Cell Surface Membrane Proteins Research Articles

Genetic dissection of early endosomal recycling highlights a TORC1‐independent role for Rag GTPases

Endocytosed cell surface membrane proteins rely on various recycling pathways for their return to the plasma membrane1. Although endosome‐to‐plasma membrane recycling is critical for many cellular processes2, much of the required machinery is unknown. We discovered that yeast also has a recycling route to the cell surface that bypasses the Golgi. Using an engineered synthetic reporter that exclusively follows this pathway, we performed a genetic screen to reveal a cohort of novel and conserved regulators of direct endosome to cell surface recycling. One critical component was the Rag GTPases, Gtr1 and Gtr2, which worked downstream of the exchange factor Vam6 and upstream of the Gtr1 effector Ltv1. Although the Rag GTPases are known to activate TORC1 on the vacuole/lysosome3–5, we find the Vam6>Gtr1‐Gtr2>Ltv1 recycling pathway is independent of TORC1, thus establishing a distinct role for these GTPases in early endosome recycling.Support or Funding InformationRO1GM058202

Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells

ABSTRACTThe Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO) cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin) were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.

Sensitive Detection of Cell Surface Membrane Proteins in Living Breast Cancer Cells Using Multicolor Fluorescence Microscopy with a Plasmonic Chip.

A plasmonic chip was applied to live cancer cell imaging. The epithelial cell adhesion molecule (EpCAM) is a surface marker that can be used to classify breast cancer cell lines into distinct differentiation states. EpCAM and the nuclei of two kinds of living breast cancer cells, MDA-MB231 and MCF-7, were stained with allophycocyanin (APC)-labeled anti-EpCAM antibody and 4',6-diamidino-2-phenylindole (DAPI), respectively, and the cells were scattered on either a plasmonic chip (metal-coated wavelength-scale grating substrate) or a control glass slide. Multicolor fluorescence microscopic imaging allowed fluorescence images of APC-EpCAM to be obtained on the plasmonic chip that were more than 10 times brighter compared with those on the glass slide. In contrast, in the fluorescence images of DAPI-stained nuclei, no difference in brightness was observed between substrates. The fluorescence enhancement of APC-EpCAM in the cell membrane in contact with the plasmonic chip is thought to be due to the excitation of APC molecules localized within the surface plasmon field. Analysis of the cross section of a fluorescence image revealed a distribution of EpCAM at a higher level of fluorescence in the center of the cell image because of contact between the cell membrane and the plasmonic chip. In contrast, fluorescence images of APC-EpCAM taken on a glass slide were so dark that only the outline of the cell was characterized. The plasmonic chip thus constitutes a simple and powerful tool for analyzing the distribution and kinetics of surface marker proteins in cell membranes contacting the chip.

Proteomic Substrate Identification for Membrane Proteases in the Brain

Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane-bound precursor proteins and control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the “a disintegrin and metalloprotease” (ADAM), the beta-site amyloid precursor protein cleaving enzymes (BACE), membrane-type matrix metalloproteases (MT-MMP) and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential not only for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer’s disease (AD), schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail, as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even in vivo from minute amounts of mouse cerebrospinal fluid (CSF). This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10 and γ-secretase, as well as ADAM17 and signal peptide peptidase like 3 (SPPL3), we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs.

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc.

Implications for Alzheimer's disease of an atomic resolution structure of amyloid-β(1-42) fibrils.

The article by Walti et al. (1) in PNAS is a milestone in biomedicine. It describes in atomic detail fibrils that form the plaques in diseased brains, first observed by Alois Alzheimer 110 y ago. These extracellular fibrils are now known to be composed of amyloid-β, a peptide of various lengths between 38 and 43 residues, proteolytically cleaved from the cell-surface membrane protein called amyloid precursor protein (APP). Amyloid-β(1–42) is more cytotoxic than its cousin, shorter by two residues, amyloid-β(1–40), for which structures have been known for several years (2). Because of its role in the etiology of Alzheimer’s disease, the structure of amyloid-β(1–42) has been eagerly awaited, and now two independent studies simultaneously describe identical, disease-relevant fibrils: the report by Walti et al. (1) is from the laboratories of Meier and Riek at Eidgenossiche Technische Hochschule Zurich and Bockmann at Lyon, and the other is from Griffin at the Massachusetts Institute of Technology and Linse at the University of Lund, Sweden (3). These structures are determined largely from distance constraints between pairs of isotopically labeled atoms measured by solid-state NMR. Computer-aided energy minimization of the amyloid-β(1–42) peptide chains yields a “bundle” of atomic models, each obeying the distance constraints. That is, the final structure is a constrained set of closely related models, rather than a single structure as yielded by a high-resolution X-ray determination or a direct image produced by cryo-EM. Although it is not possible to be certain that there is no other model that also fits the same NMR constraints, alternative models become increasingly improbable as the number of distance constraints grows. Confidence in the amyloid-β(1–42) structure is reinforced by the essentially identical results of the two independent studies. For the Walti et al. (1) structure, the rmsd of backbone atoms of the 10 lowest-energy atomic … [↵][1]1To whom correspondence should be addressed. Email: david{at}mbi.ucla.edu. [1]: #xref-corresp-1-1

Abstract 3133: Glypican-1 is a potential marker of prognosis and involved in chemoresistance of cisplatin in esophageal squamous cell cancer

Abstract [Introduction]Despite recent improvements in multi-treatment approaches, including surgery, radiotherapy and chemotherapy, the prognosis for patients with Esophageal squamous cell cancer (ESCC) remains unsatisfactory. In addition, predicting a prognosis and treatment response for ESCC had been constantly being required but remains difficult, even when using the TNM system. We identified Glypican-1(GPC1) as a novel disease specific antigen of ESCC by quantitative proteomic analysis focused on cell surface membrane proteins. GPC1 is one of the cell-surface heparan sulfate proteoglycans which has been reported as a prognostic factor in pancreatic cancer. [Objectives]This study aimed to clarify correlations between GPC1 expression and the prognosis in patient with ESCC and also reveal the association of GPC1 with the drug resistance to chemotherapeutic agents in squamous cancer cell lines and esophageal cancer patients treated with chemotherapy. [Methods]ESCC tissues were obtained from 175 patients who underwent esophagectomy at Osaka University Hospital(Osaka, Japan), from 2001 to 2013 and were subjected to immunohistochemistry. We performed immunohistochemical (IHC) assessment using anti-GPC1 antibody and investigated the association between GPC1 expression and the clinicopathological parameters. We divided the patients into two groups such as high expression group (HG) and low expression group (LG). Furthermore, we investigated associations between GPC1 expression and clinical response of neo-adjuvant chemotherapy for patients with ESCC. We established GPC1 knocked out cells of TE-14 using Crisper-Cas9 system and stable GPC1-transfected cells of LK-2(Lung squamous cell carcinoma cell line) using pcDNA3.1-GPC1 plasmid transfection. To investigate the relationship between GPC1 expression and sensitivity to cisplatin (CDDP), we examined sensitivity to CDDP by the WST-8 assay. [Results]The patients characteristics as follows, age (median) = 64yrs(39-80), gender (Male: Female) = 155:20. By the IHC analysis, 99 patients were classified into HG and 76 were LG. HG patients exhibited a poorer prognosis compared with the LG patients. (p<0.001) In multivariate analyses pT, pN, and GPC1 high expression were became significant prognostic factors (p<0.01). We clarified there was a significant negative correlation between histological responses and GPC1 expression in patient with neoadjuvant chemo therapy. Furthermore, in vitro experiments showed that the IC50 values for CDDP of GPC1 expressing cells (TE-14 and GPC1 transfected LK-2) were significantly higher than that of GPC1 no-expressing cells (GPC1 knocked out TE-14 and LK-2). [Conclusion]Our study showed that GPC1 was an independent prognostic factor in esophageal cancer. Furthermore, we showed that GPC1 was a critical molecule for chemoresistance to cisplatin. Citation Format: Takahiko Nishigaki, Tsuyoshi Takahashi, Satishi Serada, Minoru Fujimoto, Hisashi Hara, Yasuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Tetsuji Naka. Glypican-1 is a potential marker of prognosis and involved in chemoresistance of cisplatin in esophageal squamous cell cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3133.

Abstract 4998: Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of ovarian cancer

Abstract Objective:Ovarian cancer is the most lethal gynecological malignancy. Five-year survival of advanced ovarian cancer patients remains less than 50% and the mortality rate has not changed in recent years. This study is aimed to screen for novel ovarian cancer therapeutic targets using quantitative proteomic approach and to develop antibody-based medicine targeting novel ovarian cancer antigens. Method:Exhaustive quantitative proteome analysis focused on cell surface membrane proteins were performed by isobaric tags for relative and absolute quantitation(iTRAQ) using a normal ovarian surface epithelial cell line and 7 ovarian cancer cell lines. By this approach, ovarian cancer-specific membrane proteins were identified. We confirmed the expression of lipolysis-stimulated lipoprotein receptor (LSR) using immunohistochemical staining, western blotting method, and flow cytometry. By modified MTT assay, cell growth inhibition was performed in LSR-knock down cells compared with control cells. Cell adhesion assay was also performed in vitro. Anti-LSR monoclonal antibody (anti-LSR mAb) was generated to evaluate the efficacy of the LSR targeted antibody therapy in vivo. For subcutaneous xenograft experiments, ovarian cancer cell line (RMG-1) was injected subcutaneously into the CB17/SCID mice. Mice were then randomly divided into two groups and administrated intraperitoneally anti-LSR mAb or control IgG antibody twice a week for 6 times. In addition, luciferase transfected ovarian cancer cell line (A2780-luc) was implanted intraperitoneally into the CB17/SCID mice. Mice were administrated anti-LSR mAb or control IgG antibody intraperitoneally twice a week for 5 times. Result:By iTRAQ analysis, we identified 1685 proteins and one of the ovarian cancer candidates protein, LSR was identified. We confirmed the expression of LSR in 8 out of 11 ovarian cancer cell lines and all of ovarian cancer clinical specimens. We performed proliferation assay using siRNA against ovarian cancer cell lines. When expression of LSR was suppressed, a tumor growth was significantly inhibited at day 4(p<0.01). In adhesion assay, we observed significant inhibition of cell adhesion in LSR knockdown cells. These results suggested that LSR is related to cell proliferation and adhesion. A significant anti-tumour effect with 78% was observed in the anti-LSR mAb antibody administrated group compared with control IgG antibody administrated group after 25 days (p <0.001). Furthermore, in intraperitoneal xenograft model, anti-LSR antibody administration group significantly decreased signal intensities compared with the control antibody administration group at day 22 after implantation. Conclusion:We showed that the LSR is related to cell proliferation and adhesion in vitro in ovarian cancer cells and proved the antitumor effect of anti-LSR mAb. These results suggested that LSR can be a new therapeutic target of ovarian cancer. Citation Format: Satoko Matsuzaki, Kosuke Hiramatsu, Satoshi Serada, Satoshi nakagawa, Shinya Matsuzaki, Yutaka Ueda, Minoru Fujimoto, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4998.

Natural Selection in Virulence Genes of Francisella tularensis.

A fundamental tenet of evolution is that alleles that are under negative selection are often deleterious and confer no evolutionary advantage. Negatively selected alleles are removed from the gene pool and are eventually extinguished from the population. Conversely, alleles under positive selection do confer an evolutionary advantage and lead to an increase in the overall fitness of the organism. These alleles increase in frequency until they eventually become fixed in the population. Francisella tularensis is a zoonotic pathogen and a potential biothreat agent. The most virulent type of F. tularensis, Type A, is distributed across North America with Type A.I occurring mainly in the east and Type A.II appearing mainly in the west. F. tularensis is thought to be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. To test this hypothesis, we sequenced and compared whole genomes of Type A.I and A.II isolates. We analyzed a subset of virulence and housekeeping genes from several F. tularensis subspecies genomes to ascertain the presence and extent of positive selection. Eleven previously identified virulence genes were screened for positive selection along with 10 housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection at loci implicated in cell surface structures and membrane proteins, metabolism and biosynthesis, transcription, translation and cell separation, and substrate binding and transport. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolutiondriven by complex interactions between host, pathogen, and thier environment, as evidenced by several of its virulence genes which are undergoing strong, positive selection.

Generating Recombinant Antibodies to Membrane Proteins through Phage Display.

One of the most important classes of proteins in terms of drug targets is cell surface membrane proteins, and yet it is a challenging set of proteins for generating high-quality affinity reagents. In this review, we focus on the use of phage libraries, which display antibody fragments, for generating recombinant antibodies to membrane proteins. Such affinity reagents generally have high specificity and affinity for their targets. They have been used for cell staining, for promoting protein crystallization to solve three-dimensional structures, for diagnostics, and for treating diseases as therapeutics. We cover publications on this topic from the past 10 years, with a focus on the various formats of membrane proteins for affinity selection and the diverse affinity selection strategies used. Lastly, we discuss the challenges faced in this field and provide possible directions for future efforts.

Targeting the glioblastoma-initiating cell-associated antigens

Glioblastoma (GBM)-initiating cells (GICs) are a tumorigenic subpopulation that are resistant to radio/chemotherapies and are the source of recurrence, therefore it is crucial to characterize GICs and find new therapeutic targets. We have successfully established mouse and human GICs, which retain stemness characteristics and tumorigenicity. By comparing their expression profiles with those of parental cells, we have focused on two cell surface membrane proteins, Ceacam1L and Eva1, which were prominently expressed in GICs. We demonstrated that both proteins were involved in GIC characteristics, including self-renewal, stem cell gene expression and side population, and tumorigenesis, indicating that they are potential new therapeutic targets for GBM. In this research highlight, I summarize our recent reports regarding Ceacam1L and Eva1, and then discuss about Ceacam1L and Eva1 as GBM therapeutic targets.

A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells.

Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis.

Rab11a Mediates Vascular Endothelial-Cadherin Recycling and Controls Endothelial Barrier Function.

Vascular endothelial (VE)-cadherin is the predominant component of endothelial adherens junctions essential for cell-cell adhesion and formation of the vascular barrier. Endocytic recycling is an important mechanism for maintaining the expression of cell surface membrane proteins. However, little is known about the molecular mechanism of VE-cadherin recycling and its role in maintenance of vascular integrity. Using calcium-switch assay, confocal imaging, cell surface biotinylation, and flow cytometry, we showed that VE-cadherin recycling required Ras-related proteins in brain (Rab)11a and Rab11 family-interacting protein 2. Yeast 2-hybrid assay and coimmunoprecipitation demonstrated that direct interaction of VE-cadherin with family-interacting protein 2 (at aa 453-484) formed a ternary complex with Rab11a in human endothelial cells. Silencing of Rab11a or Rab11 family-interacting protein 2 in endothelial cells prevented VE-cadherin recycling and VE-cadherin expression at endothelial plasma membrane. Furthermore, inactivation of Rab11a signaling blocked junctional reannealing after vascular inflammation. Selective knockdown of Rab11a in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide or polymicrobial sepsis. Rab11a/Rab11 family-interacting protein 2-mediated VE-cadherin recycling is required for formation of adherens junctions and restoration of VE barrier integrity and hence a potential target for clinical intervention in inflammatory disease.

Abstract 4382: Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly

Abstract Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. We previously identified bone marrow stromal antigen 2 (BST2) as an endometrial cancer antigen using iTRAQ-based quantitative proteomic technology focused on cell surface membrane proteins, and also demonstrated the usefulness of an anti-BST2 monoclonal antibody (mAb) for endometrial cancer. In this study, we aimed to identify a new ovarian cancer antigen. We also aimed to develop a novel monoclonal antibody (mAb) and evaluate its preclinical efficacy against ovarian cancer. To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. As the new therapeutic target for ovarian cancer, we identified lipolysis-stimulated lipoprotein receptor (LSR) which had one of the largest significant differences in protein level between normal ovarian epithelial and ovarian cancer cell lines. Immunohistochemical analysis showed that the overall survival of ovarian serous carcinoma patients with high LSR expression was significantly shorter than those with low LSR (p = 0.0293). We newly developed anti-LSR mAb and investigated its preclinical efficacy. Anti-LSR mAb showed significant in vivo inhibition of tumor growth against a xenograft model of hLSR-positive ovarian cancer in an antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) independent manner (p = 0.0001). And anti-LSR mAb also induced G0/G1 cell cycle arrest by regulation of MEK and p44/42 MAPK activities and expression levels of cell cycle related proteins in vitro. Furthermore, anti-hLSR mAb, which crossreacts with mouse LSR, had little detectable toxicity in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition in ADCC and CDC independent manner in vivo. Anti-human LSR mAb also inhibited LSR function and showed direct tumor growth inhibition inducing G0/G1 cell cycle arrest in vitro. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Satoshi Nakagawa, Akiko Morimoto, Minoru Fujimoto, Takuhei Yokoyama, Yusuke Takahashi, Yutaka Ueda, Kiyoshi Yoshino, Eiichi Morii, Tadashi Kimura, Tetsuji Naka. Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2015-4382

Abstract 25: Loss of interferon-induced transmembrane protein 1 enhances estrogen-induced cell death in AI-resistant breast cancer cells

Abstract Estrogen deprivation using aromatase inhibitors (AIs) is the standard of care for postmenopausal women with hormone receptor-positive breast cancer. However, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. We have previously reported the development of an AI-resistant breast cancer cell line, MCF-7:5C, which was clonally derived from parental MCF-7 breast cancer cells following long term estrogen deprivation. A unique phenotype of AI-resistant MCF-7:5C cells is that they grow in the absence of estrogen; however, when they are treated with estradiol they undergo apoptosis/cell death through activation of the mitochondrial death pathway. More recently, we have also discovered that AI-resistant MCF-7:5C cells constitutively overexpress the interferon stimulated gene IFITM1. IFITM1 is a cell surface membrane protein that is most well-known for its ability to block viral replication, however, its role in breast cancer and AI-resistance is not known. We found that IFITM1 was constitutively overexpressed at the protein (> 20-fold) and mRNA (> 15-fold) level in AI-resistant MCF-7:5C breast cancer cells compared to parental MCF-7 cells and that its overexpression strongly correlated with enhanced growth and increased aggressiveness. Clinical data also indicated that IFITM1 was overexpressed in 36 out of 40 AI-resistant breast tumor samples compared to AI-sensitive tumors. In this study, we assessed the functional role of IFITM1 in AI-resistant MCF-7:5C cells. Notably, we found that knockdown of IFITM1 significantly induced cell death in MCF-7:5C cells and it reduced cell proliferation. The induction of cell death as a result of IFITM1 knockdown was associated with increases in p21, Bax, Noxa, and Puma expression; however, p53 was not altered in these cells. To validate the effect observed with IFITM1 knockdown in our resistant cells we used a second shRNA targeting IFITM1. We found that shRNA knockdown of IFITM1 induced PARP cleavage and p21, reduced cell proliferation, and induced cell death in AI-resistant MCF-7:5C cells. Moreover, we found that knockdown of IFITM1 dramatically enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells which was associated with induction of pro-apoptotic proteins and p21 via a p53-independent mechanism. Further analysis revealed that knockdown of STAT1 and STAT2, which are critical regulators of IFITM1, also significantly enhanced estradiol-induced apoptosis in MCF-7:5C cells. Taken together, these findings indicate that overexpression of IFITM1 plays a critical role in breast cancer and AI-resistance and that targeting this gene can sensitize AI-resistant cells to estrogen-induced death. Furthermore, these results suggest that IFITM1 can regulate the mitochondrial pathway through its interactions with anti-apoptotic proteins. Citation Format: Hye Joung Choi, Joan Lewis-Wambi. Loss of interferon-induced transmembrane protein 1 enhances estrogen-induced cell death in AI-resistant breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 25. doi:10.1158/1538-7445.AM2015-25

Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling.

BackgroundMonolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets.MethodsScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin.ResultsTwo primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model.ConclusionsPhenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0415-0) contains supplementary material, which is available to authorized users.

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport.

The importance of endosome-to-trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51-VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.

A Family of Tetraspans Organizes Cargo for Sorting into Multivesicular Bodies

The abundance of cell-surface membrane proteins isregulated by internalization and delivery into intralumenal vesicles (ILVs) of multivesicular bodies (MVBs). Many cargoes are ubiquitinated, allowing access to an ESCRT-dependent pathway into MVBs. Yet how nonubiquitinated proteins, such as glycosylphosphatidylinositol-anchored proteins, enter MVBs is unclear, supporting the possibility of mechanistically distinct ILV biogenesis pathways. Here we show that a family of highly ubiquitinated tetraspan Cos proteins provides a Ub signal in trans, allowing sorting of nonubiquitinated MVB cargo into the canonical ESCRT- and Ub-dependent pathway. Cos proteins create discrete endosomal subdomains that concentrate Ub cargo prior to their envelopment into ILVs, and the activity of Cos proteins is required not only for efficient sorting of canonical Ub cargo but also for sorting nonubiquitinated cargo into MVBs. Expression of these proteins increases during nutrient stress through an NAD(+)/Sir2-dependent mechanism that in turn accelerates the downregulation of a broad range of cell-surface proteins.

Novel (89)Zr cell labeling approach for PET-based cell trafficking studies.

BackgroundWith the recent growth of interest in cell-based therapies and radiolabeled cell products, there is a need to develop more robust cell labeling and imaging methods for in vivo tracking of living cells. This study describes evaluation of a novel cell labeling approach with the positron emission tomography (PET) isotope 89Zr (T1/2 = 78.4 h). 89Zr may allow PET imaging measurements for several weeks and take advantage of the high sensitivity of PET imaging.MethodsA novel cell labeling agent, 89Zr-desferrioxamine-NCS (89Zr-DBN), was synthesized. Mouse-derived melanoma cells (mMCs), dendritic cells (mDCs), and human mesenchymal stem cells (hMSCs) were covalently labeled with 89Zr-DBN via the reaction between the NCS group on 89Zr-DBN and primary amine groups present on cell surface membrane protein. The stability of the label on the cell was tested by cell efflux studies for 7 days. The effect of labeling on cellular viability was tested by proliferation, trypan blue, and cytotoxicity/apoptosis assays. The stability of label was also studied in in vivo mouse models by serial PET scans and ex vivo biodistribution following intravenous and intramyocardial injection of 89Zr-labeled hMSCs. For comparison, imaging experiments were performed after intravenous injections of 89Zr hydrogen phosphate (89Zr(HPO4)2).ResultsThe labeling agent, 89Zr-DBN, was prepared in 55% ± 5% decay-corrected radiochemical yield measured by silica gel iTLC. The cell labeling efficiency was 30% to 50% after 30 min labeling depending on cell type. Radioactivity concentrations of labeled cells of up to 0.5 MBq/106 cells were achieved without a negative effect on cellular viability. Cell efflux studies showed high stability of the radiolabel out to 7 days. Myocardially delivered 89Zr-labeled hMSCs showed retention in the myocardium, as well as redistribution to the lung, liver, and bone. Intravenously administered 89Zr-labeled hMSCs also distributed primarily to the lung, liver, and bone, whereas intravenous 89Zr(HPO4)2 distributed to the liver and bone with no activity in the lung. Thus, the in vivo stability of the radiolabel on the hMSCs was evidenced.ConclusionsWe have developed a robust, general, and biostable 89Zr-DBN-based cell labeling strategy with promise for wide applications of PET-based non-invasive in vivo cell trafficking.

Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum

The endoplasmic reticulum (ER) is an essential cellular compartment in which an enormous number of secretory and cell surface membrane proteins are synthesized and subjected to cotranslational or posttranslational modifications, such as glycosylation and disulfide bond formation. Proper maintenance of ER protein homeostasis (sometimes termed proteostasis) is essential to avoid cellular stresses and diseases caused by abnormal proteins. Accumulating knowledge of cysteine-based redox reactions catalyzed by members of the protein disulfide isomerase (PDI) family has revealed that these enzymes play pivotal roles in productive protein folding accompanied by disulfide formation, as well as efficient ER-associated degradation accompanied by disulfide reduction. Each of PDI family members forms a protein–protein interaction with a preferential partner to fulfill a distinct function. Multiple redox pathways that utilize PDIs appear to function synergistically to attain the highest quality and productivity of the ER, even under various stress conditions. This review describes the structures, physiological functions, and cooperative actions of several essential PDIs, and provides important insights into the elaborate proteostatic mechanisms that have evolved in the extremely active and stress-sensitive ER.