PTEN In Response Research Articles

Abstract MP215: Endothelial Colony Forming Cell Derived Exosomes Promote Angiogenesis And Cardiac Repair Post Myocardial Infarction

Background: Despite improvements in therapeutics, ischemic heart disease remains a leading cause of death. Cardiac remodeling after myocardial infarction (MI), predominantly due to loss of cardiomyocytes and coronary vasculature, leads to a progressive decline in cardiac function resulting in heart failure. Current therapies for cardiac repair and heart failure are of limited benefit. Cell transplantation therapy upon MI is a very promising therapeutic strategy to replace dead myocardium, reducing scarring and improving cardiac performance. Methods and Results: Our research focuses on endothelial colony-forming cell-derived exosomes (ECFC-exosomes), which are actively secreted endocytic nanovesicles (30-100 nm) that transport functional miRNAs, proteins, mRNAs, and lipids, playing a key role in paracrine intercellular communication. We identified a novel ability of ECFC-exosomes to promote angiogenesis and cardiac tissue repair. Administration of ECFCs to mice following experimental end-organ ischemia resulted in ECFC-exosome-dependent increase in angiogenesis. ECFC-derived exosomes were taken up by endothelial cells leading to their proliferation and migration, tube formation, and formation of new vessels. Administration of ECFC-exosome to a murine model of MI prevented cardiac remodeling and heart failure. The acute MI resulted in severely decreased left ventricle ejection fraction (Sham 71.2% ± 5 .87, MI+Saline 32.9% ± 2.32) and fractional shortening (Sham 29.5% ± 3.20, MI+Saline 13.6% ± 2.87), and the administration of ECFC-exosomes prevented MI-induced cardiac dysfunction (ejection fraction: MI+ECFC-Exo 64.3% ± 8.74; fractional shortening: MI+ECFC-Exo: 26.4% ± 3.13). Next generation sequencing and bioinformatics analyses identified 136 miRNAs present in ECFC-exosome cargo, and factor inhibiting HIF-1α and PTEN as their potential targets in endothelial cells. Increased nuclear HIF-1α levels in response to ECFC-exosome administration, which may aid in the transcriptional function of HIF-1α, corroborated the role of exosomal miRNA in myocardial angiogenesis. We also found decreased levels of PTEN in response to ECFC-exosome treatment, which is a key negative regulator of PI3K/Akt pathways, survival pathways of heart. We also identified the relative angiogenesis expression profile of the peri-infarcted area in response to ECFC-exosome treatment. The ECFC-exosome administration upregulated the levels of VEGF, IGFBP-1 and PDGF, among others proangiogenic factors, and downregulated the levels of angiostatic factors as IP-10 and Thrombospondin-2. Conclusion: Our findings support the view that the ECFC-exosomes represent a novel therapeutic approach to improve cardiac repair after MI.

MTMR14 protects against cerebral stroke through suppressing PTEN-regulated autophagy

The phosphoinositide phosphatase, myotubularinrelated protein 14 (MTMR14), plays a critical role in the regulating autophagy. However, its functional contribution to neuronal autophagy is still unclear. In the present study, we attempted to explore the effects of MTMR14 on ischemic stroke progression, as well as the underlying molecular mechanisms. Oxygen-glucose deprivation/reoxygenation (OGDR)-induced primary cortical neurons and pheochromocytoma (PC12) cells, and middle cerebral artery occlusion (MCAO)-operated mice were used to establish cerebral ischemia/reperfusion (I/R) injury in vitro and in vivo, respectively. OGDR treatment markedly decreased the expression of MTMR14 expression from mRNA and protein levels in the cultured primary neurons and PC12 cells. Functional analysis showed that OGDR-reduced cell viability was further accelerated by MTMR14 knockdown. On the contrary, MTMR14 over-expression significantly rescued the cell survival in OGDR-exposed cells. Moreover, autophagic markers including LC3BII and Beclin 1 were highly up-regulated in OGDR-incubated neurons and PC12 cells, while being further exacerbated by MTMR14 deletion. However, promoting MTMR14 dramatically alleviated LC3BII and Beclin 1 expression levels stimulated by OGDR. Importantly, we found that MTMR14-regulated autophagy was through its interactions with phosphatase and tensin homolog (PTEN). MTMR14 negatively modulated PTEN protein expression levels in OGDR-exposed cells. In vivo, MCAO-operated mice exhibited significantly reduced expression of MTMR14 in the ischemic penumbra tissues. After MCAO operation, MTMR14 over-expression effectively reduced infarct volume and neurological deficits scores, along with decreased activation of LC3B in neurons. Consistently, MCAO-increased PTEN, LC3BII and Beclin 1 were repressed by MTMR14 in mice. An interaction between MTMR14 and PTEN in response to MCAO was confirmed in vivo. Together, these results indicated the neuroprotective effects of MTMR14 on modulating PTEN-dependent excessive autophagy during cerebral I/R injury. Thus, targeting MTMR14 may provide feasible therapy for ischemic stroke onset and progression.

Exosomes from Adipose-Derived Stem Cells (ADSCs) Overexpressing miR-21 Promote Vascularization of Endothelial Cells

In the past few years, exosomes released from adipose-derived stem cells (abbreviated as ADSCs) have shown promises to provide therapeutic benefits in the fields of regenerative medicine. miRNAs, existing in exosomes, are endogenous, small noncoding RNAs that play important roles in a variety of cellular functions and tumor development. Emerging evidences have indicated that miR-21 is one of the important miRNAs associated with tumor angiogenesis. In this study, we identified the role of exosomes from ADSCs overexpressing miR-21 in regulating/promoting vascularization of endothelial cells. Experimental data indicated an elevated miR-21 level in exosomes released by ADSCs overexpressing miR-21. In vitro matrigel angiogenesis assay showed that exosomes secreted by ADSCs overexpressing miR-21 significantly promoted the vascularization of HUVEC cells (an endothelial cell line). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) revealed an upregulation of HIF-1α, VEGF, SDF-1, p-Akt, p-ERK1/2 and downregulation of PTEN in response to miR-21 overexpression, indicating that miR-21 enriched exosomes induced angiogenesis through Akt and ERK activation and also HIF-1α and SDF-1 expression. Our work suggests that exosomes from ADSCs that overexpressing miR-21 can potentially promote vascularization and therefore the transplantation of exosomes from their culture may be suitable for clinical effort in regenerative medicine.

SIRT4 regulates PTEN stability through IDE in response to cellular stresses.

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays a critical role in regulating cell survival, cell growth, and proliferation by antagonizing the PI3K-AKT-mTOR pathway. The regulatory mechanism of PTEN protein is still not completely understood. Here, we found that Sirtuin 4 (SIRT4) interacts with PTEN and regulates its stability. Overexpression of SIRT4 in cells causes down-regulation of PTEN. This regulation is independent of PTEN acetylation and ubiquitination. We further found that SIRT4 degrades PTEN through lysosome pathway mediated by insulin degrading enzyme (IDE). SIRT4 bridges PTEN and IDE for degradation in response to nutritional starvation stresses. Our results suggest that when cells were exposed to nutritional starvation, SIRT4 was induced and cooperated with IDE to degrade PTEN; low levels of PTEN promote cells to survive from cellular stress. Our findings provide a new regulation of PTEN in response to cellular stresses.-Liu, M., Wang, Z., Ren, M., Yang, X., Liu, B., Qi, H., Yu, M., Song, S., Chen, S., Liu, L., Zhang, Y., Zou, J., Zhu, W.-G., Yin, Y., Luo, J. SIRT4 regulates PTEN stability through IDE in response to cellular stresses.

Stearoyl-CoA desaturase-1 promotes colorectal cancer metastasis in response to glucose by suppressing PTEN

BackgroundDiabetic patients have a higher risk factor for colorectal cancer (CRC) metastasis. Stearoyl-CoA desaturase 1 (SCD1), the main enzyme responsible for producing monounsaturated fatty acids(MUFA) from saturated fatty acids, is frequently deregulated in both diabetes and CRC. The function and mechanism of SCD1 in metastasis of CRC and its relevance to glucose remains largely unknown.MethodsSCD1 expression levels were analyzed in human CRC tissues and the Cancer Browser database (https://genome-cancer.ucsc.edu/). CRC cell lines stably transfected with SCD1 shRNAs or vector were established to investigate the role of SCD1 in modulating migration and invasion of CRC cells. A glucose concentration gradient was set to investigate regulation of SCD1 in CRC relevant to diabetic conditions.ResultsThe clinical data analysis showed high expression of SCD1 in CRC tissues with a negative correlation with the prognosis of CRC. In vitro experiments revealed that SCD1 increased CRC progression through promoting epithelial–mesenchymal transition (EMT). Lipidomic analysis demonstrated that SCD1 increased MUFA levels and MUFA administration could rescue migration and invasion defect of CRC cells induced by SCD1 knockdown. Furthermore, SCD1-mediated progression of CRC was promoted by carbohydrate response-element binding protein (ChREBP) in response to high glucose. Mechanistically, hyperglycemia-SCD1-MUFA induced CRC cell migration and invasion by regulating PTEN.ConclusionsOur findings show that SCD1 promotes metastasis of CRC cells through MUFA production and suppressing PTEN in response to glucose, which may be a novel mechanism for diabetes-induced CRC metastasis.

Abstract 2565: VCP/p97 segregase-mediated regulation of chromatin-bound PTEN stability

Abstract Phosphatase and Tensin homolog (PTEN) tumor suppressor protein is associated with many human cancers. PTEN has been shown to accumulate in the nucleus and control DNA repair in response to genotoxic stress. PTEN down-regulation impairs the capacity of global genomic nucleotide excision repair by suppressing the early DNA damage sensor, Xeroderma pigmentosum C (XPC), protein. Our recent work has demonstrated that the Valosin-containing protein (VCP)/p97 segregase participates in proteolytic processing of XPC and Cockayne syndrome B (CSB) proteins. Here, we show that (i) VCP/p97 segregase, in conjunction with its adaptor proteins, specifically extracts PTEN from damaged chromatin for targeted proteasomal degradation, (ii) inhibition of VCP/p97 activity induces the accumulation of PTEN on chromatin, and (iii) VCP/p97 physically interacts with PTEN. Depletion of VCP/p97, in p97-knockout cells or siRNA-mediated ablation in normal cells, affects the stability of PTEN in response to cellular UV irradiation. Results show that compromising the proteasome and VCP/p97 functions causes the accumulation of PTEN accompanied by an increase of UBXD7, proteasomal RPN2 and Sug1 within the chromatin compartment. To our knowledge, this is the first report to reveal that VCP/p97 segregase extracts tumor suppressor PTEN from damaged chromatin. (This work was supported by NIH grants ES002388 and ES012991 to AAW). Citation Format: Jinshan He, Gulzar Wani, Qianzheng Zhu, Altaf A Wani. VCP/p97 segregase-mediated regulation of chromatin-bound PTEN stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2565. doi:10.1158/1538-7445.AM2017-2565

Regulation of phosphatase and tensin homolog on chromosome 10 in response to hypoxia

Phosphatase and tensin homolog on chromosome 10 (PTEN) is downregulated during hypertrophic and cancerous cell growth, leading to activation of the prosurvival Akt pathway. However, PTEN regulation in cardiac myocytes upon exposure to hypoxia remains unclear. We explored the role of PTEN in response to hypoxia/ischemia in the myocardium. We validated that PTEN is a transcriptional target of activating transcription factor 2 (ATF-2) and is positively regulated via a p38/ATF-2 signaling pathway. Accordingly, hypoxia-induced upregulation of phosphorylation of ATF-2 and PTEN were reversed by a dominant negative mutant p38. Inhibition of PTEN in cardiomyocytes attenuated hypoxia-induced cell death and apoptosis. Cardiac-specific knockout of PTEN resulted in increased phosphorylation of Akt and forkhead box O 1 (forkhead transcription factors), limited infarct size in animals exposed to ischemia-reperfusion injury, and ameliorated deterioration of left ventricular function and remodeling following permanent coronary artery occlusion. In addition, the activation of Bim, FASL, and caspase was coupled with PTEN activation, all of which were attenuated by PTEN inhibition. In conclusion, cardiomyocyte-specific conditional PTEN deletion limited myocardial infarct size in an in vivo model of ischemia-reperfusion injury and attenuated adverse remodeling in a model of chronic permanent coronary artery ligation.

Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses

Dictyostelium discoideum shows chemotaxis towards folic acid (FA) throughout vegetative growth, and towards cAMP during development. We determined the spatiotemporal localization of cytoskeletal and signaling molecules and investigated the FA-mediated responses in a number of signaling mutants to further our understanding of the core regulatory elements that are crucial for cell migration. Proteins enriched in the pseudopods during chemotaxis also relocalize transiently to the plasma membrane during uniform FA stimulation. In contrast, proteins that are absent from the pseudopods during migration redistribute transiently from the PM to the cytosol when cells are globally stimulated with FA. These chemotactic responses to FA were also examined in cells lacking the GTPases Ras C and G. Although Ras and phosphoinositide 3-kinase activity were significantly decreased in Ras G and Ras C/G nulls, these mutants still migrated towards FA, indicating that other pathways must support FA-mediated chemotaxis. We also examined the spatial movements of PTEN in response to uniform FA and cAMP stimulation in phospholipase C (PLC) null cells. The lack of PLC strongly influences the localization of PTEN in response to FA, but not cAMP. In addition, we compared the gradient-sensing behavior of polarized cells migrating towards cAMP to that of unpolarized cells migrating towards FA. The majority of polarized cells make U-turns when the cAMP gradient is switched from the front of the cell to the rear. Conversely, unpolarized cells immediately extend pseudopods towards the new FA source. We also observed that plasma membrane phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] levels oscillate in unpolarized cells treated with Latrunculin-A, whereas polarized cells had stable plasma membrane PtdIns(3,4,5)P3 responses toward the chemoattractant gradient source. Results were similar for cells that were starved for 4 hours, with a mixture of polarized and unpolarized cells responding to cAMP. Taken together, these findings suggest that similar components control gradient sensing during FA- and cAMP-mediated motility, but the response of polarized cells is more stable, which ultimately helps maintain their directionality.

Abstract LB-229: PTEN modulates EGFRvIII-mediated SHP-2 activation and translocation to affect glioblastoma sensitivity to tyrosine kinase inhibitors (TKI)

Abstract EGFRvIII, a constitutively active truncated mutant of epidermal growth factor receptor (EGFR), has been associated with increase neoplastic transformation and poor overall survival (OS). An earlier report showed that expression of EGFRvIII in gliomas provides resistance to chemotherapy. Co-expression of EGFRvIII and PTEN in a small subset of recurrent GBMs has been shown to increase sensitivity to tyrosine kinase inhibitors (TKI), including Erlotinib (tarceva) and Geftinib. However, the exact mechanism of EGFRvIII and PTEN interaction in response to TKI is still unresolved. Our recent reports showed that SHP-2 PTPase and its phosphatase activity are required for the EGFRvIII-mediated transformation. The aim of the present work was to investigate the molecular interaction between EGFRvIII/SHP-2 activation complex and PTEN in response to tarceva treatment and its effect on glioblastoma transformation. Our recent report demonstrated an increased SHP-2 phosphorylation in U87MG. EGFRvIII (PTEN-deficient) when compared to PTEN-intact LN229.EGFRvIII cells. In the present study, we show that tarceva treatment abolished EGFRvIII, EGFR, SHP-2 and Erk1/2, but Src family kinase (SFK), phosphorylation in LN229.EGFRvIII cells at all time intervals ranging from 30 minutes to 6 hours. On the contrary, phosphorylation of EGFRvIII, Src and Erk1/2 in U87MG. EGFRvIII cells was inhibited in early time points, but was restored in 2 to 6 hours. Surprisingly, phosphorylation of SHP-2 at Tyr542 residue was unaffected by tarceva treatment, but EGFR phosphorylation was inhibited in a time-dependent manner. PTEN deficiency resulted in resistance to tarceva treatment as assessed by MTT proliferation and soft agar transformation assays. Immunofluorescent labeling of U87MG. EGFRvIII cells with anti-phospho-SHP-2 (Tyr542) antibody showed perinuclear localization of SHP-2, whereas LN229.EGFRvIII cells exhibited membrane staining of phosphorylated SHP-2 (Tyr542). Our results showed that tarceva increased perinuclear SHP-2 Tyr542 in U87MG. EGFRvIII cells. Interestingly, stable expression of PTEN in U87MG. EGFRvIII cells conferred relocalization of phospho-SHP-2 to the membrane, a phenotype resembling LN229.EGFRvIII cells. Moreover, PTEN-expressing U87MG. EGFRvIII clones grew slower and showed a partially untransformed phenotype. These results suggest that SHP-2 is downstream effectors of PTEN and that PTEN deficiency may lead to SHP-2 activation and perinuclear localization by EGFRvIII, which may results in increased resistance to TKIs. Therefore, targeting SHP-2 may provide a new and effective therapeutic approach for treatment of glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-229.

ROCK1 functions as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability

AbstractRho kinases belong to a family of serine/threonine kinases whose role in recruitment and migration of inflammatory cells is poorly understood. We show that deficiency of ROCK1 results in increased recruitment and migration of macrophages and neutrophils in vitro and in vivo. Enhanced migration resulting from ROCK1 deficiency is observed despite normal expression of ROCK2 and a significant reduction in overall ROCK activity. ROCK1 directly binds PTEN in response to receptor activation and is essential for PTEN phosphorylation and stability. In the absence of ROCK1, PTEN phosphorylation, stability, and its activity are significantly impaired. Consequently, increased activation of downstream targets of PTEN, including PIP3, AKT, GSK-3β, and cyclin D1, is observed. Our results reveal ROCK1 as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation.

ROCK1 Functions as a Suppressor of Inflammatory Cell Migration by Regulating PTEN Phosphorylation and Stability.

Abstract 240Neutrophils and macrophages are major cellular components of the innate immune response and are recruited rapidly in large numbers to sites of infection. The small family of Rho GTPases and its downstream effectors, Rho kinases (Rho-associated, coiled-coil containing protein kinase) have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases whose role in inflammation is not known. Here we show that deficiency of ROCK1 but not ROCK2 results in increased recruitment of macrophages (3.2 fold, n=8, *p<0.01) and neutrophils (3.4 fold, n=5 *p<0.05) compared to WT controls in an in vivo model of aseptic peritonitis. In vitro, deficiency of ROCK1 in bone marrow derived macrophages shows a significant increase in haptotactic transwell migration in response to M-CSF as well as MCP-1 on fibronectin as well as an increase in migration towards the wounded area in a wound healing assay compared to controls (∼3 fold, n=3, *p<0.005). Consistently, deficiency of ROCK1 in bone marrow derived neutrophils also shows a ∼2.63 fold increase in migration in response to fMLP compared to WT bone marrow derived neutrophils (BMNs) in a chemotactic migration assay. ROCK1 deficient macrophages also demonstrate a ∼2.5 fold increase in adhesion on fibronectin (n=3, *p<0.002). The enhanced migration and adhesion in ROCK1−/− macrophages was observed in spite of comparable expression of F4/80 (WT; 85.63% vs. ROCK1−/−; 88.68%, n=4), α4β1 and α5β1 integrins (WT; 67.49% & 88.2% vs. ROCK1−/−; 71.82 % & 87.09%, n=4), while no significant difference in the phagocytosis of sheep red blood cells was observed between WT and ROCK1−/− macrophages (Phagocytic index: WT; 98% vs. ROCK1−/− 97%, n=3, p>.05). Close examination of the cytoskeleton of ROCK1 deficient macrophages using confocal microscopy revealed more F-actin content on the entire cell surface compared to wildtype controls. Consistently, flow cytometric analysis using Alexa 488-phalloidin staining revealed abundance of F-actin in ROCK1−/− macrophages compared to WT controls (WT; 46.19% vs. ROCK1−/−; 65.23%, n=3, *p<0.05). Furthermore, immunofluorescence imaging of podosomes carried out using anti-vinculin antibody revealed more pronounced and increased podosomes in ROCK1 deficient macrophages compared to WT controls (n=3, *p<0.05). Biochemical analysis of ROCK1−/− macrophages revealed that the enhanced recruitment of ROCK1 deficient macrophages and neutrophils was apparent in spite of normal expression of ROCK2 in ROCK1−/− cells and a 60% reduction in overall ROCK activity. Interestingly, although both ROCK1 and ROCK2 co-immunoprecipitate with PTEN in response to cytokine induced stimulation, only ROCK1 appeared to be essential for PTEN phosphorylation, activation and stability. In the absence of ROCK1, PTEN phosphorylation, its activity and stability were significantly impaired in spite of the presence of ROCK2 (n=3, *p<0.05). Consequently, an increase in the activation of downstream targets of PTEN including AKT, GSK-3β and cyclinD1 was observed in ROCK1 deficient macrophages relative to controls (n=3). Taken together, these studies reveal a biochemical pathway involving ROCK1 and PTEN which is involved in the recruitment of macrophages and neutrophils during acute inflammation. Thus, ROCK1 likely functions as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation. Disclosures:No relevant conflicts of interest to declare.

A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells

The production of reactive oxygen species (ROS) exerts an additional tier of control over tyrosine phosphorylation-dependent signal transduction by transiently inhibiting the catalytic activity of specific protein tyrosine phosphatases (PTPs). Hence, the ability to detect reversible oxidation of PTPs in vivo is critical to understanding the complex biological role of ROS in the control of cellular signaling. Here, we describe an assay for identifying those PTPs that are reversibly oxidized in vivo, which utilizes the unique chemistry of the invariant catalytic Cys residue in labeling the active site with biotinylated small molecules under mildly acidic conditions. We have applied this cysteinyl-labeling assay to the study of platelet-derived growth factor (PDGF) receptor signaling in an angiomyolipoma cell model. Doing so has allowed us to detect reversible oxidation of several proteins in response to sustained PDGF stimulation. As in other cell systems, we have observed the reversible oxidation of the classical PTP SHP2 and the tumor suppressor phosphatase PTEN in response to PDGF stimulation. Furthermore, we detected reversible oxidation of members of two other subclasses of PTPs, the receptor PTP LAR and the dual-specificity phosphatase MKP1. These data demonstrate the broad selectivity of the assay, allowing us to detect representatives of all of the major subgroups of the PTP superfamily. We anticipate that this cysteinyl-labeling enrichment strategy can be applied broadly to study reversible oxidation as a mechanism of harnessing PTP catalytic activity in a variety of signaling pathways.

Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway

Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. A growing body of evidences suggests that UCB plays major biological effects by inhibiting cell proliferation in cancer cell lines and eliciting cell toxicity particularly in neurons and glial cells. Early molecular events responsible for bilirubin-induced cytotoxicity remain poorly understood. Using HeLa cells and mouse embryonic fibroblasts, we found that UCB at a concentration of free pigment (Bf) of 80 nM induced oxidative stress, promoting a significant increase in intracellular reactive oxygen species (ROS) and a decreased cell survival (by the MTT test). The ROS increase activated the antioxidant cell response through APE1/Ref-1, a master redox regulator in eukaryotic cells. Activation of APE1/Ref-1 was followed by a concomitant activation of Egr-1 transcription factor and by an upregulation of PTEN tumor suppressor, an Egr-1 target gene, leading to inhibition of cell growth. Blocking ROS generation with N-acetylcysteine pretreatment, restored cell survival, limited the upregulation of PTEN in response to UCB, and prevented the inhibition of cell proliferation. HeLa cells transfected with mutants of the PTEN promoter or silenced with APE1/Ref-1 small interference RNA confirmed that UCB modulates a signaling pathway involving APE1/Ref-1, Egr-1, and PTEN. These findings describe a new molecular pathway involved in the cytotoxic effects of UCB.