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Tissues are organized into anatomical and functional units at different scales. New technologies for high-dimensional molecular profiling in situ have enabled the characterization of structure-function relationships in increasing molecular detail. However, it remains a challenge to consistently identify key functional units across experiments, tissues, and disease contexts, a task that demands extensive manual annotation. Here, we present spatial cellular graph partitioning (SCGP), a flexible method for the unsupervised annotation of tissue structures. We further present a reference-query extension pipeline, SCGP-Extension, that generalizes reference tissue structure labels to previously unseen samples, performing data integration and tissue structure discovery. Our experiments demonstrate reliable, robust partitioning of spatial data in a wide variety of contexts and best-in-class accuracy in identifying expertly annotated structures. Downstream analysis on SCGP-identified tissue structures reveals disease-relevant insights regarding diabetic kidney disease, skin disorder, and neoplastic diseases, underscoring its potential to drive biological insight and discovery from spatial datasets.
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AIMS/HYPOTHESIS: Diabetic kidney disease (DKD) is the leading cause of chronic and end-stage kidney disease in the USA and worldwide. Animal models have taught us much about DKD mechanisms, but translation of this knowledge into treatments for human disease has been slowed by the lag in our molecular understanding of human DKD. METHODS: Using our Spatial TissuE Proteomics (STEP) pipeline (comprising curated human kidney tissues, multiplexed immunofluorescence and powerful analysis tools), we imaged and analysed the expression of 21 proteins in 23 tissue sections from individuals with diabetes and healthy kidneys (n=5), compared to those with DKDIIA, IIA-B and IIB (n=2 each) and DKDIII (n=1). RESULTS: These analyses revealed the existence of 11 cellular clusters (kidney compartments/cell types): podocytes, glomerular endothelial cells, proximal tubules, distal nephron, peritubular capillaries, blood vessels (endothelial cells and vascular smooth muscle cells), macrophages, myeloid cells, other CD45+ inflammatory cells, basement membrane and the interstitium. DKD progression was associated with co-localised increases in inflammatory cells and collagen IV deposition, with concomitant loss of native proteins of each nephron segment. Cell-type frequency and neighbourhood analyses highlighted a significant increase in inflammatory cells and their adjacency to tubular and αSMA+ (α-smooth muscle actin-positive) cells in DKD. Finally, DKD progression showed marked regional variability within single tissue sections, as well as inter-individual variability within each DKD class. CONCLUSIONS/INTERPRETATION: Using the STEP pipeline, we found alterations in protein expression, cellular phenotypic composition and microenvironment structure with DKD progression, demonstrating the power of this pipeline to reveal the pathophysiology of human DKD.
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BACKGROUND: Kidney allograft rejections are orchestrated by a variety of immune cells. Because of the complex histopathologic features, accurate pathological diagnosis poses challenges even for expert pathologists. The objective of this study was to unveil novel spatial indices associated with transplant rejection by using a spatial bioinformatic approach using 36-plex immunofluorescence image data. METHODS: The image obtained from 11 T cell-mediated rejection (TCMR) and 12 antibody-mediated rejection (AMR) samples were segmented into 753 737 single cells using DeepCell's Mesmer algorithm. These cells were categorized into 13 distinct cell types through unsupervised clustering based on their biomarker expression profiles. Cell neighborhood analysis allowed us to stratify kidney tissue into 8 distinct neighborhood components consisting of unique cell type enrichment profiles. RESULTS: In contrast to TCMR samples, AMR samples exhibited a higher frequency of neighborhood components that were characterized by an enrichment of CD31+ endothelial cells. Although the overall frequency of CD68+ macrophages in AMR samples was not significantly high, CD68+ macrophages within endothelial cell-rich lesions exhibited a significantly higher frequency in AMR samples than TCMR samples. Furthermore, the frequency of interactions between CD31+ cells and CD68+ cells was significantly increased in AMR samples, implying the pivotal role of macrophages in AMR pathogenesis. Importantly, patients demonstrating a high frequency of CD31:CD68 interactions experienced significantly poorer outcomes in terms of chronic AMR progression. CONCLUSIONS: Collectively, these data indicate the potential of spatial bioinformatic as a valuable tool for aiding in pathological diagnosis and for uncovering new insights into the mechanisms underlying transplant rejection.
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Psoriasis vulgaris and other chronic inflammatory diseases improve markedly with therapeutic blockade of interleukin-23 (IL-23) signaling, but the genetic mechanisms underlying clinical responses remain poorly understood. Using single-cell transcriptomics, we profiled immune cells isolated from lesional psoriatic skin before and during IL-23 blockade. In clinically responsive patients, a psoriatic transcriptional signature in skin-resident memory T cells was strongly attenuated. In contrast, poorly responsive patients were distinguished by persistent activation of IL-17-producing T (T17) cells, a mechanism distinct from alternative cytokine signaling or resistance isolated to epidermal keratinocytes. Even in IL-23 blockade-responsive patients, we detected a recurring set of recalcitrant, disease-specific transcriptional abnormalities. This irreversible immunological state may necessitate ongoing IL-23 inhibition. Spatial transcriptomic analyses also suggested that successful IL-23 blockade requires dampening of >90% of IL-17-induced response in lymphocyte-adjacent keratinocytes, an unexpectedly high threshold. Collectively, our data establish a patient-level paradigm for dissecting responses to immunomodulatory treatments.
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Interleucina-17 , Psoríase , Humanos , Interleucina-23 , Pele , Psoríase/tratamento farmacológico , QueratinócitosRESUMO
BACKGROUND: It is unclear witch regimen is optimal as salvage chemotherapy (SCT) after immune checkpoint inhibitor (ICI) monotherapy for recurrent or metastatic head and neck cancer (RM-HNC). METHODS: This study enrolled 109 patients. Overall survival (OS) and progression-free survival 2 (PFS2) were compared between patients stratified by SCT regimen. RESULTS: Of the 109 patients, 55 underwent SCT after the failure of ICI monotherapy. The OS of these 55 patients was longer than that of patients who did not undergo SCT. The OS and PFS2 were similar between patients treated with paclitaxel (PTX) and cetuximab (Cmab) combination and those treated with PTX monotherapy. The occurrence of irAEs did not impact PFS2 nor OS. CONCLUSIONS: SCT can improve the survival outcomes of patients with RM-HNC. In addition to PTX and Cmab, PTX monotherapy is also considered an effective SCT regimen. SCT is effective regardless of the presence or absence of irAEs.
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Neoplasias de Cabeça e Pescoço , Inibidores de Checkpoint Imunológico , Recidiva Local de Neoplasia , Paclitaxel , Terapia de Salvação , Humanos , Masculino , Inibidores de Checkpoint Imunológico/uso terapêutico , Pessoa de Meia-Idade , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Recidiva Local de Neoplasia/tratamento farmacológico , Idoso , Paclitaxel/uso terapêutico , Paclitaxel/administração & dosagem , Estudos Retrospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Adulto , Cetuximab/uso terapêutico , Intervalo Livre de ProgressãoRESUMO
An acidic tumor microenvironment plays a critical role in tumor progression. However, understanding of metabolic reprogramming of tumors in response to acidic extracellular pH has remained elusive. Using comprehensive metabolomic analyses, we demonstrated that acidic extracellular pH (pH 6.8) leads to the accumulation of N1-acetylspermidine, a protumor metabolite, through up-regulation of the expression of spermidine/spermine acetyltransferase 1 (SAT1). Inhibition of SAT1 expression suppressed the accumulation of intra- and extracellular N1-acetylspermidine at acidic pH. Conversely, overexpression of SAT1 increased intra- and extracellular N1-acetylspermidine levels, supporting the proposal that SAT1 is responsible for accumulation of N1-acetylspermidine. While inhibition of SAT1 expression only had a minor effect on cancer cell growth in vitro, SAT1 knockdown significantly decreased tumor growth in vivo, supporting a contribution of the SAT1-N1-acetylspermidine axis to protumor immunity. Immune cell profiling revealed that inhibition of SAT1 expression decreased neutrophil recruitment to the tumor, resulting in impaired angiogenesis and tumor growth. We showed that antineutrophil-neutralizing antibodies suppressed growth in control tumors to a similar extent to that seen in SAT1 knockdown tumors in vivo. Further, a SAT1 signature was found to be correlated with poor patient prognosis. Our findings demonstrate that extracellular acidity stimulates recruitment of protumor neutrophils via the SAT1-N1-acetylspermidine axis, which may represent a metabolic target for antitumor immune therapy.
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Type 1 diabetes (T1D) is an autoimmune disease in which immune cells destroy insulin-producing beta cells. The aetiology of this complex disease is dependent on the interplay of multiple heterogeneous cell types in the pancreatic environment. Here, we provide a single-cell atlas of pancreatic islets of 24 T1D, autoantibody-positive and nondiabetic organ donors across multiple quantitative modalities including ~80,000 cells using single-cell transcriptomics, ~7,000,000 cells using cytometry by time of flight and ~1,000,000 cells using in situ imaging mass cytometry. We develop an advanced integrative analytical strategy to assess pancreatic islets and identify canonical cell types. We show that a subset of exocrine ductal cells acquires a signature of tolerogenic dendritic cells in an apparent attempt at immune suppression in T1D donors. Our multimodal analyses delineate cell types and processes that may contribute to T1D immunopathogenesis and provide an integrative procedure for exploration and discovery of human pancreatic function.
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Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Hormônios Pancreáticos/metabolismoRESUMO
BACKGROUND & AIMS: Significant progress has been made since the first report of inflammatory bowel disease (IBD) in 1859, after decades of research that have contributed to the understanding of the genetic and environmental factors involved in IBD pathogenesis. Today, a range of treatments is available for directed therapy, mostly targeting the overactive immune response. However, the mechanisms by which the immune system contributes to disease pathogenesis and progression are not fully understood. One challenge hindering IBD research is the heterogeneous nature of the disease and the lack of understanding of how immune cells interact with one another in the gut mucosa. Introduction of a technology that enables expansive characterization of the inflammatory environment of human IBD tissues may address this gap in knowledge. METHODS: We used the imaging mass cytometry platform to perform highly multiplex image analysis of IBD and healthy deidentified intestine sections (6 Crohn's disease compared to 6 control ileum; 6 ulcerative colitis compared to 6 control colon). The acquired images were graded for inflammation severity by analysis of adjacent H&E tissue sections. We assigned more than 300,000 cells to unique cell types and performed analyses of tissue integrity, epithelial activity, and immune cell composition. RESULTS: The intestinal epithelia of patients with IBD exhibited increased proliferation rates and expression of HLA-DR compared to control tissues, and both features were positively correlated with the severity of inflammation. The neighborhood analysis determined enrichment of regulatory T cell interactions with CD68+ macrophages, CD4+ T cells, and plasma cells in both forms of IBD, whereas activated lysozyme C+ macrophages were preferred regulatory T cell neighbors in Crohn's disease but not ulcerative colitis. CONCLUSIONS: Altogether, our study shows the power of imaging mass cytometry and its ability to both quantify immune cell types and characterize their spatial interactions within the inflammatory environment by a single analysis platform.
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Microambiente Celular , Colite Ulcerativa/patologia , Colo/patologia , Doença de Crohn/patologia , Células Epiteliais/patologia , Mucosa Intestinal/patologia , Microscopia Confocal , Adolescente , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos , Estudos de Casos e Controles , Comunicação Celular , Proliferação de Células , Criança , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Colo/imunologia , Colo/metabolismo , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Muramidase/metabolismo , Proteoma , Proteômica , Índice de Gravidade de Doença , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologiaRESUMO
Wnt5a is a non-canonical Wnt ligand that is essential for normal embryonic development in mammals. The role of Wnt5a in early intestinal development has been examined in gene ablation models, where Wnt5a-/- mice exhibit strikingly shortened intestines. However, the exact cellular source of Wnt5a has remained elusive, until a recent study found that FoxL1-expressing mesenchymal cells (FoxL1+ cells), which are localized directly beneath the intestinal epithelium, express Wnt5a. To determine whether FoxL1+ cells are a required source of Wnt5a during intestinal development, we derived FoxL1-Cre; Wnt5af/f mice, which is the first mouse model to ablate Wnt5a in a cell type-specific manner in the intestine in vivo. Our results show that Wnt5a deletion in FoxL1+ cells during fetal life causes a shortened gut phenotype in neonatal mice, and that our model is sufficient to increase rate of apoptosis in the elongating epithelium, thus explaining the shortened gut phenotype. However, in contrast to previous studies using Wnt5a null mice, we did not observe dysregulation of epithelial structure or apical-basal protein localization. Altogether, our findings establish a developmental role for FoxL1+ mesenchymal cells in controlling non-canonical Wnt signaling during midgut elongation.
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Desenvolvimento Embrionário , Fatores de Transcrição Forkhead/metabolismo , Intestinos/embriologia , Intestinos/metabolismo , Mesoderma/metabolismo , Proteína Wnt-5a/metabolismo , Animais , Apoptose , Polaridade Celular , Proliferação de Células , Células Epiteliais/metabolismo , Integrases/metabolismo , CamundongosRESUMO
Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profile open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We reveal key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrates that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells follow a more complex differentiation, where Hfn4a is associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease implicates critical cell types, developmental stages, genes, and regulatory mechanisms. The single cell multi-omics atlas reveals key chromatin remodeling events and gene expression dynamics associated with kidney development.
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Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Néfrons/crescimento & desenvolvimento , Organogênese/genética , Insuficiência Renal Crônica/genética , Animais , Comunicação Celular , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Epigenômica , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Camundongos , Néfrons/citologia , Podócitos/fisiologia , Polimorfismo de Nucleotídeo Único , RNA-Seq , Insuficiência Renal Crônica/patologia , Análise de Célula Única , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismoRESUMO
Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression.
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Regulação para Baixo/genética , Etanolaminas/metabolismo , Glutamina/metabolismo , RNA Nucleotidiltransferases/genética , Inanição/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Progressão da Doença , Células HeLa , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-ets/genética , Transcrição Gênica/genéticaRESUMO
Since the first description of 'interstitial cells of Cajal' in the mammalian gut in 1911, scientists have found structurally similar cells, now termed telocytes, in numerous tissues throughout the body. These cells have recently sparked renewed interest, facilitated through the development of a molecular handle to genetically manipulate their function in tissue homeostasis and disease. In this Primer, we discuss the discovery of telocytes, their physical properties, distribution and function, focusing on recent developments in the functional analysis of Foxl1-positive telocytes in the intestinal stem cell niche, and, finally, the current challenges of studying telocytes as a distinct cell type.
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Telócitos/fisiologia , Animais , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Homeostase/fisiologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Especificidade de Órgãos , Nicho de Células-Tronco/fisiologia , Telócitos/citologiaRESUMO
Change history: In this Letter, the surname of author Efi E. Massasa was misspelled 'Massassa'. This error has been corrected online.
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Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts1-6. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1+ telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1+ telocytes are an important source of niche signals to intestinal stem cells.
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Autorrenovação Celular , Mucosa Intestinal/citologia , Telócitos/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Aciltransferases/deficiência , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Proliferação de Células , Fatores de Transcrição Forkhead/metabolismo , Ligantes , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Our understanding of kidney disease pathogenesis is limited by an incomplete molecular characterization of the cell types responsible for the organ's multiple homeostatic functions. To help fill this knowledge gap, we characterized 57,979 cells from healthy mouse kidneys by using unbiased single-cell RNA sequencing. On the basis of gene expression patterns, we infer that inherited kidney diseases that arise from distinct genetic mutations but share the same phenotypic manifestation originate from the same differentiated cell type. We also found that the collecting duct in kidneys of adult mice generates a spectrum of cell types through a newly identified transitional cell. Computational cell trajectory analysis and in vivo lineage tracing revealed that intercalated cells and principal cells undergo transitions mediated by the Notch signaling pathway. In mouse and human kidney disease, these transitions were shifted toward a principal cell fate and were associated with metabolic acidosis.
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Rastreamento de Células/métodos , Perfilação da Expressão Gênica/métodos , Nefropatias/genética , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Análise de Célula Única/métodos , Animais , Plasticidade Celular , Marcadores Genéticos , Humanos , Camundongos , Receptores Notch/metabolismo , Análise de Sequência de RNA/métodos , Transdução de SinaisRESUMO
Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported. Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.
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Técnicas de Cultura de Células/métodos , Neoplasias/patologia , Bicarbonatos , Meios de Cultura , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico , Microambiente TumoralRESUMO
Long noncoding RNAs play a pivotal role in tumor progression, but their role in cancer cells in the nutrient-starved tumor microenvironment remains unknown. Here, we show that a nutrient starvation-responsive long noncoding RNA, JHDM1D antisense 1 (JHDM1D-AS1), promotes tumorigenesis by regulating angiogenesis in response to nutrient starvation. Expression of JHDM1D-AS1 was increased in cancer cells. In addition, expression of JHDM1D-AS1 was increased in clinical tumor samples compared to that in normal tissue. Stable expression of JHDM1D-AS1 in human pancreatic cancer (PANC-1 and AsPC-1) cells promoted cell growth in vitro Remarkably, these JHDM1D-AS1-expressing cells showed a significant increase in tumor growth in vivo that was associated with increased formation of CD31+ blood vessels and elevated infiltration of CD11b+ macrophage lineage cells into tumor tissues. Genome-wide analysis of tumor xenografts revealed that expression of genes for tumor-derived angiogenic factors such as hHGF and hFGF1 concomitant with host-derived inflammation-responsive genes such as mMmp3, mMmp9, mS100a8, and mS100a9 was increased in tumor xenografts of JHDM1D-AS1-expressing pancreatic cancer cells, leading to a poor prognosis. Our results provide evidence that increased JHDM1D-AS1 expression under nutrient starvation accelerates tumor growth by upregulating angiogenesis, thus laying the foundation for improved therapeutic strategies.
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Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/patologia , Neovascularização Patológica/genética , Inanição/genética , Animais , Calgranulina A/biossíntese , Calgranulina B/biossíntese , Linhagem Celular Tumoral , Proliferação de Células , Fator 1 de Crescimento de Fibroblastos/biossíntese , Perfilação da Expressão Gênica , Fator de Crescimento de Hepatócito/biossíntese , Humanos , Metaloproteinase 3 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos SCID , Transplante de Neoplasias , Neoplasias/genética , Interferência de RNA , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Transplante Heterólogo , Microambiente Tumoral/fisiologiaRESUMO
Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification.
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Neoplasias/metabolismo , Neoplasias/patologia , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Acetato-CoA Ligase/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Colesterol/metabolismo , Progressão da Doença , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos SCID , Regiões Promotoras Genéticas/fisiologia , Transporte Proteico/fisiologia , Regulação para Cima/fisiologiaRESUMO
Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.