RESUMEN
Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed â¼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised â¼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/metabolismo , Secuencia de Bases , Línea Celular , Emulsiones , Femenino , Humanos , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.
Asunto(s)
Colagenasas/química , Inhibidores de la Metaloproteinasa de la Matriz , Modelos Moleculares , Inhibidores Tisulares de Metaloproteinasas/química , Animales , Células CHO , Colagenasas/genética , Colagenasas/metabolismo , Cricetinae , Cricetulus , Humanos , Ratones , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismoRESUMEN
The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.
Asunto(s)
Furina/metabolismo , Metaloproteinasa 14 de la Matriz/química , Metaloproteinasa 14 de la Matriz/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Estructura Terciaria de ProteínaRESUMEN
The accurate identification and quantitation of RNA isoforms present in the cancer transcriptome is key for analyses ranging from the inference of the impacts of somatic variants to pathway analysis to biomarker development and subtype discovery. The ICGC-TCGA DREAM Somatic Mutation Calling in RNA (SMC-RNA) challenge was a crowd-sourced effort to benchmark methods for RNA isoform quantification and fusion detection from bulk cancer RNA sequencing (RNA-seq) data. It concluded in 2018 with a comparison of 77 fusion detection entries and 65 isoform quantification entries on 51 synthetic tumors and 32 cell lines with spiked-in fusion constructs. We report the entries used to build this benchmark, the leaderboard results, and the experimental features associated with the accurate prediction of RNA species. This challenge required submissions to be in the form of containerized workflows, meaning each of the entries described is easily reusable through CWL and Docker containers at https://github.com/SMC-RNA-challenge. A record of this paper's transparent peer review process is included in the supplemental information.
Asunto(s)
Neoplasias , Humanos , Neoplasias/genética , Isoformas de Proteínas/genética , ARN/genética , RNA-Seq , Análisis de Secuencia de ARNRESUMEN
Both invasion-promoting MT1-MMP and its physiological inhibitor TIMP-2 play a significant role in tumorigenesis and are identified in the most aggressive cancers. Despite its antiproteolytic effects in vitro, clinical data suggest that TIMP-2 expression is positively associated with tumor recurrence, thus emphasizing the wide-ranging role of TIMP-2 in malignancies. To shed light on this role of TIMP-2, we report that low concentrations of TIMP-2, by interacting with MT1-MMP (a specific membrane receptor of TIMP-2), induce the MEK/ERK signaling cascade in fibrosarcoma HT1080 cells which express MT1-MMP naturally. TIMP-2 binding with cell surface-associated MT1-MMP stimulates phosphorylation of MEK1/2, which is upstream of ERK1/2, and the ERK1/2 substrate p90RSK. Consistent with volumes of literature, we confirmed that the activation of ERK stimulated cell migration. Both the transcriptional silencing of MT1-MMP and the inhibition of MEK1/2 reversed the signaling effects of TIMP-2/MT1-MMP while the active site-targeting MMP inhibitor GM6001 did not. Our data suggest that both the interactions of TIMP-2 with MT1-MMP, which activate the pro-migratory ERK signaling cascade,and the conventional inhibition of MT1-MMP's catalytic activity by TIMP-2, play a role in the invasion-promoting function of MT1-MMP. The TIMP-2-induced stimulation of ERK signaling in cancer cells explains the direct, as opposed to the inverse, association of TIMP-2 expression with poor prognosis in cancer.
Asunto(s)
Movimiento Celular/fisiología , Metaloproteinasa 14 de la Matriz/metabolismo , Invasividad Neoplásica , Transducción de Señal/fisiología , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Western Blotting , Línea Celular Tumoral , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Fosforilación , ARN Interferente PequeñoRESUMEN
Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; DeltaCT) MT1-MMP-FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and DeltaCT constructs. The WT and E240A constructs also interacted with alpha-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the DeltaCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2-FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Metabolismo Energético , Femenino , Humanos , Péptido Hidrolasas/metabolismo , Mapeo de Interacción de ProteínasRESUMEN
To determine the target of the recently identified lead compound NSC130362 that is responsible for its selective anti-cancer efficacy and safety in normal cells, structure-activity relationship (SAR) studies were conducted. First, NSC13062 was validated as a starting compound for the described SAR studies in a variety of cell-based viability assays. Then, a small library of 1,4-naphthoquinines (1,4-NQs) and quinoline-5,8-diones was tested in cell viability assays using pancreatic cancer MIA PaCa-2 cells and normal human hepatocytes. The obtained data allowed us to select a set of both non-toxic compounds that preferentially induced apoptosis in cancer cells and toxic compounds that induced apoptosis in both cancer and normal cells. Anti-cancer activity of the selected non-toxic compounds was confirmed in viability assays using breast cancer HCC1187 cells. Consequently, the two sets of compounds were tested in multiple cell-based and in vitro activity assays to identify key factors responsible for the observed activity. Inhibition of the mitochondrial electron transfer chain (ETC) is a key distinguishing activity between the non-toxic and toxic compounds. Finally, we developed a mathematical model that was able to distinguish these two sets of compounds. The development of this model supports our conclusion that appropriate quantitative SAR (QSAR) models have the potential to be employed to develop anti-cancer compounds with improved potency while maintaining non-toxicity to normal cells.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neoplasias/patología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Modelos Moleculares , Modelos Teóricos , Neoplasias/tratamiento farmacológico , Relación Estructura-Actividad Cuantitativa , Células Tumorales CultivadasRESUMEN
Neoplasms have developed strategies to protect themselves against the complement-mediated host immunity. Invasion- and metastasis-promoting membrane type-1 (MT1) matrix metalloproteinase (MMP) is strongly associated with many metastatic cancer types. The relative importance of the individual functions of MT1-MMP in metastasis was, however, unknown. We have now determined that the expression of murine MT1-MMP in murine melanoma B16F1 cells strongly increased the number of metastatic loci in the lungs of syngeneic C57BL/6 mice. In contrast, MT1-MMP did not affect the number of metastatic loci in complement-deficient C57BL/6-C3-/- mice. Our results indicated, for the first time, that the anticomplement activity of MT1-MMP played a significant role in promoting metastasis in vivo and determined the relative importance of the anticomplement activity in the total metastatic effect of this multifunctional proteolytic enzyme. We believe that our results shed additional light on the functions of MT1-MMP in cancer and clearly make this protease a promising drug target in metastatic malignancies.
Asunto(s)
Complemento C3/inmunología , Proteínas Inactivadoras de Complemento/inmunología , Metaloproteinasas de la Matriz/inmunología , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/inmunología , Animales , Complemento C3/deficiencia , Complemento C3/genética , Proteínas Inactivadoras de Complemento/genética , Proteínas Inactivadoras de Complemento/metabolismo , Fibrosarcoma/enzimología , Fibrosarcoma/genética , Fibrosarcoma/inmunología , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Melanoma Experimental/enzimología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , TransfecciónRESUMEN
An elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with multiple malignancies. Recently, we discovered that recycled MT1-MMP was trafficked along the tubulin cytoskeleton into the centrosomal compartment and cleaved the integral centrosomal protein pericentrin-2. These events correlated with the induction of chromosome instability and aneuploidy in nonmalignant Madine-Darby canine kidney cells. Accordingly, we hypothesized that MT1-MMP is an oncogene that promotes malignant transformation of normal cells rather than just an enzyme that supports growth of preexisting tumors. To prove our hypothesis, we transfected normal 184B5 human mammary epithelial cells with MT1-MMP (184B5-MT1 cells). MT1-MMP was colocalized with pericentrin in the centrosomal compartment and especially in the midbody of dividing cells. 184B5-MT1 cells acquired the ability to activate MMP-2, to cleave pericentrin, and to invade the Matrigel matrix. 184B5-MT1 cells exhibited aneuploidy, and they were efficient in generating tumors in the orthotopic xenograft model in immunodeficient mice. Because of the absence of tumor angiogenesis and the resulting insufficient blood supply, the tumors then regressed with significant accompanying necrosis. Gene array studies confirmed a significant up-regulation of oncogenes and tumorigenic genes but not the angiogenesis-promoting genes in 184B5-MT1 cells. We believe that our data point to a novel function of MT1-MMP in the initial stages of malignant transformation and to new and hitherto unknown transition mechanism from normalcy to malignancy.
Asunto(s)
Aneuploidia , Mama/enzimología , Neoplasias Mamarias Experimentales/etiología , Metaloproteinasa 14 de la Matriz/fisiología , Animales , Mama/ultraestructura , Línea Celular , Inestabilidad Cromosómica , Células Epiteliales/enzimología , Células Epiteliales/ultraestructura , Femenino , Humanos , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Estrogens have many cellular functions, including their interactions with estrogen receptors alpha and beta (ERalpha and ERbeta). Earlier, we determined that the estrogen-ER complex stimulates the transcriptional activity of the matrix metalloproteinase 26 (MMP-26) gene promoter. We then determined that ERbeta is susceptible to MMP-26 proteolysis whereas ERalpha is resistant to the protease. MMP-26 targets the NH(2)-terminal region of ERbeta coding for the divergent NH(2)-terminal A/B domain that is responsible for the ligand-independent transactivation function. As a result, MMP-26 proteolysis generates the COOH-terminal fragments of ERbeta. Immunohistochemical analysis of tissue microarrays derived from 121 cancer patients corroborated these data and revealed an inverse correlation between the ERalpha-dependent expression of MMP-26 and the levels of the intact ERbeta in breast carcinomas. MMP-26 is not expressed in normal mammary epithelium. The levels of MMP-26 are strongly up-regulated in ductal carcinoma in situ (DCIS). In the course of further disease progression through stages I to III, the expression of MMP-26 decreases. In contrast to many tumor-promoting MMPs, the expression of MMP-26 in DCIS correlated with a longer patient survival. Our data suggest the existence of an MMP-26-mediated intracellular pathway that targets ERbeta and that MMP-26, a novel and valuable cancer marker, contributes favorably to the survival of the ERalpha/beta-positive cohort of breast cancer patients.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma Ductal de Mama/metabolismo , Receptor beta de Estrógeno/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Carcinoma in Situ/enzimología , Carcinoma in Situ/patología , Carcinoma Ductal de Mama/enzimología , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Humanos , Metaloproteinasas de la Matriz Secretadas , Estadificación de Neoplasias , Tasa de SupervivenciaRESUMEN
To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2740. Of the unique peptides eluted, more than 1750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, half of these immunogenic peptides were shared between different breast cancer cell lines. MHC class I binding probability was used to plot the distribution of the eluted peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines. SIGNIFICANCE: By employing proteomic analyses of eluted peptides from breast cancer cells, the current study has built an initial HLA-I-typed antigen collection for breast cancer research. It was also determined that immunogenic epitopes can be identified using established cell lines and that shared immunogenic peptides can be found in different cancer types such as breast cancer and leukemia. Importantly, out of 3196 eluted peptides that included duplicate peptides in different cells 89 peptides either contained mutation in their sequence or were derived from aberrant translation suggesting that mutation-containing epitopes are on the order of 2-3% in breast cancer cells. Finally, our results suggest that interfering with MHC class I function is one of the mechanisms of how tumor cells escape immune system attack.
Asunto(s)
Neoplasias de la Mama/inmunología , Antígenos de Histocompatibilidad Clase I/análisis , Secuencia de Aminoácidos , Presentación de Antígeno , Antígenos de Neoplasias , Neoplasias de la Mama/patología , Línea Celular Tumoral , Epítopos/genética , Antígenos HLA , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Mutación , Proteómica/métodosRESUMEN
It has been well established that invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP), a multifunctional membrane-tethered enzyme, functions in cancer cells as a mediator of pericellular proteolysis and directly cleaves several cell surface receptors, including CD44. In this report, we confirm that adhesion of diabetogenic T cells promotes the activation of endogenous MT1-MMP. Activated protease then cleaves CD44 in adherent T cells. We have validated that the T cell CD44 receptor is critical for the adhesion of diabetogenic insulin-specific, CD8-positive, K(d)-restricted cells to the matrix as well as for the subsequent transmigration of the adherent T cells through the endothelium and homing of the transmigrated T cells into the pancreatic islets. We have determined that the inhibition of MT1-MMP by low dosages of AG3340 (a subnanomolar range hydroxamate inhibitor of MMPs that has been widely tested in cancer patients) inhibited both T cell MT1-MMP activity and MT1-MMP-dependent shedding of CD44, immobilized T cells on the endothelium, repressed the homing of diabetogenic T cells into the pancreatic islets, reduced insulitis and mononuclear cell infiltration, and promoted either the recovery or the rejuvenation of the functional insulin-producing beta cells in diabetic NOD mice with freshly developed type I diabetes (IDDM). We believe our data constitute a mechanistic and substantive rationale for clinical trials of selected MT1-MMP inhibitors in the therapy of IDDM in humans.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/patología , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Animales , Adhesión Celular , Supervivencia Celular , Activación Enzimática , Citometría de Flujo , Receptores de Hialuranos/fisiología , Ratones , Ratones Endogámicos NODRESUMEN
Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9-12-mer peptides are potent inhibitors of WNV NS3 with K(i) values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.
Asunto(s)
Arginina/análisis , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/enzimología , Secuencia de Aminoácidos , Animales , Arginina/química , Células Cultivadas , Secuencia Conservada , Furina/química , Furina/metabolismo , Ratones , Datos de Secuencia Molecular , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Péptidos/química , Péptidos/farmacología , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/genética , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Serpinas/farmacología , Especificidad por Sustrato , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/aislamiento & purificación , Virus del Nilo Occidental/genéticaRESUMEN
Proteases exert control over cell behavior and affect many biological processes by making proteolytic modification of regulatory proteins. The purpose of this paper is to describe novel, important functions of matrix metalloproteinase (MMP)-26. alpha1-Antitrypsin (AAT) is a serpin, the primary function of which is to regulate the activity of neutrophil/leukocyte elastase. Insufficient antiprotease activity because of AAT deficiency in the lungs is a contributing factor to early-onset emphysema. We recently discovered that AAT is efficiently cleaved by a novel metalloproteinase, MMP-26, which exhibits an unconventional PH(81)CGVPD Cys switch motif and is autocatalytically activated in cells and tissues. An elevated expression of MMP-26 in macrophages and polymorphonuclear leukocytes supports the functional role of MMP-26 in the AAT cleavage and inflammation. We have demonstrated a direct functional link of MMP-26 expression with an estrogen dependency and confirmed the presence of the estrogen-response element in the MMP-26 promoter. Immunostaining of tumor cell lines and biopsy specimen microarrays confirmed the existence of the inverse correlations of MMP-26 and AAT in cells/tissues. An expression of MMP-26 in the estrogen-dependent neoplasms is likely to contribute to the inactivation of AAT, to the follow-up liberation of the Ser protease activity, and because of these biochemical events, to promote matrix destruction and malignant progression. In summary, we hypothesize that MMP-26, by cleaving and inactivating the AAT serpin, operates as a unique functional link that regulates a coordinated interplay between Ser and metalloproteinases in estrogen-dependent neoplasms.
Asunto(s)
Estrógenos/fisiología , Metaloproteinasas de la Matriz/fisiología , Neoplasias Hormono-Dependientes/enzimología , Neoplasias/enzimología , alfa 1-Antitripsina/metabolismo , Línea Celular Tumoral , Humanos , Macrófagos/enzimología , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/aislamiento & purificación , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Secretadas , Modelos Moleculares , Neoplasias/genética , Neutrófilos/enzimología , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin. PA is responsible for interacting with cell receptors and for the subsequent translocation of LF inside the cell compartment. A re-engineered toxin comprised of PA and a fusion chimera LF/Pseudomonas exotoxin (FP59) is a promising choice for tumor cell surface targeting. We demonstrated, however, that in vitro in cell-free system and in cultured human colon carcinoma LoVo, fibrosarcoma HT1080 and glioma U251 cells membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves both the PA83 precursor and the PA63 mature protein. Exhaustive MT1-MMP cleavage of PA83 in vitro generates several major degradation fragments with an N-terminus at Glu40, Leu48, and Gln512. In cultured cells, MT1-MMP-dependent cleavage releases the cell-bound PA83 and PA63 species from the cell surface. As a result, MT1-MMP expressing cells have less PA63 to internalize. In agreement, our observations demonstrate that MT1-MMP proteolysis of PA makes the MT1-MMP-expressing aggressive invasive cells resistant to the cytotoxic effect of a bipartite PA/FP59 toxin. We infer from our studies that synthetic inhibitors of MMPs are likely to increase the therapeutic anti-cancer effect of anthrax toxin. In addition, our study supports a unique role of furin in the activation of PA, thereby suggesting that furin inhibitors are the likely specific drugs for short-term therapy of anthrax infection.
Asunto(s)
Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Metaloendopeptidasas/metabolismo , Neoplasias/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Antígenos Bacterianos/genética , Antígenos Bacterianos/uso terapéutico , Toxinas Bacterianas/genética , Toxinas Bacterianas/uso terapéutico , Línea Celular Tumoral , Exotoxinas/genética , Exotoxinas/uso terapéutico , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Evidence suggests that stimulating apoptosis in malignant cells without inflicting collateral damage to the host's normal tissues is a promising cancer therapy. Chemo- and radiation therapies that, especially if combined, induce apoptosis in tumor cells have been used for treating cancer patients for decades. These treatments, however, are limited in their ability to discriminate between malignant and non-malignant cells and, therefore, produce substantial healthy tissue damage and subsequent toxic side-effects. In addition, as a result of these therapies, many tumor types acquire an apoptosis-resistant phenotype and become more aggressive and metastatic. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been considered a promising and reliable selective inducer of apoptosis in cancerous cells. TRAIL, however, is not uniformly effective in cancer and multiple cancer cell types are considered resistant to natural TRAIL. To overcome this deficiency of TRAIL, we have earlier constructed a yeast-human hybrid leucine zipper-TRAIL in which the yeast GCN4-pII leucine zipper was fused to human TRAIL (GCN4-TRAIL). This construct exhibited a significantly improved anti-tumor apoptotic activity and safety, but is potentially immunogenic in humans. Here, we report a novel, potent, and fully human ATF7 leucine zipper-TRAIL (ATF7-TRAIL) fusion construct that is expected to have substantially lower immunogenicity. In solution, ATF7-TRAIL exists solely as a trimer with a Tm of 80°C and is active against cancer cells both in vitro and in vivo, in a mouse tumor xenograft model. Our data suggest that our re-engineered TRAIL is a promising candidate for further evaluation as an antitumor agent.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucina Zippers/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Factores de Transcripción Activadores/química , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes de Fusión/química , Ligando Inductor de Apoptosis Relacionado con TNF/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.
Asunto(s)
Apoptosis/efectos de los fármacos , Glutatión Reductasa/metabolismo , Ensayos Analíticos de Alto Rendimiento , Estrés Oxidativo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Descubrimiento de Drogas , Glutatión/metabolismo , Glutatión Reductasa/antagonistas & inhibidores , Humanos , Ratones , Especies Reactivas de Oxígeno , Bibliotecas de Moléculas PequeñasRESUMEN
Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. The current suboptimal efficiency and selectivity drugs have therapeutic limitations and induce concomitant side effects. Recently, novel cancer therapies based on the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have emerged. TRAIL, a key component of the natural antitumor immune response, selectively kills many tumor cell types. Earlier studies with recombinant TRAIL, however, revealed its many shortcomings including a short half-life, off-target toxicity, and existence of TRAIL-resistant tumor cells. We improved the efficacy of recombinant TRAIL by redesigning its structure and the expression and purification procedures. The result is a highly stable leucine zipper (LZ)-TRAIL chimera that is simple to produce and purify. This chimera functions as a trimer in a manner that is similar to natural TRAIL. The formulation of the recombinant LZ-TRAIL we have developed has displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. Our results have shown that the half-life of LZ-TRAIL is improved and now exceeds 1 h in mice compared with a half-life of only minutes reported earlier for recombinant TRAIL. We have concluded that our LZ-TRAIL construct will serve as a foundation for a new generation of fully human LZ-TRAIL proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.
Asunto(s)
Leucina Zippers/genética , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/metabolismo , Neoplasias/patología , Ingeniería de Proteínas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Invasion-promoting MT1-MMP is directly linked to tumorigenesis and metastasis. Our studies led us to identify those genes, the expression of which is universally linked to MT1-MMP in multiple tumor types. Genome-wide expression profiling of MT1-MMP-overexpressing versus MT1-MMP-silenced cancer cells and a further data mining analysis of the preexisting expression database of 190 human tumors of 14 cancer types led us to identify 11 genes, the expression of which correlated firmly and universally with that of MT1-MMP (P < 0.00001). These genes included regulators of energy metabolism (NNT), trafficking and membrane fusion (SLCO2A1 and ANXA7), signaling and transcription (NR3C1, JAG1, PI3K delta, and CK2 alpha), chromatin rearrangement (SMARCA1), cell division (STK38/NDR1), apoptosis (DAPK1), and mRNA splicing (SNRPB2). Our subsequent extensive analysis of cultured cells, tumor xenografts, and cancer patient biopsies supported our data mining. Our results suggest that transcriptional reprogramming of the specific downstream genes, which themselves are associated with tumorigenesis, represents a distinctive "molecular signature" of the proteolytically active MT1-MMP. We suggest that the transactivation activity of MT1-MMP contributes to the promigratory cell phenotype, which is induced by this tumorigenic proteinase. The activated downstream gene network then begins functioning in unison with MT1-MMP to rework the signaling, transport, cell division, energy metabolism, and other critical cell functions and to commit the cell to migration, invasion, and, consequently, tumorigenesis.
Asunto(s)
Metaloproteinasa 14 de la Matriz/genética , Neoplasias/genética , Activación Transcripcional , Apoptosis , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Metabolismo Energético , Perfilación de la Expresión Génica , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.