PEA-15 Expression Research Articles

Macrophage-Derived Exosomes Promoted the Development and Stemness of Inflammatory Bowel Disease-Related Colorectal Cancer via nuclear paraspeckle assembly transcript 1-Mediated miRNA-34a-5p/phosphoprotein enriched in astrocytes 15 Axis.

Inflammatory bowel disease (IBD) is closely associated with the development of colorectal cancer (CRC) due to the chronic inflammatory response. Macrophages play critical roles in regulating the microenvironment to facilitate tumor progression. Exosomes are key modulators for the communication between macrophages and tumor cells. The mechanism of macrophage-derived exosomes in IBD-related CRC development remains unclear. The macrophages were isolated using fluorescence activating cell sorter (FACS). The RNA and protein expressions in exosomes and CRC cells were examined by quantitative real-time polymerase chain reaction and western blot assays, respectively. CRC cell development was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, BrdU staining, Transwell assay, and spheroid formation assay. The level of stemness was determined by detecting the proportion of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive CRC cells and the expression of LGR5, CD133, and CD44. Molecular interaction experiments were done using luciferase reporter assay and RNA immunoprecipitation assay. Xenograft tumor model in vivo and immunohistochemistry were used to observe the pathological changes. Macrophage-derived exosomes from IBD-related CRC tissues were enriched with nuclear paraspeckle assembly transcript 1 (NEAT1) and able to promote the progression and stemness of CRC both in vitro and in vivo. The exosomal NEAT1 could sponge miR-34a-5p, leading to the restoration of PEA15 expression in CRC cells and promoting the development of CRC. Inhibition of NEAT1 in exosomes could effectivity inhibit the tumor growth in the CRC xenograft model. These findings provide novel insights into how macrophages affect CRC development and highlight exosomal NEAT1 as a therapeutic target for CRC treatment.

High Expression of PEA15 Is Associated With Patient Survival in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare but very aggressive tumor that is primarily pleural in origin. The 5-year overall survival rate of patients with MPM has not improved despite therapeutic advances. Therefore, biomarker discovery to identify premalignant or early malignant tumors of the mesothelium are crucial. PEA15 is a cytoplasmic protein that is involved in various human malignancies, including MPM. However, the clinicopathological involvement of PEA15 in MPM has not yet been documented. The Oncomine database and GEPIA2 platform were used to analyze PEA15 mRNA expression and patient survival in patients with MPM. PEA15 was found to be significantly up-regulated in MPM, and this up-regulation inversely correlated with prolonged patient survival. Further, PEA15 expression was found to be increased in other cancer tissues without affecting overall survival. PEA15 may represent a new potential prognostic biomarker in MPM patients.

Phosphoprotein enriched in astrocytes (PEA)-15 is a novel regulator of adipose tissue expansion

Excessive expansion of adipose tissue in obesity typically leads to overflow and accumulation of lipids in other tissues, causing fatty liver disease and atherosclerosis. The intracellular protein, phosphoprotein enriched in astrocytes (PEA)-15 has been linked to metabolic disease but its role in lipid storage has not been examined. To delineate the role of PEA-15 in adipose tissue, we placed PEA-15−/− mice on a high fat diet. These mice developed increased body weight and greater white adipose tissue expansion compared to high fat diet-fed wild type mice. This was due to increased adipocyte cell size in PEA-15−/− mice consistent with greater lipid storage capacity. Surprisingly, PEA-15−/− mice exhibited improvements in whole body insulin sensitivity, lower hepatic weight and decreased serum triglycerides indicating a protective phenotype. To determine effects on atherosclerosis, PEA-15−/− mice were crossed with the ApoE−/− mice on a high fat diet. Strikingly, these mice were protected from atherosclerosis and had less hepatic lipid accumulation despite increased adiposity. Therefore, we reveal for the first time that PEA-15 plays a novel role in regulating the expansion of adipose tissue. Decreasing PEA-15 expression increases the sequestering of lipids in adipose tissue, protecting other tissues in obesity, thereby improving metabolic health.

The microRNA212 regulated PEA15 promotes ovarian cancer progression by inhibiting of apoptosis

PEA15 (Proliferation And Apoptosis Adaptor) is a 15kDa multifunctional phosphoprotein involved in various essential biological processes such as proliferation and apoptosis of cancer cells. Previous studies have demonstrated that PEA15 can promote the progression of many malignancies. In the present study, the expression of PEA15 in ovarian cancer and normal tissues analyzed in several databases and PEA15 was found to be significantly up-regulated in OC tissues compared to normal tissues. Immunochemical assays performed using 171 OC tissue specimens proved that the expression of PEA15 was remarkably positively correlated with the FIGO stage and associated with histologic subgroups of ovarian cancer. IHC assay for the two phosphorylation sites of PEA15 S116 and S104 was also performed. PEA15 high expression predicted a poor prognosis in OC patients analysed from K-M plot dataset. In addition, we proved knockdown of PEA15 inhibits OC cell proliferation and induces cell apoptosis by Bcl2 downregulation and Bax and cleaved Caspase-3 upregulation. Overexpression of PEA15 promotes the proliferative capacity of OC cells. Moreover, this study first discovered PEA15 expression in OC can be negatively regulated by microRNA212. Overexpression of miR-212 in ovarian cancer cells could cause downregulated the expression of PEA15 expression. Overexpression of miR-212 was found to exerted similar effects on the proliferation, and apoptosis of the ovarian cancer cells as that of PEA15 suppression. Additionally, overexpression of PEA15could at least partially abolished the effects of miR-212 on the proliferation, and apoptosis of ovarian cancer cells. In conclusion, our findings revealed PEA15 appears as a novel predictive biomarker, thus providing a valuable therapeutic target in OC treatment strategy.

Circadian expression and functional characterization of PEA-15 within the mouse suprachiasmatic nucleus.

The circadian timing system influences the functional properties of most, if not all, physiological processes. Central to themammalian timing system is the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN functions as a 'master clock' that sets the phasing of ancillary circadian oscillator populations found throughout the body. Further, via an entraining input from the retina, the SCN ensures that the clock oscillators are synchronized to the daily light/dark cycle. A critical component of the SCN timing and entrainment systems is the p44/42 mitogen-activated protein kinase (ERK/MAPK) pathway. Here, we examined the expression and function of phosphoprotein-enriched in astrocytes (PEA-15), an ERK scaffold proteinthat serves as a key regulator of MAPK signaling. A combination of immunolabeling and Western blotting approaches revealed high levels of PEA-15 within the SCN. PEA-15 expression was enriched in distinct subpopulations of SCN neurons, including arginine vasopressin (AVP)-positive neurons of the SCN shell region. Further, expression profiling detected a significant circadian oscillation in PEA-15 expression within the SCN. Brief photic stimulation during the early subjective night led to a significant increase in PEA-15 phosphorylation, an event that can trigger ERK/PEA-15 dissociation. Consistent withthis, co-immunoprecipitation assays revealed that PEA-15 is directly bound to ERK in the SCN and that photic stimulation leads to their dissociation. Finally, we show that PEA-15 regulates ERK/MAPK-dependent activation of the coreclock gene period1. Together, these data raise the prospect that PEA-15 functions as a key regulator of the SCN timing system.

The differential roles of PEA15 phosphorylations in reactive astrogliosis and astroglial apoptosis following status epilepticus

Up to this day, the roles of PEA15 expression and its phosphorylation in seizure-related events have not been still unclear. In the present study, we found that PEA15 was distinctly phosphorylated in reactive astrocytes and apoptotic astrocytes in the rat hippocampus following LiCl-pilocarpine-induced status epilepticus (SE, a prolonged seizure activity). PEA15-serine (S) 104 phosphorylation was up-regulated in reactive astrocytes following SE, although PEA15 expression and its S116 phosphorylation were unaltered. Bisindolylmaleimide (BIM), a protein kinase C (PKC) inhibitor, attenuated SE-induced reactive astrogliosis, but phorbol 12-myristate 13-acetate (PMA, a PKC activator) aggravated it. Unlike reactive astrocytes, PEA15-S116 phosphorylation was reduced in apoptotic astrocytes. However, PEA15 expression and its S104 phosphorylation were unchanged in apoptotic astrocyte. Neither BIM nor PMA affected SE-induced astroglial apoptosis. PEA15 expression and its phosphorylations were not relevant to SE-induced CA1 neuronal death. These findings indicate that PEA15-S104 and S116 phosphorylations may play a role in reactive astrogliosis and prevention of astroglial apoptosis, respectively. Therefore, we suggest that the selective manipulation of PEA15 phosphorylations may regulate apoptotic and/or proliferative signals in astrocytes.

Next-Generation CDK2/9 Inhibitors and Anaphase Catastrophe in Lung Cancer

The first generation CDK2/7/9 inhibitor seliciclib (CYC202) causes multipolar anaphase and apoptosis in lung cancer cells with supernumerary centrosomes (known as anaphase catastrophe). We investigated a new and potent CDK2/9 inhibitor, CCT68127 (Cyclacel). CCT68127 was studied in lung cancer cells (three murine and five human) and control murine pulmonary epithelial and human immortalized bronchial epithelial cells. Robotic CCT68127 cell-based proliferation screens were used. Cells undergoing multipolar anaphase and inhibited centrosome clustering were scored. Reverse phase protein arrays (RPPAs) assessed CCT68127 effects on signaling pathways. The function of PEA15, a growth regulator highlighted by RPPAs, was analyzed. Syngeneic murine lung cancer xenografts (n = 4/group) determined CCT68127 effects on tumorigenicity and circulating tumor cell levels. All statistical tests were two-sided. CCT68127 inhibited growth up to 88.5% (SD = 6.4%, P < .003) at 1 μM, induced apoptosis up to 42.6% (SD = 5.5%, P < .001) at 2 μM, and caused G1 or G2/M arrest in lung cancer cells with minimal effects on control cells (growth inhibition at 1 μM: 10.6%, SD = 3.6%, P = .32; apoptosis at 2 μM: 8.2%, SD = 1.0%, P = .22). A robotic screen found that lung cancer cells with KRAS mutation were particularly sensitive to CCT68127 ( P = .02 for IC 50 ). CCT68127 inhibited supernumerary centrosome clustering and caused anaphase catastrophe by 14.1% (SD = 3.6%, P < .009 at 1 μM). CCT68127 reduced PEA15 phosphorylation by 70% (SD = 3.0%, P = .003). The gain of PEA15 expression antagonized and its loss enhanced CCT68127-mediated growth inhibition. CCT68127 reduced lung cancer growth in vivo ( P < .001) and circulating tumor cells ( P = .004). Findings were confirmed with another CDK2/9 inhibitor, CYC065. Next-generation CDK2/9 inhibition elicits marked antineoplastic effects in lung cancer via anaphase catastrophe and reduced PEA15 phosphorylation.

MicroRNA-132 targets PEA-15 and suppresses the progression of astrocytoma in vitro.

Gliomas are highly malignant tumors, the most common of which are astrocytomas. A growing number of studies suggest that dysregulation of miRNAs is a frequent event contributing to the pathogenesis of gliomas. In this study, we found that over-expression of miR-132 inhibited cell proliferation and migration and triggered apoptosis, while knockdown of miR-132 showed opposite effects. PEA-15 was identified as a direct target of miR-132. Reintroduction of PEA-15 without 3'UTR region reversed the inhibitory effects of miR-132 on cell proliferation, migration, and apoptosis. MiR-132 was inversely correlated with the PEA-15 expression. CREB (cAMP response element binding protein) and KLF (Krüppel-like factor 8) were conformed as transcription factors of miR-132, which bidirectionally regulate the expression of miR-132. Our study suggests that miR-132 is an important tumor suppressor of astrocytoma progression by targeting PEA-15, while CREB and KLF can modulate the expression of miR-132, thus providing new insight into the molecular mechanisms underlying astrocytoma progression in vitro.

PEA-15 facilitates EGFR dephosphorylation via ERK sequestration at increased ER–PM contacts in TNBC cells

Phosphoprotein enriched in astrocytes of 15kDa (PEA-15) is known to sequester extracellular signal-regulated kinase (ERK) in the cytoplasm, inhibiting tumorigenesis of human breast cancer cells. Here, we describe how PEA-15 expression affects the dephosphorylation of epidermal growth factor receptor (EGFR) through endoplasmic reticulum (ER)–plasma membrane (PM) contacts in MDA-MB-468, triple-negative breast cancer (TNBC) cells. The increased intracellular calcium concentration resulting from increased cytoplasmic phosphorylated ERK facilitates movement of ER-anchored calcium sensors to the PM. The driving force of trans-localization of calcium-dependent proteins enhances the contact between the activated EGFR and ER-localized phosphatase, PTP1B. Consequently, our findings suggest a mechanism underneath the facilitation of EGFR dephosphorylation by cytoplasmic PEA-15 expression inside TNBC cells, which may be one of the dynamic mechanisms for down-regulation of activated EGFR in cancer cells.

Downregulation of PEA-15 reverses G1 arrest, and nuclear and chromatin changes of senescence phenotype via pErk1/2 translocation to nuclei

We previously showed that senescent cells respond to TPA with translocation of senescence associated-pErk1/2 (SA-pErk1/2) into nuclei along with reversal of senescence morphology. Here, we describe that the reversal of senescence phenotype was manifested by knockdown of cytoplasmic PEA-15 expression, a sequestrator of cytoplasmic pErk1/2. Transfection of short-interfering RNA to PEA-15 (siPEA-15) significantly induced nuclear translocation of SA-pErk1/2, and siPEA-15 with TPA co-treatment further increased the translocation. Moreover, the reversal of senescence phenotype, such as expressions of SA-β-galactosidase, p53, p21WAF1, PML body, 53BP1 and H3K9me2, was modified by either knockdown of PEA-15 or TPA treatment, indicating that nuclear translocation of SA-pErk1/2 might inhibit senescence progression. Indeed, knockdown of PEA-15 or TPA treatment significantly induced progression of G1 arrested cells to S-phase in human diploid fibroblast (HDF) senescent cells, examined by immunocytochemistry, FACS and immunoblot analyses. In conclusion, downregulation of PEA-15 expression reverses senescence phenotypes via nuclear translocation of SA-pErk1/2, which suggests in vivo maintenance of senescence phenotype by sequestration of pErk1/2 in cytoplasm.

PEA15 Regulates the DNA Damage-Induced Cell Cycle Checkpoint and Oncogene-Directed Transformation

Regulation of the DNA damage response and cell cycle progression is critical for maintaining genome integrity. Here, we report that in response to DNA damage, COPS5 deubiquitinates and stabilizes PEA15 in an ATM kinase-dependent manner. PEA15 expression oscillates throughout the cell cycle, and the loss of PEA15 accelerates cell cycle progression by activating CDK6 expression via the c-JUN transcription factor. Cells lacking PEA15 exhibit a DNA damage-induced G2/M checkpoint defect due to increased CDC25C activity and, consequentially, higher cyclin-dependent kinase 1 (CDK1)/cyclin B activity, and accordingly they have an increased rate of spontaneous mutagenesis. We find that oncogenic RAS inhibits PEA15 expression and that ectopic PEA15 expression blocks RAS-mediated transformation, which can be partially rescued by ectopic expression of CDK6. Finally, we show that PEA15 expression is downregulated in colon, breast, and lung cancer samples. Collectively, our results demonstrate that tumor suppressor PEA15 is a regulator of genome integrity and is an integral component of the DNA damage response pathway that regulates cell cycle progression, the DNA-damage-induced G2/M checkpoint, and cellular transformation.

The PEA-15/PED protein regulates cellular survival and invasiveness in colorectal carcinomas

The PEA-15/PED (phosphoprotein enriched in astrocytes 15kD/phosphoprotein enriched in diabetes) protein is a multifunctional phosphoprotein involved in various signaling pathways which determine survival, proliferation, and migration of cancer cells. Here, we investigated the expression and cellular functions of PEA-15 in colorectal carcinoma (CRC). PEA-15 is expressed in the majority of human CRC, predominantly in well differentiated tumor areas. A tissue microarray analysis of 1262 human CRC specimens from the DACHS study showed that PEA-15 expression is significantly associated with a low pT stadium as defined by limited invasion into the bowel wall. Moreover, patients with PEA-15-positive CRC exhibited a significantly longer tumor-specific survival time. To investigate the functional relevance of PEA-15 expression on a cellular level, we over-expressed PEA-15 in several CRC cell lines. Increased expression of PEA-15 resulted in a strong inhibition of clonogenicity, proliferation, and invasiveness of CRC cells. These effects were associated with a PEA-15-dependent down-regulation of integrin αvβ5 as well as with elevated levels of the phosphorylated MAP kinase ERK1/2. Moreover, expression of PEA-15 resulted in significant protection from cell death induced by cytotoxic drugs (5-FU, cisplatin), by the death ligand TRAIL, or by serum withdrawal. In conclusion, the PEA-15 protein regulates invasiveness, proliferation, and apoptosis resistance in CRC cells. PEA-15 might play an important role in chemoresistance, progression and metastasis in CRC.

Abstract 5616: Mutant PEA-15 inhibits in vitro and in vivo tumorigenesis in ovarian cancer by inhibiting beta-catenin

Abstract PEA-15 is 15-kDa serine-phosphoprotein that sequesters phospho-ERK from the nucleus to the cytoplasm. Previously, we showed that overexpression of PEA-15 had antitumor effects in both breast and ovarian cancer cells. We also reported that PEA-15 expression was associated with prolonged overall survival in patients with epithelial ovarian cancer, as found using a tissue microarray. Because PEA-15 has two major phosphorylation sites, Serine104 and Serine116, that are closely related to its biological function in regulating cell proliferation and survival, we sought to develop a more potent form of PEA-15 by modifying its two major phosphorylation sites. To determine which mutant form of PEA-15 is a more potent antitumor agent, we evaluated the function of double-mutated PEA-15 at Ser104 and Ser116; PEA-15-AA, in which residues were substituted with alanine (double-unphosphorylated form); and PEA-15-DD, in which residues were substituted with aspartic acid (double-phosphorylated form) were tested in vitro and in vivo in ovarian cancer cell lines. PEA-15-AA resulted in 85% inhibition of SKOV3.ip1 cell colony formation (P&amp;lt;0.001) under anchorage-independent conditions. Compared to vector control, PEA-15-AA reduced projection formation by about 80%-90% (P=0.01) under the 3D Matrigel culture condition and inhibition of cell migration by 60%-80% (P=0.02). Further, to determine the in vivo effect of PEA-15-AA, we injected control vector, PEA-15-AA, and PEA-15-DD stable transfectants of SKOV3.ip1 cells intraperitoneally into nude mice. We observed that PEA-15-AA strongly inhibited tumor formation and tumor size (6 of 15, P=0.02) compared to the control (9 of 10) or PEA-15-DD (16 of 16). Immunohistochemical data revealed that beta-catenin and CD-31 (endothelial cell marker) expression was significantly decreased in PEA-15-AA tumor tissues. These data show that the antitumor effect of PEA-15-AA was partially dependent on both the inhibition of beta-catenin expression and its nuclear translocalization. We next determined the clinical relevance of phosphorylated PEA-15 in patients with ovarian cancer using a human ovarian cancer tissue microarray. Our analysis showed that total double-phosphorylated PEA-15 expression at Ser104 and Ser116 was significantly higher in high-grade (II, P=0.002; III, P=0.001) ovarian tumor tissue than in adjacent normal ovarian tissue. These data indicate that PEA-15 in ovarian tumor cells exists predominantly in the double-phosphorylated form, PEA-15-DD. Further studies are warranted to determine the correlation between the phosphorylation status of PEA-15 and overall survival in patients with ovarian cancer. Taken together, our results suggest that PEA-15-AA has strong antitumor effects in vitro and in vivo that would justify its development as an effective therapeutic molecule in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5616. doi:1538-7445.AM2012-5616

Abstract 5670: Phospho-PEA-15 sensitizes ovarian cancer cells to paclitaxel by impairing the microtubule-destabilizing effect of SCLIP

Abstract Paclitaxel is a standard chemotherapeutic agent for ovarian cancer. Resistance of ovarian cancer cells to the drug has been a major obstacle in clinical practice. Thus, alternative approaches are needed to conquer the resistance. PEA-15 (phosphoprotein enriched in astrocytes-15 kDa) regulates cell proliferation and apoptosis. It is phosphorylated at S104 and S116 by Akt, PKC and CaMKII. PEA-15's functions are phosphorylation dependent. Although PEA-15 is known to mediate chemoresistance in breast cancer, the effect of PEA-15 phosphorylation status on chemosensitivity remains unknown. We hypothesized that phospho-PEA-15 (pPEA-15) enhances sensitivity of ovarian cancer cells to paclitaxel. To test our hypothesis, we silenced PEA-15 expression in HEY and OVTOKO cells using siRNA and observed a 14% reduction in apoptosis after paclitaxel exposure. To further determine if PEA-15 phosphorylation contributes to chemosensitivity, we generated SKOV3.ip1-vector (control), SKOV3.ip1-AA (AA, phosphoinhibitory at S104 and S116) and SKOV3.ip1-DD (DD, phosphomimetic at S104 and S116) stable ovarian cancer cells. Compared to the control, DD cells showed a 15% reduction in cell viability (P&amp;lt;0.01), a 92% inhibition in anchorage-independent growth (P&amp;lt;0.001), and a 15% increase in apoptosis after paclitaxel treatment. In contrast, AA cells had a 10% increase in cell viability (P&amp;lt;0.01) and a 20% decrease in apoptosis. These results indicate that pPEA-15 sensitizes ovarian cancer cells to paclitaxel. cDNA microarray analysis revealed that SCLIP, a microtubule (MT)-destabilizing phosphoprotein, was involved in pPEA-15-mediated chemosensitization. The level of SCLIP was 8.5-fold higher at mRNA and 1.6-fold higher at protein in untreated DD cells than in the control and AA cells. Interestingly, exposure to paclitaxel resulted in a 2-fold reduction in SCLIP's protein level and a dramatic increase in its phosphorylation level in DD cells. No similar changes were observed in the control and AA cells. Further, compared to the control and AA cells, higher levels of acetylated and detyrosinated α-tubulin were detected in DD cells exposed to paclitaxel, indicating that MTs are highly stabilized in treated DD cells. These results suggest that reduced expression and increased phosphorylation of SCLIP impair its MT-destabilizing effect, thereby enhancing paclitaxel sensitivity in DD cells. We demonstrate that pPEA-15 mediates chemosensitization in ovarian cancer cells by impairing the MT-destabilizing effect of SCLIP. Our findings highlight the importance of pPEA-15 as a promising target for improving the efficacy of paclitaxel-based chemotherapy in ovarian cancer. Further studies will be conducted to determine: 1) the association between pPEA-15 and SCLIP, and 2) the in vivo effectiveness of pPEA-15 in improving the therapeutic efficacy of paclitaxel in an ovarian xenograft mouse model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5670. doi:1538-7445.AM2012-5670

MEK1/2 Inhibitor Selumetinib (AZD6244) Inhibits Growth of Ovarian Clear Cell Carcinoma in a PEA-15–Dependent Manner in a Mouse Xenograft Model

Clear cell carcinoma (CCC) of the ovary tends to show resistance to standard chemotherapy, which results in poor survival for patients with CCC. Developing a novel therapeutic strategy is imperative to improve patient prognosis. Epidermal growth factor receptor (EGFR) is frequently expressed in epithelial ovarian cancer. One of the major downstream targets of the EGFR signaling cascade is extracellular signal-related kinase (ERK). PEA-15, a 15-kDa phosphoprotein, can sequester ERK in the cytoplasm. MEK1/2 plays a central role in integrating mitogenic signals into the ERK pathway. We tested the hypothesis that inhibition of the EGFR-ERK pathway suppresses tumorigenicity in CCC, and we investigated the role of PEA-15 in ERK-targeted therapy in CCC. We screened a panel of 4 CCC cell lines (RMG-I, SMOV-2, OVTOKO, and KOC-7c) and observed that the EGFR tyrosine kinase inhibitor erlotinib inhibited cell proliferation of EGFR-overexpressing CCC cell lines through partial dependence on the MEK/ERK pathway. Furthermore, erlotinib-sensitive cell lines were also sensitive to the MEK inhibitor selumetinib (AZD6244), which is under clinical development. Knockdown of PEA-15 expression resulted in reversal of selumetinib-sensitive cells to resistant cells, implying that PEA-15 contributes to selumetinib sensitivity. Both selumetinib and erlotinib significantly suppressed tumor growth (P < 0.0001) in a CCC xenograft model. However, selumetinib was better tolerated; erlotinib-treated mice exhibited significant toxic effects (marked weight loss and severe skin peeling) at high doses. Our findings indicate that the MEK-ERK pathway is a potential target for EGFR-overexpressing CCC and indicate that selumetinib and erlotinib are worth exploring as therapeutic agents for CCC.

Expression of phosphoprotein enriched in astrocytes 15 kDa (PEA-15) in astrocytic tumors: a novel approach of correlating malignancy grade and prognosis

Phosphoprotein enriched in astrocytes 15 kDa (PEA-15) is a multifunctional protein that was first identified in brain astrocytes and that has subsequently been shown to be expressed in different tissues. Despite its many important roles, the clinical significance of PEA-15 expression levels in astrocytic tumors has yet to be properly defined. We studied the PEA-15 expression pattern of 65 patients [diagnosed according to World Health Organization (WHO) criteria] with diffuse astrocytoma (WHO grade II), anaplastic astrocytoma (grade III), and glioblastoma (grade IV). PEA-15 expression levels were immunohistochemically measured and categorized as no, low, or high expression. All tumors expressed PEA-15 in our study. Twenty-three (35.4%) and 42 (64.6%) tumors expressed low and high PEA-15 levels, respectively. In grade II astrocytoma (diffuse astrocytoma) and grade III astrocytoma (anaplastic astrocytoma), 100% and 88.9% of patients expressed high PEA-15 levels, respectively, while a smaller number (50%) of patients with grade IV astrocytoma (glioblastoma) expressed high PEA-15 levels. PEA-15 expression level was inversely associated with WHO grade (P = 0.0006). Next, we evaluated prognosis and PEA-15 expression levels in 43 patients with high-grade astrocytomas based on the following parameters: age, gender, WHO grade, surgical resection extent, MIB-1 labeling index (LI), and PEA-15 expression level. Multivariable analyses revealed that high PEA-15 expression level displayed a significant correlation with longer overall survival (OS) in high-grade astrocytomas (P = 0.0024). Patients with total resection survived significantly longer (P = 0.0044) than those with lower resection extent, while patients with MIB-1 labeling index ≤25% indicated significant (P = 0.0434) correlation with OS as well. In conclusion, PEA-15 expression level was inversely associated with WHO grade and may serve as an important prognostic factor for high-grade astrocytomas.

PEA-15 Inhibits Tumorigenesis in an MDA-MB-468 Triple-Negative Breast Cancer Xenograft Model through Increased Cytoplasmic Localization of Activated Extracellular Signal-Regulated Kinase

To determine the role of PEA-15 in breast cancer. A reverse-phase protein array was used to measure PEA-15 expression levels in 320 human breast cancers; these levels were correlated with clinical and tumor characteristics. PEA-15 was overexpressed by an adenovirus vector or by stably expressing PEA-15 in different breast cancer cell lines. The effects on breast cancer cell survival and on the downstream apoptotic signaling pathway were measured in terms of cell proliferation (trypan blue for cell viability, bromodeoxyuridine incorporation for DNA synthesis), anchorage-independent growth (soft agar colony formation), and apoptosis (fluorescence-activated cell sorter analysis). The preclinical efficacy of Ad.PEA-15 given intratumorally was evaluated in nude mice bearing tumors from s.c. implanted human MDA-MB-468 triple-negative breast cancer cells. In human breast cancers, low levels of PEA-15 expression correlated with high nuclear grade (P < 0.0001) and with negative hormone receptor status (P = 0.0004). Overexpression of PEA-15 in breast cancer cells resulted in growth inhibition, reduction in DNA synthesis, and onset of caspase-8-dependent apoptosis. In athymic nude mice bearing MDA-MB-468 xenografts, tumor volumes were significantly smaller in mice treated intratumorally with Ad.PEA-15 than in control mice (P < 0.0001). Tumors from mice treated with Ad.PEA-15 had increased levels of activated (phosphorylated) extracellular signal-regulated kinase and reduced levels of Ki-67 compared with tumors from nontreated or control-adenovirus-treated mice. PEA-15 has therapeutic potential in breast cancer. Further preclinical and clinical exploration of PEA-15 as a druggable target is warranted.

Phosphoprotein Enriched in Astrocytes 15 kDa (PEA-15) Reprograms Growth Factor Signaling by Inhibiting Threonine Phosphorylation of Fibroblast Receptor Substrate 2α

Changes in cellular expression of phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) are linked to insulin resistance, tumor cell invasion, and cellular senescence; these changes alter the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway. Here, we define the mechanism whereby increased PEA-15 expression promotes and sustains ERK1/2 activation. PEA-15 binding prevented ERK1/2 membrane recruitment and threonine phosphorylation of fibroblast receptor substrate 2alpha (FRS2alpha), a key link in fibroblast growth factor (FGF) receptor activation of ERK1/2. This reduced threonine phosphorylation led to increased FGF-induced tyrosine phosphorylation of FRS2alpha, thereby enhancing downstream signaling. Conversely, short hairpin RNA-mediated depletion of endogenous PEA-15 led to reduced FRS2alpha tyrosine phosphorylation. Thus, PEA-15 interrupts a negative feedback loop that terminates growth factor receptor signaling downstream of FRS2alpha. This is the dominant mechanism by which PEA-15 activates ERK1/2 because genetic deletion of FRS2alpha blocked the capacity of PEA-15 to activate the MAP kinase pathway. Thus, PEA-15 prevents ERK1/2 localization to the plasma membrane, thereby inhibiting ERK1/2-dependent threonine phosphorylation of FRS2alpha to promote activation of the ERK1/2 MAP kinase pathway.

Proteomic differential display analysis identified upregulated astrocytic phosphoprotein PEA‐15 in human malignant pleural mesothelioma cell lines

We performed proteomic differential display analysis of human malignant pleural mesothelioma (MPM) cell lines and a human pleural mesothelial cell line by using 2-DE and LC-MS/MS. The human MPM cell lines were NCI-H28, NCI-H2052 and NCI-H2452, and the human pleural mesothelial cell line was MeT-5A. Between MeT-5A and NCI-H2052, we found 38 protein spots whose expression levels were different, from the results of 2-DE; 28 protein spots appeared higher, and 10 other protein spots lower in NCI-H2052 than in MeT-5A. These spots were analyzed by LC-MS/MS analysis and identified by a peptide sequence tag. However, from the results of 2-DE of the other cell lines, there was only one consistently upregulated protein, astrocytic phosphoprotein PEA-15, in all three MPM cell lines. Western blotting using specific antibodies against PEA-15 confirmed the elevated expression level of PEA-15 in all three MPM cell lines compared with MeT-5A cells and normal pleura tissues from patients. PEA-15 was knocked down in NCI-H2052 cells, and the proliferation of PEA-15-silenced NCI-H2052 cells was suppressed 7-15% compared with negative control cells. These results suggest that PEA-15 expression is likely to be associated with the tumorigenesis of MPM.

Vitamin D3 signalling in the brain enhances the function of phosphoprotein enriched in astrocytes – 15 kD (PEA‐15)

In spite of growing evidence linking vitamin D3 levels to mental health disorders, little is known about its direct targets in the brain. This study set out to investigate targets of vitamin D3 in a human brain stem cell line. We employed arrays with antibodies directed against more than 600 structural and signalling proteins, including phospho-variants. Over 180 proteins responded to vitamin D3, such as cyclin-dependent protein-serine kinase 1/2, epidermal growth factor receptor-tyrosine kinase, protein kinase A, protein-serine kinase Bγ and protein-serine kinase Cα. PEA-15 (phosphoprotein enriched in astrocytes-15 kD, also known as PED), known to be involved in various anti-proliferative and anti-apoptotic effects, was strongly up-regulated. In silico promoter analysis revealed conserved binding sites for vitamin D3 receptor, suggesting a strong vitamin D3 dependency of the PEA-15 promoter. PEA-15 up-regulation by vitamin D3 could be confirmed by Western blot in two different cell lines. Analysis of mRNA and protein phosphorylation status of PEA-15 suggests that increased PEA-15 promoter activity and increased protein stabilization contribute to the overall rise of PEA-15 protein. In a functional test of this novel pathway, we demonstrated that vitamin D3 was able to rescue cells from TRAIL-induced apoptosis through regulation of the PEA-15 expression and function. Summarized, our study presents novel targets of vitamin D3 relevant for apoptosis and cell proliferation, and thus strongly supports a function of vitamin D3 in the brain that impacts on processes highly relevant for major neurological disorders.