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. 2015 AACR.

Introduction In addition to its ligand-dependent signaling role, recent studies 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) 2015 American Association for Cancer Research. using a lentiviral system.

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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 were euthanized. following knockdown of b5 integrin in glioblastomas, nor was senescence induced following b3 knockdown for multiple breast, Tumor spheroids colorectal, lung, and pancreatic carcinomas (Fig. 2D). Together, 5 Multicellular spheroids were prepared by seeding 10 cells per these findings suggest that b3, but not b5, may function as a 24-well precoated with heated 1% Seaplaque agarose (Lonza) in suppressor of senescence in glioblastoma cells. Indeed, increased serum-free medium. After 7 to 10 days, spheroids were collected, SA-b-gal was observed for sh-b3 U87MG subcutaneous tumors 7 fi fi xed in 4% paraformaldehyde, and embedded in paraf n. days after implantation in mice (Fig. 2D), providing a snapshot of senescence that precedes the eventual exponential growth of b SA- -galactosidase staining control tumors (Fig. 1C). Cell senescence was measured by the Senescence b-Galactosi- avb3 ligand-binding function appears relevant for its suppres- dase Staining Kit (Cell Signaling Technology). sion of senescence, as a b3-D119A mutant unable to bind ligand could not rescue the b3-knockdown phenotype (Supplementary Immunofluorescence microscopy Fig. S3A). Furthermore, treatment with the function blocking Cells on glass coverslips were fixed with 4% paraformaldehyde avb3 antibody LM609 induced senescence in U87MG glioblas- and processed for immunofluorescence as previously described toma cells (Supplementary Fig. S3B). Together, these findings (6), for imaging on a Nikon Eclipse C1 confocal microscope with suggest that integrin avb3 may serve a specialized function in 1.4 NA 60x oil-immersion lens. Antibodies include integrin avb3 glioblastoma cells to support tumor growth by suppressing path- (LM609), p21, phospho-PAK4, and pan-methyl-histone H3 ways governing cellular senescence. (Lys9; D54; Cell Signaling Technology). avb3 allows glioblastoma cells to evade senescence by Statistical analysis suppressing p21 One-way ANOVA or t tests with P < 0.05 were performed using Senescence is a process triggered by cellular stress, leading to a Prism software (GraphPad). permanent withdrawal from the cell cycle. Exposing cells to DNA

OF2 Cancer Res; 75(21) November 1, 2015 Cancer Research

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3 3 Orthotopic braintumors(U87MGglioblastoma) Tumor volume (mm ) Tumor volume (mm ) (size) 1,000 1,500 2,000 2,500 3,000 3,500 4,000 100 200 300 400 500 600 a v 500 nvitro in 0 b Published OnlineFirstAugust21,2015;DOI:10.1158/0008-5472.CAN-15-0988 mar lolsoagrowth glioblastoma impairs 3 0 0102030 0 PANC1 (pancreatic) in vitro(U87MGglioblastoma) a sesdb rUdicroain n xrse sfl-ifrnerltv ocnrls/hN ( si/shRNA control to relative fold-difference as expressed and incorporation, BrdUrd by assessed was U87MG (GBM) cancerres.aacrjournals.org Blue: nuclearmarker Brown: Ki-67 sh- sh-CTRL 100 μm sh-β3 sh-CTRL nvitro in Days Days β3 (proliferation) n ¼ Ki-67 n n ppoi eeasse sn immunostaining using assessed were apoptosis and , s3 peod o 0dy.Shri rafrpaecnrs mgswsmaue npxl ( pixels in measured was images phase-contrast for area Spheroid days. 10 for spheroids 3D as ¼ 10, 5 – 2ec ru) ,integrin D, group). each 12 , Total efficiency x106 (cm2) P 5040302010 20 40 60 < 0 .0 o uvvlcre.C ua uo el eegona uctnostmr nnd ie Tumor mice. nude in tumors subcutaneous as grown were cells tumor human C, curve). survival for 0.005 sh-CTRL nvitro in Blue: nuclearmarker Brown: p-HH3 Tumor volume (mm3) Tumor volume (mm3) 1,000 1,000 p-Histone H3 (mitotic index) 200 400 600 800 200 400 600 800 and 0 sh- 0 * 0 1020300 β3 nvivo in on October 2,2021. ©2015 American Association forCancer LN229 (GBM) Research. A549 (lung) b ,U7Ghmngibatm el rninl xrsigsRAaantintegrin against siRNA expressing transiently cells glioblastoma human U87MG A, . a rninl sRA rsal sRA nce oni ua acrcl ie and lines cell cancer human in down knocked (shRNA) stably or (siRNA) transiently was 3 sh- sh-CTRL Days sh- sh-CTRL

Days Percent survival al nimncmrmsdmc n uo rwhmntrduigIVIS20 using monitored growth tumor and mice immunocompromised in ially 100 150 β3 50 Blue: nuclearmarker Brown: cleavedcaspase-3 β Cleaved caspase-3 0 3 (apoptosis) sh-β3 (P<0.005) sh-CTRL sh-β5 (P=0.03) 80604020 Time (days) o i6,pHsoeH,adcevdcaspase-3, cleaved and H3, p-Histone Ki-67, for D lolsoa vi eecnevaIntegrin via Senescence Avoid Glioblastomas BrdUrd incorporation Events counted per field

β Cell proliferationinvitro (si/sh- 3 vs. CTRL) 10 20 30 40 50 60 70 80604020 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Area Pancreatic * Epithelial PANC1 cancers acrRs 52)Nvme ,2015 1, November 75(21) Res; Cancer 7860 Renal si-CTRL A549 Lung % Ki-67+ * MDAMB231 Breast * * n Glioblastoma and medulloblastoma U87 (si) GBM ¼ si- * % pHH3+ β3 3 U87 (sh) GBM þ * * n GBM ahgroup; each LN229 ¼ si- * 6, U251 GBM β5 epciey( respectively * % Casp3+

, U373 GBM P *

< DAOY Medullo. 0.0001 , 0imaging P < n a 0.01). ¼ v b 9; 3 OF3 Published OnlineFirst August 21, 2015; DOI: 10.1158/0008-5472.CAN-15-0988

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A Cell-cycle analysis B Histone methylation

% G0/G1 % S % G2 100% U87MG (GBM) LN18 (GBM) β β 20 16 19 16 19 18 19 sh-CTRL sh- 3 si-CTRL si- 3 26 26 22 80% 21 20 28 15 26 26 24 34 60% 40 41

40% 62 64 59 56 56 56 % Cells per phase 51 48 20% 40 37

5 μm si-β3 si-β3 si-β3 si-β3 Green: MeH3K9 sh-β3 Blue: Hoechst nuclear dye si-CTRL si-CTRL si-CTRL si-CTRL sh-CTRL U87 (si) U87 (sh) LN18 LN229 U373

CDDNA double-strand breaks Senescence-associated β-galactosidase staining (in vitro) U87MG si-CTRL si-β3 U87MG LN18 70 14 si-b3 60 ** 12 si-b5 50 cells + nuclei

+ 40 10

sh-CTRL 30 8 20 β Blue: SA- -galactosidase μ % γ H2AX 100 m 10 6 Medullo. 0 3 3 4 β 3 si- β sh- β Breast Colon Lung Lung Pancreatic Renal 2 Breast (fold relative to si-CTRL) si-CTRL sh-CTRL Number of SA- β -Gal sh/si- U87 LN18 0 GBM GBM H23 7860

μ A549 U118 U251 U373 BT20 Green: γ-H2AX 10 m LN18 DAOY LN229 Panc-1 SW620 Blue: Hoechst nuclear dye U87MG MDA-MB-231 Glioblastoma and Epithelial cancers medulloblastoma

Senescence-associated β-galactosidase staining (in vivo) sh-CTRL

#7 #8 #9 sh- β 3 U87MG flank tumors (7 days)

#4 #5 #6

Blue: SA-β-galactosidase 100 μm

Figure 2. b3 knockdown induces senescence in glioblastoma cells. The effects of siRNA- or shRNA-mediated knockdown of b3 expression were compared in multiple glioblastoma and epithelial cancer cell lines in vitro. A, cell-cycle arrest in glioblastoma cell lines was analyzed using flow cytometry. B, the presence of senescence-associated heterochromatin foci was evaluated by immunofluorescence staining for pan-methylated Histone H3K9 (green). Cell nuclei were counterstained with Hoechst dye (blue; U87, n ¼ 10 fields; , P < 0.0001; LN18, n ¼ 5 fields; , P < 0.05). C, DNA DSBs were detected using immunofluorescence staining for g-HA2X (green). Cell nuclei were counterstained with Hoechst dye (blue). D, senescence-associated b-galactosidase staining was used to identify the frequency of senescent cells in vitro or in vivo (n ¼ 5 fields counted per group in at least two independent experiments).

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damage/attrition, oxidative stress, or oncogene activity drives Discussion antiproliferative signaling through regulatory proteins such as Whereas senescence disables proliferation of damaged cells p16, p21, p27, p53, and Rb that dictate whether a cell will senesce during development and aging, it also occurs in cells that have in response to the assault (13). Silencing of b3 induced a marked acquired a single activated oncogene (18). Oncogene-induced increase in p21 protein expression for multiple glioblastoma cells, senescence locks cells into a permanent state of arrest to prevent whereas p21 levels were unchanged or even decreased following uncontrolled proliferation and propagation of mutations (19). b3 knockdown in various epithelial cancer cells (Fig. 3A and B). During this process, tumors acquire characteristic changes in Knockdown of p21 rescued glioblastoma cells from senescence in tumor-suppressor genes that collectively halt cell-cycle progres- the absence of b3, pointing to its requirement for the observed sion, including activation of p53 and induction of p21 or p16. phenotype (Fig. 3C). Notably, other mediators of senescence such Progression to malignancy occurs when these cells alter gene as p27 (CDKN1B) and p53 (TP53) did not show consistent expression or acquire additional mutations to evade senescence. changes in response to loss of b3, and senescence was not We propose that acquisition of avb3 during progression to hindered by p53 knockdown in U87MG or LN229 glioblastomas malignancy provides glioblastomas a means to escape from (Fig. 3A and C). Together, these results implicate integrin avb3in senescence. This concept is supported by the fact that increased the protection of glioblastomas from senescence via its impact expression of avb3 and its ligand vitronectin are associated with on p21. glioblastoma progression (5). fi PAK4 implicated as mediator of senescence downstream from Our ndings raise the question of how glioblastomas and avb3 medulloblastomas develop an addiction to b3/PAK4. avb3is Integrin clustering on the cell surface regulates formation of absent on quiescent blood vessels, but becomes highly expressed focal adhesions and assembly of signaling complexes that, in on invasive endothelial cells during angiogenic remodeling (14). turn, activate various signaling cascades (14). Accordingly, Although b3 has been linked to metastasis in epithelial cancers phosphorylation of FAK (focal adhesion kinase, PTK2) and (6, 7), glioblastomas typically show high b3 expression once p130 CAS (Crk-associated substrate, BCAR1) was both tumors have become locally invasive (4), and PAK4 expression increased in b3-knockdown cells (Fig. 4A), consistent with tracks with glioma grade (20). It is, therefore, tempting to consider enrichment of focal adhesions in senescence (15). Knockdown that a major function of increasing avb3 during glioblastoma of b3, but not b5, selectively reduced Serine 474 phosphory- progression may be to continuously suppress oncogene-induced lation of PAK4 (Fig. 4A), a type II p21-activated kinase. Serine senescence to permit tumor progression. fi – 474 in the PAK4 kinase domain can be autophosphorylated or Our ndings also point to a b3 PAK4 interaction as a glio- fi phosphorylated by a number of different kinases to constitute blastoma-speci c addiction pathway. PAK proteins, serine/thre- – – the "active" form of PAK4 (16, 17). PAK4 appears to play a onine p21 activated kinases that are substrates of the small GTP functional role in suppressing senescence in glioblastoma, binding proteins Cdc42 and Rac, are important for cell migration because treating cells with the ATP-competitive PAK4 inhibitor and proliferation. In glioblastoma cells, avb3 associates with PF-03758309 increased SA-b-gal staining in U87MG cells in 2D activated PAK4 but not other PAK family members, and this culture and subcutaneous xenografts in vivo (Fig.4B).This interaction functions to suppress p21-dependent senescence. activity was observed for additional glioblastoma and medul- Although epithelial cancers can express both avb3 and PAK4, loblastoma cell lines, but not for epithelial carcinomas (Fig. they do not colocalize, which may explain why b3 knockdown fl 4B). Like knockdown of b3 itself, loss of PAK4 expression fails to in uence senescence. induced p21 expression and SA-b-gal staining selectively in Increased expression of avb3 and its ligand vitronectin have glioblastoma and medulloblastoma cells (Fig. 4C). These find- been associated with glioblastoma progression (5). The ligand- ings suggest that b3 activates PAK4 to suppress p21 in a cell- binding function of avb3 appears to be at least partially respon- type–specific manner. sible for its role in suppressing senescence, because the b3-D119A mutant unable to bind ligand did not rescue the b3-knockdown Colocalization between avb3 integrin and phosphorylated senescent phenotype (Supplementary Fig. S3), and treating PAK4 in glioblastoma U87MG cells with the av b3 function-blocking antibody Although PAK family kinases are well-established effectors of LM609-induced senescence (Supplementary Fig. S3). Despite its integrin signaling, there are likely unappreciated distinctions similarity to b3, knockdown of b5 accelerated orthotopic growth between specificintegrinab heterodimers and PAK family of U87MG glioblastomas. This could have interesting implica- kinase members that could enact specialized functions. We tions for cancer therapy, as the integrin antagonist Cilengitide reasoned that integrin b3mayinfluence the subcellular local- targets both avb3 and avb5. Although Cilengitide was effective in fi ization, and hence activation status of PAK4 in a cell-type– some patients, it did not produce a statistically signi cant survival fi specific manner providing an explanation for its role in sup- bene t in a phase III trial for glioblastoma. A selective inhibitor of pressing senescence in glioblastoma but not epithelial carci- avb3 that does not target avb5 may represent a more appropriate noma cells. Indeed, avb3 and p-PAK4 colocalized within focal targeted therapy for glioblastoma. adhesions in glioblastoma, but not epithelial cancer cells (Fig. We have unveiled a unique vulnerability of glioblastomas to 4D). b3 did not colocalize with phospho-PAK1 in these cells undergo senescence upon interference with b3/PAK4 signaling. (Supplementary Fig. S4), suggesting a specialized interaction Knockdown of b3 or PAK4, or targeting PAK4 with a pharmaco- between b3 and PAK4 in glioblastoma cells. These findings may logic inhibitor, induced p21-dependent senescence in glioblas- explain how avb3functionsspecifically via PAK4 in GBM tomas and medulloblastomas, while producing no effect for a and perhaps other brain tumors to protect them from range of histologically distinct epithelial cancers. Future studies – senescence. could delineate why b3 PAK4 interact only in glioblastomas to

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A Glioblastomas Epithelial cancers A549 PANC1 MB231 7860 U87MG LN18 LN229 Lung Pancreatic Breast Kidney sh/si: CTRL β3 CTRL β3 CTRL β3 sh: CTRL β3 CTRL β3 CTRL β3 CTRL β3

β3 β3

p21 p21

p27 Actin

p-Rb

p-p53

p53

Actin

B LN229 (glioblastoma) MDA-MB-231 (breast cancer) sh-CTRL sh- β 3

Red: p21 50 μm Blue: Hoechst nuclear dye

40 35 C U87MG 30 si-CTRL si-p21 si-p53 cells

+ 25 20 * 15 * 10 sh-CTRL % SA- β -Gal 5 0 β 3 CTRL CTRL CTRL CTRL si-p53 si-p21 si-p53 si-p21 si-p53 si-p21 si-p21 si-p53

sh- sh-CTRL sh-β3 sh-CTRL sh-β3 U87MG LN229 Blue: SA-β-galactosidase 100 μm Glioblastoma

Figure 3. Integrin avb3 suppresses p21 expression in glioblastoma cells. A, the effect of b3 knockdown on expression and phosphorylation of p21, p27, Rb, and p53 was evaluated using immunoblotting. B, immunofluorescence staining for p21 (red) confirmed accumulation in glioblastoma cells with b3 knockdown compared with sh-CTRL. C, siRNA-mediated knockdown of p21 but not p53 significantly reduced the percentage of SA-b-galactosidase–positive cells (blue) in glioblastoma cells with b3 knockdown (n ¼ 5 fields counted per group in at least two independent experiments; , P < 0.01).

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A B PAK4 inhibitor (PF-03758309) C PAK4 knockdown U87MG (GBM) shRNA: CTRL β3 β5 U87MG glioblastoma 2D culture Flank tumors p-FAK GBM Renal LN229 7860 FAK si/sh: CTRL PAK4 CTRL PAK4

p-CAS Vehicle PAK4

CAS p21

p-PAK1,2 β3

PAK2 Actin PAK inhibitor PF-03758309 p-PAK4,5,6 β Blue: SA- -galactosidase 50 μm PAK4 Glioblastoma and Glioblastoma and p-CRAF medulloblastoma Epithelial cancers medulloblastoma Epithelial cancers 80 40 Vehicle * si-CTRL RAF 60 * *

cells 30 cells PAK inhibitor si-PAK4 + + * p-Src 40 20 *

Src 20 10 % SA- β -Gal % SA- β -Gal 0 Actin 0 U87 LN229 DAOY 7860 MB231 PANC1 LN229 DAOY 7860 MB231 PANC1 GBM GBM Medullo. Renal Breast Pancreatic GBM Medullo. Renal Breast Pancreatic

D DAOY (medullo.) LN229 (GBM) Merge pPAK4 α v β 3

7860 (renal) PANC1 (pancreatic) Merge

α β

pPAK4 Green: v 3 Red: p-PAK4 Blue: Hoechst nuclear dye

α v β 3 20 μm

Figure 4. avb3 expression is required for PAK4 phosphorylation and evasion of senescence in glioblastoma. A, the effect of b3 versus b5 knockdown in U87MG cells on focal adhesion-associated proteins FAK, CAS, PAK, RAF, and Src was evaluated using immunoblotting. B, pharmacologic inhibition of PAK using PF-03758309 caused cell senescence in vitro (10–1,000 nmol/L) and in vivo (25 mg/kg/d) as measured by SA-b-Gal staining for glioblastomas, but not epithelial carcinomas. C, knockdown of PAK4 selectively induced p21 expression and SA-b-gal staining in glioblastoma cells (n ¼ 5 ﬁelds per group; , P < 0.001). D, integrin avb3 (green) and phosphorylated PAK4 (red) colocalized in glioblastoma and medulloblastoma cells, but not epithelial carcinoma cells. www.aacrjournals.org Cancer Res; 75(21) November 1, 2015 OF7

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account for the observed addiction to these signaling molecules. It Acquisition of data (provided animals, acquired and managed patients, will also be important to validate the function of this pathway in provided facilities, etc.): A. Franovic, L. Seguin, M.F. Camargo more sophisticated models of glioblastoma and medulloblasto- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Franovic, L. Seguin, S.M. Weis, D.A. Cheresh ma, including patient-derived xenografts or genetically engi- Writing, review, and/or revision of the manuscript: A. Franovic, S.M. Weis, neered mouse models of these cancers. A better understanding D.A. Cheresh of the signaling pathway triggered by b3–PAK4 may provide novel Administrative, technical, or material support (i.e., reporting or organizing therapeutic strategies to exploit the addiction to these genes for data, constructing databases): K.C. Elliott the treatment of glioblastoma in the clinic. Study supervision: A. Franovic, D.A. Cheresh

Disclosure of Potential Conﬂicts of Interest Grant Support No potential conﬂicts of interest were disclosed. This work was supported by the NIH/NCI (R01 CA45726 and R01 CA168692 to D.A. Cheresh) and the Canadian Institutes of Health Research (Post-doctoral Fellowship to A. Franovic). Authors' Contributions Conception and design: A. Franovic, S.M. Weis, D.A. Cheresh Received April 16, 2015; revised July 2, 2015; accepted July 25, 2015; Development of methodology: A. Franovic, K.C. Elliott published OnlineFirst August 21, 2015.

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OF8 Cancer Res; 75(21) November 1, 2015 Cancer Research

Glioblastomas Require Integrin αvβ3/PAK4 Signaling to Escape Senescence

Aleksandra Franovic, Kathryn C. Elliott, Laetitia Seguin, et al.

Cancer Res Published OnlineFirst August 21, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-0988

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2015/08/20/0008-5472.CAN-15-0988.DC1

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