Nitin K. Agarwal

PTEN, NHERF1 and PHLPP form a tumor suppressor network that is disabled in glioblastoma

The phosphatidylinositol-3-OH kinase (PI3K)-Akt pathway is activated in cancer by genetic or epigenetic events and efforts are under way to develop targeted therapies. phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is the major brake of the pathway and a common target for inactivation in glioblastoma, one of the most aggressive and therapy-resistant cancers. To achieve potent inhibition of the PI3K-Akt pathway in glioblastoma, we need to understand its mechanism of activation by investigating the interplay between its regulators. We show here that PTEN modulates the PI3K-Akt pathway in glioblastoma within a tumor suppressor network that includes Na+/H+ exchanger regulatory factor 1 (NHERF1) and pleckstrin-homology domain leucine-rich repeat protein phosphatases 1 (PHLPP1). The NHERF1 adaptor, previously characterized by our group as a PTEN ligand and regulator, shows also PTEN-independent Akt-modulating effects that led us to identify the PHLPP1/PHLPP2 Akt phosphatases as NHERF1 ligands. NHERF1 interacts via its PDZ domains with PHLPP1/PHLPP2 and scaffolds heterotrimeric complexes with PTEN. Functionally, PHLPP1 requires NHERF1 for membrane localization and growth-suppressive effects. PHLPP1 loss boosts Akt phosphorylation only in PTEN-negative cells and cooperates with PTEN loss for tumor growth. In a panel of low-grade and high-grade glioma patient samples, we show for the first time a significant disruption of all three members of the PTEN-NHERF1-PHLPP1 tumor suppressor network in high-grade tumors, correlating with Akt activation and patients abysmal survival. We thus propose a PTEN-NHERF1-PHLPP PI3K-Akt pathway inhibitory network that relies on molecular interactions and can undergo parallel synergistic hits in glioblastoma.

Rictor phosphorylation on the Thr-1135 site does not require mammalian target of rapamycin complex 2.

In animal cells, growth factors coordinate cell proliferation and survival by regulating the phosphoinositide 3-kinase/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K-dependent signaling kinase complex defined as mammalian target of rapamycin complex 2 (mTORC2) functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to the inhibition of PI3K, mTOR, or expression of integrin-linked kinase. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor–dependent phosphorylation of Akt, indicating that the rictor Thr-1135 phosphorylation is not critical in the regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells, suggesting that this modification might play a role in the regulation of not only mTORC2 but also the mTORC2-independent function of rictor. Mol Cancer Res; 8(6); 896–906. ©2010 AACR.

Peptidyl-prolyl cis/trans isomerase Pin1 is critical for the regulation of PKB/Akt stability and activation phosphorylation.

The serine/threonine protein kinase B (PKB, also known as Akt) plays a pivotal role in diverse cellular functions. Elevated expression of activated Akt has been detected in a wide variety of human cancers; however, the mechanism of Akt protein stability regulation remains unclear. In this study, we showed a strong correlation between the expression levels of an oncogenic peptidyl-prolyl cis/trans isomerase Pin1 and levels of Akt phosphorylation at S473 in multiple cancer types (P<0.0001). Akt-pS473 status combined with Pin1 expression levels predicted a poorer prognosis than did either one alone in patients with breast cancer (P=0.0052). We further showed that Pin1 regulated Akt stability and phosphorylation on S473 through the phosphorylated Thr-Pro motifs of Akt. These motifs are conserved evolutionary and are required for the maintenance of Akt stability and its interaction with Pin1. In addition, repressing Pin1 expression through either homologue Pin1 knockout or small interfering RNA-mediated knockingdown compromised its ability to protect Akt from degradation. Our results show how Akt protein stability is regulated by the peptidyl-prolyl cis/trans isomerase Pin1 and highlight the importance of this oncogenic network in human disease pathogenesis.

Rictor regulates cell migration by suppressing RhoGDI2

Rictor and its binding partner Sin1 are indispensable components of mTORC2 (mammalian target of rapamycin complex 2). The mTORC2 signaling complex functions as the regulatory kinase of the distinct members of AGC kinase family known to regulate cell proliferation and survival. In the early chemotaxis studies in Dictyostelium, the rictor’s ortholog has been identified as a regulator of cell migration. How rictor regulates cell migration is poorly characterized. Here we show that rictor regulates cell migration by controlling a potent inhibitor of Rho proteins known as the Rho-GDP dissociation inhibitor 2 (RhoGDI2). On the basis of on our proteomics study we identified that the rictor-dependent deficiency in cell migration is caused by upregulation of RhoGDI2 leading to a low activity of Rac and Cdc42. We found that a suppression of RhoGDI2 by rictor is not related to the Sin1 or raptor function that excludes a role of mTORC2 or mTORC1 in regulation of RhoGDI2. Our study reveals that rictor by suppressing RhoGDI2 promotes activity of the Rho proteins and cell migration.

Moesin is a glioma progression marker that induces proliferation and Wnt/β-catenin pathway activation via interaction with CD44

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/β-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/β-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/β-catenin pathway.

Trimeric G protein-CARMA1 axis links smoothened, the hedgehog receptor transducer, to NF-κB activation in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Aberrant activation of Hedgehog (Hh) and nuclear factor (NF)-κB pathways is ubiquitously observed and known to mediate tumor growth, survival, and chemoresistance in DLBCL. Here, we find that activation of Hh signaling is positively correlated with NF-κB pathway in DLBCL tumors, and that smoothened (SMO), the signal transducer subunit of Hh pathway, contributes to NF-κB activation through recruiting G protein subunits Gαi and Gα12 to activate PKCβ/CARMA1/TRAF6/NEMO signaling axis followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO. Moreover, functional inhibition of SMO enhances the cytotoxic effects of NF-κB inhibitor. Altogether, our study reveals a noncanonical Hh signaling pathway in which SMO activates trimeric G proteins and CARMA1-associated signaling complex, leading to NF-κB activation. This signaling cascade contributes to the survival of DLBCL and may serve as a potential target for combination therapies in DLBCL.

NHERF1/EBP50 Controls Morphogenesis of 3D Colonic Glands by Stabilizing PTEN and Ezrin-Radixin-Moesin Proteins at the Apical Membrane

Na(+)/H(+) exchanger 3 regulating factor 1/ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (NHERF1/EBP50), an adaptor molecule that interacts with the ERM-neurofibromatosis type 2 family of cytoskeletal proteins through its ERM-binding region and with phosphatase and tensin homolog (PTEN) and β-catenin through its PDZ domains, has been recently implicated in the progression of various human malignancies, including colorectal cancer (CRC). We report here that NHERF1 controls gland morphogenesis, as demonstrated in three-dimensional (3D) human intestinal glands developing from a single nonpolarized cell. Starting from the early two-cell developmental stage, NHERF1 concentrates at the cellular interface in a central membrane disc that marks the apical pole delimiting the forming lumen. NHERF1 depletion leads to severe disruption of the apical-basal polarity, with formation of enlarged and distorted cell spheroids devoid of a central lumen. This characteristic and the increased number of mitoses in NHERF1-depleted spheroids, including multipolar ones, mimic high-grade dysplasia lesions observed in CRC progression. NHERF1 ERM-binding or PDZ-domain mutants fail to localize apically and impair gland formation most likely by outcompeting endogenous ligands, with the latter mutant completely aborting gland development. Examination of NHERF1 ligands showed that even if both ezrin and moesin colocalized with NHERF1 at the apical membrane, moesin but not ezrin depletion disrupted morphogenesis similarly to NHERF1. NHERF1 depletion resulted also in membrane displacement of PTEN and nuclear translocation of β-catenin, events contributing to polarity loss and increased proliferation. These findings reveal an essential role of NHERF1 in epithelial morphogenesis and polarity and validate this 3D system for modeling the molecular changes observed in CRC.

Transcriptional regulation of serine/threonine protein kinase (AKT) genes by glioma-associated oncogene homolog 1.

Background: Little is known regarding the transcriptional regulation of AKT. Results: GLI1 contributes to the survival of DLBC cells by promoting transcription of AKT genes. Conclusion: AKT1 is a novel direct downstream target of the Hedgehog transcriptional factor GLI1. Significance: Identifying target genes of GLI1 provides insights into the contribution of Hedgehog signaling in the pathobiology of malignant tumors. Aberrant activation of Hedgehog signaling has been described in a growing number of cancers, including malignant lymphomas. Here, we report that canonical Hedgehog signaling modulates the transcriptional expression of AKT genes and that AKT1 is a direct transcriptional target of GLI1. We identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter, and site-directed mutagenesis assays. Moreover, we provide evidence that GLI1 contributes to the survival of diffuse large B-cell lymphoma (DLBCL) cells and that this effect occurs in part through promotion of the transcription of AKT genes. This finding is of interest as constitutive activation of AKT has been described in DLBCL, but causative factors that explain AKT expression in this lymphoma type are not completely known. In summary, we demonstrated the existence of a novel cross-talk at the transcriptional level between Hedgehog signaling and AKT with biological significance in DLBCL.

Functional inhibition of BCL2 is needed to increase the susceptibility to apoptosis to SMO inhibitors in diffuse large B-cell lymphoma of germinal center subtype

Previously, we have demonstrated that inhibition of Hedgehog pathway induces predominantly apoptosis in diffuse large B-cell lymphoma (DLBCL) cell lines of activated B-cell (ABC) type but predominantly cell cycle arrest in those of germinal center (GC). Here, we explored the possibility of overcoming the resistance to apoptosis to SMO inhibitors in five DLBCL cells of GC type using the combination of the SMO inhibitor HhAntag (Genentech Inc) with the BH3 mimetic ABT-737 (Abbott Laboratories). As controls we have used two DLBCL of ABC type (OCI-LY10 and OCI-LY3). Combinatorial treatments were performed with increasing concentrations of the HhAntag with low doses (equal or less than the IC20) of ABT-737. MTS assays were used to detect changes in cell viability and Annexin-V and PARP1 cleavage assays were used to detect apoptosis. Combining low doses of ABT-737 with increasing concentrations of HhAntag in GC DLBCL cell lines resulted in significantly increase of apoptosis in comparison to treatments with the SMO inhibitor alone. We concluded that in GC DLBCL cell lines, in contrast to those of ABC type, functional inhibition of BCL2 family members is usually needed to overcome the resistance to apoptosis to SMO inhibitors. These findings provide a rationale to explore the use of SMO and BCL2 inhibitors as adjuvant therapy for treatment of DLBCL of GC type.

Active IKKβ promotes the stability of GLI1 oncogene in diffuse large B-cell lymphoma

GLI1 oncogene has been implicated in the pathobiology of several neoplasms including diffuse large B-cell lymphoma (DLBCL). However, mechanisms underlying GLI1-increased activity in DLBCL are poorly characterized. Herein, we demonstrate that IKKβ phosphorylates GLI1 in DLBCL. IKKβ activation increased GLI1 protein levels and transcriptional activity, whereas IKKβ silencing decreased GLI1 levels and transcriptional activity. Tumor necrosis factor-α (TNFα) mediated IKKβ activation-impaired GLI1 binding with the E3 ubiquitin ligase-ITCH, leading to decreased K48-linked ubiquitination/degradation of GLI1. We found 8 IKKβ-dependent phosphorylation sites that mediate GLI1 stability. Mutating or deleting these residues facilitated GLI1-ITCH interaction and decreased the protective effect of TNFα on GLI1 stability. IKKβ-GLI1 crosstalk is significant because combined inhibition of both molecules resulted in synergistic suppression of DLBCL viability in vivo and in vitro. By linking IKKβ-mediated nuclear factor-κB activity with GLI1, we identified a crosstalk between these 2 pathways that can inform the design of novel therapeutic strategies in DLBCL.