RESUMEN
BACKGROUND: Targetable molecular drivers of gastric cancer (GC) metastasis remain largely unidentified, leading to limited targeted therapy options for advanced GC. We aimed to identify molecular drivers for metastasis and devise corresponding therapeutic strategies. METHODS: We performed an unbiased in vivo genome-wide CRISPR/Cas9 knockout (KO) screening in peritoneal dissemination using genetically engineered GC mouse models. Candidate genes were validated through in vivo transplantation assays using KO cells. We analyzed target expression patterns in GC clinical samples using immunohistochemistry. The functional contributions of target genes were studied through knockdown, KO, and overexpression approaches in tumorsphere and organoid assays. Small chemical inhibitors against Bcl-2 members and YAP were tested in vitro and in vivo. RESULTS: We identified Nf2 and Rasa1 as metastasis-suppressing genes through the screening. Clinically, RASA1 mutations along with low NF2 expression define a distinct molecular subtype of metastatic GC exhibiting aggressive traits. NF2 and RASA1 deficiency increased in vivo metastasis and in vitro tumorsphere formation by synergistically amplifying Wnt and YAP signaling in cancer stem cells (CSCs). NF2 deficiency enhanced Bcl-2-mediated Wnt signaling, conferring resistance to YAP inhibition in CSCs. This resistance was counteracted via synthetic lethality achieved by simultaneous inhibition of YAP and Bcl-2. RASA1 deficiency amplified the Wnt pathway via Bcl-xL, contributing to cancer stemness. RASA1 mutation created vulnerability to Bcl-xL inhibition, but the additional NF2 deletion conferred resistance to Bcl-xL inhibition due to YAP activation. The combined inhibition of Bcl-xL and YAP synergistically suppressed cancer stemness and in vivo metastasis in RASA1 and NF2 co-deficiency. CONCLUSION: Our research unveils the intricate interplay between YAP and Bcl-2 family members, which can lead to synthetic lethality, offering a potential strategy to overcome drug resistance. Importantly, our findings support a personalized medicine approach where combined therapy targeting YAP and Bcl-2, tailored to NF2 and RASA1 status, could effectively manage metastatic GC.
Asunto(s)
Neoplasias Gástricas , Animales , Ratones , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Mutaciones Letales Sintéticas , Proteínas Activadoras de GTPasa , Mutación , Transducción de Señal , Proteína Activadora de GTPasa p120RESUMEN
Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.
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COVID-19/metabolismo , COVID-19/virología , Interacciones Microbiota-Huesped/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Transporte Activo de Núcleo Celular , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Modelos Biológicos , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Proteínas de Complejo Poro Nuclear/antagonistas & inhibidores , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas no Estructurales Virales/genéticaRESUMEN
The molecular mechanisms underlying the pathogenesis of diffuse-type gastric cancer (DGC) have not been adequately explored due to a scarcity of appropriate animal models. A recently developed tool well suited for this line of investigation is the Pdx-1-Cre;Cdh1F/+ ;Trp53F/F ;Smad4F/F (pChe PS) mouse model that spontaneously develops metastatic DGC showing nearly complete E-cadherin loss. Here, we performed a proteogenomic analysis to uncover the molecular changes induced by the concurrent targeting of E-cadherin, p53, and Smad4 loss. The gene expression profiles of mouse DGCs and in vivo gastric phenotypes from various combinations of gene knockout demonstrated that these mutations collaborate to activate cancer-associated pathways to generate aggressive DGC. Of note, WNT-mediated epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM)-cytokine receptor interactions were prominently featured. In particular, the WNT target gene osteopontin (OPN) that functions as an ECM cytokine is highly upregulated. In validation experiments, OPN contributed to DGC stemness by promoting cancer stem cell (CSC) survival and chemoresistance. It was further found that Bcl-xL acts as a targetable downstream effector of OPN in DGC CSC survival. In addition, Zeb2 and thymosin-ß4 (Tß4) were identified as prime candidates as suppressors of E-cadherin expression from the remaining Cdh1 allele during DGC development. Specifically, Tß4 suppressed E-cadherin expression and anoikis while promoting cancer cell growth and migration. Collectively, these proteogenomic analyses broaden and deepen our understanding of the contribution of key driver mutations in the stepwise carcinogenesis of DGC through novel effectors, namely OPN and Tß4.
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Cadherinas/genética , Carcinogénesis/genética , Genoma/genética , Proteoma/genética , Proteína Smad4/genética , Neoplasias Gástricas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Antígeno CD48/genética , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/patología , Estómago/patología , Neoplasias Gástricas/patología , Transcriptoma/genética , Regulación hacia Arriba/genéticaRESUMEN
ST2 functions as a receptor for the cytokine IL-33. It has been implicated in carcinogenesis. In this study, we sought to mechanistically determine how ST2 and IL-33 function to support cancer stem cell (CSC) activity and drive gastric cancer (GC) pathogenesis. ST2+ subpopulation spontaneously arose during gastric tumorigenesis. A thorough evaluation of ST2 and IL-33 expression in gastric tumors revealed that they show an overlapping expression pattern, notably in poor differentiated GC and metastasis foci. Moreover, their expression levels are clinically correlated to cancer progression. Using a genetic model of CSC-driven gastric carcinogenesis, ST2+ subpopulation displays increased tumorigenicity, chemoresistance and metastatic potentials through increased survival fitness endowed by an elevated MAPK-regulated Bcl-xL. The IL-33/ST2 axis enhances the self-renewal and survival of GC stem cells and organoids. Importantly, we observed a synergistic cooperation between IL-33/ST2 and the canonical Wnt pathway in transactivating Wnt-dependent transcription and supporting CSC activity, a partnership that was abrogated by inhibiting Bcl-xL. Concordant with this, ST2+ subpopulation was targeted by MEK1/2 and Bcl-xL-specific inhibitors. These findings establish ST2 as a functional CSC marker that fortifies the Wnt signal while availing a novel therapeutic strategy to suppress GC progression by targeting the IL-33/ST2/Bcl-xL signaling axis.
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Neoplasias Gástricas , Vía de Señalización Wnt , Humanos , Neoplasias Gástricas/patología , Interleucina-33/genética , Interleucina-33/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/genética , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Carcinogénesis/genética , Células Madre Neoplásicas/patología , Línea Celular TumoralRESUMEN
The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain-containing ion transport regulator 3 (FXYD3), a component of the Na+/K+ pump. Accordingly, FXYD3+ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3+ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3+ CSCs were sensitive to senolytic Na+/K+ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3+ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na+/K+ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.
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Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Células Madre Neoplásicas/patología , Línea Celular Tumoral , Proteínas de la Membrana , Proteínas de Neoplasias/genéticaRESUMEN
As TNF is one of the earliest signals that can be detected in the leukocyte-derived inflammatory cascade which drives subsequent cytokine production, we are interested in determining whether TNF is one of the initiating factors controlling liver remodeling and regeneration following chronic liver damage. One of the early responses is the expression of lymphotoxin-ß by hepatic progenitor oval cells. The aim of this study was to determine whether hepatic expression of LT-ß was controlled by TNF and to understand the basis of this regulation. We previously showed that LT-ß expression is transcriptionally controlled via the TNF-induced, inflammatory NF-κB pathway in T lymphocytes. Here we show that TNF is able to upregulate LT-ß expression in hepatic cells at the transcriptional level by the binding of NF-κB p50/p65 heterodimers and Ets1 to their respective sites in the LT-ß promoter.
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Hepatocitos/metabolismo , Linfotoxina beta/genética , FN-kappa B/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Linfotoxina beta/metabolismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/genética , Factor de Transcripción Sp1/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer.
RESUMEN
SMAD4 is frequently mutated and inactivated in human gastric cancer (GC). Although the epithelial cell-autonomous functions of Smad4 have been extensively studied, its contribution to tumor immunity is largely undetermined. Here, we report that the loss of Smad4 expression in GC cells endows them with the ability to evade tumor immunity. Unlike their Smad4-proficient counterparts, Smad4-deficient stomach organoids can evade host immunity to form tumors in immunocompetent mice. Smad4-deficient GC cells show expanded CD133+ cancer stem-like cells while suppressing dendritic cell (DC) differentiation and cytotoxic T cells with granulocytic myeloid-derived suppressor cell (G-MDSC) accumulation through a secretome containing CXCL1. Moreover, Smad4 deficiency increases programmed cell death ligand-1 (PD-L1) and decreases 4-1BBL expressions, indicating a change in immunogenicity. Combinatorial immune checkpoint blockade (ICB) of anti-PD-L1 and anti-CTLA-4 or agonistic anti-4-1BB antibodies effectively treats ICB monotherapy-resistant Smad4-deficient allografts, exposing a specific vulnerability. Collectively, these data provide a rational basis for ICB strategies in treating advanced GC with Smad4 deficiency.
Asunto(s)
Neoplasias Gástricas , Humanos , Ratones , Animales , Neoplasias Gástricas/terapia , Evasión Inmune , Inmunoterapia , Linfocitos T Citotóxicos/metabolismo , Células Epiteliales/metabolismo , Antígeno B7-H1/metabolismo , Microambiente Tumoral , Quimiocina CXCL1/metabolismo , Proteína Smad4/metabolismoRESUMEN
BRAF is one of the most frequently mutated genes across a number of different cancers, with the best-characterized mutation being V600E. Despite the successes of treating BRAF mutant V600E lung cancer with BRAF pathway inhibitors, treatment strategies targeting tumors with non-V600E mutations are yet to be established. We studied cellular signaling differences between lung cancers with different BRAF mutations and determined their sensitivities to BRAF pathway inhibitors. Here, we observed that MEK inhibition induced feedback activation of the receptor tyrosine kinase (RTK) EGFR, and in some cases the RTK FGFR, resulting in transient suppression of ERK phosphorylation in BRAF non-V600E, but not BRAF V600E, mutant cells. Furthermore, we found that both EGFR and FGFR activated the MEK/ERK pathway, despite the presence of BRAF non-V600E mutations with elevated kinase activity. Moreover, in BRAF non-V600E mutants with impaired kinase activities, EGFR had even greater control over the MEK/ERK pathway, essentially contributing completely to the tonic mitogen-activated protein kinase (MAPK) signal. Accordingly, the combination of MEK inhibitor with EGFR inhibitor was effective at shrinking tumors in mouse model of BRAF non-V600E mutant lung cancer. Furthermore, the results were recapitulated with a clinically relevant dual inhibitor of EGFR and RAF, BGB-283. Overall, although BRAF V600E mutant cells are sensitive to BRAF inhibition, non-V600E mutant cancer cells are reliant on RTKs for their MAPK activation and inhibiting both MEK and RTKs are necessary in these cancers. Our findings provide evidence of critical survival signals in BRAF non-V600E mutant cancers, which could pave the way for effective treatment of these cancers.
Asunto(s)
Neoplasias Pulmonares/genética , Sistema de Señalización de MAP Quinasas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Sustitución de Aminoácidos , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ácido Glutámico/genética , Células HT29 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Desnudos , Proteínas Mutantes/genética , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Tumorales Cultivadas , Valina/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Studies of genomic instability have historically focused on intrinsic mechanisms rather than extrinsic mechanisms based in the tumor microenvironment (TME). TGFß is the most abundantly secreted cytokine in the TME, where it imparts various aggressive characteristics including invasive migration, drug resistance, and epithelial-to-mesenchymal transition (EMT). Here we show that TGFß also promotes genomic instability in the form of DNA double strand breaks (DSB) in cancer cells that lack the tumor suppressor gene RUNX3 Loss of RUNX3 resulted in transcriptional downregulation of the redox regulator heme oxygenase-1 (HO-1 or HMOX1). Consequently, elevated oxidative DNA damage disrupted genomic integrity and triggered cellular senescence, which was accompanied by tumor-promoting inflammatory cytokine expression and acquisition of the senescence-associated secretory phenotype (SASP). Recapitulating the above findings, tumors harboring a TGFß gene expression signature and RUNX3 loss exhibited higher levels of genomic instability. In summary, RUNX3 creates an effective barrier against further TGFß-dependent tumor progression by preventing genomic instability. These data suggest a novel cooperation between cancer cell-extrinsic TGFß signaling and cancer cell-intrinsic RUNX3 inactivation as aggravating factors for genomic instability.Significance: RUNX3 inactivation in cancer removes an antioxidant barrier against DNA double strand breaks induced by TGFß expressed in the tumor microenvironment. Cancer Res; 78(1); 88-102. ©2017 AACR.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Inestabilidad Genómica , Factor de Crecimiento Transformador beta/metabolismo , Células A549 , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Senescencia Celular/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Daño del ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Genes p53 , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Reporter assays are widely used in applications that require measurement of changes in gene expression over time (e.g. drug screening). With standard reporter vectors, the measurable effect of a treatment or compound (altered reporter activity) is substantially diluted and delayed, compared with its true effect (altered transcriptional activity). This problem is caused by the relatively long half-lives of both the reporter protein and its mRNA. As a result, the activities of compounds, ligands or treatments that have a relatively minor effect, or a substantial but transient effect, often remain undetected. To circumvent this problem, we introduced modular protein- and mRNA-destabilizing elements into a range of commonly used reporters. Our data show that both elements are required for maximal responses to both increases and decreases in transcriptional activity. The double-destabilized reporter vectors showed markedly improved performance in drug screening, kinetic assays and dose-response titrations.
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Secuencias de Aminoácidos , Genes Reporteros , Secuencias Reguladoras de Ácido Ribonucleico , Transcripción Genética , Animales , Células CHO , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Vectores Genéticos , Semivida , Células HeLa , Humanos , Proteínas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Terminología como Asunto , TransfecciónRESUMEN
Carcinomas are phenotypically arrayed along an epithelial-mesenchymal transition (EMT) spectrum, a developmental program currently exploited to understand the acquisition of drug resistance through a re-routing of growth factor signaling. This review collates the current approaches employed in developing therapeutics against cancer-associated EMT, and provides an assessment of their respective strengths and drawbacks. We reflect on the close relationship between EMT and chemoresistance against current targeted therapeutics, with a special focus on the epigenetic mechanisms that link these processes. This prompts the hypothesis that carcinoma-associated EMT shares a common epigenetic pathway to cellular plasticity as somatic cell reprogramming during tissue repair and regeneration. Indeed, their striking resemblance suggests that EMT in carcinoma is a pathological adaptation of an intrinsic program of cellular plasticity that is crucial to tissue homeostasis. We thus propose a revised approach that targets the epigenetic mechanisms underlying pathogenic EMT to arrest cellular plasticity regardless of upstream cancer-driving mutations.
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Resistencia a Antineoplásicos/genética , Epigénesis Genética , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias , Transducción de Señal/genética , Animales , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapiaRESUMEN
Early detection of gastric cancers saves lives, but remains a diagnostic challenge. In this study, we aimed to identify cell-surface biomarkers of early gastric cancer. We hypothesized that a subset of plasma membrane proteins induced by the Helicobacter pylori oncoprotein CagA will be retained in early gastric cancers through non-oncogene addiction. An inducible system for expression of CagA was used to identify differentially upregulated membrane protein transcripts in vitro. The top hits were then analyzed in gene expression datasets comparing transcriptome of gastric cancer with normal tissue, to focus on markers retained in cancer. Among the transcripts enriched upon CagA induction in vitro, a significant elevation of CEACAM6 was noted in gene expression datasets of gastric cancer. We used quantitative digital immunohistochemistry to measure CEACAM6 protein levels in tissue microarrays of gastric cancer. We demonstrate an increase in CEACAM6 in early gastric cancers, when compared to matched normal tissue, with an AUC of 0.83 for diagnostic validity. Finally, we show that a fluorescently conjugated CEACAM6 antibody binds avidly to freshly resected gastric cancer xenograft samples and can be detected by endoscopy in real time. Together, these results suggest that CEACAM6 upregulation is a cell surface response to H. pylori CagA, and is retained in early gastric cancers. They highlight a novel link between CEACAM6 expression and CagA in gastric cancer, and suggest CEACAM6 to be a promising biomarker to aid with the fluorescent endoscopic diagnosis of early neoplastic lesions in the stomach.
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Antígenos Bacterianos/fisiología , Antígenos CD/análisis , Proteínas Bacterianas/fisiología , Biomarcadores de Tumor/análisis , Moléculas de Adhesión Celular/análisis , Neoplasias Gástricas/diagnóstico , Animales , Técnica del Anticuerpo Fluorescente , Proteínas Ligadas a GPI/análisis , Infecciones por Helicobacter/metabolismo , Humanos , Ratones , Regulación hacia ArribaRESUMEN
Lymphotoxin-beta (LT-beta) is a transmembrane protein expressed mainly on cells of the lymphoid lineage. It associates with LT-alpha on the cell surface to form the heterotrimeric LTalpha1,beta2 complex, which binds the LT-beta receptor. Membrane lymphotoxin is a crucial signal for the appropriate development of lymph nodes and Peyer's patches, and in the formation of B and T cell compartments in the spleen. In this study we report the characterization of mechanisms governing both basal as well as PMA- and TNF-inducible regulation of the human LT-beta promoter. Using a Jurkat T cell line, induction with either PMA or TNF resulted in an increase in mRNA levels compared with uninduced values. This induction corresponded to an increase in transcriptional activity of the human LT-beta promoter. Mutational and deletion analysis demonstrated the importance of Ets and NF-kappaB motifs in the regulation of basal transcription. Furthermore, the ability of PMA to induce activity was lost in the Ets mutant constructs. Interestingly, the same mutation had little effect on the ability of TNF to induce transcription of the LT-beta promoter. TNF inducibility was localized to the NF-kappaB site positioned at -83 of the promoter sequence. Thus, it appears that the Ets site, although playing a major role in PMA induction, did not mediate TNF inducibility. Therefore, our study suggests that alternative signaling pathways may be present to induce the expression of LT-beta in response to different immunological or inflammatory stimuli.
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Linfotoxina-alfa/biosíntesis , Proteínas de la Membrana/biosíntesis , FN-kappa B/fisiología , Proteínas Proto-Oncogénicas/fisiología , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Secuencia de Bases , Huella de ADN , Desoxirribonucleasa I/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Células Jurkat , Linfotoxina-alfa/antagonistas & inhibidores , Linfotoxina-alfa/genética , Linfotoxina beta , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Familia de Multigenes/inmunología , Mutagénesis Sitio-Dirigida , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/inmunología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/farmacología , Eliminación de Secuencia/inmunología , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción ReIA , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21(WAF1) 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21(WAF1) 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21(WAF1) protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21(WAF1) protein expression is mediated largely by stabilization of p21(WAF1) mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21(WAF1) expression or growth inhibition in this system. CP1 is a novel p21(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21(WAF1) mRNA stability.
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Regiones no Traducidas 3'/genética , Neoplasias de la Mama/genética , Ciclinas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Poli C/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN , Factor de Crecimiento Epidérmico/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Cinética , Luciferasas/genética , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The androgen receptor (AR) mediates androgen action and plays a central role in the proliferation of specific cancer cells. We demonstrated recently that AR mRNA stability is a major determinant of AR gene expression in prostate and breast cancer cells and that androgens differentially regulate AR mRNA decay dependent on cell type (Yeap, B. B., Kreuger, R. G., Leedman, P. J. (1999) Endocrinology 140, 3282-3291). Here, we have identified a highly conserved UC-rich region in the 3-untranslated region of AR mRNA that contains a 5'-C(U)(n)C motif and a 3'-CCCUCCC poly(C)-binding protein motif. In transfection studies with LNCaP human prostate cancer cells, the AR UC-rich region reduced expression of a luciferase reporter gene. The AR UC-rich region was a target for cytoplasmic and nuclear RNA-binding proteins from human prostate and breast cancer cells as well as human testicular and breast cancer tissue. One of these proteins is HuR, a ubiquitously expressed member of the Elav/Hu family of RNA-binding proteins involved in the stabilization of several mRNAs. Poly(C)-binding protein-1 and -2 (CP1 and CP2), previously implicated in the control of mRNA turnover and translation, also bound avidly to the UC-rich region. Mutational analysis of the UC-rich region identified specific binding motifs for both HuR and the CPs. HuR and CP1 bound simultaneously to the UC-rich RNA and in a cooperative manner. Immunoprecipitation studies confirmed that each of these proteins associated with AR mRNA in prostate cancer cells. In summary, we have identified and characterized a novel complex of AR mRNA-binding proteins that target the highly conserved UC-rich region. The binding of HuR, CP1, and CP2 to AR mRNA suggests a role for each of these proteins in the post-transcriptional regulation of AR expression in cancer cells.