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Glioblastomas Require Integrin Avb3/PAK4 Signaling to Escape Senescence Aleksandra Franovic1,2, Kathryn C

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Glioblastomas Require Integrin Avb3/PAK4 Signaling to Escape Senescence Aleksandra Franovic1,2, Kathryn C Published OnlineFirst August 21, 2015; DOI: 10.1158/0008-5472.CAN-15-0988 Cancer Priority Report Research Glioblastomas Require Integrin avb3/PAK4 Signaling to Escape Senescence Aleksandra Franovic1,2, Kathryn C. Elliott1,2, Laetitia Seguin1,2, M. Fernanda Camargo1,2, Sara M. Weis1,2, and David A. Cheresh1,2 Abstract Integrin avb3 has been implicated as a driver of aggressive and cells did not exhibit a similar requirement for either other integrins metastatic disease, and is upregulated during glioblastoma pro- or additional PAK family members. Moreover, avb3/PAK4 depen- gression. Here, we demonstrate that integrin avb3 allows glioblas- dence was not found to be critical in epithelial cancers. Taken toma cells to counteract senescence through a novel tissue-specific together, our findings established that glioblastomas are selectively effector mechanism involving recruitment and activation of the addicted to this pathway as a strategy to evade oncogene-induced cytoskeletal regulatory kinase PAK4. Mechanistically, targeting senescence, with implications that inhibiting the avb3–PAK4 either avb3 or PAK4 led to emergence of a p21-dependent, p53- signaling axis may offer novel therapeutic opportunities to target independent cell senescence phenotype. Notably, glioblastoma this aggressive cancer. Cancer Res; 75(21); 1–8. Ó2015 AACR. Introduction In addition to its ligand-dependent signaling role, recent stud- ies suggest that avb3 has noncanonical cell biologic functions that Glioblastoma multiforme, or GBM, is the most aggressive and are ligand independent (6, 7, 11). Because avb3 expression malignant form of astrocytoma characterized by highly invasive correlates with glioblastoma progression, we silenced b3ina tumor cells. Although these tumors are treated using a combina- variety of human glioblastoma cells and assessed their growth in tion of surgery, radiotherapy, and chemotherapy, only 5% of vivo and in vitro to evaluate the net contribution of this integrin's patients survive for longer than 5 years after diagnosis. Large-scale ligand-dependent and -independent functions to glioblastoma efforts have recently provided new clues into gliomagenesis and biology. To our surprise, glioblastoma cells demonstrated an alterations that characterize this disease (1). Despite identification addiction to avb3 as a means to avoid p21 (CDKN1A)-dependent of new biomarkers and important molecular pathways (2), tar- cellular senescence, whereas b3 knockdown did not trigger this geted therapies have not yet elicited durable clinical responses (3). effect in a range of histologically distinct epithelial cancers. Loss of The expression of integrin avb3 (ITGAV/ITGB3) and its ligand avb3 led to a concomitant decrease in PAK4 activation, whereas vitronectin increases during the transition from low-grade astro- PAK4 knockdown increased p21 and senescence. These findings glial-derived tumors to advanced glioblastoma (4, 5), and we and reveal a new cell type-specific function for integrin avb3, and others have identified avb3 as a driver of an aggressive and highlight a particular vulnerability of glioblastoma cells for com- metastatic tumor phenotype (6, 7). In GBM biopsy samples, avb3 ponents of this pathway. expression is prominent in both tumor microvessels and glial tumor cells, and is the most prevalent in highly proliferating and Materials and Methods infiltrating areas (8). Cilengitide, designed to target the ligand- binding properties of avb3 and other av integrins (9), was tested Cell lines and regents in combination with temozolomide chemoradiotherapy in a All cells were purchased from the ATCC within the past 5 years: randomized phase III trial for patients with newly diagnosed Glioblastoma (U87MG, LN229, LN18, U373, U118, and U251), glioblastoma with methylated MGMT promoters (Clinical- medulloblastoma (DAOY), renal (7860), colorectal (SW620), Trials.gov NCT00689221). Although some patients responded, pancreatic (PANC1), breast (MDA-MB-231 and BT20), and lung Cilengitide failed to meet its primary endpoint of a significant (A549 and H23). Cell line authentication was performed by the fi survival advantage (10). ATCC using short tandem repeat DNA pro les. Upon receipt, each cell line was expanded, cryopreserved as low-passage stocks, and tested routinely for Mycoplasma. The ATP-competitive PAK4 inhib- – 1 itor PF-03758309 (Chemieitek) was used at 10 1000 nmol/L Department of Pathology, University of California, San Diego, La Jolla, in vitro in vivo California. 2Moores Cancer Center, University of California, San Diego, and 25 mg/kg/day . La Jolla, California. RNA interference and expression constructs Note: Supplementary data for this article are available at Cancer Research For transient knockdown, cells were transfected using the Online (http://cancerres.aacrjournals.org/). HiPerFect (Qiagen) with AllStars siRNA (Qiagen) for negative Corresponding Author: David A. Cheresh, Moores UCSD Cancer Center, 3855 control (1027280), ITGB3 (SI00004585), ITGB5 (SI02780617), Health Sciences Drive #0803, La Jolla, CA 92093-0803. Phone: 858-822-2232; PAK4 (SI00082341), CDKN1A (SI00008547), or TP53 Fax: 858-822-2630; E-mail: [email protected] (SI00011655). For stable knockdown, cells were infected with doi: 10.1158/0008-5472.CAN-15-0988 shRNA-targeting ITGB3 (Open Biosystems; TRCN0000003234) Ó2015 American Association for Cancer Research. using a lentiviral system. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst August 21, 2015; DOI: 10.1158/0008-5472.CAN-15-0988 Franovic et al. Immunoblotting Results fi Lysates made in 4% SDS were quanti ed using the Pierce BCA a b in vitro fi Glioblastomas depend on integrin v 3 for growth and Kit (Thermo Fisher Scienti c), and 25 to 50 mg protein loaded in vivo onto a denaturing SDS-polyacrylamide gel, transferred to poly- Because we and others have established integrin avb3asa vinylidene difluoride membranes, blotted with horseradish per- mediator of glioblastoma progression (4, 9, 12), we assessed the oxidase–conjugated secondary antibodies (Bio-Rad), and bands effect of b3 knockdown on growth of U87MG human glioblas- detected by enhanced chemiluminescence (Advansta). Antibo- toma cells. U87MG cells with siRNA-mediated knockdown of b3 dies include b3 (Abcam); b-actin (Sigma); FAK-pY861 (Invitro- (si-b3) produced smaller 3D spheroids with less cell proliferation, gen); FAK and p130Cas (BD Trandsduction Laboratories); b5, p21 a lower mitotic index, but no difference in apoptosis compared Waf1/Cip1, p27 Kip1 (D69C12), p53-pS392), p53 (7F5), Rb- with control (si-CTRL) or b5 (ITGB5; si-b5) siRNA (Fig. 1A). pS795, Rb (D20), PAK4, p130Cas-pY410, PAK4-pS474/PAK5- When implanted as intracranial orthotopic xenografts in mice, pS602/PAK6-pS560, PAK1, PAK2, and PAK1-pS144/PAK2-pS141 U87MG cells with stable shRNA-mediated knockdown of b3 (Cell Signaling Technology). failed to form detectable tumors by 75 days, whereas knockdown of b5 accelerated tumor growth and decreased survival time (Fig. Proliferation and cell cycle 1B). This b3 dependence for in vivo tumor growth was also The colorimetric BrdUrd (bromodeoxyuridine) Cell Prolifera- observed for LN229 glioblastoma cells, but not for PANC1 tion Assay Kit (Millipore) was used with absorbance at 450/550 pancreatic or A549 lung carcinoma cells expressing similar levels nm relative to control. Cells were stained using propidium iodide of b3 (Fig. 1C; Supplementary Fig. S1). Furthermore, b3 knock- and subjected to flow-cytometry analysis for cell cycle. down suppressed proliferation of multiple glioblastoma cell lines and the DAOY medulloblastoma cell line, but not b3-positive Animals carcinomas from pancreas, breast, and lung (Fig. 1D). Together, Animal protocols were approved by the UCSD Institutional these findings suggest that glioblastomas and perhaps other brain Animal Care and Use Committee. 6- to 8-week-old female athy- tumors are unique in their requirement for integrin avb3. mic nu/nu mice were purchased from the UCSD Animal Care To understand why b3-knockdown suppressed glioblastoma Program. growth, we examined their growth properties and morphology in vitro. Following b3-knockdown, the cells stopped proliferating Flank tumor xenografts fl 6 well before reaching con uence, yet they remained attached to the Mice were injected s.c. with 10 tumor cells in 200 mL PBS. plate and adopted a large flat appearance consistent with cells Tumor size was measured weekly with calipers. undergoing senescence. Accordingly, silencing b3 but not b5 resulted in G0–G1 arrest with no increase in the sub-G1 population Orthotopic brain tumor xenografts (Fig. 2A; Supplementary Fig. S2), suggesting cell-cycle arrest Mice were anesthetized by intramuscular injection of ketamine, without apoptosis. b3 knockdown also led to enhanced methyl- dexmedetomidine, and buprenorphine. Using a stereotaxic frame ation of histone H3 in senescence-associated heterochromatin (Stoelting Co.), a small burr hole was made in the skull 2 mm foci (Fig. 2B), increased g-H2AX–positive nuclei indicative of anterior and 2 mm lateral to the bregma. A 31-gauge Hamilton DNA double-strand breaks (DSB; Fig. 2C), and accumulation of needle/syringe was inserted 3 mm, and 0.25 mL/min was dis- 5 senescence-associated acidic b-galactosidase (SA-b-gal) activity pensed (10 tumor cells in 2 mL media). Animals were monitored (Fig. 2D). Interestingly, no changes in SA-b-gal were observed daily and those exhibiting signs of morbidity
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