PTEN-null Cell Lines Research Articles

Abstract 5451: Testing a combination of AKT inhibitor (AZD5363) with PARP inhibitor olaparib plus carboplatin in TNBC

Abstract Background: AKT, a hallmark of cancer in a wide range of solid tumors, is a serine/threonine protein kinase. Inhibition of AKT is known to cause the perturbation of cell proliferation signals and induction of apoptosis in several preclinical in vitro and in vivo models. AKT inhibitor AZD5363 shows promise in different solid tumors in patients with activating mutations of AKT (E17K) (Hyman D. M. et al., JCO 2017), the efficacy is not substantial in patients with PIK3CA mutations (Banerji. U, CCR 2017). PI3K-PTEN-AKT pathway alterations are very common in TNBC. Alterations of AKT1/2/3 (15%), PTEN (29%) and PIK3CA (25%) mediated upregulation of the PI3K pathway are common in TNBC (De et al., 2016; cBioPortal). In our Avera cohort (January 2014-June 2017) of TNBC patients (n=93), we have recorded a total of 16.5 % alteration in AKT1/2/3. We have also identified an integral role of the PI3K-AKT pathway in the DDR-mediated antitumor action of PARP inhibitor in TNBC (De et al., 2014). Aim: Here we tested the role of genetic background in determining the effectiveness of a combination of AZD5363 with PARP inhibitor Olaparib (O, Lynparza) plus carboplatin (C) in TNBC. Methods: Using cell line based models of TNBC with defined genetic backgrounds, we studied proliferation (Incucyte) and cell cycle by PI (Accuri). Mechanistic induction of apoptosis was evaluated by live-cell triple immuno-fluorescence (IF) (MitoView Blue+NucView488 Casp3 substrate + CF 594 AnnexinV) with a simultaneous quantification of Annexin (Accuri). Long-term clonogenic response to triple combination was tested using 3D On-Top Matrigel assay. Result: AZD5363 treatment in PTEN-null SUM149 cells with BRCA1 mutation [2288delT (fs>735X)] responded synergistically to O and C with a dose-dependent increase apoptosis but with limited G1 increase. AZD5363 treatment in PTEN-null MDA-MB468 cells with wt BRCA1 led to an additive effect in slowing proliferation when combined with O and C and a very strong G1 increase but limited induction of apoptosis. AZD5363 as a single agent slowed proliferation and increased G1 in PIK3CA (P539R/H1074R) mutated BT20 cells with a concomitant increase in apoptosis. Mechanistically AZD5363 inhibited pophoyrlation of AKT substrates PRAS40 and GSKβ in TNBC cell lines. As expected KRAS/BRAF mutated (G13D/G464V), BRCA1 wt MDA-MB231 cells showed no effect on either proliferation or apoptosis. Best effect on the inhibition of clonogenic growth (reducing 3D colony formation and size as compared to control over single or double treatment) was recorded in SUM149 compared to MDA468 > BT20, > HCC1937. Summary: Here we present the effect of the combination of AZD5363 and O plus C in several TNBC cell line based models. Our data demonstrate that maximum effect of the triple combination is seen in PTEN-null cell line harboring BRCA1 mutations followed by PTEN-null cell BRCA1wt. Mechanistic work utlizing AKT1-E17K transfected cells is ongoing. Citation Format: Jennifer H. Carlson, Mark J. O'Connor, Casey Williams, Pradip De, Nandini Dey, Brian Leyland-Jones. Testing a combination of AKT inhibitor (AZD5363) with PARP inhibitor olaparib plus carboplatin in TNBC [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 5451.

Abstract 5802: Efficacy of PI3Kβ inhibitor AZD8186 in PTEN-deficient triple-negative breast cancer

Abstract Background: Phosphatase and tensin homologue (PTEN) loss is a frequently disrupted tumor suppressor in cancer. PTEN serves as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway to promote balanced cell proliferation, survival and differentiation. Consequently, loss of PTEN function increases cell proliferation, decreases cell death and contributes to tumor initiation. PTEN loss occurs across a variety of cancer subtypes, and these PTEN deficient tumors are dependent on PI3Kβ activity. As a result, therapeutic approaches that focus on the inhibition of PI3Kβ isoform in PTEN-null cells are of interest. We evaluate the efficacy of PI3Kβ inhibitors focused on triple negative breast cancers with loss of PTEN function. Methods: We studied the effects of p110β inhibitor AZD8186 in a panel of ten TNBC cell lines, which included four PTEN-mutant cell lines. Three of four cell lines were confirmed to have PTEN loss on western blot. In vitro, cell proliferation assays were performed to determine the half maximal inhibitory concentration (IC50) after 3 days of treatment and to test the effects in combination with chemotherapy. We confirmed the sensitivity of the PTEN-null cell lines to AZD8186 with colony formation assays. Western blot analysis was performed to assess PTEN expression and PI3K pathway activation in MDA-MB-436 and MDA-MB-468 cell lines, and to evaluate effects of AZD8186 on PI3K signaling. Results: Cell lines with PTEN loss were significantly more sensitive to AZD8186 in vitro (p= 0.008). AZD8186 inhibited PI3K signaling. Western blot revealed decreased activation of pAKT, pGSK3β, pPRAS40 and pS6. Activation of other pathways were evaluated with no activation of the MAPK/ERK or MEK pathways appreciated. AZD8186 treatment resulted in increased apoptosis but did not have a significant effect on cell-cycle progression in PTEN-deficient cell lines. AZD8186 was not synergistic with eribulin, paclitaxel or carboplatin; combinations with novel targeted therapies in vitro and in vivo are ongoing. Conclusion: AZD8186 had single agent efficacy in PTEN-deficient triple negative breast cancer cell lines in vitro. Further study is needed to identify rationale combinations and in vivo efficacy. Citation Format: Nicci Owusu-Brackett, Ming Zhao, Argun Akcakanat, Kurt W. Evans, Erkan Yuca, Funda Meric-Bernstam. Efficacy of PI3Kβ inhibitor AZD8186 in PTEN-deficient triple-negative breast cancer [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 5802.

Inhibiting PI3Kβ with AZD8186 Regulates Key Metabolic Pathways in PTEN-Null Tumors.

Purpose: PTEN-null tumors become dependent on the PI3Kβ isoform and can be targeted by molecules such as the selective PI3Kβ inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, the consequences of inhibiting PI3Kβ are poorly defined.Experimental Design: To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models.Results: AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kβ also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis.Conclusions: This study identifies novel mechanistic biomarkers of PI3Kβ inhibition in PTEN-null tumors supporting the concept that targeting PI3Kβ may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents. Clin Cancer Res; 23(24); 7584-95. ©2017 AACR.

Abstract 1780: Identifying preclinical predictive biomarkers to taselisib in breast cancer cell lines

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in cancer, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (GDC0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013). A panel of 50 breast cancer cell lines were profiled for sensitivity to taselisib using viability assays and subsequently correlated with PIK3CA mutations and expression of ER, PR, HER2 and PTEN. The PIK3CA mutant cell lines displayed an average of 30-fold greater sensitivity in IC50 compared to the PIK3CA wild-type cell lines. PTEN null cell lines were largely refractory to taselisib. Within the PIK3CA wild-type cell lines, the HER2 amplified cells showed an average of 18-fold greater sensitivity in IC50 over the HER2 wild-type cell lines. No differences in taselisib sensitivity were seen between the kinase, helical, or the C2 domain of the PIK3CA gene within the PIK3CA mutant cell lines. In HER2 amplified PIK3CA wild-type cell lines, taselisib did not fully suppress pAKT and pS6 signaling, however full suppression was observed in combination with the HER2 inhibitor, lapatinib. Increased apoptosis was observed with the combination of taselisib and lapatinib in the HER2 amplified, PIK3CA mutant cells, but not in HER2 amplified, PIK3CA wild-type cells. In PIK3CA mutant luminal cell lines, taselisib induced ESR1 transcriptional activity, but not in PIK3CA wild-type luminal lines, as assessed using PR, GREB1 and IGFBP4 gene expression. This induction was ER- and estradiol-dependent, and was suppressed using the estrogen degrader, fulvestrant. Lastly, as up to 40% of ER+, second line PIK3CA mutant metastatic tumors have been shown to harbor a mutation in the ESR1 gene (Spoerke JM et al., Nat Commun, 2016), we assessed the crosstalk between the PI3K and ER pathways in PIK3CA mutant MCF-7 cells that overexpress two ESR1 hotspot mutations. These ESR1 mutant cell lines displayed increased ER transcriptional activity, which was further activated by taselisib, an observation that was reversed with fulvestrant co-treatment. These data suggest that the combination of taselisib and an estrogen degrader can suppress crosstalk between PI3K and ER in both an ESR1 mutant and wild-type background, and supports the ongoing SANDPIPER study of taselisib in combination with fulvestrant in metastatic ER+, PIK3CA mutant cancers that have progressed following an aromatase inhibitor therapy (NCT02340221). Citation Format: Heidi M. Savage, Heather M. Moore, Carol L. O'Brien, Wei Zhou, Ciara Metcalfe, Mark R. Lackner, Timothy R. Wilson. Identifying preclinical predictive biomarkers to taselisib in breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1780. doi:10.1158/1538-7445.AM2017-1780

Anti-EGFR monoclonal antibodies enhance sensitivity to DNA-damaging agents in BRCA1-mutated and PTEN-wild-type triple-negative breast cancer cells.

Increased epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer (TNBC) is recognized as a promising therapeutic target, specifically through the use of selective EGFR inhibitors combined with chemotherapies. TNBC is characterized by genetic instability that leads to increased sensitivity to cytotoxic agents. We analyzed the effect of anti-EGFR monoclonal antibodies (mAbs; cetuximab and panitumumab) in combination with chemotherapeutic agents (docetaxel, cisplatin, and epirubicin) on EGFR-expressing TNBC cell lines that have different mutation statuses for one oncogene (KRAS) and two tumor suppressor genes (PTEN and BRCA1). Both mAbs failed to improve the cytotoxic effect of chemotherapies in the KRAS mutant cell line (MDA-MB-231) and PTEN-null cell lines (HCC-1937 and MDA-MB-468). In contrast, mAbs combined with DNA-damaging agents (cisplatin or epirubicin) had a synergistic effect in the BRCA1-mutant cell line SUM-1315 (wild-type KRAS and PTEN). The reintroduction of wild-type BRCA1 into SUM-1315 cells abolished this synergism. The improved effect of combination therapy was associated with cell cycle arrest at G1 phase and inhibition of the phosphorylation of EGFR and ERK1/2 proteins. These results suggest that patients with BRCA1-associated TNBC without genetic alterations in the PTEN and KRAS genes may have improved therapeutic responses to anti-EGFR mAbs combined with DNA-damaging agents. © 2017 Wiley Periodicals, Inc.

The use of whole genome copy number variation (CNV) to measure genomic instability in mCRPC CTCs.

307 Background: AR targeted therapies in combination with PARP inhibitors have recently shown efficacy in mCRPC patients with specific DNA repair gene mutations in metastatic tissue biopsies. Homologous recombination DNA repair deficiencies (HRD), associated with response to PARP inhibitors, can also be assessed by genomic instability/scarring. Accurate genomic scarring measurement in tumors can be confounded by intra-tumor heterogeneity and/or non-tumor genome contamination. To better identify PARPi sensitivity in mCRPC patients, we developed a genomic instability and scarring assay starting from single CTCs. Methods: VCaP, LnCaP or PC3 cell lines were spiked into healthy donor blood. Individual spiked cells were identified and recovered for genomic analysis using the standard Epic CTC assay. Post recovery, cells were lysed, whole genome amplified, constructed into shotgun libraries and sequenced to ~2M 2x150bp PE reads. Following alignment, whole genome copy number and instability analysis was performed to identify large scale transitions (LST, n of chromosomal breaks between adjacent regions of at least 10 Mb), % of genome altered (percentage of 1Mbp bins with copy number alterations), as well as specific tumor suppressor/oncogene copy number alterations. The association of PTEN loss with increases in genomic instability and scaring was performed. Results: Loss of PTEN function was previously shown to be associated with genomic instability. Our assessment of PTEN deletion was confirmed in PC3, while at least one genomic copy was observed in LnCaP and VCaP. The number of LSTs and % of genome altered was higher in PC3 (n = 19 +/- 3; 9.3% +/- 2.6%) than both VCaP and LnCap (n = 8 +/- 2; 6.1% +/- 0.4%). Conclusions: The association of higher genomic instability in a PTEN null cell line (PC3) vs. those with either heterozygous (LnCaP) or wild type (VCaP) PTEN status, matches published reports associating PTEN loss with increased genomic instability. Although, PC3 only demonstrates mild PARPi sensitivity in vitro, the detection of increased genomic scarring vs. cells with at least 1 functional PTEN allele confirms the assays ability to quantify genome instability at the single cell level from CTCs in a liquid biopsy.

Abstract 1949: Oncogenic signaling in amphiregulin- and EGFR-expressing PTEN-null human breast cancer

Abstract A subset of triple negative breast cancer (TNBC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) and loss of PTEN, and patients with these factors have a poor prognosis. In addition, approximately half of all basal breast cancers show autocrine activation of EGFR by amphiregulin (AREG), resulting in altered receptor trafficking and downstream signaling. However, despite the strong rationale for targeting EGFR in the treatment of TNBC, EGFR inhibitors have performed poorly in clinical trials. To further analyze the signaling processes active in this subset of TNBC, we performed reverse-phase protein array (RPPA) analysis on SUM149 cells, a highly tumorigenic and metastatic PTEN-null cell line that expresses high levels of AREG and EGFR, and MCF10A cells, a non-transformed mammary epithelial cell line cultured in the presence of either EGF or AREG. This analysis identified a number of signaling components mediated specifically by amphiregulin, including marked changes in integrin expression and increased levels of fibronectin, which is known to be a mediator of invasive capacity via interaction with integrin β1. RPPA analysis also revealed elevated AKT activity in SUM-149 cells, which was not surprising given the lack of PTEN expression. Interestingly, this AKT activity is not abrogated by treatment with the EGFR inhibitor gefitinib, nor by treatment with PI3K inhibitors. These findings and the results of preliminary experiments in SUM149 cells re-expressing PTEN suggest that AKT activity in these cells is, at least partly, independent of EGFR and PI3K activity. In addition, expression profiling demonstrated that AREG-activated EGFR regulates gene expression differently than EGF-activated EGFR, and functional analysis via genome-scale shRNA screening identified a set of genes, including PLK1 and BIRC5, that are regulated by EGFR in MCF10A cells but uncoupled from EGFR signaling in SUM149 cells, although they are essential for the survival of SUM-149 cells. Thus, our results demonstrate that in cells with constitutive EGFR activation and PTEN loss, critical survival genes are uncoupled from regulation by EGFR, a mechanism that is likely to mediate resistance to EGFR inhibitors. Citation Format: Christiana S. Kappler, Stephen T. Guest, Ericka L. Smith, Jonathan C. Irish, Elizabeth Garrett-Mayer, Zachary Kratche, Stephen Ethier. Oncogenic signaling in amphiregulin- and EGFR-expressing PTEN-null human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2015-1949

Switch in signaling control of mTORC1 activity after oncoprotein expression in thyroid cancer cell lines.

Thyroid growth is regulated by TSH and requires mammalian target of rapamycin (mTOR). Thyroid cancers frequently exhibit mutations in MAPK and/or phosphoinositol-3-kinase-related kinase effectors. The objective of the study was to explore the contribution of RET/PTC, RAS, and BRAF to mTOR regulation and response to mTOR inhibitors. PCCL3 cells conditionally expressing RET/PTC3, HRAS(G12V), or BRAF(V600E) and human thyroid cancer cells harboring mutations of these genes were used to test pathways controlling mTOR and its requirement for growth. TSH/cAMP-induced growth of PCCL3 cells requires mTOR, which is stimulated via protein kinase A in a MAPK kinase (MEK)- and AKT-independent manner. Expression of RET/PTC3, HRAS(G12V), or BRAF(V600E) in PCCL3 cells induces mTOR but does not entirely abrogate the cAMP-mediated control of its activity. Acute oncoprotein-induced mTOR activity is regulated by MEK and AKT, albeit to differing degrees. By contrast, mTOR was not activated by TSH/cAMP in human thyroid cancer cells. Tumor genotype did not predict the effects of rapamycin or the mTOR kinase inhibitor AZD8055 on growth, with the exception of a PTEN-null cell line. Selective blockade of MEK did not influence mTOR activity of BRAF or RAS mutant cells. Combined MEK and mTOR kinase inhibition was synergistic on growth of BRAF- and RAS-mutant thyroid cancer cells in vitro and in vivo. Thyroid cancer cells lose TSH/cAMP dependency of mTOR signaling and cell growth. mTOR activity is not decreased by the MEK or AKT inhibitors in the RAS or BRAF human thyroid cancer cell lines. This may account for the augmented effects of combining the mTOR inhibitors with selective antagonists of these oncogenic drivers.

The positive correlation between DJ‐1 and β‐catenin expression shows prognostic value for patients with glioma

The relationship between DJ-1 and β-catenin, and its impact on the prognosis for glioma patients has not been fully understood. This study determined the effect of DJ-1 on β-catenin and the prognostic significance of this interaction in glioma patients. We collected tumor specimens from 88 glioma patients and determined the expression of DJ-1, β-catenin and PTEN by using immunohistochemical staining. The involvement of DJ-1 and β-catenin in glioma cell lines was evaluated by immunohistochemistry and Western blotting. High DJ-1 expression (37.5%) and high β-catenin expression (34.1%) in glioma specimens were significantly associated with high grade and poor prognosis in glioma patients. However, only high levels of DJ-1 (P = 0.014) was a strong independent prognostic factor, correlated with a reduced overall survival time. In vitro DJ-1 expression was positively correlated with the expression levels of β-catenin and p-Akt, and negatively correlated with PTEN expression in U87, U251 MG, SWO-38 and SHG44 human glioma cell lines. After the knockdown of DJ-1, Akt, p-Akt or β-catenin expression levels were not affected in the PTEN-null cell lines (U87 and U251 MG). However, in the SWO-38 cell line, which has wild-type PTEN protein, the level of PTEN increased while Akt/p-Akt and β-catenin levels were reduced. Furthermore, β-catenin staining weakened in SWO-38 cells after DJ-1 levels decreased according to immunocytochemical analysis. In conclusion, DJ-1 and β-catenin may contribute to the development and recurrence of glioma and are valuable prognostic factors for glioma patients. DJ-1 may regulate β-catenin expression via PTEN and p-Akt.

Abstract B12: PTEN modulates sensitivity to a novel ERK inhibitor in BRAFV600E-mutant melanomas

Abstract The presence of activating BRAF mutations in approximately 50% of melanomas, the majority of which are BRAFV600E, has prompted the development of selective inhibitors of the BRAF/MAPK/ERK pathway for targeted therapy. RAF inhibitors, including vemurafenib and GSK2118436, have shown striking results in clinical trials, highlighting this pathway as a suitable target for melanoma therapy. A number of other pharmacological agents targeting this pathway are currently in pre-clinical or clinical development. However, therapy with MAPK pathway inhibitors results in a wide range of response patterns in patients with BRAF-mutant tumors. Furthermore, even in patients with measurable responses, tumors eventually develop resistance and patients relapse. It has been previously reported that activation of the PI3K pathway may play a role modulating the response to RAF and MEK inhibitors. Here we report that melanoma cells which harbor the BRAFV600E mutation and express PTEN have low levels of phospho-AKT and are sensitive to a novel ATP-competitive ERK inhibitor. In contrast, BRAFV600E mutant melanoma cell lines that do not express PTEN have higher levels of phospho-AKT and are less sensitive to ERK inhibition. Importantly, we have confirmed these findings in an in vivo xenotransplantation model of melanoma. Additionally, we found that siRNA-mediated PTEN silencing in a PTEN wild-type cell line modestly decreases sensitivity to ERK inhibition. Conversely, PTEN overexpression in a PTEN-null cell line partially sensitizes cells to the ERK small molecule inhibitor. Consistent with these findings, targeting PI3K in melanoma cells lacking functional PTEN sensitizes them to ERK inhibition. Our studies suggest that the presence or absence of functional PTEN can modulate response to ERK inhibitors and warrant further evaluation of combination strategies to treat melanomas refractory to inhibitors of the MAPK pathway.

Characterization of a novel PTEN mutation in MDA-MB-453 breast carcinoma cell line

BackgroundCowden Syndrome (CS) patients with germ line point mutations in the PTEN gene are at high risk for developing breast cancer. It is believed that cells harboring these mutant PTEN alleles are predisposed to malignant conversion. This article will characterize the biochemical and biological properties of a mutant PTEN protein found in a commonly used metastatic breast cancer cell line.MethodsThe expression of PTEN in human breast carcinoma cell lines was evaluated by Western blotting analysis. Cell line MDA-MB-453 was selected for further analysis. Mutation analysis of the PTEN gene was carried out using DNA isolated from MDA-MB-453. Site-directed mutagenesis was used to generate a PTEN E307K mutant cDNA and ectopic expressed in PC3, U87MG, MCF7 and Pten-/- mouse embryo fibroblasts (MEFS). Histidine (His)-tagged PTEN fusion protein was generated in Sf9 baculovirus expression system. Lipid phosphatase and ubiquitination assays were carried out to characterize the biochemical properties of PTEN E307K mutant. The intracellular localization of PTEN E307K was determined by subcellular fractionation experiments. The ability of PTEN E307K to alter cell growth, migration and apoptosis was analyzed in multiple PTEN-null cell lines.ResultsWe found a mutation in the PTEN gene at codon 307 in MDA-MB-453 cell line. The glutamate (E) to lysine (K) substitution rendered the mutant protein to migrate with a faster mobility on SDS-PAGE gels. Biochemically, the PTEN E307K mutant displayed similar lipid phosphatase and growth suppressing activities when compared to wild-type (WT) protein. However, the PTEN E307K mutant was present at higher levels in the membrane fraction and suppressed Akt activation to a greater extent than the WT protein. Additionally, the PTEN E307K mutant was polyubiquitinated to a greater extent by NEDD4-1 and displayed reduced nuclear localization. Finally, the PTEN E307K mutant failed to confer chemosensitivity to cisplatinum when re-expressed in Pten-/- MEFS.ConclusionsMutation at codon 307 in PTEN C2 loop alters its subcellular distribution with greater membrane localization while being excluded from the cell nucleus. This mutation may predispose breast epithelial cells to malignant transformation. Also, tumor cells harboring this mutation may be less susceptible to the cytotoxic effects of chemotherapeutics.

939 poster EFFECTS OF RADIATION-INDUCED ALPHAVBETA3 INTEGRIN MODULATION ON CILENGITIDE EFFICIENCY IN PEDIATRIC GLIOMA CELLS

The efficacy of PARP inhibition under treatment conditions of RT plus temozolomide (TMZ) for glioblastoma (GBM) has not been previously reported, therefore we sought to determine the effects of PARP inhibition as well as the molecular determinants of response to PARP inhibitors in combination with standard treatment of GBM. Materials: In the present study, PJ-34, a highly potent PARP inhibitor, was used to investigate the effects of PTEN status on PARP inhibition in combination with RT and TMZ using in vitro and in vivo models of GBM. First, two primary GBM cell lines that lack PTEN expression and two primary GBM cells lines that express PTEN were utilized to determine if PTEN status affects the efficacy of PARP inhibition in combination with RT and TMZ. Next, DNA damage and ATP levels were measured after the addition of a PARP inhibitor to RT and TMZ with respect to PTEN status. The role of glucose metabolism and PI3K/ AKT signaling in the response to PARP inhibition was then characterized in correlation to PTEN status. Finally, a U87-MG xenograft model was used to validate our in vitro findings. Results: Cell lines lacking PTEN were sensitized to PJ-34 in the setting of RT plus TMZ, whereas PTEN expression was associated with resistance. AKT activation was higher in resistant cell lines and was associated with increased ATP levels. Additionally, forced expression of PTEN conferred resistance to PJ-34. Treatment with the glycolytic inhibitor 2-deoxyglucose (2-DG) or the phosphatidylinosotide-3 kinase (PI3K) inhibitor LY294002 sensitized resistant PTEN-expressing cell lines to PJ-34 suggesting PI3K/AKT signaling is likely responsible for resistance in PTEN positive cells. The therapeutic benefits of PARP inhibition observed in PTEN-null cell lines may be dependent on RT, as treatment with TMZ alone prevented sensitization. Conclusions: In summary, PTEN status may be a promising marker to identify tumors that will respond to PARP inhibition when administered with TMZ and RT. Our findings suggest that the ability of PARP inhibitors, such as PJ34, to enhance the anti-tumor effects of RT and TMZ is highly dependent on the equilibrium between PARP’s pro-survival (increased PI3K/AKT signaling) and pro-death (increased DNA damage) functions. To this end, we are the first to report that the ability of PARP inhibition to enhance TMZ and RT response depends on the proteomic profile of GBM tumors, especially PTEN status.

Abstract B87: Genomic complexity and BRAF/MEK-dependence in V600E BRAF mutant melanoma

Abstract Constitutive ERK activation, often the result of BRAF mutation, is a common finding in human cancer. BRAF mutations occur in approximately 8% of human tumors, with the highest frequency observed in melanoma (40–70%). Supporting its classification as an oncogene, V600E BRAF stimulates ERK signaling, induces proliferation and is capable in model systems of promoting transformation. BRAF mutations are, however, common in nevi suggesting that BRAF mutation alone is insufficient for tumorigenesis. These observations suggest that additional alterations cooperate with BRAF in promoting melanoma formation and that inhibition of oncogenic BRAF may not be sufficient in many tumors to induce apoptosis in some BRAF mutant melanomas. Prior studies have demonstrated that loss of PTEN is a common event in melanoma and PTEN has been shown to cooperate with BRAF mutation in accelerating invasive melanoma formation in genetic murine models. With the goal of identifying model systems in which to study the the role played by PTEN in promoting tumorigenesis, we performed MALDI-TOF mass spectrometry genotyping on a panel of 116 melanoma cell lines. Seventy-two (62%) harbored BRAF mutations (66 V600E, 3 V600K, 3 in exon 11). Proteomic profiling of the class of V600E BRAF mutant cell lines demonstrated significant variability in the expression of PTEN and pAKT. Nine cells lines harboring V600E BRAF mutations were found to express no PTEN protein and high pAKT by immunoblotting. The majority of these cell lines had small (1–2) base-pair insertions and deletions leading to a frame shift and early truncation. Array CGH confirmed focal homozygous deletion of the PTEN gene in three of the models. These deletions were small and in one case detected by the Agilent 1M but not the 244K platform. We have previously reported that mutations of BRAF are associated with enhanced and selective sensitivity to MEK inhibition when compared to either receptor tyrosine kinase-driven cells or cells harboring a RAS mutation. To determine the MAPK pathway dependence of V600E BRAF mutant cell lines as a function of PTEN expression, we used PD0325901, a selective allosteric inhibitor of MEK1/2. A subset of the V600E BRAF mutant, PTEN null or mutant cells were dependent upon MEK for proliferation with IC50 values ranging from 1.1 to >500nM. These data suggest that PTEN loss is insufficient to confer absolute resistance to MAPK-pathway inhibition. MEK inhibition was, however, cytostatic in all V600E BRAF, PTEN null cell lines whereas MEK inhibition induced significant apoptosis in a subset of V600E BRAF, PTEN wild-type models. PTEN null, BRAF mutant cell lines were sensitive to two selective PI3 kinase inhibitors and the combination of MEK and PI3 kinase inhibition was more effective than either alone in a subset of models. In summary, our data suggest that PTEN is one of several concurrent genetic/epigenetic alterations that condition MEK-dependence in melanoma cell lines harboring V600E BRAF mutations. Given the promising preliminary data with BRAF and MEK selective inhibitors in early stage clinical trials, these data may aid in the identification of those patients most likely to benefit from selective inhibitors of the MAPK pathway. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B87.

Primary chemoprevention of endometrial hyperplasia with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone in the PTEN heterozygote murine model

PTEN mutations have been implicated in the development of endometrial hyperplasia and subsequent cancer. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists have demonstrated antineoplastic and chemopreventive effects. The purpose of this study was to evaluate the effects of the PPAR-gamma agonist rosiglitazone on both PTEN wild type and PTEN null cell lines and in the PTEN heterozygote((+/-)) murine model. Hec-1-A (PTEN wild type) and Ishikawa (PTEN null) cells were treated with rosiglitazone. Thirty-five female PTEN(+/-) mice were genotyped and placed into one of four groups for treatment for 18 weeks: A) PTEN wild type with 4 mg/kg rosiglitazone, B) PTEN(+/-) mice with vehicle, C) PTEN(+/-) mice with 4 mg/kg rosiglitazone, and D) PTEN(+/-) mice with 8 mg/kg rosiglitazone. Proliferation and apoptosis were measured by bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragmentation sites assay. Rosiglitazone caused cell growth inhibition in both Hec-1-A and Ishikawa in a dose-dependent manner (P < 0.02 and P < 0.03, respectively). Rosiglitazone also induced apoptosis in both Hec-1-A (P < .001) and Ishikawa (P < .001) cells in a dose-dependent manner. In the murine model, rosiglitazone decreased proliferation of the endometrial hyperplastic lesions (B vs C; 39.7% vs 9.3% and B vs D; 39.7% vs 4.2%; P < 0.0001) and increased apoptosis of glandular endometrial epithelial cells (B vs C; 2.8% vs 22.4%; P < 0.0001 and B vs D; 2.8% vs 30.2%; P = 0.003). PPAR-gamma agonist rosiglitazone inhibits proliferation and induces apoptosis in both PTEN intact and PTEN null cancer cell lines and in hyperplastic endometrial lesions in the PTEN(+/)(-)murine model.

MAGI-2 Inhibits cell migration and proliferation via PTEN in human hepatocarcinoma cells

MAGI-2, a multidomain scaffolding protein, contains nine potential protein–protein interaction modules, including a GuK domain, two WW domains and six PDZ domains. In this study, we examined eight human hepatocarcinoma cell lines (HHCCs) and found that MAGI-2 was expressed only in 7721 cells. After 7721, 7404 and 97H cells were transfected with myc-MAGI-2 plasmid, their migration and proliferation was significantly inhibited, which was associated with downregulation of p-FAK and p-Akt. It is known that p-FAK is a substrate of PTEN and p-Akt can be regulated by PTEN via PIP3. We demonstrated that PTEN was upregulated after myc-MAGI-2 transfection, which was due to the enhancement of PTEN protein stability rather than mRNA levels. Furthermore, MAGI-2-induced inhibition of cell migration and proliferation was attenuated in 7721 cells with PTEN silence or in PTEN-null cell line U87MG, and PTEN transfection could restore the effect of MAGI-2 in U87MG cells. Finally, the molecular association between PTEN and MAGI-2 was confirmed. Our results suggested that PTEN played a critical role in MAGI-2-induced inhibition of cell migration and proliferation in HHCCs.

Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain

The regulation of PTEN intrinsic biochemical properties has not been fully elucidated. In this report, we investigated the role of the PTEN carboxyl-terminal tail domain in regulating its membrane targeting and catalytic functions. Characterization of a panel of PTEN phosphorylation site mutants revealed that mutating Ser-385 to alanine (S385A) promoted membrane localization in vivo and phosphatase activity in vitro. Furthermore, S385A mutation was associated with a substantial reduction in the phosphorylation of the Ser-380/Thr-382/Thr-383 cluster. Therefore, Ser-385 could prime additional dephosphorylation events to regulate PTEN catalytic activity. Moreover, substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutation. Next, we further defined the underlying mechanisms responsible for the COOH-terminal tail region in modulating PTEN biological activity. We have identified an interaction between the 71-amino acid carboxyl-terminal tail region and the CBRIII motif of the C2 domain, which has been implicated in membrane binding. In addition, a synthetic phosphomimic peptide encompassing the phosphorylation site cluster between amino acids 368 and 390 within the tail region mediated the suppression of PTEN catalytic activity in vitro. This same peptide when expressed in cultured cells also impeded PTEN membrane localization and enhanced phospho-Akt levels. Thus, our data suggest that the COOH-terminal tail can act as an autoinhibitory domain to control both PTEN membrane recruitment and phosphatase activity.

Loss of Phosphatase and Tensin Homologue Increases Transforming Growth Factor β–Mediated Invasion with Enhanced SMAD3 Transcriptional Activity

In normal epithelial tissues, the multifunctional cytokine transforming growth factor-beta (TGF-beta) acts as a tumor suppressor through growth inhibition and induction of differentiation whereas in advanced cancers, TGF-beta promotes tumor progression through induction of tumor invasion, neoangiogenesis, and immunosuppression. The molecular mechanisms through which TGF-beta shifts from a tumor suppressor to a tumor enhancer are poorly understood. We now show a role for the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in repressing the protumorigenic effects of TGF-beta. The TGF-beta effector SMAD3 inducibly interacts with PTEN on TGF-beta treatment under endogenous conditions. RNA interference (RNAi) suppression of PTEN expression enhances SMAD3 transcriptional activity and TGF-beta-mediated induction of SMAD3 target genes whereas reconstitution of PTEN in a null cancer cell line represses the expression of TGF-beta-regulated target genes. Targeting PTEN expression through RNAi in a PTEN wild-type cell line increases TGF-beta-mediated invasion but does not affect TGF-beta-mediated growth inhibition. Reconstitution of PTEN expression in a PTEN-null cell line blocks TGF-beta-induced invasion but does not modulate TGF-beta-mediated growth regulation. These effects are distinct from Akt and Forkhead family members that also interact with SMAD3 to regulate apoptosis or proliferation, respectively. Pharmacologic inhibitors targeting TGF-beta receptors and phosphatidylinositol 3-kinase signaling downstream from PTEN cooperate to block TGF-beta-mediated invasion. Thus, the loss of PTEN expression in human cancers may contribute to a role for TGF-beta as a tumor enhancer with specific effects on cellular motility and invasion.

Physiological levels of PTEN control the size of the cellular Ins(1,3,4,5,6)P 5 pool

To understand how a signaling molecule's activities are regulated, we need insight into the processes controlling the dynamic balance between its synthesis and degradation. For the Ins(1,3,4,5,6)P 5 signal, this information is woefully inadequate. For example, the only known cytosolic enzyme with the capacity to degrade Ins(1,3,4,5,6)P 5 is the tumour-suppressor PTEN [J.J. Caffrey, T. Darden, M.R. Wenk, S.B. Shears, FEBS Lett. 499 (2001) 6 [24]], but the biological relevance has been questioned by others [E.A. Orchiston, D. Bennett, N.R. Leslie, R.G. Clarke, L. Winward, C.P. Downes, S.T. Safrany, J. Biol. Chem. 279 (2004) 1116 [1]]. The current study emphasizes the role of physiological levels of PTEN in Ins(1,3,4,5,6)P 5 homeostasis. We employed two cell models. First, we used a human U87MG glioblastoma PTEN-null cell line that hosts an ecdysone-inducible PTEN expression system. Second, the human H1299 bronchial cell line, in which PTEN is hypomorphic due to promoter methylation, has been stably transfected with physiologically relevant levels of PTEN. In both models, a novel consequence of PTEN expression was to increase Ins(1,3,4,5,6)P 5 pool size by 30–40% ( p < 0.01); this response was wortmannin-insensitive and, therefore, independent of the PtdIns 3-kinase pathway. In U87MG cells, induction of the G129R catalytically inactive PTEN mutant did not affect Ins(1,3,4,5,6)P 5 levels. PTEN induction did not alter the expression of enzymes participating in Ins(1,3,4,5,6)P 5 synthesis. Another effect of PTEN expression in U87MG cells was to decrease InsP 6 levels by 13% ( p < 0.02). The InsP 6-phosphatase, MIPP, may be responsible for the latter effect; we show that recombinant human MIPP dephosphorylates InsP 6 to d/ l-Ins(1,2,4,5,6)P 5, levels of which increased 60% ( p < 0.05) following PTEN expression in U87MG cells. Overall, our data add higher inositol phosphates to the list of important cellular regulators [Y. Huang, R.P. Wernyj, D.D. Norton, P. Precht, M.C. Seminario, R.L. Wange, Oncogene, 24 (2005) 3819 [57]] the levels of which are modulated by expression of the highly pleiotropic PTEN protein.

PTEN Regulates Mdm2 Expression through the P1 Promoter

MDM2 is an oncoprotein that controls tumorigenesis through both p53-dependent and -independent mechanisms. Mdm2 mRNA level is transcriptionally regulated by p53 in response to stress such as DNA damage, and its protein level and subcellular localization are post-translationally modulated by the AKT serine/threonine kinase. Previous studies showed that PTEN, a dual specificity phosphatase that antagonizes phosphatidylinositol 3-kinase/AKT signaling, is capable of blocking MDM2 nuclear translocation and destabilizing the MDM2 protein. Results from our current study demonstrate an additional role for PTEN in regulating MDM2 functions; PTEN modulates Mdm2 transcription and isoform selection by negatively regulating its P1 promoter. In Pten-null cell lines and prostate cancer tissues, Mdm2 P1 promoter activity is up-regulated, resulting in increased L-Mdm2 expression and enhanced p90(MDM2) isoform production. Furthermore, PTEN controls Mdm2 P1 promoter activity through its lipid phosphatase activity, independent of p53. Thus, our results provide a novel mechanism for PTEN in controlling MDM2 oncoprotein functions.