ABSTRACT
The surfaceome is critical because surface proteins provide a gateway for internal signals and transfer of molecules into cells, and surfaceome differences can influence therapy response. We have used a surfaceome analysis method, based on comparing RNA-seq data between normal and abnormal cells (Surfaceome DataBase Mining or Surfaceome DBM), to identify sets of upregulated cell surface protein mRNAs in an LMO2-mediated T-ALL mouse model and corroborated by protein detection using antibodies. In this model the leukemia initiating cells (LICs) comprise pre-leukaemic, differentiation inhibited thymocytes allowing us to provide a profile of the LIC surfaceome in which GPR56, CD53 and CD59a are co-expressed with CD25. Implementation of cell surface interaction assays demonstrates fluid interaction of surface proteins and CD25 is only internalized when co-localized with other proteins. The Surfaceome DBM approach to analyse cancer cell surfaceomes is a way to find targetable surface biomarkers for clinical conditions where RNA-seq data from normal and abnormal cell are available.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Biomarkers, Tumor/metabolism , LIM Domain Proteins/metabolism , Leukemia, Lymphoid/genetics , Proto-Oncogene Proteins/metabolism , Transcriptome , Adaptor Proteins, Signal Transducing/genetics , Animals , Biomarkers, Tumor/genetics , CD59 Antigens/genetics , CD59 Antigens/metabolism , Cell Membrane/metabolism , Cells, Cultured , HEK293 Cells , Humans , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/metabolism , LIM Domain Proteins/genetics , Leukemia, Lymphoid/metabolism , Leukemia, Lymphoid/pathology , Mice , Neoplastic Stem Cells/metabolism , Proto-Oncogene Proteins/genetics , RNA-Seq , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Tetraspanin 25/genetics , Tetraspanin 25/metabolismABSTRACT
BACKGROUND: The molecular heterogeneity of autoimmune and inflammatory diseases has been one of the main obstacles to the development of safe and specific therapeutic options. Here, we evaluated the diagnostic and clinical value of a robust, inexpensive, immunoassay detecting the circulating soluble form of the monocyte-specific surface receptor sialic acid binding Ig-like lectin 1 (sSIGLEC-1). METHODS: We developed an immunoassay to measure sSIGLEC-1 in small volumes of plasma/serum from systemic lupus erythematosus (SLE) patients (n = 75) and healthy donors (n = 504). Samples from systemic sclerosis patients (n = 99) were studied as an autoimmune control. We investigated the correlation between sSIGLEC-1 and both monocyte surface SIGLEC-1 and type I interferon-regulated gene (IRG) expression. Associations of sSIGLEC-1 with clinical features were evaluated in an independent cohort of SLE patients (n = 656). RESULTS: Plasma concentrations of sSIGLEC-1 strongly correlated with expression of SIGLEC-1 on the surface of blood monocytes and with IRG expression in SLE patients. We found ancestry-related differences in sSIGLEC-1 concentrations in SLE patients, with patients of non-European ancestry showing higher levels compared to patients of European ancestry. Higher sSIGLEC-1 concentrations were associated with lower serum complement component 3 and increased frequency of renal complications in European patients, but not with the SLE Disease Activity Index clinical score. CONCLUSIONS: Our sSIGLEC-1 immunoassay provides a specific and easily assayed marker for monocyte-macrophage activation, and interferonopathy in SLE and other diseases. Further studies can extend its clinical associations and its potential use to stratify patients and as a secondary endpoint in clinical trials.
Subject(s)
Biomarkers/blood , Interferon-alpha/biosynthesis , Lupus Erythematosus, Systemic/blood , Sialic Acid Binding Ig-like Lectin 1/blood , Adult , Aged , Female , Humans , Immunoassay/methods , Lupus Erythematosus, Systemic/ethnology , Lupus Erythematosus, Systemic/immunology , Lupus Nephritis/blood , Lupus Nephritis/ethnology , Lupus Nephritis/immunology , Male , Middle Aged , Transcriptome , Young AdultABSTRACT
Haemangioblastoma is a rare malignancy of the CNS where vascular proliferation causes lesions due to endothelial propagation. We found that conditionally expressing mutant Kras, using Rag1-Cre, gave rise to CNS haemangioblastoma in the cortex and cerebellum in mice that present with highly vascular tumours with stromal cells similar to human haemangioblastomas. The aberrant haemangioblastoma endothelial cells do not express mutant Kras but rather the mutant oncogene is expressed in CNS interstitial cells, including neuronal cells and progeny. This demonstrates a non-cell autonomous origin of this disease that is unexpectedly induced via Rag1-Cre expression in CNS interstitial cells. This is the first time that mutant RAS has been shown to stimulate non-cell autonomous proliferation in malignancy and suggests that mutant RAS can control endothelial cell proliferation in neo-vascularisation when expressed in certain cells.
Subject(s)
Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/pathology , Genes, ras , Hemangioblastoma/genetics , Hemangioblastoma/pathology , Mutation , Animals , Cerebellar Neoplasms/mortality , Disease Models, Animal , Gene Expression , Genes, Reporter , Hemangioblastoma/mortality , Humans , Incidence , Mice , Mice, TransgenicABSTRACT
AIMS: We have previously shown in a phase I trial that nelfinavir (NFV) is safe with chemoradiation in PDAC with good signs for efficacy. Reverse translationally, we aimed to test the influence of PSCs on nelfinavir mediated radiosensitization to PDAC preclinically, because PDAC is very rich in desmoplasia and PSCs are known to mediate radioresistance. METHODS: In a direct co-culture model of several PDAC cell lines with PSC we performed clonogenic assays +/- nelfinavir. This was repeated exposing cells to hypoxic conditions. In xenograft PDAC tumors we tested radiation +/- nelfinavir +/- PSC. RESULTS: NFV sensitized both, PDAC only and PDAC cocultured with PSC (PDAC: Panc-1, MiaPaCa-2, PSN-1). In Panc-1 and PSN-1 this effect was larger +PSC compared to -PSC. Human pancreatic stellate cells (hPSC) were also sensitized by NFV which reduced p-FAK levels in hPSC, an effect that we previously found to sensitize specifically PDAC/PSC coculture. Contrarily, LY294002 reduced p-Akt in PSC (hPSC and LTC-14) but had no impact on PSC radiation survival. In vitro, nelfinavir sensitized Panc-1 and PSN-1 under normoxic and hypoxic conditions. In PSN-1 xenografts, +PSC led to faster tumor regrowth after radiation vs -PSC. The regrowth delay effect of nelfinavir after radiation was dramatically larger +PSC vs -PSC (time to reach 250mm(3) 183% vs 22%). CONCLUSION: NFV mediated radiosensitization in PDAC with stroma is partly mediated by p-FAK inhibition (Chen et al., 2013). In vitro, NFV sensitizes both normoxic and hypoxic PDAC +/- PSC to a roughly similar extent. The dramatic increased effect of xenograft regrowth inhibition by nelfinavir in tumors with PSC is attributed to vascular normalization (Brunner et al., 2014) rather than direct modification of hypoxia as shown by the tumor regrowth after gemcitabine with NFV.
Subject(s)
HIV Protease Inhibitors/pharmacology , Nelfinavir/pharmacology , Pancreatic Neoplasms/radiotherapy , Pancreatic Stellate Cells/drug effects , Radiation-Sensitizing Agents/pharmacology , Animals , Cell Line, Tumor , Disease Models, Animal , Female , Mice , Mice, Nude , Pancreatic Neoplasms/pathologyABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic reaction driven by pancreatic stellate cells (PSCs) that contributes to tumor progression. Here we sought to characterize the interactions between pancreatic cancer cells (PCCs) and PSCs that affect the inflammatory and immune response in pancreatic tumors. Conditioned media from mono- and cocultures of PSCs and PCCs were assayed for expression of cytokines and growth factors. IP-10/CXCL10 was the most highly induced chemokine in coculture of PSCs and PCCs. Its expression was induced in the PSCs by PCCs. IP-10 was elevated in human PDAC specimens, and positively correlated with high stroma content. Furthermore, gene expression of IP-10 and its receptor CXCR3 were significantly associated with the intratumoral presence of regulatory T cells (Tregs). In an independent cohort of 48 patients with resectable pancreatic ductal adenocarcinoma, high IP-10 expression levels correlated with decreased median overall survival. Finally, IP-10 stimulated the ex vivo recruitment of CXCR3+ effector T cells as well as CXCR3+ Tregs derived from patients with PDAC. Our findings suggest that, in pancreatic cancer, CXCR3+ Tregs can be recruited by IP-10 expressed by PSCs in the tumor stroma, leading to immunosuppressive and tumor-promoting effects.
Subject(s)
Carcinoma, Pancreatic Ductal/immunology , Chemokine CXCL10/biosynthesis , Pancreatic Neoplasms/immunology , T-Lymphocytes, Regulatory/immunology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , Chemokine CXCL10/genetics , Chemokine CXCL10/immunology , HEK293 Cells , Humans , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Stellate Cells/immunology , Pancreatic Stellate Cells/metabolism , Pancreatic Stellate Cells/pathology , Stromal Cells/immunology , Stromal Cells/metabolism , Stromal Cells/pathology , Survival Analysis , T-Lymphocytes, Regulatory/pathologyABSTRACT
BACKGROUND AND PURPOSE: Progression of pancreatic ductal adenocarcinoma (PDAC) is promoted by desmoplasia induced by pancreatic stellate cells (PSC). Contributory to this progression is epithelial mesenchymal transition (EMT), which shares many characteristics with the cancer stem cell (CSC) hypothesis. We investigated the role of these processes on the radioresponse and tumorigenicity of pancreatic cancer cells. MATERIALS AND METHODS: We used an in vitro sphere model and in vivo xenograft model to examine the role of PSC in EMT and CSC processes. RESULTS: We demonstrated that PSC enhanced the CSC phenotype and radioresistance of pancreatic cancer cells. Furthermore, the expression of several EMT and CSC markers supported enhanced processes in our models and that translated into remarkable in vivo tumorigenicity. Multi-dose TGFß neutralizing antibody inhibited the EMT and CSC processes, sensitized cells to radiation and reduced in vivo tumorigenicity. A proteomic screen identified multiple novel factors that were regulated by PSC in pancreatic cells. CONCLUSION: These results are critical in highlighting the role of PSC in tumor progression and radioresistance by manipulating the EMT and CSC processes. TGFß and the novel factors identified are important targets for better therapeutic outcome in response to PSC mediated mechanisms.
Subject(s)
Adenocarcinoma/pathology , Carcinoma, Pancreatic Ductal/pathology , Epithelial-Mesenchymal Transition/physiology , Neoplastic Stem Cells/pathology , Pancreatic Neoplasms/pathology , Pancreatic Stellate Cells/physiology , Radiation Tolerance/physiology , Adenocarcinoma/radiotherapy , Antibodies, Neutralizing/pharmacology , Biomarkers, Tumor/analysis , Carcinoma, Pancreatic Ductal/radiotherapy , Cell Survival/physiology , Cell Survival/radiation effects , Fibroblasts/pathology , Humans , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/radiation effects , Pancreatic Neoplasms/radiotherapy , Phenotype , Transforming Growth Factor beta/metabolism , Tumor Cells, CulturedABSTRACT
The concept of cancer stem cells is generally accepted in different malignancies. We have previously shown that the MDA-MB231 breast cancer cells were more radiation resistant when sorted for the two stem cell markers CD24 and ESA. In this study, we examined a possible mechanism that might underlie this phenotype by looking at cell cycle profile and the effect this has on DNA repair pathways. The cell cycle profile showed that there were more CD24(-) ESA(+) sorted MDA-MB231 cells in the S- and G(2)-phases compared with the unsorted cells, 60 and 38% respectively. Cyclin D and E protein levels supported the cell cycle profile and highlighted the possible involvement of homologous recombination (HR) repair in the radioresistant phenotype. To further support this, CD24(-) ESA(+) sorted MDA-MB231 cells demonstrated statistically significant more RAD51 and less γ-H2AX foci 2 h post 4Gy ionising radiation, compared with the unsorted population. Inhibition of the HR pathway effectively sterilised the CD24(- ) ESA(+) sorted MDA-MB231 cells but had no effect on the unsorted cells or MDA468 control breast cancer cell line. Although the changes we saw were specific to MDA-MB231, these results merit further investigation and can be crucial in identifying a mechanism responsible for cancer stem cells treatment resistance in primary tumors.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/physiopathology , Neoplastic Stem Cells/pathology , Recombination, Genetic/genetics , Benzofurans/pharmacology , Cell Cycle , Cell Line, Tumor , Female , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Neoplastic Stem Cells/cytology , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Radiation Tolerance/drug effects , Recombination, Genetic/drug effectsABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a strong desmoplastic reaction where the stromal compartment often accounts for more than half of the tumor volume. Pancreatic stellate cells (PSC) are a central mediator of desmoplasia. There is increasing evidence that desmoplasia is contributing to the poor therapeutic response of PDAC. We show that PSCs promote radioprotection and stimulate proliferation in pancreatic cancer cells (PCC) in direct coculture. Our in vivo studies show PSC-dependent radioprotection in response to a single dose and to fractionated radiation. Abrogating ß1-integrin signaling abolishes the PSC-mediated radioprotection in PCCs. Furthermore, this effect is independent of PI3K (phosphoinositide 3-kinase) but dependent on FAK. Taken together, we show for the first time that PSCs promote radioprotection of PCCs in a ß1-integrin-dependent manner.
Subject(s)
Integrin beta1/genetics , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/radiotherapy , Pancreatic Stellate Cells/cytology , Animals , Cell Line, Tumor , Cell Proliferation , Coculture Techniques , Female , Humans , Mice , Mice, Nude , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , RNA, Small Interfering/metabolism , Signal TransductionABSTRACT
PURPOSE: A subpopulation of cancer stem-like cells (CSLC) is hypothesized to exist in different cancer cell lines and to mediate radioresistance in solid tumors. METHODS AND MATERIALS: Cells were stained for CSLC markers and sorted (fluorescence-activated cell sorter/magnetic beads) to compare foci and radiosensitivity of phosphorylated histone H2AX at Ser 139 (gamma-H2AX) in sorted vs. unsorted populations in eight cell lines from different organs. CSLC properties were examined using anchorage-independent growth and levels of activated Notch1. Validation consisted of testing tumorigenicity and postirradiation enrichment of CSLC in xenograft tumors. RESULTS: The quantity of CSLC was generally in good agreement with primary tumors. CSLC from MDA-MB-231 (breast) and Panc-1 and PSN-1 (both pancreatic) cells had fewer residual gamma-H2AX foci than unsorted cells, pointing to radioresistance of CSLC. However, only MDA-MB-231 CSLC were more radioresistant than unsorted cells. Furthermore, MDA-MB-231 CSLC showed enhanced anchorage-independent growth and overexpression of activated Notch1 protein. The expression of cancer stem cell surface markers in the MDA-MB-231 xenograft model was increased after exposure to fractionated radiation. In contrast to PSN-1 cells, a growth advantage for MDA-MB-231 CSLC xenograft tumors was found compared to tumors arising from unsorted cells. CONCLUSIONS: CSLC subpopulations showed no general radioresistant phenotype, despite the quantities of CSLC subpopulations shown to correspond relatively well in other reports. Likewise, CSLC characteristics were found in some but not all of the tested cell lines. The reported problems in testing for CSLC in cell lines may be overcome by additional techniques, beyond sorting for markers.
Subject(s)
Histones/analysis , Membrane Proteins/analysis , Neoplastic Stem Cells/chemistry , Neoplastic Stem Cells/radiation effects , Radiation Tolerance , Receptor, Notch1/analysis , Animals , Biomarkers/analysis , Cell Count , Cell Line, Tumor/metabolism , Cell Line, Tumor/radiation effects , Cell Separation , Cell Survival , DNA Breaks , Female , Flow Cytometry/methods , Humans , Mice , Mice, SCID , Neoplastic Stem Cells/pathology , Radiation Tolerance/genetics , Spheroids, Cellular/pathology , Tumor Stem Cell Assay/methodsABSTRACT
PURPOSE: Pancreatic stellate cells (PSC) drive desmoplasia in pancreatic cancer. Our study analyzed both tumor and PSC, since interaction of these cell types may promote tumor progression. RESULTS: SPARC was expressed predominantly in the peritumoral and distal stroma. SPARC in distal stroma correlated inversely with overall survival of the patients with LAPC (p = 0.013) with a relative hazard of 2.23 (95% CI, 1.05 to 4.72; p = 0.036). TGFbeta1 in the tumor was also a negative prognostic factor (p = 0.03). Within the tumor cells, phospho-Akt correlated with TGFbeta1, SPARC and survivin. Tumor phospho-Akt correlated with stroma phospho-Akt, tumor TGFbeta1 correlated with stroma TGFbeta1 and alpha-SMA, tumor survivin correlated with stroma survivin and distal SPARC. Within the stroma, SPARC and TGFbeta1 correlated with alpha-SMA. Peritumoral SPARC correlated with distal SPARC. In vitro, SPARC was highly expressed in hPSC but not in Panc-1 cells. Exogenous SPARC did not change radiation resistance but increased the invasion of Panc-1 cells both in monoculture and in coculture with hPSC. EXPERIMENTAL DESIGN: Immunohistochemical expression of SPARC, CTGF, TGFbeta1, phospho-Akt, survivin and alpha-SMA was analyzed prior to chemoradiation in 58 locally advanced pancreatic cancer (LAPC) biopsy specimens. Fisher's exact test served to detect associations between tumor and PSC expression of markers. Kaplan-Meier analysis and multivariate analysis were used to evaluate the association of marker expression with overall survival. SPARC expression was analyzed in human pancreatic cancer cells (Panc-1) and in human PSC (hPSC) and the effect of SPARC on the invasion of Panc-1 cells was measured in monoculture or in coculture with hPSC. CONCLUSIONS: Our hypothesis of a detrimental effect of PSC on patient survival in LAPC after chemoradiation is supported by the inverse correlation of SPARC in distal stromal cells with patients survival. Furthermore in vitro data indicate that paracrine SPARC from PSC increases the invasion of pancreatic cancer cells.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma/radiotherapy , Osteonectin/biosynthesis , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/radiotherapy , Biopsy , Coculture Techniques , Cohort Studies , Disease Progression , Gene Expression Profiling , Humans , Immunohistochemistry/methods , Immunophenotyping , Prognosis , Proto-Oncogene Proteins c-akt/metabolism , Time Factors , Transforming Growth Factor beta1/metabolismABSTRACT
Transformation of the indolent follicular lymphoma (FL) to the aggressive diffuse large B-cell lymphoma (DLBCL) results in resistance to therapy with shortened survival. It has been demonstrated that the 12q12-14 region was mainly amplified in DLBCL cases but not in their FL counterparts. Therefore, we examined the DNA copy number and protein expression profiles for CDK2, CDK4 and GADD153, three genes that map to 12q12-14, in a set of 44 paired FL/DLBCL samples from 22 patients. The concordant amplification of these genes occurred in seven of 22 (32%) of FL cases, compared with 15 of 22 (68%) of DLBCL cases. At the protein level, 15 of 22 of the DLBCL samples (68%) showed strong staining for the CDK2 protein, compared with five of 21 of FL samples (24%). The majority of the DLBCL samples (16/22, 72%) expressed the CDK4 protein, whereas the majority of the FL samples (12/21, 57%) showed no expression of this protein. Except for one DLBCL case, no expression of the GADD153 protein could be detected. The deregulation of the CDK2 and CDK4 genes at the genetic and protein levels suggest a functional role for these genes in the transformation process and could potentially provide targets for prognostic tests or therapeutic interventions.
Subject(s)
Biomarkers, Tumor/genetics , Chromosomes, Human, Pair 12/genetics , Lymphoma, B-Cell/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Biomarkers, Tumor/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , DNA, Neoplasm/genetics , Disease Progression , Humans , Lymphoma, B-Cell/metabolism , Lymphoma, Follicular/genetics , Lymphoma, Follicular/metabolism , Lymphoma, Large B-Cell, Diffuse/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Polymerase Chain Reaction/methods , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolism , Tumor Cells, CulturedABSTRACT
The corresponding author submitted this article 1 to Molecular Cancer on the assumption that the co-author had agreed to the submission. Since this is not the case, the authors are retracting the article. The corresponding author is deeply sorry for any inconvenience this may have caused to the editorial and publishing staff. An apology is also extended to the readers.
ABSTRACT
c-Myc oncogene is an important regulator of cell cycle and apoptosis, and its dysregulated expression is associated with many malignancies. Myc is instrumental in directly or indirectly regulating the progression through the G1 phase and G1/S transition, and transformation by Myc results in perturbed cell cycle. Also contributory to the control of G1 is the Ras effector pathway Raf/MEK/ERK MAP kinase. Together with GSK3, ERK plays an important role in the critical hierarchical phosphorylation of S62/T58 controlling Myc protein levels. Therefore, our main aim was to examine the levels of MAPK in Myc transformed cells in light of the roles of ERK in cell cycle and control of Myc protein levels. We found that active forms of ERK were barely detectable in v-Myc (MC29) transformed cells. Furthermore, we could only detect reduced levels of activated ERK in c-Myc transformed cells compared to the non-transformed primary chick embryo fibroblast cells. The addition of LiCl inhibited GSK3 and successfully restored the levels of ERK in v-Myc and c-Myc transformed cells to those found in non-transformed cells. In addition, LiCl stabilised Myc protein in the non-transformed and c-Myc transformed cells but not in v-Myc transformed cells. These results can provide an important insight into the role of MAPK in the mechanism of Myc induced transformation and carcinogenesis.
Subject(s)
Glycogen Synthase Kinase 3/antagonists & inhibitors , Lithium Chloride/pharmacology , MAP Kinase Signaling System/drug effects , Oncogene Protein p55(v-myc)/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , Cell Line, Transformed , Cell Transformation, Neoplastic , Chick Embryo , Culture Media, Serum-Free/pharmacology , Fibroblasts , Genes, myc/genetics , Genes, myc/physiology , Glycogen Synthase Kinase 3/metabolism , Half-Life , Mitogen-Activated Protein Kinase 1/metabolism , Oncogene Protein p55(v-myc)/genetics , Phosphorylation/drug effectsABSTRACT
Mammalian DNA polymerase beta(beta-pol) is a single polypeptide chain enzyme of 39kDa. beta-pol has enzymatic activities appropriate for roles in base excision repair and other DNA metabolism events involving gap-filling DNA synthesis. Many crystal structures of beta-pol complexed with dNTP and DNA substrates have been solved, and mouse fibroblast cell lines deleted in the beta-pol gene have been examined. These approaches have enhanced our understanding of structural and functional aspects of beta-pol's role in protecting genomic DNA.