PTEN-deficient Glioblastoma Cells Research Articles

CSIG-11. KINASE-DIRECTED INHIBITION OF PYRIMIDINE BIOSYNTHESIS IN PTEN-DEFICIENT GLIOBLASTOMA

Abstract Targeting pyrimidine biosynthesis has been a mainstay of chemotherapy in oncology, including frontline treatment of pancreatic, breast, and colorectal carcinomas. In glioblastoma, the targeting pyrimidine biosynthesis is a promising emerging approach for counteracting the effects of PTEN-deficiency in glioblastoma. PTEN loss triggers the activation of mTORC1, which in turn phosphorylates and activates the ribosomal protein kinases S6K1 and S6K2. We have previously shown that combination treatment of inhibitors targeting S6K1 and the TYRO3-AXL-MERTK receptor tyrosine kinases (TAM-RTKs) triggers cytotoxic responses in PTEN-deficient glioblastoma cells. Here we show brain-penetrant inactivation of S6K1 and TAM-RTKs using the S6K1 inhibitor LY-2584702 and the TAM-RTK inhibitor BMS-777607, which reduced glioblastoma tumor growth. Pharmacogenetic analysis of signal transduction indicated a key role for S6K2 in sustaining survival signaling in PTEN-deficient glioblastoma cells. Steady-state metabolomics revealed that combined inactivation of S6K1 and TAM-RTKs resulted in decreased nucleotide biosynthesis, and flux analysis indicated reduced flux of glucose to pyrimidines. Altogether the results indicate a kinase-directed therapeutic strategy for targeting S6K1 and TAM-RTKs to reduce pyrimidine biosynthesis and glioblastoma tumor growth.

Targeting the Synthetic Vulnerability of PTEN-Deficient Glioblastoma Cells with MCL1 Inhibitors.

PTEN deletion or mutation occurs in 30% to 60% of patients with glioblastoma (GBM) and is associated with poor prognosis. Efficacious therapy for this subgroup of patients is currently lacking. To identify potential target(s) to selectively suppress PTEN-deficient GBM growth, we performed a three-step synthetic lethal screen on LN18 PTEN wild-type (WT) and knockout (KO) isogeneic GBM cell lines using a library containing 606 target-selective inhibitors. A MCL1 inhibitor UMI-77 identified in the screen exhibited excellent suppression on the proliferation, colony formation, 3D spheroid, and neurosphere formation of PTEN-deficient GBM cells. Mechanistically, loss of PTEN in GBM cells led to upregulation of MCL1 in posttranslational level via inhibition of GSK3β, and consequently confer cells resistance to apoptosis. Pharmacologic inhibition or knockdown of MCL1 blocked this PI3K-GSK3β-MCL1 axis and caused reduction of several antiapoptotic proteins, finally induced massive caspase-3 cleavage and apoptosis. In both subcutaneous and orthotopic GBM models, knockdown of MCL1 significantly impaired the in vivo growth of PTEN-deficient xenografts. Moreover, the combination of UMI-77 and temozolomide synergistically killed PTEN-deficient GBM cells. Collectively, our work identified MCL1 as a promising target for PTEN-deficient GBM. For future clinical investigations, priority should be given to the development of a selective MCL1 inhibitor with efficient brain delivery and minimal in vivo toxicity.

A DNA Repair Inhibitor Isolated from an Ecuadorian Fungal Endophyte Exhibits Synthetic Lethality in PTEN-Deficient Glioblastoma.

Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine (1), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.

MicroRNA-29a activates a multi-component growth and invasion program in glioblastoma

BackgroundGlioblastoma is a malignant brain tumor characterized by rapid growth, diffuse invasion and therapeutic resistance. We recently used microRNA expression profiles to subclassify glioblastoma into five genetically and clinically distinct subclasses, and showed that microRNAs both define and contribute to the phenotypes of these subclasses. Here we show that miR-29a activates a multi-faceted growth and invasion program that promotes glioblastoma aggressiveness.MethodsmicroRNA expression profiles from 197 glioblastomas were analyzed to identify the candidate miRNAs that are correlated to glioblastoma aggressiveness. The candidate miRNA, miR-29a, was further studied in vitro and in vivo.ResultsMembers of the miR-29 subfamily display increased expression in the two glioblastoma subclasses with the worst prognoses (astrocytic and neural). We observed that miR-29a is among the microRNAs that are most positively-correlated with PTEN copy number in glioblastoma, and that miR-29a promotes glioblastoma growth and invasion in part by targeting PTEN. In PTEN-deficient glioblastoma cells, however, miR-29a nevertheless activates AKT by downregulating the metastasis suppressor, EphB3. In addition, miR-29a robustly promotes invasion in PTEN-deficient glioblastoma cells by repressing translation of the Sox4 transcription factor, and this upregulates the invasion-promoting protein, HIC5. Indeed, we identified Sox4 as the most anti-correlated predicted target of miR-29a in glioblastoma. Importantly, inhibition of endogenous miR-29a decreases glioblastoma growth and invasion in vitro and in vivo, and increased miR-29a expression in glioblastoma specimens correlates with decreased patient survival.ConclusionsTaken together, these data identify miR-29a as a master regulator of glioblastoma growth and invasion.

Abstract 117: Lysyl oxidase secreted by PTEN-deficient glioblastoma cells recruits macrophages and promotes malignant growth

Abstract Glioblastoma (GBM) is the most lethal form of brain cancer in adults. The median survival of GBM patients is only about one year after initial diagnosis. Genomic profiling has stratified GBM into various subgroups, which are driven by specific genetic alternations of core signaling pathways, including RTK/RAS/PI3K, P53 and RB pathways. Loss of PTEN, TP53 and CDKN2A tumor suppressors, and amplification of EGFR are frequently observed in GBM. However, targeted therapies, such as therapy against EGFR, have failed in the clinic, and no effective therapeutic drugs available to target tumor suppressors. The main reason for this failure is tumor-cell genetic heterogeneity, which induces aberrant activation of multiple signaling pathways. Stromal/immune cells in the tumor microenvironment (TME) are genetically stable, which not only play a pivotal role in GBM progression by affecting multiple cancer hallmarks, but can also be educated by cancer cells. However, whether and how the behavior and function of specific stromal/immune cells in the TME are regulated by cancer cell genetic alternations in GBM remain relatively undefined. Here, we show that PTEN deficiency in GBM specifically triggers immune response by promoting macrophage recruitment, while without affecting macroglia and other immune cells. Using unbiased transcriptome profiling, we identified that lysyl oxidase (LOX) is preferentially secreted by PTEN-deficient cancer cells, and is a potent macrophage chemoattractant. Transcriptome profiling following Gene Set Enrichment Analysis (GSEA) and functional validation demonstrated that activation of SRC and AKT signaling pathways drives LOX upregulation in PTEN-deficient cancer cells via the YAP1-TEAD complex. Upon the secretion from PTEN-deficient cancer cells, LOX can be uptaken by macrophages via integrin β1 signaling. Using the phospho-kinase antibody array, we identified PYK2 as the main downstream of LOX that is responsible for macrophage migration. Genetic and pharmacological inhibition of LOX in PTEN-deficient cancer cells does not affect tumor cell proliferation in vitro, but markedly inhibits macrophage density and tumor growth in vivo. Bioinformatics analysis in clinical samples demonstrated that PTEN-YAP1-LOX-integrin β1 axis is enriched in GBM patients with higher macrophage density, and that these patients show lower survival. Together, our findings highlight the significance of PTEN-YAP1-LOX-integrin β1 axis in macrophage infiltration in GBM, and demonstrate a possibility of improving GBM treatment by targeting this axis-mediated macrophage recruitment. Citation Format: Peiwen Chen, Alan Y. Wang, Ronald A. DePinho. Lysyl oxidase secreted by PTEN-deficient glioblastoma cells recruits macrophages and promotes malignant growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 117.

Pharmacologic Targeting of S6K1 in PTEN-Deficient Neoplasia

Genetic S6K1 inactivation can induce apoptosis in PTEN-deficient cells. We analyzed the therapeutic potential of S6K1 inhibitors in PTEN-deficient Tcell leukemia and glioblastoma. Results revealed that the S6K1 inhibitor LY-2779964 was relatively ineffective as a single agent, while S6K1-targeting AD80 induced cytotoxicity selectively in PTEN-deficient cells. Invivo, AD80 rescued 50% of mice transplanted with PTEN-deficient leukemia cells. Cells surviving LY-2779964 treatment exhibited inhibitor-induced S6K1 phosphorylation due to increased mTOR-S6K1 co-association, which primed the rapid recovery of S6K1 signaling. In contrast, AD80 avoided S6K1 phosphorylation and mTOR co-association, resulting in durable suppression of S6K1-induced signaling and protein synthesis. Kinome analysis revealed that AD80 coordinately inhibits S6K1 together with the TAM family tyrosine kinase AXL. TAM suppression by BMS-777607 or genetic knockdown potentiated cytotoxic responses to LY-2779964 in PTEN-deficient glioblastoma cells. These results reveal that combination targeting of S6K1 andTAMs is a potential strategy for treatment of PTEN-deficient malignancy.

ATPS-66INVESTIGATING S6K1 INHIBITORS FOR TARGETING PTEN-DEFICIENT GLIOBLASTOMA

PTEN-inactivation in glioblastoma (GBM) triggers the hyperactivation of protein kinases, including mTOR and its downstream target, the ribosomal protein S6 kinase 1 (S6K1). Previous studies have demonstrated the potential efficacy of mTOR inhibitors for GBM therapy. We explored S6K1 inhibitors for potential cytotoxic effects in PTEN-deficient GBM cells. All S6K1 inhibitors were effective in reducing the phosphorylation of S6K1 substrates. However, analysis of the phosphorylation of the inhibitor-bound S6K1 revealed important differences: some S6K1 inhibitors induced the phosphorylation of inhibitor-bound kinase, while others had little evidence of this effect. Induced phosphorylation of the inhibitor-bound kinase has been termed inhibitor hijacking. Our analysis in GBM cells suggests that S6K1 inhibitor hijacking is correlated with induced proximity to the upstream kinase, mTORC1. Evidence of inhibitor hijacking was inversely correlated with cytotoxic activity. We propose selective development of S6K1 inhibitors that avoid the hijacking effect for counteracting oncogenic signals in PTEN-deficient glioblastoma.

KAP regulates ROCK2 and Cdk2 in an RNA-activated glioblastoma invasion pathway

Aberrant splicing of the cyclin-dependent kinase-associated phosphatase, KAP, promotes glioblastoma invasion in a Cdc2-dependent manner. However, the mechanism by which this occurs is unknown. Here we show that miR-26a, which is often amplified in glioblastoma, promotes invasion in phosphatase and tensin homolog (PTEN)-competent and PTEN-deficient glioblastoma cells by directly downregulating KAP expression. Mechanistically, we find that KAP binds and activates ROCK2. Thus, RNA-mediated downregulation of KAP leads to decreased ROCK2 activity and this, in turn, increases Rac1-mediated invasion. In addition, the decrease in KAP expression activates the cyclin-dependent kinase, Cdk2, and this directly promotes invasion by increasing retinoblastoma phosphorylation, E2F-dependent Cdc2 expression and Cdc2-mediated inactivation of the actomyosin inhibitor, caldesmon. Importantly, glioblastoma cell invasion mediated by this pathway can be antagonized by Cdk2/Cdc2 inhibitors in vitro and in vivo. Thus, two distinct RNA-based mechanisms activate this novel KAP/ROCK2/Cdk2-dependent invasion pathway in glioblastoma.

PTEN restoration and PIK3CB knockdown synergistically suppress glioblastoma growth in vitro and in xenografts

Glioblastoma is the most frequent and malignant glioma in adults. To develop an effective gene therapy strategy for glioblastoma, we investigated the anti-proliferative effects of phosphatase and tensin homolog (PTEN) restoration and siRNAs specifically targeting PIK3CB and PIK3CA on PTEN-deficient glioblastoma cells in vitro and in subcutaneous xenografts. Restoration of PTEN or knockdown of PIK3CB, but not PIK3CA, in glioblastoma cells markedly down-regulates the phosphorylation level of AKT, inhibits cell proliferation and colony formation, arrests the cell cycle at the G0/G1 stage, and promotes caspase-dependent apoptosis. Combined treatment with PTEN restoration and PIK3CB knockdown shows strong synergy. PTEN restoration or PIK3CB knockdown is also able to efficiently inhibit the growth of human U251 glioblastoma xenografts in nude mice, while tumor growth is entirely suppressed by a combination of the two treatments. In addition, we found that the mRNA levels of inhibitors of apoptosis proteins (IAPs) are reduced in U251 cells by PTEN restoration, suggesting that combined antitumor effects may also be partly attributed to the inhibition of the IAP pathway by PTEN restoration. Collectively, our results demonstrate that PI3 K isoforms play specific roles in tumorigenesis, and that combined treatment of PTEN restoration and PIK3CB siRNA is a promising gene therapy strategy for PTEN-deficient gliomas.

Integrative genome analysis reveals an oncomir/oncogene cluster regulating glioblastoma survivorship

Using a multidimensional genomic data set on glioblastoma from The Cancer Genome Atlas, we identified hsa-miR-26a as a cooperating component of a frequently occurring amplicon that also contains CDK4 and CENTG1, two oncogenes that regulate the RB1 and PI3 kinase/AKT pathways, respectively. By integrating DNA copy number, mRNA, microRNA, and DNA methylation data, we identified functionally relevant targets of miR-26a in glioblastoma, including PTEN, RB1, and MAP3K2/MEKK2. We demonstrate that miR-26a alone can transform cells and it promotes glioblastoma cell growth in vitro and in the mouse brain by decreasing PTEN, RB1, and MAP3K2/MEKK2 protein expression, thereby increasing AKT activation, promoting proliferation, and decreasing c-JUN N-terminal kinase-dependent apoptosis. Overexpression of miR-26a in PTEN-competent and PTEN-deficient glioblastoma cells promoted tumor growth in vivo, and it further increased growth in cells overexpressing CDK4 or CENTG1. Importantly, glioblastoma patients harboring this amplification displayed markedly decreased survival. Thus, hsa-miR-26a, CDK4, and CENTG1 comprise a functionally integrated oncomir/oncogene DNA cluster that promotes aggressiveness in human cancers by cooperatively targeting the RB1, PI3K/AKT, and JNK pathways.

Epidermal Growth Factor Receptor and PTEN Modulate Tissue Factor Expression in Glioblastoma through JunD/Activator Protein-1 Transcriptional Activity

Hypoxia and necrosis are fundamental features of glioblastoma (GBM) and their emergence is critical for the rapid biological progression of this fatal tumor; yet, underlying mechanisms are poorly understood. We have suggested that vaso-occlusion following intravascular thrombosis could initiate or propagate hypoxia and necrosis in GBM. Tissue factor (TF), the main cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Epidermal growth factor receptor (EGFR) amplification and PTEN loss are two common genetic alterations seen in GBM but not in lower-grade astrocytomas that could be responsible for TF up-regulation. The most frequent EGFR mutation in GBM involves deletion of exons 2 to 7, resulting in the expression of a constitutively active receptor, EGFRvIII. Here, we show that overexpression of EGFR or EGFRvIII in human glioma cells causes increased basal TF expression and that stimulation of EGFR by its ligand, EGF, leads to a marked dose-dependent up-regulation of TF. In all cases, increased TF expression led to accelerated plasma coagulation in vitro. EGFR-mediated TF expression depended most strongly on activator protein-1 (AP-1) transcriptional activity and was associated with c-Jun NH(2)-terminal kinase (JNK) and JunD activation. Restoration of PTEN expression in PTEN-deficient GBM cells diminished EGFR-induced TF expression by inhibiting JunD/AP-1 transcriptional activity. PTEN mediated this effect by antagonizing phosphatidylinositol 3-kinase activity, which in turn attenuated both Akt and JNK activities. These mechanisms are likely at work in vivo, as EGFR expression was highly correlated with TF expression in human high-grade astrocytoma specimens.

Deregulation of a STAT3-Interleukin 8 Signaling Pathway Promotes Human Glioblastoma Cell Proliferation and Invasiveness

Inactivation of the tumor suppressor phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) is recognized as a major event in the pathogenesis of the brain tumor glioblastoma. However, the mechanisms by which PTEN loss specifically impacts the malignant behavior of glioblastoma cells, including their proliferation and propensity for invasiveness, remain poorly understood. Genetic studies suggest that the transcription factor signal transducers and activators of transcription 3 (STAT3) harbors a PTEN-regulated tumor suppressive function in mouse astrocytes. Here, we report that STAT3 plays a critical tumor suppressive role in PTEN-deficient human glioblastoma cells. Endogenous STAT3 signaling is specifically inhibited in PTEN-deficient glioblastoma cells. Strikingly, reactivation of STAT3 in PTEN-deficient glioblastoma cells inhibits their proliferation, invasiveness, and ability to spread on myelin. We also identify the chemokine interleukin 8 (IL8) as a novel target gene of STAT3 in human glioblastoma cells. Activated STAT3 occupies the endogenous IL8 promoter and directly represses IL8 transcription. Consistent with these results, IL8 is upregulated in PTEN-deficient human glioblastoma tumors. Importantly, IL8 repression mediates STAT3 inhibition of glioblastoma cell proliferation, invasiveness, and spreading on myelin. Collectively, our findings uncover a novel link between STAT3 and IL8, the deregulation of which plays a key role in the malignant behavior of PTEN-deficient glioblastoma cells. These studies suggest that STAT3 activation or IL8 inhibition may have potential in patient-tailored treatment of PTEN-deficient brain tumors.

Combination of all-trans retinoic acid and interferon-gamma upregulated p27kip1 and down regulated CDK2 to cause cell cycle arrest leading to differentiation and apoptosis in human glioblastoma LN18 (PTEN-proficient) and U87MG (PTEN-deficient) cells

Deletion or mutation of phosphatase and tensin homolog located on chromosome ten (PTEN) occurs in as high as 80% glioblastoma. All-trans retinoic acid (ATRA) induces differentiation in cancer cells. Interferon-gamma (IFN-gamma) induces apoptosis in many cancers including glioblastoma. We used the combination of ATRA and IFN-gamma to control growth of human glioblastoma LN18 (PTEN-proficient) and U87MG (PTEN-deficient) cells and explored any advantage of having PTEN in the cells. LN18 and U87MG cells were treated with ATRA (1 microM) for 7 days and then IFN-gamma (5 ng/ml) for 1 day. Methylene blue staining indicated astrocytic differentiation. Wright staining and ApopTag assay showed characteristic features of apoptosis. Western blotting demonstrated the levels of specific proteins. ATRA and IFN-gamma alone and in combination could induce apoptosis in LN18 cells; while ATRA alone induced differentiation only, IFN-gamma alone induced apoptosis, and ATRA plus IFN-gamma increased apoptosis in U87MG cells. The variation in induction of apoptosis by ATRA alone might be attributed to difference in PTEN expression in the two cell lines. Compared with control cells, IFN-gamma alone and ATRA plus IFN-gamma increased PTEN expression in LN18 cells while there was no PTEN expression or induction in U87MG cells after treatments with ATRA alone and ATRA plus IFN-gamma. Apoptosis in both cell lines was associated with increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c into the cytosol, and calpain and caspase-3 activities. Treatments elevated p27(kip1) and decreased CDK2 levels in both cell lines, indicating cell cycle arrest at G(1)/S phase. The combination of ATRA and IFN-gamma could control the growth of both PTEN-proficient and PTEN-deficient glioblastoma cells by arresting cell division and inducing differentiation and apoptosis. Thus, our study indicated that the growth of both PTEN-proficient and PTEN-deficient glioblastoma cells could effectively be controlled by treatment with the combination of ATRA and IFN-gamma.

The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region.

PTEN is a recently identified tumor suppressor inactivated in a variety of cancers such as glioblastoma and endometrial and prostate carcinoma. It contains an amino-terminal phosphatase domain and acts as a phosphatidylinositol 3,4,5-trisphosphate phosphatase antagonizing the activity of the phosphatidylinositol 3-OH kinase. PTEN also contains a carboxyl-terminal domain, and we addressed the role of this region that, analogous to the amino-terminal phosphatase domain, is the target of many mutations identified in tumors. Expression of carboxyl-terminal mutants in PTEN-deficient glioblastoma cells permitted the anchorage-independent growth of the cells that otherwise was suppressed by wild-type PTEN. The stability of these mutants in cells was reduced because of rapid degradation. Although the carboxyl-terminal region contains regulatory PEST sequences and a PDZ-binding motif, these specific elements were dispensable for the tumor-suppressor function. The study of carboxyl-terminal point mutations affecting the stability of PTEN revealed that these were located in strongly predicted beta-strands. Surprisingly, the phosphatase activity of these mutants was affected in correlation with the degree of disruption of these structural elements. We conclude that the carboxyl-terminal region is essential for regulating PTEN stability and enzymatic activity and that mutations in this region are responsible for the reversion of the tumor-suppressor phenotype. We also propose that the molecular conformational changes induced by these mutations constitute the mechanism for PTEN inactivation.