RESUMEN
In the present study, we have tested the hypothesis that fusion between an altered cell and a mesenchymal stem cell produces a hybrid cell with enhanced characteristics associated with metastatic cancer cells, and we have developed a flexible model for investigating the mechanisms of metastasis. Human HepG2 cells with low metastatic potential were induced to fuse with rat bone marrow mesenchymal stem cells, and the progeny were compared with the parental cells for possession of enhanced in vitro and in vivo characteristics of malignant cells. Compared to the parental cells, the fused cells exhibited enhanced expression of E-cadherin, vimentin, Twist, Snail, matrix metalloproteinase 2 and 9 activities, aneuploidy and enhanced in vitro invasion and migration. In an in vivo xenograft assay, the fused cells generated increased numbers of metastatic liver and lung lesions. This model system is a flexible tool for investigation of the mechanisms of stem cell fusion in carcinogenesis and metastasis and for the discovery of new therapeutic targets to inhibit metastasis.
Asunto(s)
Neoplasias Hepáticas/patología , Neoplasias Pulmonares/patología , Células Madre Mesenquimatosas/metabolismo , Aneuploidia , Animales , Células de la Médula Ósea/citología , Fusión Celular , Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Ratas , Ratas Sprague-DawleyRESUMEN
PURPOSE: The goal was to determine if prostate tumor cells containing a mutant alpha6 integrin would be defective in tumor re-population following clinically relevant fractionated ionizing radiation (IR) treatments. MATERIAL AND METHODS: Human prostate cancer cells derived from PC3N cells were used which conditionally expressed a cleavable, wild type form of alpha6 integrin (PC3N-alpha6-WT) or a mutated non-cleavable form of alpha6 integrin (PC3N-alpha6-RR). The resulting tumor growth before, during and after fractionated doses of IR (3 Gyx10 days) was analyzed using the endpoints of tumor growth inhibition (T/C), tumor growth delay (T-C), tumor doubling time (Td) and tumor cell kill (Log(10) cell kill). RESULTS: The T/C values were 36.1% and 39.5%, the T-C values were 20.5 days and 28.5 days and the Td values were 5.5 and 10.5 days for the irradiated PC3N-alpha6-WT and PC3N-alpha6-RR cells, respectively. The Log(10) was 1.1 for the PC3N-alpha6-WT cells and 0.8 for the PC3N-alpha6-RR cells. The tumor response to IR was altered in tumors expressing the mutant alpha6 integrin as indicated by a significant increase in tumor growth inhibition, an increase in tumor growth delay, an increase in tumor doubling time and an increase in tumor cell kill. CONCLUSIONS: Blocking integrin cleavage in vivo may be efficacious for increasing the IR responsiveness of slow growing, pro-metastatic human prostate cancer.
Asunto(s)
Integrina alfa6/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/radioterapia , Animales , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN/genética , Humanos , Integrina alfa6/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Mutación , Trasplante de Neoplasias , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Tolerancia a Radiación/genética , Tolerancia a Radiación/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Trasplante HeterólogoRESUMEN
Human prostate tumor cell invasion and metastasis are dependent in part on cell adhesion to extracellular matrix proteins and cell migration. We previously identified a synthetic D-amino acid tumor cell adhesion peptide called HYD1 (kikmviswkg) that supported adhesion of tumor cells derived from breast, prostate, ovary and pancreas tissue. Alanine substitution analysis and a peptide deletion strategy were used to determine the minimal element of HYD1 necessary for bioactivity in a prostate cancer cell line called PC3N. Bioactivity was measured by assays of cell adhesion, migration and ERK signaling. The most potent element of HYD1 necessary to support cell adhesion was kmvixw, the block to migration required xkmviswxx and activation of ERK signaling required ikmviswxx. The shortest sequence active in all three assays was iswkg. The HYD1 peptide contains overlapping elements required for adhesion, blocking migration and the activation of ERK signaling. These linear peptide sequences provide the starting point for development of novel compounds to target cancer cell adhesion and migration.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Oligopéptidos/farmacología , Neoplasias de la Próstata/fisiopatología , Secuencia de Aminoácidos , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Masculino , Oligopéptidos/síntesis química , Oligopéptidos/química , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Cell motility is partially dependent on interactions between the integrins and the extracellular matrix. Our previous studies have identified synthetic D-amino acid cell adhesion peptides using a combinatorial screening approach. In this study, we demonstrate that HYD1 (kikmviswkg) completely blocks random haptotactic migration and inhibits invasion of prostate carcinoma cells on laminin-5. This effect is adhesion independent and reversible. The inhibition of migration by HYD1 involves a dramatic remodeling of the actin cytoskeleton resulting in increased stress fiber formation and actin colocalization with cortactin at the cell membrane. HYD1 interacts with alpha6beta1 (not alpha6beta4) and alpha3beta1 integrins and surprisingly elevates laminin-5-dependent intracellular signals including focal adhesion kinase, mitogen-activated protein kinase kinase and extracellular signal-regulated kinase. HYD1 does not contain a previously characterized binding sequence for integrins. A scrambled derivative of HYD1, called HYDS (wiksmkivkg), does not interact with the alpha6 or alpha3 integrin subunits and is not biologically active. Taken together, these results indicate that HYD1 is a biologically active integrin-targeting peptide that reversibly inhibits tumor cell migration on laminin-5 and uncouples phosphotyrosine signaling from cytoskeletal-dependent migration.
Asunto(s)
Aminoácidos/farmacología , Moléculas de Adhesión Celular/metabolismo , Oligopéptidos/farmacología , Péptidos/farmacología , Actinas/química , Aminoácidos/química , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Citoesqueleto/metabolismo , Humanos , Integrina alfa3beta1/metabolismo , Integrina alfa6beta1/metabolismo , Masculino , Oligopéptidos/química , Péptidos/química , Fosforilación , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , KalininaRESUMEN
During human prostate cancer progression, the integrin alpha6beta1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases. We previously identified a novel structural variant of the alpha6 integrin called alpha6p. This variant was produced on the cell surface and was missing the beta-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen activator (uPA) function-blocking antibody (3689), we showed that uPA, acting as a protease, is responsible for production of alpha6p. We also showed that addition of uPA in the culture media of cells that do not produce alpha6p, resulted in a dose-dependent alpha6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using alpha2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the alpha6 integrin directly. Further, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of alpha6p, and this induction was abolished by PAI-1 but not alpha2-antiplasmin. Finally, the alpha6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon. These data, taken together, suggest that this is a novel function of uPA, that is, to remove the beta-barrel ligand-binding domain of the integrin while preserving its heterodimer association.