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In the original publication of the article, the spelling of the sixth author's given name was incorrect. The corrected author name should read as "Wadie David". The original article has been corrected.
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PURPOSE: Studies have identified several estrogen receptor α (ERα) ligand-binding domain (LBD) somatic mutations in endocrine therapy resistant, metastatic ER-positive breast cancers. The most common mutations, Tyr537Ser (Y537S) and Asp538Gly (D538G), are detected in ~ 30% of endocrine resistant metastatic breast cancer patients. These ESR1 mutations induce the agonist conformation of ERα, confer an estrogen-independent phenotype, and promote drug resistance to antiestrogens. METHODS: ER-positive, estrogen-dependent MCF-7 cells were engineered to express either the Y537S or D538G mutants using CRISPR knock-in (cY537S and cD538G). These cells were used to screen several estrogen receptor degrader (ERD) compounds synthesized using the Proteolysis Targeting Chimeras (PROTAC) method to induce degradation of ERα via the ubiquitin-proteasome pathway. RESULTS: Wild-type MCF-7 and ERα LBD mutant cells were treated with ERD-148 (10 pM-1 µM) and assayed for cellular proliferation using the PrestoBlue cell viability assay. ERD-148 attenuated ER-dependent growth with IC50 values of 0.8, 10.5, and 6.1 nM in MCF-7, cY537S, and cD538G cells, respectively. Western blot analysis showed that MCF-7 cells treated with 1 nM ERD-148 for 24 h exhibited reduced ERα protein expression as compared to the mutants. The ER-regulated gene, GREB1, demonstrated significant downregulation in parental and mutant cells after 24 h of ERD-148 treatment at 10 nM. Growth of the ER-negative, estrogen-independent MDA-MB-231 breast cancer cells was not inhibited by ERD-148 at the ~ IC90 observed in the ER-positive cells. CONCLUSION: ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ERα with comparable potency to Fulvestrant with marginal non-specific toxicity.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/genética , Estrógenos/farmacología , Mutación , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Proteolisis , Células Tumorales CultivadasRESUMEN
Cytochrome P450 17A1 (CYP17A1) is the requisite enzyme for synthesis of sex steroids, including estrogens and androgens. As such, inhibition of CYP17A1 is a target for inhibiting the growth of hormone-dependent cancers including prostate and breast cancer. Abiraterone, is a first in class potent and selective CYP17A1 inhibitor that has been approved for the treatment of castration-resistant prostate cancer. Given that, androgens are the precursors for estrogen production, it has been proposed that abiraterone could be an effective form of treatment for estrogen receptor (ER)-positive breast cancer, though its utility in this context has yet to be established. Abiraterone has a core steroid-like chemical structure, and so we hypothesized that it may bind to nuclear steroid receptors including ER and have estrogenic activity. We tested this hypothesis by investigating abiraterone's ability to directly modulate ER signaling in breast cancer cell line models. We show that abiraterone directly activates ER, induces ER-target gene expression, and elicits estrogen-response-element reporter activity in the ER-positive cell lines MCF-7 and T47D. Abiraterone also induced cell proliferation by ~2.5-fold over vehicle in both MCF-7 and T47D cells. Importantly, abiraterone-induced cell proliferation and ER-activity was blocked by the selective estrogen receptor downregulator (SERD) fulvestrant, confirming that abiraterone directly acts at the ER. These data suggest that abiraterone should be combined with other ER antagonists when used for the clinical management of ER-positive breast cancer.
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Androstenos/farmacología , Neoplasias de la Mama/metabolismo , Estradiol/análogos & derivados , Receptores de Estrógenos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estradiol/farmacología , Femenino , Fulvestrant , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Proteínas de Neoplasias/genéticaRESUMEN
Acquired mutations in the ligand-binding domain (LBD) of the gene encoding estrogen receptor α (ESR1) are common mechanisms of endocrine therapy resistance in patients with metastatic ER+ breast cancer. The ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1,200 Federal Drug Administration-approved (FDA-approved) drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib + fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with WT cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant-induced endocrine therapy resistance in breast cancer.
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Neoplasias de la Mama , Receptor alfa de Estrógeno/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Fulvestrant/farmacología , Fulvestrant/uso terapéutico , Humanos , Mutación , Dominios Proteicos , Transcripción GenéticaRESUMEN
BACKGROUND: Manual skill proficiency is not currently employed in selecting residents for general surgery training programs. The study objective was to assess whether the technical skill levels of applicants to a general surgery residency program are higher than those of internal medicine residents. MATERIAL AND METHODS: Forty-two applicants to a community general surgery program underwent manual skill testing on interview day. Four laparoscopic tasks on a virtual reality (VR) simulator (LapSim, Goteborg, Sweden) were tested. Performance scores were computer-generated. Participants' previous experience with other manual dexterity activities was assessed via a questionnaire. Applicants' self-perception of their surgical skills was correlated with their skill dexterity scores on the simulator. Candidates' simulator scores were also compared with those of a group of internal medicine interns (n = 9) and a group of mid-level surgical residents, PGY 2-3 (n = 7). RESULTS: Simulator scores of the applicants were significantly lower than those of mid-level surgical residents in all VR tasks (P < 0.05). The internal medicine interns scored higher that the surgery candidates in three of four simulator tasks. Participation in other manual dexterity activities was not associated with increased dexterity scores. CONCLUSION: This study suggests that surgical dexterity levels do not correlate with the self-assessed skill levels or with previous experience with other manual dexterity activities. Moreover, there appears to be no self-selection of applicants for surgery residency based on actual surgical skills. Selection criteria for surgical training, which incorporate technical proficiency skills, may potentially better discriminate those applicants with an aptitude for a surgical specialty.
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Pruebas de Aptitud , Evaluación Educacional/métodos , Cirugía General/educación , Internado y Residencia/normas , Destreza Motora , Aptitud , Simulación por Computador , Femenino , Humanos , Laparoscopía/educación , Masculino , Encuestas y Cuestionarios , Interfaz Usuario-ComputadorRESUMEN
PURPOSE: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor-positive (ER+) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models. EXPERIMENTAL DESIGN: Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy. RESULTS: Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21-1.52) and led to a decrease in the surviving fraction of cells at 2 Gy (P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08-1.68) and abemaciclib-treated (rER: 1.19-2.05) cells. Combination treatment decreased RAD51 foci formation (P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94-1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth. CONCLUSIONS: These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence.
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Neoplasias de la Mama/radioterapia , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Receptores de Estrógenos/metabolismo , Animales , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Quimioradioterapia , Femenino , Humanos , Ratones , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The aromatase inhibitors (AIs) are used to treat estrogen receptor-positive (ER+) breast tumors in post-menopausal women, and function by blocking the conversion of adrenal androgens to estrogens by the enzyme CYP19 aromatase. Breast cancer patients receiving AI therapy have circulating estrogen levels below the level of detection; however, androgen concentrations remain unchanged. We were interested in studying the effects of androgens on breast cancer cell proliferation under profound estrogen-deprived conditions. Using in vitro models of estrogen-dependent breast cancer cell growth we show that the androgens testosterone and 5alpha-dihydrotestosterone induce the growth of MCF-7, T47D and BT-474 cells in the absence of estrogen. Furthermore, we demonstrate that under profound estrogen-deprived conditions these breast cancer cells up-regulate steroidogenic enzymes that can metabolize androgens to estrogens. Lastly, we found that the downstream metabolite of 5alpha-dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), is estrogenic in breast cancer cells, and induces growth and ER-signaling via activation of ERalpha. In conclusion, our results show that breast cancer cells deprived of estrogen up-regulate steroidogenic enzymes and metabolize androgens to estrogen-like steroids. The generation of estrogen-like steroids represents a potential mechanism of resistance to aromatase inhibitors.
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Androstano-3,17-diol/metabolismo , Inhibidores de la Aromatasa/uso terapéutico , Neoplasias de la Mama/metabolismo , Proliferación Celular , Resistencia a Antineoplásicos/fisiología , Receptor alfa de Estrógeno/metabolismo , Andrógenos/metabolismo , Andrógenos/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Femenino , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients.Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110-22. ©2017 AACR.
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Neoplasias de la Mama/genética , Variaciones en el Número de Copia de ADN/genética , Células Neoplásicas Circulantes/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Mutación , Células Neoplásicas Circulantes/patologíaRESUMEN
Fulvestrant is a dose dependent selective estrogen receptor (ER) down-regulator (SERD) used in ER-positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch(®) system. Clinical data are limited to a subset of patients with ER-positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B-cell lymphoma (BCL2) using the CellSearch(®) CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC-expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC-ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene (ESR1). All seven ER positive patients progressing on AIs had heterogeneous CTC-ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non-ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies.
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Resistencia a Antineoplásicos/efectos de los fármacos , Estradiol/análogos & derivados , Células Neoplásicas Circulantes/metabolismo , Receptores de Estrógenos/metabolismo , Inhibidores de la Aromatasa/farmacología , Inhibidores de la Aromatasa/uso terapéutico , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Estradiol/farmacología , Fulvestrant , Humanos , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/patología , Resultado del TratamientoRESUMEN
BACKGROUND: Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors. METHODS: Polymerase chain reaction (PCR) and Western blot analysis were used to determine PXR mRNA and protein expression, respectively. Real-time PCR and CYP3A catalytic activity using 7-benzyl-trifluoromethyl coumarin (BFC) as the probe substrate were used to measure the induction of P450 3A4 or MDR1. siRNA transfections were performed for PXR and cytotoxicity determined by a colorimetric based assay or Annexin v-Fitc staining. RESULTS: Differences were observed in the molecular size of the PXR protein expressed in sarcoma cell lines when compared with the wildtype PXR expressed in normal liver, kidney, or small intestine. A polyclonal PXR antibody raised against the N-terminus of the wildtype PXR did not detect PXR expressed in these sarcoma cell lines. In the osteosarcoma cell lines, etoposide and doxorubicin were better inducers of P450 3A4 and MDR1 than rifampin. siRNA against PXR down-regulated P450 3A4 expression only in the osteosarcoma cell line. Cytotoxicity assays showed that the resistance of the osteosarcoma cell lines to etoposide correlated with PXR protein expression levels and activation of P450 3A4 and could be prevented by ketoconazole. CONCLUSION: The results suggest that PXR plays a critical role in the regulation of P450 3A4 expression in osteosarcoma and that its expression and activation in these tumors may influence the effect of chemotherapeutic agents on the induction of target genes implicated in drug resistance.
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Resistencia a Antineoplásicos/fisiología , Osteosarcoma/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Doxorrubicina/farmacología , Etopósido/farmacología , Humanos , Osteosarcoma/genética , Receptor X de Pregnano , Isoformas de Proteínas/efectos de los fármacos , ARN Mensajero/análisis , ARN Interferente Pequeño , Receptores de Esteroides/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rifampin/farmacologíaRESUMEN
BACKGROUND: Estrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors. RESULTS: We generated DNA microarray-based gene expression profiles from three estrogen receptor alpha (ERalpha)-positive breast cancer cell lines stimulated by 17beta-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from our preclinical models were co-expressed with ERalpha in a panel of ERalpha+ breast tumor mRNA profiles, when corrections were made for patient age, as well as with progesterone receptor. Furthermore, the E2-regulated genes were significantly enriched for transcriptional targets of the myc oncogene and were found to be coordinately expressed with Myc in human tumors. CONCLUSION: Our results provide significant validation of a widely used in vitro model of estrogen signaling as being pathologically relevant to breast cancers in vivo.
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Neoplasias de la Mama/genética , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica , Factores de Edad , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Receptor alfa de Estrógeno/análisis , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/metabolismo , Receptores de Progesterona/biosíntesis , Receptores de Progesterona/genética , Activación Transcripcional , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Gene regulated in breast cancer 1 (GREB1) is a novel estrogen-regulated gene shown to play a pivotal role in hormone-stimulated breast cancer growth. GREB1 is expressed in the prostate and its putative promoter contains potential androgen receptor (AR) response elements. METHODS: We investigated the effects of androgens on GREB1 expression and its role in androgen-dependent prostate cancer growth. RESULTS: Real-time PCR demonstrated high level GREB1 expression in benign prostatic hypertrophy (BPH), localized prostate cancer (L-PCa), and hormone refractory prostate cancer (HR-PCa). Androgen treatment of AR-positive prostate cancer cells induced dose-dependent GREB1 expression, which was blocked by anti-androgens. AR binding to the GREB1 promoter was confirmed by chromatin immunoprecipitation (ChIP) assays. Suppression of GREB1 by RNA interference blocked androgen-stimulated LNCaP cell proliferation. CONCLUSIONS: GREB1 is expressed in proliferating prostatic tissue and prostate cancer, is regulated by androgens, and suppression of GREB1 blocks androgen-induced growth suggesting GREB1 may be critically involved in prostate cancer proliferation.
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Andrógenos/farmacología , Proliferación Celular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/fisiopatología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/genética , Relación Dosis-Respuesta a Droga , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Proteínas de Neoplasias/análisis , Neoplasias Hormono-Dependientes/química , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/patología , Neoplasias Hormono-Dependientes/fisiopatología , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/química , Neoplasias de la Próstata/patología , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Estrogen plays a central role in breast cancer pathogenesis and many potent risk factors for the development of the disease can be explained in terms of increased lifetime exposure to estrogen. Although estrogen regulated genes have been identified, those critically involved in growth regulation remain elusive.METHODS. To identify candidate genes involved in estrogen stimulated breast cancer growth, DNA microarray based gene expression profiles were generated from three estrogen receptor alpha (ER alpha) positive breast cancer cell lines grown under multiple stimulatory and inhibitory conditions. RESULTS: Only three genes were significantly induced by 17beta-estradiol (E2) relative to control in all three cell lines: GREB 1, stromal cell-derived factor 1 (SDF-1) and trefoil factor 1 (pS2). Quantitative real-time PCR assays confirmed that in all three cell lines, GREB 1 was induced by E2, but not by the antiestrogens tamoxifen (TAM) or ICI 182,780. GREB 1 expression level was strongly correlated with ER alpha positivity in 39 breast cancer cell lines of known ER alpha expression status. GREB 1 induction by E2 was rapid (7.3 fold by 2 h for MCF-7) and mirrored the fraction of cells entering S-phase when released from an estrogen deprivation induced cell arrest. Suppression of GREB 1 using siRNA blocked estrogen induced growth in MCF-7 cells and caused a paradoxical E2 induced growth inhibition. CONCLUSION: These data suggest that GREB 1 is critically involved in the estrogen induced growth of breast cancer cells and has the potential of being a clinical marker for response to endocrine therapy as well as a potential therapeutic target.
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Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Estradiol , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Distribución AleatoriaRESUMEN
Myofibroblast differentiation and activation by transforming growth factor-beta1 (TGF-beta1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-beta1 and adhesion-dependent signals. TGF-beta1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-beta receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-beta1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-beta1-induced alpha-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of alpha-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-beta1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-beta1/Smad signaling.