RESUMO
Aggressive cancers are characterized by hypoxia, which is a key driver of tumor development and treatment resistance. Proteins specifically expressed in the hypoxic tumor microenvironment thus represent interesting candidates for targeted drug delivery strategies. Carbonic anhydrase (CAIX) has been identified as an attractive treatment target as it is highly hypoxia specific and expressed at the cell-surface to promote cancer cell aggressiveness. Here, we find that cancer cell internalization of CAIX is negatively regulated by post-translational modification with chondroitin or heparan sulfate glycosaminoglycan chains. We show that perturbed glycosaminoglycan modification results in increased CAIX endocytosis. We hypothesized that perturbation of CAIX glycosaminoglycan conjugation may provide opportunities for enhanced drug delivery to hypoxic tumor cells. In support of this concept, pharmacological inhibition of glycosaminoglycan biosynthesis with xylosides significantly potentiated the internalization and cytotoxic activity of an antibody-drug conjugate (ADC) targeted at CAIX. Moreover, cells expressing glycosaminoglycan-deficient CAIX were significantly more sensitive to ADC treatment as compared with cells expressing wild-type CAIX. We find that inhibition of CAIX endocytosis is associated with an increased localization of glycosaminoglycan-conjugated CAIX in membrane lipid raft domains stabilized by caveolin-1 clusters. The association of CAIX with caveolin-1 was partially attenuated by acidosis, i.e. another important feature of malignant tumors. Accordingly, we found increased internalization of CAIX at acidic conditions. These findings provide first evidence that intracellular drug delivery at pathophysiological conditions of malignant tumors can be attenuated by tumor antigen glycosaminoglycan modification, which is of conceptual importance in the future development of targeted cancer treatments.
RESUMO
Hypoxia and acidosis are inherent stress factors of the tumor microenvironment and have been linked to increased tumor aggressiveness and treatment resistance. Molecules involved in the adaptive mechanisms that drive stress-induced disease progression constitute interesting candidates of therapeutic intervention. Here, we provide evidence of a novel role of heparan sulfate proteoglycans (HSPG) in the adaptive response of tumor cells to hypoxia and acidosis through increased internalization of lipoproteins, resulting in a lipid-storing phenotype and enhanced tumor-forming capacity. Patient glioblastoma tumors and cells under hypoxic and acidic stress acquired a lipid droplet (LD)-loaded phenotype, and showed an increased recruitment of all major lipoproteins, HDL, LDL, and VLDL. Stress-induced LD accumulation was associated with increased spheroid-forming capacity during reoxygenation in vitro and lung metastatic potential in vivo On a mechanistic level, we found no apparent effect of hypoxia on HSPGs, whereas lipoprotein receptors (VLDLR and SR-B1) were transiently upregulated by hypoxia. Importantly, however, using pharmacologic and genetic approaches, we show that stress-mediated lipoprotein uptake is highly dependent on intact HSPG expression. The functional relevance of HSPG in the context of tumor cell stress was evidenced by HSPG-dependent lipoprotein cell signaling activation through the ERK/MAPK pathway and by reversal of the LD-loaded phenotype by targeting of HSPGs. We conclude that HSPGs may have an important role in the adaptive response to major stress factors of the tumor microenvironment, with functional consequences on tumor cell signaling and metastatic potential. Cancer Res; 76(16); 4828-40. ©2016 AACR.
Assuntos
Endocitose/fisiologia , Heparina/análogos & derivados , Lipoproteínas/metabolismo , Invasividade Neoplásica/patologia , Proteoglicanas/metabolismo , Microambiente Tumoral/fisiologia , Acidose/metabolismo , Adaptação Fisiológica/fisiologia , Western Blotting , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Cromatografia Líquida , Heparina/metabolismo , Humanos , Microdissecção e Captura a Laser , Espectrometria de Massas , Microscopia Eletrônica , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologiaRESUMO
The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling.
Assuntos
Caveolina 1/metabolismo , Endocitose , Exossomos/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Sistema de Sinalização das MAP Quinases , Microdomínios da Membrana/metabolismo , Animais , Transporte Biológico , Butadienos/farmacologia , Células CHO , Células COS , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Citoesqueleto/metabolismo , Endossomos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioblastoma/metabolismo , Células HeLa , Proteínas de Choque Térmico , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Chaperonas Moleculares , Nitrilas/farmacologia , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development.
Assuntos
Vasos Sanguíneos/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica/patologia , Exossomos/metabolismo , Glioma/irrigação sanguínea , Glioma/patologia , Animais , Comunicação Autócrina , Neoplasias Encefálicas/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Camundongos , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Comunicação Parácrina , Pericitos/metabolismo , Pericitos/patologia , Proteoma/metabolismo , Transdução de Sinais/genética , Doadores de Tecidos , Transcriptoma/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Among markers of glioblastoma initiating cells, AC133 has been shown to be associated with glioblastoma resistance and malignancy. Recently, it was demonstrated that increasing oxygen tension (pO2) down-regulated AC133 expression in glioblastoma cells in vitro. In order to better understand extrinsic factor regulation of AC133, this work aimed to investigate the relationship between cell culture pO2, AC133 expression, and tumor development and phenotype. Using treatments with CoCl2 and HIF-1α shRNA knockdowns on non-sorted human primary glioblastoma cells cultured at low (3%) versus high (21%) oxygen tension, we established a responsibility for low pO2 in the maintenance of high levels of AC133 expression, with a major but non-exclusive role for HIF-1α. We also demonstrated that human glioblastoma cells previously cultured under high oxygen tension can lose part of their aggressiveness when orthotopically engrafted in SCID mice or lead to tumors with distinct phenotypes and no re-expression of AC133. These observations showed that the specific pO2 microenvironment irreversibly impacts glioblastoma cell phenotypes, highlighting the pertinence of culture conditions when extrapolating data from xenogenic models to human cells in their source environment. They also raised AC133 as a marker of non-exposure to oxygenated areas rather than a marker of aggressiveness or low pO2 niches.
Assuntos
Antígenos CD/biossíntese , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Glicoproteínas/biossíntese , Oxigênio/administração & dosagem , Antígeno AC133 , Animais , Antígenos CD/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Glicoproteínas/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos SCID , Pressão Parcial , Peptídeos/genética , Transplante HeterólogoRESUMO
As the pentaspan stem cell marker CD133 was shown to bind cholesterol and to localize in plasma membrane protrusions, we investigated a possible function for CD133 in endocytosis. Using the CD133 siRNA knockdown strategy and non-differentiated human colon cancer Caco-2 cells that constitutively over-expressed CD133, we provide for the first time direct evidence for a role of CD133 in the intracellular accumulation of fluorescently labeled extracellular compounds. Assessed using AC133 monoclonal antibody, CD133 knockdown was shown to improve Alexa488-transferrin (Tf) uptake in Caco-2 cells but had no impact on FITC-dextran or FITC-cholera-toxin. Absence of effect of the CD133 knockdown on Tf recycling established a role for CD133 in inhibiting Tf endocytosis rather than in stimulating Tf exocytosis. Use of previously identified inhibitors of known endocytic pathways and the positive impact of CD133 knockdown on cellular uptake of clathrin-endocytosed synthetic lipid nanocapsules supported that CD133 impact on endocytosis was primarily ascribed to the clathrin pathway. Also, cholesterol extraction with methyl-ß-cyclodextrine up regulated Tf uptake at greater intensity in the CD133(high) situation than in the CD133(low) situation, thus suggesting a role for cholesterol in the inhibitory effect of CD133 on endocytosis. Interestingly, cell treatment with the AC133 antibody down regulated Tf uptake, thus demonstrating that direct extracellular binding to CD133 could affect endocytosis. Moreover, flow cytometry and confocal microscopy established that down regulation of CD133 improved the accessibility to the TfR from the extracellular space, providing a mechanism by which CD133 inhibited Tf uptake. As Tf is involved in supplying iron to the cell, effects of iron supplementation and deprivation on CD133/AC133 expression were investigated. Both demonstrated a dose-dependent down regulation here discussed to the light of transcriptional and post-transciptional effects. Taken together, these data extend our knowledge of the function of CD133 and underline the interest of further exploring the CD133-Tf-iron network.