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Background and rationale: Liver derived messenger ribonucleic acid (mRNA) transcripts were reported to be elevated in the circulation of hepatocellular carcinoma (HCC) patients. We now report the detection of high-risk mRNA variants exclusively in the circulation of HCC patients. Numerous genomic alleles such as single nucleotide polymorphisms (SNPs), nucleotide insertions and deletions (called Indels), splicing variants in many genes, have been associated with elevated risk of cancer. Our findings potentially offer a novel non-invasive platform for HCC surveillance and early detection. Approach: RNAseq analysis was carried out in the plasma of 14 individuals with a diagnosis of HCC, 8 with LC and no HCC, and 6 with no liver disease diagnosis. RNA from 6 matching tumors and 5 circulating extracellular vesicle (EV) samples from 14 of those with HCC was also analyzed. Specimens from two cholangiocarcinoma (CCA) patients were also included in our study. HCC specific SNPs and Indels referred as "variants" were identified using GATK HaplotypeCaller and annotated by SnpEff to filter out high risk variants. Results: The variant calling on all RNA samples enabled the detection of 5.2 million SNPs, 0.91 million insertions and 0.81 million deletions. RNAseq analyses in tumors, normal liver tissue, plasma, and plasma derived EVs led to the detection of 5480 high-risk tumor specific mRNA variants in the circulation of HCC patients. These variants are concurrently detected in tumors and plasma samples or tumors and EVs from HCC patients, but none of these were detected in normal liver, plasma of LC patients or normal healthy individuals. Our results demonstrate selective detection of concordant high-risk HCC-specific mRNA variants in free plasma, plasma derived EVs and tumors of HCC patients. The variants comprise of splicing, frameshift, fusion and single nucleotide alterations and correspond to cancer and tumor metabolism pathways. Detection of these high-risk variants in matching specimens from same subjects with an enrichment in circulating EVs is remarkable. Validation of these HCC selective ctmRNA variants in larger patient cohorts is likely to identify a predictive set of ctmRNA with high diagnostic performance and thus offer a novel non-invasive serology-based biomarker for HCC.
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Cells are known to release different types of vesicles such as small extracellular vesicles (sEVs) and large extracellular vesicles (LEVs). sEVs and LEVs play important roles in intercellular communication, pre-metastatic niche formation, and disease progression; both can be detected cell culture media and biological fluids. sEVs and LEVs contain a variety of protein and RNA cargo, and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins. It has recently been established that standard isolation techniques, such as differential ultracentrifugation, yield a mixed population of EVs. However, density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris. Here, we describe the most common methods used to isolate sEVs from cell culture medium, mouse and human plasma, and a new technique for isolating sEVs from tissues as well. This article also provides detailed procedures to isolate LEVs.
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The human circulation contains cell-free DNA and non-coding microRNA (miRNA). Less is known about the presence of messenger RNA (mRNA). This report profiles the human circulating mRNA transcriptome in people with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) to determine whether mRNA analytes can be used as biomarkers of liver disease. Using RNAseq and RT-qPCR, we investigate circulating mRNA in plasma from HCC and LC patients and demonstrate detection of transcripts representing more than 19,000 different protein coding genes. Remarkably, the circulating mRNA expression levels were similar from person to person over the 21 individuals whose samples were analyzed by RNAseq. Liver derived circulating transcripts such as albumin (ALB), apolipoprotein (APO) A1, A2 & H, serpin A1 & E1, ferritin light chain (FTL) and fibrinogen like 1 (FGL1) were significantly upregulated in HCC patient samples. Higher levels of some of these liver-specific transcripts in the plasma of HCC patients were confirmed by RT-qPCR in another cohort of 20 individuals. Several less abundant circulating transcripts associated with cancer were detected in most HCC samples, but not in healthy subjects. Liver specificity of circulating transcripts was confirmed by investigating their expression in HCC tumor and liver cancer cell lines. Liver specific mRNA sequences in the plasma were predominantly present outside circulating extracellular vesicles. Conclusions: The circulating "mRNA" transcriptome is remarkably consistent in diversity and expression from person to person. Detection of transcripts corresponding to disease selective polypeptides suggests the possibility that circulating mRNA can work as a biomarker analyte for cancer detection.
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The ß1 integrins, known to promote cancer progression, are abundant in extracellular vesicles (EVs). We investigated whether prostate cancer (PrCa) EVs affect anchorage-independent growth and whether ß1 integrins are required for this effect. Specifically using a cell-line-based genetic rescue and an in vivo PrCa model, we show that gradient-purified small EVs (sEVs) from either cancer cells or blood from tumor-bearing TRAMP (transgenic adenocarcinoma of the mouse prostate) mice promote anchorage-independent growth of PrCa cells. In contrast, sEVs from cultured PrCa cells harboring a short hairpin RNA to ß1, from wild-type mice or from TRAMP mice carrying a ß1 conditional ablation in the prostatic epithelium (ß1pc-/-), do not. We find that sEVs, from cancer cells or TRAMP blood, are functional and co-express ß1 and sEV markers; in contrast, sEVs from ß1pc-/-/TRAMP or wild-type mice lack ß1 and sEV markers. Our results demonstrate that ß1 integrins in tumor-cell-derived sEVs are required for stimulation of anchorage-independent growth.
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The αvß3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvß3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvß3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvß3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvß3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to ß3-negative recipient cells. We also demonstrate the intracellular localization of ß3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvß3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvß3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvß3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvß3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvß3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvß3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.
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Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Exosomas/metabolismo , Integrina alfaVbeta3/genética , Neoplasias de la Próstata/genética , Adenocarcinoma/sangre , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos/farmacología , Benzamidas , Biomarcadores de Tumor/sangre , Exosomas/química , Expresión Génica , Humanos , Integrina alfaVbeta3/sangre , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Nitrilos , Células PC-3 , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Tetraspanina 29/sangre , Tetraspanina 29/genética , Tetraspanina 30/sangre , Tetraspanina 30/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Radiation therapy is an effective cancer treatment modality although tumors invariably become resistant. Using the transgenic adenocarcinoma of mouse prostate (TRAMP) model system, we report that a hypofractionated radiation schedule (10 Gy/day for 5 consecutive days) effectively blocks prostate tumor growth in wild type (ß1wt /TRAMP) mice as well as in mice carrying a conditional ablation of ß1 integrins in the prostatic epithelium (ß1pc-/- /TRAMP). Since JNK is known to be suppressed by ß1 integrins and mediates radiation-induced apoptosis, we tested the effect of SP600125, an inhibitor of c-Jun amino-terminal kinase (JNK) in the TRAMP model system. Our results show that SP600125 negates the effect of radiation on tumor growth in ß1pc-/- /TRAMP mice and leads to invasive adenocarcinoma. These effects are associated with increased focal adhesion kinase (FAK) expression and phosphorylation in prostate tumors in ß1pc-/- /TRAMP mice. In marked contrast, radiation-induced tumor growth suppression, FAK expression and phosphorylation are not altered by SP600125 treatment of ß1wt /TRAMP mice. Furthermore, we have reported earlier that abrogation of insulin-like growth factor receptor (IGF-IR) in prostate cancer cells enhances the sensitivity to radiation. Here we further explore the ß1/IGF-IR crosstalk and report that ß1 integrins promote cell proliferation partly by enhancing the expression of IGF-IR. In conclusion, we demonstrate that ß1 integrin-mediated inhibition of JNK signaling modulates tumor growth rate upon hypofractionated radiation.
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Adenocarcinoma/radioterapia , Integrina beta1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias de la Próstata/radioterapia , Hipofraccionamiento de la Dosis de Radiación , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Animales , Antracenos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Terapia Combinada , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Integrina beta1/genética , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Masculino , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Carga Tumoral/efectos de la radiaciónRESUMEN
Transforming growth factor (TGF) ß1 activity depends on a complex signalling cascade that controls expression of several genes. Among others, TGFß1 regulates expression of matrix metalloproteinases (MMPs) through activation of Smads. In the present study, we demonstrate for the first time that the αvß6 integrin interacts with TGFß receptor II (TßRII) through the ß6 cytoplasmic domain and promotes Smad3 activation in prostate cancer (PrCa) cells. Another related αv integrin, αvß5, as well as the αvß6/3 integrin, which contains a chimeric form of ß6 with a ß3 cytoplasmic domain, do not associate with TßRII and fail to show similar responses. We provide evidence that αvß6 is required for up-regulation of MMP2 by TGFß1 through a Smad3-mediated transcriptional programme in PrCa cells. The functional relevance of these results is underscored by the finding that αvß6 modulates cell migration in an MMP2-dependent manner on an αvß6-specific ligand, latency-associated peptide (LAP)-TGFß. Overall, these mechanistic studies establish that expression of a single integrin, αvß6, is sufficient to promote activation of Smad3, regulation of MMP2 levels and consequent catalytic activity, as well as cell migration. Our study describes a new TGFß1-αvß6-MMP2 signalling pathway that, given TGFß1 pro-metastatic activity, may have profound implications for PrCa therapy.
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Antígenos de Neoplasias/metabolismo , Regulación Enzimológica de la Expresión Génica , Integrinas/metabolismo , Metaloproteinasa 2 de la Matriz/biosíntesis , Factor de Crecimiento Transformador beta1/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Humanos , MasculinoRESUMEN
Dynamic crosstalk between growth factor receptors, cell adhesion molecules and extracellular matrix is essential for cancer cell migration and invasion. Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization. They also mediate signal transduction to regulate cell proliferation and survival. The type 1 insulin-like growth factor receptor (IGF-IR) mediates tumor cell growth, adhesion and inhibition of apoptosis in several types of cancer. We have previously demonstrated that ß1 integrins regulate anchorage-independent growth of prostate cancer (PrCa) cells by regulating IGF-IR expression and androgen receptor-mediated transcriptional functions. Furthermore, we have recently reported that IGF-IR regulates the expression of ß1 integrins in PrCa cells. We have dissected the mechanism through which IGF-IR regulates ß1 integrin expression in PrCa. Here we report that IGF-IR is crucial for PrCa cell growth and that ß1 integrins contribute to the regulation of proliferation by IGF-IR. We demonstrate that ß1 integrin regulation by IGF-IR does not occur at the mRNA level. Exogenous expression of a CD4 - ß1 integrin cytoplasmic domain chimera does not interfere with such regulation and fails to stabilize ß1 integrin expression in the absence of IGF-IR. This appears to be due to the lack of interaction between the ß1 cytoplasmic domain and IGF-IR. We demonstrate that IGF-IR stabilizes the ß1 subunit by protecting it from proteasomal degradation. The α5 subunit, one of the binding partners of ß1, is also downregulated along with ß1 upon IGF-IR knockdown while no change is observed in the expression of the α2, α3, α4, α6 and α7 subunits. Our results reveal a crucial mechanistic role for the α5ß1 integrin, downstream of IGF-IR, in regulating cancer growth.
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Integrina alfa5beta1/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptor IGF Tipo 1/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño/genética , Receptor Cross-Talk , Receptor IGF Tipo 1/deficiencia , Receptor IGF Tipo 1/genéticaRESUMEN
Normal cellular phenotypes that serve an oncogenic function during tumorigenesis are potential candidates for cancer targeting drugs. Within a subset of invasive primary breast carcinoma, we observed relatively abundant expression of Tetherin, a cell surface protein encoded by the Bone Marrow Stromal Cell Antigen (BST2) known to play an inhibitory role in viral release from infected immune cells of the host. Using breast cancer cell lines derived from low and intermediate histopathologic grade invasive primary tumors that maintain growth-suppressive TGFß signaling, we demonstrate that BST2 is negatively regulated by the TGFß axis in epithelial cells. Binding of the transcription factor AP2 to the BST2 promoter was attenuated by inhibition of the TGFß pathway thereby increasing BST2 expression in tumor cells. In contrast, inherent TGFß resistance characteristic of high grade breast tumors is a key factor underlying compromised BST2 regulation, and consequently its constitutive overexpression relative to non-malignant breast epithelium, and to most low and intermediate grade cancer cells. In both 2-dimensional and 3-dimensional growth conditions, BST2-silenced tumor cells displayed an enhancement in tamoxifen or staurosporine-induced apoptotic cell death together with a reduction in the S-phase fraction compared to BST2 overexpressing counterparts. In a subset of breast cancer patients treated with pro apoptotic hormonal therapy, BST2 expression correlated with a trend for poor clinical outcome, further supporting its role in conferring an anti apoptotic phenotype. Similar to the effects of gene manipulation, declining levels of endogenous BST2 induced by the phytoalexin - resveratrol, restored apoptotic function, and curbed cell proliferation. We provide evidence for a direct approach that diminishes aberrant BST2 expression in cancer cells as an early targeting strategy to assist in surmounting resistance to pro apoptotic therapies.
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Antígenos CD/genética , Apoptosis , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Antígenos CD/metabolismo , Antineoplásicos Hormonales/farmacología , Secuencia de Bases , Sitios de Unión , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/mortalidad , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Humanos , Estimación de Kaplan-Meier , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Modelos de Riesgos Proporcionales , Unión Proteica , Resveratrol , Estilbenos/farmacología , Tamoxifeno/farmacología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
This study was carried out to dissect the mechanism by which ß1 integrins promote resistance to radiation. For this purpose, we conditionally ablated ß1 integrins in the prostatic epithelium of transgenic adenocarcinoma of mouse prostate (TRAMP) mice. The ability of ß1 to promote resistance to radiation was also analyzed by using an inhibitory antibody to ß1 , AIIB2, in a xenograft model. The role of ß1 integrins and of a ß1 downstream target, c-Jun amino-terminal kinase 1 (JNK1), in regulating radiation-induced apoptosis in vivo and in vitro was studied. We show that ß1 integrins promote prostate cancer (PrCa) progression and resistance to radiation in vivo. Mechanistically, ß1 integrins are shown here to suppress activation of JNK1 and, consequently apoptosis, in response to irradiation. Downregulation of JNK1 is necessary to preserve the effect of ß1 on resistance to radiation in vitro and in vivo. Finally, given the established crosstalk between ß1 integrins and type1 insulin-like growth factor receptor (IGF-IR), we analyzed the ability of IGF-IR to modulate ß1 integrin levels. We report that IGF-IR regulates the expression of ß1 integrins, which in turn confer resistance to radiation in PrCa cells. In conclusion, this study demonstrates that ß1 integrins mediate resistance to ionizing radiation through inhibition of JNK1 activation.
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Integrina beta1/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/radioterapia , Tolerancia a Radiación/fisiología , Animales , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Caspasa 3/metabolismo , Línea Celular Tumoral , Humanos , Integrina beta1/genética , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Ratones Transgénicos , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Trasplante HeterólogoRESUMEN
Despite the findings that ß1 integrins play a vital role in the regulation of cell proliferation and survival, the mechanisms through which they operate and lead to cancer progression remain elusive. Previously, our laboratory has shown that ß(1A) integrins support insulin-like growth factor 1 (IGFI)-mediated mitogenic and transforming activities. Here, we report that ß(1A) integrins regulate basal levels of IGF-IR, although they are not critical for maintaining cancer cell morphology. Upon transfection of ß(1A) siRNA and consequent downregulation of IGF-IR, we show inhibition of anchorage-independent growth of prostate cancer cells, a function which is dependent on IGF-IR expression. In addition, we demonstrate that IGFI-mediated activation of androgen receptor (AR), known to occur in prostate cancer cells, requires expression of ß(1A) integrins as evaluated by luciferase reporter assays and immunoblotting analysis. Since ß(1A) integrin levels are increased by R1881 or dihydrotestosterone (DHT), our results imply that ß(1A) integrins support an androgen-enhanced feedback loop that regulates the expression of IGF-IR. ß(1A) integrins also regulate inducible levels of IGF-IR in cells stimulated by androgen or by a combination of androgen and IGFI, as evaluated by flow cytometric analysis and immunoblotting. Furthermore, upon transfection of ß(1A) siRNA and consequent downregulation of IGF-IR, neither activation of AKT, an effector of IGF-IR, nor AR levels are affected. We conclude that ß(1A) integrin expression is critical for maintaining the regulatory crosstalk between IGF-IR and AR.
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Regulación de la Expresión Génica/fisiología , Factor I del Crecimiento Similar a la Insulina/farmacología , Integrina beta1/metabolismo , Receptores Androgénicos/metabolismo , Andrógenos , Animales , Línea Celular Tumoral , Retroalimentación Fisiológica/fisiología , Humanos , Integrina beta1/genética , Ratones , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Prostate-specific antigen (PSA) is a pivotal downstream target gene of the androgen receptor (AR), and a serum biomarker to monitor prostate cancer (PrCa) progression. It has been reported that PSA transactivates AR, but the mechanistic requirements of this response have not been investigated. METHODS: We studied the localization of PSA, AR, and Src in intracellular compartments of synthetic androgen (R1881)-stimulated LNCaP and C4-2B PrCa cells, using immunofluorescence and subcellular fractionation approaches. We also investigated the effect of downregulation of PSA on AR expression by immunoblotting and real-time PCR using short hairpin RNA (shRNA) and small interfering RNA (siRNA). Src activity was analyzed by immunoblotting. RESULTS: R1881 stimulation induced nuclear localization of both PSA and AR in LNCaP and C4-2B PrCa cells as well as increased phosphorylation of Src. Stable shRNA or transient siRNA knockdown of PSA resulted in reduced AR protein levels as well as AR mRNA levels in C4-2B cells. Similar to C4-2B cells, ablation of AR levels upon silencing of PSA was also confirmed in VCaP cells, another androgen-independent cell line. Silencing of PSA did not cause significant changes in Src activation; besides, Src regulation by integrins did not appear to affect AR transcriptional activity. CONCLUSIONS: PSA localizes to nuclei of androgen-stimulated PrCa cells, and controls AR mRNA and protein levels. This regulatory loop is specific for PSA, does not involve known AR activators such as Src and AKT, and may contribute to AR signaling under conditions of increasing PSA levels in patients.
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Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/biosíntesis , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Masculino , Metribolona/farmacología , Neoplasias Hormono-Dependientes/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismoRESUMEN
Whether stromal components facilitate growth, invasion, and dissemination of cancer cells or suppress neoplastic lesions from further malignant progression is a continuing conundrum in tumor biology. Conceptualizing a dynamic picture of tumorigenesis is complicated by inter-individual heterogeneity. In the post genomic era, unraveling such complexity remains a challenge for the cancer biologist. Towards establishing a functional association between cellular crosstalk and differential cancer aggressiveness, we identified a signature of malignant breast epithelial response to stromal signaling. Proximity to fibroblasts resulted in gene transcript alterations of >2-fold for 107 probes, collectively designated as Fibroblast Triggered Gene Expression in Tumor (FTExT). The hazard ratio predicted by the FTExT classifier for distant relapse in patients with intermediate and high grade breast tumors was significant compared to routine clinical variables (dataset 1, nâ=â258, HR--2.11, 95% CI 1.17-3.80, p-value 0.01; dataset 2, nâ=â171, HR--3.07, 95% CI 1.21-7.83, p-value 0.01). Biofunctions represented by FTExT included inflammatory signaling, free radical scavenging, cell death, and cell proliferation. Unlike genes of the 'proliferation cluster', which are overexpressed in aggressive primary tumors, FTExT genes were uniquely repressed in such cases. As proof of concept for our correlative findings, which link stromal-epithelial crosstalk and tumor behavior, we show a distinctive differential in stromal impact on prognosis-defining functional endpoints of cell cycle progression, and resistance to therapy-induced growth arrest and apoptosis in low vs. high grade cancer cells. Our experimental data thus reveal aspects of 'paracrine cooperativity' that are exclusively contingent upon the histopathologically defined grade of interacting tumor epithelium, and demonstrate that epithelial responsiveness to the tumor microenvironment is a deterministic factor underlying clinical outcome. In this light, early attenuation of epithelial-stromal crosstalk could improve the management of cases prone to be clinically challenging.
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Neoplasias de la Mama/patología , Transducción de Señal , Células del Estroma/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patologíaRESUMEN
Inherent cancer phenotypes that are independent of fluctuating cross-talk with the surrounding tissue matrix are highly desirable candidates for targeting tumor cells. Our novel study design uses epithelial cell lines derived from low versus high histologic grade primary breast cancer to effectively diminish the breadth of transient variability generated within the tumor microenvironment of the host, revealing a "paracrine-independent expression of grade-associated" (PEGA) gene signature. PEGA members extended beyond "proliferation-driven" signatures commonly associated with aggressive, high-grade breast cancer. The calcium-binding protein S100P was prominent among PEGA genes overexpressed in high-grade tumors. A three-member fingerprint of S100P-correlated genes, consisting of GPRC5A, FXYD3, and PYCARD, conferred poor outcome in multiple breast cancer data sets, irrespective of estrogen receptor status but dependent on tumor size (P < 0.01). S100P silencing markedly diminished coregulated gene transcripts and reversed aggressive tumor behavior. Exposure to pathway-implicated agents, including the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, phenothiazine, and chlorpromazine, resulted in rapid apoptotic cell death in high-grade tumor cells resistant to the chemotherapeutic drug cisplatin. This is the first comprehensive study describing molecular phenotypes intimately associated with histologic grade whose expression remains relatively fixed despite an unavoidably changing environment to which tumor cells are invariably exposed.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adulto , Anciano , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Tumorales CultivadasRESUMEN
Breast cancer outcome is highly variable. Whether inadvertent exposure to environmental xenobiotics evokes a biological response promoting cancer aggressiveness and a higher probability of tumor recurrence remains unknown. To determine specific molecular alterations which arise in high-risk breast tissue in the presence of the ubiquitous xenoestrogen, bisphenol A (BPA), we used nonmalignant random periareolar fine-needle aspirates in a novel functional assay. Early events induced by BPA in epithelial-stromal cocultures derived from the contralateral tissue of patients with breast cancer included gene expression patterns which facilitate apoptosis evasion, endurance of microenvironmental stress, and cell cycle deregulation without a detectable increase in cell numbers. This BPA response profile was significantly associated with breast tumors characterized by high histologic grade (P < 0.001) and large tumor size (P = 0.002), resulting in decreased recurrence-free patient survival (P < 0.001). Our assays show a biological "fingerprint" of probable prior exposure to endocrine-disrupting agents, and suggest a scenario in which their presence in the microenvironmental milieu of high-risk breast tissue could play a deterministic role in establishing and maintaining tumor aggressiveness and poor patient outcome.
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Neoplasias de la Mama/patología , Fenoles/farmacología , Adulto , Compuestos de Bencidrilo , Biopsia con Aguja Fina , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Células Epiteliales/patología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
Estrogen receptor alpha (ERalpha) and tumor suppressor protein p53 exert opposing effects on cellular proliferation. As a transcriptional regulator, p53 is capable of activating or repressing various target genes. We have previously reported that ERalpha binds directly to p53, leading to down-regulation of transcriptional activation by p53. In addition to transcriptional activation, transcriptional repression of a subset of target genes by p53 plays important roles in diverse biological processes, such as apoptosis. Here, we report that ERalpha inhibits p53-mediated transcriptional repression. Chromatin immunoprecipitation assays reveal that ERalpha interacts in vivo with p53 bound to promoters of Survivin and multidrug resistance gene 1, both targets for transcriptional repression by p53. ERalpha binding to p53 leads to inhibition of p53-mediated transcriptional regulation of these genes in human cancer cells. Transcriptional derepression of Survivin by ERalpha is dependent on the p53-binding site on the Survivin promoter, consistent with our observation that p53 is necessary for ERalpha to access the promoters. Importantly, mutagenic conversion of this site to an activation element enabled ERalpha to repress p53-mediated transcriptional activation. Further, RNA interference-mediated knockdown of ERalpha resulted in reduced Survivin expression and enhanced the propensity of MCF-7 cells to undergo apoptosis in response to staurosporine treatment, an effect that was blocked by exogenous expression of Survivin. These results unravel a novel mechanism by which ERalpha opposes p53-mediated apoptosis in breast cancer cells. The findings could have translational implications in developing new therapeutic and prevention strategies against breast cancer.
Asunto(s)
Apoptosis/fisiología , Receptor alfa de Estrógeno/metabolismo , Activación Transcripcional/fisiología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Apoptosis/efectos de los fármacos , Sitios de Unión , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Regulación hacia Abajo , Factor de Transcripción E2F1/metabolismo , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Regiones Promotoras Genéticas , Unión Proteica , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Estaurosporina/farmacología , Survivin , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Toward the goal of identifying early genetic losses, which mediate the release of human breast epithelium from replicative suppression leading to cellular immortalization, we have used a newly developed in vitro model system. This system consists of epithelial cultures derived from noncancerous breast tissue, treated with the chemical carcinogen N-ethyl-N-nitrosourea, and continuously passaged to yield cell populations culminating in the immortal phenotype. Genome-wide allelotyping of early passage N-ethyl-N-nitrosourea-exposed cell populations revealed aberrations at >10% (18 of 169) loci examined. Allelic losses encompassing chromosomes 6q24-6q27, implicating immortalization-associated candidate genes, hZAC and SEN6, occurred in two independently derived cell lines before the Hayflick limit. Additional LOH sites were present in one cell line at 3p11-3p26, 11p15, and 20p12-13. Allelic losses reported in this cell line preceded detectable levels of telomerase activity and the occurrence of p53-related aberrations. Information gained from the search for early immortalization-associated genetic deletions in cultured cells was applied in a novel approach toward the analysis of morphologically normal terminal ductal lobular units microdissected from 20 cases of ductal carcinoma in situ. Notably, clonal allelic losses at chromosome 3p24 and 6q24 were an early occurrence in adjoining terminal ductal lobular units of a proportion of primary tumors, which displayed loss of heterozygosity (3 of 11 and 3 of 6, respectively). The biological insights provided by the new model system reported here strongly suggest that early allelic losses delineated in immortalized cultures and validated in vivo could serve as surrogate endpoints to assist in the identification and intervention of high-risk benign breast tissue, which sustains the potential for continuous proliferation.