PTEN Protein Stability Research Articles

Post-Translational Modification of PTEN Protein: Quantity and Activity.

Post-translational modifications play crucial roles in regulating protein functions and stabilities. PTEN is a critical tumor suppressor involved in regulating cellular proliferation, survival, and migration processes. However, dysregulation of PTEN is common in various human cancers. PTEN stability and activation/suppression have been extensively studied in the context of tumorigenesis through inhibition of the PI3K/AKT signaling pathway. PTEN undergoes various post-translational modifications, primarily including phosphorylation, acetylation, ubiquitination, SUMOylation, neddylation, and oxidation, which finely tune its activity and stability. Generally, phosphorylation modulates PTEN activity through its lipid phosphatase function, leading to altered power of the signaling pathways. Acetylation influences PTEN protein stability and degradation rate. SUMOylation has been implicated in PTEN localization and interactions with other proteins, affecting its overall function. Neddylation, as a novel modification of PTEN, is a key regulatory mechanism in the loss of tumor suppressor function of PTEN. Although current therapeutic approaches focus primarily on inhibiting PI3 kinase, understanding the post-translational modifications of PTEN could help provide new therapeutic strategies that can restore PTEN's role in PIP3-dependent tumors. The present review summarizes the major recent developments in the regulation of PTEN protein level and activity. We expect that these insights will contribute to better understanding of this critical tumor suppressor and its potential implications for cancer therapy in the future.

MiR-2110 induced by chemically synthesized cinobufagin functions as a tumor-metastatic suppressor via targeting FGFR1 to reduce PTEN ubiquitination degradation in nasopharyngeal carcinoma.

Tumor cell metastasis is the key cause of death in patients with nasopharyngeal carcinoma (NPC). MiR-2110 was cloned and identified in Epstein-Barr virus (EBV)-positive NPC, but its role is unclear in NPC. In this study, we investigated the effect of miR-2110 on NPC metastasis and its related molecular basis. In addition, we also explored whether miR-2110 can be regulated by cinobufotalin (CB) and participate in the inhibition of CB on NPC metastasis. Bioinformatics, RT-PCR, and in situ hybridization were used to observe the expression of miR-2110 in NPC tissues and cells. Scratch, Boyden, and tail vein metastasis model of nude mouse were used to detect the effect of miR-2110 on NPC metastasis. Western blot, Co-IP, luciferase activity, colocalization of micro confocal and ubiquitination assays were used to identify the molecular mechanism of miR-2110 affecting NPC metastasis. Finally, miR-2110 induced by CB participates in CB-stimulated inhibition of NPC metastasis was explored. The data showed that increased miR-2110 significantly suppresses NPC cell migration, invasion, and metastasis. Suppressing miR-2110 markedly restored NPC cell migration and invasion. Mechanistically, miR-2110 directly targeted FGFR1 and reduced its protein expression. Decreased FGFR1 attenuated its recruitment of NEDD4, which downregulated NEDD4-induced phosphatase and tensin homolog (PTEN) ubiquitination and degradation and further increased PTEN protein stability, thereby inactivating PI3K/AKT-stimulated epithelial-mesenchymal transition signaling and ultimately suppressing NPC metastasis. Interestingly, CB, a potential new inhibitory drug for NPC metastasis, significantly induced miR-2110 expression by suppressing PI3K/AKT/c-Jun-mediated transcription inhibition. Suppression of miR-2110 significantly restored cell migration and invasion in CB-treated NPC cells. Finally, a clinical sample assay indicated that reduced miR-2110 was negatively correlated with NPC lymph node metastasis and positively related to NPC patient survival prognosis. In summary, miR-2110 is a metastatic suppressor involving in CB-induced suppression of NPC metastasis.

Correction: High Frequency of PIK3R1 and PIK3R2 Mutations in Endometrial Cancer Elucidates a Novel Mechanism for Regulation of PTEN Protein Stability.

In the original version of this article (1), Fig. 3D was mistakenly replaced with Fig. 3C by the compositor during the production of the paper. Figure 3D has been corrected in the latest online HTML and PDF versions of the article. The publisher regrets the error.

Pentamidine Inhibits Ovarian Cancer Cell Proliferation and Migration by Maintaining Stability of PTEN in vitro.

PurposePentamidine is an anti-protozoal cationic aromatic diamidine drug and has been reported to exhibit anticancer properties. We aimed to identify the effect of pentamidine on proliferation and migration of human ovarian cancer (OC) cell lines and the related mechanisms.MethodsHO8910 and Caov3 ovarian cancer cells were treated with pentamidine. MTS and colony formation assays were used to detect the proliferation ability of cells. The migration of cells was detected using wound healing and transwell assays. The protein levels of PTEN, phosphorylated Akt, Akt, N-cadherin, E-cadherin and snail were detected by Western blotting. Immunoprecipitation and Western blotting were used to detect ubiquitination levels of PTEN.ResultsOur findings revealed that pentamidine inhibited both proliferation and migration of OC cells. Further investigation found that pentamidine increased the protein expression of PTEN and reduced phosphorylation levels of AKT in OC cells. Pentamidine treatment modulated PTEN stability through the ubiquitin/proteasome pathway. In addition, pentamidine inhibited the expression of N-cadherin and snail, and increased E-cadherin expression in a dose-dependent manner.ConclusionPentamidine is involved in the maintenance of PTEN protein stability and suppresses proliferation and migration of OC cells.

BGL3 inhibits papillary thyroid carcinoma progressionvia regulating PTEN stability.

BGL3, a novel long non-coding RNA (lncRNA) that plays a crucial role in several human malignancies. However, the clinical significance and biological function of BGL3 in papillary thyroid carcinoma (PTC) have not been explored. Herein, we aimed to investigate the role of BGL3 in human PTC. A total of 85 pairs of PTC and normal tissues were collected for clinicopathological analysis. Expression of BGL3 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of BGL3 on PTC cells ware determined by CCK-8, colony formation, EdU and wound healing assays. The molecular mechanism underlying BGL3 was tested by ChIP, Co-IP, RNA pull-down and luciferase reporter assays. In vivo experiments were conducted using xenografts in nude mice. BGL3 was significantly decreased in PTC tissues compared to adjacent normal thyroid tissues, and it was transcriptionally repressed by oncogene Myc. Low BGL3 is positively related to larger tumor size, lymph node metastasis, later TNM stage and poor prognosis. Overexpression of BGL3 inhibited PTC cell proliferation and migration in vitro, and reduced tumor size and lung metastasis nodules in vivo. BGL3 was mainly located in the cytoplasm, in which interacted with PTEN and recruited OTUD3, enhancing the de-ubiquitination effect of OTUD3 on PTEN, resulting in increasing PTEN protein stability and inactivating carcinogenic PI3K/AKT signaling. Our data underscore the critical tumor-inhibiting role of BGL3 in PTC via post-translational regulation of PTEN protein stability, which may serve as a novel therapeutic target and prognostic biomarker in human PTC.

YY1-regulated LINC00152 promotes triple negative breast cancer progression by affecting on stability of PTEN protein

Thousands of lncRNAs have been identified but few have been functionally characterized in triple negative breast cancer (TNBC). LINC00152 was known as cytoskeleton regulator RNA (CYTOR) and expressed in various cancers including breast cancer. But the underlying molecular mechanism of LINC00152 in pathogenesis of TNBC have not been elucidated. In our study, we identified that LINC00152 expression was dramatically elevated in TNBC tissue and cells. Inhibition or overexpression of LINC00152 obviously increased or suppressed PTEN protein expression but did not affect the mRNA expression level. Our further experiments showed up-regulated LINC00152 in TNBC obviously enhanced NEDD4-1 mediated ubiquitination and degradation of PTEN protein. Finally, we demonstrated that YY1 bound with LINC00152 promotor and mostly inhibited the transcription of LINC00152. Furthermore, analysis of clinical samples resource retrieved from databases suggested high LINC00152 expression was correlated with ER or PR negative expression, late TNM stage and lymphatic invasion, as well as shorter overall survival time in patients. Consequently, this study firstly reveals that up-regulated LINC00152 mediates PTEN protein stability attenuation in TNBC.

Deubiquitylase OTUD3 regulates PTEN stability and suppresses tumorigenesis.

PTEN is one of the most frequently mutated tumour suppressors and reduction in PTEN protein stability also plays a role in tumorigenesis. Although several ubiquitin ligases for PTEN have been identified, the deubiquitylase for de-polyubiquitylation and stabilization of PTEN is less defined. Here, we report OTUD3 as a deubiquitylase of PTEN. OTUD3 interacts with, de-polyubiquitylates and stabilizes PTEN. Depletion of OTUD3 leads to the activation of Akt signalling, induction of cellular transformation and cancer metastasis. OTUD3 transgenic mice exhibit higher levels of the PTEN protein and are less prone to tumorigenesis. Reduction of OTUD3 expression, concomitant with decreased PTEN abundance, correlates with human breast cancer progression. Furthermore, we identified loss-of-function OTUD3 mutations in human cancers, which either abolish OTUD3 catalytic activity or attenuate the interaction with PTEN. These findings demonstrate that OTUD3 is an essential regulator of PTEN and that the OTUD3-PTEN signalling axis plays a critical role in tumour suppression.

Post-translational regulation of PTEN catalytic function and protein stability in the hibernating 13-lined ground squirrel

BackgroundThe insulin signaling pathway functions as a major regulator of many metabolic and cellular functions, and has been shown to be reversibly suppressed in many species during hibernation. This study characterized the regulation of PTEN phosphatase, a negative regulator of the insulin receptor network, over the torpor–arousal cycle of hibernation in the skeletal muscle of Ictidomys tridecemlineatus. MethodsWestern blotting and RT-PCR were used to analyze post-translational and transcriptional regulations of PTEN respectively. Enzymatic activities were determined by the malachite green assay, while protein stability was assessed the using pulse-proteolysis method. ResultsDuring torpor, the ratio of non-phosphorylated PTEN (S380/T382/T383) was significantly elevated by 1.4-fold during late torpor compared with euthermic controls; this was coupled with an increase in substrate affinity for PIP3 (by 56%) in late torpor. Two proteolytic cleavage PEST motifs were identified in the C-terminus that overlapped with the phosphorylation sites of PTEN; pulse-proteolysis analysis of PTEN protein showed a decrease in protein stability during late torpor (Cm of urea decreased by 21%). Furthermore, the increase in PTEN activity observed was correlated with a decrease in PDK-1 phosphorylation by 32%, suggesting a downstream effect of PTEN activation during torpor. Transcriptional analysis showed that mRNA expression of pten and pdk-1 remain unchanged during hibernation, suggesting post-translation modification as the primary regulatory mechanism of PTEN function. ConclusionPhosphorylation plays an important role in the regulation of PTEN enzymatic activity and protein stability. General significanceActivation of PTEN during torpor can regulate insulin signaling during periods of low energy state.

Abstract A69: MCT-1 expression promotes neoplastic multinucleation through the Src/p190B signaling activation in triple-negative breast cancer cells

Abstract Multinucleation is associated with malignant neoplasm; however, the molecular mechanism underlying the nuclear abnormality remains unclear. Loss or mutation of PTEN is frequently implicated in the development of malignancies. We now demonstrate that increased expression of the oncogene MCT-1 antagonizes PTEN gene expression, protein stability and activation, thereby further promoting PI3K/AKT signaling and survival rate of the PTEN-deficient cells. MCT-1 interacts with p190B and Src in the triple negative breast cancer cells, supporting they are in proximity of the signaling complexes in vivo. MCT-1 overexpression and PTEN loss synergistically augmented the Src/p190B signaling function leading to inhibition of RhoA activity. Under such condition, the incidence of mitotic catastrophes including spindle multipolarity and cytokinesis failure were enhanced, driving a Src/p190B-dependent neoplastic multinucleation. Reduced MCT-1 expression by shRNA dramatically repressed the Src/p190B function and improved the nuclear structure, as well as suppressed tumorigenicity of the PTEN-null triple negative breast cancer cells. Consistent with the oncogenic effects in vitro, clinical evidence has confirmed that MCT-1 and p190B genes are highly expressed in human breast cancers. Accordingly, MCT-1 gene induction is recognized as a potentially indicator of breast tumor development. Abrogating MCT-1 function may be a promising strategy for treatment of the triple negative breast cancer involving aberrant activation of Src and/or dysfunction of PTEN. Citation Format: Meng-Hsun Wu, Yen-An Chen, Hsiao-Huei Chen, Ko-Wei Chang, I-Shou Chang, Lu-Hai Wang, Hsin-Ling Hsu. MCT-1 expression promotes neoplastic multinucleation through the Src/p190B signaling activation in triple-negative breast cancer cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A69. doi:10.1158/1538-7445.CHTME14-A69

Poly-ADP ribosylation of PTEN by tankyrases promotes PTEN degradation and tumor growth.

PTEN [phosphatidylinositol (3,4,5)-trisphosphate phosphatase and tensin homolog deleted from chromosome 10], a phosphatase and critical tumor suppressor, is regulated by numerous post-translational modifications, including phosphorylation, ubiquitination, acetylation, and SUMOylation, which affect PTEN localization and protein stability. Here we report ADP-ribosylation as a new post-translational modification of PTEN. We identified PTEN as a novel substrate of tankyrases, which are members of the poly(ADP-ribose) polymerases (PARPs). We showed that tankyrases interact with and ribosylate PTEN, which promotes the recognition of PTEN by a PAR-binding E3 ubiquitin ligase, RNF146, leading to PTEN ubiquitination and degradation. Double knockdown of tankyrase1/2 stabilized PTEN, resulting in the subsequent down-regulation of AKT phosphorylation and thus suppressed cell proliferation and glycolysis in vitro and tumor growth in vivo. Furthermore, tankyrases were up-regulated and negatively correlated with PTEN expression in human colon carcinomas. Together, our study revealed a new regulation of PTEN and highlighted a role for tankyrases in the PTEN-AKT pathway that can be explored further for cancer treatment.

MCT-1 expression and PTEN deficiency synergistically promote neoplastic multinucleation through the Src/p190B signaling activation.

Multinucleation is associated with malignant neoplasms; however, the molecular mechanism underlying the nuclear abnormality remains unclear. Loss or mutation of PTEN promotes the development of malignant tumors. We now demonstrate that increased expression of the oncogene MCT-1 (multiple copies in T-cell malignancy 1) antagonizes PTEN gene presentation, PTEN protein stability and PTEN functional activity, thereby further promoting phosphoinositide 3 kinase/AKT signaling, survival rate and malignancies of the PTEN-deficient cells. In the PTEN-null cancer cells, MCT-1 interacts with p190B and Src in vivo, supporting that they are in proximity of the signaling complexes. MCT-1 overexpression and PTEN loss synergistically augments the Src/p190B signaling function that leads to inhibition of RhoA activity. Under such a condition, the incidence of mitotic catastrophes including spindle multipolarity and cytokinesis failure is enhanced, driving an Src/p190B/RhoA-dependent neoplastic multinucleation. Targeting MCT-1 by the short hairpin RNA markedly represses the Src/p190B function, improves nuclear structures and suppresses xenograft tumorigenicity of the PTEN-null breast cancer cells. Consistent with the oncogenic effects in vitro, clinical evidence has confirmed that MCT-1 gene stimulation is correlated with p190B gene promotion and PTEN gene suppression in human breast cancer. Accordingly, MCT-1 gene induction is recognized as a potential biomarker of breast tumor development. Abrogating MCT-1 function may be a promising stratagem for management of breast cancer involving Src hyperactivation and/or PTEN dysfunction.

PIASxα Ligase Enhances SUMO1 Modification of PTEN Protein as a SUMO E3 Ligase

The tumor suppressor PTEN plays a critical role in the regulation of multiple cellular processes that include survival, cell cycle, proliferation, and apoptosis. PTEN is frequently mutated or deleted in various human cancer cells to promote tumorigenesis. PTEN is regulated by SUMOylation, but the SUMO E3 ligase involved in the SUMOylation of PTEN remains unclear. Here, we demonstrated that PIASxα is a SUMO E3 ligase for PTEN. PIASxα physically interacted with PTEN both in vitro and in vivo. Their interaction depended on the integrity of phosphatase and C2 domains of PTEN and the region of PIASxα comprising residues 134-347. PIASxα enhanced PTEN protein stability by reducing PTEN ubiquitination, whereas the mutation of PTEN SUMO1 conjugation sites neutralized the effect of PIASxα on PTEN protein half-life. Functionally, PIASxα, as a potential tumor suppressor, negatively regulated the PI3K-Akt pathway through stabilizing PTEN protein. Overexpression of PIASxα led to G0/G1 cell cycle arrest, thus triggering cell proliferation inhibition and tumor suppression, whereas PIASxα knockdown or deficiency in catalytic activity abolished the inhibition. Together our studies suggest that PIASxα is a novel SUMO E3 ligase for PTEN, and it positively regulates PTEN protein level in tumor suppression.

The deubiquitylase Ataxin-3 restricts PTEN transcription in lung cancer cells

The phosphatidylinositol-3-kinase (PI3K) pathway is commonly hyperactivated in cancer. One mechanism by which this occurs is by silencing of the phosphatase and tensin homolog (PTEN), a tumor suppressor and major antagonist of the pathway, through genetic, epigenetic or posttranscriptional mechanisms. Here, we used an unbiased siRNA screen in non-small-cell lung cancer cells to identify deubiquitylases (DUBs) that have an impact on PI3K signaling by regulating the abundance of PTEN. We found that PTEN expression was induced by depleting any of three members of the Josephin family DUBs: ataxin 3 (ATXN3), ataxin 3-like (ATXN3L) and Josephin domain containing 1 (JOSD1). However, this effect is not mediated through altered PTEN protein stability. Instead, depletion of each DUB increases expression of both the PTEN transcript and its competing endogenous RNA, PTENP1. In ATXN3-depleted cells, under conditions of transcriptional inhibition, PTEN and PTENP1 mRNAs rapidly decay, suggesting that ATXN3 acts primarily by repressing their transcription. Importantly, the PTEN induction observed in response to ATXN3 siRNA is sufficient to downregulate Akt phosphorylation and hence PI3K signaling. Histone deacetylase inhibitors (HDACi) have been suggested as potential mediators of PTEN transcriptional reactivation in non-small-cell lung cancer. Although PTEN exhibits a very limited response to the broad-spectrum HDACi Vorinostat (SAHA) in A549 cells, we find that combination with ATXN3 depletion enhances PTEN induction in an additive manner. Similarly, these interventions additively decrease cell viability. Thus, ATXN3 provides an autonomous, complementary therapeutic target in cancers with epigenetic downregulation of PTEN.

Deubiquitylation and stabilization of PTEN by USP13

The tumor suppressor PTEN is frequently lost in human cancers. In addition to gene mutations and deletions, recent studies have revealed the importance of post-translational modifications, such as ubiquitination, in the regulation of PTEN stability, activity and localization. However, the deubiquitinase that regulates PTEN poly-ubiquitination and protein stability remains unknown. Here we screened a total of 30 deubiquitinating enzymes (DUBs) and identified five DUBs that physically associate with PTEN. One of them, USP13, stabilizes PTEN protein via direct binding and deubiquitination of PTEN. Loss of USP13 in breast cancer cells promotes AKT phosphorylation, cell proliferation, anchorage-independent growth, glycolysis and tumor growth through downregulation of PTEN. Conversely, overexpression of USP13 suppresses tumorigenesis and glycolysis in PTEN-positive but not PTEN-null breast cancer cells. Importantly, USP13 protein is downregulated in human breast tumors and correlates with PTEN protein levels. These findings identify USP13 as a tumor-suppressing protein that functions through deubiquitination and stabilization of PTEN.

PD-1 Increases PTEN Phosphatase Activity While Decreasing PTEN Protein Stability by Inhibiting Casein Kinase 2

Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.

PD-1 Decreases PTEN Protein Stability While Increasing PTEN Phosphatase Activity by Inhibiting CK2.

Abstract 2145Maintenance of peripheral tolerance is essential for homeostasis of the immune system. The newer pathway of the B7:CD28 family consisting of the receptor PD-1 (CD279) and its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), plays a vital role in the induction and maintenance of T cell anergy and peripheral tolerance. This pathway also regulates the balance between stimulatory and inhibitory signals needed for effective immunity and maintenance of T cell homeostasis but can also compromise anti-tumor and anti-viral immunity. The precise mechanisms via which PD-1 mediates its inhibitory effects in T cells remain elusive. One of the critical signaling pathways targeted by PD-1 is the PI3K/Akt pathway, via which PD-1 mediates inhibitory effects on T cell proliferation, cytokine production and glucose uptake. In contrast to CTLA-4, which affects PI3/Akt signaling by targeting Akt through PP2A activation, PD-1 targets the PI3K/Akt pathway by directly inhibiting activation of PI3K. It has been proposed that PD-1 inhibits PI3K by upregulating PTEN (phosphatase and tensin homologue deleted on chromosome 10), a phosphoinositole 3,4,5-trisphosphatase important for antagonizing PI3K signaling, but the mechanism remains unclear. In the present study we sought to dissect the effects of PD-1 mediated signaling on the regulation of PTEN expression and function. We used highly purified primary human CD4+ T cells and magnetic beads conjugated with monoclonal antibodies (mAbs) against CD3 and CD28 with or without agonist mAb against PD-1. As determined by assessment of PTEN lipid phosphatase activity, stimulation of T cells via TCR/CD3, CD28 and PD-1 resulted in prominent increase of PTEN phosphatase activity compared to T cells stimulated via TCR/CD3 and CD28 without PD-1 signals. Surprisingly, although T cells receiving PD-1 signals displayed increased PTEN phosphatase activity they expressed lower levels of PTEN compared to T cells stimulated via TCR/CD3 and CD28, as determined by both western blot and by flow cytometry. In other cell types, the abundance of PTEN can be regulated transcriptionally and through phosphorylation-dependent modulation of protein stability. Assessment of PTEN mRNA by real time qPCR indicated that PTEN mRNA levels were comparable between T cells stimulated via TCR/CD3/CD28 and T cells stimulated via TCR/CD3/CD28 and PD-1. PTEN possesses a C-terminal non-catalytic regulatory domain, which plays an important role in its biological activity. A cluster of serine/threonine residues within this region is phosphorylated by the serine/threonine kinase casein kinase 2 (CK2). CK2-mediated phosphorylation stabilizes PTEN by preventing its ubiquitin-dependent degradation while decreasing PTEN phosphatase activity. In contrast, impaired phosphorylation in this region results in decreased PTEN protein stability but increased phosphatase activity. Because PD-1 signaling resulted in reduced PTEN protein expression but increased phosphatase activity, we examined whether PD-1 might alter CK2 activity and CK2-mediated phosphorylation of PTEN. In CD4+ T cells stimulated via TCR/CD3 and CD28, CK2 activation was robust and resulted in sustained phosphorylation on PTEN S380/T382/383 cluster. In contrast, T cells receiving PD-1 signals displayed impaired CK2 activity and significantly diminished phosphorylation of PTEN in the S380/T382/383 cluster. To determine whether downregulation of PTEN protein expression via PD-1 was mediated via proteasome-dependent degradation we used the proteasome inhibitor MG132. Stimulation of T cells via TCR/CD3, CD28 and PD-1 in the presence of MG132 prevented PD-1-mediated decrease of PTEN and resulted in reduced PTEN activity. Our results identify CK2 as a new target of PD-1-mediated signaling and reveal an unexpected mechanism via which PD-1 regulates PTEN phosphatase activity and inhibition of the PI3K/Akt pathway. Disclosures:No relevant conflicts of interest to declare.

Correction: High Frequency of PIK3R1 and PIK3R2 Mutations in Endometrial Cancer Elucidates a Novel Mechanism for Regulation of PTEN Protein Stability

Correction: High Frequency of <i>PIK3R1</i> and <i>PIK3R2</i> Mutations in Endometrial Cancer Elucidates a Novel Mechanism for Regulation of PTEN Protein Stability

High Frequency of PIK3R1 and PIK3R2 Mutations in Endometrial Cancer Elucidates a Novel Mechanism for Regulation of PTEN Protein Stability

We demonstrate that phosphatidylinositol 3-kinase (PI3K) pathway aberrations occur in >80% of endometrioid endometrial cancers, with coordinate mutations of multiple PI3K pathway members being more common than predicted by chance. PIK3R1 (p85α) mutations occur at a higher rate in endometrial cancer than in any other tumor lineage, and PIK3R2 (p85β), not previously demonstrated to be a cancer gene, is also frequently mutated. The dominant activation event in the PI3K pathway appears to be PTEN protein loss. However, in tumors with retained PTEN protein, PI3K pathway mutations phenocopy PTEN loss, resulting in pathway activation. KRAS mutations are common in endometrioid tumors activating independent events from PI3K pathway aberrations. Multiple PIK3R1 and PIK3R2 mutations demonstrate gain of function, including disruption of a novel mechanism of pathway regulation wherein p85α dimers bind and stabilize PTEN. Taken together, the PI3K pathway represents a critical driver of endometrial cancer pathogenesis and a novel therapeutic target.

Abstract 2881: Posttranslational inactivation of PTEN promoted by loss of Notch1 signaling in T-ALL

Abstract T-cell acute lymphoblastic leukemias (T-ALL) are frequently associated with mutations in Notch 1, many of which lead to increased susceptibilities to cleavage by gamma secretase and generation of ICN1, promoting enhanced Notch1 signaling. Increased Notch1 activity in T-ALL was reported to indirectly repress transcription of PTEN, the major tumor suppressor involved in antagonizing PIP3-dependent cell signaling and in maintaining tight control over the pro-survival AKT pathway. While use of gamma secretase inhibitors (GSIs) prevents Notch1-activating cleavage and restores PTEN protein levels, a link between GSI treatments and PTEN posttranslational inactivation was also implied (Haematologica 95: 674-678, 2010). Phosphorylation by casein kinase II (CK2) of the S380/T382/3 cluster in the carboxyl-terminal region of PTEN is an efficient means of regulating PTEN activity, and of maintaining tight control over cell viability. PTEN phosphorylation impacts its cytosolic versus membrane localization, and PTEN protein stability and levels. However, the relative impact of Notch1 and GSIs on PTEN levels resulting from effects on PTEN transcription versus potential PTEN posttranslational effects has not been established. To begin to address this important question, we used conditional ectopic PTEN expression under control of a tetracycline-inducible promoter in PTEN-null Jurkat cells (designated Jkt/tet-onPten) to assess whether Notch1 can regulate PTEN posttranslationally. Cells were treated with doxycycline to induce PTEN, then with compound E (CompE), a potent GSI. CompE treatment increased phosphorylation of PTEN and AKT (S473 and T308), leading to elevated phosphorylation of GSK3αβ and S6RP downstream, consistent with inactivation of PTEN. This was accompanied by a moderate increase in cell proliferation. Analogous results were seen in Jkt/tet-onPten cells in which Notch1 was knocked down with a shRNA lentiviral vector (Jkt/tet-onPtenKD cells). Knockdown of Notch1 was accompanied by increased cytosolic localization of PTEN, consistent with its phosphorylation and inactivation. Treatment of Jkt/tet-onPtenKD cells with a CK2 inhibitor (tetrabromobenzimidazole) reduced PTEN phosphorylation and restored AKT activation, establishing that the effects of Notch1 on PTEN phosphorylation were via CK2. Our results suggest a mechanism whereby reduced Notch1 signaling in T-ALL cells increases PTEN posttranslational inactivation and AKT signaling, through an effect on CK2-mediated phosphorylation of PTEN. Although GSI treatment should transcriptionally increase PTEN levels, our data suggest that GSI treatment could potentially result in a more aggressive T-ALL due to increased PTEN inactivation and increased AKT signaling. Thus, combined use of a GSI with a downstream inhibitor to CK2, AKT or mTOR could provide significant benefit in treating T-ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2881. doi:10.1158/1538-7445.AM2011-2881

Oncogenic Role of the E3 Ubiquitin Ligase NEDD4-1, a PTEN Negative Regulator, in Non-Small-Cell Lung Carcinomas

Loss of the PTEN tumor suppressor gene occurs frequently in non-small-cell lung carcinoma (NSCLC), although neither genetic alterations nor epigenetic silencing are significant predictors of PTEN protein levels. Since recent reports implicated neural precursor cell expressed, developmentally down-regulated 4-1 (NEDD4-1) as the E3 ubiquitin ligase that regulates PTEN stability, we investigated the role of NEDD4-1 in the regulation of PTEN expression in cases of NSCLC. Our findings indicate that NEDD4-1 plays a critical role in the development of NSCLC and provides novel insight on the mechanisms that contribute to inactivate PTEN in lung cancer. Immunohistochemical analysis on tissue microarrays containing 103 NSCLC resections revealed NEDD4-1 overexpression in 80% of tumors, which correlated with the loss of PTEN protein (n=98; P<0.001). Accordingly, adoptive NEDD4-1 expression in NSCLC cells decreased PTEN protein stability, whereas knock-down of NEDD4-1 expression decreased PTEN ubiquitylation and increased PTEN protein levels. In 25% of cases, NEDD4-1 overexpression was due to gene amplification at 15q21. In addition, manipulation of NEDD4-1 expression in different lung cell systems demonstrated that suppression of NEDD4-1 expression significantly reduced proliferation of NSCLC cells in vitro and tumor growth in vivo, whereas NEDD4-1 overexpression facilitated anchorage-dependent and independent growth in vitro of nontransformed lung epithelial cells that lack pRB and TP53 (BEAS-2B). NEDD4-1 overexpression also augmented the tumorigenicity of lung cancer cells that have an intact PTEN gene (NCI-H460 cells).