Moesin Is a Glioma Progression Marker That Induces Proliferation and Wnt/B-Catenin Pathway Activation Via Interaction with CD44

Published OnlineFirst December 5, 2012; DOI: 10.1158/0008-5472.CAN-12-1040

Cancer Molecular and Cellular Pathobiology Research

Moesin Is a Glioma Progression Marker That Induces Proliferation and Wnt/b-Catenin Pathway Activation via Interaction with CD44

Xiaoping Zhu1, Fabiana C. Morales1, Nitin Kumar Agarwal1, Turgut Dogruluk1, Mihai Gagea2, and Maria-Magdalena Georgescu1,3

Abstract Moesin is an ERM family protein that connects the actin cytoskeleton to transmembrane receptors. With the identification of the ERM family protein NF2 as a tumor suppressor in glioblastoma, we investigated roles for other ERM proteins in this malignancy. Here, we report that overexpression of moesin occurs generally in high-grade glioblastoma in a pattern correlated with the stem cell marker CD44. Unlike NF2, moesin acts as an oncogene by increasing cell proliferation and stem cell neurosphere formation, with its ectopic overexpression sufficient to shorten survival in an orthotopic mouse model of glioblastoma. Moesin was the major ERM member activated by phosphorylation in glioblastoma cells, where it interacted and colocalized with CD44 in membrane protrusions. Increasing the levels of moesin competitively displaced NF2 from CD44, increasing CD44 expression in a positive feedback loop driven by the Wnt/b-catenin signaling pathway. Therapeutic targeting of the moesin–CD44 interaction with the small-molecule inhibitor 7-cyanoquinocarcinol (DX-52-1) or with a CD44-mimetic peptide specifically reduced the proliferation of glioblastoma cells overexpressing moesin, where the Wnt/b-catenin pathway was activated. Our findings establish moesin and CD44 as progression markers and drugable targets in glioblastoma, relating their oncogenic effects to activation of the Wnt/b-catenin pathway. Cancer Res; 73(3); 1142–55. 2012 AACR.

Introduction which MGMT levels are elevated show a poor response to this Gliomas, or tumors with features of glial differentiation, chemotherapy (4). The relatively high incidence of glioblasto- account for more than 50% of all brain tumors (1). Among ma, its resistance to treatment, and the dismal patient prog- these, glioblastoma (WHO grade 4) is the most aggressive and nosis rendered this tumor a forefront for the drug discovery commonly occurring primary intracranial tumor in adults, efforts. fi with an average incidence of 4 to 5 per 100,000 persons per Ezrin, radixin, moesin (ERM), and neuro bromatosis type 2 year. Glioblastoma either arises de novo or progresses over time (NF2) proteins form a distinct subfamily of cytoskeletal mem- from lower grade tumors. The median survival is 12 to 15 bers, belonging to the larger family of band 4.1 proteins (5). The months, with survival rates of 20% and 5% at 2 and 5 years, ERM-NF2 proteins share a conserved structure, consisting in a respectively (2). The conventional treatment consists of sur- an amino (N)-terminal FERM domain, an extended -helical gical resection followed by radiotherapy and adjuvant chemo- intermediate region, and an actin-binding carboxyl (C)-termi- fi therapy with temozolomide, an oral alkylating agent (3). Some nal region, the latter being modi ed in NF2. The amino acid glioblastoma tumor cells express an enzyme called O-6-methyl- identity between ezrin and radixin, moesin, or NF2 is 86%, 86%, guanine-DNA methyltransferase (MGMT) that repairs the type and 62%, respectively, in the FERM domain, and 63%, 60%, and of DNA damage induced by temozolomide, and tumors in 25%, respectively, in the rest of the molecule. It is apparent that the homology within the FERM domain is higher than in the other regions, and indeed the ERM-NF2 proteins share com- Authors' Affiliations: 1Department of Neuro-Oncology and 2Veterinary mon ligands that bind to 2 defined pockets in the FERM Medicine, University of Texas MD Anderson Cancer Center, Houston; and domain (6). One pocket accommodates the juxtamembrane 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas polybasic motif of some transmembrane receptors, such as CD44 and NEP/CD10, or a different consensus motif in ICAM2 Note: Supplementary data for this article are available at Cancer Research – Online(http://cancerres.aacrjournals.org/). and CD43 (7 10). A nonoveralpping neighbouring pocket binds to the Na/H exchanger 3 regulatory factors (NHERF) 1 and 2 Corresponding Author: M.-M. Georgescu, The University of Texas South- western Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390. Phone: adaptor proteins (6). The FERM domain pockets are also the 214-648-7174; Fax: 214-648-6323; E-mail: binding site for the C-terminal self-association region of the [email protected] ERM proteins (11), and this head-to-tail interaction can be doi: 10.1158/0008-5472.CAN-12-1040 either intramolecular or intermolecular, resulting in "closed" 2012 American Association for Cancer Research. monomers or in dimers, respectively. In the monomeric

1142 Cancer Res; 73(3) February 1, 2013

The Moesin-CD44 Oncogenic Complex: New Target in Glioblastoma

"closed" conformation, the binding sites for FERM ligands, as sphere formation assays are detailed in Supplementary well as for actin, are masked, and the inactive ERM proteins are Material. most likely localized in the cytoplasm (5, 12, 13). At least 2 events have been reported to induce a molecular opening, Moesin–CD44 inhibitors phosphorylation of a conserved C-terminal Thr residue An inhibitory peptide targeting the moesin interaction site (Thr567/564/558 for ezrin/radixin/moesin, respectively; on CD44 (CD44pep) was designed, SRRRCGQKKKLVINSGN- ref. 14), and binding of a phosphatidyl inositol-4,5-bispho- GAV, and subsequently custom synthesized with amidated C- sphate (PIP2) molecule in yet another area of the FERM domain terminus (Biosynthesis). The quinocarmycin analog DX-52-1 (15, 16). In open conformation, the ERM proteins are able to previously shown to bind ERM proteins and inhibit the inter- function by connecting plasma membrane proteins to the actin action of radixin with CD44 (25, 26) was obtained from the cytoskeleton (7). National Cancer Institute (Bethesda, MD). Briefly, 2 103 cells The physiologic roles of ERM proteins are related to the in 100 mL Dulbecco's Modified Eagle's Medium containing 5% structuring of the cell cortex, being involved in cell differen- FBS were plated in triplicate in 96-well plates, allowed to attach tiation and morphogenesis (7). In cancer, except for NF2, which overnight, and subsequently treated with dimethyl sulfoxide has been incontestably established as a tumor suppressor (17, vehicle (0) and serial concentrations of DX-52-1 or CD44pep. 18), the role of the other ERM proteins is not entirely clear. We For GSC neurosphere assays, the inhibitors were added at show here that moesin is distinctively upregulated in glioblas- plating (day 0). toma and acts as an oncoprotein in this malignancy. The dissection of moesin's pro-oncogenic mechanism of action Orthotopic tumorigenicity assay identified CD44, a hyaluronan transmembrane receptor and The orthotopic tumorigenicity assay was performed in stem cell marker, as a transducer of signals upstream the Wnt/ severe combined immunodeficient (SCID) mice by inoculating b-catenin pathway. Importantly, we show that this oncogenic 106 LN229 cells as described (19). pathway can be drugged by specifically targeting the interaction between moesin and CD44. Protein analysis and antibodies Cell lysis, immunoprecipitation, immunoblotting, and cel- Materials and Methods lular fractionation were previously described (21). Antibodies Human tissue specimens are listed in Supplementary Material. Frozen glioma specimens classified as based on World Health Organization (WHO) criteria were previously described Luciferase assay (19). The paraffin-embedded human normal brain, grade 2 TOP/FOP b-catenin luciferase reporter assays were con- (oligodendroglioma, low grade astrocytoma, and mixed oli- ducted as described (27). goastrocytoma), grade 3 (anaplastic astrocytoma and anaplastic oligodendroglioma), and grade 4 (glioblastoma) glioma RNA extraction and quantitative real-time PCR tissue microarray (TMA) was also described (20, 21). microarray analysis The transcription target microarray protocol is provided in Immunohistochemistry and immunoflorescence Supplementary Material. Immunohistochemistry and confocal immunofluorescence were conducted as described (refs. 20, 21; see also Supplemen- Statistical analysis tary Material). The statistical analysis is detailed in Supplementary Material. Plasmids and short hairpin RNAs Human moesin cDNA was obtained from Dr. Vijaya Ramesh Results (Addgene Inc.) and the constructs used are described in Moesin is progressively increased in gliomas and Supplementary Material. behaves as an oncoprotein. To analyze the expression of ERM-NF2 proteins in tumors of Cell lines, glioblastoma stem cells, retroviral infections, increasing malignant phenotype, we initially examined 17 proliferation, and neurosphere formation assays glioma samples by Western blot analysis (Fig. 1A). Distinctive- The glioblastoma cell lines LN229, LN18, LN308, LN373, ly, moesin showed a marked expression increase in glioblas- LN428, U87-MG, D54, A172, MO59J, and U251-MG were pre- toma compared with lower grade gliomas (Fig. 1A). NF2 had an viously described (20). The normal human astrocyte (NHA) cell opposite trend, confirming our previous observations (21), line was obtained from Dr. TJ Liu (MD Anderson Cancer whereas ezrin and radixin did not show significant differences Center, Houston, TX). Patient-derived GSC2, GSC11, and (Fig. 1B). Exploration of the same samples for putative ERM GSC23 were kindly provided by Drs. Howard Colman and Fred ligands revealed increased CD44 expression and decreased Lang (MD Anderson Cancer Center), and propagated as neuro- NHERF1 expression in glioblastoma (Fig. 1B). Importantly, the spheres, as described (22, 23). All the cells lines were tested and CD44 and moesin expression patterns exhibited strong direct authenticated (20). Transfections and retroviral infections correlation (Fig. 1B, bottom graph). The expression of the EGF were described (24). The MTT proliferation assay was con- receptor (EGFR) and EGFRv3 constitutive active splice variant ducted as described (20). The glioblastoma cell (GSC) neuro- in a subset of the glioblastoma samples (Fig. 1B, red arrow)

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Figure 1. Moesin is a progression marker in glioma. A and B, immunoblot with indicated antibodies of tissue extracts (8–10 mg protein) from 10 lower grade glioma and 7 glioblastoma cases. The mean patient survival is indicated. LN229 with moesin knockdown (shMoe) or without (shV) and D54 glioblastoma cell lines were included as controls. The red arrow indicates EGFRv3. Actin-normalized ERM-NF2 and CD44 levels are shown in the top graph as individual brain tumor specimen values distributed around the mean. The bottom table indicates high correlation between moesin, CD44, and NF2 levels across the 17 samples. C, immunohistochemistry (200) with moesin antibody of glioma TMA. The diagram shows the number of the specimens and the intensity of moesin staining (mean SD) per glioma category. C, Western blot analysis of cell extracts (20 mg protein) from NHA and 10 human glioblastoma cell lines showing moesin and CD44 expression. The graph shows actin-normalized moesin and CD44 levels.

conﬁrmed the high grade of the tumors, but did not correlate in gliomas of increasing severity (Fig. 1C). Additional expres- with moesin expression. Subsequent immunohistochemistry sion analysis of a panel of 10 glioblastoma cell lines in com- analysis of a glioma TMA containing normal brain and grades 2 parison with a normal human astrocyte (NHA) cell line showed to 4 gliomas, conﬁrmed the gradual increase of moesin staining increased moesin levels in all glioblastoma lines and higher

1144 Cancer Res; 73(3) February 1, 2013 Cancer Research

The Moesin-CD44 Oncogenic Complex: New Target in Glioblastoma

CD44 levels in the majority (Fig. 1D). These combined results knockdown (Fig. 3B), indicating that moesin is the ERM indicate moesin and CD44 as glioma progression markers, with protein phosphorylated in these cells. Similarly, in U251, a highly correlated expression levels. stronger faster band disappeared following moesin knock- To define the role of moesin as oncoprotein in glioma, we down, and a lighter top band disappeared following ezrin examined the effect of moesin overexpression on tumor cell knockdown (Supplementary Fig. S3B). This suggested that growth. Stable overexpression of Myc-tagged moesin in NHA both moesin and ezrin are phosphorylated in these cells, and LN229 cells that express lower levels of endogenous moesin being the main phosphorylated species. Radixin was moesin than other glioblastoma cells increased cell prolifera- not phosphorylated in either cell line. We confirmed these tion relatively to vector control cells (Fig. 2A). To compare results by cellular fractionation (not shown) and by immuno- moesin's growth-promoting effects with those of another ERM fluorescence with phospho-ERM antibody of ERM-depleted protein, we established stable Myc-tagged ezrin-overexpres- cells with a second set of shRNAs (Supplementary Fig. S4), and sing cells (Fig. 2A). The proliferation of these cells was inter- observed that phosphorylated moesin is localized in micro- mediate between that of moesin-expressing cells and control spike-like plasma membrane protrusions (Supplementary Figs. cells, suggesting a stronger oncogenic effect of moesin. Alter- S4 and S5). We also investigated the ERM protein phosphor- natively, stable depletion of moesin by 2 different short hairpin ylation in cells with Myc-moesin overexpression, and noted RNAs (shRNA) in LN229 and NHA cells did not significantly that it increased, and corresponded to the membrane fraction alter cell proliferation (Fig. 2B), suggesting possible compen- of overexpressed moesin (Fig. 3C). Overall, these results indi- sation by endogenous ezrin. cate that moesin is the ERM protein preferentially phosphor- As established cell lines are less representative of human ylated in glioblastoma cells. gliomas than primary tumor cells propagated as neurospheres To appreciate the importance of ERM phosphorylation in that retain stem cell characteristics such as self-renewal (28, glioma, the glioma TMA was examined by immunofluores- 29), we examined the effect of moesin on 2 patient-derived cence with phospho-ERM antibody (Fig. 3D). Strong positivity primary GSC neurosphere lines (Fig. 2C). Moesin overexpres- was observed in approximately 50% of glioblastoma samples, sion significantly increased neurosphere size in both GSC2 and in contrast with mild positivity in 10% to 15% of lower grade GSC23 and also the neurosphere self-renewal ability in GSC2. gliomas. Both atypical glial and microvascular endothelial cells As a global measure of GSC growth, the differences in the total were P-ERM positive in glioblastoma (Fig. 3D inset). These number of cells were quantified in an MTT assay (Supplemen- results revealed high levels of ERM phosphorylation, most tary Fig. S1). As for cell lines, partial knockdown of moesin in likely of moesin, specifically present in glioblastoma samples, GSC11, which express higher levels of endogenous moesin than indicating an activated ERM state in tumors. other GSCs, did not significantly alter their proliferation (Sup- plementary Fig. S2). Collectively, these results showed that Moesin enhances the expression of CD44 and competes moesin has marked growth-promoting consequences on GSCs. with NF2 for CD44 binding To confirm the proliferative effect of moesin overexpression In phosphorylated active conformation, ERM proteins inter- in vivo, we used our previously described brain tumor model act with their ligands (Fig. 3A). Because moesin levels paral- (19). In this orthotopic model, the moesin-everexpressing leled CD44 levels in glioma samples (Fig. 1A), we examined the LN229 cells were highly proliferative and induced rapidly effect of moesin overexpression on CD44 expression. In LN229 developing tumors that significantly shortened the animal cells, endogenous and overexpressed moesin colocalized with survival (Fig. 2D). These results relate moesin overexpression CD44 at plasma membrane microspike protrusions (Fig. 4A). to a fast-proliferating glioma phenotype, characteristic for At higher magnification, the colocalization was concentrated glioblastoma. at the base of the microspikes and in discrete areas along these structures (Supplementary Fig. S6). Overexpressed moesin also Moesin is constitutively phosphorylated in glioblastoma enhanced CD44 expression in these cells (Fig. 4A). To quantify cells the latter effect, a time course mitogenic stimulation of LN229 To investigate the mechanism underlying the specific effect cells with and without moesin overexpression showed marked of moesin on glioblastoma growth, we first analyzed the increase of CD44 expression levels in moesin-overexpressing activation status and subcellular localization of ERM proteins. cells (Fig. 4B). As observed, in the absence of serum, CD44 As mentioned, ERM proteins reside in inactive closed confor- expression was undetectable in LN229 cells (Fig. 4B, top). mation in the cytosolic compartment, and following activation Similarly, in GSCs growing in serum-free conditions, CD44 through phosphorylation and PIP2 binding, interact with their expression was undetectable, in contrast to that of 4 other ligands at the plasma membrane in an active open conforma- neural stem cell markers investigated (Figs. 4C and Supple- tion (Fig. 3A; refs. 5, 7). To examine ERM phosphorylation and mentary Fig. S7). Moesin overexpression considerably raised localization, we developed shRNAs for all ERM proteins (Sup- CD44 expression in both LN229 cells and GSCs (Fig. 4B and C) plementary Fig. S3A). The phosphorylation of the Thr567/564/ and also Sox2 expression in GSCs (Fig. 4C), consistent with a 558 site was examined in cytosolic and membrane fractions shift towards a more undifferentiated and malignant from LN229 and U251 glioblastoma cells depleted of individual phenotype. ERM proteins (Fig. 3B and Supplementary Fig. S3B). The An important aspect of the interaction between CD44 and phospho-ERM antibody detected a band in the membrane ERM-NF2 proteins is that whereas ezrin has been reported to fractions of LN229 cells that disappeared following moesin enhance CD44 growth signaling (30), NF2 has been shown to

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Figure 2. Moesin increases cell proliferation and tumorigenicity. NHA and LN229 cells with stable expression of Myc-Moesin (MycMoe) and Myc-Ezrin (MycEz; A) or stable moesin knockdown by two shRNAs (sh4 and sh7; B) were subjected to MTT proliferation assay for the indicated time periods. Data are mean SD (n ¼ 3). C, establishment of Myc-Moesin–expressing GSC neurosphere lines (images at 100). Neurosphere size and numbers were recorded. Data are mean SEM from 20 microscope 10 ﬁelds. D, control and Myc-Moesin–overexpressing LN229 cells were injected intracranially in 6-week-old SCID female mice. Mice were sacriﬁced at the indicated periods. Arrows highlight the brain tumors stained by hematoxylin and eosin (H&E; images 20). Ki-67 staining of tumors from dying mice is shown (images 200). The Kaplan–Meier curve and the median survival of mice (n ¼ 5) are shown.

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The Moesin-CD44 Oncogenic Complex: New Target in Glioblastoma

Figure 3. Moesin is constitutively phosphorylated in glioblastoma. A, simplified view of the activation of ERM proteins, featuring the domains and the 2 events that lead to the "open" active conformation. B, purification of cytoplasmic (C) and membrane (M) fractions from control (shV), ezrin (sh8Ez), radixin (sh8Rad), or moesin (sh3Moe) knockdown LN229 cells shows the phospho-Thr567/564/558-ERM (P- ERM) species corresponding to moesin in the membrane compartment. Arrows indicate ERM proteins. Ns, nonspecific band. ERK and N-cadherin (N-cad) were used as cytoplasmic- and membrane- fraction markers, respectively. C, immunofluorescence (400) with P-ERM and moesin antibodies of control and Myc-Moesin– overexpressing LN229 cells shows signal overlap in membrane protrusions. D, confocal immunofluorescence (400) of the glioma TMA with P-ERM antibody and ToPro-3 nuclear staining, with quantification of P-ERM–positive specimens per glioma type. The inset shows P-ERM staining of endothelial cell membrane (arrowhead) and erythrocytes (green) inside the lumen.

repress it (31). As in approximately one-third of glioblastoma The moesin–CD44 interaction constitutes a potential tumors, NF2 expression is maintained at normal high levels therapeutic target in glioblastoma. (21), we examined the possibility of a competition between To examine whether the oncogenic effect of moesin is moesin, ezrin, and NF2 for CD44 occupancy (Fig. 4D). Immu- mediated through the interaction with CD44, we treated cells noprecipitation of CD44 from lysates of vector, Myc-moesin, overexpressing moesin with a CD44-derived peptide, CD44pep, and Myc-ezrin cells, as well as from vector and moesin kndock- known to strongly interact in vitro with the FERM domain of down cells, showed that moesin overexpression displaced NF2 ERM proteins (9, 32). At a concentration of 0.5 mmol/L, and ezrin from CD44, whereas moesin depletion had an CD44pep had no effect on the proliferation of control or opposite effect (Fig. 4D, graphs). Ezrin overexpression dis- Myc-moesin–overexpressing cells (Fig. 5A, left graph). Remark- placed moesin and, to a lesser extent NF2, from CD44. These ably, at 10 mmol/L, CD44pep speciﬁcally suppressed the pro- results suggested that moesin overexpression has a double liferation of Myc-moesin–overexpressing cells to levels slightly impact on CD44 signaling, ﬁrst, by enhancing its expression, under those of control cells (Fig. 5A, right graph). This result and second, by decreasing its occupancy for NF2. indicated that the effects of moesin on proliferation are

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Figure 4. Moesin enhances CD44 expression and competes with NF2 for CD44 binding. A, immunoﬂuorescence (400) of control and Myc-Moesin– overexpressing LN229 cells with moesin and CD44 antibodies. B, time-course stimulation of serum-deprived vector and Myc-Moesin–overexpressing LN229 cells with 10% FBS for the indicated periods. Duplicate samples are shown. Random indicates unsynchronized growing cells in 10% FBS. Actin-normalized CD44 levels are plotted as mean SD (n ¼ 2). C, CD44 and Sox2 expression are upregulated in Myc-Moesin–overexpressing GSCs. Actin-normalized Sox2 and Olig2 levels are plotted as mean SD (n ¼ 2). D, coimmunoprecipitation of moesin, ezrin, and NF2 with CD44 and control IgG antibodies from protein lysates of vector, Myc-Moesin, Myc-Ezrin, shRNA vector (shV), and moesin knockdown (sh7Moe) LN229 cells shows competition between moesin, ezrin, and NF2 for CD44 binding. The levels of coimmunoprecipitated moesin, ezrin, and NF2 were normalized to immunoprecipitated CD44 levels and expressed as percentage from the levels of the vector control coimmunoprecipitated proteins that were set to 100%.

1148 Cancer Res; 73(3) February 1, 2013 Cancer Research

The Moesin-CD44 Oncogenic Complex: New Target in Glioblastoma

Figure 5. The moesin–CD44 interaction is a potential therapeutic target. A and B, proliferation of vector and Myc-Moesin–expressing LN229 cells treated with 2 concentrations of CD44pep (A) or with serial concentrations of DX-52-1 (B). Note specific growth suppression of Myc-Moesin–expressing cells at 10 mmol/L CD44pep and 100 nmol/L DX-52-1. C–E, treatment with CD44pep (C and D) and DX-52-1 (E) of control and Myc-Moesin–overexpressing GSC2 neurospheres. The global neurosphere growth is quantified as in Supplementary Fig. S1. Images and neurosphere size quantification are presented as in Fig. 2C. mediated through the interaction with CD44, and that this that DX-52-1 binds tightly to radixin and less to ezrin and interaction can be specifically targeted for inhibition. moesin (25), and is able to disrupt the interaction between The DX-52-1 drug has been developed for melanoma treat- radixin and CD44 (26). On the basis of these observations, we ment in the 1990s (33–35). Kahsai and colleagues have shown treated vector control and moesin-overexpressing cells with

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serial concentration of DX-52-1 (Fig. 5B). A sharp and specific phosphorylation and intracellular localization of b-catenin. decline of moesin-overexpressing cell proliferation to control b-catenin phosphorylation on residue Ser552 by Akt has been levels was apparent at 100 nmol/L. Higher concentrations shown to induce b-catenin nuclear translocation and tran- stopped proliferation in both control and moesin-overexpres- scriptional activation (41, 42). This residue was phosphorylated sing cells. by moesin more than by ezrin overexpression in LN229 cells To facilitate a more rapid translation into the clinic, these (Fig. 6E). The fractionation and immunofluorescence analysis results were confirmed in GSCs (Fig. 5C–E). Both inhibitors of moesin-overexpressing cells showed that a fraction of significantly reduced the cell growth of moesin-overexpressing b-catenin translocated from the membrane to the nuclear neurospheres in 3-day proliferation assays (Fig. 5C–E, left compartment in these cells (Fig. 6F). Overall, this comprehen- graphs). Interestingly, the growth of vector control GSCs, sive analysis showed that moesin, through binding to CD44, which do not express CD44, was unchanged, supporting inhi- induces b-catenin nuclear translocation and increased tran- bition specificity for the moesin–CD44 interaction. In 5-day scriptional activity, which can be inhibited by specifically neurosphere formation assays, the size of moesin-overexpres- targeting the moesin–CD44 interaction. sing neurospheres was reduced by both inhibitors to levels similar to vector neurospheres (Fig. 5C–E, right graphs). The Discussion neurosphere count was not significantly changed (not shown), Glioblastoma is one of the relatively frequent cancers with suggesting that moesin might increase self-renewal indepen- very poor prognosis due to its invasiveness, tumor heteroge- dently of CD44. Mechanistically, treatment with CD44pep neity, and resistance to chemotherapy. Efforts are focused on decreased the levels of moesin, CD44 and Sox2 (Fig. 5D), characterizing the molecular signature of these tumors to implicating the stability of the moesin-CD44 complex as key diagnose them and define new targets amenable to therapy. regulator of GSC growth. Collectively, these experiments clear- Our previous studies have defined a network of interacting ly showed that the oncogenic effects of moesin overexpression tumor suppressors specifically disrupted in glioblastoma that in glioblastoma, most likely through the interaction with CD44, include NF2 and NHERF1 (19–21). Continuing this analysis could be targeted with therapeutic drugs. with a comprehensive examination of the other three ERM- NF2 members, we found that the expression levels of moesin, Large-scale analysis identifies the Wnt/b-catenin but not of ezrin or radixin, are significantly upregulated in pathway as specific signaling downstream of the glioblastoma. Moesin could thus be used as reliable marker of moesin–CD44 interaction. grade 4 glioblastoma, and we pursue an extensive marker Signaling through CD44 has been previously investigated, validation analysis for diagnostic purposes. and downstream ERK and PI3K-Akt pathway activation or The ERM proteins have been implicated in the motility of Hippo cascade inhibition were described (36–40). Moesin cells and different aspects of metastasis (7, 43). We show here overexpression in glioblastoma and NHA cells activated the that moesin overexpression increases glioma cell growth, and PI3K-Akt, ERK, and p38 MAPK pathways (Fig. 6A). To gain a this effect is translated in vivo by a significantly decreased better insight on the signaling triggered by moesin in glioblas- animal survival due to massive tumor cell proliferation. Inter- toma, we interrogated a transcription target microarray con- estingly, moesin depletion did not consistently decrease pro- taining direct targets of transcription factors downstream of liferation, suggesting that moesin is an oncogenic protein when signaling pathways involved in tumor growth (Fig. 6B and overexpressed, similarly to EGFR growth factor receptors, for Supplementary Table S1). This profiling revealed that: (i) example. A compensatory effect of ezrin in moesin-depleted growth and metastasis-promoting factors were upregulated cells and GSCs could also explain the lack of phenotype change by moesin, whereas the inhibitors of growth were generally not (also see below). Analyzing the EGFR expression that is upre- modified, and (ii) Wnt/b-catenin, a major growth pathway not gulated in roughly 40% of glioblastoma samples (44), we did not previously implicated downstream of CD44 signaling, was find a correlation with moesin levels that were consistently activated by moesin. The profiled PI3K-Akt, ERK, JNK, NF-kB, elevated in the majority of cases. In contrast, CD44, a trans- or Hippo pathways were engaged at various extents, as deter- membrane receptor that interacts directly with ERM-NF2 mined by FOXO, AP1, NF-kB, and YAP/TAZ target variations, proteins, showed similar expression trend as moesin. This led respectively (Fig. 6B). us to hypothesize that the proliferative signals of moesin are We confirmed the Wnt/b-catenin activation by showing that dependent of its interaction with CD44. The findings of specific c-Jun, the highest activated transcription target from the large- phosphorylation of moesin in glioblastoma on the conserved scale analysis, was specifically upregulated in moesin-over- Thr558 residue that dictates a permissive conformational state expressing cells but not in ezrin-overexpressing cells (Fig. 6C). for interactions between the FERM domain and CD44 sup- We next used the luciferase-reporter system with b-catenin– ported this model (Fig. 7). responsive (TOP) or mutated (FOP) elements and observed CD44 is a hyaluronic acid receptor that has an intracellular that b-catenin transcriptional activity was significantly elevat- tail with nonoverlapping binding sites for ERM-NF2 and ed in moesin-overexpressing cells (Fig. 6D). Importantly, this ankyrin (38). Because all ERM-NF2 members bind to the same activation was abolished when moesin-overexpressing cells site but have opposite effects on CD44 signal transduction, it were treated with CD44pep (Fig. 6D), indicating that the has been hypothesized that they may compete for CD44 moesin-CD44 interaction specifically triggers the activation binding, depending on the cell proliferative status, which of b-catenin transcriptional activity. We further examined the would determine their activation by phosphorylation (38). We

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The Moesin-CD44 Oncogenic Complex: New Target in Glioblastoma

Figure 6. Moesin activates Wnt/b-catenin pathway. A, immunoblot of protein extracts with indicated antibodies shows pathway analysis in Myc-Moesin– overexpressing cells. B, quantitative SYBER-green–based RT-PCR array showing changes in transcript expression of indicated signaling pathways target genes in Myc-Moesin–expressing LN229 cells relatively to vector control. Gene expression level was normalized to GAPDH expression set to 1. C, immunoblot conﬁrming increased c-Jun expression in Myc-Moesin-expressing cells. D, b-catenin TOP/FOP luciferase reporter assay with or without CD44pep treatment of vector and moesin-overexpressing LN229 cells. The luciferase activity was normalized to Renilla activity. Data are mean SD (n ¼ 3). E, immunoblot of whole protein extracts shows increased b-catenin phosphorylation on Ser552 in Myc-Moesin–overexpressing cells. F, increased b-catenin nuclear localization in Myc-Moesin cells shown by nuclear (N)/cytoplasm (C)/membrane (M) fractionation (left) and immunoﬂuorescence (right) of LN229 cells. PARP and N-cadherin were used as nuclear and membrane-fraction markers, respectively. The graph shows nuclear PARP-normalized b-catenin levels.

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Figure 7. Model of moesin-CD44 signaling and inhibitory targeting in glioblastoma. It is assumed here that CD44-RTK complexes trigger CD44 signaling. LGG, lower grade gliomas (II and III). Note a positive feedback loop in which CD44 is the upstream activator and the downstream transcriptional target of both ERK and Wnt/b-catenin pathways.

brought evidence that moesin, ezrin, and NF2 compete for CD44 does not have a catalytic activity and the mechanisms CD44 binding in glioblastoma, suggesting that not only the cell by which it signals relate to its interactions with receptor proliferative status is important for selective CD44 occupancy tyrosine kinases (RTK), such as Met or the EGFR family (38). by ERM-NF2 members but also the expression levels of the Although several pathways have been described downstream various ERM-NF2 proteins. Accordingly, NF2 acts as a natural CD44 coreceptor function (38), we profiled a transcription inhibitor of CD44 signaling, with effects proportional to its factor target real-time (RT)-PCR microarray that included expression levels. In glioblastoma, its levels are undetectable or outputs of known pathways but also other pathways involved decreased in two-thirds of cases (21, 45), thus favoring an in proliferation but not previously described for CD44 signal- enhanced signaling through complexes between CD44 and ing. Surprisingly, the Wnt/b-catenin pathway was prominently other ERM members. Interestingly, moesin is overexpressed and specifically activated downstream of the moesin–CD44 in almost all glioblastoma cases, suggesting that its effect is not interaction. A possible mechanism of activation of this path- limited only to displacing the traces of NF2 from CD44. Indeed, way by CD44 could transit through the activation of the PI3K by it seems that moesin is required for sustaining a CD44 positive CD44-associated RTKs, with subsequent phosphorylation of feedback loop, in which the signaling through CD44 augments b-catenin by Akt on residue Ser552, which has been shown to CD440s own expression. ERK activation has been previously direct b-catenin to the nucleus (41, 42). In moesin-overexpres- shown to enhance CD44 transcription (46), and we have shown sing cells, all these modifications were present, strongly sup- that serum stimulation of LN229 cells increases CD44 levels. porting this mechanism (Fig. 7). Other moesin-independent However, moesin overexpression dramatically upregulates but converging influences on the activation of Wnt-b-catenin CD44, with consecutive activation of CD44-dependent down- pathway could come from the loss of NHERF1, which has been stream pathways, including ERK, indicating that moesin is a shown to destabilize b-catenin from the membrane and facil- limiting factor for signaling through CD44. The question of why itate its nuclear translocation (27). moesin, and not ezrin or radixin, is the preferred ERM protein An extensive literature on glioma transcriptomes from upregulated in glioblastoma could open several avenues for public microarray databases is available (47–51). To examine exploration. The cell-type specific regulation of transcription whether moesin upregulation in glioblastoma is due to in glioblastoma cells could be an explanation for increased increased gene transcription, we reviewed these studies and moesin expression (see below). However, the interesting obser- found that moesin is on the short list of genes with mRNA levels vation that moesin is the major ERM protein phosphorylated consistently elevated in glioblastoma versus anaplastic astro- and thus activated in glioblastoma opens the possibility of a cytoma (48, 50). Moreover, moesin transcript is part of the broader signaling network in which the kinase responsible for signature that differentiates glioblastoma-rich from oligoden- ERM phosphorylation has more specificity for moesin than for droglioma-rich groups (49). mRNA glioblastoma subgroups ezrin. Taken together, these observations suggest a model in have been also described, including the proneural subtype with which moesin is preferentially transcribed and phosphorylated better prognosis and the mesenchymal subtype with increased in glioblastoma, with major activating consequences on CD44 aggressiveness (47, 51). Although moesin transcript was not signaling (Fig. 7). part of the mesenchymal subtype, it is present on the list of 884

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The Moesin-CD44 Oncogenic Complex: New Target in Glioblastoma

genes in TCGA Worst Prognosis Signature from the glioblas- action. These drugs could not only be beneficial in glioblastoma TCGA dataset (47), suggesting that it is highly enriched in toma, but also in tumors with ezrin overexpression, such as the poor prognosis subsets of all glioblastoma subtypes. Sim- osteosarcoma or rhabdomyosarcoma, or in tumors with NF2- ilarly, CD44 expression, even if not present in the latter analysis, inactivating mutations, such as meningiomas, schwannomas, has been associated with poorer prognosis in a subset of and mesotheliomas. glioblastoma (52, 53). While deemed a stem cell marker, CD44 In conclusion, we described here moesin as a marker for is not consistently expressed in GSC neurospheres (22, 53). We glioma progression that induces oncogenic growth through a hint that serum deprivation might underlie this. Importanly, mechanism involving the interaction with CD44 and subse- moesin boosts CD44 expression in serum-deprived GSCs and quent activation of a host of downstream signaling cascades, also increases the expression of the neural stem cell marker including the Wnt/b-catenin pathway. We described for the Sox2, supporting a functional shift towards an aggressive stem- first time a new therapy target consisting of the moesin–CD44 like phenotype in glioblastoma. interface, whose disruption represses both the proliferation With the advent of personalized cancer therapy, the discov- and b-catenin–dependent signaling. This study adds moesin ery of markers for disease staging, progression, and response to and CD44 on the map of relevant molecular changes in therapy is front line. Although glioblastoma is a rapidly fatal glioblastoma and provides a drugable target for glioblastoma disease, some patients benefit from temozolomide, whereas therapy. those with MGMT overexpression are poor responders (4). As we found moesin as a glioma progression marker in conjunc- Disclosure of Potential Conflicts of Interest tion with CD44, we also tested whether it could constitute a No potential conflicts of interest were disclosed. therapeutic target. Although CD44 has been previously investigated as therapeutic target in various malignancies (54), the Authors' Contributions Conception and design: X. Zhu, T. Dogruluk, M.-M. Georgescu options considered for its inhibition were either eliminating its Development of methodology: X. Zhu, F. Morales, T. Dogruluk, M.-M. expression by siRNAs or interfering with the binding of ligands Georgescu to its extracellular domain by a variety of blocking antibodies Acquisition of data (provided animals, acquired and managed patients, fi provided facilities, etc.): X. Zhu, F. Morales, N. Agarwal, T. Dogruluk, M. Gagea, and peptides (55, 56). In this study, we show for the rst time a M.-M. Georgescu new modality of efficient CD44 inhibition by disrupting its Analysis and interpretation of data (e.g., statistical analysis, biostatistics, interaction with moesin (Fig. 7). In principle, NF2 is the natural computational analysis): X. Zhu, F. Morales, N. Agarwal, T. Dogruluk, M. Gagea, M.-M. Georgescu inhibitor of this interaction, and we have previously shown that Writing, review, and/or revision of the manuscript: X. Zhu, F. Morales, N. NF2 overexpression suppresses the proliferation of glioblas- Agarwal, T. Dogruluk, M. Gagea, M.-M. Georgescu fi Administrative, technical, or material support (i.e., reporting or orga- toma cells (21). Replacing NF2 with a speci c CD44 peptide nizing data, constructing databases): X. Zhu, F. Morales, N. Agarwal, T. induced a potent inhibition of cell proliferation, comparable Dogruluk, M.-M. Georgescu with that achieved by NF2 overexpression (data not shown). Study supervision: M.-M. Georgescu Importantly, the DX-52-1 quinocarmycin analog that was shown to disrupt the binding between radixin and CD44 Grant Support This study was supported by grants NCI-CA107201 and ARRA supplement, (26), had a similar effect on cell growth in moesin-overexpres- and G.S. Hogan Gastrointestinal Research Fund awards (to M.-M. Georgescu). sing cells, including GSCs, providing a proof-of-principle that a The costs of publication of this article were defrayed in part by the drug can accomplish the same effect as the specific peptide. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this DX-52-1 has been previously used in a clinical trial that was fact. interrupted because of high toxicity (33). However, our data prompt to the design and/or high-throughput detection of new Received March 19, 2012; revised November 6, 2012; accepted November 21, and less toxic compounds targeting the CD44–moesin inter- 2012; published OnlineFirst December 5, 2012.

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Moesin Is a Glioma Progression Marker That Induces Proliferation and Wnt/ β-Catenin Pathway Activation via Interaction with CD44

Xiaoping Zhu, Fabiana C. Morales, Nitin Kumar Agarwal, et al.

Cancer Res 2013;73:1142-1155. Published OnlineFirst December 5, 2012.

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