Phospho-enriched protein in astrocytes (PEA-15) is a 15-kDa phosphoprotein that slows cell proliferation by binding to and sequestering extracellular signal-regulated kinase (ERK) in the cytoplasm, thereby inhibiting ERK-dependent transcription and proliferation. In previous studies of E1A human gene therapy for ovarian cancer, we discovered that PEA-15 induced the antitumor effect of E1A by sequestering activated ERK in the cytoplasm of cancer cells. Here, we investigated the role of PEA-15 in ovarian cancer tumorigenesis, the expression levels of PEA-15 in human ovarian cancer, and whether PEA-15 expression correlated with overall survival in women with ovarian cancer. We overexpressed PEA-15 in low-PEA-15-expressing cells and knocked down PEA-15 in high-PEA-15-expressing cells and analyzed the effects on proliferation, anchorage-independent growth, and cell cycle progression. We then assessed PEA-15 expression in an annotated tissue microarray of tumor samples from 395 women with primary epithelial ovarian cancer and tested whether PEA-15 expression was linked with overall survival. PEA-15 expression inhibited proliferation, and cell cycle analysis did not reveal apoptosis but did reveal autophagy, which was confirmed by an increase in LC3 cleavage. Inhibition of the ERK1/2 pathway decreased PEA-15-induced autophagy. These findings suggest that the antitumor activity of PEA-15 is mediated, in part, by the induction of autophagy involving activation of the ERK1/2 pathway. Multivariable analyses indicated that the women with high-PEA-15-expressing tumors survived longer than those with low-PEA-15-expressing tumors (hazard ratio, 1.973; P = 0.0167). Our findings indicate that PEA-15 expression is an important prognostic marker in ovarian cancer. [Cancer Res 2008;68(22):9302-10]

Purpose: To determine the role of PEA-15 in breast cancer. Experimental Design: 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.Results: 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-adenovirustreated mice.Conclusion: PEA-15 has therapeutic potential in breast cancer. Further preclinical and clinical exploration of PEA-15 as a druggable target is warranted. Clin Cancer Res; 16(6); 1802-11. ©2010 AACR.Breast cancer is the most common cancer in women in the United States and most of the Western world (1). In 2008, 182,460 cases are expected to be diagnosed (26% of all cancers in women), and 40,480 women are expected to die of the disease (accounting for 15% of all cancer deaths in women, making this disease the second most common cause of cancer death among women). Breast cancer that is negative for estrogen receptor, progesterone receptor, and HER-2 is known as triple receptor-negative breast cancer. It is the only major type of breast cancer for which no specific targeted therapy is available to improve patient outcomes (2). A deeper understanding of the molecular mechanisms responsible for breast cancer is imperative if more effective treatments are to be discovered.PEA-15 (also called PED) is an acidic, serine-phosphorylated, 15-kDa phosphoprotein that contains a death effector domain and is associated with microtubules (3). In primary astrocytes (the first cells in which this protein was identified), PEA-15 was shown to regulate the nuclear localization of extracellular signal-regulated kinase (ERK) and consequently affect the transcription of ERK-dependent targets such as transcription factor Elk. This function was shown...

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 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 four 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. Further, 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, 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.

Paclitaxel is a standard chemotherapeutic agent for ovarian cancer. PEA-15 (phosphoprotein enriched in astrocytes-15 kDa) regulates cell proliferation, autophagy, apoptosis, and glucose metabolism and also mediates AKT-dependent chemoresistance in breast cancer. PEA-15's functions are tightly regulated by its phosphorylation status at Ser104 and Ser116. However, the effect of PEA-15 phosphorylation status on chemosensitivity of cancer cells remains unknown. Here, we tested the hypothesis that PEA-15 phosphorylated at both Ser104 and Ser116 (pPEA-15) sensitizes ovarian cancer cells to paclitaxel. We first found that knockdown of PEA-15 in PEA-15-high-expressing HEY and OVTOKO ovarian cancer cells resulted in paclitaxel resistance, whereas re-expression of PEA-15 in these cells led to paclitaxel sensitization. We next found that SKOV3.ip1-DD cells (expressing phosphomimetic PEA-15) were more sensitive to paclitaxel than SKOV3.ip1-AA cells (expressing nonphosphorylatable PEA-15). Compared to SKOV3.ip1-vector and SKOV3.ip1-AA cells, SKOV3.ip1-DD cells displayed reduced cell viability, inhibited anchorage-independent growth, and augmented apoptosis when treated with paclitaxel. Furthermore, HEY and OVTOKO cells displayed enhanced paclitaxel sensitivity when transiently overexpressing phosphomimetic PEA-15 and reduced paclitaxel sensitivity when transiently overexpressing nonphosphorylatable PEA-15. These results indicate that pPEA-15 sensitizes ovarian cancer cells to paclitaxel. cDNA microarray analysis suggested that SCLIP (SCG10-like protein), a microtubule (MT)-destabilizing protein, is involved in pPEA-15-mediated chemosensitization. We found that reduced expression and possibly posttranslational modification of SCLIP following paclitaxel treatment impaired SCLIP's MT-destabilizing effect, thereby promoting induction of mitotic arrest and apoptosis by paclitaxel. Our findings highlight the importance of pPEA-15 as a promising target for improving the efficacy of paclitaxel-based therapy in ovarian cancer.

Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin β-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the β-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin β-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02–4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1β and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.

#2042 Background: Inflammatory breast cancer (IBC) is a rare subtype well known for its propensity for rapid metastasis. The cause of this rapid metastasis in IBC is unknown. An immunohistochemical analysis of 44 cases of IBC showed HER2 overexpression in 48% of patients and epidermal growth factor receptor (EGFR) overexpression in 30% of patients. EGFR overexpression was the only poor prognostic factor: the 5-year overall survival rate was significantly lower for women with EGFR-positive disease than for women with EGFR-negative disease (P=0.01). HER2 overexpression was not a prognostic factor. The association between EGFR overexpression and increased risk of death indicated that EGFR may represent a potential therapeutic target in IBC. We hypothesized that suppression of the EGFR pathway inhibits proliferation and metastasis of IBC.&#x2028; Methods: SUM149 IBC cells, which express low levels of HER2 and high levels of EGFR, were treated with siRNA against EGFR and with the EGFR tyrosine kinase inhibitor erlotinib.&#x2028; Results: EGFR siRNA knockdown (but not non-targeting siRNA control) inhibited the proliferation of SUM149 cells. SUM149 cells were sensitive to EGFR tyrosine kinase inhibitor erlotinib in a 2-dimensional (2-D) culture system (median inhibitory concentration [IC50] = 0.90 µM). When we activated ERK by transfecting constitutively active MEK1 in SUM149 cells, the cells showed more resistance to erlotinib. Moreover, ERK siRNA knockdown sensitized SUM149 cells to erlotinib. Further, when we cultured the SUM149 cells in matrigel by using the 3-D culture system (100% matrigel in the bottom layer and cultured medium with 5% FBS and 2% matrigel in the top layer), erlotinib treatment changed the molecular phenotype of SUM149 cells from mesenchymal (a phenotype characterized by low beta-catenin expression and high vimentin and fibronectin expression) to epithelial (recovery of beta-catenin to the sites of cell-cell contacts; downregulation of fibronectin). This reversal of the mesenchymal phenotype, a hallmark of inhibition of epithelial-to-mesenchymal transition (EMT), was ERK dependent. Interestingly, the erlotinib concentration that inhibited the mesenchymal phenotype (0.1 mcM) was one log lower than the concentration that inhibited proliferation (1 mcM).&#x2028; Conclusion: Inhibition of tumor growth and EMT in SUM149 IBC cell lines is dependent on the ERK pathway through the EGFR pathway. The erlotinib dose needed to produce an anti-mesenchymal effect is much lower than the cytotoxic dose. Thus, we speculate ERK pathway to be important in inhibiting metastasis in IBC. We are currently investigating the effects of erlotinib in a xenograft model of IBC. Our study provides a rationale for developing novel treatment strategies targeting the EGFR and ERK pathways to inhibit the growth and metastasis of IBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2042.

Selumetinib is a highly selective allosteric inhibitor of MEK1/2. Multiple clinical trials of Selumetinib are ongoing in patients with different types of cancer. However, the therapeutic role of Selumetinib in breast cancer has not been well defined. We sought to determine the effect of targeted inhibition of the MEK-ERK pathway by Selumetinib in triple-negative breast cancer (TNBC). We studied the effect of Selumetinib on 2 TNBC cell lines, MDA-MB-231 and SUM-149. In MDA-MB-231 cells, in 2-dimensional (2D) culture, Selumetinib at 0.1μM did not reduce cell viability, but in a 3-dimensional (3D) cell culture model, which mimics the human microenvironment, Selumetinib at 0.1 μM and 1 μM inhibited epithelial to mesenchymal transition (EMT). This result was confirmed by western blotting: expression of the mesenchymal markers fibronectin and vimentin was inhibited, and the epithelial marker beta-catenin was diffusely expressed in both the cytoplasm and the nucleus before treatment but was localized at the plasma membrane after treatment. Selumetinib in 3D cell culture also inhibited projections/filopodia formation, suggesting reversal to a more epithelial phenotype. Results were similar in SUM-149 cells: Selumetinib at 0.1 μM had minimal impact on cell viability in 2D culture, but Selumetinib at 0.1 μM and 1 μM inhibited projections/filopodia in 3D culture. Inhibition of ERK phosphorylation by Selumetinib correlated with a slight increase in the epithelial marker E-cadherin and loss of vimentin. These results suggest that treating TNBC with Selumetinib induces mesenchymal to epithelial transition (MET). In addition, in MDA-MB-231 cells, Selumetinib significantly inhibited anchorage-independent growth, an indicator of in vivo tumorigenicity. Previously, the ERK2 isoform was shown to induce EMT in epithelial cells. We therefore examined whether ERK1 and 2 expression levels correlate with Selumetinib's effect on EMT regulation in TNBC cells. We used shRNA specifically targeting ERK1 and ERK2 (shERK1 and 2). Compared with parental MDA-MB-231 cells, stable clones that constitutively expressed shERK1 or shERK2 showed no difference in growth rate in 2D culture or projection formation in 3D culture. However, in 3D culture, treatment with Selumetinib inhibited spindle-shaped cell morphology and reduced scattering of the parental, vector-transfected, and shERK2 clones but did not inhibit mesenchymal filamentous structures in the shERK1 clones. Our data demonstrate that ERK1 may be necessary for Selumetinib -induced mesenchymal to epithelial transition in TNBC. We are planning in vivo studies to determine if low-dose Selumetinib can inhibit EMT, leading to a reduction of metastasis in a TNBC xenograft model. We will further confirm if ERK1 can serve as a biomarker for MEK inhibitor therapy in TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3368. doi:10.1158/1538-7445.AM2011-3368

Background: PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) has tumor suppressor properties in both breast and ovarian cancer cells. Overexpression of PEA-15 in MDA-MB-468 triple-negative breast cancer cells using adenoviral vector (Ad.PEA-15) inhibited cell growth by inducing apoptosis. Treatment with Ad.PEA-15 led to significant regression of tumors in mice, indicating that PEA-15 could have therapeutic potential in breast cancer. PEA-15 exerts its antitumor activity by binding to ERK in the cytoplasm and preventing its translocation into the nucleus, thereby inhibiting ERK-dependent transcription and cell proliferation. Because ERK inhibitors have been shown to be toxic, in this study, we developed PEA-15 mimetic peptides as a therapeutic agent in breast cancer. These peptides, which are expected to be less toxic because of their ability to mimic PEA-15, are proposed to inhibit cell proliferation by sequestering ERK in the cytoplasm. Material and Methods: PEA-15 mimetic peptides were designed and synthesized based on structural analysis of linear segments from the C-terminal end of PEA-15, which is one of the two regions that are presumably critical for ERK binding, using nuclear magnetic resonance spectroscopy. These synthetic peptides were labeled with FAM to enable determination of their subcellular localization. The PULSin reagent was used as a delivery reagent for peptide penetration into cancer cells. The penetration ability of these peptides into HeLa and MDA-MB-468 cells was analyzed by fluorescence microscope and fluorescence-activated cell sorting. Their effect on ERK subcellular localization was examined by cell-fractionation analysis. Their impact on cell survival was determined by trypan blue viability assay and propidium iodide staining. Results: Ten peptides were designed and synthesized based on the C-terminal segments of PEA-15. Three of these FAM-labeled synthetic peptides delivered with the PULSin reagent were able to penetrate HeLa and MDA-MB-468 cells with 80-90% and 30-40% efficiency, respectively. Further, these peptides redirected ERK into the cytoplasm from the nucleus in MDA-MB-468 cells and caused 31% reduction in viability of HeLa cells as compared to the untreated control. Ongoing studies are evaluating PEA-15 peptides for their ability to interact with ERK using ELISA. In the future, we will also test their therapeutic efficacy and safety using a xenograft breast cancer model. Discussion: Previous studies revealed that PEA-15 has antitumor activity against breast and ovarian cancer cells by sequestering ERK in the cytoplasm. Due to the toxicity of ERK enzymatic inhibitors, we developed PEA-15 mimetic peptides and demonstrated their therapeutic potential for breast cancer, which may provide opportunities for breast cancer patients who currently have limited treatment options. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-20.