PDF file - 357K, Supplementary Figure 1. Chemical structure of paclitaxel: (2α,4α,5β,7β,10β,13α)-4,10-bis(acetyloxy)-13-{(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyloxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-yl benzoate. Supplementary Figure 2. Overexpression of PEA-15 phosphorylated at both Ser104 and Ser116 enhanced the apoptotic effect of paclitaxel in ovarian cancer cells. Supplementary Figure 3. The addition of caspase 8 and 9 inhibitors reduced paclitaxel-induced apoptosis in SKOV3.ip1-AA and SKOV3.ip1-DD cells. Supplementary Figure 4. Quantification of SCLIP mRNA levels in SKOV3.ip1 stable cells. Supplementary Figure 5. SCLIP mediated paclitaxel resistance in ovarian cancer cells.

Supplementary Figures 1-2 from PEA-15 Induces Autophagy in Human Ovarian Cancer Cells and Is Associated with Prolonged Overall Survival

Abstract 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. The functions of PEA-15 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 with 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-destabilizing protein, is involved in pPEA-15–mediated chemosensitization. We found that reduced expression and possibly posttranslational modification of SCLIP following paclitaxel treatment impaired the microtubule-destabilizing effect of SCLIP, 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. Mol Cancer Ther; 12(6); 1099–111. ©2013 AACR.

Taxanes are among the drugs most commonly used for preoperative chemotherapy for breast cancer. Taxanes induce mitotic arrest and subsequent apoptosis. The spindle-assembly checkpoint (SAC) is known to be activated during mitosis, along with cyclin-dependent kinase-1 (CDK1), and is required for taxane-induced cell death. We hypothesized that CDK1 activity predicts response to taxane-containing chemotherapy. This study included breast cancer patients who received preoperative chemotherapy- taxane-containing treatment followed by anthracycline-based treatment-and then underwent surgery. Before starting taxane-containing chemotherapy, patients underwent fine-needle aspiration biopsy, and the biopsy samples were incubated in paclitaxel solution to measure CDK activity. Clinical were evaluated after taxane therapy, and pathological resposes were evaluated after completion of all preoperative chemotherapy. Thirty five patients were eligible for analysis of clinical response to taxane-containing therapy. Twenty-six patients had taxane-sensitive and 9 taxane-resistant tumors. Using a cut-off of CDK activity determined by the ROC analysis, patients were classified into SAC function and dysfunction groups. Univariate logistic regression analysis with clinicopathologic parameters showed that only CDK-based SAC functionality was significantly correlated with clinical response (P =0.017). No significant correlation was observed between SAC functionality and pathologic response. CDK-based SAC functionality significantly predicted clinical response (P =.0072, overall agreement = 71.4%), and this is a unique mechanism-based marker for predicting taxane chemosensitivity. Further, large prospective study is needed to determine CDK-based SAC functionality could be developed as a predictive biomarker.

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<0.01), a 92% inhibition in anchorage-independent growth (P<0.001), and a 15% increase in apoptosis after paclitaxel treatment. In contrast, AA cells had a 10% increase in cell viability (P<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

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.

Abstract 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

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-adenovirus treated 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. (C) 2010 AACR.

Abstract 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.

Abstract Ovarian clear cell carcinoma (CCC) has shown resistance to the current standard chemotherapy used for epithelial ovarian cancer (EOC). Therefore, developing a novel therapeutic strategy is imperative to improve the prognosis for patients with CCC. Epidermal growth factor receptor (EGFR) is frequently expressed in EOC. PEA-15 is a 15-kDa phosphoprotein that slows cell proliferation by binding to and sequestering ERK in the cytoplasm. The purpose of the current study was to determine whether the EGFR-ERK pathway is a therapeutic target in CCC and whether PEA-15 modulates ERK-targeted therapy in CCC. We screened a panel of four CCC cell lines (RMG-I, SMOV-2, OVTOKO and KOC-7c) and performed Western blotting to detect the expression levels of EGFR, pEGFR, ERK and pERK. RMG-I and SMOV-2 showed high EGFR expression, while OVTOKO and KOC-7c showed lower expression. We next performed WST-1 assay and showed that erlotinib inhibited cell proliferation in high EGFR-expressing cell lines (IC50 = 0.1 μM for RMG-I and 1 μM for SMOV-2) by suppressing pEGFR and pERK expression and inducing G1 arrest. Further, transfection with constitutive active MEK1 significantly reduced the sensitivity to erlotinib in RMG-I cells (p<0.005), suggesting that erlotinib sensitivity is partially dependent on ERK. Therefore, we hypothesized that ERK inhibition suppresses tumorigenicity in CCC. We examined the sensitivity of CCC to MEK inhibitor AZD6244 (ARRY-142886) and observed that the erlotinib-sensitive cell lines were also sensitive to AZD6244 (IC50 = 0.79 μM for RMG-I and 0.85 μM for SMOV-2) by suppressing pERK expression and inducing G1 arrest. We next investigated the possible mechanistic role of PEA-15 in the therapeutic effect of the ERK-targeted therapy with AZD6244. Phosphorylated PEA-15 at ser116 [pPEA-15(S116)] levels increased after AZD6244 treatment in the sensitive cell lines. Transient transfection with phosphomimetic mutant PEA-15 (S116D) reduced cell viability, sensitizing the insensitive cell lines to AZD6244, implying that pPEA-15 (S116) confers sensitivity to AZD6244 in CCC cells. Further, both AZD6244 (50mg/kg/d) and erlotinib (100mg/kg/d) significantly suppressed tumor growth (p < 0.05) in a CCC xenograft model. As expected, the levels of pERK1/2 and Ki-67 expression were markedly reduced in the AZD6244 treated mice groups. The expression of both PEA-15 and pPEA-15 (S116) expressions was also increased in the AZD6244 treatment groups in vivo. However, at the doses used, AZD6244 was better tolerated than erlotinib in terms of weight loss and skin effects (severe skin peeling). We concluded that the MEK/ERK pathway is a potential therapeutic target for CCC and that AZD6244 is worth exploring as a therapeutic agent for patients with CCC. Further studies are warranted to determine if PEA-15 may be useful to predict sensitivity to ERK-targeted therapy in CCC. 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 1619.

Abstract Abstract #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. 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. 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). 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.

Abstract 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]