase activation as well as human prostate cancer invasion.

Low response rate and recurrence are common issues in lung cancer, thus, identifying a potential compound for these patients is essential. Utilizing an in silico screening method, we identified withaferin A (WA), a cell-permeable steroidal lactone initially extracted from Withania somnifera, as a potential anti&ndash, lung cancer and anti&ndash, lung cancer stem-like cell (CSC) agent. First, we demonstrated that WA exhibited potent cytotoxicity in several lung cancer cells, as evidenced by low IC50 values. WA concurrently induced autophagy and apoptosis and the activation of reactive oxygen species (ROS), which plays an upstream role in mediating WA-elicited effects. The increase in p62 indicated that WA may modulate the autophagy flux followed by apoptosis. In vivo research also demonstrated the anti-tumor effect of WA treatment. We subsequently demonstrated that WA could inhibit the growth of lung CSCs, decrease side population cells, and inhibit lung cancer spheroid-forming capacity, at least through downregulation of mTOR/STAT3 signaling. Furthermore, the combination of WA and chemotherapeutic drugs, including cisplatin and pemetrexed, exerted synergistic effects on the inhibition of epidermal growth factor receptor (EGFR) wild-type lung cancer cell viability. In addition, WA can further enhance the cytotoxic effect of cisplatin in lung CSCs. Therefore, WA alone or in combination with standard chemotherapy is a potential treatment option for EGFR wild-type lung cancer and may decrease the occurrence of cisplatin resistance by inhibiting lung CSCs.

Thioridazine (THD) is a common phenothiazine antipsychotic drug reported to suppress growth in several types of cancer cells. We previously showed that THD acts as an antiglioblastoma and anticancer stem-like cell agent. However, the signaling pathway underlying autophagy and apoptosis induction remains unclear. THD treatment significantly induced autophagy with upregulated AMPK activity and engendered cell death with increased sub-G1 in glioblastoma multiform (GBM) cell lines. Notably, through whole gene expression screening with THD treatment, frizzled (Fzd) proteins, a family of G-protein-coupled receptors, were found, suggesting the participation of Wnt/β-catenin signaling. After THD treatment, Fzd-1 and GSK3β-S9 phosphorylation (inactivated form) was reduced to promote β-catenin degradation, which attenuated P62 inhibition. The autophagy marker LC3-II markedly increased when P62 was released from β-catenin inhibition. Additionally, the P62-dependent caspase-8 activation that induced P53-independent apoptosis was confirmed by inhibiting T-cell factor/β-catenin and autophagy flux. Moreover, treatment with THD combined with temozolomide (TMZ) engendered increased LC3-II expression and caspase-3 activity, indicating promising drug synergism. In conclusion, THD induces autophagy in GBM cells by not only upregulating AMPK activity, but also enhancing P62-mediated autophagy and apoptosis through Wnt/β-catenin signaling. Therefore, THD is a potential alternative therapeutic agent for drug repositioning in GBM.

Cancer stem cell (CSC) theory has drawn much attention, with evidence supporting the contribution of stem cells to tumor initiation, relapse, and therapy resistance. In this study, we focus on screening drugs that target CSCs to improve the current treatment outcome and overcome drug resistance in patients with lung cancer. We used publicly available embryonic stem cell and CSC-associated gene signatures to query the Connectivity Map (CMap) for potential drugs that can, at least in part, reverse the gene expression profile of CSCs. High scores were noted for several phenothiazine-like antipsychotic drugs, including trifluoperazine and perphenazine. The combination of gemcitabine with either trifluoperazine or perphenazine shows synergistic effects in anti-NSCLC cells. By using L1000 and CMap analysis, we identified withaferin A (WA) as a potential drug, which shares similar gene expression patterns to trifluoperazine or perphenazine combine with gemcitabine and to reverse differential gene expression of stem-like cell vs. differential cell. Further experiments demonstrated that WA significantly suppressed cancer stemness associated phenotypes, shown by decreasing side population and tumor spheroid formation. Furthermore, WA was accompanied by down-regulation of JAK2/STAT3 signaling pathway. The combination of WA and cisplatin significantly reduced the number of cancer spheroids and attenuated the proportion of ALDH+ cell population in CL97 lung spheroids, which was resistant to cisplatin treatment alone. In vivo study also demonstrated the anti-tumor effect of WA treatment. In conclusion, WA was identified as a potential anti-cancer stem cell inhibitor, which could also inhibit tumorigenesis and overcome cisplatin resistance in lung cancer. Citation Format: Tai-shan Cheng, Yu-Chen Tsai, Alexander T. H. Wu, Peter M. H. Chang, Wei-Hsiang Hsu, Jin-Mei Lai, Chi-Ying Huang. Identification of withaferin A as a novel cancer stem cell inhibitor to overcome drug resistance in non-small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-32.

Ethnopharmacological relevance Sijunzi decoction is a well-known traditional Chinese medicine (TCM) commonly used for invigorating vital energy and for the enhancement of immunity. Modified Sijunzi decoctions have been extensively used to treat cachexia and improve the quality of life of cancer patients undergoing chemotherapy. Aim of the study This study was aimed to provide comprehensive evidence for the anti-cachectic effect of a modified Sijunzi decoction (Zhen-Qi; ZQ-SJZ) and characterize its anti-cachectic mechanism, especially in cisplatin-induced muscle atrophy. Materials and methods We employed a Lewis lung carcinoma (LLC)-induced cancer cachectic mouse model to demonstrate the anti-cachectic effect of ZQ-SJZ. Moreover, we provided an in vitro C2C12 myotube formation model to investigate the effect of ZQ-SJZ in hampering cisplatin-induced muscle atrophy. Results The administration of ZQ-SJZ can recover tumor- and/or cisplatin-induced body weight loss, intestinal mucosal damage, as well as forelimb grip strength and myofiber size. The administration of ZQ-SJZ also significantly prolonged the survival of LLC-induced cachectic mice under cisplatin treatment. Mechanistically, ZQ-SJZ increased the levels of myogenic proteins, such as myosin heavy chain (MyHC) and myogenin, and decreased the atrophy-related protein, atrogin-1, in cisplatin-treated C2C12 myotubes in vitro. In addition, cisplatin-induced mitochondria dysfunction could be hampered by the co-administration of ZQ-SJZ, by which it recovered the cisplatin-mediated decrease in PGC-1α and PKM1 levels. Conclusions The administration of ZQ-SJZ can recover tumor- and/or cisplatin-induced cachectic conditions and significantly prolong the survival of LLC-induced cachectic mice under cisplatin treatment. The profound effect of ZQ-SJZ in hampering tumor- and/or cisplatin-induced cachexia may be due to its modulation of the mitochondrial function and subsequent myogenesis. Taken together, these results demonstrated the anti-cachectic mechanism of ZQ-SJZ and its potential use as a palliative strategy to improve the efficacy of chemotherapy.

Dysregulation of pericellular proteolysis is often required for tumor invasion and cancer progression. It has been shown that down-regulation of hepatocyte growth factor activator inhibitor-2 (HAI-2) results in activation of matriptase (a membrane-anchored serine protease), human prostate cancer cell motility and tumor growth. In this study, we further characterized if HAI-2 was a cognate inhibitor for matriptase and identified which Kunitz domain of HAI-2 was required for inhibiting matriptase and human prostate cancer cell motility. Our results show that HAI-2 overexpression suppressed matriptase-induced prostate cancer cell motility. We demonstrate that HAI-2 interacts with matriptase on cell surface and inhibits matriptase proteolytic activity. Moreover, cellular HAI-2 harnesses its Kunitz domain 1 (KD1) to inhibit matriptase activation and prostate cancer cell motility although recombinant KD1 and KD2 of HAI-2 both show an inhibitory activity and interaction with matriptase protease domain. The results together indicate that HAI-2 is a cognate inhibitor of matriptase, and KD1 of HAI-2 plays a major role in the inhibition of cellular matritptase activation as well as human prostate cancer invasion.

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser115. The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

In this study, a dual-layer of Al/Al2O3 films was deposited on the Mg–10Li–0.5Zn substrate using both techniques of magnetron sputtering and atomic layer deposition (ALD). The pre-sputtered Al interlayer has a crystalline structure and the ALD-Al2O3 film is amorphous. The Al interlayer could effectively obstruct the diffusion out of Li atoms from the Mg–10Li–0.5Zn substrate during the deposition of ALD-Al2O3 film. The Mg–10Li–0.5Zn specimen with a dual-layer of Al/Al2O3 films exhibits a much better corrosion resistance than those specimens with a single layer of sputtered Al or ALD-Al2O3.

Al2O3 films were deposited by the atomic layer deposition (ALD) technique onto pure copper at temperatures in the range 100–200 °C. The chemical composition, microstructure, and mechanic properties of the ALD-deposited Al2O3 films were systematically analyzed. The variations in the film characteristics with substrate temperature were observed. Oxidation trials revealed that 20-nm-thick Al2O3 films deposited at a substrate temperature as low as 100 °C suppress oxidative attack on pure copper. The Al2O3 films also showed excellent durability of adhesion strength, according to predictions using the Coffin–Manson model based on the results of accelerated temperature cycling tests. These features indicate that ALD-deposited Al2O3 film is a very promising candidate to be a protective coating for pure copper.

Ketamine, a dissociative anesthetic, is misused and abused worldwide as an illegal recreational drug. In addition to its neuropathic toxicity, ketamine abuse has numerous effects, including renal failure; however, the underlying mechanism is poorly understood. The process called epithelial phenotypic changes (EPCs) causes the loss of cell-cell adhesion and cell polarity in renal diseases, as well as the acquisition of migratory and invasive properties. Madin-Darby canine kidney cells, an in vitro cell model, were subjected to experimental manipulation to investigate whether ketamine could promote EPCs. Our data showed that ketamine dramatically decreased transepithelial electrical resistance and increased paracellular permeability and junction disruption, which were coupled to decreased levels of apical junctional proteins (ZO-1, occludin, and E-cadherin). Consistent with the downregulation of epithelial markers, the mesenchymal markers N-cadherin, fibronectin, and vimentin were markedly upregulated following ketamine stimulation. Of the E-cadherin repressor complexes tested, the mRNA levels of Snail, Slug, Twist, and ZEB1 were elevated. Moreover, ketamine significantly enhanced migration and invasion. Ketamine-mediated changes were at least partly caused by the inhibition of GSK-3β activity through Ser-9 phosphorylation by the PI3K/Akt pathway. Inhibiting PI3K/Akt with LY294002 reactivated GSK-3β and suppressed ketamine-enhanced permeability, EPCs, and motility. These findings were recapitulated by the inactivation of GSK-3β using the inhibitor 3F8. Taken together, these results provide evidence that ketamine induces renal distal tubular EPCs through the downregulation of several junction proteins, the upregulation of mesenchymal markers, the activation of Akt, and the inactivation of GSK-3β.

The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3beta. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits gamma-tubulin to centrosomal or noncentrosomal hNinein-containing sites, implying that the gamma-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the gamma-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for gamma-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full -length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit gamma-tubulin at centrosomal and noncentrosomal sites.

To explore more hNinein interacting proteins, the yeast two-hybrid screening using ninein C-terminal domain as bait protein was performed. One novel gene, CGI-99, was demonstrated to associate with hNinein in the yeast two-hybrid method and in vitro GST pull-down assay. Molecular characterization also showed that CGI-99 possessed a transcriptional activity at the N-terminal. In addition, CGI-99 formed a dimer with the C-terminal, which overlapped with hNinein binding site. In kinase assay, CGI-99 binds to hNinein and completely blocks the phosphorylation of hNinein by GSK3β. Moreover, CGI-99 was highly expressed in all brain tumors which is in agreement with the Northern blot analysis. Taken together, we have isolated a novel protein CGI-99, which may be involved in the functional regulation of human ninein in the centrosome structure and may also be important in brain development and tumorigenesis.

The human dynamin-like protein, HdynIV, has recently been cloned and shown to be involved in the formation and trafficking of coated vesicles. In particular, one of the HdynIV variant overexpressions has been suggested to contribute to the pathogenesis of brain tumors. In this paper, we report on the genomic organization of the human HdynIV gene. The gene was found to correspond to 20 exons of genomic sequence on human chromosome 12, distributed over 64 kb of genomic DNA. The two exons, numbers 15 and 16, are subjected to differential splicing, generating four different transcripts of a perfect match to our recent report on the four different spliced HdynIV variants [DNA Cell Biol. 19 (2000) 189]. We have also characterized the 5′ regulatory region of the HdynIV gene in order to understand the molecular mechanisms regulating its expression. The transcriptional initiation site was identified by 5′-RACE. The 5′-flanking sequence of the HdynIV gene contains three GC boxes that concatenate Ap2- and Sp1-binding motifs, but that does not contain either the TATA or CAAT consensus sequence. A region between −140 and +92 contributed to high promoter activity. Deletion analysis demonstrated that the minimal promoter activity required the region of −110 to −100. Electrophoretic mobility shift assay demonstrated that a putative transcriptional factor bound to the region of −119 to −90. Site-directed mutagenesis analysis of this region revealed that nucleotides at −108 to −100 were essential for transactivation mediated by this transcriptional factor. In conclusion, we have characterized the minimal HdynIV promoter and shown that CTCCCAGCA (−108 to −100) sequence may act as a novel transcriptional element for regulating HdynIV gene expression.

The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. The major need now is to explore this disparate data to find biologically relevant interactions and pathways. Thus, in the post-genomic era, there is clearly a need for the development of algorithms that can accurately predict novel protein-protein interaction networks in silico. The evolutionarily conserved Aurora family kinases have been chosen as a model for the development of a method to identify novel biological networks by a comparison of human and various model organisms. Our search methodology was designed to predict and prioritize molecular targets for Aurora family kinases, so that only the most promising are subjected to empirical testing. Four potential Aurora substrates and/or interacting proteins, TACC3, survivin, Hec1, and hsNuf2, were identified and empirically validated. Together, these results justify the timely implementation of in silico biology in routine wet-lab studies and have also allowed the application of a new approach to the elucidation of protein function in the post-genomic era.

SH3 domains are found in many signal transduction proteins where they mediate protein-protein binding by recognizing specific peptides rich in proline. Based on the analysis of sequence alignment data, the NADPH oxidase p67(phox) C-terminal SH3 domain possesses a typical compact beta-barrel consisting of five beta-strands arranged in two antiparallel beta-sheets of three and two beta-strands. Multiple amino acid substitutions were made at beta e and its flanking residues to determine the role of the boundary sequences in binding activity and conformational specificity of the domain. Analysis of amino acid P55 indicated that all mutants were completely abolished in their binding activities. The substitution of F58 with Y58 showed no effect of the binding, whereas substitution with stop codon abolished activity. Furthermore, when amino acid V59 was substituted with stop codon, activity was also completely abolished. Substitution of E60 with stop codon showed no effect of binding. Moreover, our data show that V59 particularly could not be replaced by Leu. Taken together, these data suggest that V59 may not only contribute the exact boundary site but also play on the specificity for protein-protein interactions in phagocyte NADPH oxidase.

Dysregulation of cell surface proteolysis has been strongly implicated in tumorigenicity and metastasis. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer (PCa) cell migration, invasion, tumorigenicity and metastasis using a human PCa progression model (103E, N1, and N2 cells) and xenograft models. N1 and N2 cells were established through serial intraprostatic propagation of 103E human PCa cells and isolation of the metastatic cells from nearby lymph nodes. The invasion capability of these cells was revealed to gradually increase throughout the serial isolations (103E

Curcumin has been shown to possess potent chemopreventive and antitumor effects on prostate cancer. However, the molecular mechanism involved in curcumin's ability to suppress prostate cancer cell invasion, tumor growth, and metastasis is not yet well understood. In this study, we have shown that curcumin can suppress epidermal growth factor (EGF)- stimulated and heregulin-stimulated PC-3 cell invasion, as well as androgen-induced LNCaP cell invasion. Curcumin treatment significantly resulted in reduced matrix metalloproteinase 9 activity and downregulation of cellular matriptase, a membrane-anchored serine protease with oncogenic roles in tumor formation and invasion. Our data further show that curcumin is able to inhibit the induction effects of androgens and EGF on matriptase activation, as well as to reduce the activated levels of matriptase after its overexpression, thus suggesting that curcumin may interrupt diverse signal pathways to block the protease. Furthermore, the reduction of activated matriptase in cells by curcumin was also partly due to curcumin's effect on promoting the shedding of matriptase into an extracellular environment, but not via altering matriptase gene expression. In addition, curcumin significantly suppressed the invasive ability of prostate cancer cells induced by matriptase overexpression. In xenograft model, curcumin not only inhibits prostate cancer tumor growth and metastasis but also downregulates matriptase activity in vivo. Overall, the data indicate that curcumin exhibits a suppressive effect on prostate cancer cell invasion, tumor growth, and metastasis, at least in part via downregulating matriptase function. Cancer Prev Res; 6(5); 495–505. ©2013 AACR.

Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human prostate cancer (PCa) progression. In this communication, we investigated the roles of both ErbB-2 signaling in matriptase zymogen activation and matriptase in ErbB-2-induced PCa malignancy. In a human PCa cell progression model, we observed that advanced PCa C-81 LNCaP cells exhibited an aggressive phenotype with increased cell migration and invasion capacity; these cells concurrently showed both enhanced ErbB-2 phosphorylation and increased matriptase zymogen activation compared with parental C-33 LNCaP cells. Moreover, ErbB2 activation, both ligand-dependent (eg, epidermal growth factor treatment) and ligand-independent (eg, overexpression), was able to induce matriptase zymogen activation in this cell line. Inhibition of ErbB-2 activity by either the specific inhibitor, AG825, in epidermal growth factor-treated C-33 LNCaP cells or ErbB-2 knockdown in C-81 LNCaP cells, reduced matriptase activation. These observations were confirmed by similar studies using both DU145 and PC3 cells. Together, these data suggest that ErbB-2 signaling plays an important role in matriptase zymogen activation. ErbB-2-enhanced matriptase activation was suppressed by a phosphatidylinositol 3-kinase inhibitor (ie, LY294002) but not by a MEK inhibitor (ie, PD98059). Suppression of matriptase expression by small hairpin RNA knockdown in ErbB-2-overexpressing LNCaP cells dramatically suppressed cancer cell invasion. In summary, our data indicate that ErbB-2 signaling via the phosphatidylinositol 3-kinase pathway results in up-regulated matriptase zymogen activity, which contributes to PCa cell invasion.

Aurora-A is involved in chromosome alignment, centrosome maturation, mitotic spindle assembly and regards to an oncogene. Aurora-A is also known to bind to several other proteins affecting its up-regulation or down-regulation and localization. However, how these different binding signals work together to regulate Aurora-A is not properly known. To explore more Aurora-A interacting proteins, the low-copy yeast two-hybrid screening using Aurora-A as bait protein was performed. One novel gene, AIBp, was demonstrated to associate with Aurora-A by the yeast two-hybrid method and in vitro GST pull-down assay. Molecular characterization showed that AIBp possessed a binding site at the C-terminal with Aurora-A (kinase domain). Interestingly, AIBp also interacts with hNinein at the N-terminal, which overlaps with a previously reported hNinein and GSK3beta binding site. Using a kinase assay, AIBp interacts with the Aurora-A kinase domain functions as a positive regulator, whereas AIBp binding to hNinein appears to block the phosphorylation of hNinein by both Aurora-A and GSK3beta. siRNA-mediated elimination of AIBp from HeLa cells, results in a doughnut-like shape, asymmetrical spindle pole and multiple spindle pole formation. We also demonstrated that both AIBp and Aurora-A are co-overexpressed in various brain tumors. These studies demonstrate that AIBp may not only be required for the dynamic movement of Aurora-A at the centrosomes and spindle apparatus during the cell cycle, but may also be important during brain tumorigenesis.

Hepatitis delta antigen (HDAg) is a nuclear protein that is intimately involved in hepatitis delta virus (HDV) RNA replication. HDAg consists of two protein species, the small form (S-HDAg) and the large form (L-HDAg). Previous studies have shown that posttranslational modifications of S-HDAg, such as phosphorylation, acetylation, and methylation, can modulate HDV RNA replication. In this study, we show that S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein. Mapping data showed that multiple lysine residues are SUMO1 acceptors within S-HDAg. Using a genetic fusion strategy, we found that conjugation of SUMO1 to S-HDAg selectively enhanced HDV genomic RNA and mRNA synthesis but not antigenomic RNA synthesis. This result supports our previous proposition that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that for genomic RNA synthesis and mRNA transcription, requiring different modified forms of S-HDAg. Sumoylation represents a new type of modification for HDAg.

BACKGROUND: Lung adenocarcinoma is the leading cause of cancer-related deaths among both men and women in the world. Despite recent advances in diagnosis and treatment, the mortality rates with an overall 5-year survival of only 15%. This high mortality is probably attributable to early metastasis. Although several well-known markers correlated with poor/metastasis prognosis in lung adenocarcinoma patients by immunohistochemistry was reported, the molecular mechanisms of lung adenocarcinoma development are still not clear. To explore novel molecular markers and their signaling pathways will be crucial for aiding in treatment of lung adenocarcinoma patients. METHODOLOGY/PRINCIPAL FINDINGS: To identify novel lung adenocarcinoma-associated /metastasis genes and to clarify the underlying molecular mechanisms of these targets in lung cancer progression, we created a bioinformatics scheme consisting of integrating three gene expression profile datasets, including pairwise lung adenocarcinoma, secondary metastatic tumors vs. benign tumors, and a series of invasive cell lines. Among the novel targets identified, FLJ10540 was overexpressed in lung cancer tissues and is associated with cell migration and invasion. Furthermore, we employed two co-expression strategies to identify in which pathway FLJ10540 was involved. Lung adenocarcinoma array profiles and tissue microarray IHC staining data showed that FLJ10540 and VEGF-A, as well as FLJ10540 and phospho-AKT exhibit positive correlations, respectively. Stimulation of lung cancer cells with VEGF-A results in an increase in FLJ10540 protein expression and enhances complex formation with PI3K. Treatment with VEGFR2 and PI3K inhibitors affects cell migration and invasion by activating the PI3K/AKT pathway. Moreover, knockdown of FLJ10540 destabilizes formation of the P110-alpha/P85-alpha-(PI3K) complex, further supporting the participation of FLJ10540 in the VEGF-A/PI3K/AKT pathway. CONCLUSIONS/SIGNIFICANCE: This finding set the stage for further testing of FLJ10540 as a new therapeutic target for treating lung cancer and may contribute to the development of new therapeutic strategies that are able to block the PI3K/AKT pathway in lung cancer cells.

Emerging evidence shows that glycogen synthase kinase 3beta (GSK3beta) is involved in mitotic division and that inhibiting of GSK3beta kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3beta involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3beta interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3beta and is phosphorylated at Thr-111, Thr-937 ((S/T)P motif) and Ser-974/Thr-978 ((S/T)XXX(S/T)-p motif; p is a phosphorylatable residue). Inhibition of GSK3beta impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3beta. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3beta. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3beta-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3beta differentially increases GSK3beta-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3beta interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3beta-Astrin complex may also facilitate further physiological and pathological phosphorylation.

Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus, an interaction between arsenic and cigarette smoking in lung carcinogenesis was suspected. p53 dysfunction or mutation in lung epithelial cells was frequently observed in cigarette smokers. Our present study was to explore the differential effects by arsenic on H1355 cells (human lung adenocarcinoma cell line with mutation in p53), BEAS-2B (immortalized lung epithelial cell with functional p53) and pifithrin-alpha-treated BEAS-2B cells (p53-inhibited cells). These cells were treated with different doses of sodium arsenite (0, 0.1, 1, 5 and 10 microM) for 48 h. A greater reduction in cell viability was observed in the BEAS-2B cells vs. p53 compromised cells (H1355 or p53-inhibited BEAS-2B). Similar observation was also made on 7-day cell survival (growth) study. TUNEL analysis confirmed that there was indeed a significantly reduced arsenite-induced apoptosis found in p53-compromised cells. Centrosomal abnormality has been attributed to eventual chromosomal missegregation, aneuploidy and tumorigenesis. In our present study, reduced p21 and Gadd45a expressions and increased centrosomal abnormality (atopic and multiple centrosomes) were observed in both arsenite-treated H1355 and p53-inhibited BEAS-2B cells as compared with similarly treated BEAS-2B cells. Increased anchorage-independent growth (colony formation) of BEAS-2B cells co-treated with pifithrin-alpha and 5 microM sodium arsenite was also observed in soft agar. Our present investigation demonstrated that arsenic would act specifically on p53 compromised cells (either with p53 dysfunction or inhibited) to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenic, especially under the condition of p53 dysfunction.

Human Ninein (hNinein) is implicated in centrosomal microtubule nucleation and microtubule anchoring in interphase cells and may act as a scaffold protein, but its direct interaction partners remain unexplored in the centrosome. In this report, we show clearly that a spindle-associated protein, Astrin, interacts and co-localizes with hNinein at the centrosome during the S and G2 phases, and this complex may dissociate in the M phase. We also demonstrate that the truncated forms of hNinein, which could interfere with gamma-tubulin and function as dominant-negative mutants, are able to affect Astrin localization to the centrosome. Moreover, siRNA-mediated knockdown of hNinein in HeLa cells causes Astrin to fail to target to the centrosome, whereas hNinein can localize at the centrosome in the absence of Astrin. In addition, reduction in hNinein protein levels causes mislocalization of Astrin with the spindle apparatus and results in the formation of an aberrant mitotic spindle. Collectively, these data suggest that hNinein is required for targeting Astrin to the centrosome during the S and G2 phases. We therefore propose a model wherein hNinein regulates the dynamic movement of Astrin throughout the cell cycle and this interaction, in turn, is required for maintenance of centrosome/spindle pole integrity.

Although prominent FRAT/GBP exhibits a limited degree of homology to Axin, the binding sites on GSK3 for FRAT/GBP and Axin may overlap to prevent the effect of FRAT/GBP in stabilizing beta-catenin in the Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein, GSK3beta interaction protein (GSKIP), which binds to GSK3beta. We have defined a 25-amino acid region in the C-terminus of GSKIP that is highly similar to the GSK3beta interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that GSKIP is a good GSK3beta substrate, and both the full-length protein and a C-terminal fragment of GSKIP can block phosphorylation of primed and nonprimed substrates in different fashions. Similar to Axin GID(381-405) and FRATtide, synthesized GSKIPtide is also shown to compete with and/or block the phosphorylation of Axin and beta-catenin by GSK3beta. Furthermore, our data indicate that overexpression of GSKIP induces beta-catenin accumulation in the cytoplasm and nucleus as visualized by immunofluorescence. A functional assay also demonstrates that GSKIP-transfected cells have a significant effect on the transactivity of Tcf-4. Collectively, we define GSKIP as a naturally occurring protein that is homologous with the GSK3beta interaction domain of Axin and is able to negatively regulate GSK3beta of the Wnt signaling pathway.

A centrosomal-associated protein, ninein is a microtubules minus end capping, centrosome position, and anchoring protein, but the underlying structure and physiological functions are still unknown. To identify the molecules that regulate the function of human ninein in centrosome, we performed yeast two-hybrid screen and isolated the SUMO-conjugating E2 enzyme, Ubc9, and SUMOylation enhancing enzymes, including PIAS1 and PIASxalpha, as binding partners of hNinein. These interactions as well as the interaction between hNinein and SUMO-1 are also confirmed by a glutathione S-transferase (GST) pull-down experiment. Furthermore, the C-terminal region of hNinein can be SUMOylated in vitro and in HeLa cells transfected with a plasmid expressing GFP-hNinein. Our findings firstly place SUMOylation target on the centrosome structure protein, hNinein, which results in the switch localization from centrosome to nucleus, suggesting the importance of the SUMOylation of hNinein and probably other centrosomal proteins may also be involved in the centrosome activity.

To explore more possible roles for GSK3beta function, the yeast two-hybrid screening using GSK3beta as a bait protein was performed. In this study, we demonstrated that two variants of CABYR (281 and 379) interacted with GSK3beta in the yeast two-hybrid and GST pull down assay. Molecular characterization showed that CABYR variants formed a dimer with a proline-rich extensin-like domain, which slightly overlapped with GSK3beta-binding site. In kinase assay, we also showed that CABYR variants act as an ideal substrate for GSK3beta within the extensin-like domain and phosphorylation sites on CABYR were mapped. Interestingly, Northern blot showed that CABYR transcripts were expressed more distinctly in the fetal brain than in the adult brain, suggesting that this protein may play a role during brain development. Moreover, differential expression of CABYR variants may exhibit aberrant expression in brain tumors and cancer cell lines.

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes and the lytic cycle. This work identifies Ubc9 and PIAS1 as binding partners of Rta in a yeast two-hybrid screen. These bindings are verified by glutathione S-transferase pull-down assay, coimmunoprecipitation, and confocal microscopy. The interactions appear to cause Rta sumoylation, because not only can Rta be sumoylated in vitro but also sumoylated Rta can be detected in P3HR1 cells following lytic induction and in 293T cells after transfecting plasmids that express Rta and SUMO-1. Moreover, PIAS1 stimulates conjugation of SUMO-1 to Rta, thus acting as an E3 ligase. Furthermore, transfecting plasmids that express Ubc9, PIAS1, and SUMO-1 increases the capacity of Rta to transactivate the promoter that includes an Rta response element, indicating that the modification by SUMO-1 increases the transactivation activity of Rta. This study reveals that Rta is sumoylated at the Lys-19, Lys-213, and Lys-517 residues and that SUMO-1 conjugation at the Lys-19 residue is crucial for enhancing the transactivation activity of Rta. These results indicate that sumoylation of Rta may be important in EBV lytic activation.

Rac3 may play an important role in tumor growth but little is known about its expression and mutation in human tumor tissues. We examined the expression of Rac3 using RT-PCR and mutation of the Rac3 gene by DNA sequencing. Overexpression of the Rac3 gene occurred in 19% (5/26) of brain tumors; 3 of 9 (33%) meningiomas, 1 of 11 (9%) astrocytomas and 1 of 6 (17%) pituitary adenomas. Two of the 3 meningiomas with Rac3 overexpression were recurrent meningiomas. The only astrocytoma with Rac3 overexpression was a glioblastoma multiforme. Mutation of the Rac3 gene occurred in 63% (12/19) of brain tumours; 4 of 7 (57.1%) meningiomas, 4 of 5 (80%) pituitary adenomas and 4 of 7 (57.1%) astrocytomas. Except in one astrocytoma, the other four tumors with Rac3 overexpression (3 meningiomas and one pituitary adenoma) did not have Rac3 mutations. Our data is the first report of the frequency of Rac3 overexpression and mutation in human brain tumors. Overexpression may be associated with aggressive tumor behavior. The relationship between Rac3 expression and mutation requires further investigation.

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303–2096) co-localized with γ-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783–1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.

Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. E-cadherin was discovered as a protein associated with beta-catenin which plays a crucial role in cell-cell adhesion. To further understand the molecular basis of Wnt signaling pathway and E-cadherin in brain tumorigenesis, the expression of four Wnt genes (Wnt1, Wnt5a, Wnt10b and Wnt13) and E-cadherin were analyzed by reverse transcriptase-polymerase chain reaction. In addition, their downstream effector, beta-catenin, was also investigated. The results showed that the expression of Wnt5a (41/45), Wnt10b (37/45), and Wnt13 (35/45) were found in brain tumors, whereas Wnt1 (6/45) was shown to be less related. Interestingly, E-cadherin was only expressed in a few cases of astrocytoma (2/16), whereas it was expressed in most meningioma (14/15) and pituitary adenoma tumors (12/14). There was no apparent difference of beta-catenin expression profile in brain tumors; however, the sequencing data of beta-catenin showed two mutations on speculative phosphorylation sites, S73F and S23G in astrocytoma. Furthermore, an in vitro functional assay showed that S73F and S23G mutants of beta-catenin did not affect transcriptional activity in TCF-4-leuciferase reporter construct, suggesting that they may need more complex factors to participate in astrocytoma. Taken together, our data suggest that the mutations of beta-catenin together with E-cadherin and Wnt signaling might be involved in brain tumorigenesis.

Curcumin has been shown with potent chemopreventive and antitumor effects on prostate cancer. However, the molecular mechanism how curcumin suppresses prostate cancer cell migration and invasion is not well understood. In this study we showed that curcumin could significantly suppress prostate cancer PC3 cell migration and invasion. Moreover, our data also showed that curcumin was able to suppress EGF- or heregulin-stimulated PC-3 cell invasion, as well as androgen-induced LNCaP cell invasion. Curcumin treatment resulted in not only significant reductions on MMP-9 and MMP-2 activities, but also on activated level of matriptase which is a membrane-bound serine protease and has been proposed to play important roles in tumor invasion and malignancy. Matriptase is synthesized as a full length zymogen, which is rapidly stepped into the maturation process, mature form through a cleavage within the SEA domain to from a latent matriptase. Our data further showed that curcumin can inhibit the latent matriptase formation in the maturation process to affect matriptase activity. Furthermore, the reduction of activated matriptase by curcumin was also partly due to curcumin promoting the shedding of activated matriptase into extracellular environment, but not due to curcumin effect on matriptase gene expression. In addition, we established the stable pools of CWR22Rv1 cells with matriptase overexpression and found that expression of matriptase in CWR22Rv1 cells enhanced their invasive ability. With curcumin treatment, the invasive ability induced by matriptase overexpression was significantly suppressed. In summary, the data indicated that curcumin exhibits a suppressive effect on prostate cancer cell invasion, at least in part by down-regulating matriptase activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2000. doi:1538-7445.AM2012-2000

Dysregulation of pericellular proteolysis has been strongly involved in tumorigenesis. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer cell invasion and tumorigenicity within a human prostate cancer progression model (103E, N1, and N2 cells). This model was newly established by a serial of intraprostatic propagation of a human prostate cancer 103E cell line and isolation of the metastatic cells from nearby lymph nodes. The invasion capability of these cells was gradually increased after the serial isolations (103E Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A54.

Dysregulation of proteolysis on cell surface has been strongly implicated in cancer cell migration and invasion. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer cell migration and invasion within a human prostate cancer progression model, established by a serial of intraprostatic injections of prostate cancer cells and isolation of the invasive cells from nearby lymph nodes. The invasion capability of these cells was increased after the serial isolations. Interestingly, the expression of HAI-2 but not HAI-1 was dramatically decreased following the progression and concurrent with an increase of activated matriptase. Overexpression of HAI-2 reduced prostate cancer cell migration and invasion, at least in part due to its inhibitory role in matriptase. Following the orthotopic tumor growth in mice, the level of matriptase was increased while HAI-2 protein level was decreased. These results indicated that during the progression of human prostate cancer, HAI-2 expression was reduced, leading to constitutive matriptase activation, increased cell migration and invasion. Thus, HAI-2 exerted an inhibitory role in prostate cancer progression partly through inhibiting matripase activity. Imbalance between HAI-2 and matriptase may result in prostate tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2011-1413