Michael De Lay

MicroRNA-451 Regulates LKB1/AMPK Signaling and Allows Adaptation to Metabolic Stress in Glioma Cells

To sustain tumor growth, cancer cells must be able to adapt to fluctuations in energy availability. We have identified a single microRNA that controls glioma cell proliferation, migration, and responsiveness to glucose deprivation. Abundant glucose allows relatively high miR-451 expression, promoting cell growth. In low glucose, miR-451 levels decrease, slowing proliferation but enhancing migration and survival. This allows cells to survive metabolic stress and seek out favorable growth conditions. In glioblastoma patients, elevated miR-451 is associated with shorter survival. The effects of miR-451 are mediated by LKB1, which it represses through targeting its binding partner, CAB39 (MO25 alpha). Overexpression of miR-451 sensitized cells to glucose deprivation, suggesting that its downregulation is necessary for robust activation of LKB1 in response to metabolic stress. Thus, miR-451 is a regulator of the LKB1/AMPK pathway, and this may represent a fundamental mechanism that contributes to cellular adaptation in response to altered energy availability.

Gene Expression Profile Identifies Tyrosine Kinase c-Met as a Targetable Mediator of Antiangiogenic Therapy Resistance

Purpose: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts. Experimental Design: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s). Results: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met–activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts. Conclusion: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance. Clin Cancer Res; 19(7); 1773–83. ©2012 AACR.

Hypoxia-induced tumor cell autophagy mediates resistance to anti-angiogenic therapy

While anti-angiogenic therapy was initially greeted enthusiastically by the cancer community, initial successes with this therapeutic modality were tempered by the failure of angiogenesis inhibitors to produce sustained clinical responses in most patients, with resistance to the inhibitors frequently developing. We recently reported that hypoxia increases after the devascularization caused by anti-angiogenic therapy, consistent with the goals of these therapies, but that some tumor cells become resistant and survive the hypoxic insult elicited by anti-angiogenic therapy through autophagy by activating both AMPK and HIF1A pathways. These findings suggest that modulating the autophagy pathway may someday allow anti-angiogenic therapy to fulfill its therapeutic potential. However, further work will clearly be needed to develop more potent and specific autophagy inhibitors and to better understand the regulators of autophagy in malignant cells.

GLUT3 upregulation promotes metabolic reprogramming associated with antiangiogenic therapy resistance

Clinical trials revealed limited response duration of glioblastomas to VEGF-neutralizing antibody bevacizumab. Thriving in the devascularized microenvironment occurring after antiangiogenic therapy requires tumor cell adaptation to decreased glucose, with 50% less glucose identified in bevacizumab-treated xenografts. Compared with bevacizumab-responsive xenograft cells, resistant cells exhibited increased glucose uptake, glycolysis, 13C NMR pyruvate to lactate conversion, and survival in low glucose. Glucose transporter 3 (GLUT3) was upregulated in bevacizumab-resistant versus sensitive xenografts and patient specimens in a HIF-1α-dependent manner. Resistant versus sensitive cell mitochondria in oxidative phosphorylation-selective conditions produced less ATP. Despite unchanged mitochondrial numbers, normoxic resistant cells had lower mitochondrial membrane potential than sensitive cells, confirming poorer mitochondrial health, but avoided the mitochondrial dysfunction of hypoxic sensitive cells. Thin-layer chromatography revealed increased triglycerides in bevacizumab-resistant versus sensitive xenografts, a change driven by mitochondrial stress. A glycogen synthase kinase-3β inhibitor suppressing GLUT3 transcription caused greater cell death in bevacizumab-resistant than -responsive cells. Overexpressing GLUT3 in tumor cells recapitulated bevacizumab-resistant cell features: survival and proliferation in low glucose, increased glycolysis, impaired oxidative phosphorylation, and rapid in vivo proliferation only slowed by bevacizumab to that of untreated bevacizumab-responsive tumors. Targeting GLUT3 or the increased glycolysis reliance in resistant tumors could unlock the potential of antiangiogenic treatments.

Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer

Significance Invasion is a major cause of cancer mortality, as exemplified by metastatic spread of peripheral malignancies or local intracranial invasion of glioblastoma. While individual mediators of invasion are identified, functional or structural interactions between these mediators remain undefined. We identified a structural cross-activating c-Met/β1 integrin complex that promotes breast cancer metastases and invasive resistance of glioblastoma to the antiangiogenic therapy bevacizumab. We show that tumor cells adapt to their microenvironmental stressors by usurping c-Met and β1 integrin, with c-Met displacing α5 integrin from β1 integrin to form a c-Met/β1 complex with far greater fibronectin affinity than α5β1 integrin. These findings challenge conventional thinking about integrin–ligand interactions and define a molecular target for disrupting metastases or invasive oncologic resistance. The molecular underpinnings of invasion, a hallmark of cancer, have been defined in terms of individual mediators but crucial interactions between these mediators remain undefined. In xenograft models and patient specimens, we identified a c-Met/β1 integrin complex that formed during significant invasive oncologic processes: breast cancer metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/β1 complex formation through an engineered inducible heterodimerization system promoted features crucial to overcoming stressors during metastases or antiangiogenic therapy: migration in the primary site, survival under hypoxia, and extravasation out of circulation. c-Met/β1 complex formation was up-regulated by hypoxia, while VEGF binding VEGFR2 sequestered c-Met and β1 integrin, preventing their binding. Complex formation promoted ligand-independent receptor activation, with integrin-linked kinase phosphorylating c-Met and crystallography revealing the c-Met/β1 complex to maintain the high-affinity β1 integrin conformation. Site-directed mutagenesis verified the necessity for c-Met/β1 binding of amino acids predicted by crystallography to mediate their extracellular interaction. Far-Western blotting and sequential immunoprecipitation revealed that c-Met displaced α5 integrin from β1 integrin, creating a complex with much greater affinity for fibronectin (FN) than α5β1. Thus, tumor cells adapt to microenvironmental stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, movement toward concentrations of environmental chemoattractants, and haptotaxis, movement controlled by the relative strengths of peripheral adhesions. Tumor cells then redirect these receptors away from their conventional binding partners, forming a powerful structural c-Met/β1 complex whose ligand-independent cross-activation and robust affinity for FN drive invasive oncologic processes.

Abstract 2312: Targeting beta1 integrin potentiates antiangiogenic therapy and inhibits growth of bevacizumab-resistant glioblastoma multiforme.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC INTRODUCTION: Antiangiogenic therapies like bevacizumab hold promise for cancer treatment. However, the development of acquired resistance, including an aggressive mesenchymal phenotype, limit utility of these agents. We previously published a report in which microarray and PCR analysis revealed beta1 integrin to be upregulated in bevacizumab-resistant glioblastomas (BRGs). Because the beta1 integrin subunit mediates tumor-microenvironment interactions, we further investigated its role in mesenchymal-type resistance to anti-angiogenic therapy. METHODS: Immunostaining of BRG specimens and genetic or pharmacologic beta1 targeting in BRG cells and BRG-derived xenografts were used to study the role of beta1 integrin in anti-angiogenic therapy resistance in vitro and in vivo. Cells were transduced to express beta1-GFP fusion protein to measure beta1 integrin turnover in focal adhesions using fluorescence recovery after photobleaching (FRAP). RESULTS: Beta1 integrin and downstream beta1-effector focal adhesion kinase immunostaining were upregulated in 75% and 86% of BRGs, resepectively, compared to pre-treatment paired specimens. Flow cytometry revealed 8-fold upregulation of beta1 integrin in BRG-derived primary GBM cells compared to cells from bevacizumab-naive GBMs (P<0.05). FRAP revealed more rapid beta1 integrin turnover in focal adhesion kinases in BRG-derived cells than in bevacizumab-naive GBM cells, with 4-fold more time to achieve 37% recovery in bevacizumab-naive GBM cells than in BRG-derived cells expressing a beta1-GFP fusion protein (95 versus 398 seconds; P<0.05). Hypoxia, which was increased 80% (as evidenced by CA9 immunostaining) (P<0.05) after bevacizumab resistance, increased beta1 integrin expression 30% in cultured bevacizumab-naive GBM cells (P<0.01). Two hours of incubation with 10 and 100 ng/mL VEGF reduced adhesion of GBM cells to beta1 ligands fibronectin, collagen IV, and laminin by 10-50% in a dose-dependent fashion (P<0.05). BRGs demonstrated aggressive mesenchymal-like phenotype in vitro and growth of subcutaneous BRG xenografts was attenuated by beta1 antibody OS2966 (P<0.05). Addition of OS2966 allowed 20-fold dose reduction of bevacizumab per cycle in subcutaneous GBM-derived xenografts (P<0.05). Intracranial delivery of OS2966 through osmotic pumps in mice carrying intracranial BRG-derived xenografts over 28 days increased tumor cell apoptosis, decreased tumor cell invasiveness, and altered tumor cell morphology (P<0.05). CONCLUSIONS: Beta1 integrin expression and turnover in focal adhesion kinases is upregulated in BRGs due to 2 possible mechanisms - hypoxic upregulation and bevacizumab-induced VEGF depletion eliminating VEGF-mediated inhibition of beta1 activity. Beta1 inhibition is well tolerated in vivo and holds promise for disrupting anti-angiogenic therapy resistance. Citation Format: W. Shawn Carbonell, Michael De Lay, Arman Jahangiri, Catherine C. Park, Manish Aghi. Targeting beta1 integrin potentiates antiangiogenic therapy and inhibits growth of bevacizumab-resistant glioblastoma multiforme. [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 2312. doi:10.1158/1538-7445.AM2013-2312

Abstract 1610: Serial treatment-transplant xenograft model of Bevacizumab-resistance reveals temporal induction of markers associated with disease progression.

INTRODUCTION: Acquired resistance threatens the potential of anti-angiogenic therapy. Xenograft models of acquired anti-angiogenic therapy resistance are lacking and often fail to reflect changes human tumors acquire during prolonged serial treatment with anti-angiogenic therapy because of the significantly shorter lifespan of tumor-bearing mice. To create a xenograft model of bevacizumab-resistant GBM allowing temporal definition of changes during resistance to VEGF blockade, we created a model whereby serially passaged xenografts were continually exposed to bevacizumab versus IgG control antibody over 5 generations in vivo. METHODS: We treated 5 subcutaneous U87 glioma cell line-derived xenografts with bevacizumab or IgG, explanted the least responsive xenograft and reimplanted it subcutaneously, then repeated this pattern of treatment followed by explantation and reimplantation of the least responsive xenograft for 4 more generations, generating 5 generations of U87-IgG and U87-BevR xenografts. Western blot assessed temporal changes. RESULTS: Subcutaneous fifth generation U87-BevR were unresponsive to bevacizumab versus IgG (P=0.8). Bevacizumab caused U87-IgG to regress (P CONCLUSIONS: Our novel model of acquired anti-angiogenic therapy resistance showed that acquired resistance increases parenchymal and perivascular invasion with some changes (increased c-Met, VEGFR2, and phosphorylated STAT3) occurring early and lasting throughout resistance, while other changes like alpha5beta1 integrin upregulation peaked later and decreased over time. The ability of this model to replicate prolonged anti-angiogenic therapy duration seen in patients should generate a molecular profile of anti-angiogenic therapy resistance, and should define which resistance-associated changes are driver versus passenger alterations. Citation Format: Arman Jahangiri, Michael De Lay, W. Shawn Carbonell, Liane Miller, Manish K. Aghi. Serial treatment-transplant xenograft model of Bevacizumab-resistance reveals temporal induction of markers associated with disease progression. [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 1610. doi:10.1158/1538-7445.AM2013-1610