RESUMEN
Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor-activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF-ß regulates the transcription of target genes in a cell context-dependent manner by screening a double-stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti-Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF-ß. The preference of the activated Smad complexes for conventional Smad-binding motifs such as Smad-binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor-binding motifs other than SBE or CAGA, including the Fos/Jun-binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor-binding motifs together with Smad-binding motifs indicated that some of the motifs may be involved in cell type-dependent transcriptional activation by TGF-ß. The results suggest that the CASTing method is useful for elucidating the molecular basis of context-dependent Smad signaling.
Asunto(s)
ADN , Transducción de Señal , Factor de Crecimiento Transformador beta , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Células Hep G2 , ADN/metabolismo , Unión Proteica , Proteína smad3/metabolismo , Proteína Smad2/metabolismo , Células A549 , Células HaCaT , Proteínas Smad/metabolismoRESUMEN
Epstein-Barr virus (EBV) is one of the major drivers of gastric carcinogenesis. EBV infection is established before tumour initiation and is generally maintained throughout tumour development; however, the significance of EBV in tumour maintenance and progression remains to be elucidated. Here, we report eight cases of EBV-associated gastric carcinoma (EBVaGC) with intratumoural heterogenous expression of EBV-encoded small RNA (EBER), a highly expressed latent gene of EBV, and demonstrate clinicopathological characteristics of these rare cases. By performing detailed histological assessment of EBER-positive and -negative components of each case, detection of EBV genome in tumour cells by fluorescence in situ hybridisation, TP73 methylation analysis, whole exome sequencing, and targeted gene panel sequencing, we identified tumours in two patients to be collision tumours of different origins. In the other six patients, some genetic/epigenetic alterations were shared between EBER-positive and -negative components, suggesting that EBV was eliminated from tumour cells during progression. Interestingly, in both tumour types, programmed death ligand 1 and intratumoural infiltration of CD8+ T lymphocytes were lower in EBER-negative than in EBER-positive components, suggesting an immunogenic role of EBV. To the best of our knowledge, this study is the first to demonstrate the detailed histological features and genetic/epigenetic alterations in EBVaGC with heterogenous EBER expression; the loss of EBV may benefit tumour progression and immune evasion and might be clinically important for selecting treatment strategies for such cancers. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Carcinoma , Infecciones por Virus de Epstein-Barr , Neoplasias Gástricas , Humanos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/patología , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Genoma Viral , Carcinoma/genética , ARN Viral/genética , Microambiente TumoralRESUMEN
Transforming growth factor ß (TGF-ß) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-ß also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-ß confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-ß, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-ß generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-ß-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis.
Asunto(s)
Neoplasias de la Boca , Factor de Crecimiento Transformador beta , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal/genética , Puntos de Control de la Fase G1 del Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Queratinas/metabolismo , Neoplasias de la Boca/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The activation of RIG-I-like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand-mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)-ß that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple-negative breast cancer cells (TNBC) attenuates transforming growth factor-ß (TGF-ß) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF-ß signaling by RLR ligands with respect to IFN-ß-mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN-ß, which inhibits the growth of the surrounding cancer cells. In addition, IFN-ß-induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN-ß expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF-ß that suppresses IFN-ß expression is suggested when RLR-mediated cancer treatment is used in TNBC.
Asunto(s)
Interferón beta/metabolismo , Poli I-C/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 3 Regulador del Interferón/metabolismo , Poli I-C/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína smad3/genética , Proteína smad3/metabolismo , Transfección , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Smad proteins transduce signals downstream of transforming growth factor-ß (TGF-ß) and are one of the factors that regulate the expression of genes related to diseases affecting the skin. In the present study, we identified MAB21L4, also known as male abnormal 21 like 4 or C2orf54, as the most up-regulated targets of TGF-ß and Smad3 in differentiated human progenitor epidermal keratinocytes using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). We found that TGF-ß induced expression of the barrier protein involucrin (encoded by the IVL gene). Transcriptional activity of the IVL promoter induced by TGF-ß was inhibited by MAB21L4 siRNAs. Further analysis revealed that MAB21L4 siRNAs also down-regulated the expression of several target genes of TGF-ß. MAB21L4 protein was located mainly in the cytosol, where it was physically bound to Smad3 and a transcriptional corepressor c-Ski. siRNAs for MAB21L4 did not inhibit the binding of Smad3 to their target genomic regions but down-regulated the acetylation of histone H3 lys 27 (H3K27ac), an active histone mark, near the Smad3 binding regions. These findings suggest that TGF-ß-induced MAB21L4 up-regulates the gene expression induced by TGF-ß, possibly through the inhibition of c-Ski via physical interaction in the cytosol.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta , Humanos , Queratinocitos/metabolismo , Masculino , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Glioma-initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum-free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP-induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133-positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx-1b, but not the shorter isoform, pmx-1a, induces GIC differentiation. Upon BMP stimulation, pmx-1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133-positive GIC population. Thus, pmx-1b promotes loss of stem cell-like properties of GICs through region-specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , ADN Metiltransferasa 3A , Epigénesis Genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Neoplásicas/metabolismoRESUMEN
DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5-aza-deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome-wide using methylation array in reference to RNA-sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor-suppressive role of UQCRH. Moreover, 5-aza-deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B-induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Apoptosis/genética , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Metilación de ADN/genética , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Neoplasias Renales/patologíaRESUMEN
We previously reported that bone morphogenetic protein (BMP) signaling promotes tumorigenesis in gynecologic cancer cells. BMP2 enhances proliferation of ovarian and endometrial cancer cells via c-KIT induction, and triggers epithelial-mesenchymal transition (EMT) by SNAIL and/or SLUG induction, leading to increased cell migration. However, the downstream effectors of BMP signaling in gynecological cancer cells have not been clearly elucidated. In this study, we performed RNA-sequencing of Ishikawa endometrial and SKOV3 ovarian cancer cells after BMP2 stimulation, and identified TNFRSF12A, encoding fibroblast growth factor-inducible 14 (FN14) as a common BMP2-induced gene. FN14 knockdown suppressed BMP2-induced cell proliferation and migration, confirmed by MTS and scratch assays, respectively. In addition, FN14 silencing augmented chemosensitivity of SKOV3 cells. As a downstream effector of BMP signaling, FN14 modulated both c-KIT and SNAIL expression, which are important for growth and migration of ovarian and endometrial cancer cells. These results support the notion that the tumor promoting effects of BMP signaling in gynecological cancers are partially attributed to FN14 induction.
Asunto(s)
Neoplasias Endometriales , Neoplasias Ováricas , Transducción de Señal , Proteína Morfogenética Ósea 2/farmacología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Transición Epitelial-Mesenquimal , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Receptor de TWEAKRESUMEN
Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor ß (TGF-ß) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-ß family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration of mono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.
RESUMEN
Transforming growth factor-ß (TGF-ß) signaling promotes cancer progression. In particular, the epithelial-mesenchymal transition (EMT) induced by TGF-ß is considered crucial to the malignant phenotype of cancer cells. Here, we report that the EMT-associated cellular responses induced by TGF-ß are mediated by distinct signaling pathways that diverge at Smad3. By expressing chimeric Smad1/Smad3 proteins in SMAD3 knockout A549 cells, we found that the ß4 region in the Smad3 MH1 domain is essential for TGF-ß-induced cell motility, but is not essential for other EMT-associated responses including epithelial marker downregulation. TGF-ß was previously reported to enhance cell motility by activating Rac1 via phosphoinositide 3-kinase. Intriguingly, TGF-ß-dependent signaling mediated by Smad3's ß4 region causes the downregulation of multiple mRNAs that encode GTPase activating proteins that target Rac1 (ARHGAPs), thereby attenuating Rac1 inactivation. Therefore, two independent pathways downstream of TGF-ß type I receptor contribute cooperatively to sustained Rac1 activation, thereby leading to enhanced cell motility.
Asunto(s)
Movimiento Celular , Transición Epitelial-Mesenquimal , Proteínas Activadoras de GTPasa/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Proteína de Unión al GTP rac1/metabolismo , Células A549 , Proteínas Activadoras de GTPasa/genética , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína smad3/genética , Proteína de Unión al GTP rac1/genéticaRESUMEN
Development of innovative therapeutic modalities would address an unmet clinical need in the treatment of triple negative breast cancer (TNBC). Activation of retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) such as melanoma differentiation-associated gene 5 (MDA5) and RIG-I in cancer cells is suggested to suppress tumor progression by inducing cell death. Transfection of polyI:C, a conventionally used synthetic double-stranded RNA (dsRNA) analogue that activates RLRs, has been evaluated in clinical trials. However, detailed mechanisms of tumor suppression by RLRs, especially interactions with other signaling pathways, remain elusive. Here, we showed that transfection of polyI:C suppressed transforming growth factor-ß (TGF-ß) signaling in a MDA5- and RIG-I-dependent manner. We found that suppression of TGF-ß signaling by polyI:C promoted cancer cell death, which was attenuated by forced expression of constitutively active Smad3. More detailed analysis suggested that cell death by polyI:C transfection exhibited characteristics of pyroptosis, which is distinct from apoptosis. Therapeutic efficacy of polyI:C transfection was also demonstrated using a mouse model. These results indicated that intratumor administration of polyI:C and related dsRNA analogues may be promising treatments for TNBC through inhibition of the anti-pyroptotic function of TGF-ß.
Asunto(s)
Piroptosis , ARN Bicatenario/farmacología , Factor de Crecimiento Transformador beta/farmacología , Neoplasias de la Mama Triple Negativas/patología , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Poli I-C/farmacología , Poli I-C/uso terapéutico , Piroptosis/efectos de los fármacos , Piroptosis/genética , ARN Bicatenario/síntesis química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células THP-1 , Factor de Crecimiento Transformador beta/fisiología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/terapia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BMP signaling has been found to have tumor-promoting as well as tumor-suppressing effects in different types of tumors. In this study, we investigated the effects of BMP signaling and of BMP inhibitors on ovarian cancer (OC) cells in vitro and in vivo. High expression of BMP receptor 2 (BMPR2) correlated with poor overall survival of OC patients in the TCGA dataset. Both BMP2 and BMPR2 enhanced OC cell proliferation, whereas BMP receptor kinase inhibitors inhibited OC cell growth in cell culture as well as in a mouse model. BMP2 also augmented sphere formation, migration, and invasion of OC cells, and induced EMT. High BMP2 expression was observed after chemotherapy of OC patients in the GSE109934 dataset. In accordance, carboplatin, used for the treatment of OC patients, increased BMP2 secretion from OC cells, and induced EMT partially via activation of BMP signaling. Our data suggest that BMP signaling has tumor-promoting effects in OC, and that BMP inhibitors might be useful therapeutic agents for OC patients. Considering that carboplatin treatment augmented BMP2 secretion, the possibility to use a combination of BMP inhibitors and carboplatin in the treatment of OC patients, would be worth exploring.
RESUMEN
Adult bone structural integrity is maintained by remodeling via the coupling of osteoclastic bone resorption and osteoblastic bone formation. Osteocytes or osteoblasts express receptor activator of nuclear factor κ-B ligand (Rankl) or osteoprotegerin (Opg) to promote or inhibit osteoclastogenesis, respectively. Bone morphogenetic protein (BMP) is a potent bone inducer, but its major role in adult bone is to induce osteocytes to upregulate sclerostin (Sost) and increase the Rankl/Opg expression ratio, resulting in promotion of osteoclastogenesis. However, the precise effect of BMP-target gene(s) in osteoblasts on the Rankl/Opg expression ratio remains unclear. In the present study, we identified atonal homolog 8 (Atoh8), which is directly upregulated by the BMP-Smad1 axis in osteoblasts. In vivo, Atoh8 was detected in osteoblasts but not osteocytes in adult mice. Although global Atoh8-knockout mice showed only a mild phenotype in the neonate skeleton, the bone volume was decreased and osteoclasts were increased in the adult phase. Atoh8-null marrow stroma cells were more potent than wild-type cells in inducing osteoclastogenesis in marrow cells. Atoh8 loss in osteoblasts increased Runx2 expression and the Rankl/Opg expression ratio, while Runx2 knockdown normalized the Rankl/Opg expression ratio. Moreover, Atoh8 formed a protein complex with Runx2 to inhibit Runx2 transcriptional activity and decrease the Rankl/Opg expression ratio. These results suggest that bone remodeling is regulated elaborately by BMP signaling; while BMP primarily promotes bone resorption, it simultaneously induces Atoh8 to inhibit Runx2 and reduce the Rankl/Opg expression ratio in osteoblasts, suppressing osteoclastogenesis and preventing excessive BMP-mediated bone resorption.
RESUMEN
Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-ß (TGF-ß) is secreted by various cell types residing in the TME and promotes tumor progression by inducing the epithelial-to-mesenchymal transition (EMT) of cancer cells and tumor angiogenesis. TGF-ß comprises three isoforms, TGF-ß1, -ß2, and -ß3, and transduces intracellular signals via TGF-ß type I receptor (TßRI) and TGF-ß type II receptor (TßRII). For the purpose of designing ligand traps that reduce oncogenic signaling in the TME, chimeric proteins comprising the ligand-interacting ectodomains of receptors fused with the Fc portion of immunoglobulin are often used. For example, chimeric soluble TßRII (TßRII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-ß ligands, but several lines of evidence indicate that TßRII-Fc more effectively traps TGF-ß1 and TGF-ß3 than TGF-ß2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-ß receptor containing both TßRI and TßRII (TßRI-TßRII-Fc) and found that TßRI-TßRII-Fc trapped all TGF-ß isoforms, leading to inhibition of both the TGF-ß signal and TGF-ß-induced EMT of oral cancer cells, whereas TßRII-Fc failed to trap TGF-ß2. Furthermore, we found that TßRI-TßRII-Fc suppresses tumor growth and angiogenesis more effectively than TßRII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-ß expression. These results suggest that TßRI-TßRII-Fc may be a promising tool for targeting all TGF-ß isoforms in the TME.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias de la Boca/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Receptores Fc/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Carcinoma de Células Escamosas/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Boca/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Receptores Fc/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Factor de Crecimiento Transformador beta/metabolismo , Microambiente TumoralRESUMEN
Advanced clear cell renal cell carcinoma (ccRCC) frequently causes systemic inflammation. Recent studies have shown that cancer cells reshape the immune landscape by secreting cytokines or chemokines. This phenotype, called cancer-cell-intrinsic inflammation, triggers a metastatic cascade. Here, we identified the functional role and regulatory mechanism of inflammation driven by advanced ccRCC cells. The inflammatory nature of advanced ccRCC was recapitulated in a preclinical model of ccRCC. Amplification of cancer-cell-intrinsic inflammation during ccRCC progression triggered neutrophil-dependent lung metastasis. Massive expression of inflammation-related genes was transcriptionally activated by epigenetic remodelling through mechanisms such as DNA demethylation and super-enhancer formation. A bromodomain and extra-terminal motif inhibitor synchronously suppressed C-X-C-type chemokines in ccRCC cells and decreased neutrophil-dependent lung metastasis. Overall, our findings provide insight into the nature of inflammatory ccRCC, which triggers metastatic cascades, and suggest a potential therapeutic strategy.
Asunto(s)
Carcinoma de Células Renales/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/secundario , Línea Celular Tumoral , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Inflamación , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Metástasis Linfática , Masculino , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patología , Pronóstico , Análisis de Supervivencia , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Thyroid transcription factor-1 (TTF-1, encoded by the NKX2-1 gene) is highly expressed in small-cell lung carcinoma (SCLC) and lung adenocarcinoma (LADC), but how its functional roles differ between SCLC and LADC remains to be elucidated. Here, we compared the genome-wide distributions of TTF-1 binding regions and the transcriptional programs regulated by TTF-1 between NCI-H209 (H209), a human SCLC cell line, and NCI-H441 (H441), a human LADC cell line, using chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq). TTF-1 binding regions in H209 and H441 cells differed by 75.0% and E-box motifs were highly enriched exclusively in the TTF-1 binding regions of H209 cells. Transcriptome profiling revealed that TTF-1 is involved in neuroendocrine differentiation in H209 cells. We report that TTF-1 and achaete-scute homolog 1 (ASCL1, also known as ASH1, an E-box binding basic helix-loop-helix transcription factor, and a lineage-survival oncogene of SCLC) are coexpressed and bound to adjacent sites on target genes expressed in SCLC, and cooperatively regulate transcription. Furthermore, TTF-1 regulated expression of the Bcl-2 gene family and showed antiapoptotic function in SCLC. Our findings suggest that TTF-1 promotes SCLC growth and contributes to neuroendocrine and antiapoptotic gene expression by partly coordinating with ASCL1.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Proliferación Celular/genética , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células Pequeñas/genética , Factor Nuclear Tiroideo 1/metabolismo , Apoptosis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Línea Celular Tumoral , Supervivencia Celular/genética , Secuenciación de Inmunoprecipitación de Cromatina , Regulación Neoplásica de la Expresión Génica/genética , Ontología de Genes , Estudio de Asociación del Genoma Completo , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Motivos de Nucleótidos , Pronóstico , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , RNA-Seq , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/mortalidad , Análisis de Matrices TisularesRESUMEN
Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) is implicated in vascular diseases such as pulmonary arterial hypertension (PAH). Here, we showed that the transcription factor ATOH8 was a direct target of SMAD1/5 and was induced in a manner dependent on BMP but independent of Notch, another critical signaling pathway in ECs. In zebrafish and mice, inactivation of Atoh8 did not cause an arteriovenous malformation-like phenotype, which may arise because of dysregulated Notch signaling. In contrast, Atoh8-deficient mice exhibited a phenotype mimicking PAH, which included increased pulmonary arterial pressure and right ventricular hypertrophy. Moreover, ATOH8 expression was decreased in PAH patient lungs. We showed that in cells, ATOH8 interacted with hypoxia-inducible factor 2α (HIF-2α) and decreased its abundance, leading to reduced induction of HIF-2α target genes in response to hypoxia. Together, these findings suggest that the BMP receptor type II/ALK-1/SMAD/ATOH8 axis may attenuate hypoxic responses in ECs in the pulmonary circulation and may help prevent the development of PAH.
Asunto(s)
Receptores de Activinas Tipo II/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/prevención & control , Hipoxia/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Receptores de Activinas Tipo II/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células HEK293 , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipoxia/genética , Hipoxia/patología , Ratones , Ratones Noqueados , Proteínas Smad/genética , Pez CebraRESUMEN
Smad proteins are transcriptional regulators activated by TGF-ß. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5'-GTCTAGAC-3') and CAGA motifs (5'-AGCCAGACA-3' or 5'-TGTCTGGCT-3'). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis-acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-ß. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo.
Asunto(s)
Proteína smad3/química , Proteína smad3/metabolismo , Proteína Smad4/química , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Secuencias de Aminoácidos , Secuencia de Bases , Sitios de Unión , Humanos , Unión Proteica , Elementos de Respuesta , Proteína Smad2/química , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína Smad4/genética , Activación TranscripcionalRESUMEN
Cyclin-dependent kinase (CDK) 4 and CDK6 inhibitors are effective therapeutic options for hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. Although CDK4/6 inhibitors mainly target the cyclin D-CDK4/6-retinoblastoma tumor suppressor protein (RB) axis, little is known about the clinical impact of inhibiting phosphorylation of other CDK4/6 target proteins. Here, we focused on other CDK4/6 targets, SMAD proteins. We showed that a CDK4/6 inhibitor palbociclib and activin-SMAD2 signaling cooperatively inhibited cell cycle progression of a luminal-type breast cancer cell line T47D. Palbociclib enhanced SMAD2 binding to the genome by inhibiting CDK4/6-mediated linker phosphorylation of the SMAD2 protein. We also showed that cyclin G2 plays essential roles in SMAD2-dependent cytostatic response. Moreover, comparison of the SMAD2 ChIP-seq data of T47D cells with those of Hs578T (triple-negative breast cancer cells) indicated that palbociclib augmented different SMAD2-mediated functions based on cell type, and enhanced SMAD2 binding to the target regions on the genome without affecting its binding pattern. In summary, palbociclib enhances the cytostatic effects of the activin-SMAD2 signaling pathway, whereas it possibly strengthens the tumor-promoting aspect in aggressive breast cancer.
Asunto(s)
Activinas/farmacología , Piperazinas/farmacología , Piridinas/farmacología , Receptores de Estrógenos/metabolismo , Proteína Smad2/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Citostáticos/farmacología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteína Smad2/genéticaRESUMEN
Transforming growth factor (TGF)-ß regulates a wide variety of cellular responses, including cell growth arrest, apoptosis, cell differentiation, motility, invasion, extracellular matrix production, tissue fibrosis, angiogenesis, and immune function. Although tumor-suppressive roles of TGF-ß have been extensively studied and well-characterized in many cancers, especially at early stages, accumulating evidence has revealed the critical roles of TGF-ß as a pro-tumorigenic factor in various types of cancer. This review will focus on recent findings regarding epithelial-mesenchymal transition (EMT) induced by TGF-ß, in relation to crosstalk with some other signaling pathways, and the roles of TGF-ß in lung and pancreatic cancers, in which TGF-ß has been shown to be involved in cancer progression. Recent findings also strongly suggested that targeting TGF-ß signaling using specific inhibitors may be useful for the treatment of some cancers. TGF-ß plays a pivotal role in the differentiation and function of regulatory T cells (Tregs). TGF-ß is produced as latent high molecular weight complexes, and the latent TGF-ß complex expressed on the surface of Tregs contains glycoprotein A repetitions predominant (GARP, also known as leucine-rich repeat containing 32 or LRRC32). Inhibition of the TGF-ß activities through regulation of the latent TGF-ß complex activation will be discussed.