RESUMO
Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.
Assuntos
Neoplasias da Mama/patologia , Autorrenovação Celular/fisiologia , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/fisiologia , Superóxido Dismutase/fisiologia , Acetilação , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Neoplasias da Mama/metabolismo , Reprogramação Celular , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Peróxido de Hidrogênio/metabolismo , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/enzimologia , Invasividade Neoplásica , Proteínas de Neoplasias/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/químicaRESUMO
INTRODUCTION: Transforming growth factor-ßs (TGF-ßs) play a dual role in breast cancer, with context-dependent tumor-suppressive or pro-oncogenic effects. TGF-ß antagonists are showing promise in early-phase clinical oncology trials to neutralize the pro-oncogenic effects. However, there is currently no way to determine whether the tumor-suppressive effects of TGF-ß are still active in human breast tumors at the time of surgery and treatment, a situation that could lead to adverse therapeutic responses. METHODS: Using a breast cancer progression model that exemplifies the dual role of TGF-ß, promoter-wide chromatin immunoprecipitation and transcriptomic approaches were applied to identify a core set of TGF-ß-regulated genes that specifically reflect only the tumor-suppressor arm of the pathway. The clinical significance of this signature and the underlying biology were investigated using bioinformatic analyses in clinical breast cancer datasets, and knockdown validation approaches in tumor xenografts. RESULTS: TGF-ß-driven tumor suppression was highly dependent on Smad3, and Smad3 target genes that were specifically enriched for involvement in tumor suppression were identified. Patterns of Smad3 binding reflected the preexisting active chromatin landscape, and target genes were frequently regulated in opposite directions in vitro and in vivo, highlighting the strong contextuality of TGF-ß action. An in vivo-weighted TGF-ß/Smad3 tumor-suppressor signature was associated with good outcome in estrogen receptor-positive breast cancer cohorts. TGF-ß/Smad3 effects on cell proliferation, differentiation and ephrin signaling contributed to the observed tumor suppression. CONCLUSIONS: Tumor-suppressive effects of TGF-ß persist in some breast cancer patients at the time of surgery and affect clinical outcome. Carefully tailored in vitro/in vivo genomic approaches can identify such patients for exclusion from treatment with TGF-ß antagonists.
Assuntos
Neoplasias da Mama/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Efrinas/metabolismo , Feminino , Humanos , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno , Receptor EphA2/metabolismo , Proteína Smad2/genética , Proteína Smad3/biossíntese , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/biossíntese , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAFV600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.
Assuntos
Imidazóis , Oximas , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Camundongos , Animais , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Proteínas Proto-Oncogênicas B-raf/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , MutaçãoRESUMO
Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer cell proliferation and CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H, induce redox imbalance, dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function. Significance: ClpP agonists disrupt mitochondrial homeostasis by activating mitochondrial matrix protease ClpP. We report that ClpP agonists inhibit cell growth and cancer stem cell functions in breast cancer models by modulating multiple metabolic pathways essential to cancer stem cell function.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Peptídeo Hidrolases/metabolismo , NAD/metabolismo , Recidiva Local de Neoplasia/metabolismo , Mitocôndrias , Homeostase , Endopeptidases/metabolismo , Células-Tronco Neoplásicas , Endopeptidase Clp/metabolismoRESUMO
Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.
Assuntos
Neoplasias da Mama/imunologia , Macrófagos/imunologia , Células-Tronco Neoplásicas/citologia , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Microscopia Intravital , Camundongos , Camundongos SCID , Metástase Neoplásica , Células Neoplásicas Circulantes/imunologia , Células-Tronco Neoplásicas/imunologia , Receptores Notch/genética , Receptores Notch/imunologia , Transdução de Sinais , Microambiente Tumoral/imunologiaRESUMO
INTRODUCTION: Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference (RNAi)-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFß/Smad pathway as an example. METHODS: We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse) which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. A panel of conditionally immortalized mammary epithelial cell (IMEC) cultures were derived from the virgin mammary glands of offspring of these crosses and used to assess the Smad dependency of different biological responses to TGFß. RESULTS: IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFß. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFß-induced apoptotic, growth inhibitory and EMT responses, whereas either Smad can support TGFß-induced invasion as long as a threshold level of total Smad is exceeded. CONCLUSIONS: This work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium.
Assuntos
Células Epiteliais/metabolismo , Glândulas Mamárias Animais/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Apoptose/genética , Diferenciação Celular , Movimento Celular , Células Cultivadas , Transição Epitelial-Mesenquimal/genética , Feminino , Técnicas de Inativação de Genes , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Transgênicos , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Peptidylarginine deiminases (PADI) catalyze posttranslational modification of many target proteins and have been suggested to play a role in carcinogenesis. Citrullination of histones by PADI4 was recently implicated in regulating embryonic stem and hematopoietic progenitor cells. Here, we investigated a possible role for PADI4 in regulating breast cancer stem cells. PADI4 activity limited the number of cancer stem cells (CSC) in multiple breast cancer models in vitro and in vivo. Mechanistically, PADI4 inhibition resulted in a widespread redistribution of histone H3, with increased accumulation around transcriptional start sites. Interestingly, epigenetic effects of PADI4 on the bulk tumor cell population did not explain the CSC phenotype. However, in sorted tumor cell populations, PADI4 downregulated expression of master transcription factors of stemness, NANOG and OCT4, specifically in the cancer stem cell compartment, by reducing the transcriptionally activating H3R17me2a histone mark at those loci; this effect was not seen in the non-stem cells. A gene signature reflecting tumor cell-autonomous PADI4 inhibition was associated with poor outcome in human breast cancer datasets, consistent with a tumor-suppressive role for PADI4 in estrogen receptor-positive tumors. These results contrast with known tumor-promoting effects of PADI4 on the tumor stroma and suggest that the balance between opposing tumor cell-autonomous and stromal effects may determine net outcome. Our findings reveal a novel role for PADI4 as a tumor suppressor in regulating breast cancer stem cells and provide insight into context-specific effects of PADI4 in epigenetic modulation. SIGNIFICANCE: These findings demonstrate a novel activity of the citrullinating enzyme PADI4 in suppressing breast cancer stem cells through epigenetic repression of stemness master transcription factors NANOG and OCT4.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas , Células MCF-7 , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína-Arginina Desiminase do Tipo 4/antagonistas & inibidores , Proteína-Arginina Desiminase do Tipo 4/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
PURPOSE: TGFßs are overexpressed in many advanced cancers and promote cancer progression through mechanisms that include suppression of immunosurveillance. Multiple strategies to antagonize the TGFß pathway are in early-phase oncology trials. However, TGFßs also have tumor-suppressive activities early in tumorigenesis, and the extent to which these might be retained in advanced disease has not been fully explored. EXPERIMENTAL DESIGN: A panel of 12 immunocompetent mouse allograft models of metastatic breast cancer was tested for the effect of neutralizing anti-TGFß antibodies on lung metastatic burden. Extensive correlative biology analyses were performed to assess potential predictive biomarkers and probe underlying mechanisms. RESULTS: Heterogeneous responses to anti-TGFß treatment were observed, with 5 of 12 models (42%) showing suppression of metastasis, 4 of 12 (33%) showing no response, and 3 of 12 (25%) showing an undesirable stimulation (up to 9-fold) of metastasis. Inhibition of metastasis was immune-dependent, whereas stimulation of metastasis was immune-independent and targeted the tumor cell compartment, potentially affecting the cancer stem cell. Thus, the integrated outcome of TGFß antagonism depends on a complex balance between enhancing effective antitumor immunity and disrupting persistent tumor-suppressive effects of TGFß on the tumor cell. Applying transcriptomic signatures derived from treatment-naïve mouse primary tumors to human breast cancer datasets suggested that patients with breast cancer with high-grade, estrogen receptor-negative disease are most likely to benefit from anti-TGFß therapy. CONCLUSIONS: Contrary to dogma, tumor-suppressive responses to TGFß are retained in some advanced metastatic tumors. Safe deployment of TGFß antagonists in the clinic will require good predictive biomarkers.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Pulmonares/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Resultado do TratamentoRESUMO
Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA (LINC00675) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP (FOXA1-Regulated Conserved Small Protein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells.
Assuntos
Apoptose/genética , Carcinogênese/genética , Neoplasias Colorretais/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , RNA Longo não Codificante/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Trato Gastrointestinal/metabolismo , Genes Reporter , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Anotação de Sequência Molecular , Especificidade de Órgãos , RNA Longo não Codificante/genéticaRESUMO
The transforming growth factor-beta (TGF-beta) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-beta is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-beta has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-beta reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-beta receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-beta involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-beta. These data suggest new roles for the TGF-beta pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation.
Assuntos
Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/patologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Proteína 1 Inibidora de Diferenciação/biossíntese , Proteína 1 Inibidora de Diferenciação/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/deficiência , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Fator de Crescimento Transformador beta/deficiência , Transplante HeterólogoRESUMO
The C-terminal binding protein (CtBP) is an NADH-dependent dimeric family of nuclear proteins that scaffold interactions between transcriptional regulators and chromatin-modifying complexes. Its association with poor survival in several cancers implicates CtBP as a promising target for pharmacological intervention. We employed computer-assisted drug design to search for CtBP inhibitors, using quantitative structure-activity relationship (QSAR) modeling and docking. Functional screening of these drugs identified 4 compounds with low toxicity and high water solubility. Micro molar concentrations of these CtBP inhibitors produces significant de-repression of epigenetically silenced pro-epithelial genes, preferentially in the triple-negative breast cancer cell line MDA-MB-231. This epigenetic reprogramming occurs through eviction of CtBP from gene promoters; disrupted recruitment of chromatin-modifying protein complexes containing LSD1, and HDAC1; and re-wiring of activating histone marks at targeted genes. In functional assays, CtBP inhibition disrupts CtBP dimerization, decreases cell migration, abolishes cellular invasion, and improves DNA repair. Combinatorial use of CtBP inhibitors with the LSD1 inhibitor pargyline has synergistic influence. Finally, integrated correlation of gene expression in breast cancer patients with nuclear levels of CtBP1 and LSD1, reveals new potential therapeutic vulnerabilities. These findings implicate a broad role for this class of compounds in strategies for epigenetically targeted therapeutic intervention.
Assuntos
Oxirredutases do Álcool/genética , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Feminino , HumanosRESUMO
Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Sialoglicoproteínas/biossíntese , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/fisiologia , Colágeno/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Sialoproteína de Ligação à Integrina , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Sialoglicoproteínas/genética , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/imunologiaRESUMO
The TGF-beta signaling network plays a complex role in carcinogenesis because it has the potential to act as either a tumor suppressor or a pro-oncogenic pathway. Currently, it is not known whether TGF-beta can switch from tumor suppressor to pro-oncogenic factor during the course of carcinogenic progression in a single cell lineage with a defined initiating oncogenic event or whether the specific nature of the response is determined by cell type and molecular etiology. To address this question, we have introduced a dominant negative type II TGF-beta receptor into a series of genetically related human breast-derived cell lines representing different stages in the progression process. We show that decreased TGF-beta responsiveness alone cannot initiate tumorigenesis but that it can cooperate with an initiating oncogenic lesion to make a premalignant breast cell tumorigenic and a low-grade tumorigenic cell line histologically and proliferatively more aggressive. In a high-grade tumorigenic cell line, however, reduced TGF-beta responsiveness has no effect on primary tumorigenesis but significantly decreases metastasis. Our results demonstrate a causal role for loss of TGF-beta responsiveness in promoting breast cancer progression up to the stage of advanced, histologically aggressive, but nonmetastatic disease and suggest that at that point TGF-beta switches from tumor suppressor to prometastatic factor.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Mamárias Experimentais/prevenção & controle , Fator de Crescimento Transformador beta/farmacologia , Animais , Transformação Celular Neoplásica , Progressão da Doença , Feminino , Humanos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
TGF-betas play diverse and complex roles in many biological processes. In tumorigenesis, they can function either as tumor suppressors or as pro-oncogenic factors, depending on the stage of the disease. We have developed transgenic mice expressing a TGF-beta antagonist of the soluble type II TGF-beta receptor:Fc fusion protein class, under the regulation of the mammary-selective MMTV-LTR promoter/enhancer. Biologically significant levels of antagonist were detectable in the serum and most tissues of this mouse line. The mice were resistant to the development of metastases at multiple organ sites when compared with wild-type controls, both in a tail vein metastasis assay using isogenic melanoma cells and in crosses with the MMTV-neu transgenic mouse model of metastatic breast cancer. Importantly, metastasis from endogenous mammary tumors was suppressed without any enhancement of primary tumorigenesis. Furthermore, aged transgenic mice did not exhibit the severe pathology characteristic of TGF-beta null mice, despite lifetime exposure to the antagonist. The data suggest that in vivo the antagonist may selectively neutralize the undesirable TGF-beta associated with metastasis, while sparing the regulatory roles of TGF-betas in normal tissues. Thus this soluble TGF-beta antagonist has potential for long-term clinical use in the prevention of metastasis.
Assuntos
Fragmentos Fc das Imunoglobulinas/fisiologia , Imunoglobulina G/fisiologia , Neoplasias Hepáticas/secundário , Neoplasias Mamárias Animais/prevenção & controle , Melanoma Experimental/prevenção & controle , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Feminino , Vetores Genéticos , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Neoplasias Mamárias Animais/patologia , Vírus do Tumor Mamário do Camundongo , Camundongos , Camundongos Transgênicos , Metástase Neoplásica/prevenção & controle , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Solubilidade , Células Tumorais CultivadasRESUMO
Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinase/genética , RNA Mensageiro/análise , Fatores de Transcrição SOXB1/genética , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Antígenos CD , Antineoplásicos/farmacologia , Caderinas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virologia , Divisão Celular , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Papillomaviridae/isolamento & purificação , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Retinal Desidrogenase , Fatores de Transcrição SOXB1/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Esferoides Celulares , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Células Tumorais CultivadasRESUMO
The role of transforming growth factor beta in breast cancer is controversial with tumor suppressor and pro-oncogenic activities having been demonstrated. To address whether the same or different signal transduction pathways mediate these opposing activities, we manipulated the Smad2/3 signaling pathway in cells of common origin but differing degrees of malignancy derived from MCF10A human breast cells. We show that interference with endogenous Smad2/3 signaling enhances the malignancy of xenografted tumors of premalignant and well-differentiated tumor cells but strongly suppresses lung metastases of more aggressive carcinoma cells after tail vein injection. Overexpression of Smad3 in the same cells has opposite effects. The data demonstrate that the Smad2/3 signaling pathway mediates tumor suppressor and prometastatic signals, depending on the cellular context.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/fisiologia , Transativadores/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Transplante de Neoplasias , Fosforilação , Transdução de Sinais , Proteína Smad2 , Proteína Smad3 , Transativadores/antagonistas & inibidores , Transativadores/biossíntese , Transativadores/genética , Transfecção , Fator de Crescimento Transformador beta/fisiologia , Transplante HeterólogoRESUMO
Many tumors are hierarchically organized with a minority cell population that has stem-like properties and enhanced ability to initiate tumorigenesis and drive therapeutic relapse. These cancer stem cells (CSCs) are typically identified by complex combinations of cell-surface markers that differ among tumor types. Here, we developed a flexible lentiviral-based reporter system that allows direct visualization of CSCs based on functional properties. The reporter responds to the core stem cell transcription factors OCT4 and SOX2, with further selectivity and kinetic resolution coming from use of a proteasome-targeting degron. Cancer cells marked by this reporter have the expected properties of self-renewal, generation of heterogeneous offspring, high tumor- and metastasis-initiating activity, and resistance to chemotherapeutics. With this approach, the spatial distribution of CSCs can be assessed in settings that retain microenvironmental and structural cues, and CSC plasticity and response to therapeutics can be monitored in real time.
Assuntos
Expressão Gênica , Genes Reporter , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/farmacologia , Divisão Celular Assimétrica , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Rastreamento de Células , Transformação Celular Neoplásica/genética , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Ordem dos Genes , Vetores Genéticos , Xenoenxertos , Humanos , Imunofenotipagem , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Elementos de Resposta , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Transforming growth factor-ß (TGF-ß) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-ß signaling occurs through Smad2/3-Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-ß may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-ß. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-ß1. Mixed Smad complexes were also prominent in 15-16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes.
Assuntos
Neoplasias da Mama/metabolismo , Camundongos/embriologia , Transdução de Sinais , Proteínas Smad/análise , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/análise , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Técnicas de Preparação Histocitológica , Humanos , Imuno-Histoquímica/métodos , Camundongos Transgênicos , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas/métodos , Alinhamento de Sequência , Fator de Crescimento Transformador beta/genética , Regulação para CimaRESUMO
Smad3, a major intracellular mediator of TGFß signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFß, the TGFß receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFß-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFß responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer.
Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Transição Epitelial-Mesenquimal/genética , Proteína Smad3/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Fosforilação/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Carcinoma are complex societies of mutually interacting cells in which there is a progressive failure of normal homeostatic mechanisms, causing the parenchymal component to expand inappropriately and ultimately to disseminate to distant sites. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumor microenvironment and then to normal fibroblasts as it traverses the underlying connective tissue towards the bloodstream. The interaction of tumor cells with stromal fibroblasts influences tumor biology by mechanisms that are not yet fully understood. Here, we report a role for normal stroma fibroblasts in the progression of invasive tumors to metastatic tumors. Using a coculture system of human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts, we found that medium conditioned by cocultures of the two cell types (CoCM) increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. Transient treatment of MCF10CA1a cells with CoCM in vitro accelerated tumor growth at orthotopic sites in vivo, and resulted in an expanded pattern of metastatic engraftment. The effects of CoCM on MCF10CA1a cells were dependent on small amounts of active TGF-beta1 secreted by fibroblasts under the influence of the tumor cells, and required intact ALK5-, p38-, and JNK signaling in the tumor cells. In conclusion, these results demonstrate that transient interactions between tumor cells and normal fibroblasts can modify the acellular component of the local microenvironment such that it induces long-lasting increases in tumorigenicity and alters the metastatic pattern of the cancer cells in vivo. TGF-beta appears to be a key player in this process, providing further rationale for the development of anti-cancer therapeutics that target the TGF-beta pathway.