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1.
Proc Natl Acad Sci U S A ; 121(30): e2408109121, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39028694

RESUMO

The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.


Assuntos
COVID-19 , Pulmão , Organoides , SARS-CoV-2 , Internalização do Vírus , Humanos , SARS-CoV-2/fisiologia , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/virologia , Pulmão/virologia , Pulmão/imunologia , Pulmão/patologia , Organoides/virologia , Tratamento Farmacológico da COVID-19 , Células-Tronco Pluripotentes Induzidas/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , Inflamação , Citocinas/metabolismo , Apoptose
2.
Subcell Biochem ; 98: 15-40, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35378701

RESUMO

Macropinocytosis is an evolutionarily conserved endocytic pathway that mediates the nonselective acquisition of extracellular material via large endocytic vesicles known as macropinosomes. In addition to other functions, this uptake pathway supports cancer cell metabolism through the uptake of nutrients. Cells harboring oncogene or tumor suppressor mutations are known to display heightened macropinocytosis, which confers to the cancer cells the ability to survive and proliferate despite the nutrient-scarce conditions of the tumor microenvironment. Thus, macropinocytosis is associated with cancer malignancy. Macropinocytic uptake can be induced in cancer cells by different stress stimuli, acting as an adaptive mechanism for the cells to resist stresses in the tumor milieu. Here, we review the cellular stresses that are known to promote macropinocytosis, as well as the underlying molecular mechanisms that drive this process.


Assuntos
Neoplasias , Pinocitose , Transporte Biológico , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Nutrientes , Pinocitose/fisiologia , Transdução de Sinais , Microambiente Tumoral
3.
Gastroenterology ; 159(5): 1882-1897.e5, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32768595

RESUMO

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. METHODS: We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. RESULTS: EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. CONCLUSIONS: HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Movimento Celular , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Hipóxia Tumoral , Microambiente Tumoral , Proteína Supressora de Tumor p53/genética , Regulação para Cima
4.
Biochem Biophys Res Commun ; 533(3): 424-428, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-32972751

RESUMO

Nutrient stress driven by glutamine deficiency activates EGFR signaling in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cells. EGFR signaling in the context of glutamine starvation is thought to be instigated by the transcriptional upregulation of EGFR ligands and functions as an adaptation mechanism to allow PDAC cells to maintain metabolic fitness. Having a clear view of the intricate signaling cascades potentiated by the metabolic induction of EGFR is important in understanding how these effector pathways influence cancer progression. In this study, we examined the complex signaling that occurs in PDAC cells when EGFR is activated by glutamine deprivation. We elucidate that the metabolic activation of EGFR is principally mediated by HB-EGF, and that other members of the ErbB receptor tyrosine kinase family are not activated by glutamine starvation. Additionally, we determine that glutamine depletion-driven EGFR signaling is associated with a specific receptor phosphorylation known to participate in a feedback loop, a process that is dependent on Erk. Lastly, we determine that K-Ras is required for glutamine depletion-induced Erk activation, as well as EGFR feedback phosphorylation, but is dispensable for Akt activation. These data provide important insights into the regulation of EGFR signaling in the context of metabolic stresses.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Carcinoma Ductal Pancreático/enzimologia , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Glutamina/fisiologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Pancreáticas/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo
5.
Biochem Biophys Res Commun ; 533(3): 437-441, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-32972756

RESUMO

The interplay between nutrient scarcity and signal transduction circuits is an important aspect of tumorigenesis that regulates many aspects of cancer progression. Glutamine is a critical nutrient for cancer cells, as it contributes to biosynthetic reactions that sustain cancer proliferation and growth. In tumors, because nutrient utilization can often outpace supply, glutamine levels can become limiting and oncogene-mediated metabolic rewiring triggers signaling cascades that support nutrient stress survival. Recently, we identified that in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine depletion can trigger p21-activated kinase (Pak) activation through EGFR signaling as a means to circumvent metabolic stress. Here, we elucidate that glutamine starvation, as well EGF stimulation, can enhance the presence of many different Pak phosphoforms, and that this activation only occurs in a subset of PDAC cells. Pak is a well-established effector of Rac1, and while Rac1 mutant variants can modulate the metabolic induction of Pak phosphorylation, Rac1 inhibition only partially attenuates Pak activation upon glutamine depletion. We decipher that in order to efficiently suppress metabolic activation of Pak, both EGFR and Rac1 signaling must be inhibited. These results provide a mechanistic understanding of how glutamine-regulated signal transduction can control Pak activation in PDAC cells.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Estresse Fisiológico , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Carcinoma Ductal Pancreático/enzimologia , Ativação Enzimática , Glutamina/fisiologia , Humanos , Isoenzimas/metabolismo , Nutrientes , Neoplasias Pancreáticas/enzimologia , Fosforilação , Células Tumorais Cultivadas
6.
Nature ; 497(7451): 633-7, 2013 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-23665962

RESUMO

Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells through large, heterogeneous vesicles known as macropinosomes. Oncogenic Ras proteins have been shown to stimulate macropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown. Here we show that Ras-transformed cells use macropinocytosis to transport extracellular protein into the cell. The internalized protein undergoes proteolytic degradation, yielding amino acids including glutamine that can enter central carbon metabolism. Accordingly, the dependence of Ras-transformed cells on free extracellular glutamine for growth can be suppressed by the macropinocytic uptake of protein. Consistent with macropinocytosis representing an important route of nutrient uptake in tumours, its pharmacological inhibition compromises the growth of Ras-transformed pancreatic tumour xenografts. These results identify macropinocytosis as a mechanism by which cancer cells support their unique metabolic needs and point to the possible exploitation of this process in the design of anticancer therapies.


Assuntos
Aminoácidos/metabolismo , Transformação Celular Neoplásica , Proteína Oncogênica p21(ras)/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pinocitose , Animais , Transporte Biológico , Carbono/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Feminino , Glutamina/metabolismo , Camundongos , Camundongos Nus , Células NIH 3T3 , Proteína Oncogênica p21(ras)/genética , Neoplasias Pancreáticas/genética , Proteólise
7.
bioRxiv ; 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38293142

RESUMO

Macropinocytosis has emerged as a nutrient-scavenging pathway that cancer cells exploit to survive the nutrient-deprived conditions of the tumor microenvironment. Cancer cells are especially reliant on glutamine for their survival, and in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine deficiency can enhance the stimulation of macropinocytosis, allowing the cells to escape metabolic stress through the production of extracellular-protein-derived amino acids. Here, we identify the atypical protein kinase C (aPKC) enzymes, PKCζ and PKCι as novel regulators of macropinocytosis. In normal epithelial cells, aPKCs are known to regulate cell polarity in association with the scaffold proteins Par3 and Par6, controlling the function of several targets, including the Par1 kinases. In PDAC cells, we identify that each of these cell polarity proteins are required for glutamine stress-induced macropinocytosis. Mechanistically, we find that the aPKCs are regulated by EGFR signaling or by the transcription factor CREM to promote the relocation of Par3 to microtubules, facilitating macropinocytosis in a dynein-dependent manner. Importantly, we determine that cell fitness impairment caused by aPKC depletion is rescued by the restoration of macropinocytosis and that aPKCs support PDAC growth in vivo. These results identify a previously unappreciated role for cell polarity proteins in the regulation of macropinocytosis and provide a better understanding of the mechanistic underpinnings that control macropinocytic uptake in the context of metabolic stress.

8.
Nat Cancer ; 5(1): 100-113, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37814011

RESUMO

In pancreatic ductal adenocarcinoma (PDAC), glutamine is a critical nutrient that drives a wide array of metabolic and biosynthetic processes that support tumor growth. Here, we elucidate how 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that broadly inhibits glutamine metabolism, blocks PDAC tumor growth and metastasis. We find that DON significantly reduces asparagine production by inhibiting asparagine synthetase (ASNS), and that the effects of DON are rescued by asparagine. As a metabolic adaptation, PDAC cells upregulate ASNS expression in response to DON, and we show that ASNS levels are inversely correlated with DON efficacy. We also show that L-asparaginase (ASNase) synergizes with DON to affect the viability of PDAC cells, and that DON and ASNase combination therapy has a significant impact on metastasis. These results shed light on the mechanisms that drive the effects of glutamine mimicry and point to the utility of cotargeting adaptive responses to control PDAC progression.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Glutamina/metabolismo , Asparagina/metabolismo , Linhagem Celular Tumoral , Asparaginase/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Processos Neoplásicos
9.
bioRxiv ; 2024 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-38979336

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers and thus identifying more effective therapies is a major unmet need. In this study we characterized the super enhancer (SE) landscape of human PDAC to identify novel, potentially targetable, drivers of the disease. Our analysis revealed that MICAL2 is a super enhancer-associated gene in human PDAC. MICAL2 is a flavin monooxygenase that induces actin depolymerization and indirectly promotes SRF transcription by modulating the availability of serum response factor coactivators myocardin related transcription factors (MRTF-A and MRTF-B). We found that MICAL2 is overexpressed in PDAC and correlates with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and EMT signaling pathways, contributing to tumor growth and metastasis. In loss and gain of function experiments in human and mouse PDAC cells, we observed that MICAL2 promotes both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 expression also inhibited macropinocytosis. Through in vitro phenotypic analyses, we show that MICAL2, MRTF-A and MRTF-B influence PDAC cell proliferation, migration and promote cell cycle progression. Importantly, we demonstrate that MICAL2 is essential for in vivo tumor growth and metastasis. Interestingly, we find that MRTF-B, but not MRTF-A, phenocopies MICAL2-driven phenotypes in vivo . This study highlights the multiple ways in which MICAL2 impacts PDAC biology and suggests that its inhibition may impede PDAC progression. Our results provide a foundation for future investigations into the role of MICAL2 in PDAC and its potential as a target for therapeutic intervention.

10.
J Exp Med ; 220(3)2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36729077

RESUMO

In this issue of JEM, Chu and An et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20221316) describe the role of the tricyclic antidepressant nortriptyline in inhibition of fatty acid uptake. Nortriptyline promotes cell acidification and suppresses macropinocytosis, providing a link between fatty acid uptake and tumor progression.


Assuntos
Neoplasias , Nortriptilina , Humanos , Neoplasias/tratamento farmacológico , Ácidos Graxos , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico
11.
bioRxiv ; 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36747824

RESUMO

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.

12.
Dev Cell ; 13(6): 783-95, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18061562

RESUMO

The Notch signaling pathway, which plays a critical role in cell-fate decisions throughout development, is regulated by endocytosis of both the ligand and receptor. Endocytosis of the Drosophila ligands, Delta and Serrate, is required in the signaling cell for signal initiation and requires one of two ubiquitin ligases, Neuralized or Mind bomb. Through in vitro binding assays we have identified an interaction between Neuralized and phosphoinositides, modified membrane lipids that mediate membrane trafficking and signaling. We show that interactions between phosphoinositides and Neuralized contribute to the membrane localization of Neuralized in the absence of Delta, and that the phosphoinositide-binding motif is required for Neuralized to endocytose Delta downstream of Delta ubiquitination. Lastly, we provide evidence that this interaction may also be important for vertebrate Neuralized function. These results demonstrate that, through interactions with Neuralized, phosphoinositides may regulate Delta endocytosis and, by extension, Notch signal transduction.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Fosfatidilinositóis/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Animais Geneticamente Modificados , Western Blotting , Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Imunofluorescência , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/genética , Mutagênese , Receptores Notch/genética , Receptores Notch/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
13.
Front Cell Dev Biol ; 9: 664295, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34055797

RESUMO

Cancer cells exhibit increased glycolytic flux and adenosine triphosphate (ATP) hydrolysis. These processes increase the acidic burden on the cells through the production of lactate and protons. Nonetheless, cancer cells can maintain an alkaline intracellular pH (pHi) relative to untransformed cells, which sets the stage for optimal functioning of glycolytic enzymes, evasion of cell death, and increased proliferation and motility. Upregulation of plasma membrane transporters allows for H+ and lactate efflux; however, recent evidence suggests that the acidification of organelles can contribute to maintenance of an alkaline cytosol in cancer cells by siphoning off protons, thereby supporting tumor growth. The Golgi is such an acidic organelle, with resting pH ranging from 6.0 to 6.7. Here, we posit that the Golgi represents a "proton sink" in cancer and delineate the proton channels involved in Golgi acidification and the ion channels that influence this process. Furthermore, we discuss ion channel regulators that can affect Golgi pH and Golgi-dependent processes that may contribute to pHi homeostasis in cancer.

14.
J Vis Exp ; (174)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34515683

RESUMO

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.


Assuntos
Endossomos , Pinocitose , Endocitose , Microscopia de Fluorescência , Reprodutibilidade dos Testes
15.
Cancer Discov ; 11(7): 1808-1825, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33653692

RESUMO

Although pancreatic ductal adenocarcinoma (PDAC) cells are exposed to a nutrient-depleted tumor microenvironment, they can acquire nutrients via macropinocytosis, an endocytic form of protein scavenging that functions to support cancer metabolism. Here, we provide evidence that macropinocytosis is also operational in the pancreatic tumor stroma. We find that glutamine deficiency triggers macropinocytic uptake in pancreatic cancer-associated fibroblasts (CAF). Mechanistically, we decipher that stromal macropinocytosis is potentiated via the enhancement of cytosolic Ca2+ and dependent on ARHGEF2 and CaMKK2-AMPK signaling. We elucidate that macropinocytosis has a dual function in CAFs-it serves as a source of intracellular amino acids that sustain CAF cell fitness and function, and it provides secreted amino acids that promote tumor cell survival. Importantly, we demonstrate that stromal macropinocytosis supports PDAC tumor growth. These results highlight the functional role of macropinocytosis in the tumor stroma and provide a mechanistic understanding of how nutrient deficiency can control stromal protein scavenging. SIGNIFICANCE: Glutamine deprivation drives stromal macropinocytosis to support CAF cell fitness and provide amino acids that sustain PDAC cell survival. Selective disruption of macropinocytosis in CAFs suppresses PDAC tumor growth.This article is highlighted in the In This Issue feature, p. 1601.


Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Células Estromais , Microambiente Tumoral , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Carcinoma Ductal Pancreático/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/patologia , Pinocitose , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais
16.
Mol Biol Cell ; 18(1): 1-13, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17065551

RESUMO

Notch signaling, which is crucial to metazoan development, requires endocytosis of Notch ligands, such as Delta and Serrate. Neuralized is a plasma membrane-associated ubiquitin ligase that is required for neural development and Delta internalization. Neuralized is comprised of three domains that include a C-terminal RING domain and two neuralized homology repeat (NHR) domains. All three domains are conserved between organisms, suggesting that these regions of Neuralized are functionally important. Although the Neuralized RING domain has been shown to be required for Delta ubiquitination, the function of the NHR domains remains elusive. Here we show that neuralized, a well-characterized neurogenic allele, exhibits a mutation in a conserved residue of the NHR1 domain that results in mislocalization of Neuralized and defects in Delta binding and internalization. Furthermore, we describe a novel isoform of Neuralized and show that it is recruited to the plasma membrane by Delta and that this is mediated by the NHR1 domain. Finally, we show that the NHR1 domain of Neuralized is both necessary and sufficient to bind Delta. Altogether, our data demonstrate that NHR domains can function in facilitating protein-protein interactions and in the case of Neuralized, mediate binding to its ubiquitination target, Delta.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Endocitose , Endossomos/metabolismo , Ácido Glutâmico/genética , Glutamina/genética , Glicina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Mutação/genética , Neurônios/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico
17.
Cancer Discov ; 10(6): 822-835, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32200349

RESUMO

Cancer cells reprogram their metabolism to meet elevated energy demands and favor glycolysis for energy production. This boost in glycolytic flux supports proliferation, but also generates acid in the form of hydrogen ions that must be eliminated from the cytoplasm to maintain the alkaline intracellular pH (pHi) associated with transformation. To cope with acid production, tumor cells employ ion transport systems, including the family of sodium-hydrogen exchangers (NHE). Here, we identify NHE7 as a novel regulator of pHi in pancreatic ductal adenocarcinoma (PDAC). We determine that NHE7 suppression causes alkalinization of the Golgi, leading to a buildup of cytosolic acid that diminishes tumor cell fitness mainly through the dysregulation of actin. Importantly, NHE7 knockdown in vivo leads to the abrogation of tumor growth. These results identify Golgi acidification as a mechanism to control pHi and point to the regulation of pHi as a possible therapeutic vulnerability in PDAC. SIGNIFICANCE: NHE7 regulates cytosolic pH through Golgi acidification, which points to the Golgi as a "proton sink" for metabolic acid. Disruption of cytosolic pH homeostasis via NHE7 suppression compromises PDAC cell viability and tumor growth.See related commentary by Ward and DeNicola, p. 768.This article is highlighted in the In This Issue feature, p. 747.


Assuntos
Carcinoma Ductal Pancreático/patologia , Complexo de Golgi/metabolismo , Neoplasias Pancreáticas/patologia , Trocadores de Sódio-Hidrogênio/metabolismo , Homeostase , Humanos , Concentração de Íons de Hidrogênio
18.
J Exp Med ; 217(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32510550

RESUMO

Tumor cells rely on glutamine to fulfill their metabolic demands and sustain proliferation. The elevated consumption of glutamine can lead to intratumoral nutrient depletion, causing metabolic stress that has the potential to impact tumor progression. Here, we show that nutrient stress caused by glutamine deprivation leads to the induction of epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, we demonstrate that glutamine deficiency regulates EMT through the up-regulation of the EMT master regulator Slug, a process that is dependent on both MEK/ERK signaling and ATF4. We find that Slug is required in PDAC cells for glutamine deprivation-induced EMT, cell motility, and nutrient stress survival. Importantly, we decipher that Slug is associated with nutrient stress in PDAC tumors and is required for metastasis. These results delineate a novel role for Slug in the nutrient stress response and provide insight into how nutrient depletion might influence PDAC progression.


Assuntos
Transição Epitelial-Mesenquimal , Glutamina/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fatores de Transcrição da Família Snail/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Neoplasias Pancreáticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição da Família Snail/genética , Estresse Fisiológico
19.
Philos Trans R Soc Lond B Biol Sci ; 374(1765): 20180153, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30967003

RESUMO

In tumour cells, macropinocytosis functions as an amino acid supply route and supports cancer cell survival and proliferation. Initially demonstrated in oncogenic KRAS-driven models of pancreatic cancer, macropinocytosis triggers the internalization of extracellular proteins via discrete endocytic vesicles called macropinosomes. The incoming protein cargo is targeted for lysosome-dependent degradation, causing the intracellular release of amino acids. These protein-derived amino acids support metabolic fitness by contributing to the intracellular amino acid pools, as well as to the biosynthesis of central carbon metabolites. In this way, macropinocytosis represents a novel amino acid supply route that tumour cells use to survive the nutrient-poor conditions of the tumour microenvironment. Macropinocytosis has also emerged as an entry mechanism for a variety of nanomedicines, suggesting that macropinocytosis regulation in the tumour setting can be harnessed for the delivery of anti-cancer therapeutics. A slew of recent studies point to the possibility that macropinocytosis is a pervasive feature of many different tumour types. In this review, we focus on the role of this important uptake mechanism in a variety of cancers and highlight the main molecular drivers of macropinocytosis in these malignancies. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.


Assuntos
Neoplasias/metabolismo , Pinocitose/fisiologia , Microambiente Tumoral , Humanos , Neoplasias/fisiopatologia
20.
Trends Cancer ; 5(6): 332-334, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31208695

RESUMO

Macropinocytosis is an important nutrient-scavenging pathway in numerous cancer types, including pancreatic, lung, prostate, and bladder. This Forum highlights recent work identifying the key regulators of macropinocytosis that support tumor cell fitness in different contexts, providing a unique framework for strategies to target macropinocytosis in the treatment of cancer.


Assuntos
Neoplasias/etiologia , Neoplasias/metabolismo , Pinocitose/genética , Pinocitose/imunologia , Transdução de Sinais , Animais , Biomarcadores Tumorais , Modelos Animais de Doenças , Xenoenxertos , Humanos , Neoplasias/patologia
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