RESUMO
Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest-enriched gene MOXD1 associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that MOXD1 expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify MOXD1 as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neuroblastoma , Peixe-Zebra , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Linhagem da Célula/genética , Crista Neural/metabolismo , Crista Neural/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , Neuroblastoma/metabolismo , Células de Schwann/metabolismo , Células de Schwann/patologia , Peixe-Zebra/genéticaRESUMO
Therapeutic resistance and recurrence remain core challenges in cancer therapy. How therapy resistance arises is currently not fully understood with tumors surviving via multiple alternative routes. Here, we demonstrate that a subset of cancer cells survives therapeutic stress by entering a transient state characterized by whole-genome doubling. At the onset of the polyploidization program, we identified an upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF2α. We found altered chromatin accessibility, ablated expression of retinoblastoma protein (RB1), and enrichment of AP-1 motif accessibility. We demonstrate that AP-1 and HIF2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF2α reduced the number of surviving cells following chemotherapy treatment. The role of AP-1 and HIF2α in stress response by polyploidy suggests a novel avenue for tackling chemotherapy-induced resistance in cancer. SIGNIFICANCE: In response to cisplatin treatment, some surviving cancer cells undergo whole-genome duplications without mitosis, which represents a mechanism of drug resistance. This study presents mechanistic data to implicate AP-1 and HIF2α signaling in the formation of this surviving cell phenotype. The results open a new avenue for targeting drug-resistant cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fator de Transcrição AP-1/genética , Regulação para Cima , Transdução de Sinais , Neoplasias/tratamento farmacológicoRESUMO
Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine tumors. PGLs can further be divided into sympathetic (sPGLs) and head-and-neck (HN-PGLs). There are virtually no treatment options, and no cure, for metastatic PCCs and PGLs (PPGLs). Here, we composed a tissue microarray (TMA) consisting of 149 PPGLs, reflecting clinical features, presenting as a useful resource. Mutations in the pseudohypoxic marker HIF-2α correlate to an aggressive tumor phenotype. We show that HIF-2α localized to the cytoplasm in PPGLs. This subcompartmentalized protein expression differed between tumor subtypes, and strongly correlated to proliferation. Half of all sPGLs were metastatic at time of diagnosis. Cytoplasmic HIF-2α was strongly expressed in metastatic sPGLs and predicted poor outcome in this subgroup. We propose that higher cytoplasmic HIF-2α expression could serve as a useful clinical marker to differentiate paragangliomas from pheochromocytomas, and may help predict outcome in sPGL patients.
Assuntos
Neoplasias das Glândulas Suprarrenais , Paraganglioma , Neoplasias do Sistema Nervoso Periférico , Feocromocitoma , Humanos , Neoplasias das Glândulas Suprarrenais/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citoplasma/metabolismo , Paraganglioma/diagnóstico , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/genéticaRESUMO
Cancer is a multifactorial, complex disease exhibiting extraordinary phenotypic plasticity and diversity. One of the greatest challenges in cancer treatment is intratumoral heterogeneity, which obstructs the efficient eradication of the tumor. Tumor heterogeneity is often associated with the presence of cancer stem cells (CSCs), a cancer cell sub-population possessing a panel of stem-like properties, such as a self-renewal ability and multipotency potential. CSCs are associated with enhanced chemoresistance due to the enhanced efflux of chemotherapeutic agents and the existence of powerful antioxidant and DNA damage repair mechanisms. The distinctive characteristics of CSCs make them ideal targets for clinical therapeutic approaches, and the identification of efficient and specific CSCs biomarkers is of utmost importance. Aldehyde dehydrogenases (ALDHs) comprise a wide superfamily of metabolic enzymes that, over the last years, have gained increasing attention due to their association with stem-related features in a wide panel of hematopoietic malignancies and solid cancers. Aldehyde dehydrogenase 1B1 (ALDH1B1) is an isoform that has been characterized as a marker of colon cancer progression, while various studies suggest its importance in additional malignancies. Here, we review the basic concepts related to CSCs and discuss the potential role of ALDH1B1 in cancer development and its contribution to the CSC phenotype.
RESUMO
BACKGROUND: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development. RESULTS: Here, we show that HIF-2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF-2α in vivo causes developmental delays, induces proliferation, and self-renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF-2α reveal enrichment of genes associated with cancer, invasion, epithelial-to-mesenchymal transition, and growth arrest. CONCLUSIONS: Taken together, these results suggest that expression levels of HIF-2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Crista Neural/embriologia , Animais , Fator de Transcrição CDX2/metabolismo , Sistemas CRISPR-Cas , Embrião de Galinha , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica no Desenvolvimento , Fator 1-beta Nuclear de Hepatócito/metabolismo , Humanos , Crista Neural/metabolismo , Fatores de Transcrição SOX9/metabolismoRESUMO
Lung cancer is the worldwide leading cause of death from cancer. Epigenetic modifications such as methylation and changes in chromatin accessibility are major gene regulatory mechanisms involved in tumorigenesis and cellular lineage commitment. We aimed to characterize these processes in the context of neuroendocrine (NE) lung cancer. Illumina 450K DNA methylation data were collected for 1407 lung cancers including 27 NE tumors. NE differentially methylated regions (NE-DMRs) were identified and correlated with gene expression data for 151 lung cancers and 31 human tissue entities from the Genotype-Tissue Expression (GTEx) consortium. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were performed on eight lung cancer cell lines, including three NE cell lines, to identify neuroendocrine specific gene regulatory elements. We identified DMRs with methylation patterns associated with differential gene expression and an NE tumor phenotype. DMR-associated genes could further be split into six functional modules, including one highly specific gene module for NE lung cancer showing high expression in both normal and malignant brain tissue. The regulatory potential of NE-DMRs was further validated in vitro using paired ATAC- and RNA-seq and revealed both proximal and distal regulatory elements of canonical NE-marker genes such as CHGA, NCAM1, INSM1, as well as a number of novel candidate markers of NE lung cancer. Using multilevel genomic analyses of both tumor bulk tissue and lung cancer cell lines, we identified a large catalogue of gene regulatory elements related to the NE phenotype of lung cancer.
RESUMO
Patients with estrogen receptor α positive (ERα+) breast cancer can respond to endocrine therapy, but treatment resistance is common and associated with downregulation of ERα expression in the dormant residual cells. Here we show, using long-term NSG xenograft models of human breast cancer and primary human monocytes, in vitro primary cell cultures and tumors from breast cancer patients, that macrophage derived tumor necrosis factor alpha (TNFα) downregulates ERα in breast cancer cells via inactivation of the transcription factor Forkhead box O transcription factor 3a (FOXO3a). Moreover, presence of tumor associated macrophages in the primary tumor of breast cancer patients, was associated with ERα negativity, and with worse prognosis in patients with ERα+ tumors. We propose that pro-inflammatory macrophages, despite being tumoricidal, may have direct effects on tumor progression and endocrine resistance in breast cancer patients. Our findings suggest that TNFα antagonists should be evaluated for treatment of ERα+ breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/genética , Proteína Forkhead Box O3/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Neoplasias da Mama/genética , Células Cultivadas , Regulação para Baixo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Macrófagos/citologia , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Células Precursoras de Monócitos e Macrófagos/citologia , Células Precursoras de Monócitos e Macrófagos/metabolismo , Células Precursoras de Monócitos e Macrófagos/transplanteRESUMO
BACKGROUND: Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. METHODS: Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor growth in vivo. Two-sided student t-test was used. RESULTS: We detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2α expression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. In coherence, RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2α protein knockdown resulted in profound downregulation of target genes. CONCLUSIONS: The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2α expression at normoxia suggest that HIF-2α have additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2α has transcriptional and non-transcriptional roles in neuroblastoma.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indanos/farmacologia , Neuroblastoma/patologia , Sulfonas/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Apoptose , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/genética , Neuroblastoma/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The PI3K pathway is a major driver of cancer progression. However, clinical resistance to PI3K inhibition is common. IBL-302 is a novel highly specific triple PIM, PI3K, and mTOR inhibitor. Screening IBL-302 in over 700 cell lines representing 47 tumor types identified neuroblastoma as a strong candidate for PIM/PI3K/mTOR inhibition. IBL-302 was more effective than single PI3K inhibition in vitro, and IBL-302 treatment of neuroblastoma patient-derived xenograft (PDX) cells induced apoptosis, differentiated tumor cells, and decreased N-Myc protein levels. IBL-302 further enhanced the effect of the common cytotoxic chemotherapies cisplatin, doxorubicin, and etoposide. Global genome, proteome, and phospho-proteome analyses identified crucial biological processes, including cell motility and apoptosis, targeted by IBL-302 treatment. While IBL-302 treatment alone reduced tumor growth in vivo, combination therapy with low-dose cisplatin inhibited neuroblastoma PDX growth. Complementing conventional chemotherapy treatment with PIM/PI3K/mTOR inhibition has the potential to improve clinical outcomes and reduce severe late effects in children with high-risk neuroblastoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neuroblastoma/tratamento farmacológico , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tiofenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Tiofenos/uso terapêutico , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma is a neural crest-derived childhood tumor of the peripheral nervous system in which MycN amplification is a hallmark of poor prognosis. Here we show that MycN is expressed together with phosphorylation-stabilizing factor CIP2A in regions of the neural plate destined to form the CNS, but MycN is excluded from the neighboring neural crest stem cell domain. Interestingly, ectopic expression of MycN or CIP2A in the neural crest domain biases cells toward CNS-like neural stem cells that express Sox2. Consistent with this, some forms of neuroblastoma have been shown to share transcriptional resemblance with CNS neural stem cells. As high MycN/CIP2A levels correlate with poor prognosis, we posit that a MycN/CIP2A-mediated cell-fate bias may reflect a possible mechanism underlying early priming of some aggressive forms of neuroblastoma. In contrast to MycN, its paralogue cMyc is normally expressed in the neural crest stem cell domain and typically is associated with better overall survival in clinical neuroblastoma, perhaps reflecting a more "normal" neural crest-like state. These data suggest that priming for some forms of aggressive neuroblastoma may occur before neural crest emigration from the CNS and well before sympathoadrenal specification.
Assuntos
Autoantígenos/fisiologia , Proteínas de Membrana/fisiologia , Proteína Proto-Oncogênica N-Myc/fisiologia , Crista Neural/citologia , Células-Tronco Neurais/fisiologia , Neuroblastoma/etiologia , Autoantígenos/análise , Movimento Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/análise , Proteína Proto-Oncogênica N-Myc/análise , Neuroblastoma/patologia , Fatores de Transcrição SOXB1/análiseRESUMO
Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) - mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Imunoprecipitação da Cromatina/métodos , DNA/metabolismo , Engenharia Genética , Proteínas de Homeodomínio/metabolismo , Espectrometria de Massas/métodos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição/genéticaRESUMO
Hypoxia (i.e., low oxygen levels) is a known feature of aggressive tumors. Cells, including tumor cells, respond to conditions of insufficient oxygen by activating a transcriptional program mainly driven by hypoxia-inducible factors (HIF)-1 and HIF-2. Both HIF-1α and HIF-2α expression levels have been shown to correlate to patient outcome in various tumor forms and in neuroblastoma, a solid childhood tumor of the sympathetic nervous system, in particular, HIF-2α marks a subpopulation of immature neural crest-like perivascularly located cells and associates with aggressive disease and distant metastasis. It has for long been recognized that the HIF-α subunits are oxygen-dependently regulated at the post-translational level, via ubiquitination and proteasomal degradation. Evidence of oxygen-independent mechanisms of regulation, transcriptional control of EPAS1/HIF2A and possible cytoplasmic activities of HIF-2α has also emerged during recent years. In this review, we discuss these non-conventional actions of HIF-2α, its putative role as a therapeutic target and the constraints it carries, as well as the importance of HIF-2 activity in a vascularized setting, the so-called pseudo-hypoxic state.
Assuntos
Fator 1 Induzível por Hipóxia/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Animais , Hipóxia Celular , Humanos , Neovascularização Patológica/metabolismo , Neuroblastoma/irrigação sanguínea , Neuroblastoma/genética , Oxigênio/metabolismo , Resultado do TratamentoRESUMO
We previously demonstrated that small cell lung carcinoma (SCLC) cells lack HIF-2α protein expression, whereas HIF-1α in these cells is expressed at both acute and prolonged hypoxia. Here we show that low HIF2A expression correlates with high expression of MYC genes. Knockdown of HIF1A expression had no or limited effect on cell survival and growth in vitro. Unexpectedly, hypoxic ATP levels were not affected by HIF-1α knockdown and SCLC cell viability did not decrease upon glucose deprivation. In line with these in vitro data, xenograft tumor-take and growth were not significantly affected by repressed HIF1A expression. Glutamine withdrawal drastically decreased SCLC cell proliferation and increased cell death at normoxia and hypoxia in a HIF-independent fashion and the dependence on glutaminolysis was linked to amplification of either MYC or MYCL. Downregulation of GLS expression, regulating the first step of the glutaminolysis pathway, in MYC/MYCL overexpressing SCLC cells resulted in both impaired growth and increased cell death. Our results suggest that MYC/MYCL overexpression in SCLC cells overrides the need of HIF-1 activity in response to hypoxia by inducing glutaminolysis and lipogenesis. Targeting the glutaminolysis pathway might hence be a novel approach to selectively kill MYC amplified SCLC cells in vivo.
Assuntos
Genes myc , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Amplificação de Genes , Expressão Gênica , Técnicas de Silenciamento de Genes , Glutamina/metabolismo , Glicólise , Xenoenxertos , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Camundongos , Modelos BiológicosRESUMO
Tumor hypoxia correlates to aggressive disease, and while this is explained by a variety of factors, one clue to understand this phenomena was the finding that hypoxia induces a de-differentiated, stem cell-like phenotype in neuroblastoma and breast tumor cells. The hypoxia inducible transcription factors (HIFs) are regulated at the translational level by fluctuating oxygen concentrations, but emerging data reveal that both HIF-1α and HIF-2α expression can be induced by aberrantly activated growth factor signaling independently of oxygen levels. Furthermore, HIF-2α is regulated by hypoxia also at the transcriptional level in neuroblastoma and glioma cells. In cultured tumor cells, HIF-2α is stabilized at physiological oxygen concentrations followed by induced expression of classical hypoxia-driven genes, resulting in a pseudohypoxic phenotype. In addition, in neuroblastoma and glioma specimens, a small subset of HIF-2α positive, HIF-1α negative, tumor cells is found adjacent to blood vessels, i.e. in areas with presumably adequate oxygenation. These tumor niches are thus pseudohypoxic, and the HIF-2α expressing cells present immature features. We have postulated that this niche in neuroblastomas encompass the tumor stem cells. Oncogenes or tumor suppressor genes associated with pseudohypoxia are frequently mutated or deleted in the germline, implicating that the pseudohypoxic phenotype indeed is tumorigenic. In summary, the hypoxic and pseudohypoxic phenotypes of solid tumors are attractive therapeutic targets.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Hipóxia Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Hipóxia/metabolismo , Neuroblastoma/metabolismo , Animais , HumanosRESUMO
Triple-negative (TN) breast cancers (ER-PR-HER2-) are highly metastatic and associated with poor prognosis. Within this subtype, invasive, stroma-rich tumours with infiltration of inflammatory cells are even more aggressive. The effect of myeloid cells on reactive stroma formation in TN breast cancer is largely unknown. Here, we show that primary human monocytes have a survival advantage, proliferate in vivo and develop into immunosuppressive myeloid cells expressing the myeloid-derived suppressor cell marker S100A9 only in a TN breast cancer environment. This results in activation of cancer-associated fibroblasts and expression of CXCL16, which we show to be a monocyte chemoattractant. We propose that this migratory feedback loop amplifies the formation of a reactive stroma, contributing to the aggressive phenotype of TN breast tumours. These insights could help select more suitable therapies targeting the stromal component of these tumours, and could aid prediction of drug resistance.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Quimiocina CXCL16/metabolismo , Fatores Quimiotáticos/farmacologia , Monócitos/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Colágeno/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Imunossupressão , Camundongos Nus , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Células Mieloides/metabolismo , Células Mieloides/patologia , Solubilidade , Células Estromais/patologia , Neoplasias de Mama Triplo Negativas/genética , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hypoxia-inducible factor (HIF) is a master regulator of cellular responses to oxygen deprival with a critical role in mediating the angiogenic switch in solid tumors. Differential expression of the HIF subunits HIF1α and HIF2α occurs in many human tumor types, suggesting selective implications to biologic context. For example, high expression of HIF2α that occurs in neuroblastoma is associated with stem cell-like features, disseminated disease, and poor clinical outcomes, suggesting pivotal significance for HIF2 control in neuroblastoma biology. In this study, we provide novel insights into how HIF2α expression is transcriptionally controlled by hypoxia and how this control is abrogated by inhibition of insulin-like growth factor-1R/INSR-driven phosphoinositide 3-kinase (PI3K) signaling. Reducing PI3K activity was sufficient to decrease HIF2α mRNA and protein expression in a manner with smaller and less vascularized tumors in vivo. PI3K-regulated HIF2A mRNA expression was independent of Akt or mTORC1 signaling but relied upon mTORC2 signaling. HIF2A mRNA was induced by hypoxia in neuroblastoma cells isolated from metastatic patient-derived tumor xenografts, where HIF2A levels could be reduced by treatment with PI3K and mTORC2 inhibitors. Our results suggest that targeting PI3K and mTORC2 in aggressive neuroblastomas with an immature phenotype may improve therapeutic efficacy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Complexos Multiproteicos/metabolismo , Neovascularização Patológica/genética , Neuroblastoma/irrigação sanguínea , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Nus , Neovascularização Patológica/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Receptor IGF Tipo 1 , Receptor IGF Tipo 2/genética , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Proteína Regulatória Associada a mTOR , Transdução de Sinais/fisiologia , Transcrição Gênica/genéticaRESUMO
Hypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leads to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neuroblastoma/metabolismo , Receptores de Estrogênio/fisiologia , Transcrição Gênica/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Humanos , Neuroblastoma/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Resultado do Tratamento , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease often have a poor outcome. Here we describe the establishment of neuroblastoma patient-derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high risk neuroblastoma into immunodeficient mice. In vivo tumour growth was monitored by magnetic resonance imaging and fluorodeoxyglucose-positron emission tomography. Neuroblastoma PDXs retained the undifferentiated histology and proliferative capacity of their corresponding patient tumours. The PDXs expressed neuroblastoma markers neural cell adhesion molecule, chromogranin A, synaptophysin and tyrosine hydroxylase. Whole genome genotyping array analyses demonstrated that PDXs retained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Thus, neuroblastoma PDXs recapitulate the hallmarks of high-risk neuroblastoma in patients. PDX-derived cells were cultured in serum-free medium where they formed free-floating neurospheres, expressed neuroblastoma gene markers MYCN, CHGA, TH, SYP and NPY, and retained tumour-initiating and metastatic capacity in vivo. PDXs showed much higher degree of infiltrative growth and distant metastasis as compared to neuroblastoma SK-N-BE(2)c cell line-derived orthotopic tumours. Importantly, the PDXs presented with bone marrow involvement, a clinical feature of aggressive neuroblastoma. Thus, neuroblastoma PDXs serve as clinically relevant models for studying and targeting high-risk metastatic neuroblastoma.