RESUMEN
Bladder, colon, gastric, prostate, and uterine cancers originate in organs surrounded by laminin-coated smooth muscle. In human prostate cancer, tumors that are organ confined, without extracapsular extension through muscle, have an overall cancer survival rate of up to 97% compared with 32% for metastatic disease. Our previous work modeling extracapsular extension reported the blocking of tumor invasion by mutation of a laminin-binding integrin called α6ß1. Expression of the α6AA mutant resulted in a biophysical switch from cell-ECM (extracellular matrix) to cell-cell adhesion with drug sensitivity properties and an inability to invade muscle. Here we used different admixtures of α6AA and α6WT cells to test the cell heterogeneity requirements for muscle invasion. Time-lapse video microscopy revealed that tumor mixtures self-assembled into invasive networks in vitro, whereas α6AA cells assembled only as cohesive clusters. Invasion of α6AA cells into and through live muscle occurred using a 1:1 mixture of α6AA and α6WT cells. Electric cell-substrate impedance sensing measurements revealed that compared with α6AA cells, invasion-competent α6WT cells were 2.5-fold faster at closing a cell-ECM or cell-cell wound, respectively. Cell-ECM rebuilding kinetics show that an increased response occurred in mixtures since the response was eightfold greater compared with populations containing only one cell type. A synthetic cell adhesion cyclic peptide called MTI-101 completely blocked electric cell-substrate impedance sensing cell-ECM wound recovery that persisted in vitro up to 20 h after the wound. Treatment of tumor-bearing animals with 10 mg/kg MTI-101 weekly resulted in a fourfold decrease of muscle invasion by tumor and a decrease of the depth of invasion into muscle comparable to the α6AA cells. Taken together, these data suggest that mixed biophysical phenotypes of tumor cells within a population can provide functional advantages for tumor invasion into and through muscle that can be potentially inhibited by a synthetic cell adhesion molecule.
Asunto(s)
Extensión Extranodal , Laminina , Masculino , Animales , Humanos , Laminina/química , Laminina/genética , Laminina/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Adhesión Celular , Músculos/metabolismo , FenotipoRESUMEN
Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual ) of 3.25 min-1 , threefold faster than α3 integrin (1.0 min-1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min-1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min-1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6ß4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6ß1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Integrina alfa3/metabolismo , Integrina alfa6/metabolismo , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Movimiento Celular , Endosomas/genética , Endosomas/metabolismo , Silenciador del Gen , Humanos , Integrina alfa3/genética , Integrina alfa6/genética , Masculino , Neoplasias de la Próstata/genética , Transporte de ProteínasRESUMEN
Human pancreatic and prostate cancers metastasize along nerve axons during perineural invasion. The extracellular matrix laminin class of proteins is an abundant component of both myelinated and non-myelinated nerves. Analysis of human pancreatic and prostate tissue revealed both perineural and endoneural invasion with Schwann cells surrounded or disrupted by tumor, respectively. Tumor and nerve cell co-culture conditions were used to determine if myelinating or non-myelinating Schwann cell (S16 and S16Y, respectively) phenotype was equally likely to promote integrin-dependent cancer cell invasion and migration on laminin. Conditioned medium from S16 cells increased tumor cell (DU145, PC3, and CFPAC1) invasion into laminin approximately 1.3-2.0 fold compared to fetal bovine serum (FBS) treated cells. Integrin function (e.g., ITGA6p formation) increased up to 1.5 fold in prostate (DU145, PC3, RWPE-1) and pancreatic (CFPAC1) cells, and invasion was dependent on ITGA6p formation and ITGB1 as determined by function-blocking antibodies. In contrast, conditioned medium isolated from S16Y cells (non-myelinating phenotype) decreased constitutive levels of ITGA6p in the tumor cells by 50% compared to untreated cells and decreased ITGA6p formation 3.0 fold compared to S16 treated cells. Flow cytometry and western blot analysis revealed loss of ITGA6p formation as reversible and independent of overall loss of ITGA6 expression. These results suggest that the myelinating phenotype of Schwann cells within the tumor microenvironment increased integrin-dependent tumor invasion on laminin.
Asunto(s)
Integrina alfa6/metabolismo , Integrina beta1/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/patología , Células de Schwann/fisiología , Línea Celular Tumoral , Movimiento Celular , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Invasividad Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias de la Próstata/metabolismoRESUMEN
Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent "inside-out" signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent "inside-out" signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype.
Asunto(s)
Actinas/genética , Neoplasias de la Mama/genética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Integrina alfa6/genética , Neoplasias de la Próstata/genética , Proteínas Serina-Treonina Quinasas/genética , Actinas/metabolismo , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Variación Genética , Humanos , Integrina alfa6/análisis , Integrina alfa6/metabolismo , Masculino , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Regulación hacia ArribaRESUMEN
In 2023, approximately 288,300 new diagnoses of prostate cancer will occur, with 34,700 disease-related deaths. Death from prostate cancer is associated with metastasis, enabled by progression of tumor phenotypes and successful extracapsular extension to reach Batson's venous plexus, a specific route to the spine and brain. Using a mouse-human tumor xenograft model, we isolated an aggressive muscle invasive cell population of prostate cancer, called DU145J7 with a distinct biophysical phenotype, elevated histone H3K27, and increased matrix metalloproteinase 14 expression as compared to the non-aggressive parent cell population called DU145WT. Our goal was to determine the sensitivities to known chemotherapeutic agents of the aggressive cells as compared to the parent population. High-throughput screening was performed with 5,578 compounds, comprising of approved and investigational drugs for oncology. Eleven compounds were selected for additional testing, which revealed that vorinostat, 5-azacitidine, and fimepinostat (epigenetic inhibitors) showed 2.6-to-7.5-fold increases in lethality for the aggressive prostate cancer cell population as compared to the parent, as judged by the concentration of drug to inhibit 50% cell growth (IC50). On the other hand, the DU145J7 cells were 2.2-to-4.0-fold resistant to mitoxantrone, daunorubicin, and gimatecan (topoisomerase inhibitors) as compared to DU145WT. No differences in sensitivities between cell populations were found for docetaxel or pirarubicin. The increased sensitivity of DU145J7 prostate cancer cells to chromatin modifying agents suggests a therapeutic vulnerability occurs after tumor cells invade into and through muscle. Future work will determine which epigenetic modifiers and what combinations will be most effective to eradicate early aggressive tumor populations.
RESUMEN
Integrin α6ß4 is an essential, dynamic adhesion receptor for laminin 332 found on epithelial cells, required for formation of strong cell-extracellular matrix (ECM) adhesion and induced migration, and coordinated by regions of the ß4C cytoplasmic domain. ß4E, a unique splice variant of ß4 expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique sequence of 114 amino acids instead of ß4C's canonical 1089 amino acids. We determined the distribution of α6ß4E within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. Canonical α6ß4C expressed in all basal cells, as expected, while α6ß4E expressed within a subset of luminal cells. α6ß4E expression was induced by three-dimensional culture conditions, activated Src, was reversible, and was stabilized by bortezomib, a proteasome inhibitor. α6ß4C expressed in all cells during induced migration, whereas α6ß4E was restricted to a subset of cells with increased kinetics of cell-cell and cell-ECM resistance properties. Interestingly, α6ß4E presented in "ringlike" patterns measuring â¼1.75 × 0.72 microns and containing actin and CD9 at cell-ECM locations. In contrast, α6ß4C expressed only within hemidesmosome-like structures containing BP180. Integrin α6ß4E is an inducible adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Integrina alfa6beta4/fisiología , Actinas/metabolismo , Actinas/fisiología , Línea Celular Tumoral , Desmosomas/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Hemidesmosomas/metabolismo , Humanos , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Isoformas de Proteínas , Tetraspanina 29/metabolismo , Tetraspanina 29/fisiologíaRESUMEN
Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6ß1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 µmol/L S1P challenge as compared with the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific α6 integrin extracellular region.
Asunto(s)
Comunicación Celular , Edición Génica , Integrina alfa6/genética , Neoplasias de los Músculos/patología , Neoplasias de la Próstata/patología , Animales , Apoptosis , Adhesión Celular , Movimiento Celular , Proliferación Celular , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Humanos , Integrina alfa6/química , Integrina alfa6/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de los Músculos/genética , Neoplasias de los Músculos/metabolismo , Invasividad Neoplásica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The aim of this study was to identify biomarkers that may be predictive for the clinical activity of the redox-active antitumor agent imexon. EXPERIMENTAL DESIGN: cDNA microarray and quantitative real-time PCR were used to identify global changes in gene expression in peripheral blood mononuclear cells (PBMC) collected from patients treated with imexon during a phase I trial. Electrophoretic mobility shift assays and Western blot analysis were done using the RPMI8226 myeloma cell line grown in vitro and PBMCs treated ex vivo to investigate the molecular mechanism responsible for these gene changes. RESULTS: Both cDNA microarray and quantitative real-time PCR showed the up-regulation of many antioxidant genes, including thioredoxin reductase-1, glutaredoxin-2, and peroxiredoxin-3 in PBMCs collected from patients treated with imexon. Studies in PBMCs treated ex vivo and RPMI8226 myeloma cells showed that imexon increased binding to the activator protein-1 consensus sequence measured by electrophoretic mobility shift assay. Supershift analysis showed that the majority of the activator protein-1 DNA binding activity was c-Jun, with minor contribution of Jun-D. Nuclear translocation of the nuclear factor (erythroid-derived 1)-like 2 transcription factor and its binding to the antioxidant response element was also increased after imexon treatment, which correlated with an increase in the message levels for nuclear factor (erythroid-derived 1)-like 2/antioxidant response element-regulated antioxidant genes. CONCLUSIONS: Together, these results show that a predominant biological effect of imexon is a change in redox state that can be detected in surrogate normal tissues as increased redox-sensitive transcription factor binding and increased antioxidant gene expression.
Asunto(s)
Antioxidantes/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hexanonas/farmacología , Biomarcadores de Tumor , Línea Celular Tumoral , Ensayos Clínicos como Asunto , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Leucocitos Mononucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción , Estrés Oxidativo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismoRESUMEN
The laminin-binding integrins, α3ß1 and α6ß1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. ß1 integrins share the Rab11 recycling machinery, but the trafficking of α3ß1 and α6ß1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6ß1 trafficking. Rab11-FIP5 and its membrane-binding domain were required for α6ß1 recycling, without affecting the other laminin-binding integrin (i.e., α3ß1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6ß1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6ß1 recycling in cell-cell locations. Taken together, these data demonstrate that α6ß1 is distinct from α3ß1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location.Implications: Rab11-FIP5-dependent α6ß1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Mol Cancer Res; 16(8); 1319-31. ©2018 AACR.
Asunto(s)
Integrina alfa5beta1/metabolismo , Neoplasias de la Próstata/genética , Proteínas de Unión al GTP rab/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/metabolismoRESUMEN
Chordoma is a rare, radiation-resistant, skull-base and spinal tumor with high local recurrence containing mixed cell-adhesion phenotypes. We characterized DNA damage response (DDR) signaling (γH2AX, pKAP1, pATM) and survival response to ionizing radiation (IR) in human chordoma samples (42 resections, 23 patients) to test if blocking cell adhesion sensitizes U-CH1 tumor cells to IR. U-CH1 cells expressed brachyury, YAP, and laminin adhesion receptors (CD49c, CD49f, CD44), and approximately 15% to 20% of U-CH1 cells featured an α6 integrin-dependent (CD49f) cohesive cluster phenotype, which confers therapeutic resistance and aids metastasis. DDR to IR in U-CH1 cells was compared to normal prostate epithelial (PrEC) and tumor cells (DU145). Flow cytometry showed a dose- and time-dependent increase in γH2AX and pKAP1 expression in all cell lines. However, nearly 50% of U-CH1 cells exhibited nonresponsive phenotype to IR (measured by γH2AX and pKAP1) independent of cell cycle status. Immunofluorescence microscopy verified that only 15% of U-CH1 clustered cells were γH2AX or pKAP1 positive (versus 80% of nonclustered cells) 2 hours following 2-Gy IR. Conversely, both tumor cell lines were uniformly defective in pATM response. HYD1, a synthetic ECM ligand, inhibited DDR through an unresolved γH2AX response. ß1 integrin-blocking antibody (AIIB2) decreased cell survival 50% itself and approximately doubled the IR-induced cell kill at all IR doses observed at 2 and 4 weeks posttreatment. These results suggest that a heterogeneity of DDR to IR exists within a chordoma population. Blocking integrin function alone and/or as an adjuvant to IR may eradicate chordomas containing the cohesive cluster phenotype.
Asunto(s)
Cordoma/metabolismo , Integrina beta1/metabolismo , Fenotipo , Radiación Ionizante , Sacro/metabolismo , Neoplasias de la Columna Vertebral/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Cordoma/patología , Cordoma/radioterapia , Humanos , Sacro/patología , Neoplasias de la Columna Vertebral/patología , Neoplasias de la Columna Vertebral/radioterapia , Resultado del TratamientoRESUMEN
Bone is a favored site for solid tumor metastasis, especially among patients with breast, lung or prostate carcinomas. Micro CT is a powerful and inexpensive tool that can be used to investigate tumor progression in xenograft models of human disease. Many previous studies have relied on terminal analysis of harvested bones to document metastatic tumor activity. The current protocol uses live animals and combines sequential micro CT evaluation of lesion development with matched histopathology at the end of the study. The approach allows for both rapid detection and evaluation of bone lesion progression in live animals. Bone resident tumors are established either by direct (intraosseous) or arterial (intracardiac) injection, and lesion development is evaluated for up to eight weeks. This protocol provides a clinically relevant method for investigating bone metastasis progression and the development of osteotropic therapeutic strategies for the treatment of bone metastases.
RESUMEN
Akt, a serine/threonine kinase that promotes cell survival, is activated by binding of its pleckstrin homology (PH) domain to membrane phosphatidylinositol (PtdIns)-3-phosphates formed by PtdIns-3-kinase. D-3-Deoxy-phosphatidyl-myo-inositols that cannot be phosphorylated on the 3-position of the myo-inositol group are inhibitors of the Akt PH domain. The most active compound is D-3-deoxy-phosphatidyl-myo-inositol 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate] (PX-316). PX-316 administered intraperitoneally to mice at 150 mg/kg inhibits Akt activation in HT-29 human tumor xenografts up to 78% at 10 h with recovery to 34% at 48 h. Phosphorylation of GSK-3beta, a downstream target of Akt, is also inhibited. There is no decrease in PtdIns(3,4,5)-trisphosphate levels by PX-316, showing it is not an inhibitor of PtdIns-3-K in vivo. Gene expression profiling of HT-29 tumor xenografts shows many similarities between the effects of PX-316 and the PtdIns-3-K inhibitor wortmannin, with downregulation of several ribosomal-related genes, while PX-316 uniquely increases the expression of a group of mitochondrial-related genes. PX-316 has antitumor activity against early human MCF-7 breast cancer and HT-29 colon cancer xenografts in mice. PX-316 formulated in 20% hydroxypropyl-beta-cyclodextrin for intravenous administration is well tolerated in mice and rats with no hemolysis and no hematological toxicity. Thus, PX-316 is the lead compound of a new class of potential agents that inhibit Akt survival signaling.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Fosfatos de Inositol/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , 2-Hidroxipropil-beta-Ciclodextrina , Androstadienos/farmacología , Animales , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Activación Enzimática , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Mitocondrias/metabolismo , Modelos Químicos , Modelos Moleculares , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt , Serina/química , Transducción de Señal , Factores de Tiempo , Wortmanina , beta-Ciclodextrinas/químicaRESUMEN
The mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase signaling cascades have been implicated in a number of human cancers. The tumor suppressor gene tuberous sclerosis-2 (Tsc-2) functions as a negative regulator of mTOR. Critical proteins in both pathways are activated following treatment of Eker rats (Tsc-2(EK/+)) with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ), which also results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. Western blot analysis of kidney tumors formed following treatment of Tsc-2(EK/+) rats with TGHQ for 8 months revealed increases in B-Raf, Raf-1, pERK, cyclin D1, 4EBP1, and p-4EBP1-Ser65, -Thr70, and -Thr37/46 expression. Similar changes are observed following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (quinol-thioether rat renal epithelial [QTRRE] cells) that are also null for tuberin. These cells exhibit high ERK, B-Raf, and Raf-1 kinase activity and increased expression of all p-4EBP1s and cyclin D1. Treatment of the QTRRE cells with the Raf kinase inhibitor, sorafenib, or the MEK1/2 kinase inhibitor, PD 98059, produced a significant decrease in the protein expression of all p-4EBP1s and cyclin D1. Following siRNA knockdown of Raf-1, Western blot analysis revealed a significant decrease in Raf-1, cyclin D1, and all p-4EBP1 forms noted above. In contrast, siRNA knockdown of B-Raf resulted in a nominal change in these proteins. The data indicate that Raf-1/MEK/ERK participates in crosstalk with 4EBP1, which represents a novel pathway interaction leading to increased protein synthesis, cell growth, and kidney tumor formation.
Asunto(s)
Carcinoma de Células Renales/inducido químicamente , Proteínas Portadoras/metabolismo , Ciclina D1/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glutatión/análogos & derivados , Hidroquinonas/toxicidad , Neoplasias Renales/inducido químicamente , Fosfoproteínas/metabolismo , Receptor Cross-Talk , Proteínas Supresoras de Tumor/genética , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Proteínas Portadoras/genética , Técnicas de Cultivo de Célula , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Ciclina D1/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Glutatión/toxicidad , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Pérdida de Heterocigocidad , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Fosfoproteínas/genética , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf , ARN Interferente Pequeño/genética , Ratas , Ratas Mutantes , Receptor Cross-Talk/efectos de los fármacos , Transducción de Señal , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
The laminin-binding integrin α6ß1 plays a major role in determining the aggressive phenotype of tumor cells during metastasis. Our previous work has shown that cleavage of the α6ß1 integrin to produce the structural variant α6pß1 on tumor cell surfaces is mediated by the serine protease urokinase plasminogen activator (uPA). Cleavage of α6ß1 increases tumor cell motility, invasion, and prostate cancer metastasis, and blockage of uPA inhibits α6pß1 production. In human tumors, uPA and uPAR are expressed in tumor cells and tumor-associated macrophages (TAM). TAMs localize to solid tumors and contribute to increased tumor growth and the metastatic phenotype. In this study, we utilized a coculture system of PC-3 prostate tumor cells and macrophages [12-O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human leukemia HL-60 cells] to investigate the hypothesis that macrophages stimulate the production of the prometastatic variant α6pß1 on human prostate cancer cells via the uPA/uPAR axis. Our results indicate that adherent macrophages cocultured with PC-3 cells increased PC-3 uPAR mRNA, uPAR cell surface protein expression and α6 integrin cleavage. The stimulation does not require macrophage/tumor cell contact because macrophage conditioned medium is sufficient for increased uPAR transcription and α6 cleavage-dependent PC-3 cell invasion. The increased cleavage was dependent on uPAR because production was blocked by silencing RNA-targeting uPAR. These results indicate that macrophages can stimulate uPA/uPAR production in tumor cells which results in α6 integrin cleavage. These data suggest that TAMs promote prometastatic integrin-dependent pericellular proteolysis.
Asunto(s)
Integrina alfa6beta1/metabolismo , Macrófagos/metabolismo , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Células HL-60 , Humanos , Macrófagos/patología , Masculino , Invasividad Neoplásica , Neoplasias de la Próstata/patología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Existing clinical imaging procedures lack sensitivity and specificity in detecting early prostate cancer bone metastatic lesions. In this study, we developed a highly reproducible bone metastasis xenograft model and identified possible molecular imaging candidates for detecting early bone metastatic lesions. Bone trophic human prostate cells (PC-3B1) were isolated and characterized for their ability to reach bone after intracardiac injection into SCID mice. The appearances of skeletal metastases were evaluated using digital radiographic imaging and confirmed by necropsy and histology. The PC-3B1 cells retain a bone homing phenotype after long term propagation in tissue culture and exhibit progressive bone lesions within 3 weeks following intracardiac injection. Comparative transcription signatures of PC-3 and PC-3B1 cells were determined using a cancer specific microarray and confirmed by RT-PCR analysis. The analysis identified increased expression of four cell surface molecules in PC-3B1 cells that may be suitable as molecular imaging candidates to detect bone micro metastases.