DRR Regulates AKT Activation to Drive Brain Cancer Invasion

ORIGINAL ARTICLE DRR regulates AKT activation to drive brain cancer invasion

A Dudley1, M Sater1,PULe1, G Trinh1, MS Sadr1, J Bergeron1, GF Deleavey2, B Bedell1, MJ Damha2 and K Petrecca1

Glioblastoma (GBM) is the most common and invasive adult brain cancer. The rapid invasion of cancer cells into the normal brain is a major cause of treatment failure, yet the mechanisms that regulate this process are poorly understood. We have identiﬁed a novel mechanism of brain cancer invasion. We show that downregulated in renal cell carcinoma (DRR), which is newly expressed in invasive gliomas, recruits AKT to focal adhesions. This DRR- induced pathological relocalization of AKT bypasses commonly altered upstream signaling events and leads to AKT activation and invasion. We also developed an oligonucleotide therapeutic that reduces DRR expression and prevents glioma invasion in an in vivo preclinical model of the disease. Our ﬁndings identify DRR as a novel GBM target and show that oligonucleotides targeting DRR is a novel therapeutic approach for the treatment of DRR-positive GBMs.

Oncogene (2014) 33, 4952–4960; doi:10.1038/onc.2013.436; published online 21 October 2013 Keywords: brain cancer; glioblastoma; invasion; AKT; DRR

INTRODUCTION localization to the cell membrane can be sufficient for its Glioblastoma (GBM) is the most common adult brain cancer. The activation. median survival with current therapy is 14.6 months.1,2 Cancer cell Downregulated in renal cell carcinoma (DRR) is a recently 21 invasion into the normal brain is a major contributor to this rapid identified driver of glioma invasion. It has been reported to 22–24 treatment failure, yet the mechanisms governing this phenomenon function as a putative tumor suppressor protein. Normally remain poorly understood. expressed in the neurons, DRR has been shown to bundle actin 25 The epidermal growth factor receptor (EGFR)/phosphatidylino- and regulate neurite outgrowth. In the context of gliomas, it is sitol 3-kinase (PI3K)/AKT pathway is a major driver of GBM expressed in both low- and high-grade gliomas but not in normal 21 invasion.3 EGFR activates the lipid kinase PI3K, which then glia. DRR localizes along stress fibers, at focal adhesions (FAs) phosphorylates PI(4,5)P converting it to PI(3,4,5)P . PIP and within the nucleus, and its expression promotes FA 2 3 3 21 (phosphatidylinositol (3,4,5)-triphosphate) in turn binds the turnover. Despite the evidence that DRR participates in glioma pleckstrin homology domain of AKT thereby recruiting it to the invasion, the underlying mechanism is unknown. cell membrane. Once there, AKT is activated by phosphorylation at As the EFGR/PI3K-PTEN/AKT pathway is a driver of GBM invasion Thr308 and Ser473 by 3-Phosphoinositide-dependent protein that is altered in the large majority of GBMs, and DRR is kinase 1 and mammalian target of rapamycin complex 2, overexpressed in invasive gliomas, we tested whether DRR is respectively.4–8 Activated AKT translocates from the membrane involved in this pathway. We found that DRR expression leads to to the cytosol and the nucleus, where it drives downstream elevated AKT activation by recruiting AKT to FAs in a manner pathways affecting cell proliferation, survival, metabolism and dependent on SRC-family kinases (SFKs) and cell adhesion. We invasion.9–11 This cascade can be terminated by phosphatase and also found that reduction of DRR expression using antisense tensin homolog (PTEN), which interrupts PIP3 signaling by oligonucleotides (AOs) prevents glioma invasion in an in vivo 3,8,10 dephosphorylating PIP3 to PIP2. xenograft mouse model. Therefore, our study reveals that DRR is a Oncogenic alterations of EGFR, PIK3CA and PTEN have been novel GBM target and that AOs targeting DRR is a promising new identified in GBM.12 Combined, this pathway is deregulated in therapeutic approach to prevent brain cancer invasion. over 80% of these tumors, and elevated phosphorylated AKT (pAKT) levels are found in 85% of GBMs.12–14 EGFR is thus an attractive therapeutic target, yet clinical trials testing EGFR RESULTS inhibitors in GBM have yielded disappointing results.15–17 DRR induces AKT phosphorylation and this event is independent Importantly, AKT can also be activated independently of of EGFR signaling receptor tyrosine kinase (RTK) or PI3K activity. The viral oncogene The EGFR/PI3K-PTEN/AKT signaling pathway is altered in over 80% v-akt is created by the addition of a myristoylation signal to the of GBMs, and pAKT is elevated in over 85% of GBMs.12–14 In this amino terminus of AKT; this confers constitutive activation to AKT, study, we used the prototypical human GBM-derived U251 cells allowing it to associate with the cell membrane without the need (CTL) as a model to create a stable DRR-overexpressing (DRRov) for upstream RTK or PI3K involvement.18,19 Similarly, a mutation in cell line. We previously characterized these cell lines and found the pleckstrin homology domain of AKT leads to the association of that upon DRR overexpression cells become more invasive and AKT with the cell membrane and constitutive activation in breast, the rate of FA turnover is increased.21 To determine whether DRR colorectal and ovarian cancers.20 Thus, events promoting AKT affects AKT activation, we assessed pAKT levels in both CTL and

1Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada and 2Department of Chemistry, McGill University, Montreal, Quebec, Canada. Correspondence: Dr K Petrecca, Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, 3801 University Ave, Suite 109c, Montreal H3A 2B4, Quebec, Canada. E-mail: [email protected] Received 28 March 2013; revised 2 August 2013; accepted 12 August 2013; published online 21 October 2013 DRR activates AKT to drive brain cancer invasion A Dudley et al 4953 DRRov cells. Gain of DRR expression was associated with also observed a decrease in phosphotyrosine levels in DRRov cells significantly elevated AKT phosphorylation at both the Thr308 after AG1478 treatment, whereas the levels of pAKT did not and Ser473 sites compared with CTL cells, whereas total AKT change (Figure 1e). Gefitinib, while effectively blocking EGF- expression was similar in both cell lines (Figure 1a). pAKT induced EGFR phosphorylation in both cell lines, also did not immunolabeling also revealed significantly elevated pAKT expres- decrease pAKT levels in DRRov cells (Supplementary Figure 1c). sion in DRRov cells compared with CTL cells (Figure 1b). As EGFR Consistent with this observation, we found that EGFR inhibition signaling is altered in 45–55% of GBMs and is a well-characterized did not significantly reduce DRRov cell invasion in a three- activator of the PI3K/AKT pathway under normal and pathophy- dimensional invasion assay (Figures 1f, g). Thus, although siological conditions,12–14 we investigated EGFR protein EGFR/phosphorylated EGFR expression is elevated in DRRov cells, expression and phosphorylated EGFR levels in the context of it is not involved in DRR-induced AKT phosphorylation or cell DRR expression. We found that DRRov cells express significantly invasion. higher levels of EGFR and activated EGFR, as indicated by phosphotyrosine (Figure 1c) and phosphorylated EGFR (Tyr 845) (Supplementary Figure 1a), as well as cell surface EGFR compared DRR-induced AKT phosphorylation is SFK- and integrin-dependent with CTL cells (Figure 1d). We analyzed EGFR mRNA levels by Activating inputs that can lead to AKT phosphorylation include quantitative real-time PCR in DRRov and CTL cells and did not RAS/RAF/MEK/ERK kinases, Rho-GTPases, integrin-linked kinase observe a significant difference between cell lines, suggesting (ILK), SFKs, FA kinase (FAK) and PI3K.14,28–32 We tested each of post-transcriptional regulation (Supplementary Figure 1b). these inputs in order to determine how DRR regulates AKT We used the EGFR kinase inhibitors AG1478 26 and gefitinib27 to activation. determine whether this increased EGFR/phosphorylated EGFR The MEK inhibitor U0126 is a non-ATP competitive inhibitor that expression was responsible for the DRR-induced AKT targets MEK1 and MEK2 and effectively inhibits downstream ERK phosphorylation. We treated CTL cells with EGF and observed phosphorylation33 (see Supplementary Figure 2a). We found that an increase in phosphotyrosine and pAKT levels, which were MEK inhibition did not reduce pAKT levels in DRRov cells inhibited after treatment with EGF in the presence of AG1478. We (Figure 2a).

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Figure 1. Phospho-AKT is elevated in DRR-overexpressing cells and DRR-induced AKT phosphorylation is independent of EGFR signaling. (a) Western blot showing the expression of DRR, Threonine 308 and Serine 473 AKT phosphorylation, total AKT, in DRRov and CTL cells. Tubulin is shown as a loading control. pAKT Ser473 band densitometry indicated. n ¼ 3, DRRov pAKT vs CTL pAKT Po0.05 (t-test). (b) Quantification of pAKT (Ser473) fluorescence intensity in DRRov and CTL cells. Represented as mean±s.e.m with total fluorescence intensity normalized to CTL cells. n ¼ 9, **Po0.0001 (t-test). (c) Western blot showing elevated EGFR and phosphotyrosine (pTyr) levels in DRRov cells compared with CTL cells. EGFR band densitometry indicated. (d) Surface membrane EGFR expression in DRRov and CTL cells. Scale bar ¼ 20 mm. (e) Western blot showing that EGFR inhibition with AG1478 does not reduce pAKT (Ser473) expression levels in DRRov cells. (f) Three dimension (3D) invasion assay of DRRov cells untreated (unt) or treated with AG1478. (g) Quantification of invasion. Represented as mean±s.e.m. n ¼ 3, P40.05 (ANOVA). EGFR inhibition does not significantly reduce DRR-induced cell invasion.

& 2014 Macmillan Publishers Limited Oncogene (2014) 4952 – 4960 DRR activates AKT to drive brain cancer invasion A Dudley et al 4954 DRRov CTL μ DRRov CTL U0126( M) 005 10dmso 00 5 10 dmso EGF -+++-++ + ++ unt C3 unt C3 pAKT 60 pAKT 60 tubulin 55 tubulin 55

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DRRov DRRov CTL 30min 1.5hr LY294002(μM) 0051020dmso 00 510 20 dmso EGF - +++-++ + ++++ unt FN FN+LY unt FN FN+LY pTyr 175 pAKT 60 60 pAKT 60 AKT pFAK 125 60 AKT FAK 125 tubulin 55 tubulin 55 (pAKT) 1.01.0 0.3 Figure 2. DRR-induced AKT phosphorylation is SFK and adhesion-dependent. western blots showing (a) pAKT (Ser473) levels in DRRov and CTL cells untreated or treated with MEK inhibitor U0126 or with dimethyl sulfoxide (DMSO) in the absence or presence of EGF. (b) pAKT (Ser473) levels in response to the Rho inhibitor, C3 transferase, in DRRov and CTL cells. (c) pAKT (Ser473) levels in DRRov cells transfected for 48 or 72 h with small interfering RNA (siRNA) (si) against ILK or control scrambled siRNA (scr). (d) pAKT (Ser473) and total AKT levels in response to the SFK inhibitor PP2 and its inactive analog PP3 in DRRov cells. pAKT band densitometry indicated. n ¼ 3, PP2 vs unt Po0.05 (t-test). PP3 vs unt P40.05 (t-test). (e) pAKT (Ser473) and AKT levels in response to PP2 treatment in DRRov and CTL cells seeded on ﬁbronectin (FN) for 30 min or 4 h. pAKT band densitometry indicated. n ¼ 3, pAKT DRRov FN þ PP2 vs DRRov unt or DRRov FN Po0.05 (analysis of variance, ANOVA, Bonferroni). (f) pFAK and pAKT (Ser473) levels after FAK inhibition with PF-228 (PF). (g) pAKT (Ser473) levels in response to PI3K inhibition with LY294002 in the absence or presence of EGF. pTyr. (h) pAKT (Ser473) levels in response to LY294002 (LY) in DRRov cells seeded on ﬁbronectin for 30 min or 1.5 h. pAKT band densitometry indicated. n ¼ 3, pAKT DRRov FN þ LY vs DRRov unt or DRRov FN Po0.05 (ANOVA, Bonferroni). For all western blots, tubulin is shown as a loading control.

Rho-family GTPases are involved in AKT regulation and also act (Figure 2d). We next seeded DRRov cells on the integrin ligand downstream of AKT to regulate cytoskeletal dynamics and cell fibronectin in order to generate physiologically relevant FAs. In movement.29,34–36 Application of the Rho A, B and C inhibitor, this culture environment, we treated DRRov cells with the SFK exoenzyme C3 transferase, to DRRov cells predictably altered inhibitors PP2 (10 mM) (Figure 2e) and SU6656 (5 mM) cytoskeletal architecture and cell morphology by reducing stress (Supplementary Figure 3a) and observed reduced pAKT levels; fiber levels and collapsing cell structure (see Supplementary suggesting that SFKs are regulating DRR- induced AKT activation Figure 2b). However, this treatment did not affect pAKT levels in a cell-matrix adhesion-dependent manner. (Figure 2b). Activation of FAK is an early event following adhesion, is ILK operates between integrins and RTKs and is an important dependent upon SFK activity and leads to AKT phosphoryla- activator of AKT.31,37 ILK expression was abolished in DRRov cells tion.38,39 To test FAK involvement in AKT phosphorylation, we using an ILK-targeted small interfering RNA oligonucleotide and blocked FAK activity by treating DRRov cells with the FAK inhibitor was unaltered after transfection with a scrambled sequence PF-228 for 1 h before seeding cells on fibronectin. This treatment (Figure 2c). However, silencing ILK expression did not decrease effectively prevented the phosphorylation of FAK at Y397, yet pAKT levels (Figure 2c). pAKT levels were not reduced (Figure 2f), suggesting that DRR- SFKs are well-characterized effectors of integrin signaling and induced AKT phosphorylation occurs independently of FAK are key regulators of FA dynamics.38 Application of the SFK activity. inhibitor PP2 (5 mM) to DRRov cells reduced pAKT levels, whereas To determine whether PI3K is involved, we treated DRRov cells the inactive analog PP3 had no significant effect on pAKT levels with the PI3K inhibitors LY294002 (5, 10 and 20 mM) and

Oncogene (2014) 4952 – 4960 & 2014 Macmillan Publishers Limited DRR activates AKT to drive brain cancer invasion A Dudley et al 4955 wortmannin (100 nM). Although AKT phosphorylation was effec- whether reduction of DRR expression could prevent brain cancer tively inhibited in CTL cells after treatment with both inhibitors, invasion in vivo. Direct application of AOs to brain cancers could neither LY294002 nor wortmannin prevented AKT phosphoryla- be an effective treatment modality.42 We designed AOs targeting tion in DRRov cells (Figure 2g, Supplementary Figure 3b). DRR after having previously shown that malignant glioma cells Following our observation that SFK inhibition of AKT phosphor- expressing negligible levels of DRR displayed decreased invasive ylation was adhesion-dependent, we examined whether PI3K properties in vivo compared with those overexpressing DRR.21 inhibition would be effective in reducing pAKT levels in an GBM stem cell lines cultured in the absence of serum closely adhesion-dependent context. By repeating the experiments on reproduce the pathological features of human GBMs in in vivo 43 fibronectin, we found that even low concentrations (5 mM)of intracranial models. First, we assessed DRR levels in a human LY294002 could decrease AKT phosphorylation (Figure 2h). These GBM stem cell line (hGSC) and found that it was expressed findings reveal the importance of integrin-mediated adhesion in (Figure 5a). Upon treatment with DRR-targeted AOs, we were able DRR-induced AKT phosphorylation. to efficiently knock down DRR levels and observed a simultaneous Integrins interact with the extracellular-matrix through the RGD decrease in pAKT compared with scramble AOs (Figure 5a). These (arginine–glycine–aspartic acid) amino-acid sequence.40 We used findings indicate that DRR regulates AKT signaling in a glioma a modified RGD peptide that competes with the RGD domain of stem cell model and confirm our data from the DRRov cells. the extracellular-matrix for integrin binding to directly test the We extended these findings to an in vivo model. hGSCs treated adhesion dependence we have observed. We treated cells in with Cy5-conjugated control AOs and AOs targeting DRR were suspension with the modified RGD peptide for 30 min before implanted in the striatum of immuno-compromised CD1 mice. The seeding cells on fibronectin. This treatment gave rise to a non- stem cell marker Sox-2 was used to identify all implanted cells, adherent population of cells. In this non-integrin binding cell whereas Cy5 labeling was used to identify tumor cells that had population there was a loss of AKT phosphorylation (Figures 3a, b). been treated with AOs. Whereas cancer cells treated with Cy5- Similarly, AKT was not phosphorylated in DRRov cells that were conjugated control AOs readily invaded the peritumoral brain well maintained in suspension without RGD treatment (Figures 3a, b). beyond the tumor site (Figure 5b upper panels, c), cancer cells treated with anti-DRR AOs did not, but instead remained within the well circumscribed tumor mass (Figure 5b lower panels, c). DRR recruits AKT to FAs These findings show that targeting DRR in vivo is an efficient We have previously showed that within the cytoplasm, DRR is 21 approach to reduce brain cancer invasion and suggest that AOs localized at FAs and along stress fibers. Our results showing that are an effective method for reducing DRR levels. DRR regulates AKT phosphorylation in an adhesion- and SFK- dependent manner suggest that DRR may be recruiting AKT to FAs. We tested this by immunolabeling AKT and pAKT in DRRov DISCUSSION and CTL cells seeded on fibronectin. For each time point studied Our studies identify a novel mechanism for AKT activation and (30 m to 6 h), both AKT and pAKT shared an overlapping invasion in brain cancer. We have also developed a promising expression pattern with vinculin in DRRov cells (Figure 3c). In anti-invasive therapeutic to prevent brain cancer invasion. We contrast, AKT and pAKT were expressed diffusely in the cytoplasm show that DRR, which is newly expressed in invasive gliomas,21 of CTL cells (Figure 3c). recruits AKT to FAs in a SFK- and adhesion-dependent manner. We As both DRR and SFKs regulate FA dynamics, we next tested also show that the inhibition of SFKs, and to a lesser extent PI3K, whether SFKs are involved in DRR-induced AKT recruitment to FAs. prevents AKT phosphorylation. Finally, we show that the reduction DRRov cells seeded on fibronectin and treated with 10 mM PP2 of DRR expression using AOs targeting DRR prevents invasion in a developed FAs containing vinculin, yet pAKT was not observed at xenograft mouse model of the disease. these FAs (Figure 3d). To further explore the possibility that DRR Mutations in AKT that target it to the cell surface, which bypass recruits AKT to FAs, we utilized a mutant form of DRR, DRRDPEPE, 21 upstream signaling events (such as RTK/PI3K activation), can be which does not localize to FAs. We found that when this form of sufficient for full AKT phosphorylation,18 and these alterations DRR is expressed, we did not observe colocalization between have been described in cancers.20 Our findings support a model in pAKT and FAs (Figure 3e), and pAKT levels were not elevated which DRR pathologically relocalizes AKT to FAs in a SFK- (Figure 3f). dependent manner. AKT is phosphorylated at FAs and this elevated level of activated AKT remains detectable for at least SFK and PI3K inhibitors prevent DRR-induced invasion 6 h, suggesting that under these conditions AKT is hyper-active. Having identified a mechanistic link between adhesion, DRR, SFK/ Furthermore, we observed that PI3K inhibition only reduces AKT PI3K activity and AKT, we next tested whether this signaling axis phosphorylation upon integrin activation, suggesting that directly affected the invasive properties of DRRov cells. To this diffusely localized PI3K does not have a role in AKT end, we tested whether SFK or PI3K inhibition could prevent phosphorylation in the presence of DRR. These results support a DRRov cells from invading in a three-dimensional invasion assay. novel mode of AKT activation following its recruitment to integrin- based FAs. We found that treatment of DRRov spheroids with either 10 mM AKT is activated in the large majority of GBMs, and emerging PP2 or LY294002 significantly reduced the invasiveness of these 44 cells at 48 and 96 h (Figures 4a, b), whereas CTL spheroids showed evidence suggests that AKT has a role in GBM invasion. As AKT is significant reductions only at 96 h (Figures 4a, c). We also observed not frequently mutated in GBM, its activation is presumed to be reduced invasion of DRRov spheroids in the presence of LY294002 under the control of upstream RTKs. Our findings that AKT and 5-Fluorouracil, an inhibitor of cell division41 (Supplementary activation is not under the control of EGFR in DRR-positive glioma cells may provide an explanation for the negative results of EGFR Figure 4). This observation suggests that the SFK/PI3K/AKT 45,46 pathway is implicated in DRR-mediated invasion. inhibitors in clinical trials. In contrast, our studies point to DRR as an important molecular player in GBM invasion, making DRR a valuable target for therapeutics as well as a potentially useful Antisense oligonucleotide-mediated DRR ablation prevents biomarker for EGFR-independent brain cancer invasion. invasion in a GBM mouse model Our results highlight the importance of SFKs and integrins as We have shown that DRR regulates AKT phosphorylation to drive targets to prevent GBM invasion. Dasatinib is a potent SRC and invasion, and that treatment with SFK and PI3K inhibitors can SFK inhibitor that has been shown to inhibit migration and significantly reduce this invasion. Our next step was to test invasion of GBM cells.47,48 These data correlate well with our

& 2014 Macmillan Publishers Limited Oncogene (2014) 4952 – 4960 DRR activates AKT to drive brain cancer invasion A Dudley et al 4956 DRRoV

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Figure 3. DRR recruits AKT to FAs. (a) Western blot showing that pAKT (Ser473) levels are reduced in the presence of the integrin blocker RGD. Lane 1-cells grown on FN, lane 2-cells in the non-adherent (non-adh) fraction after RGD treatment, lane 3-cells in suspension without RGD treatment. (b) Quantiﬁcation of pAKT (Ser473) band densitometry corrected for loading control. Represented as mean±s.e.m with values measured relative to the FN condition. n ¼ 3, **Po0.05 (ANOVA, Bonferroni). (c) Co-immunolabeling of AKT (green) or pAKT (green) with the FA marker, vinculin (red) in DRRov and CTL cells grown on FN. (d) Co-immunolabeling of pAKT (green) and vinculin (red) in DRRov cells treated with PP2. (e) Co-immunolabeling of pAKT (green) and vinculin (red) in DRRDPEPE showing that pAKT is no longer localized at FAs. All pAKT immunolabeling is speciﬁc for Ser473. Scale bars ¼ 20 mm. (f) Western blot showing that pAKT (Ser473) is not elevated in cells expressing DRRDPEPE.

Oncogene (2014) 4952 – 4960 & 2014 Macmillan Publishers Limited DRR activates AKT to drive brain cancer invasion A Dudley et al 4957 DRRov CTL 0h 48h 96h 0h 48h 96h

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Invasion distance ( 0 0 48 96 48 96 Time (h) Time (h) Figure 4. SFK and PI3K inhibitors prevent DRR-induced invasion. (a) 3D invasion assays of DRRov and CTL spheroids untreated or treated with 10 mM PP2 or LY294002. Invasion front is demarcated by a dashed circle. (b) Quantiﬁcation of 3D invasion of DRRov spheroids with indicated treatment. Represented as mean±s.e.m. n ¼ 3, **Po0.01, ****Po0.0001 (ANOVA, Bonferroni). (c) Quantiﬁcation of 3D invasion of CTL spheroids with indicated treatment. Represented as mean±s.e.m. n ¼ 3, ***Po0.001, ****Po0.0001 (ANOVA, Bonferroni).

findings and suggest that SFK inhibitors may be useful in Signaling Technology, Cat. nos. 6528 & 6201) (Boston, MA, USA). Anti-EGFR preventing DRR-induced brain cancer invasion. A phase I/II trial (Santa-Cruz Technology, Dallas, TX, USA). Anti-phosphotyrosine, anti-FAK involving patients with newly diagnosed GBM is currently testing (Millipore, Billerica, MA, USA). Anti-phospho-FAK (Tyr397) (Invitrogen, dasatinib combined with radiotherapy and concomitant Burlington, ON, Canada). Anti-a-tubulin, anti-a-vinculin fibronectin from temozolomide, followed by adjuvant dasatinib plus bovine plasma, 5-fluorouracil (Sigma, St Louis, MO, USA). AG1478, U0126, LY294002, wortmannin, PP2, SU6656 PF-228, GRGDSP peptide temozolomide.49,50 The integrin inhibitor Cilengitide is also 51 (Calbiochem, Billerica, MA, USA). Gefitinib (Cayman Chemical, Ann Arbor, being tested as a first-line therapy in GBM. In these trials MI, USA). C3 transferase (Cytoskeleton Inc., Denver, CO, USA). Recombinant progression-free survival and overall survival are primary end human EGF (Invitrogen). PureCol Bovine Collagen Solution Type 1 points. Given our findings, it would be relevant to assess the (Advanced BioMatrix, San Diego, CA, USA). SYBR Green PCR Master Mix extent of tumor invasion on surveillance imaging as another (Roche, Ann Arbor, MI, USA). important biomarker of efficacy. AOs delivered to the resection cavity of GBM patients via Cell culture implanted catheters, thereby bypassing the need for systemic DRRov cells: AmphoPack 293 cells were first transfected with the DRR delivery is currently in Phase II/III clinical trials. Clinical trials testing plasmid pLib (Clontech, Mountain View, CA, USA) using Lipofectamine AOs targeting transforming growth factor b2 have shown reagent according to the manufacturer’s protocol. Forty-eight and seventy- encouraging results.42 We have developed an AO that targets two hours post transfection, retrovirus was collected and added to the DRR expression and prevents brain cancer invasion in a xenograft human GBM-derived U251 cell line. Stable cell lines were selected with G418 media. U251 brain cancer cells and DRR-overexpressing cells were model. As GBM invasion into normal brain is a local event in over 21 52 cultured as previously described. Human GBM stem cells (hGSCs) 85% of patients, delivery of AOs targeting invasion directly into were kindly provided by Dr Samuel Weiss (University of Calgary). hGSCs the resection cavity provides treatment to the most relevant area were isolated as previously performed53 and expanded in neurosphere with minimized toxicity. Upon tumor resection, catheters can be cultures. Spheres were cultured in complete Neurocult-NS-A proliferation placed directly into the resection cavity providing a simple medium (Neurocult basal medium containing: Neurocult-NS-A mechanism for routine AO delivery. Given that this therapeutic differentiation supplement at a concentration of 1/10 dilution, 20 ng/ml modality is currently being investigated, our findings suggest that rh EGF, 20 ng/ml rh bFGF and 2 mg/ml Heparin) from Stemcell Technologies anti-DRR AOs could be a promising and practical therapy to treat (Vancouver, BC, Canada). When spheres appear large enough for passaging DRR-positive GBM patients, with a reduction in invasion as a (o300 mm in diameter), they were collected in tubes and spun at treatment endpoint. 1200 r.p.m. for 3 min. To dissociate the spheres, 700 ul of Accumax (Millipore) was added to the cell pellet and incubated for 5 min at 37 1C, they were then washed with phosphate-buffered saline, centrifuged and resuspended in complete Neurocult-NS-A proliferation medium and MATERIALS AND METHODS seeded at a concentration of 200 000 cells/flask. Antibodies and reagents Anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), anti-AKT, anti- Pharmacological inhibition assays phospho-p44/42 MAPK (Thr202/Tyr204), Anti-p44/42 MAPK, anti-ILK1, anti- Cells were treated overnight with 1 mM AG1478 or 5 mM gefitinib before phospho-EGFR (Tyr 845), Signal Silence ILK1 and scramble siRNA II kit (Cell being treated with 50 ng/ml EGF either alone or in the presence of 1 mM

& 2014 Macmillan Publishers Limited Oncogene (2014) 4952 – 4960 DRR activates AKT to drive brain cancer invasion A Dudley et al 4958 Ø scr AO DRR 18

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cell number 5 0 scr AO Figure 5. Antisense oligonucleotide-mediated DRR ablation prevents invasion in a mouse model. (a) Western blot showing DRR and pAKT (Ser473) expression levels in human GBM stem cells untransfected (Ø), transfected with control non-targeting AO (scr) or with DRR targeting AO (AO). Decreased DRR expression level using AOs in hGSC cells correlates with decreased pAKT expression level. Tubulin is shown as a loading control. (b) Mouse brain sections stained with hematoxylin and eosin (H&E), human Sox-2 to identify all implanted cancer cells (red), Cy5 ﬂuorescence revealing cells treated with AOs (green). Dashed line demarcates tumor core. Box in inset indicates tumor location. Arrowheads indicate cells that have escaped the implanted tumor mass and invaded the normal peritumoral brain. (c) Quantiﬁcation of invasion. Represented as mean±s.e.m. n ¼ 3, scr vs AO Po0.05 (t-test).

AG1478 or 5 mM gefitinib for 10 min at 37 1C; with 5–20 mM U0126 wortmannin for 10 min at 37 1C before lysis. For the experiments in which for2hat371C, then stimulated with EGF alone or in the presence of fibronectin was used, 24-well plates were coated with 50 mg/ml U0126 for 10 min at 37 1Cbeforelysis;with2mg/ml C3 transferase for 5 h fibronectin overnight at 4 1C. Fibronectin was then removed and the at 37 1C before lysis; with 5 or 10 mM PP2 (or PP3) or 5 mM SU6656 for 5 h at plate was allowed to air dry at room temperature for B1h before 37 1C before lysis; with 200 nM PF-228 for 1 h at 37 1C before lysis; with 5– the addition of cells. All inhibitors were dissolved in dimethyl 20 mM LY294002 or 100 nM wortmannin for 2 h or 1 h, respectively, and sulfoxide therefore an equivalent volume of dimethyl sulfoxide was then stimulated with EGF alone or in the presence of LY294002 or used as a control.

Oncogene (2014) 4952 – 4960 & 2014 Macmillan Publishers Limited DRR activates AKT to drive brain cancer invasion A Dudley et al 4959 RGD assay Statistical analysis Plates were coated with fibronectin. Cells were treated with 500 mM RGD in Each experiment was performed at least three times (n ¼ 3) from suspension on a rotating platform at room temperature for 30 min. A total independent biological replicates. Data were analyzed with the statistical of 200 000 cells/well were plated onto fibronectin for 30 min. Cells that did software GraphPad Prism 5 and has been presented as mean±standard not adhere were removed and lysed. error of mean (s.e.m). Po0.05 was considered statistically significant.

Immunocytochemistry Cells were grown to B70% confluency on glass coverslips (with or without CONFLICT OF INTEREST fibronectin) in supplemented Dulbecco’s Modified Eagle Medium. Cells The authors declare no conflict of interest. were fixed with 3% paraformaldehyde, then permeabilized with 0.5% Triton X-100 before being incubated with indicated primary and secondary antibodies. Images were captured with the LSM 700 confocal microscope using a Â 60 oil immersion objective. Where indicated, fluorescence was ACKNOWLEDGEMENTS quantified using ImageJ (http://rsbweb.nih.gov). This work was supported in part by grants from Industry Canada, Centre of Excellence for Commercialization (KP), Canadian Institutes for Health Research—Proof-of- Principle Grant (KP and MJD) and Canadian Institutes for Health Research— Three-dimension invasion assay Operating Grant (MJD). AD was supported by a McGill University Faculty of Medicine 21 Invasion assays were performed as previously described. Briefly, 25 000 Scholarship. GT was supported by a McGill University Faculty of Medicine Scholarship. cells/drop were plated onto the lid of a 10 cm Petri dish and hung to allow GFD was supported by a Vanier Canada Graduate Scholarship. spheroid formation. On the third day, spheroids were transferred to 2% agar. On the sixth day, spheroids were implanted into a collagen type I matrix and appropriate media was added (regular Dulbecco’s Modified Eagle Medium±1 mM 5-Fluorouracil or supplemented with 10 mM PP2/ REFERENCES LY294002/AG1478±1 mM 5-Fluorouracil). Spheroids were imaged over 24 h 1 Villavicencio AT, Burneikiene S, Romanelli P, Fariselli L, McNeely L, Lipani JD et al. intervals with a Zeiss light microscope Â 5 objective. Four spheroids/ Survival following stereotactic radiosurgery for newly diagnosed and recurrent condition from three independent experiments were used to quantify glioblastoma multiforme: a multicenter experience. Neurosurg Rev 2009; 32: invasion. 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Oncogene (2014) 4952 – 4960 & 2014 Macmillan Publishers Limited