Hypoxic Induction of Exosome Uptake through Proteoglycan-Dependent Endocytosis Fuels the Lipid Droplet Phenotype in Glioma.

As an adaptive response to hypoxic stress, aggressive tumors rewire their metabolic phenotype into increased malignant behavior through extracellular lipid scavenging and storage in lipid droplets (LD). However, the underlying mechanisms and potential lipid source retrieved in the hypoxic tumor microenvironment remain poorly understood. Here, we show that exosome-like extracellular vesicles (EV), known as influential messengers in the tumor microenvironment, may also serve anabolic functions by transforming hypoxic, patient-derived human glioblastoma cell lines into the LD+ phenotype. EVs were internalized via a hypoxia-sensitive, endocytic mechanism that fueled LD formation through direct lipid transfer, and independently of fatty acid synthase activity. EVs can enter cells through multiple and yet ill-defined pathways. On a mechanistic level, we found that hypoxia-mediated EV uptake depends on increased heparan sulfate proteoglycan (HSPG) endocytosis that preferentially followed the lipid raft pathway. The functional relevance of HSPG was evidenced by the reversal of EV-mediated LD loading by targeting of HSPG receptor function. IMPLICATIONS: Together, our data extend the multifaceted role of EVs in cancer biology by showing their LD-inducing capacity in hypoxic glioma cells. Moreover, these findings highlight a potential function for HSPG-mediated endocytosis as a salvage pathway for EV retrieval during tumor stress conditions.

Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity.

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.

Metastasis Stimulation by Hypoxia and Acidosis-Induced Extracellular Lipid Uptake Is Mediated by Proteoglycan-Dependent Endocytosis.

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.

Global profiling of metabolic adaptation to hypoxic stress in human glioblastoma cells.

Oncogenetic events and unique phenomena of the tumor microenvironment together induce adaptive metabolic responses that may offer new diagnostic tools and therapeutic targets of cancer. Hypoxia, or low oxygen tension, represents a well-established and universal feature of the tumor microenvironment and has been linked to increased tumor aggressiveness as well as resistance to conventional oncological treatments. Previous studies have provided important insights into hypoxia induced changes of the transcriptome and proteome; however, how this translates into changes at the metabolite level remains to be defined. Here, we have investigated dynamic, time-dependent effects of hypoxia on the cancer cell metabolome across all families of macromolecules, i.e., carbohydrate, protein, lipid and nucleic acid, in human glioblastoma cells. Using GC/MS and LC/MS/MS, 345 and 126 metabolites were identified and quantified in cells and corresponding media, respectively, at short (6 h), intermediate (24 h), and prolonged (48 h) incubation at normoxic or hypoxic (1% O2) conditions. In conjunction, we performed gene array studies with hypoxic and normoxic cells following short and prolonged incubation. We found that levels of several key metabolites varied with the duration of hypoxic stress. In some cases, metabolic changes corresponded with hypoxic regulation of key pathways at the transcriptional level. Our results provide new insights into the metabolic response of glioblastoma cells to hypoxia, which should stimulate further work aimed at targeting cancer cell adaptive mechanisms to microenvironmental stress.

Exosome and microvesicle mediated phene transfer in mammalian cells.

Extracellular vesicles (EVs), e.g. exosomes and microvesicles, emerge as new signaling organelles in the exchange of information between cells at the paracrine and systemic level. It is clear that these virus like particles carry complex biological information that can elicit a pleiotropic response in recipient cells with potential relevance in physiology as well as in cancer and other pathological conditions. Numerous studies convincingly show that the molecular composition of EVs closely reflects their cell or tissue of origin. Thus, the signaling status of donor cells, more specifically their endosomal compartments, may largely determine the biological output in recipient cells, a process that we then may conceptualize as vesicle mediated phene transfer. Whereas more conventional modes of cell-cell communication mostly depend on extracellular ligand concentration and cell-surface receptor availability, the magnitude of the EV signaling response relies on the capture and uptake by target cells, allowing release of the EV content. Numerous reports point at the intriguing possibility that, among thousands of mRNAs, miRNAs, and proteins, single EV constituents effectuate the biological response, e.g. stimulation of angiogenesis and cancer cell metastasis, in recipient cells; however, we find it conceivable that strategies targeted at general mechanisms of EV function should provide more rational avenues for therapeutic intervention directed at the EV system. Such strategies include manipulation of EV formation in the endolysosomal system, EV stability in the extracellular milieu, and EV entry into target cells. Here, we provide important insights into potential mechanisms of EV transport in mammalian cells and how these may be targeted.

Heparan sulfate proteoglycan as a cell-surface endocytosis receptor.

How various macromolecules are exchanged between cells and how they gain entry into recipient cells are fundamental questions in cell biology with important implications e.g. non-viral drug delivery, infectious disease, metabolic disorders, and cancer. The role of heparan sulfate proteoglycan (HSPG) as a cell-surface receptor of diverse macromolecular cargo has recently been manifested. Exosomes, cell penetrating peptides, polycation-nucleic acid complexes, viruses, lipoproteins, growth factors and morphogens among other ligands enter cells through HSPG-mediated endocytosis. Key questions that partially have been unraveled over recent years include the respective roles of HSPG core protein and HS chain structure specificity for macromolecular cargo endocytosis, the down-stream intracellular signaling events involved in HSPG-dependent membrane invagination and vesicle formation, and the biological significance of the HSPG transport pathway. Here, we discuss the intriguing role of HSPGs as a major entry pathway of macromolecules in mammalian cells with emphasis on recent in vitro and in vivo data that provide compelling evidence of HSPG as an autonomous endocytosis receptor.

Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity.

Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosome-associated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1.

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.

Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development.

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.

ScFv anti-heparan sulfate antibodies unexpectedly activate endothelial and cancer cells through p38 MAPK: implications for antibody-based targeting of heparan sulfate proteoglycans in cancer.

Tumor development requires angiogenesis and anti-angiogenic therapies have been introduced in the treatment of cancer. In this context, heparan sulfate proteoglycans (HSPGs) emerge as interesting targets, owing to their function as co-receptors of major, pro-angiogenic factors. Accordingly, previous studies have suggested anti-tumor effects of heparin, i.e. over-sulfated HS, and various heparin mimetics; however, a significant drawback is their unspecific mechanism of action and potentially serious side-effects related to their anticoagulant properties. Here, we have explored the use of human ScFv anti-HS antibodies (αHS) as a more rational approach to target HSPG function in endothelial cells (ECs). αHS were initially selected for their recognition of HS epitopes localized preferentially to the vasculature of patient glioblastoma tumors, i.e. highly angiogenic brain tumors. Unexpectedly, we found that these αHS exhibited potent pro-angiogenic effects in primary human ECs. αHS were shown to stimulate EC differentiation, which was associated with increased EC tube formation and proliferation. Moreover, αHS supported EC survival under hypoxia and starvation, i.e. conditions typical of the tumor microenvironment. Importantly, αHS-mediated proliferation was efficiently counter-acted by heparin and was absent in HSPG-deficient mutant cells, confirming HS-specific effects. On a mechanistic level, binding of αHS to HSPGs of ECs as well as glioblastoma cells was found to trigger p38 MAPK-dependent signaling resulting in increased proliferation. We conclude that several αHS that recognize HS epitopes abundant in the tumor vasculature may elicit a pro-angiogenic response, which has implications for the development of antibody-based targeting of HSPGs in cancer.

Chondroitin sulfate expression predicts poor outcome in breast cancer.

Experimental studies have established that the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate act as co-receptors of cytokines and growth factors that drive the malignant cell phenotype and the remodelling of the surrounding tumor stroma. However, the clinical relevance of these studies remains ill-defined. The present study investigates the significance of chondroitin sulfate expression in malignant cells and the stroma, respectively, of tumors from two independent cohorts of breast cancer patients (cohort I: 144 patients, 130 evaluable samples; cohort II: 498 patients, 469 evaluable samples; ER-positive patients ~86% in both cohorts). Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the relationship between chondroitin sulfate and recurrence-free and overall survival. High chondroitin sulfate expression in malignant cells was shown to predict shorter recurrence-free survival (P=0.007, cohort I; P=0.024, cohort II) and overall survival (cohort I: P=0.044; cohort II: P<0.001) in both cohorts. In multivariate analysis, high chondroitin sulfate in malignant cells was shown to be an independent, predictive factor of poor overall survival (cohort I: hazard ratio 2.28: 95% confidence interval 1.08-4.81, P=0.031; cohort II: hazard ratio 1.71: 95% confidence interval 1.23-2.38, P=0.001). However, chondroitin sulfate in the stroma showed no correlation with known markers of tumor aggressiveness or with clinical outcome in either cohort. Our data suggest that high chondroitin sulfate expression in malignant cells is associated with an adverse outcome in patients with primary breast cancer, supporting the idea of a functional and potentially targetable role of chondroitin sulfate in tumor disease.

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells.

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.