Gaelle Muller-Greven

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.

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.

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.

Abstract 3276: The tissue and cellular destination of therapeutic IgGs in glioblastoma

Most patients with recurrent glioblastoma (GBM) are treated with bevacizumab, a humanized monoclonal antibody (mAb) that binds VEGF-A and inhibits its binding to VEGFR. Approximately 30% of GBM patients are non-responsive to bevacizumab and the underlying mechanism for the lack of response is not known. It has been assumed that bevacizumab solely targets circulating VEGF-A in blood. We hypothesized that bevacizumab and human IgGs in general gain access to the perivascular niche that contains cancer stem cells (CSCs) in GBM. We found that bevacizumab gains access to the perivascular tumor area through leaky blood vessels and was internalized by tumor cells in an orthotopic xenograft mouse model of GBM. In vitro, CSCs (CD133+) from GBM rapidly internalized either bevacizumab or human IgG into membrane protrusions that contained actin and internalization was significantly inhibited by a macropinocytosis inhibitor (EIPA), suggesting CSCs internalize bevacizumab or human IgG via macropinocytosis. Furthermore, bevacizumab or human IgG was largely detected in the Rab4+ “fast” recycling compartment at 5 min, and both were largely detected in the LAMP1+ compartment (late endosome/lysosome) at 3 hr in the CSCs. CSCs (CD133+) from GBM do not express the neonatal Fc receptor, the canonical pathway for recycling of IgG. Administration of bevacizumab to an orthotopic xenograft mouse model of established GBM showed that bevacizumab was partially co-localized with Rab4+ or with LAMP1+ in perivascular tumor cells, consistent with our in vitro findings. Taken together, our data show that in GBM, humanized IgG, including bevacizumab, gains access to the perivascular tumor space and is then macropinocytosed by CSCs and trafficked to a recycling compartment or to the late endosome/lysosome. These data suggest that alterations in endocytosis or recycling in the CSCs could impact the fate of therapeutic IgGs like bevacizumab and ultimately influence a patients’ response to GBM therapy. Citation Format: Gaelle Muller-Greven, Cathleen Carlin, Steven Toms, Manmeet Ahluwalia, Markus Bredel, Justin Lathia, Jeremy Rich, Petra Hamerlik, Candece L. Gladson. The tissue and cellular destination of therapeutic IgGs in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3276.

Abstract LB-340: Mechanism of bevacizumab internalization and fate by brain endothelial cells .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Antiangiogenic immunotherapy shows great promise for treatment of malignancies, including cancer. Bevacizumab is a humanized monoclonal antibody that inactivates vascular endothelial growth factor-A (VEGF-A), thereby inhibiting angiogenesis. It has received FDA approval for patients with recurrent glioblastoma (GBM); however, more than 30% of patients are non-responsive. The internalization and fate of bevacizumab in endothelial cells (ECs) may play a critical role in the response to therapy, i.e., bevacizumab may be degraded, recycled to the vessel lumen, or transcytosed to the sub-basal EC where tumor cell VEGF-A is found. We examined the internalization of bevacizumab by normal brain ECs in different conditions and found that internalization increased in a time-dependent manner (30 min - 24 hrs). Epidermal growth factor (EGF) stimulated internalization, suggesting bevacizumab enters the cells by an endocytic pathway that is induced by growth factors, such as macropinocytosis. Also, internalized bevacizumab was compartmentalized into vesicles that in part co-localized with the EGF receptor. Treatment with Amiloride (inhibitor of macropinocytosis) decreased the number of bevacizumab-containing vesicles per cell, suggesting macropinocytosis may be one mechanism of internalization. Treatment with Bafilomycin A1 (inhibitor of lysosomal acidification) caused accumulation of bevacizumab, suggesting one fate is lysosomal degradation. Ongoing experiments will determine the endocytic compartment(s) containing bevacizumab, its trafficking, and whether it is transcytosed across normal brain and tumor-associated ECs. Understanding the mechanism of internalization and trafficking of bevacizumab in brain ECs will positively impact the design and modification of monoclonal antibody therapy for GBM patients and for other cancers. Citation Format: Gaelle M. Muller-Greven, Cathleen Carlin, Candece L. Gladson. Mechanism of bevacizumab internalization and fate by brain 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 LB-340. doi:10.1158/1538-7445.AM2013-LB-340