Nuclear PTEN Research Articles

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN–SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

MicroRNA-21 expression and its pathogenetic significance in cutaneous melanoma.

Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratify patients for molecular therapy. MicroRNAs are promising as tissue biomarkers because they can be assayed directly from formalin-fixed paraffin-embedded clinical samples. We previously reported that microRNA-21 (miR-21) was strongly expressed in melanoma relative to naevi and now sought to further assess the significance of this by assessing its relationship with its putative target, PTEN. Clinical melanoma samples were analysed by immunohistochemical analysis for PTEN, stem-loop qRT-PCR for miR-21 and PCR for BRAF/NRAS mutation status. Cell lines were investigated for the effect of anti-miR-21 on PTEN. A total of 81 clinical melanocytic tumour samples were investigated, with uniformly high PTEN expression in the nucleus and cytoplasm of naevi and with preferential loss of PTEN expression in the nucleus of melanoma cells. miR-21 expression was inversely associated with nuclear PTEN expression but not with cytoplasmic PTEN expression. An anti-miR-21 preferentially altered nuclear PTEN in melanoma cell lines. The presence of a BRAF or NRAS mutation had no significant effect on miR-21 expression. These data suggest miR-21 may exert an oncogenic effect in melanoma by favouring redistribution of PTEN to the nucleus.

Abstract B12: Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC

Abstract Background: TNBC is a highly aggressive form of BRCA-associated BC subtype for which chemotherapy still remains a major form of treatment to which patients initially respond yet a majority of them inevitably relapse. PARP1 has been identified as a target in BRCA-defined cancers and inhibitors of PARP1 has been recently approved by FDA as targeted agents in BRCA-defined cancers. The loss of PTEN is the most common “first event” associated with basal-like subtype (Martins et al., 2012) and this PI3K-pathway activating event (deletion/mutation/loss of PTEN) occurs more frequently (35%) than PIK3CA mutations in this subtype of BC (Ellis & Perou, 2013). In the cytoplasm, PTEN-loss mediated upregulation of PI3K/AKT signal is known to depend on the PI3Kbeta-isoform. In contrast nuclear PTEN controls DNA repair (Bassi et al., 2013). A failure to repair the damaged DNA upon inhibition of PARP causes accumulation of DNA double-strand breaks and leads to apoptosis of cancer cells. Thus we hypothesized that a combination of p110beta-isoform specific inhibitor and PARP inhibitor will sensitize PARP inhibitors in PTEN-null TNBC model. Methods: Five PARP inhibitors, Talazoparib (BMN673, B), Niraparib (N), Olaparib (O), Rucaparib (R) and Veliparib (V) were tested in four PTEN-null TNBC cell lines, MDA-MB468, HCC70 (p.F90fs*9), BT549 (p.V275fs*1) and SUM149 cell lines in combination with p110beta specific inhibitor, AZD6482. Proliferative, apoptotic and PARylation signals following drug combinations were demonstrated by WB in a dose and time dependent manner. Pro-apoptotic and anti-proliferative effects were verified using complementary 3D ON-TOP assay, real time proliferation (Incucyte), AnnexinV and cl-caspase3 analyses. In order to understand the initial effects of isoform-specific inhibitors of PI3K we also compared anti-proliferative signals of GDC-0032 (p110 beta sparing inhibitor) and GDC-0941 (pan PI3K inhibitor) with that of AZD6482 at 3/6 hours in MDA-MB468 and SUM149 cells. Results: In PTEN null TNBC cells 500 nM BMN673 alone was effective in slowing cell proliferation and inducing apoptosis while AZD6482 (both 5-10 uM) did not have anti-proliferative or pro-apoptotic effects at 48 and 72 hours. Combinations of different PARPi (100nM of B, 1uM of N, 10uM of O, 10uM of R and 10uM of V) with AZD6482 blocked 3D growth of PTEN null BT549 and MDA-MB468 TNBT cells in a time dependent manner (96 hours & 7 days). PARP inhibitors abrogated PAR signals either alone or in combination with carboplatin in both BRCA1/2 WT and mutated cells. The most pronounced anti-tumor effect was observed with the combination of B and AZD6482 which was mechanistically explained by the robust increase of AnnexinV positive cells following a single 500nM dose of B at both 48 and 72 hours. In both MDA-MB468 and SUM149 cells although panPI3K inhibitor, GDC-0941 downregulated pAKT (S473, T308), pP70S6K (T389), pS6RP and p4EBP1 (T37/46) levels, p110 alpha-specific inhibitor GDC-0032 failed to perturb these signals while AZD6482 blocked them all. In summary, we demonstrate a remarkable sensitivity of tumor cells to PARP inhibition in PTEN-defined TNBC models and identify that PTEN-nullness as a potential predictive context for a possible co-targeting of PI3K pathway to further sensitize TNBC to PARP inhibitors. Significance: Our findings squarely place PTEN expression at the nexus of oncogenic signals in TNBC and indicate that TNBC patients with no PTEN activity in their tumors might benefit from combined p110beta-isoform specific inhibitor and PARP inhibitor. Citation Format: Nandini Dey, Jennifer H. Carlson, Pradip De, Brian Leyland-Jones. Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B12.

The PTEN tumor suppressor gene and its role in lymphoma pathogenesis.

The phosphatase and tensin homolog gene PTEN is one of the most frequently mutated tumor suppressor genes in human cancer. Loss of PTEN function occurs in a variety of human cancers via its mutation, deletion, transcriptional silencing, or protein instability. PTEN deficiency in cancer has been associated with advanced disease, chemotherapy resistance, and poor survival. Impaired PTEN function, which antagonizes phosphoinositide 3-kinase (PI3K) signaling, causes the accumulation of phosphatidylinositol (3,4,5)-triphosphate and thereby the suppression of downstream components of the PI3K pathway, including the protein kinase B and mammalian target of rapamycin kinases. In addition to having lipid phosphorylation activity, PTEN has critical roles in the regulation of genomic instability, DNA repair, stem cell self-renewal, cellular senescence, and cell migration. Although PTEN deficiency in solid tumors has been studied extensively, rare studies have investigated PTEN alteration in lymphoid malignancies. However, genomic or epigenomic aberrations of PTEN and dysregulated signaling are likely critical in lymphoma pathogenesis and progression. This review provides updated summary on the role of PTEN deficiency in human cancers, specifically in lymphoid malignancies; the molecular mechanisms of PTEN regulation; and the distinct functions of nuclear PTEN. Therapeutic strategies for rescuing PTEN deficiency in human cancers are proposed.

BMET-28ROLE OF NUCLEAR PTEN IN THE BRAIN METASTASES MECHANISM

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a well-known tumor suppressor, then early studies proposed that PTEN was exclusively cytoplasmic. However, recent reports demonstrate that nuclear PTEN has important tumor suppressive function. Meanwhile recent report mentioned that PTEN mRNA expression was significantly downregulated in brain metastases compared with primary breast cancer. We studied the expression pattern of nuclear and cytoplasmic PTEN in 34 patients with primary tumors and brain metastases. These patients underwent surgical interventions at Hiroshima University Hospital, Japan, between 1995 and 2013. Clinical characteristics of patients are as follows. Sex: male14, female14. Mean age: primary 59.7y, metastases 62.1y. Primary tumors contained gastric, esophageal, colorectal, breast, ovarian, cervical, and prostate cancer. PTEN expression levels were immunohistochemically measured and categorized as negative (-), positive (+), or strong positive (++) expression. PTEN expression levels were compared using Mann-Whitney U test. In all analyses, differences with P < 0.05 were considered statistically significant. Results are as follows. −/+/++ (%): cytoplasmic primary 3:21:76, cytoplasmic metastases 26:62:12, nuclear primary 6:32:62, nuclear metastases 79:15:6. Study results demonstrate that PTEN expression levels are lower in brain metastases compared with in primary tumors. Especially, nuclear PTEN expression level was significantly downregulated in various brain metastases, so our findings confirm that decreased PTEN function by nuclear PTEN expression loss is associated with brain metastases.

PTEN Controls the DNA Replication Process through MCM2 in Response to Replicative Stress.

PTEN is a tumor suppressor frequently mutated in human cancers. PTEN inhibits the phosphatidylinositol 3-kinase (PI3K)-AKT cascade, and nuclear PTEN guards the genome by multiple mechanisms. Here, we report that PTEN physically associates with the minichromosome maintenance complex component 2 (MCM2), which is essential for DNA replication. Specifically, PTEN dephosphorylates MCM2 at serine 41 (S41) and restricts replication fork progression under replicative stress. PTEN disruption results in unrestrained fork progression upon replication stalling, which is similar to the phenotype of cells expressing the phosphomimic MCM2 mutant S41D. Moreover, PTEN is necessary for prevention of chromosomal aberrations under replication stress. This study demonstrates that PTEN regulates DNA replication through MCM2 and loss of PTEN function leads to replication defects and genomic instability. We propose that PTEN plays a critical role in maintaining genetic stability through a replication-specific mechanism, and this is a crucial facet of PTEN tumor suppressor activity.

PRL-3 Promotes the Malignant Progression of Melanoma via Triggering Dephosphorylation and Cytoplasmic Localization of NHERF1

Phosphatase of regenerating liver-3 (PRL-3) has been reported to have a critical role in metastatic progression of cancers. Here, we investigate how PRL-3 increases the malignant degree of melanoma cells. The expression of PRL-3 increased gradually during the malignant progression of melanoma. The phosphorylation of Akt was elevated in highly malignant melanoma cells, which was accompanied by a decrease in nuclear phosphatase and tensin homolog (PTEN). The phosphorylation of NHERF1 in the serine site was regulated by PRL-3 and showed cytoplasmic translocation upon dephosphorylation, which resulted in a decrease in nuclear PTEN. The co-translocation of NHERF1 and PTEN from the nucleus to the cytoplasm was observed during the malignant progression of melanoma cells. Tumor growth was inhibited significantly, and the survival was prolonged upon knockdown of cytoplasmic NHERF1 in B16BL6 cells prior to the inoculation into mice. Taken together, to our knowledge previously unreported, we have identified NHERF1 as a potential substrate of PRL-3. Its phosphorylation status as well as its change in cellular localization and association with PTEN correlated with the malignant progression of melanoma. Our data provide an explanation for how PRL-3 promotes the malignant progression of melanoma, as well as a diagnostic marker or therapeutic target for malignant melanoma.

BMI1, ATM and DDR.

BMI1, ATM and DDR.

Abstract IA10: Nuclear PTEN in regulation of genome integrity

Abstract Loss of PTEN function has been implicated in the etiology of numerous human malignancies, including sporadic brain, prostate, thyroid, breast, uterine and kidney tumors. PTEN germline have also been found in patients with PHTS, an autosomal-dominant cancer predisposition syndrome. Despite a thorough understanding of the inhibitory role for PTEN in the PI3K pathway, acting as a 3′PI phosphatase and directly antagonizing PI3'K, relatively little is known about PTEN's functions in the nucleus, a cellular compartment where it also resides. Our recent work has uncovered a nuclear function of PTEN in tumor suppression, which is independent from its 3′PI-phosphatase activity, but relies on PTEN protein phosphatase activity. Nuclear PTEN, which is SUMOylated, plays a prominent role in homologous recombination-based DNA repair, acting downstream of the protein kinase ATM. PTEN-deficient cells and tumors are impaired in their response to DNA damage and display heightened sensitivity to genotoxic agents, which may have significant implications for the treatment of PTEN-deficient cancers. Citation Format: Vuk Stambolic. Nuclear PTEN in regulation of genome integrity. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA10.

Activation of nuclear PTEN by inhibition of Notch signaling induces G2/M cell cycle arrest in gastric cancer.

Mutation in PTEN has not yet been detected, but its function as a tumor suppressor is inactivated in many cancers. In this study we determined that, activated Notch signaling disables PTEN by phosphorylation and thereby contributes to gastric tumorigenesis. Notch inhibition by small interfering RNA or γ-secretase inhibitor (GSI) induced mitotic arrest and apoptosis in gastric cancer cells. Notch inhibition induced dephosphorylation in the C-terminal domain of PTEN, which led to PTEN nuclear localization. Overexpression of activated Notch1-induced phosphorylation of PTEN and reversed GSI-induced mitotic arrest. Dephosphorylated nuclear PTEN caused prometaphase arrest by interaction with the cyclin B1-CDK1 complex, resulting in their accumulation in the nucleus and subsequent apoptosis. We found a correlation between high expression levels of Notch1 and low survival rates and, similarly, between reduced nuclear PTEN expression and increasing the TNM classification of malignant tumours stages in malignant tissues from gastric cancer patients. The growth of Notch1-depleted gastric tumors was significantly retarded in xenografted mice, and in addition, PTEN deletion restored growth similar to control tumors. We also demonstrated that combination treatment with GSI and chemotherapeutic agents significantly reduced the orthotopically transplanted gastric tumors in mice without noticeable toxicity. Overall, our findings suggest that inhibition of Notch signaling can be employed as a PTEN activator, making it a potential target for gastric cancer therapy.

Ndfip1 represses cell proliferation by controlling Pten localization and signaling specificity.

Pten controls a signaling axis that is implicated to regulate cell proliferation, growth, survival, migration, and metabolism. The molecular mechanisms underlying the specificity of Pten responses to such diverse cellular functions are currently poorly understood. Here we report the control of Pten activity and signaling specificity during the cell cycle by Ndfip1 regulation of Pten spatial distribution. Genetic deletion of Ndfip1 resulted in a loss of Pten nuclear compartmentalization and increased cell proliferation, despite cytoplasmic Pten remaining active in regulating PI3K/Akt signaling. Cells lacking nuclear Pten were found to have dysregulated levels of Plk1 and cyclin D1 that could drive cell proliferation. In vivo, transgene expression of Ndfip1 in the developing brain increased nuclear Pten and lengthened the cell cycle of neuronal progenitors, resulting in microencephaly. Our results show that local partitioning of Pten from the cytoplasm to the nucleus represents a key mechanism contributing to the specificity of Pten signaling during cell proliferation.

Lung neuroendocrine tumors: correlation of ubiquitinylation and sumoylation with nucleo-cytosolic partitioning of PTEN.

BackgroundThe tumor suppressor phosphatase and tensin homolog (PTEN) is a pleiotropic enzyme, inhibiting phosphatidyl-inositol-3 kinase (PI3K) signaling in the cytosol and stabilizing the genome in the nucleus. Nucleo-cytosolic partitioning is dependent on the post-translational modifications ubiquitinylation and sumoylation. This cellular compartmentalization of PTEN was investigated in lung neuroendocrine tumors (lung NET).MethodsTumor tissues from 192 lung NET patients (surgical specimens = 183, autopsies = 9) were investigated on tissue microarrays. PTEN was H-scored by two investigators in nucleus and cytosol using the monoclonal antibody 6H2.1. Results were correlated with immunoreactivity for USP7 (herpes virus-associated ubiquitin-specific protease 7) and SUMO2/3 (small ubiquitin-related modifier protein 2/3) as well as PTEN and p53 FISH gene status. Clinico-pathologic data including overall survival, proliferation rate and diagnostic markers (synaptophysin, chromogranin A, Mib-1, TTF-1) were recorded.ResultsThe multicentre cohort included 58 typical carcinoids (TC), 42 atypical carcinoids (AC), 32 large cell neuroendocrine carcinomas (LCNEC) and 60 small cell lung carcinomas (SCLC). Carcinoids were smaller in size and had higher synaptophysin and chromogranin A, but lower TTF-1 expressions. Patients with carcinoids were predominantly female and 10 years younger than patients with LCNEC/SCLC. In comparison to the carcinoids, LCNEC/SCLC tumors presented a stronger loss of nuclear and cytosolic PTEN associated with a loss of PTEN and p53. Concomitantly, a loss of nuclear USP7 but increase of nuclear and cytosolic SUMO2/3 was found. Loss of nuclear and cytosolic PTEN, loss of nuclear USP7 and increase of cytosolic SUMO2/3 thus correlated with poor survival. Among carcinoids, loss of cytosolic PTEN was predominantly found in TTF1-negative larger tumors of male patients. Among SCLC, loss of both cytosolic and nuclear PTEN but not proliferation rate or tumor size delineated a subgroup with poorer survival (all p-values <0.05).ConclusionsCellular ubiquitinylation and sumoylation likely influence the functional PTEN loss in high grade lung NET. Both nuclear and cytosolic PTEN immunoreactivity should be considered for correlation with clinico-pathologic parameters.

Cell autonomous phosphoinositide 3-kinase activation in oocytes disrupts normal ovarian function through promoting survival and overgrowth of ovarian follicles.

In this study, we explored the effects of oocytic phosphoinositide 3-kinase (PI3K) activation on folliculogensis by generating transgenic mice, in which the oocyte-specific Cre-recombinase induces the expression of constitutively active mutant PI3K during the formation of primordial follicles. The ovaries of neonatal transgenic (Cre+) mice showed significantly reduced apoptosis in follicles, which resulted in an excess number of follicles per ovary. Thus, the elevation of phosphatidylinositol (3,4,5)-trisphosphate levels within oocytes promotes the survival of follicles during neonatal development. Despite the increase in AKT phosphorylation, primordial follicles in neonatal Cre+ mice remained dormant demonstrating a nuclear accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These primordial follicles containing a high level of nuclear PTEN persisted in postpubertal females, suggesting that PTEN is the dominant factor in the maintenance of female reproductive lifespan through the regulation of primordial follicle recruitment. Although the oocytic PI3K activity and PTEN levels were elevated, the activation of primordial follicles and the subsequent accumulation of antral follicles with developmentally competent oocytes progressed normally in prepubertal Cre+ mice. However, mature Cre+ female mice were anovulatory. Because postnatal day 50 Cre+ mice released cumulus-oocyte complexes with developmentally competent oocytes in response to super-ovulation treatment, the anovulatory phenotype was not due to follicular defects but rather endocrine abnormalities, which were likely caused by the excess number of overgrown follicles. Our current study has elucidated the critical role of oocytic PI3K activity in follicular function, as well as the presence of a PTEN-mediated mechanism in the prevention of immature follicle activation.

PTEN expression in colorectal adenomas: Relationship to morphology and cell heterogeneity

IntroductionColorectal adenomas are the most frequent benign colorectal tumors. These tumors are characterized by dysplasia, low and high grade, the latter having the potential to evolve to adenocarcinoma.The aim of this study was to study the expression patterns of PTEN protein in a series of colorectal adenomas and the relationships to cell proliferation and CD133, marker of stem phenotype. MethodsColorectal adenomas were studied for the immunohistochemical expression of PTEN on tissue microarrays. PTEN expression was analysed with regard to morphological features and with regard to the Ki67 and CD133-positive cell compartments by using the Kendall rank-correlation test. ResultsPTEN was expressed in 92% adenomas, either in a cytoplasmic or nuclear pattern. Cytoplasmic PTEN was correlated to cytoplasmic CD133 (p=0.02, tau 0.191) while nuclear PTEN to decreased adenoma size and to tubular architecture (p=0.01, τ−0.184 and p=0.01, τ−0.183). Nuclear PTEN was also correlated to low grade intraepithelial neoplasia, while global PTEN (nuclear or cytoplasmic) was correlated to the presence of a decreased Ki67-positive component but with marginal significance (p=0.06, τ−0.144 and p=0.07, τ−0.213). ConclusionThe results of this study suggest a role for PTEN in colorectal adenoma morphogenesis and cell protein heterogeneity, being correlated to decreased size, tubular architecture and a high CD133-positive component.

Abstract 2402: Antitumor effect of a poly (ADP-ribose) polymerase (PARP) inhibitor in endometrial carcinoma cell lines

Abstract The loss of PTEN is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in the nucleus. Poly (ADP-ribose) polymerase (PARP) plays a key role in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in a panel of endometrial cancer cell lines. Methods The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN introduction on the sensitivity of cells to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN+ (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. Results The SF50 values were 100 nM or less in six of the 16 (38%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in five of the 16 (31%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 995 ± 1,096 nM; Wild-type [n = 4]: 144 ± 91 nM, p = 0.17 by Student'st test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN+ cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN+ cells. Conclusions Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. The inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer. Citation Format: Aki Miyasaka, Katsutoshi Oda, Yuji Ikeda, Tomoko Kashiyama, Takahiro Koso, Kanako Inaba, Tomohiko Fukuda, Chinami Makii, Kayo Asada, Reiko Kurikawa, Osamu Wada Hiraike, Kenbun Sone, Yuriko Uehara, Yoko Matsumoto, Takahide Arimoto, Hiroyuki Kuramoto, Tetsu Yano, Kei Kawana, Yutaka Osuga, Tomoyuki Fujii. Antitumor effect of a poly (ADP-ribose) polymerase (PARP) inhibitor in endometrial carcinoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2402. doi:10.1158/1538-7445.AM2014-2402

Abstract 2389: PTEN localizes to the nucleus and interacts with Rad52 in response to DNA damage

Abstract PTEN is a tumor suppressor with a dual phosphatase activity. Mutations of PTEN gene has been associated with many sporadic cancers and heritable neoplastic syndromes. PTEN also exhibits nuclear localization. However, the exact function of nuclear PTEN remains unclear. Here we show that PTEN exhibits a time-dependent nuclear translocation after DNA damage, co-localizing with DNA damage foci on chromatin, and that the nuclear translocation of PTEN is closely associated with its phosphorylation on S366/T370. Deletional analysis reveals that C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3β are involved in the phosphorylation of S366/T370, as well as PTEN's association with chromatin during DNA damage response. Significantly, PTEN specifically interacts with Rad52 and positively regulates its sumoylation. Taken together, these observations strongly suggest that PTEN may mediate DNA damage repair through interacting with and regulating Rad52 in the nucleus. Citation Format: Byeong Hyeok Choi, Wei Dai. PTEN localizes to the nucleus and interacts with Rad52 in response to DNA damage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2389. doi:10.1158/1538-7445.AM2014-2389

Abstract 406: A potential new mechanism for PTEN to maintain genome stability

Abstract Purpose: PTEN (phosphatase and tensin homolog) is among the most mutated or lost tumor suppressors. Recent studies revealed that nuclear PTEN represses tumorigenesis by stabilizing the centromere, a highly condensed heterochromatin domain featured with long array of DNA repeats (namely, satellite DNA), repressive histones, such as trimethylated Histone H3 at Lys9 residue (H3K9me3), and heterochromatin proteins. Our studies are aimed to explore the molecular mechanism that PTEN regulates this heterochromatin structure and thus maintaining genome stability. Methods and Results: Co-immunoprecipitation (Co-IP) assay showed that PTEN and the heterochromatin proteins directly interact. Chromatin immunoprecipitation (ChIP) assay showed that PTEN is enriched in satellite locus. Depletion of PTEN leads to abnormal heterochromatin structure, indicated by reduced H3K9m3 foci (detected by immunofluorescent staining) and occupancy to satellite locus (ChIP assay), decreased heterochromatin protein levels (Westernblot analysis), and overexpression of satellite transcripts (quantitative RT-PCR). Through a knockdown-and-mutant rescue strategy, we found that the C-terminal of PTEN is essential for its repressive function on satellite transcription. Furthermore, overexpression of the C-terminal truncated PTEN into PTEN-null BT549 cells, as compared to wild-type PTEN overexpression, lost its function to suppress cell growth (MTT assay), cell cycle progression (flow cytometry analysis) and protection from DNA damage (MTT and colony formation assays). Conclusions: Our finding showed that PTEN guards the genome stability through maintaining the normal structure of heterochromatin domain. Citation Format: Lili Gong, Shiaw-Yih Lin. A potential new mechanism for PTEN to maintain genome stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2014-406

A 9-protein biomarker molecular signature for predicting histologic type in endometrial carcinoma by immunohistochemistry.

Histologic typing may be difficult in a subset of endometrial carcinoma (EC) cases. In these cases, interobserver agreement improves when immunohistochemistry (IHC) is used. A series of endometrioid type (EEC) grades 1, 2, and 3 and serous type (SC) were immunostained for p53, p16, estrogen receptor, PTEN, IMP2, IMP3, HER2, cyclin B2 and E1, HMGA2, FolR1, MSLN, Claudins 3 and 4, and NRF2. Nine biomarkers showed significant differences with thresholds in IHC value scale between both types (p53 ≥ 20, IMP2 ≥ 115, IMP3 ≥ 2, cyclin E1 ≥ 220, HMGA2 ≥ 30, FolR1 ≥ 50, p16 ≥ 170, nuclear PTEN ≥ 2 and estrogen receptor ≤ 50; P < .005). This combination led to increased discrimination when considering cases satisfying 0 to 5 conditions predicted as EEC and those satisfying 6 to 9 conditions predicted as SC. This signature correctly predicted all 48 EEC grade 1-2 cases and 18 SC cases, but 3 SC cases were wrongly predicted as EEC. Sensitivity was 86% (95% confidence interval [CI], 64%-97%), and specificity was 100% (95% CI, 89%-100%). The classifier correctly predicted all 28 EEC grade 3 cases but only identified the EEC and SC components in 4 of 9 mixed EEC-SC. An independent validation series (29 EEC grades 1-2, 28 EEC grade 3, and 31 SC) showed 100% sensitivity (95% CI, 84%-100%) and 83% specificity (95% CI, 64%-94%). We propose an internally and externally validated 9-protein biomarker signature to predict the histologic type of EC (EEC or SC) by IHC. The results also suggest that mixed EEC-SC is molecularly ambiguous.

Cowden syndrome-associated germline SDHD variants alter PTEN nuclear translocation through SRC-induced PTEN oxidation.

Germline mutations in the PTEN tumor-suppressor gene and germline variations in succinate dehydrogenase subunit D gene (SDHD-G12S, SDHD-H50R) are associated with a subset of Cowden syndrome and Cowden syndrome-like individuals (CS/CSL) and confer high risk of breast, thyroid and other cancers. However, very little is known about the underlying crosstalk between SDHD and PTEN in CS-associated thyroid cancer. Here, we show SDHD-G12S and SDHD-H50R lead to impaired PTEN function through alteration of its subcellular localization accompanied by resistance to apoptosis and induction of migration in both papillary and follicular thyroid carcinoma cell lines. Other studies have shown elevated proto-oncogene tyrosine kinase (SRC) activity in invasive thyroid cancer cells; so, we explore bosutinib, a specific inhibitor for SRC, to explore SRC as a mediator of SDH-PTEN crosstalk in this context. We show that SRC inhibition could rescue SDHD dysfunction-induced cellular phenotype and tumorigenesis only when wild-type PTEN is expressed, in thyroid cancer lines. Patient lymphoblast cells carrying either SDHD-G12S or SDHD-H50R also show increased nuclear PTEN and more oxidized PTEN after hydrogen peroxide treatment. Like in thyroid cells, bosutinib decreases oxidative PTEN in patient lymphoblast cells carrying SDHD variants, but not in patients carrying both SDHD variants and PTEN truncating mutations. In summary, our data suggest a novel mechanism whereby SDHD germline variants SDHD-G12S or SDHD-H50R induce thyroid tumorigenesis mediated by PTEN accumulation in the nucleus and may shed light on potential treatment with SRC inhibitors like bosutinib in PTEN-wild-type SDHD-variant/mutation positive CS/CSL patients and sporadic thyroid neoplasias.

Loss of nuclear PTEN in HCV-infected human hepatocytes.

BackgroundHepatitis C virus (HCV) infection is a major risk factor for chronic hepatitis and hepatocellular carcinoma (HCC); however, the mechanism of HCV-mediated hepatocarcinogenesis is not well understood. Insufficiency of PTEN tumor suppressor is associated with more aggressive cancers, including HCC. We asked whether viral non-coding RNA could initiate oncogenesis in HCV infected human hepatocytes. The results presented herein suggest that loss of nuclear PTEN in HCV-infected human hepatocytes results from depletion of Transportin-2, which is a direct target of viral non-coding RNA, vmr11.MethodsThe intracellular distribution of PTEN in HCV-infected cells was monitored by immunostaining and Western blots of nuclear and cytoplasmic proteins. Effects of PTEN depletion were examined by comparing expression arrays of uninfected cells with either HCV-infected or vmr11-transfected cells. Target genes suggested by array analyses were validated by Western blot. The influence of nuclear PTEN deficiency on virus production was determined by quantitative analysis of HCV genomic RNA in culture media of infected hepatocytes.ResultsImport of PTEN to the nucleus relies on the interaction of Transportin-2 and PTEN proteins; we show that depletion of Transportin-2 by HCV infection or by the introduction of vmr11 in uninfected cells results in reduced nuclear PTEN. In turn, nuclear PTEN insufficiency correlates with increased virus production and the induction of γ-H2AX, a marker of DNA double-strand breaks and genomic instability.ConclusionAn HCV-derived small non-coding RNA inhibits Transportin-2 and PTEN translocation to the nucleus, suggesting a direct viral role in hepatic oncogenesis.