Expression of LC3B and FIP200/Atg17 in brain metastases of breast cancer.

BACKGROUND: Macroautophagy/autophagy is considered to play key roles in tumor cell evasion of therapy and establishment of metastases in breast cancer. High expression of LC3, a residual autophagy marker, in primary breast tumors has been associated with metastatic disease and poor outcome. FIP200/Atg17, a multi-functional pro-survival molecule required for autophagy, has been implicated in brain metastases in experimental models. However, expression of these proteins has not been examined in brain metastases from patients with breast cancer. METHODS: In this retrospective study, specimens from 44 patients with brain metastases of infiltrating ductal carcinoma of the breast (IDC), unpaired samples from 52 patients with primary IDC (primary-BC) and 16 matched-paired samples were analyzed for LC3 puncta, expression of FIP200/Atg17, and p62 staining. RESULTS: LC3-puncta+ tumor cells and FIP200/Atg17 expression were detected in greater than 90% of brain metastases but there were considerable intra- and inter-tumor differences in expression levels. High numbers of LC3-puncta+ tumor cells in brain metastases correlated with a significantly shorter survival time in triple-negative breast cancer. FIP200/Atg17 protein levels were significantly higher in metastases that subsequently recurred following therapy. The percentages of LC3 puncta+ tumor cells and FIP200/Atg17 protein expression levels, but not mRNA levels, were significantly higher in metastases than primary-BC. Meta-analysis of gene expression datasets revealed a significant correlation between higher FIP200(RB1CC1)/Atg17 mRNA levels in primary-BC tumors and shorter disease-free survival. CONCLUSIONS: These results support assessments of precision medicine-guided targeting of autophagy in treatment of brain metastases in breast cancer patients.

Correlation of higher levels of soluble TNF-R1 with a shorter survival, independent of age, in recurrent glioblastoma.

The circulating levels of soluble tumor necrosis factor receptor-1 (sTNF-R1) and sTNF-R2 are altered in numerous diseases, including several types of cancer. Correlations with the risk of progression in some cancers, as well as systemic manifestations of the disease and therapeutic side-effects, have been described. However, there is very little information on the levels of these soluble receptors in glioblastoma (GBM). Here, we report on an exploratory retrospective study of the levels of sTNF-Rs in the vascular circulation of patients with GBM. Banked samples were obtained from 112 GBM patients (66 untreated, newly-diagnosed patients and 46 with recurrent disease) from two institutions. The levels of sTNF-R1 in the plasma were significantly lower in patients with newly-diagnosed or recurrent GBM than apparently healthy individuals and correlated with the intensity of expression of TNF-R1 on the tumor-associated endothelial cells (ECs) in the corresponding biopsies. Elevated levels of sTNF-R1 in patients with recurrent, but not newly-diagnosed GBM, were significantly associated with a shorter survival, independent of age (p = 0.02) or steroid medication. In contrast, the levels of circulating sTNF-R2 were significantly higher in recurrent GBM than healthy individuals and there was no significant correlation with expression of TNF-R2 on the tumor-associated ECs or survival time. The results indicate that larger, prospective studies are warranted to determine the predictive value of the levels of sTNF-R1 in patients with recurrent GBM and the factors that regulate the levels of sTNF-Rs in the circulation in GBM patients.

Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5ß1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with ß1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5ß1 integrin and uPAR drive the translocation of α5ß1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Cancer stem cell-specific scavenger receptor CD36 drives glioblastoma progression.

Glioblastoma (GBM) contains a self-renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self-renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient-derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self-renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self-renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non-CSCs, increased with exposure to oxidized low-density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages.

FAK-related nonkinase is a multifunctional negative regulator of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease whose underlying molecular mechanisms are largely unknown. Herein, we show that focal adhesion kinase-related nonkinase (FRNK) plays a key role in limiting the development of lung fibrosis. Loss of FRNK function in vivo leads to increased lung fibrosis in an experimental mouse model. The increase in lung fibrosis is confirmed at the histological, biochemical, and physiological levels. Concordantly, loss of FRNK function results in increased fibroblast migration and myofibroblast differentiation and activation of signaling proteins that drive these phenotypes. FRNK-deficient murine lung fibroblasts also have an increased capacity to produce and contract matrix proteins. Restoration of FRNK expression in vivo and in vitro reverses these profibrotic phenotypes. These data demonstrate the multiple antifibrotic actions of FRNK. More important, FRNK expression is down-regulated in human IPF, and down-regulation of FRNK in normal human lung fibroblasts recapitulates the profibrotic phenotype seen in FRNK-deficient cells. The effect of loss and gain of FRNK in the experimental model, when taken together with its down-regulation in human IPF, suggests that FRNK acts as an endogenous negative regulator of lung fibrosis by repressing multiple profibrotic responses.

Laminin alpha 2 enables glioblastoma stem cell growth.

OBJECTIVE: Glioblastomas (GBMs) are lethal cancers that display cellular hierarchies parallel to normal brain. At the apex are GBM stem cells (GSCs), which are relatively resistant to conventional therapy. Interactions with the adjacent perivascular niche are an important driver of malignancy and self-renewal in GSCs. Extracellular matrix (ECM) cues instruct neural stem/progenitor cell-niche interactions, and the objective of our study was to elucidate its composition and contribution to GSC maintenance in the perivascular niche. METHODS: We interrogated human tumor tissue for immunofluorescence analysis and derived GSCs from tumor tissues for functional studies. Bioinformatics analyses were conducted by mining publicly available databases. RESULTS: We find that laminin ECM proteins are localized to the perivascular GBM niche and inform negative patient prognosis. To identify the source of laminins, we characterized cellular elements within the niche and found that laminin α chains were expressed by nonstem tumor cells and tumor-associated endothelial cells (ECs). RNA interference targeting laminin α2 inhibited GSC growth and self-renewal. In co-culture studies of GSCs and ECs, laminin α2 knockdown in ECs resulted in decreased tumor growth. INTERPRETATION: Our studies highlight the contribution of nonstem tumor cell-derived laminin juxtracrine signaling. As laminin α2 has recently been identified as a molecular marker of aggressive ependymoma, we propose that the brain vascular ECM promotes tumor malignancy through maintenance of the GSC compartment, providing not only a molecular fingerprint but also a possible therapeutic target.

Tissue factor is a critical regulator of radiation therapy-induced glioblastoma remodeling.

Radiation therapy (RT) provides therapeutic benefits for patients with glioblastoma (GBM), but inevitably induces poorly understood global changes in GBM and its microenvironment (TME) that promote radio-resistance and recurrence. Through a cell surface marker screen, we identified that CD142 (tissue factor or F3) is robustly induced in the senescence-associated ß-galactosidase (SA-ßGal)-positive GBM cells after irradiation. F3 promotes clonal expansion of irradiated SA-ßGal+ GBM cells and orchestrates oncogenic TME remodeling by activating both tumor-autonomous signaling and extrinsic coagulation pathways. Intratumoral F3 signaling induces a mesenchymal-like cell state transition and elevated chemokine secretion. Simultaneously, F3-mediated focal hypercoagulation states lead to activation of tumor-associated macrophages (TAMs) and extracellular matrix (ECM) remodeling. A newly developed F3-targeting agent potently inhibits the aforementioned oncogenic events and impedes tumor relapse in vivo. These findings support F3 as a critical regulator for therapeutic resistance and oncogenic senescence in GBM, opening potential therapeutic avenues.

Integrin α3ß1 promotes vessel formation of glioblastoma-associated endothelial cells through calcium-mediated macropinocytosis and lysosomal exocytosis.

Therapeutic targeting of angiogenesis in glioblastoma has yielded mixed outcomes. Investigation of tumor-associated angiogenesis has focused on the factors that stimulate the sprouting, migration, and hyperproliferation of the endothelial cells. However, little is known regarding the processes underlying the formation of the tumor-associated vessels. To address this issue, we investigated vessel formation in CD31+ cells isolated from human glioblastoma tumors. The results indicate that overexpression of integrin α3ß1 plays a central role in the promotion of tube formation in the tumor-associated endothelial cells in glioblastoma. Blocking α3ß1 function reduced sprout and tube formation in the tumor-associated endothelial cells and vessel density in organotypic cultures of glioblastoma. The data further suggest a mechanistic model in which integrin α3ß1-promoted calcium influx stimulates macropinocytosis and directed maturation of the macropinosomes in a manner that promotes lysosomal exocytosis during nascent lumen formation. Altogether, our data indicate that integrin α3ß1 may be a therapeutic target on the glioblastoma vasculature.

Histidine-rich glycoprotein modulates the anti-angiogenic effects of vasculostatin.

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein expressed on glial cells within the brain. Its expression is dramatically down-regulated in many glioblastomas, consistent with its functional ability to inhibit angiogenesis and tumor growth in vivo. We have shown that the soluble anti-angiogenic domain of BAI1 (termed Vstat120) requires CD36, a cell surface glycoprotein expressed on microvascular endothelial cells (MVECs), for it to elicit an anti-angiogenic response. We now report that Vstat120 binding to CD36 on MVECs activates a caspase-mediated pro-apoptotic pathway, and this effect is abrogated by histidine-rich glycoprotein (HRGP). HRGP is a circulating glycoprotein previously shown to function as a CD36 decoy to promote angiogenesis in the presence of thrombospondin-1 or -2. Data here show that Vstat120 specifically binds HRGP. Under favorable MVEC growth conditions this interaction allows chemotactic-directed migration as well as endothelial tube formation to persist in in vitro cellular systems, and increased tumor growth in vivo as demonstrated in both subcutaneous and orthotopic brain tumor models, concomitant with an increase in tumor vascularity. Finally, we show that HRGP expression is increased in human brain cancers, with the protein heavily localized to the basement membrane of the tumors. These data help define a novel angiogenic axis that could be exploited for the treatment of human cancers and other diseases where excess angiogenesis occurs.

Downregulation of FAK-related non-kinase mediates the migratory phenotype of human fibrotic lung fibroblasts.

Fibroblast migration plays an important role in the normal wound healing process; however, dysregulated cell migration may contribute to the progressive formation of fibrotic lesions in the diseased condition. To examine the role of focal-adhesion-kinase (FAK)-related non-kinase (FRNK) in regulation of fibrotic lung fibroblast migration, we examined cell migration, FRNK expression, and activation of focal adhesion kinase (FAK) and Rho GTPase (Rho and Rac) in primary lung fibroblasts derived from both idiopathic pulmonary fibrosis (IPF) patients and normal human controls. Fibrotic (IPF) lung fibroblasts have increased cell migration when compared to control human lung fibroblasts. FRNK expression is significantly reduced in IPF lung fibroblasts, while activation of FAK, Rho and Rac is increased in IPF lung fibroblasts. Endogenous FRNK expression is inversely correlated with FAK activation and cell migration rate in IPF lung fibroblasts. Forced exogenous FRNK expression abrogates the increased cell migration, and blocked the activation of FAK and Rho GTPase (Rho and Rac), in IPF lung fibroblasts. These data for the first time provide evidence that downregulation of endogenous FRNK plays a role in promoting cell migration through FAK and Rho GTPase in fibrotic IPF lung fibroblasts.

Thrombospondin-1-induced apoptosis of brain microvascular endothelial cells can be mediated by TNF-R1.

Thrombospondin-1 (TSP-1) treatment of dermal microvascular endothelial cells (MvEC) has been shown to upregulate Fas ligand (FasL) and to induce apoptosis by a mechanism that requires caspase-8 activity. We have examined the potential anti-angiogenic effects of TSP-1 on primary human brain MvEC. The addition of TSP-1 to primary human brain MvEC cultured as monolayers on type 1 collagen, induced cell death and apoptosis (evidenced by caspase-3 cleavage) in a dose- (5-30 nM) and time-dependent (maximal at 17 h) manner. TSP-1 treatment for 17 h induced caspase-3 cleavage that required caspase-8 activity and the tumor necrosis factor receptor 1 (TNF-R1). We did not find a requirement for Fas, or the tumor necrosis-related apoptosis-inducing ligand receptors (TRAIL-R) 1 and 2. We confirmed the findings using caspase inhibitors, blocking antibodies and small interfering RNA (siRNA). Further analysis indicated that the TSP-1 induction of caspase-3 cleavage of primary human brain MvEC adherent to collagen required the synthesis of new message and protein, and that TSP-1 induced the expression of TNFalpha mRNA and protein. Consistent with these findings, when the primary human brain MvEC were propagated on collagen gels mAb anti-TNF-R1 reversed the inhibitory effect, in part, of TSP-1 on tube formation and branching. These data identify a novel mechanism whereby TSP-1 can inhibit angiogenesis-through induction of apoptosis in a process mediated by TNF-R1.

Urokinase receptor mediates lung fibroblast attachment and migration toward provisional matrix proteins through interaction with multiple integrins.

Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibits a dependence on the urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the urokinase-type plasminogen activator cellular receptor (u-PAR)-integrin interactions and to determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR colocalizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin, and collagens. Inhibitory antibody studies have revealed that human lung fibroblasts utilize alpha(v)beta(5) to attach to vitronectin, predominantly alpha(5)beta(1) (and alpha(v)beta(3)) to attach to fibronectin, and alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) to attach to collagen. Blocking studies with alpha-integrin subunit decoy peptides and u-PAR neutralizing antibodies indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these decoy peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple alpha-integrins but with a preference for alpha(3). Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of fibroblast invasion would be expected to lead to amelioration of fibroproliferative diseases of the lung.

New molecular targets in angiogenic vessels of glioblastoma tumours.

Antiangiogenesis approaches have the potential to be particularly effective in the treatment of glioblastoma tumours. These tumours exhibit extremely high levels of neovascularisation, which may contribute to their extremely aggressive behaviour, not only by providing oxygenation and nutrition, but also by establishing a leaky vasculature that lacks a blood-brain barrier. This leaky vasculature enables migration of tumour cells, as well as the build up of fluid, which exacerbates tissue damage due to increased intracranial pressure. Here, we discuss the considerable progress that has been made in the identification of the pro- and antiangiogenic factors produced by glioblastoma tumours and the effects of these molecules in animal models of the disease. The safety and efficacy of some of these approaches have now been demonstrated in clinical trials. However, the ability of tumours to overcome these therapies and to re-establish angiogenesis requires further clinical research regarding potential multimodality therapies, as well as basic research into the regulation of angiogenesis by as yet unidentified factors. Optimisation of noninvasive procedures for monitoring of angiogenesis would greatly facilitate such research.

ABT-510, a modified type 1 repeat peptide of thrombospondin, inhibits malignant glioma growth in vivo by inhibiting angiogenesis.

Anti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown to inhibit angiogenesis in non-glioma tumor models and a modified TSR peptide, ABT-510, has now entered into Phase II clinical trials of its efficacy in non-glioma tumors. As microvascular endothelial cells (MvEC) exhibit heterogeneity, we evaluated the ability of the modified TSR peptide (NAcSarGlyValDallolleThrNvalleArgProNHE, ABT-510) to inhibit malignant glioma growth in vivo and to induce apoptosis of brain microvessel endothelial cells (MvEC) propagated in vitro. We found that daily administration of ABT-510 until euthanasia (days 7 to 19), significantly inhibited the growth of human malignant astrocytoma tumors established in the brain of athymic nude mice. The microvessel density was significantly lower and the number of apoptotic MvEC was significantly higher (3-fold) in the tumors of the ABT-510-treated animals. Similar results were found using a model in which the established tumor is an intracerebral malignant glioma propagated in a syngeneic mouse model. ABT-510 treatment of primary human brain MvEC propagated as a monolayer resulted in induction of apoptosis in a dose- and time-dependent manner through a caspase-8-dependent mechanism. It also inhibited tubular morphogenesis of MvEC propagated in collagen gels in a dose- and caspase-8 dependent manner through a mechanism that requires the TSP-1 receptor (CD36) on the MvEC. These findings indicate that ABT-510 should be evaluated as a therapeutic option for patients with malignant glioma.

Low-density lipoprotein receptor-related protein contributes to the antiangiogenic activity of thrombospondin-2 in a murine glioma model.

Host antiangiogenesis factors defend against tumor growth. The matricellular protein, thrombospondin-2 (TSP-2), has been shown to act as an antiangiogenesis factor in a carcinogen-induced model of skin cancer. Here, using an in vivo malignant glioma model in which the characteristics of the tumors formed after intracerebral implantation of GL261 mouse glioma cells are assessed, we found that tumor growth and microvessel density were significantly enhanced in tumors propagated in TSP-2(-/-) mice. Mechanistically, matrix metalloproteinase (MMP)-2 has been associated with neoangiogenesis and it has been proposed that the levels of available MMP-2 may be down-regulated by formation of a complex with TSP-2 that is internalized by low-density lipoprotein receptor-related protein 1 (LRP1). We found elevated expression of MMP-2 and MMP-9 in tumors propagated in TSP-2(-/-) mice, with a preferential localization in the microvasculature. In wild-type mice, MMP-2 was coexpressed with TSP-2 in the tumor microvasculature. In vitro, addition of recombinant (rec) TSP-2 to mouse brain microvessel endothelial cells reduced MMP-2 levels and invasion through mechanisms that could be inhibited by a competitive inhibitor of ligand binding to LRP1 or by siLRP1. Thus, the antiangiogenic activity of TSP-2 is capable of inhibiting the growth of gliomas in part by reducing the levels of MMP-2 in the tumor microvasculature. This mechanism is mediated by LRP1.

Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells.

Cellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. As we have found that Lyn, but not Fyn, activity promotes migration of glioblastoma cells in response to the cooperative signal generated by platelet-derived growth factor receptor beta and integrin alpha(v)beta3, we compared the activity and expression of Lyn and Fyn in glioblastoma (grade IV) tumor biopsy samples with that in anaplastic astrocytoma (grade III) tumors, nonneoplastic brain, and normal autopsy brain samples. Lyn kinase activity was significantly elevated in glioblastoma tumor samples. Notably, the Lyn kinase activity accounted for >90% of pan-Src kinase activity in glioblastoma samples but only approximately 30% of pan-Src kinase activity in the other groups. The levels of phosphorylation of the autophosphorylation site were consistent with significantly higher Lyn activity in glioblastoma tumor tissue than nonneoplastic brain. Although the normalized levels of Lyn protein and the relative levels of Lyn message were significantly higher in glioblastoma samples than nonneoplastic brain, the normalized levels of Lyn protein did not correlate with Lyn activity in the glioblastoma samples. There was no significant difference in the normalized levels of c-Src and Fyn protein and message in the glioblastoma and nonneoplastic brain. Immunostaining revealed that Lyn is located primarily in the glioblastoma cells in the tumor biopsies. These data indicate that Lyn kinase activity is significantly elevated in glioblastoma tumors and suggest that it is the Lyn activity that promotes the malignant phenotype in these tumors.

Macropinocytosis of Bevacizumab by Glioblastoma Cells in the Perivascular Niche Affects their Survival.

Purpose: Bevacizumab, a humanized monoclonal antibody to VEGF, is used routinely in the treatment of patients with recurrent glioblastoma (GBM). However, very little is known regarding the effects of bevacizumab on the cells in the perivascular space in tumors.Experimental Design: Established orthotopic xenograft and syngeneic models of GBM were used to determine entry of monoclonal anti-VEGF-A into, and uptake by cells in, the perivascular space. Based on the results, we examined CD133+ cells derived from GBM tumors in vitro Bevacizumab internalization, trafficking, and effects on cell survival were analyzed using multilabel confocal microscopy, immunoblotting, and cytotoxicity assays in the presence/absence of inhibitors.Results: In the GBM mouse models, administered anti-mouse-VEGF-A entered the perivascular tumor niche and was internalized by Sox2+/CD44+ tumor cells. In the perivascular tumor cells, bevacizumab was detected in the recycling compartment or the lysosomes, and increased autophagy was found. Bevacizumab was internalized rapidly by CD133+/Sox2+-GBM cells in vitro through macropinocytosis with a fraction being trafficked to a recycling compartment, independent of FcRn, and a fraction to lysosomes. Bevacizumab treatment of CD133+ GBM cells depleted VEGF-A and induced autophagy thereby improving cell survival. An inhibitor of lysosomal acidification decreased bevacizumab-induced autophagy and increased cell death. Inhibition of macropinocytosis increased cell death, suggesting macropinocytosis of bevacizumab promotes CD133+ cell survival.Conclusions: We demonstrate that bevacizumab is internalized by Sox2+/CD44+-GBM tumor cells residing in the perivascular tumor niche. Macropinocytosis of bevacizumab and trafficking to the lysosomes promotes CD133+ cell survival, as does the autophagy induced by bevacizumab depletion of VEGF-A. Clin Cancer Res; 23(22); 7059-71. ©2017 AACR.

Inhibition of cystine uptake disrupts the growth of primary brain tumors.

Glial cells play an important role in sequestering neuronally released glutamate via Na+-dependent transporters. Surprisingly, these transporters are not operational in glial-derived tumors (gliomas). Instead, gliomas release glutamate, causing excitotoxic death of neurons in the vicinity of the tumor. We now show that glutamate release from glioma cells is an obligatory by-product of cellular cystine uptake via system xc-, an electroneutral cystine-glutamate exchanger. Cystine is an essential precursor for the biosynthesis of glutathione, a major redox regulatory molecule that protects cells from endogenously produced reactive oxygen species (ROS). Glioma cells, but not neurons or astrocytes, rely primarily on cystine uptake via system xc- for their glutathione synthesis. Inhibition of system xc- causes a rapid depletion of glutathione, and the resulting loss of ROS defense causes caspase-mediated apoptosis. Glioma cells can be rescued if glutathione status is experimentally restored or if glutathione is substituted by alternate cellular antioxidants, confirming that ROS are indeed mediators of cell death. We describe two potent drugs that permit pharmacological inhibition of system xc-. One of these drugs, sulfasalazine, is clinically used to treat inflammatory bowel disease and rheumatoid arthritis. Sulfasalazine was able to reduce glutathione levels in tumor tissue and slow tumor growth in vivo in a commonly used intracranial xenograft animal model for human gliomas when administered by intraperitoneal injection. These data suggest that inhibition of cystine uptake into glioma cells through the pharmacological inhibition of system xc- may be a viable therapeutic strategy with a Food and Drug Administration-approved drug already in hand.

A novel technique to quantify glioma tumor invasion using serial microscopy sections.

Here we present a new technique to quantitatively characterize malignant glioma invasion in a syngeneic mouse model. The GL261 mouse malignant glioma cell line was injected intracerebrally into the C57B1/6 black mouse and allowed to propagate for 10 or 17 days, followed by euthanasia of the animal, harvesting of the brain, fixation, and serial sectioning. Histologic examination was performed and the primary tumor mass and discontinuous sites of tumor invasion were traced on digital images of serial microscopy sections, followed by analysis of the invasion characteristics using a custom-written MATLAB program. We found a significant increase in the number of discontinuous tumor invasion sites and in the distance of these sites from the tumor centroid in mice that were euthanized at 17 days post-tumor cell injection, as compared to mice euthanized at 10 days. Furthermore, a scatter plot analyses indicated that the invasion site data could be grouped based on the characteristics of area and distance from the tumor centroid to reveal significant differences between the two experimental groups of mice. This quantitative method will allow a future in vivo analysis of invasion characteristics in glioma cells expressing altered levels or function of invasion genes, and of new therapy targeting invading glioma cells.

Focal adhesion kinase enhances signaling through the Shc/extracellular signal-regulated kinase pathway in anaplastic astrocytoma tumor biopsy samples.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that on activation generates signals that can modulate crucial cell functions, including cell proliferation, migration, and survival. In vitro, overexpression of FAK has been shown to promote cell proliferation by signaling through the Ras/mitogen-activated protein kinase cascade in several cell types. We have shown previously that overexpression of exogenous FAK lacking alternative splicing in malignant astrocytoma clones injected intracerebrally into SCID mouse brains promotes tumor cell proliferation. Here, we show that in anaplastic astrocytoma biopsy samples, FAK is expressed as an unspliced variant and migrates with a faster mobility similar to that observed in embryonic brain. Compared with nonneoplastic adult brain biopsies, the levels of FAK protein are elevated as are its levels of activation as assessed by autophosphorylation and overall tyrosine phosphorylation. The activity of Src kinase in these tumors is also elevated, as well as the activity of Src kinase associated with FAK; the latter may result in enhanced Src kinase phosphorylation of FAK. Phosphorylated Shc is associated with FAK in the anaplastic astrocytoma biopsy samples and in astrocytoma cells overexpressing FAK in vitro but not in nonneoplastic brain biopsy samples. Elevated extracellular signal-regulated kinase-2 activation and elevated expression of cyclins D and E are also found in anaplastic astrocytoma biopsy samples. These data provide evidence that the increased FAK activity in these tumors contributes to phosphorylation of Shc and likely to the promotion of Ras activity, extracellular signal-regulated kinase-2 activation, and cell proliferation in vivo.