Targeting Of PTEN Research Articles

Methods for PTEN in Stem Cells and Cancer Stem Cells.

PTEN (phosphatase and tensin homologue) is the first tumor suppressor identified to have phosphatase activity and its gene is the second most frequently deleted or mutated tumor-suppressor gene associated with human cancers. Germline PTEN mutations are the cause of three inherited autosomal dominant disorders. Phosphatidylinositol 3,4,5,-triphosphate (PIP3), the product of the PI3 kinase, is one of the key intracellular targets of PTEN's phosphatase activity, although PTEN's phosphatase-independent activities have also been identified. PTEN is critical for stem cell maintenance, which contributes to its controlled tumorigenesis. PTEN loss leads the development of cancer stem cells (CSCs) that share properties with somatic stem cells, including the capacity for self-renewal and multi-lineage differentiation. Methods to isolate and functionally test stem cells and CSCs are important for understanding PTEN functions and the development of therapeutic approaches to target CSCs without having adverse effects on normal stem cells. Here, we describe protocols for the isolation and functional analysis of PTEN deficient embryonic stem cells, hematopoietic stem cells and leukemia-initiating cells (LICs), neural stem cells, and prostate stem cells and CSCs.

PI3K/AKT Pathway and Its Mediators in Thyroid Carcinomas.

Thyroid malignancies are the most common endocrine system carcinomas, with four histopathological forms. The phosphoinositide 3-kinase-protein kinase B/AKT (PI3K-PKB/AKT) pathway is one of the most critical molecular signaling pathways implicated in key cellular processes. Its continuous activation by several aberrant receptor tyrosine kinases (RTKs) and genetic mutations in its downstream effectors result in high cell proliferation in a broad number of cancers, including thyroid carcinomas. In this review article, the role of different signaling pathways of PI3K/AKT in thyroid cancers, with the emphasis on the PI3K/AKT/mammalian target of rapamycin (mTOR), PI3K/AKT/forkhead box O (FOXO) and PI3K/AKT/phosphatase and tensin homolog deleted on chromosome ten (PTEN) pathways, and various therapeutic strategies targeting these pathways have been summarized. In most of the in vitro studies, agents inhibiting mTOR in monotherapy or in combination with chemotherapy for thyroid malignancies have been introduced as promising anticancer therapies. FOXOs and PTEN are two outstanding downstream targets of the PI3K/AKT pathway. At the present time, no study has been undertaken to consider thyroid cancer treatment via FOXOs and PTEN targeting. According to the critical role of these proteins in cell cycle arrest, it seems that a treatment strategy based on the combination of FOXOs or PTEN activity induction with PI3K/AKT downstream mediators (e.g., mTOR) inhibition will be beneficial and promising in thyroid cancer treatment.

Distinct pathways in the pathogenesis of sebaceous carcinomas implicated by differentially expressed microRNAs.

The molecular-genetic alterations contributing to the pathogenesis of sebaceous carcinoma and sebaceous adenoma remain poorly understood. Given that sebaceous carcinoma is associated with substantial morbidity and mortality, there is a critical need to delineate the pathways driving sebaceous carcinoma and candidate molecules for targeted therapy. To describe differentially expressed microRNAs (miRNAs) in a series of periocular sebaceous carcinomas compared with sebaceous adenomas in order to identify pathways driving the pathogenesis of sebaceous carcinoma. Thirty sebaceous carcinomas and 23 sebaceous adenomas (including 11 that were confirmed to be related to Muir-Torre syndrome and 6 that were confirmed to be sporadic) were obtained from archives (from 48 patients) of 2 institutions (University of Texas MD Anderson Cancer Center, Houston, and Siriraj Hospital, Mahidol University, Bangkok, Thailand) and profiled. Expression of miRNAs was determined using total RNA from formalin-fixed, paraffin-embedded tissue and real-time reverse transcription-polymerase chain reaction performed in a microfluidics card containing 378 unique miRNAs. Fold change was determined using the ΔΔCt method (reference probe, RNU48). Median centering was used to normalize the data. Two-sample t tests were used to identify differentially expressed miRNAs. The false discovery rate was assessed by β-uniform mixture analysis of P values from the t statistics. Significance was defined by this estimated false discovery rate. Serial testing and validation confirmed overexpression of 2 miRNAs previously reported to be oncogenic, miR-486-5p (4.4-fold; P = 2.4 × 10-8) and miR-184 (3.5-fold; P = 1.7 × 10-6), in sebaceous carcinoma compared with sebaceous adenoma and downregulation of 2 miRNAs previously reported to have tumor-suppressive properties, miR-211 (-5.8-fold; P = 2.3 × 10-9) and miR-518d (-4.5-fold; 6.7 × 10-5), in sebaceous carcinoma compared with sebaceous adenoma. Sebaceous carcinoma exhibits an miRNA expression profile distinct from that of sebaceous adenoma, implicating dysregulation of NF-κB and PTEN (targets of miR-486-5p) and TGF-β signaling (target of miR-211) in the pathogenesis of sebaceous carcinoma. The identification of miRNAs whose expression is altered in sebaceous carcinoma compared with sebaceous adenoma provides a novel entry point for a more comprehensive understanding of the molecular-genetic alterations pivotal to the development of sebaceous carcinoma.

MicroRNA-181a regulates epithelial-mesenchymal transition by targeting PTEN in drug-resistant lung adenocarcinoma cells.

Chemoresistance is an inevitable occurrence in lung adenocarcinoma, which has been associated with decreased expression of the phosphatase and tensin homolog deleted on chromosome ten (PTEN). Therefore, it is important to identify novel molecular mechanisms to suppress chemoresistance in lung adenocarcinoma cells. Paclitaxel- and cisplatin-resistant A549 lung carcinoma cell derivatives were developed by long-term serial culture. The metastatic properties of the cells were assessed using wound-healing assays, migration assays, invasion assays, morphological examination, and western blot analysis/RT-PCR of genes associated with the epithelial-mesenchymal transition (EMT). To identify novel regulators of EMT in A549 cells, differentially expressed miRNAs in drug-resistant cells were identified by microarray analysis. The role of miR-181a was established by transfection with specific mimic and inhibitor followed by functional assays. Luciferase assays were performed to assess the ability of miR-181a to target the PTEN promoter, and regulation of PTEN expression by miR-181a was assessed by western blot analysis and RT-PCR. Paclitaxel- and cisplatin-resistant A549 cells acquired metastatic properties and EMT phenotype and had reduced PTEN expression as compared to sensitive cells. miR‑181a was identified as a differentially expressed miRNA in drug-resistant A549 cells, and miR-181a mimic and inhibitor were shown to affect migration, invasion, morphology and expression of EMT-associated genes. PTEN was identified as a direct target of miR-181a. Our findings demonstrate that miR-181a expression in lung adenocarcinoma is associated with EMT progression, potentially through targeting of PTEN. Regulation of miR-181a may provide a novel strategy for overcoming resistance to paclitaxel and cisplatin in lung adenocarcinoma.

MicroRNA-93 promotes cell proliferation via targeting of PTEN in Osteosarcoma cells.

BackgroundAberrant microRNA (miRNA) expression plays an essential role in osteosarcoma (OS) pathogenesis. Recent studies have shown that dysregulation of miRNA expression is associated with increased tumorigenesis and poor prognosis in several types of cancers, including OS. The aim of this study was to investigate the relevant microRNAs involved in the development of OS.MethodsTo explore possible oncogenic factors in OS, we used a microarray-based approach to profile changes in the expression of miRNAs and their target mRNAs in five OS cell lines and human mesenchymal stem cells (hMSCs). An miRNA, miR-93, was significantly up-regulated, whereas phosphatase and tensin homologue (PTEN) expression was significantly down-regulated in all tested OS cells, when compared with hMSCs.ResultsWhen anti-miR-93 was transfected into OS cell lines, PTEN expression was greatly increased, suggesting that PTEN might be a target of miR-93 in ES cells. The expression of phosphorylated Akt protein, which is known to be inversely correlated with that of PTEN, was significantly down-regulated in anti-miR-93-transfected cells. Furthermore, transfection of anti-miR-93 inhibited the proliferation and cell cycle progression of ES cells. In addition, the down-regulation of miR-93 in these cells significantly suppressed tumor growth in vivo.ConclusionEctopic expression of miR-93 decreased PTEN protein levels. Furthermore, miR-93 increased proliferation and decreased apoptosis in OS cells, whereas its silencing in these cells inhibited such carcinogenic processes. Taking these observations together, miR-93 can be seen to play a critical role in carcinogenesis through suppression of PTEN, and may serve as a therapeutic target for the treatment of OS.

Tumor-derived microRNA-494 promotes angiogenesis in non-small cell lung cancer.

Angiogenesis, a crucial step in tumor growth and metastasis, is regulated by various pro- or anti-angiogenic factors. Recently, microRNAs have been shown to modulate angiogenic processes by modulating the expression of critical angiogenic factors. However, roles of tumor-derived microRNAs in regulating tumor vascularization remain to be elucidated. In this study, we found that delivery of miR-494 into human vascular endothelial cells (ECs) enhanced the EC migration and promoted angiogenesis. The angiogenic effect of miR-494 was mediated by the targeting of PTEN and the subsequent activation of Akt/eNOS pathway. Importantly, co-culture experiments demonstrated that a lung cancer cell line, A549, secreted and delivered miR-494 into ECs via a microvesicle-mediated route. In addition, we found that the expression of miR-494 was induced in the tumor cells in response to hypoxia, likely via a HIF-1α-mediated mechanism. Furthermore, a specific miR-494 antagomiR effectively inhibited angiogenesis and attenuated the growth of tumor xenografts in nude mice. Taken together, these results demonstrated that miR-494 is a novel tumor-derived paracrine signal to promote angiogenesis and tumor growth under hypoxic condition.

MicroRNA-494 Promotes Cyclosporine-Induced Nephrotoxicity and Epithelial to Mesenchymal Transition by Inhibiting PTEN

A major complication associated with cyclosporine (CsA) treatment is nephrotoxicity. In this study, we examined whether microRNAs play a role in cyclosporine-induced nephrotoxicity. Treatment of mice with CsA resulted in nephrotoxicity that was associated with an early increase in expression of microRNA mmu-miR-494 (miR-494). Similarly, tubular epithelial cell epithelial-mesenchymal transition (EMT) induced by CsA toxicity resulted in the upregulation of microRNA-494 and a decrease in PTEN levels in vitro. miR-494 directly targeted Pten and negatively regulated its expression. Preventing Pten targeting by miR-494 was sufficient to prevent CsA induced EMT. Knockdown of miR-494 prevented the downregulation of PTEN in tubular epithelial cells following CsA treatment and also prevented CsA induced EMT. Thus, miR-494 plays a major role in promoting CsA induced nephrotoxicity through its ability to target Pten thereby contributing to EMT. We suggest that manipulating miR-494 expression may represent a novel approach to preventing EMT associated with CsA induced nephrotoxicity.

Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism.

Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

Cutting short the path to murine liver cancer models.

Abstract The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

Abstract 3539: The noncoding PTEN pseudogene influences PI3K signaling and inhibits breast cancer progression

Abstract The PTEN pseudogene is a non-coding RNA, reported to function as a decoy for microRNAs (miRNAs) degrading PTEN mRNA. The purpose of this study was to examine PTENP1 expression and its influence on PTEN expression and PI3K-Akt-mTOR signaling in human breast cancer. Tumor samples from 30 patients with locally advanced breast cancer were examined for PTEN and PTENP1 expression before and after treatment, and the results were correlated to tumor response rates. Furthermore, the human PTENP1 positive MDA-MB231 and the murine PTENP1 negative C3HBA breast cancer cell lines were lentivirally transfected with PTENP1 to assess the effects of PTENP1 overexpression on cell proliferation in vitro, tumor growth in mice, as well as its influence on PTEN and PI3K-Akt-mTOR signaling. Knockdown of PTENP1 by siRNA in MDA-MB231 cells was performed to examine how down regulated gene expression of the pseudogene would affect PTEN. To evaluate PTENP1-associated miRNAs, RNA sequencing from MDA-MB231 transfected with PTENP1 was performed. PTENP1 was present genomically in all human breast cancer samples. PTEN and PTENP1 mRNA expression was concordant in the breast cancer samples before and after 16 weeks of doxorubicin treatment. When comparing the response group, tumor samples from patients with progressive disease (PD) had the lowest PTEN mRNA levels, whereas tumors with stable disease (SD) or partial response (PR) had a higher PTEN expression. PD tumors had a minor change in PTEN and PTENP1 levels after treatment, whereas PTEN and PTENP1 increased in SD and PR tumors. PTENP1 overexpression significantly reduced C3HBA tumor growth in C3H mice. MDA-MB231 primary tumor growth was not affected, but a reduced occurrence of lung metastases was observed in NOD/SCID mice. Cell proliferation was not affected in neither cell line in vitro. This disparate response in vitro vs. in vivo suggests that PTENP1 influences tumor-host interactions in vivo. Lentiviral PTENP1 transfection increased PTEN mRNA and protein expression in murine C3HBA, but with increased pAkt and S6K protein levels indicative of non-PI3K dependent activation. In MDA-MB231 tumors, PTENP1 did not affect PTEN mRNA, but PTEN protein expression increased slightly. The MDA-MB231 tumors were found negative for pAKT and with a low S6K protein expression. PTENP1 knockdown decreased PIK3CA, PTEN, S6K and mTOR gene expression in MDA-MB231, suggesting that PTENP1 facilitates PI3K pathway signaling. While PTENP1 had an effect on PTEN gene and protein expression, it had no obvious effect on PTEN targeting miRNAs in MDA-MB231. Thus, the mechanism behind PTEN regulation mediated by PTENP1 in breast cancer needs to be elaborated on further. In conclusion, PTENP1 is present in human breast cancers and reacts concordantly with PTEN when tumors are exposed to doxorubicin. Up- and down regulation of PTENP1 influences PI3K signaling, and PTENP1 transfection inhibits tumor progression in mice. Citation Format: Synnøve Yndestad, Eilin Austreid, Hans Petter Eikesdal. The noncoding PTEN pseudogene influences PI3K signaling and inhibits breast cancer progression. [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 3539. doi:10.1158/1538-7445.AM2014-3539

Investigation of key miRNAs and target genes in bladder cancer using miRNA profiling and bioinformatic tools.

Despite the association of several miRNAs with bladder cancer, little is known about the miRNAs' regulatory networks. In this study, we aimed to construct potential networks of bladder-cancer-related miRNAs and their known target genes using miRNA expression profiling and bioinformatics tools and to investigate potential key molecules that might play roles in bladder cancer regulatory networks. Global miRNA expression profiles were obtained using microarray followed by RT-qPCR validation using two randomly selected miRNAs. Known targets of deregulated miRNAs were utilized using DIANA-TarBase database v6.0. The incorporation of deregulated miRNAs and target genes into KEGG pathways were utilized using DIANA-mirPath software. To construct potential miRNA regulatory networks, the overlapping parts of three selected KEGG pathways were visualized by Cytoscape software. We finally gained 19 deregulated miRNAs, including 5 ups- and 14 down regulated in 27 bladder-cancer tissue samples and 8 normal urothelial tissue samples. The enrichment results of deregulated miRNAs and known target genes showed that most pathways were related to cancer or cell signaling pathways. We determined the hub CDK6, BCL2, E2F3, PTEN, MYC, RB, and ERBB3 target genes and hub hsa-let-7c, hsa-miR-195-5p, hsa-miR-141-3p, hsa-miR-26a-5p, hsa-miR-23b-3p, and hsa-miR-125b-5p miRNAs of the constructed networks. These findings provide new insights into the bladder cancer regulatory networks and give us a hypothesis that hsa-let-7c, hsa-miR-195-5p, and hsa-miR-125b-5p, along with CDK4 and CDK6 genes might exist in the same bladder cancer pathway. Particularly, hub miRNAs and genes might be potential biomarkers for bladder cancer clinics.

CRISPR-mediated direct mutation of cancer genes in the mouse liver.

The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem (ES) cells1. Here we describe a new method of cancer model generation using the CRISPR/Cas system in vivo in wild-type mice. We have used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs)2–4 to the liver and directly target the tumor suppressor genes Pten5 and p536, alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology7, 8. Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumor tissue revealed insertion or deletion (indel) mutations of the tumor suppressor genes, including bi-allelic mutations of both Pten and p53 in tumors. Furthermore, co-injection of Cas9 plasmids harboring sgRNAs targeting the β-Catenin gene (Ctnnb1) and a single-stranded DNA (ssDNA) oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-Catenin. This study demonstrates the feasibility of direct mutation of tumor suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

MicroRNA and skeletal muscle function: novel potential roles in exercise, diseases, and aging.

microRNA and skeletal muscle function: novel potential roles in exercise, diseases, and aging.

Alteration in Mir-21/PTEN expression modulates gefitinib resistance in non-small cell lung cancer.

Resistance to TKI treatment is a major obstacle in effective treatment of NSCLC. Besides EGFR mutation status, the mechanisms involved are largely unknown. Some evidence supports a role for microRNA 21 in modulating drug sensitivity of chemotherapy but its role in NSCLC TKI resistance still remains unexplored. This study aimed to investigate whether NSCLC miR-21 mediated resistance to TKIs also results from Pten targeting. Here, we show miR-21 promotes cancer by negatively regulating Pten expression in human NSCLC tissues: high miR-21 expression levels were associated with shorter DFS in 47 NSCLC patients; high miR-21/low Pten expression levels indicated a poor TKI clinical response and shorter overall survival in another 46 NSCLC patients undergoing TKI treatment. In vitro assays showed that miR-21 was up-regulated concomitantly to down-regulation of Pten in pc-9/GR cells in comparison with pc-9 cells. Moreover, over-expression of miR-21 significantly decreased gefitinib sensitivity by down-regulating Pten expression and activating Akt and ERK pathways in pc-9 cells, while miR-21 knockdown dramatically restored gefitinib sensitivity of pc-9/GR cells by up-regulation of Pten expression and inactivation of AKT and ERK pathways, in vivo and in vitro. We propose alteration of miR-21/Pten expression as a novel mechanism for TKI resistance in NSCLC cancer. Our findings provide a new basis for using miR 21/Pten-based therapeutic strategies to reverse gefitinib resistance in NSCLC.

Subcellular targeting and dynamic regulation of PTEN: implications for neuronal cells and neurological disorders.

PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5)P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum (ER), the mitochondria, or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein–protein interactions, or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i) recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii) current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.

Unraveling natalizumab effects on deregulated miR-17 expression in CD4+ T cells of patients with relapsing-remitting multiple sclerosis.

MicroRNAs (miRNAs) are a family of noncoding RNAs that play critical roles in the posttranscriptional regulation of gene expression. Accumulating evidence supports their involvement in the pathogenesis of multiple sclerosis (MS). Here, we compare miR-17 expressions in CD4+ T cells from relapsing-remitting (RR) MS patients treated with natalizumab versus untreated patients. miR-17 was downregulated under natalizumab treatment and upregulated during relapse, therefore supporting a possible role of miR-17 in MS immunopathogenesis. Downregulation of miR-17 was associated with upregulation of PTEN, BIM, E2F1, and p21 target genes. In vitro miR-17 inhibition was associated with upregulation of the same targets and resulted in impaired CD4+ T cell activation and proliferation. We further describe deregulated TGFBR2 expression in untreated patients versus healthy volunteers (HVs) and confirm in vitro the link between miR-17 and TGFBR2 expressions. These findings support an effect of natalizumab on expression of specific miRNA and subsequent expression of genes involved in proliferation and control of the cell cycle.

MADD Is a Downstream Target of PTEN in Triggering Apoptosis

Mitogen-activated kinase activating death domain containing protein (MADD) is abundantly expressed in cancer cells and necessary for maintaining cancer cell survival. However, this survival function of MADD is dependent upon its phosphorylation by protein kinase B (Akt). The tumour suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. The downstream targets of PTEN in triggering apoptosis have not yet been completely identified. Here, we report that MADD can act as a pro-apoptotic factor to initiate TRAIL-induced apoptosis when its phosphorylation is attenuated by PTEN. Our data show that tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) induced a reduction in MADD phosphorylation with a concomitant up-regulation of PTEN. Knock down of PTEN using a specific siRNA prevented TRAIL-induced reduction in pMADD levels. Surprisingly, Akt non-phosphorylated MADD translocated from the plasma membrane to cytoplasm where it bound to 14-3-3 and displaced 14-3-3 associated Bax, which translocated to mitochondria resulting in cytochrome c release. Taken together, our data reveal that PTEN can convey the death signal by preventing MADD phosphorylation by Akt.

Metformin inhibits lung cancer cells proliferation through repressing microRNA-222

Metformin, which is commonly used as an oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis. However, the mechanism by which metformin affects various cancers, including lung cancer, remains unknown. MiR-222 induces cell growth and cell cycle progression via direct targeting of p27, p57 and PTEN in cancer cells. In the present study, we used A549 and NCI-H358 human lung cancer cell lines to study the effects and mechanisms of metformin. Metformin treatment reduced expression of miR-222 in these cells (p < 0.05). As a result, protein abundance of p27, p57 and PTEN were increased in cells exposed to metformin. Therefore, these data provide novel evidence for a mechanism that may contribute to the anti-neoplastic effects of metformin suggested by recent population studies and justifying further work to explore potential roles for it in lung cancer treatment.

Identification of PTEN at the ER and MAMs and its regulation of Ca2+ signaling and apoptosis in a protein phosphatase-dependent manner

The tumor suppressor activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10) is thought to be largely attributable to its lipid phosphatase activity. PTEN dephosphorylates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate to directly antagonize the phosphoinositide 3-kinase-Akt pathway and prevent the activating phosphorylation of Akt. PTEN has also other proposed mechanisms of action, including a poorly characterized protein phosphatase activity, protein-protein interactions, as well as emerging functions in different compartment of the cells such as nucleus and mitochondria. We show here that a fraction of PTEN protein localizes to the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs), signaling domains involved in calcium ((2+)) transfer from the ER to mitochondria and apoptosis induction. We demonstrate that PTEN silencing impairs ER Ca(2+) release, lowers cytosolic and mitochondrial Ca(2+) transients and decreases cellular sensitivity to Ca(2+)-mediated apoptotic stimulation. Specific targeting of PTEN to the ER is sufficient to enhance ER-to-mitochondria Ca(2+) transfer and sensitivity to apoptosis. PTEN localization at the ER is further increased during Ca(2+)-dependent apoptosis induction. Importantly, PTEN interacts with the inositol 1,4,5-trisphosphate receptors (IP3Rs) and this correlates with the reduction in their phosphorylation and increased Ca(2+) release. We propose that ER-localized PTEN regulates Ca(2+) release from the ER in a protein phosphatase-dependent manner that counteracts Akt-mediated reduction in Ca(2+) release via IP3Rs. These findings provide new insights into the mechanisms and the extent of PTEN tumor-suppressive functions, highlighting new potential strategies for therapeutic intervention.

Effect of PTEN Antisense Oligonucleotide on Oesophageal Squamous Cell Carcinoma Cell Lines

To investigate the effects of a phosphatase and tensin homologue (PTEN) antisense oligonucleotide on PTEN and mammalian target of rapamycin (mTOR) mRNA and protein, cell proliferation and apoptosis in oesophageal squamous cell carcinoma (OCSS) cell lines. EC9706 and EC1 cells were transfected with PTEN antisense oligonucleotide, sense oligonucleotide or nonsense oligonucleotide. Cell proliferation and apoptosis were quantified. Immuno cyto chemistry and in situ hybridization were used to determine PTEN and mTOR protein and mRNA levels, respectively. Transfection with PTEN antisense oligonucleotide dose- and time-dependently enhanced cell proliferation and inhibited apoptosis in both EC9706 and EC1 cells. PTEN mRNA and protein were significantly downregulated, and mTOR protein and mRNA were significantly upregulated. These data suggest that PTEN is an important tumour suppressor gene in the development of OSCC.