RESUMO
A wide range of neurological manifestations have been associated with the development of COVID-19 following SARS-CoV-2 infection. However, the etiology of the neurological symptomatology is still largely unexplored. Here, we used state-of-the-art multiplexed immunostaining of human brains (n = 6 COVID-19, median age = 69.5 years; n = 7 control, median age = 68 years) and demonstrated that expression of the SARS-CoV-2 receptor ACE2 is restricted to a subset of neurovascular pericytes. Strikingly, neurological symptoms were exclusive to, and ubiquitous in, patients that exhibited moderate to high ACE2 expression in perivascular cells. Viral dsRNA was identified in the vascular wall and paralleled by perivascular inflammation, as signified by T cell and macrophage infiltration. Furthermore, fibrinogen leakage indicated compromised integrity of the blood-brain barrier. Notably, cerebrospinal fluid from additional 16 individuals (n = 8 COVID-19, median age = 67 years; n = 8 control, median age = 69.5 years) exhibited significantly lower levels of the pericyte marker PDGFRß in SARS-CoV-2-infected cases, indicative of disrupted pericyte homeostasis. We conclude that pericyte infection by SARS-CoV-2 underlies virus entry into the privileged central nervous system space, as well as neurological symptomatology due to perivascular inflammation and a locally compromised blood-brain barrier.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Encéfalo/virologia , COVID-19/fisiopatologia , Encefalite Viral/virologia , Pericitos/virologia , Enzima de Conversão de Angiotensina 2/genética , Animais , Barreira Hematoencefálica , Encéfalo/patologia , COVID-19/etiologia , Estudos de Casos e Controles , Encefalite Viral/patologia , Fibrinogênio/metabolismo , Humanos , Imuno-Histoquímica/métodos , Camundongos , Pericitos/metabolismo , Pericitos/patologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/líquido cefalorraquidianoRESUMO
Cancer cells sustain their metabolic needs through nutrients and oxygen supplied by the bloodstream. The requirement for tumor angiogenesis has been therapeutically exploited in the clinical setting mainly by means of inhibition of the vascular endothelial growth factor family of ligands and receptors. Despite promising results in preclinical models, the benefits for patients proved to be limited. Inadequate efficacy similarly halted the development of agents impinging on the activity of the activin receptor-like kinase (ALK)1, a member of the transforming growth factor-ß superfamily. Notwithstanding its characterization as an endothelial cell marker, the full spectrum of biological processes associated with ALK1 is essentially unexplored. Here, we present data revealing the genetic network associated with ACVRL1 (the gene encoding for ALK1) expression in human cancer tissues. Computational analysis unveiled a hitherto unknown role for ACVRL1 in relation to genes modulating the functionality of the immune cell compartment. Moreover, we generated a signature of 8 genes co-expressed with ACVRL1 across different tumor types and characterized the c-type lectin domain containing protein (CLEC)14A as a potential downstream target of ACVRL1. Considering the lack of reagents for ALK1 detection that has hampered the field to date, our work provides the opportunity to validate the 8-gene signature and CLEC14A as biomarkers for ALK1 activity. Ultimately, this may help revisit the clinical development of already existing ALK1-blocking compounds as precision medicines for cancer.
Assuntos
Receptores de Activinas Tipo II/imunologia , Biomarcadores Tumorais/imunologia , Moléculas de Adesão Celular/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Lectinas Tipo C/imunologia , Neoplasias/imunologia , Transcrição Gênica/imunologia , Receptores de Activinas Tipo II/genética , Animais , Biomarcadores Tumorais/genética , Moléculas de Adesão Celular/genética , Feminino , Humanos , Lectinas Tipo C/genética , Masculino , Camundongos , Neoplasias/genética , Neoplasias/patologiaRESUMO
Intratumoral heterogeneity is an inherent feature of most human cancers and has profound implications for cancer therapy. As a result, there is an emergent need to explore previously unmapped mechanisms regulating distinct subpopulations of tumor cells and to understand their contribution to tumor progression and treatment response. Aberrant platelet-derived growth factor receptor beta (PDGFRß) signaling in cancer has motivated the development of several antagonists currently in clinical use, including imatinib, sunitinib, and sorafenib. The discovery of a novel ligand for PDGFRß, platelet-derived growth factor (PDGF)-DD, opened the possibility of a previously unidentified signaling pathway involved in tumor development. However, the precise function of PDGF-DD in tumor growth and invasion remains elusive. Here, making use of a newly generated Pdgfd knockout mouse, we reveal a functionally important malignant cell heterogeneity modulated by PDGF-DD signaling in pancreatic neuroendocrine tumors (PanNET). Our analyses demonstrate that tumor growth was delayed in the absence of signaling by PDGF-DD. Surprisingly, ablation of PDGF-DD did not affect the vasculature or stroma of PanNET; instead, we found that PDGF-DD stimulated bulk tumor cell proliferation by induction of paracrine mitogenic signaling between heterogeneous malignant cell clones, some of which expressed PDGFRß. The presence of a subclonal population of tumor cells characterized by PDGFRß expression was further validated in a cohort of human PanNET. In conclusion, we demonstrate a previously unrecognized heterogeneity in PanNET characterized by signaling through the PDGF-DD/PDGFRß axis.
Assuntos
Linfocinas/genética , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , Fator de Crescimento Derivado de Plaquetas/genética , Animais , Proliferação de Células/genética , Camundongos , Neovascularização Patológica , Tumores Neuroendócrinos/irrigação sanguínea , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/genéticaRESUMO
Angiogenesis is a hallmark of cancer and is now a validated therapeutic target in the clinical setting. Despite the initial success, anti-angiogenic compounds impinging on the vascular endothelial growth factor (VEGF) pathway display limited survival benefits in patients and resistance often develops due to activation of alternative pathways. Thus, finding and validating new targets is highly warranted. Activin receptor-like kinase (ALK)1 is a transforming growth factor beta (TGF-ß) type I receptor predominantly expressed in actively proliferating endothelial cells (ECs). ALK1 has been shown to play a pivotal role in regulating angiogenesis by binding to bone morphogenetic protein (BMP)9 and 10. Two main pharmacological inhibitors, an ALK1-Fc fusion protein (Dalantercept/ACE-041) and a fully human antibody against the extracellular domain of ALK1 (PF-03446962) are currently under clinical development. Herein, we briefly recapitulate the role of ALK1 in blood vessel formation and the current status of the preclinical and clinical studies on inhibition of ALK1 signalling as an anti-angiogenic strategy. Future directions in terms of new combination regimens will also be presented.
Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Inibidores da Angiogênese/uso terapêutico , Neoplasias/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Neoplasias/irrigação sanguínea , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
Advanced breast cancers represent a major therapeutic challenge due to their refractoriness to treatment. Cancer-associated fibroblasts (CAFs) are the most abundant constituents of the tumor microenvironment and have been linked to most hallmarks of cancer. However, the influence of CAFs on therapeutic outcome remains largely unchartered. Here, we reveal that spatial coincidence of abundant CAF infiltration with malignant cells was associated with reduced estrogen receptor (ER)-α expression and activity in luminal breast tumors. Notably, CAFs mediated estrogen-independent tumor growth by selectively regulating ER-α signaling. Whereas most prototypical estrogen-responsive genes were suppressed, CAFs maintained gene expression related to therapeutic resistance, basal-like differentiation, and invasion. A functional drug screen in co-cultures identified effector pathways involved in the CAF-induced regulation of ER-α signaling. Among these, the Transforming Growth Factor-ß and the Janus kinase signaling cascades were validated as actionable targets to counteract the CAF-induced modulation of ER-α activity. Finally, genes that were downregulated in cancer cells by CAFs were predictive of poor response to endocrine treatment. In conclusion, our work reveals that CAFs directly control the luminal breast cancer phenotype by selectively modulating ER-α expression and transcriptional function, and further proposes novel targets to disrupt the crosstalk between CAFs and tumor cells to reinstate treatment response to endocrine therapy in patients.
Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Feminino , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Microambiente Tumoral/genéticaRESUMO
Deregulated expression of the MYC oncogene is a frequent event during tumorigenesis and generally correlates with aggressive disease and poor prognosis. While MYC is a potent inducer of apoptosis, it often suppresses cellular senescence, which together with apoptosis is an important barrier against tumor development. For this latter function, MYC is dependent on cyclin-dependent kinase 2 (CDK2). Here, we utilized a MYC/BCL-XL-driven mouse model of acute myeloblastic leukemia (AML) to investigate whether pharmacological inhibition of CDK2 can inhibit MYC-driven tumorigenesis through induction of senescence. Purified mouse hematopoietic stem cells transduced with MYC and BCL-XL were transplanted into lethally irradiated mice, leading to the development of massive leukemia and subsequent death 15-17 days after transplantation. Upon disease onset, mice were treated with the selective CDK2 inhibitor CVT2584 or vehicle either by daily intraperitoneal injections or continuous delivery via mini-pumps. CVT2584 treatment delayed disease onset and moderately but significantly improved survival of mice. Flow cytometry revealed a significant decrease in tumor load in the spleen, liver and bone marrow of CVT2584-treated compared to vehicle-treated mice. This was correlated with induced senescence evidenced by reduced cell proliferation, increased senescence-associated ß-galactosidase activity and heterochromatin foci, expression of p19ARF and p21CIP1, and reduced phosphorylation (activation) of pRb, while very few apoptotic cells were observed. In addition, phosphorylation of MYC at Ser-62 was decreased. In summary, inhibition of CDK2 delayed MYC/BCL-XL-driven AML linked to senescence induction. Our results suggest that CDK2 is a promising target for pro-senescence cancer therapy, in particular for MYC-driven tumors, including leukemia.
Assuntos
Senescência Celular/genética , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Leucemia/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína bcl-X/metabolismo , Animais , Apoptose/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Feminino , Humanos , Leucemia/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação/genéticaRESUMO
Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, although their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. Here, following a negative selection strategy combined with single-cell RNA sequencing of 768 transcriptomes of mesenchymal cells from a genetically engineered mouse model of breast cancer, we define three distinct subpopulations of CAFs. Validation at the transcriptional and protein level in several experimental models of cancer and human tumors reveal spatial separation of the CAF subclasses attributable to different origins, including the peri-vascular niche, the mammary fat pad and the transformed epithelium. Gene profiles for each CAF subtype correlate to distinctive functional programs and hold independent prognostic capability in clinical cohorts by association to metastatic disease. In conclusion, the improved resolution of the widely defined CAF population opens the possibility for biomarker-driven development of drugs for precision targeting of CAFs.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer , Análise de Sequência de RNA , Transcriptoma , Tecido Adiposo/metabolismo , Animais , Biomarcadores Tumorais/genética , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/classificação , Ciclo Celular/genética , Linhagem Celular Tumoral , Análise por Conglomerados , Progressão da Doença , Epitélio/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Junções Intercelulares/genética , Modelos Logísticos , Camundongos , Camundongos Transgênicos , Prognóstico , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
Breast tumors of the basal-like, hormone receptor-negative subtype remain an unmet clinical challenge, as there is high rate of recurrence and poor survival in patients following treatment. Coevolution of the malignant mammary epithelium and its underlying stroma instigates cancer-associated fibroblasts (CAFs) to support most, if not all, hallmarks of cancer progression. Here we delineate a previously unappreciated role for CAFs as determinants of the molecular subtype of breast cancer. We identified paracrine crosstalk between cancer cells expressing platelet-derived growth factor (PDGF)-CC and CAFs expressing the cognate receptors in human basal-like mammary carcinomas. Genetic or pharmacological intervention of PDGF-CC activity in mouse models of cancer resulted in conversion of basal-like breast cancers into a hormone receptor-positive state that enhanced sensitivity to endocrine therapy in previously resistant tumors. We conclude that specification of breast cancer to the basal-like subtype is under microenvironmental control and is therapeutically actionable.
Assuntos
Neoplasias da Mama/patologia , Linfocinas/metabolismo , Comunicação Parácrina , Fator de Crescimento Derivado de Plaquetas/metabolismo , Microambiente Tumoral , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/irrigação sanguínea , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Fibrose , Humanos , Linfocinas/deficiência , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neovascularização Patológica/patologia , Fator de Crescimento Derivado de Plaquetas/deficiência , Prognóstico , Modelos de Riscos Proporcionais , Transdução de Sinais , Células Estromais/patologia , Análise de Sobrevida , Resultado do TratamentoRESUMO
Angiogenesis occurs early in tumor development, sustains primary tumor growth and provides a route for metastatic escape. The TGF-ß family receptors modulate angiogenesis via endothelial-cell specific pathways. Here we investigate the interaction of two such receptors, ALK1 and endoglin, in pancreatic neuroendocrine tumors (PanNET). Independently, ALK1 and endoglin deficiencies exhibited genetically divergent phenotypes, while both highly correlate to an endothelial metagene in human and mouse PanNETs. A concurrent deficiency of both receptors synergistically decreased tumor burden to a greater extent than either individual knockdown. Furthermore, the knockout of Gdf2 (BMP9), the primary ligand for ALK1 and endoglin, exhibited a mixed phenotype from each of ALK1 and endoglin deficiencies; overall primary tumor burden decreased, but hepatic metastases increased. Tumors lacking BMP9 display a hyperbranching vasculature, and an increase in vascular mesenchymal-marker expression, which may be implicit in the increase in metastases. Taken together, our work cautions against singular blockade of BMP9 and instead demonstrates the utility of dual blockade of ALK1 and endoglin as a strategy for anti-angiogenic therapy in PanNET.
Assuntos
Receptores de Ativinas Tipo I/genética , Endoglina/genética , Neovascularização Patológica/genética , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Fator de Crescimento Transformador beta/genética , Receptores de Ativinas Tipo I/deficiência , Receptores de Activinas Tipo II , Animais , Endoglina/deficiência , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fator 2 de Diferenciação de Crescimento/deficiência , Fator 2 de Diferenciação de Crescimento/genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neovascularização Patológica/metabolismo , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Carga Tumoral/genéticaRESUMO
Exploration of new strategies for the prevention of breast cancer metastasis is justifiably at the center of clinical attention. In this study, we combined a computational biology approach with mechanism-based preclinical trials to identify inhibitors of activin-like receptor kinase (ALK) 1 as effective agents for blocking angiogenesis and metastasis in breast cancer. Pharmacologic targeting of ALK1 provided long-term therapeutic benefit in mouse models of mammary carcinoma, accompanied by strikingly reduced metastatic colonization as a monotherapy or part of combinations with chemotherapy. Gene-expression analysis of breast cancer specimens from a population-based nested case-control study encompassing 768 subjects defined endothelial expression of ALK1 as an independent and highly specific prognostic factor for metastatic manifestation, a finding that was corroborated in an independent clinical cohort. Overall, our results suggest that pharmacologic inhibition of endothelial ALK1 constitutes a tractable strategy for interfering with metastatic dissemination of breast cancer.
Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Receptores de Activinas Tipo II/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endotélio/enzimologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Metástase NeoplásicaRESUMO
The activated RAS/RAF cascade plays a crucial role in lung cancer, but is also known to induce cellular senescence, a major barrier imposed on tumor cells early in tumorigenesis. MYC is a key factor in suppression of RAS/BRAF(V600E)-induced senescence in vitro. However, it is still unclear whether MYC has the same role during tumor development in vivo. Using a conditional, compound knock-in model of Cre-activated BRAF(V600E) and tamoxifen-regulatable MycER, we show that tamoxifen-induced activation of MYC accelerated the onset and increased the number and size of BRAF(V600E)-driven adenomas in a dose-dependent manner, resulting in reduced survival. Furthermore, MYC activation leads to reduced expression of the senescence markers p16(INK4A), p21(CIP1), and H3K9me3-containing heterochromatin foci, and an increased percentage of Ki67(+) tumor cells. This suggests that MYC already early during tumor formation suppresses a BRAF(V600E)-induced senescence-like state. Initial activation of MYC followed by tamoxifen withdrawal still resulted in an increased number of tumors and reduced survival. However, these tumors were of smaller size, showed increased expression of p16(INK4A) and p21(CIP1), and reduced number of Ki67(+) cells, indicating that MYC inactivation restores BRAF(V600E)-induced senescence. Surprisingly, MYC activation did not promote adenoma to carcinoma progression. This suggests that senescence suppression by MYC is a discrete step in tumor development important for sustained tumor growth but preceding malignant transformation and that additional oncogenic events are required for carcinoma development and metastasis. These findings contribute to our understanding of the neoplastic transformation process, with implications for future treatment strategies.
Assuntos
Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Processos de Crescimento Celular/fisiologia , Senescência Celular , Estudos de Coortes , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-myc/genética , Tamoxifeno/farmacologia , Ativação TranscricionalRESUMO
Studies in primary and tumor cells suggest that MYC plays an important role in regulating cellular senescence, thereby impacting on tumor development. Here we describe different common methods to measure senescence in cell cultures and in tissues. These include measurement of senescence-associated ß-galactosidase activity (SA-ß-gal), senescence-associated heterochromatin foci (SAHFs), proliferative arrest, morphological changes, and expression and activity of proteins involved in the senescence process, such as p53 and Rb pathway proteins and secretory proteins. It is important to note that there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers that together determine whether cells are senescent or not. Measurement of senescence is an important aspect of studies of MYC biology and will improve our understanding of MYC function and regulation both in preclinical and clinical settings. This may form the basis for new concepts of pro-senescence therapy to combat MYC in cancer.