Antibody specificity against highly conserved membrane protein Claudin 6 driven by single atomic contact point.

The tight junction protein claudin 6 (CLDN6) is differentially expressed on cancer cells with almost no expression in healthy tissue. However, achieving therapeutic MAb specificity for this 4 transmembrane protein is challenging because it is nearly identical to the widely expressed CLDN9, with only 3 extracellular amino acids different. Most other CLDN6 MAbs, including those in clinical development are cross-reactive with CLDN9, and several trials have now been stopped. Here we isolated rare MAbs that bind CLDN6 with up to picomolar affinity and display minimal cross-reactivity with CLDN9, 22 other CLDN family members, or across the human membrane proteome. Amino acid-level epitope mapping distinguished the binding sites of our MAbs from existing clinical-stage MAbs. Atomic-level epitope mapping identified the structural mechanism by which our MAbs differentiate CLDN6 and CLDN9 through steric hindrance at a single molecular contact point, the γ carbon on CLDN6 residue Q156.

Antigenicity, stability, and reproducibility of Zika reporter virus particles for long-term applications.

The development of vaccines against flaviviruses, including Zika virus (ZIKV) and dengue virus (DENV), continues to be a major challenge, hindered by the lack of efficient and reliable methods for screening neutralizing activity of sera or antibodies. To address this need, we previously developed a plasmid-based, replication-incompetent DENV reporter virus particle (RVP) production system as an efficient and safe alternative to the Plaque Reduction Neutralization Test (PRNT). As part of the response to the 2015-2016 ZIKV outbreak, we developed pseudo-infectious ZIKV RVPs by modifying our DENV RVP system. The use of ZIKV RVPs as critical reagents in human clinical trials requires their further validation using stability and reproducibility metrics for large-scale applications. In the current study, we validated ZIKV RVPs using infectivity, neutralization, and enhancement assays with monoclonal antibodies (MAbs) and human ZIKV-positive patient serum. ZIKV RVPs are antigenically equivalent to live virus based on binding ELISA and neutralization results and are nonreplicating based on the results of live virus replication assays. We demonstrate reproducible neutralization titer data (NT50 values) across different RVP production lots, volumes, time frames, and laboratories. We also show RVP stability across experimentally relevant time intervals and temperatures. Our results demonstrate that ZIKV RVPs provide a safe, high-throughput, and reproducible reagent for large-scale, long-term studies of neutralizing antibodies and sera, which can facilitate large-scale screening and epidemiological studies to help expedite ZIKV vaccine development.

Snorkel: an epitope tagging system for measuring the surface expression of membrane proteins.

Tags are widely used to monitor a protein's expression level, interactions, protein trafficking, and localization. Membrane proteins are often tagged in their extracellular domains to allow discrimination between protein in the plasma membrane from that in internal pools. Multipass membrane proteins offer special challenges for inserting a tag since the extracellular regions are often composed of small loops and thus inserting an epitope tag risks perturbing the structure, function, or location of the membrane protein. We have developed a novel tagging system called snorkel where a transmembrane domain followed by a tag is appended to the cytoplasmic C-terminus of the membrane protein. In this way the tag is displayed extracellularly, but structurally separate from the membrane protein. We have tested the snorkel tag system on a diverse panel of membrane proteins including GPCRs and ion channels and demonstrated that it reliably allows for monitoring of the surface expression.

KISS1 over-expression suppresses metastasis of pancreatic adenocarcinoma in a xenograft mouse model.

Identifying molecular targets for treatment of pancreatic cancer metastasis is critical due to the high frequency of dissemination prior to diagnosis of this lethal disease. Because the KISS1 metastasis suppressor is expressed at reduced levels in advanced pancreatic cancer, we hypothesized that re-expression of KISS1 would reduce metastases. Highly metastatic S2VP10 cells expressing luciferase (S2VP10L) were transfected with a FLAG-tagged version of KISS1 (KFM), KFMΔSS (with deleted secretion signal sequence), or pcDNA3 control plasmid (CP) and expression was confirmed by RTQ-PCR. SCID mice were implanted orthotopically with S2VP10L cells or transfectants and tumor growth and metastases were monitored using bioluminescence imaging. Mice with S2VP10L-KISS1 tumors developed fewer liver (98%) and lung (99%) metastases than S2VP10L. Unexpectedly, mice with S2VP10L-KFMΔSS tumors also had reduced liver and lung metastases, but had more metastases than mice with S2VP10L-KISS. KISS1 protein was found in the cytoplasm of both KFMΔSS and KISS1-expressing orthotopic tumors by immunohistochemistry. Metastases were not found in lungs of mice with S2VP10L-KISS1 tumors; whereas, KFMΔSS lung sections had regions of concentrated KISS1 staining, suggesting that secretion of KISS1 is needed to reduce metastasis significantly. These data suggest induction of KISS1 expression has potential as an adjuvant treatment for pancreatic cancer.

Breast cancer metastasis suppressor 1 coordinately regulates metastasis-associated microRNA expression.

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple tumor types without blocking tumorigenesis. BRMS1 forms complexes with SIN3, histone deacetylases and selected transcription factors that modify metastasis-associated gene expression (e.g., EGFR, OPN, PI4P5K1A, PLAU). microRNA (miRNA) are a recently discovered class of regulatory, noncoding RNA, some of which are involved in neoplastic progression. Based on these data, we hypothesized that BRMS1 may also exert some of its antimetastatic effects by regulating miRNA expression. MicroRNA arrays were done comparing small RNAs that were purified from metastatic MDA-MB-231 and MDA-MB-435 and their nonmetastatic BRMS1-transfected counterparts. miRNA expression changed by BRMS1 were validated using SYBR Green RT-PCR. BRMS1 decreased metastasis-promoting (miR-10b, -373 and -520c) miRNA, with corresponding reduction of their downstream targets (e.g., RhoC which is downstream of miR-10b). Concurrently, BRMS1 increased expression of metastasis suppressing miRNA (miR-146a, -146b and -335). Collectively, these data show that BRMS1 coordinately regulates expression of multiple metastasis-associated miRNA and suggests that recruitment of BRMS1-containing SIN3:HDAC complexes to, as yet undefined, miRNA promoters might be involved in the regulation of cancer metastasis.

KiSS1 suppresses TNFalpha-induced breast cancer cell invasion via an inhibition of RhoA-mediated NF-kappaB activation.

Tumor necrosis factor-alpha (TNFalpha) induces cancer development and metastasis, which is prominently achieved by nuclear factor-kappa B (NF-kappaB) activation. TNFalpha-induced NF-kappaB activation enhances cellular mechanisms including proliferation, migration, and invasion. KiSS1, a key regulator of puberty, was initially discovered as a tumor metastasis suppressor. The expression of KiSS1 was lost or down-regulated in different metastatic tumors. However, it is unclear whether KiSS1 regulates TNFalpha-induced NF-kappaB activation and further tumor cell migration. In this study, we demonstrate that KiSS1 suppresses the migration of breast cancer cells by inhibiting TNFalpha-induced NF-kappaB pathway and RhoA activation. Both KiSS1 overexpression and KP10 (kisspeptin-10) stimulation inhibited TNFalpha-induced NF-kappaB activity, suppressed TNFalpha-induced cell migration and cell attachment to fibronectin in breast cancer cells while KP10 has little effect on cancer cell proliferation. Furthermore, KP10 inhibited TNFalpha-induced cell migration and RhoA GTPase activation. Therefore, our data demonstrate that KiSS1 inhibits TNFalpha-induced NF-kappaB activation via downregulation of RhoA activation and suppression of breast cancer cell migration and invasion.

Morelloflavone blocks injury-induced neointimal formation by inhibiting vascular smooth muscle cell migration.

BACKGROUND: In-stent restenosis, or renarrowing within a coronary stent, is the most ominous complication of percutaneous coronary intervention, caused by vascular smooth muscle cell (VSMC) migration into and proliferation in the intima. Although drug-eluting stents reduce restenosis, they delay the tissue healing of the injured arteries. No promising alternative anti-restenosis treatments are currently on the horizon. METHODS: In endothelium-denudated mouse carotid arteries, oral morelloflavone-an active ingredient of the Thai medicinal plant Garcinia dulcis-significantly decreased the degree of neointimal hyperplasia, without affecting neointimal cell cycle progression or apoptosis as evaluated by Ki-67 and TUNEL staining, respectively. At the cellular level, morelloflavone robustly inhibited VSMC migration as shown by both scratch wound and invasion assays. In addition, morelloflavone prevented VSMCs from forming lamellipodia, a VSMC migration apparatus. Mechanistically, the inhibition by morelloflavone of VSMC migration was through its negative regulatory effects on several migration-related kinases, including FAK, Src, ERK, and RhoA. Consistently with the animal data, morelloflavone did not affect VSMC cell cycle progression or induce apoptosis. RESULTS: These data suggest that morelloflavone blocks injury-induced neointimal hyperplasia via the inhibition of VSMC migration, without inducing apoptosis or cell cycle arrest. GENERAL SIGNIFICANCE: We propose morelloflavone to be a viable oral agent for the prevention of restenosis, without compromising effects on the integrity and healing of the injured arteries.

Breast cancer metastasis suppressor-1 differentially modulates growth factor signaling.

That metastatic tumor cells grow in selective non-native environments suggests an ability to differentially respond to local microenvironments. BRMS1, like other metastasis suppressors, halts ectopic growth (metastasis) without blocking orthotopic tumor formation. BRMS1-expressing tumor cells reach secondary sites but do not colonize distant tissues, compelling the hypothesis that BRMS1 selectively restricts the ability of tumor cells to respond to exogenous regulators in different tissues. Here we report that BRMS1 expression in metastatic human breast cancer cells leads to a selective reduction in epidermal growth factor receptor expression and downstream (AKT) signaling. Signaling through another receptor tyrosine kinase, hepatocyte growth factor receptor (c-Met), remains unaltered despite reduced levels of the signaling intermediate phosphatidylinositol (4,5)-bisphosphate. Interestingly, reduced downstream calcium signaling is observed following treatment with platelet-derived growth factor, consistent with decreased phosphatidylinositol (4,5)-bisphosphate. However, platelet-derived growth factor receptor expression is unaltered. Thus, BRMS1 differentially attenuates cellular responses to mitogenic signals, not only dependent upon the specific signal received, but at varying steps within the same signaling cascade. Specific modulation of signaling responses received from the microenvironment may ultimately dictate which environments are permissive/restrictive for tumor cell growth and provide insights into the biology underlying metastasis.

Metastasis suppressors genes in cancer.

The major problem for cancer patients is metastasis of the cancer from the primary tumor to secondary sites. Metastasis is the process by which tumor cells disseminate from the primary tumor, migrate through the basement membrane, survive in the circulatory system, invade into a secondary site, and start to proliferate. In the past, research had concentrated on the biology, taking more of a global view instead of a molecular view. More recently, the focus has been determining the molecular underpinnings, looking at genes that induce or inhibit metastasis. Metastasis suppressors, by definition, inhibit metastasis at any step of the metastatic cascade without blocking primary tumor growth. The expanding list of metastasis suppressors exist with every cellular compartment and have been shown to work by regulating signaling pathways that inhibit proliferation, cell migration and growth at the secondary site. Still, the biochemical basis of their inhibition is not completely known. Here we review the known metastasis suppressors and summarize the suspected mechanisms by which they inhibit metastasis.

GCIP/CCNDBP1, a helix-loop-helix protein, suppresses tumorigenesis.

Deletions and/or loss of heterozygosity (LOH) on chromosome 15 (15q15 and 15q21) have been found in several human tumors, including carcinomas of the colorectum, breast, lung, prostate, and bladder, suggesting the presence of potential tumor suppressor gene(s) in this particular region of chromosome 15. GCIP also called CCNDBP1, DIP1, or HHM, localized at chromosome 15q15, is a recently identified helix-loop-helix leucine zipper (HLH-ZIP) protein without a basic region like the Id family of proteins. In this study, we reported that the expression of GCIP was significantly downregulated in several different human tumors, including breast tumor, prostate tumor, and colon tumors. In human colon tumors, both mRNA and protein expression levels of GCIP were decreased significantly compared to the normal tissues. Treatment of colon cancer cells SW480 with sodium butyrate (NaB), which induces colon cancer cell differentiation, can induce the upregulation of GCIP expression, suggesting that the protein functions as a negative regulator in cell proliferation. Overexpression of GCIP in SW480 colon cancer cell line resulted in a significant inhibition on tumor cell colony formation, while silencing of GCIP expression by siRNA can promote cell colony formation. Furthermore, overexpression of GCIP inhibited the transcriptional activity of cyclin D1 promoter and the expression of cyclin D1 protein in the cell. Finally, we demonstrate that GCIP specifically interacts with one of the class III HDAC proteins, SirT6, which is important for maintaining genome stability. Together, our data suggest a possible function of GCIP in tumor suppression.

Regulation of KiSS-1 metastasis suppressor gene expression in breast cancer cells by direct interaction of transcription factors activator protein-2alpha and specificity protein-1.

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2alpha (AP-2alpha) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2alpha binding elements, AP-2alpha-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2alpha into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2alpha wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2alpha regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2alpha directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2alpha and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2alpha in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.

Modulation of muscle regeneration, myogenesis, and adipogenesis by the Rho family guanine nucleotide exchange factor GEFT.

Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases. However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage. In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo. In order to molecularly characterize this regenerative effect, we extrapolate the mechanism of action by examining the consequence of GEFT expression in multipotent cell lines capable of differentiating into a number of cell types, including muscle and adipocyte lineages. Our data demonstrate that endogenous GEFT is transcriptionally upregulated during myogenic differentiation and downregulated during adipogenic differentiation. Exogenous expression of GEFT promotes myogenesis of C2C12 cells via activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, while a dominant-negative mutant of GEFT inhibits this process. Moreover, we show that GEFT inhibits insulin-induced adipogenesis in 3T3L1 preadipocytes. In summary, we provide the first evidence that the Rho family signaling pathways act as potential regulators of skeletal muscle regeneration and provide the first reported molecular mechanism illustrating how a mammalian Rho family GEF controls this process by modulating mesenchymal cell fate decisions.

G-protein-activated phospholipase C-beta, new partners for cell polarity proteins Par3 and Par6.

Cell polarity and asymmetric cell division are fundamental traits of all living cells and play an essential role in embryonic development, neuronal cell chirality formation, and maintenance of mammalian epithelial cell morphology. Heterotrimeric GTP-binding proteins (G proteins) are involved in directing cell polarity and asymmetric cell division in different organisms. However, the mechanism for G-protein-mediated cell polarity and asymmetric cell division is poorly understood. In this study, we have demonstrated that G-protein-activated phospholipase C-beta (PLC-beta) interacts with cell polarity proteins Par3 and Par6 (Par: partition-defective) to form protein complexes and to mediate downstream signal transduction. The interactions between PLC-beta and Par proteins are direct and require the extreme C-terminal-specific sequence motifs of PLC-beta and the PDZ (PSD95/Dlg/ZO-1) domains of Par proteins. Binding of Par proteins with PLC-beta stimulates PLC-beta enzymatic activity, leading to the hydrolysis of phosphatidylinositol-4,5-bisphosphate, and the production of diacylglycerol and inositol 1,4,5-triphosphate, important mediators in cell polarity and cell asymmetric division processes. Furthermore, we have shown that coexpression of PLC-beta with Par proteins induces transcriptional activation coupled to intracellular Ca2+ and the Wnt signaling pathway. Therefore, our data suggest that the interaction of PLC-beta with cell polarity Par proteins may serve as a nexus to transduce extracellular signals to transcriptional regulation through G-protein-mediated signaling pathway in cell polarity and cell asymmetric division.