Tumor Suppressor Phosphatase Research Articles

PTEN Expression Is Decreased in Pediatric Burkitt Lymphoma Tissues but Does Not Correlate with AKT Activation.

AbstractAbstract 2664 Background:We have recently shown that a subset of pediatric Burkitt lymphoma (pBL) show gain of 13q31, containing the microRNA (miRNA) MIR-17–92 locus, as well as associated increased expression of miRNA from that locus. PTEN, a tumor suppressor phosphatase that negatively regulates the PI3K/AKT/mTOR pro-survival pathway, is negatively regulated by MIR-17–92. Therefore, we hypothesized that PTEN protein and mRNA expression would be decreased in pBL cases with 13q31 locus gain and high expression of MIR-17–92. Because PTEN negatively regulates the PI3K/AKT/mTOR pathway, we also assessed AKT activation in pBL tissues. Methods:We assessed PTEN protein expression by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) pBL tissues from a cohort of patients (n=25) where MIR-17–92 expression levels and status of the 13q31 locus have been previously determined (Schiffman/Miles, Brit J Haematol 155:477, 2011). From FFPE tissues, we assayed PTEN mRNA levels by quantitative RT-PCR. The results of protein and mRNA expression were then correlated with presence or absence of a 13q31 gain and the expression level of MIR17. To evaluate AKT activation in pBL, we assayed for phospho-(p)-AKT by IHC. In addition, PTEN, AKT, and p-AKT protein levels were evaluated by western blotting in Raji and Ramos BL cell lines. Results:PTEN protein was weakly expressed in 5/25 cases (20%) of pBL. PTEN protein showed moderate nuclear and cytoplasmic staining in a subset of paracortical and germinal center lymphocytes in reactive lymph node control tissues, and vascular endothelial cells provided internal positive control in tumor tissue. pBL tissues showed decreased relative PTEN mRNA expression compared to reactive lymph node controls (1.56 vs. 2.61, p<3E-07). There was no significant difference in PTEN mRNA levels between PTEN protein positive and negative cases (1.27 vs. 1.61, p=0.22); cases with or without 13q31gain (1.49 vs. 1.57, p=0.60); or cases with high or low expression of MIR17. p-AKT was detected by IHC in 1/25 cases (4%). The BL cell lines Raji and Ramos showed very low levels of PTEN protein but no detectable p-AKT; total AKT was readily detectable. Discussion:pBL shows decreased PTEN mRNA, and most cases are negative for PTEN protein expression. Unexpectedly, higher MIR-17–92 expression level or 13q31 gain did not show an inverse relationship with PTEN expression at the protein or mRNA level. Despite decreased PTEN expression, p-AKT was detected in only 1/25 pBL cases and was not detected in BL cell lines. These findings suggest that decreased PTEN expression does not lead to AKT activation in pBL. Ongoing studies will investigate the potential of alternate PTEN targets and/or alternate targets for MIR-17–92 in pBL. Disclosures:No relevant conflicts of interest to declare.

Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor

Deregulation of receptor tyrosine kinases (RTKs) is linked to a broad range of cancers, stressing the necessity of studying their regulatory pathways. We and others demonstrated previously that c-Cbl is necessary for the lysosomal degradation of erythropoietin-producing hepatocellular B1 (EphB1) carcinoma and epidermal growth factor receptor (EGFR) RTKs. Moreover, the tumor suppressor phosphatase and tensin homolog (PTEN) was shown to modulate c-Cbl-dependent EGFR degradation. We therefore investigated the involvement of PTEN in EphB1 signaling and degradation. We used PTEN mutants, PTEN, and NHERF1 small interfering RNA in CHO-EphB1 and SW480 cells endogenously expressing EphB1 to delineate EphB1-PTEN interactions. PTEN was constitutively associated with c-Cbl, protecting it from degradation. EphB1 stimulation triggered ∼50% serine-threonine PTEN dephosphorylation and PTEN-Cbl complex disruption, a process requiring PTEN protein phosphatase activity. Both proteins independently translocated to EphB1, with PTEN in association with the scaffold protein NHERF1. Biologically, PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis. This study demonstrates for the first time in mammalian cells that the Eph receptor and PTEN associate and influence their signaling. Moreover, it contributes to the emerging concept that PTEN regulates expression of RTKs through modulation of their degradation. Finally, it reveals a new role for PTEN protein phosphatase activity involved in this process.

PTEN Mutations as a Cause of Constitutive Insulin Sensitivity and Obesity

BackgroundEpidemiologic and genetic evidence links type 2 diabetes, obesity, and cancer. The tumor-suppressor phosphatase and tensin homologue (PTEN) has roles in both cellular growth and metabolic signaling. Germline PTEN mutations cause a cancer-predisposition syndrome, providing an opportunity to study the effect of PTEN haploinsufficiency in humans.MethodsWe measured insulin sensitivity and beta-cell function in 15 PTEN mutation carriers and 15 matched controls. Insulin signaling was measured in muscle and adipose-tissue biopsy specimens from 5 mutation carriers and 5 well-matched controls. We also assessed the effect of PTEN haploinsufficiency on obesity by comparing anthropometric indexes between the 15 patients and 2097 controls from a population-based study of healthy adults. Body composition was evaluated by means of dual-emission x-ray absorptiometry and skinfold thickness.ResultsMeasures of insulin resistance were lower in the patients with a PTEN mutation than in controls (e.g., mean fasting plasma insulin level, 29 pmol per liter [range, 9 to 99] vs. 74 pmol per liter [range, 22 to 185]; P=0.001). This finding was confirmed with the use of hyperinsulinemic euglycemic clamping, showing a glucose infusion rate among carriers 2 times that among controls (P=0.009). The patients' insulin sensitivity could be explained by the presence of enhanced insulin signaling through the PI3K-AKT pathway, as evidenced by increased AKT phosphorylation. The PTEN mutation carriers were obese as compared with population-based controls (mean body-mass index [the weight in kilograms divided by the square of the height in meters], 32 [range, 23 to 42] vs. 26 [range, 15 to 48]; P<0.001). This increased body mass in the patients was due to augmented adiposity without corresponding changes in fat distribution.ConclusionsPTEN haploinsufficiency is a monogenic cause of profound constitutive insulin sensitization that is apparently obesogenic. We demonstrate an apparently divergent effect of PTEN mutations: increased risks of obesity and cancer but a decreased risk of type 2 diabetes owing to enhanced insulin sensitivity. (Funded by the Wellcome Trust and others.)

Enhancer of zeste homolog 2 is overexpressed and contributes to epigenetic inactivation of p21 and phosphatase and tensin homolog in B-cell acute lymphoblastic leukemia

Enhancer of zeste homolog 2 (EZH2) is crucially involved in epigenetic silencing by acting as a histone methyltransferase. Although EZH2 is overexpressed in many solid cancers, the role of EZH2 in B-cell acute lymphoblastic leukemia (B-ALL) remains largely unexplored. In a microarray experiment, we found that EZH2 was significantly upregulated in Nalm-6 cells and this was associated with the silencing of tumor suppressor genes p21, p53 and phosphatase and tensin homolog (PTEN). The abnormal expression of these genes was further confirmed by quantitative realtime polymerase chain reaction and Western blot analysis on Nalm-6 cells. Chromatin immunoprecipitation assay showed that EZH2 and H3K27me3 were both enriched in the promoter region of PTEN and p21 in Nalm-6 cells but not in normal B cells. Functional analysis showed that siRNA-mediated EZH2 knockdown led to decreased proliferation and increased apoptosis of Nalm-6 cells, accompanied by the reactivation of PTEN and p21 expression. Furthermore, we found that EZH2 inhibitor deazaneplanocin A promoted vincristine sulfate-induced apoptosis of Nalm-6 cells. Taken together, our data suggest that EZH2 is overexpressed in B-ALL and promotes the progression of B-ALL by directly mediating the inactivation of tumor suppressor genes p21 and PTEN, and could serve as a potential epigenetic target for B-ALL therapy.

Effects of PTEN inhibition on regulation of tau phosphorylation in an okadaic acid-induced neurodegeneration model

One of pathological hallmarks of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs) consisting of abnormally hyperphosphorylated tau. The molecular mechanisms underlying the regulation of tau hyperphosphorylation remain largely unclear. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated in the pathogenesis of AD, however, potential functions and role of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in AD pathogenesis have not been fully explored. Here, we report that okadaic acid (OA)-induced tau phosphorylation is accompanied by PTEN induction, knockdown of PTEN reduces the tau hyperphosphorylation by OA in SH-SY5Y cells and increases cell proliferation and survival. The effect of PTEN suppression on tau dephosphorylation appeared to be mediated by inhibition of glycogen synthase kinase 3 while enhancing the Akt activity. Reduction of tau phosphorylation was also observed in the OA-induced parental SH-SY5Y cells co-treated with bisperoxovanadate (bpv), a potent PTEN inhibitor. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis and give some insight into the mechanisms through which suppression of PTEN expression may contribute towards the amelioration of tauopathy, implying that pharmacological intervention of PTEN may be a new therapeutic approach for the treatment of AD.

The soybean peptide lunasin promotes apoptosis of mammary epithelial cells via induction of tumor suppressor PTEN: similarities and distinct actions from soy isoflavone genistein

Breast cancer is the leading cause of cancer deaths in women. Diet and lifestyle are major contributing factors to increased breast cancer risk. While mechanisms underlying dietary protection of mammary tumor formation are increasingly elucidated, there remains a dearth of knowledge on the nature and precise actions of specific bioactive components present in foods with purported health effects. The 43-amino acid peptide lunasin (LUN) is found in soybeans, is bioavailable similar to the isoflavone genistein (GEN), and thus may mediate the beneficial effects of soy food consumption. Here, we evaluated whether LUN displays common and distinct actions from those of GEN in non-malignant (mouse HC11) and malignant (human MCF-7) mammary epithelial cells. In MCF-7 cells, LUN up-regulated tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN) promoter activity, increased PTEN transcript and protein levels and enhanced nuclear PTEN localization, similar to that shown for GEN in mammary epithelial cells. LUN-induced cellular apoptosis, akin to GEN, was mediated by PTEN, but unlike that for GEN, was p53-independent. LUN promoted E-cadherin and β-catenin non-nuclear localization similar to GEN, but unlike GEN, did not influence the proliferative effects of oncogene Wnt1 on HC11 cells. Further, LUN did not recapitulate GEN inhibitory effects on expansion of the cancer stem-like/progenitor population in MCF-7 cells. Results suggest the concerted actions of GEN and LUN on cellular apoptosis for potential mammary tumor preventive effects and highlight whole food consumption rather than intake of specific dietary supplements with limited biological effects for greater health benefits.

3′ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ] by voltage-sensing phosphatase (VSP)

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.

Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting.

This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening. Furthermore, the proliferation, differentiation and self-renewal biological features of brain glioma stem cells were identified. Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated. Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability, and expressed the stem cell-specific markers CD133 and nestin. After incubation with fetal bovine serum, the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly, indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons. Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells. These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.

Abstract LB-143: ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers

Abstract Tyrosine kinases, like the ErbB and Src families, can mediate potent oncogenic growth and are successful therapeutic targets of human cancers. However, many cancer cells overcome or successfully modulate these downstream signaling cascades in the absence of growth factor or kinase over-expression. We have previously identified a cellular phosphatase, PTPN13 that significantly modulates mitogenic signaling and can function as tumor suppressors. Decreased PTPN13 expression enhances ErbB2-mediated MAP Kinase activation, and is also associated with decreased survival in breast cancer. Here we show that PTPN13 regulates a signaling complex consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation, proximity ligation assay and immunofluoroscence studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further potentiates these changes. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in cancers harboring ErbB2-activating mutations and decreased PTPN13 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-143. doi:1538-7445.AM2012-LB-143

Targeting Nonclassical Oncogenes for Therapy in T-ALL

Constitutive phosphoinositide 3-kinase (PI3K)/Akt activation is common in Tcell acute lymphoblastic leukemia (T-ALL). Although four distinct class I PI3K isoforms (α, β, γ, δ) could participate in T-ALL pathogenesis, none has been implicated in this process. We report that in the absence of PTEN phosphatase tumor suppressor function, PI3Kγ or PI3Kδ alone can support leukemogenesis, whereas inactivation of both isoforms suppressed tumor formation. The reliance of PTEN null T-ALL on the combined activities of PI3Kγ/δ was further demonstrated by the ability of a dual inhibitor to reduce disease burden and prolong survival in mice as well as prevent proliferation and promote activation of proapoptotic pathways in human tumors. These results support combined inhibition of PI3Kγ/δ as therapy for T-ALL.

Abstract A19: Blocking autophagy using lysosomotropic agents sensitizes resistant prostate tumor cells to the novel Akt inhibitor AZD5363

Abstract Introduction and objective: Prostate cancer development is often associated with deletion or silencing of tumor suppressor phosphatase and tensin homologue (PTEN), a negative regulator of the PI3K-Akt pathway, leading to resistance to various therapies in both preclinical and clinical trials. Therefore, the PI3K/Akt pathway plays a central role in various cellular processes promoting survival signaling that can contribute to the malignant phenotype, and, consequently is an attractive pharmacological target. However, as single agents, AKT inhibitors have yet to demonstrate satisfactory results in the treatment of prostate cancer. Although therapeutically relevant concentrations of AKT inhibitors can effectively induce apoptosis as single agents in some prostate cancer cell lines, such as LNCaP, they induce minimal cell death in others, suggesting that there are intrinsic resistance mechanisms that limit their activity in these models. Methods: We investigated the effects of the Akt inhibitor AZD5363 on cell proliferation, cell cycle, apoptosis and Akt downstream pathway. Survival mechanisms induced by AZD5363 were investigated. Then, we examined the impacts of pharmacologic or genetic inhibition of autophagy in combination with AZD5363 on cell proliferation and apoptosis. Furthermore, the anti-cancer activity of combination treatment of a lysosomotropic inhibitor of autophagy (chloroquine) with the Akt inhibitor AZD5363 was evaluated in PC-3 prostate cancer xenograft model. Results: Here, we show that Akt inhibitor AZD5363 affected the Akt downstream pathway by reducing p-mTOR, p-P70S6K and p-S6K protein levels. Moreover, Akt inhibitor AZD5363 alone caused G2 growth arrest failed to induce significant apoptosis and increased autophagy in the PC3 and DU-145 prostate cancer cell lines. Blocking autophagy using pharmacological inhibitors (3-methyladenine, chloroquine and bafilomycin A) or genetic inhibitors (siRNA targeting Atg3 and Atg7) enhanced cell death induced by Akt inhibitor AZD5363 in these tumor prostate cell lines. Importantly, the combination of AZD5363 with chloroquine significantly reduced tumor volume by 84.9% compared with the control group and by 77.5% compared with either drug alone in the PTEN null prostate cancer PC3 xenograft model. Conclusion: Taken together, these data demonstrate that Akt inhibitor AZD5363 synergized with the lysosomotropic inhibitor of autophagy, chloroquine, to induce apoptosis and delay tumor progression in a xenograft model of prostate cancer which was relatively resistant to monotherapy AZD5363. Hence, combination of an AKT inhibitor with an agent that inhibits autophagy provides a novel therapeutic approach which merits further evaluation. Citation Format: Francois Lamoureux, Christian Thomas, Masafumi Kumano, Fan Zhang, Martin Gleave, Amina Zoubeidi. Blocking autophagy using lysosomotropic agents sensitizes resistant prostate tumor cells to the novel Akt inhibitor AZD5363 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A19.

ErbB2, EphrinB1, Src Kinase and PTPN13 Signaling Complex Regulates MAP Kinase Signaling in Human Cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.

Repression of mammosphere formation of human breast cancer cells by soy isoflavone genistein and blueberry polyphenolic acids suggests diet-mediated targeting of cancer stem-like/progenitor cells

Mammary stem cells are undifferentiated epithelial cells, which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we addressed whether dietary factors selectively target mammary epithelial cells that display stem-like/progenitor subpopulations with previously recognized tumor-initiating potential. Using estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 human breast cancer cell lines and freshly isolated epithelial cells from MMTV-Wnt-1 transgenic mouse mammary tumors, we demonstrate that sera of adult mice consuming soy isoflavone genistein (GEN) or blueberry (BB) polyphenol-containing diets alter the population of stem-like/progenitor cells, as measured by their functional ability to self-renew and form anchorage-independent spheroid cultures in vitro at low frequency (1-2%). Serum effects on mammosphere formation were dose-dependently replicated by GEN (40 nM >2 μM) and targeted the basal stem-like CD44+/CD24-/ESA+ and the luminal progenitor CD24+ subpopulations in MDA-MB-231 and MCF-7 cells. GEN inhibition of mammosphere formation was mimicked by the Akt inhibitor perifosine and was associated with enhanced tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) expression. In contrast, a selected mixture of BB phenolic acids was only active in MDA-MD-231 cells and its CD44+/CD24-/ESA+ subpopulation, and this activity was independent of induction of PTEN expression. These findings delineate a novel and selective function of distinct dietary factors in targeting stem/progenitor cell populations in estrogen receptor-dependent and -independent breast cancers.

SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane

The membrane association of the tumour suppressor phosphatase and tensin homologue (PTEN) is required to oppose the phosphatidylinositol-3-kinase/AKT pathway by dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3). How cytosolic PTEN interacts with its main substrate, PIP3, localized at the inner face of plasma membrane remains unclear. Here we show that PTEN is covalently modified by SUMO1 at both K(266) and K(254) sites in the C2 domain of PTEN. SUMO1 modification at K(266) located in the CBR3 loop, which has a central role in PTEN membrane association, mainly facilitates cooperative binding of PTEN to the plasma membrane by electrostatic interactions. This results in the downregulation of the phosphatidylinositol-3 kinase/AKT pathway and consequently, suppression of anchorage-independent cell proliferation and tumour growth in vivo. Our data demonstrate a molecular mechanism whereby SUMO1 modification is required for PTEN tumour suppressor function by controlling PTEN membrane association and regulation of the phosphatidylinositol-3 kinase/AKT pathway.

PTEN Regulates PDGF Ligand Switch for β-PDGFR Signaling in Prostate Cancer

Platelet-derived growth factor (PDGF) family members are potent growth factors that regulate cell proliferation, migration, and transformation. Clinical studies have shown that both PDGF receptor β (β-PDGFR) and its ligand PDGF D are up-regulated in primary prostate cancers and bone metastases, whereas PDGF B, a classic ligand for β-PDGFR, is not frequently detected in clinical samples. In this study, we examined the role of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of PDGF expression levels using both a prostate-specific, conditional PTEN-knockout mouse model and mouse prostate epithelial cell lines established from these mice. We found an increase in PDGF D and β-PDGFR expression levels in PTEN-null tumor cells, accompanied by a decrease in PDGF B expression. Among Akt isoforms, increased Akt3 expression was most prominent in mouse PTEN-null cells, and phosphatidylinositol 3-kinase/Akt activity was essential for the maintenance of increased PDGF D and β-PDGFR expression. In vitro deletion of PTEN resulted in a PDGF ligand switch from PDGF B to PDGF D in normal mouse prostate epithelial cells, further demonstrating that PTEN regulates this ligand switch. Similar associations between PTEN status and PDGF isoforms were noted in human prostate cancer cell lines. Taken together, these results suggest a mechanism by which loss of PTEN may promote prostate cancer progression via PDGF D/β-PDGFR signal transduction.

Inhibitors of Mtor and PI3K Synergistically Block Plasmacytoma Cell Growth

Abstract 2903 Introduction:Both, the phosphoinositide-3-kinase (PI3K)-AKT pathway as well as its nutrient-dependent downstream target, the mToR (mammalian target of rapamycin) kinase, are essential for the growth and survival of malignant plasma cells. PI3K-AKT can be activated by the loss of the tumor suppressor phosphatase and tensin homolog (PTEN) or by stimulation with cytokines such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). Inhibitors of the mToR pathway like sirolimus, everolimus and temsirolimus are approved for immunosuppression and treatment of renal cell cancer. In fact, they show activity in multiple myeloma patients. However, the clinical activity of mToR inhibitors may be limited by feedback loops in tumor cells that lead to activation of AKT upon inhibition of the rapamycin-sensitive Raptor complex. Selective PI3K inhibitors (Ly294002, NVP-BKM120) as well as dual PI3K-mToR inhibitors (NVP-BEZ235) are in clinical development. Here, the inhibitory effect of a panel of mToR and PI3K inhibitors, alone and in combination, was evaluated in malignant plasma cell lines and primary samples from myeloma patients. Results:In five human plasma cell lines, rapamycin, everolimus, Ly294002, NVP-BKM120 and NVP-BEZ235 (kindly provided by Novartis) induced a dose-dependent growth inhibition as measured by MTS assay. Despite the observed strong anti-myeloma activity of the mToR inhibitors rapamycin and everolimus with IC50 values in the nm range, the AKT pathway was activated as indicated by increased phosphorylation at Ser473. This observation was also made in explanted plasma cell tumors of INA-6 xenografted SCID mice that were treated with rapamycin, indicating that this feedback loop is also active in vivo. Therefore, combining mToR and PI3K inhibitors could be an effective strategy to overcome rapamycin-induced AKT activation. In fact, rapamycin in combination with the selective PI3K inhibitor Ly294002 or NVP-BKM120 led to synergistic growth inhibition in plasma cell lines, as calculated by the Calcusyn™ software (Biosoft). This abrogation of AKT activation was also seen in Western blot analysis. Combined treatment also enhanced the induction of apoptosis in cell lines as well as in plasmacytoma cells. Interestingly, the activity of the dual inhibitor NVP-BEZ235 could be even further enhanced by the addition of rapamycin. Conclusion:First clinical trials with mToR inhibitors showed anti-tumor activity and an acceptable safety profile in patients with multiple myeloma. The data presented here suggest that a combination of mToR inhibitors with PI3K targeting compounds or the use of dual inhibitors may have a favorable therapeutic potential. Disclosures:Guenther:Novartis, Celgene: Consultancy, Research Funding. Gramatzki:Novartis, Celgene: Consultancy, Research Funding.

PTEN reconstitution alters glioma responses to c-Met pathway inhibition

Mutations/deletions of the tumor-suppressor phosphatase and tensin homolog PTEN result in PI3K/Akt pathway hyperactivation and potentially alter oncogenic responses to targeted receptor tyrosine kinase inhibitors. We previously showed that hepatocyte growth factor (HGF):c-Met pathway inhibition decreases tumor growth and oncogenic signaling responses in PTEN-null/Met+ gliomas. Here, we use two tet-on PTENwt-inducible glioma cell lines and xenograft models to examine the influence of PTEN on oncogenic signaling responses to HGF:c-Met pathway inhibitors. Reconstitution of PTEN inhibited Akt by more than 80% and inhibited cell growth by approximately 70-75% in both cell lines in vitro. C-Met inhibition alone inhibited in-vitro cell growth by approximately 80-85% and the magnitude of growth inhibition was not altered by combining PTEN reconstitution with c-Met inhibition. Combining PTEN reconstitution with Met inhibition arrested a higher percentage of cells in G(1)/G(0) phase of the cell cycle when compared with either PTEN reconstitution or c-Met inhibition alone. Both PTEN reconstitution alone and inhibiting autocrine HGF:c-Met signaling alone, using anti-HGF mAb, robustly inhibited the growth of subcutaneous and intracranial glioma xenografts. Combining anti-HGF therapy with PTEN reconstitution did not significantly alter the magnitude of xenograft growth inhibition. Semiquantitative immunohistopathological analyses revealed that the inhibition of glioma xenograft angiogenesis and cell proliferation by anti-HGF mAb was greatest in conjunction with PTEN reconstitution. In contrast, xenograft cell apoptosis was greatest in response to anti-HGF therapy alone and PTEN reconstitution abrogated the apoptotic response to anti-HGF therapy. These results provide new insights into how PTEN modulates glioma responses to the inhibition of HGF:c-Met signaling and possibly other receptor tyrosine kinase pathways.

Relapsed Diffuse Large B-Cell Lymphoma: Clinical Utility of Cell of Origin

Relapsed Diffuse Large B-Cell Lymphoma: Clinical Utility of Cell of Origin

Identification of microprocessor-dependent cancer cells allows screening for growth-sustaining micro-RNAs

Micro-RNAs are deregulated in cancer cells, and some are either tumor suppressive or oncogenic. In addition, a link has been established between decreased expression of micro-RNAs and transformation, and several proteins of the RNA interference pathway have been shown to be haploinsufficient tumor suppressors. Oncogenic micro-RNAs (oncomiRs) could represent new therapeutic targets, and their identification is therefore crucial. However, structural and functional redundancy between micro-RNAs hampers approaches relying on individual micro-RNA inhibition. We reasoned that in cancer cells that depend on oncomiRs, impairing the micro-RNA pathway could lead to growth perturbation rather than increased tumorigenesis. Identifying such cells could allow functional analyses of individual micro-RNAs by complementation of the phenotypes observed upon global micro-RNA inhibition. Therefore, we developed episomal vectors coding for small hairpin RNAs targeting either Drosha or DGCR8, the two components of the microprocessor, the nuclear micro-RNA maturation complex. We identified cancer cell lines in which both vectors induced colony growth arrest. We then screened for individual micro-RNAs complementing this growth arrest, and identified miR-19a, miR-19b, miR-20a and miR-27b as major growth-sustaining micro-RNAs. However, the effect of miR-19a and miR-19b was only transient. In addition, embryonic stem cell-derived micro-RNAs with miR-20a seeds were much less efficient than miR-20a in sustaining cancer cell growth, a finding that contrasted with results obtained in stem cells. Finally, we showed that the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10, a shared target of miR-19 and miR-20, was functionally involved in the growth arrest induced by microprocessor inhibition. We conclude that our approach allowed to identify microprocessor-dependent cancer cells, which could be used to screen for growth-sustaining micro-RNAs. This complementation screen unveiled functional differences between homologous micro-RNAs. Phenotypic characterization of the complemented cells will allow precise functional studies of these micro-RNAs.

Inactivation of Rac1 reduces Trastuzumab resistance in PTEN deficient and insulin-like growth factor I receptor overexpressing human breast cancer SKBR3 cells

Drug resistance remains to be a big challenge in applying anti-HER2 monoclonal antibody Trastuzumab for treating breast cancer with HER2 overexpression. Amplification of insulin-like growth factor I receptor (IGF-IR) and deletion of tumor suppressor phosphatase and tensin homolog (PTEN) are implicated in Trastuzumab resistance, however, the underlying mechanisms have not been clearly defined. Activation of Rac1, a member of Rho GTPase family, is capable of causing cytoskeleton reorganization, regulating gene expression and promoting cell proliferation. To investigate the mechanism of Trastuzumab resistance, PTEN knockdown and IGF-IR overexpressing stable cell lines were generated in HER2 overexpression human breast cancer SKBR3 cells. Rac1 was highly activated in PTEN deficient and IGF-IR overexpressing Trastuzumab-resistant cells in a HER2-independent manner. Inactivation of Rac1 by using a Rac1 inhibitor NSC23766 or siRNA knocking down the expression of Tiam1, a guanine nucleotide exchange factor for Rac, significantly reduced Trastuzumab resistance in SKBR3 cells. Inhibition of Rac1 had no effect on the levels of phosphor-HER2 and phosphor-Akt, but significantly decreased the levels of cyclin D1 in Trastuzumab-resistant cells. Inhibition of Akt with an Akt inhibitor also significantly reduced Trastuzumab resistance. However, simultaneous inhibition of both Rac1 and Akt resulted in a significantly more decrease of Trastuzumab resistance than inactivation of Rac1 or Akt alone. These results suggest that Rac1 activation is critically involved in Trastuzumab resistance caused by PTEN deletion or IGF-IR overexpression. Simultaneous inhibition of Rac1 and Akt may represent a promising strategy in reducing Trastuzumab resistance in HER2 overexpression breast cancer.