Monica E. Burgett

Lyn facilitates glioblastoma cell survival under conditions of nutrient deprivation by promoting autophagy.

Members of the Src family kinases (SFK) can modulate diverse cellular processes, including division, death and survival, but their role in autophagy has been minimally explored. Here, we investigated the roles of Lyn, a SFK, in promoting the survival of human glioblastoma tumor (GBM) cells in vitro and in vivo using lentiviral vector-mediated expression of constitutively-active Lyn (CA-Lyn) or dominant-negative Lyn (DN-Lyn). Expression of either CA-Lyn or DN-Lyn had no effect on the survival of U87 GBM cells grown under nutrient-rich conditions. In contrast, under nutrient-deprived conditions (absence of supplementation with L-glutamine, which is essential for growth of GBM cells, and FBS) CA-Lyn expression enhanced survival and promoted autophagy as well as inhibiting cell death and promoting proliferation. Expression of DN-Lyn promoted cell death. In the nutrient-deprived GBM cells, CA-Lyn expression enhanced AMPK activity and reduced the levels of pS6 kinase whereas DN-Lyn enhanced the levels of pS6 kinase. Similar results were obtained in vitro using another cultured GBM cell line and primary glioma stem cells. On propagation of the transduced GBM cells in the brains of nude mice, the CA-Lyn xenografts formed larger tumors than control cells and autophagosomes were detectable in the tumor cells. The DN-Lyn xenografts formed smaller tumors and contained more apoptotic cells. Our findings suggest that on nutrient deprivation in vitro Lyn acts to enhance the survival of GBM cells by promoting autophagy and proliferation as well as inhibiting cell death, and Lyn promotes the same effects in vivo in xenograft tumors. As the levels of Lyn protein or its activity are elevated in several cancers these findings may be of broad relevance to cancer biology.

Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM). Here, we show that angiogenesis also can be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and endothelial cells (ECs). As shown in vitro, this direct interaction is mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. It promotes both EC network formation and enhances directed migration toward basic fibroblast growth factor. Activation of αvβ3 and bone marrow tyrosine kinase on chromosome X (BMX) is required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells and CSCs in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting.

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.

Abstract 3898: Glioma stem cells promote brain endothelial cell motility: a new mechanism for the direct interaction of glioma stem cells with endothelial cells.

Angiogenesis, the development of a neovasculature, is a key histologic feature of malignant glioma. It occurs through multiple mechanisms, including sprouting and migration of existing endothelial cells (ECs), and is driven by signals in the microenvironment. Glioma stem cells (GSC) inhabit a region near ECs (perivascular niche). Here we examined the hypothesis that GSCs interact directly with ECs and promote EC motility. In 4 of 5 glioblastoma biopsies we found that ∼40% of GSCs were localized within 25 μm of ECs, based on double-labeling for Sox2 and vWf. We then examined the effect of GSCs on EC motility when plated on a 2D laminin substrate, and found a significant increase in EC motility in the presence of GSCs as well as a direct interaction. In a 3D assay, EC migration was enhanced by GSC-conditioned media, but the physical presence and interaction between ECs and GSCs further increased EC migration through a laminin-coated, 3-μm pore filter, as compared to GSC-conditioned media. Integrin αvβ3 and L1CAM have been reported to be upregulated on ECs and GSCs, respectively, in GBM tumors, and integrin αvβ3 can bind the RGD-peptide in L1CAM. We examined the effect of blocking or downregulating integrin αvβ3 or L1CAM on EC interaction with GSCs. In a cell-cell binding assay, blocking or downregulating the integrin β3 subunit significantly inhibited EC-GSC direct contact, as did an RGD peptide and an antibody to L1CAM. Furthermore, downregulation of integrin β3 on ECs significantly reduced EC migration in the presence of GSCs in the transwell migration assay and reduced EC association with GSCs in mouse brain slices in organotypic culture, analyzed by 2-Photon-LSM. These data suggest GSCs promote angiogenesis in part by promoting EC motility, and that the EC-GSC direct interaction mediated by integrin αvβ3 on ECs and L1CAM on GSCs also stimulates EC motility. Citation Format: Monica E. Burgett, Ping Huang, Russell S. Tipps, Amit Vasanji, Justin D. Lathia, Meizhang Li, Shideng Bao, Jeremy N. Rich, Candece L. Gladson. Glioma stem cells promote brain endothelial cell motility: a new mechanism for the direct interaction of glioma stem cells with endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2013-3898

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

BackgroundMacroautophagy/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.MethodsIn 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.ResultsLC3-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.ConclusionsThese 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.

Abstract 5288: Glioma stem cells stimulate the motility of brain endothelial cells: Identification of cell-adhesion molecules mediating motility and direct interaction

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Angiogenesis is a prominent characteristic of malignant glioma. Glioma stem cells (GSCs) reside adjacent to blood vessels in the perivascular niche, and recent evidence suggests a role for GSCs in promoting angiogenesis. Here we examined the interplay between GSCs and endothelial cells (ECs) on a laminin substrate, and the potential for GSC promotion of EC motility. In an in vitro 2D motility assay, we found that GSCs dramatically stimulated EC migration, and ECs also stimulated GSC migration. An antibody to integrin α6 blocked in part the motility of both GSCs and ECs. We also found that GSCs directly interact with ECs; therefore, we investigated which cell-adhesion molecules mediated the interaction. In a cell-cell binding assay, blocking antibodies to both L1-cell adhesion molecule (L1CAM) and integrin αvβ3 inhibited in part the direct contact between GSCs and ECs. In the motility assay, the combination of antibodies to L1CAM and αvβ3 significantly inhibited the motility and interaction of both cell types. These data suggest GSCs can promote angiogenesis by promoting EC migration, and indicate a role for the cell-adhesion receptors L1CAM and αvβ3 in the direct interaction between GSCs and ECs that may promote EC motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5288. doi:1538-7445.AM2012-5288

Expression (Exp) of FIP200 and Rb in breast cancer (BreastCA) metastasis (met) to the brain and effect on survival.

e21152 Background: BreastCA is the most common non-cutaneous cancer and the 2nd leading cause of cancer deaths. Although the outcome for BreastCA patients (pts) with brain met remains poor, there is significant variation in overall survival (OS). However, there are no markers or to predict OS. FIP200 is a signaling node regulating several pathways. It inhibits cell proliferation when localized to the nucleus by promoting Rb-1 and p21, and it promotes cell survival when localized to the cytoplasm through inhibition of Pyk2 and regulation of autophagy. FIP200 gene is deleted or mutated in approximately 20 % of primary BreastCA biopsies, and the level of Exp of FIP200 and Rb1 are correlated in BreastCA cell lines. The Exp of FIP200 and its cellular localization have not been examined in brain met of any cancer. METHODS In a single institution retrospective analysis brain tissues on 21 pts with metastatic BreastCA, obtained between 8/2000 and 3/2010 were evaluated for Exp of FIP200 and Rb1. These biopsies were selected based on the availability of adequate tissue in the blocks and a prior diagnosis (Dx) of invasive ductal BreastCA. Immunohistochemistry was used to evaluate the localization and levels of Exp of FIP200 and Rb1 which in turn were tested as predictors of prolonged OS. RESULTS 37% of primary breastCA were triple negative, 25% were ER negative/HER2 positive. Median time from Dx to met in the 21 pts was 23 (0-245) months (mos), and the median OS from Dx of BreastCA was 43 (6-264) mos. Nuclear Exp of Rb1 was detected in < 30% vs. ≥ 30% of cells in 11 vs. 10 pts. The median OS was 39 (range 6-264) mos in pts with Rb1 Exp < 30% of cells and 47 (20-190) mo in those with Rb1 Exp in ≥ 30% of cells. Nuclear Exp of FIP200 was detected in <20% vs. ≥ 20% of cells in 13 vs. 8 pts. The median OS was 39 (6-264) mos in pts with FIP200 nuclear Exp in <20% of cells and 45 (43-122) mos in those with FIP200 Exp in ≥ 20% of cells. Cytoplasmic Exp of Fip200 was detected in ≥ 20% of cells in 20 of 21 cases. CONCLUSIONS The pattern of nuclear Exp of Rb1 is different in pts with brain met from invasive intra-ductal BreastCA with a longer OS. An expanded study is underway to determine whether nuclear Rb1 and/or FIP200 predict OS and define the role of these proteins in brain met from BreastCA.