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1.
Oncoimmunology ; 5(7): e1171434, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27622021

ABSTRACT

Antibody-drug conjugates (ADC), combining the specificity of tumor recognition by monoclonal antibodies (mAb) and the powerful cytotoxicity of anticancer drugs, are currently under growing interest and development. Here, we studied the potential of Chi-Tn, a mAb directed to a glyco-peptidic tumor-associated antigen, to be used as an ADC for cancer treatment. First, we demonstrated that Chi-Tn specifically targeted tumor cells in vivo. Also, using flow cytometry and deconvolution microscopy, we showed that the Chi-Tn mAb is rapidly internalized - condition necessary to ensure the delivery of conjugated cytotoxic drugs in an active form, and targeted to early and recycling endosomes. When conjugated to saporin (SAP) or to auristatin F, the Chi-Tn ADC exhibited effective cytotoxicity to Tn-positive tumor cells in vitro, which correlated with the level of tumoral Tn expression. Furthermore, the Chi-Tn mAb conjugated to auristatin F also exhibited efficient antitumor activity in vivo, validating for the first time the use of an anti-Tn antibody as an effective ADC.

2.
PLoS One ; 8(8): e72708, 2013.
Article in English | MEDLINE | ID: mdl-23977342

ABSTRACT

Milk Fat Globule--EGF--factor VIII (MFGE8), also called lactadherin, is a secreted protein, which binds extracellularly to phosphatidylserine and to αvß3 and αvß5 integrins. On human and mouse cells expressing these integrins, such as endothelial cells, phagocytes and some tumors, MFGE8/lactadherin has been shown to promote survival, epithelial to mesenchymal transition and phagocytosis. A protumoral function of MFGE8 has consequently been documented for a few types of human cancers, including melanoma, a subtype of breast cancers, and bladder carcinoma. Inhibiting the functions of MFGE8 could thus represent a new type of therapy for human cancers. Here, we show by immunohistochemistry on a collection of human ovarian cancers that MFGE8 is overexpressed in 45% of these tumors, and we confirm that it is specifically overexpressed in the triple-negative subtype of human breast cancers. We have established new in vitro assays to measure the effect of MFGE8 on survival, adhesion and migration of human ovarian and triple-negative breast cancer cell lines. Using these assays, we could identify new MFGE8-specific monoclonal antibodies, which efficiently blocked these three tumor-promoting effects of MFGE8. Our results suggest future use of MFGE8-blocking antibodies as new anti-cancer therapeutics in subgroups of ovarian carcinoma, and triple-negative breast carcinoma patients.


Subject(s)
Antibodies, Blocking/pharmacology , Antigens, Surface/immunology , Cell Movement/drug effects , Milk Proteins/immunology , Molecular Targeted Therapy , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Animals , Antigens, Surface/metabolism , Biological Assay , Biopsy , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Female , Humans , Mice , Milk Proteins/metabolism , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology
3.
J Biol Chem ; 279(44): 46104-12, 2004 Oct 29.
Article in English | MEDLINE | ID: mdl-15319436

ABSTRACT

Translationally controlled tumor protein (TCTP) is cytoplasmic and structurally related to guanine-nucleotide free chaperones. TCTP (also called histamine-releasing factor) has been described previously as a secreted protein that participates in inflammatory responses by promoting the release of histamine. How TCTP is eventually exported out of the cell to promote such activities is unknown. Here we show that TCTP secretion was insensitive to either brefeldin A or monensin, suggesting that it proceeds via an endoplasmic reticulum/Golgi-independent or nonclassical pathway. Moreover, our analyses also suggest that secreted TCTP originates from pre-existing pools. TSAP6, a p53-inducible 5-6 transmembrane protein, was found to interact with TCTP in a yeast two-hybrid hunt. GST pull down assays confirmed their direct interaction, and immunofluorescence analysis revealed their partial co-distribution to vesicular-like structures at the plasma membrane and around the nucleus. Functionally, the overexpression of TSAP6 consistently leads to enhanced secretion of both endogenously and exogenously expressed TCTP. Finally, we found TCTP in preparations of small secreted vesicles called exosomes, which have been suggested as a possible pathway for nonclassical secretion. Overexpression of TSAP6 also increased TCTP levels in exosome preparations. Altogether, these data identify a novel role for TSAP6 in the export of TCTP and indicate that this multipass membrane protein could have a general role in the regulation of vesicular trafficking and secretion.


Subject(s)
Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Oncogene Proteins/physiology , Biomarkers, Tumor , Cell Cycle Proteins , Cell Line , Cycloheximide/pharmacology , Humans , Neoplasm Proteins/analysis , Nuclear Proteins/analysis , Oncogene Proteins/analysis , Oxidoreductases , Protein Transport , Secretory Vesicles/chemistry , Tumor Protein, Translationally-Controlled 1
4.
J Biol Chem ; 279(28): 28989-97, 2004 Jul 09.
Article in English | MEDLINE | ID: mdl-15123706

ABSTRACT

Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic beta(2) receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade.


Subject(s)
Carrier Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Angiotensin, Type 2/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins/genetics , Cell Division/physiology , Chlorocebus aethiops , Cloning, Molecular , Dimerization , Enzyme Activation , Fibroblast Growth Factor 2/metabolism , Humans , Insulin/metabolism , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Molecular Sequence Data , Protein Structure, Tertiary , Receptor, Angiotensin, Type 2/chemistry , Receptor, Angiotensin, Type 2/genetics , Sequence Alignment , Tissue Distribution , Tumor Suppressor Proteins/genetics , Two-Hybrid System Techniques
5.
Proc Natl Acad Sci U S A ; 100(24): 13892-7, 2003 Nov 25.
Article in English | MEDLINE | ID: mdl-14623968

ABSTRACT

Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bbeta, as TCTP-interacting partners. In vitro and in vivo studies confirmed that TCTP bound specifically eEF1Bbeta and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bbeta. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.


Subject(s)
Biomarkers, Tumor/metabolism , Guanine Nucleotide Dissociation Inhibitors/metabolism , Guanine Nucleotides/metabolism , Peptide Elongation Factor 1/metabolism , Biomarkers, Tumor/genetics , Drug Stability , Guanine Nucleotide Dissociation Inhibitors/genetics , Guanosine Diphosphate/metabolism , Humans , In Vitro Techniques , Kinetics , Peptide Elongation Factor 1/genetics , Protein Biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tumor Protein, Translationally-Controlled 1 , Two-Hybrid System Techniques
6.
Proc Natl Acad Sci U S A ; 100(5): 2284-9, 2003 Mar 04.
Article in English | MEDLINE | ID: mdl-12606722

ABSTRACT

The p53 tumor suppressor protein plays a crucial role in tumorigenesis by controlling cell-cycle progression and apoptosis. We have previously described a transcript designated tumor suppressor activated pathway-6 (TSAP6) that is up-regulated in the p53-inducible cell line, LTR6. Cloning of the murine and human full-length TSAP6 cDNA revealed that it encodes a 488-aa protein with five to six transmembrane domains. This gene is the murine and human homologue of the recently published rat pHyde. Antibodies raised against murine and human TSAP6 recognize a 50- to 55-kDa band induced by p53. Analysis of the TSAP6 promoter identified a functional p53-responsive element. Functional studies demonstrated that TSAP6 antisense cDNA diminished levels of the 50- to 55-kDa protein and decreased significantly the levels of p53-induced apoptosis. Furthermore, TSAP6 small interfering RNA inhibited apoptosis in TSAP6-overexpressing cells. Yeast two-hybrid analysis followed by GST/in vitro-transcribed/translated pull-down assays and in vivo coimmunoprecipitations revealed that TSAP6 associated with Nix, a proapoptotic Bcl-2-related protein and the Myt1 kinase, a negative regulator of the G(2)/M transition. Moreover, TSAP6 enhanced the susceptibility of cells to apoptosis and cooperated with Nix to exacerbate this effect. Cell-cycle studies indicated that TSAP6 could augment Myt1 activity. Overall, these data suggest that TSAP6 may act downstream to p53 to interface apoptosis and cell-cycle progression.


Subject(s)
Apoptosis , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/physiology , Oncogene Proteins , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins , Amino Acid Sequence , Animals , Blotting, Northern , Blotting, Western , Cell Cycle , Cell Cycle Proteins , Cloning, Molecular , DNA, Complementary/metabolism , Flow Cytometry , G1 Phase , G2 Phase , Glutathione Transferase/metabolism , HeLa Cells , Humans , Luciferases/metabolism , Mice , Mitosis , Molecular Sequence Data , Oligonucleotides, Antisense/pharmacology , Oxidoreductases , Phosphorylation , Precipitin Tests , Promoter Regions, Genetic , Protein Binding , Protein Biosynthesis , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/metabolism , S Phase , Sequence Homology, Amino Acid , Time Factors , Transcription, Genetic , Tumor Cells, Cultured , Two-Hybrid System Techniques
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