Hitomi Saso

TIG1 Promotes the Development and Progression of Inflammatory Breast Cancer through Activation of Axl Kinase

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer, but the basis for its aggressive properties are not fully understood. In this study, we report that high tumoral expression of TIG1 (RARRES1), a functionally undefined membrane protein, confers shorter survival in patients with IBC. TIG1 depletion decreased IBC cell proliferation, migration, and invasion in vitro and inhibited tumor growth of IBC cells in vivo. We identified the receptor tyrosine kinase, Axl, as a TIG1-binding protein. TIG1 interaction stablilized Axl by inhibiting its proteasome-dependent degradation. TIG1-depleted IBC cells exhibited reduced Axl expression, inactivation of NF-κB, and downregulation of matrix metalloproteinase-9, indicating that TIG1 regulates invasion of IBC cells by supporting the Axl signaling pathway in IBC cells. Consistent with these results, treatment of IBC cells with the Axl inhibitor SGI-7079 decreased their malignant properties in vitro. Finally, TIG1 expression correlated positively with Axl expression in primary human IBC specimens. Our findings establish that TIG1 positively modifies the malignant properties of IBC by supporting Axl function, advancing understanding of its development and rationalizing TIG1 and Axl as promising therapeutic targets in IBC treatment.

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. Mol Cancer Ther; 11(2); 360–9. ©2011 AACR.

MEK Inhibitor Selumetinib (AZD6244; ARRY-142886) Prevents Lung Metastasis in a Triple-Negative Breast Cancer Xenograft Model

Patients with triple-negative breast cancer (TNBC) have a poor prognosis because TNBC often metastasizes, leading to death. Among patients with TNBC, those with extracellular signal-regulated kinase 2 (ERK2)-overexpressing tumors were at higher risk of death than those with low-ERK2-expressing tumors (hazard ratio, 2.76; 95% confidence interval, 1.19–6.41). The MAPK pathway has been shown to be a marker of breast cancer metastasis, but has not been explored as a potential therapeutic target for preventing TNBC metastasis. Interestingly, when we treated TNBC cells with the allosteric MEK inhibitor selumetinib, cell viability was not reduced in two-dimensional culture. However, in three-dimensional culture, selumetinib changed the mesenchymal phenotype of TNBC cells to an epithelial phenotype. Cells that undergo epithelial–mesenchymal transition (EMT) are thought to contribute to the metastatic process. EMT leads to generation of mesenchymal-like breast cancer cells with stem cell–like characteristics and a CD44+CD24−/low expression pattern. We tested the hypothesis that targeted inhibition of the MAPK pathway by selumetinib inhibits acquisition of the breast cancer stem cell phenotype and prevents lung metastasis of TNBC. TNBC cells treated with selumetinib showed inhibition of anchorage-independent growth, an indicator of in vivo tumorigenicity (P < 0.005), and decreases in the CD44+CD24−/low fraction, ALDH1 activity, and mammosphere-forming efficiency. Mice treated with selumetinib formed significantly fewer lung metastases than control mice injected with vehicle (P < 0.05). Our data demonstrate that MEK inhibitors can inhibit breast cancer stem cells and may have clinical potential for the prevention of metastasis in certain cases in which tumors are MAPK dependent. Mol Cancer Ther; 14(12); 2773–81. ©2015 AACR.

PEA-15 unphosphorylated at both serine 104 and serine 116 inhibits ovarian cancer cell tumorigenicity and progression through blocking β-catenin.

Ovarian cancer is a major cause of death among women; there remains an urgent need to develop new effective therapies to target this cancer. Phosphoprotein enriched in astrocytes (PEA-15) is a 15-kDa phosphoprotein that is known to bind ERK1/2, thus blocking cell proliferation. The physiological activity of PEA-15 is dependent on the phosphorylation status of serine 104 (Ser104) and Ser116. However, little is known about the impact of PEA-15 phosphorylation on tumor progression. We have previously shown that overexpression of PEA-15 has an antitumor effect against both breast and ovarian cancer cells. Here, we report that using a human ovarian cancer tissue microarray, we found that tissues from patients with ovarian cancer were significantly more likely than adjacent normal tissues to express PEA-15 phosphorylated at both sites. Using phosphomimetic and nonphosphorylatable mutants of PEA-15, we found that mutant double-unphosphorylated PEA-15 in which Ser104 and Ser116 were substituted with alanine (PEA-15-AA) had a more potent antitumorigenic effect in ovarian cancer than did phosphomimetic PEA-15 in which Ser104 and Ser116 were substituted with aspartic acid (PEA-15-DD). Further, we observed that the antitumorigenic effect of PEA-15-AA was a result of inhibition of the migration capacity of cells and inhibition of in vivo angiogenesis. This inhibition was partially dependent on inhibition of β-catenin expression and nuclear translocalization. Taken together, our results suggest that phosphorylated PEA-15 is an important contributor to the aggressiveness of ovarian cancer and justify the development of PEA-15-AA as an effective therapeutic molecule in the treatment of ovarian cancer.

Abstract 1624: Silencing of ERK2 reverses EMT and suppresses the CSC phenotype, inhibiting lung metastasis in triple-negative breast cancer

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking estrogen receptor, progesterone receptor, and HER2 overexpression. Patients with TNBC have a generally poor prognosis due to metastasis, high rates of recurrence, and lack of FDA-approved targeted therapies. We previously showed using functional proteomics that patients with high-ERK2-expressing TNBC tumors had a higher risk of death than those with low-ERK2-expressing tumors. Moreover, ERK2 but not ERK1 plays an important role in epithelial-mesenchymal transition (EMT) and is required for acquisition of stem cell-like characteristics. Compared to other breast cancer subtypes, TNBC has a higher proportion of cancer stem cells (CSCs) and is linked to EMT, two critical features associated with breast cancer progression, metastasis, and recurrence in patients. The MAPK signaling pathway is activated in TNBC, but the roles of ERK isoforms in tumor progression and metastasis are not well defined. We hypothesized that ERK2 but not ERK1 promotes EMT, the CSC phenotype, and metastasis in TNBC. Methods and Results: Knockdown of ERK2 in SUM149 and BT549 TNBC cells significantly inhibited anchorage-independent colony formation (p Conclusions and Future Directions: Our findings support our hypothesis, indicating that ERK2 promotes EMT and the CSC phenotype through EGR1 and mediates metastasis in TNBC. Future studies will determine ERK activity and pathway engagement using a novel peptide sensor based on the Sox fluorophore. We will pursue a therapeutic approach using siRNA against ERK2 incorporated in a DOTAP:cholesterol liposome. Citation Format: Mary Kathryn Pitner, Hitomi Saso, Richard Larson, Rachel M. Sammons, Huiqin Chen, Caimiao Wei, Gaurav Chauhan, Kimie Kondo, Naoto T. Ueno, Kevin Dalby, Bisrat G. Debeb, Chandra Bartholomeusz. Silencing of ERK2 reverses EMT and suppresses the CSC phenotype, inhibiting lung metastasis in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1624.

Abstract 858: ERK2 rather than ERK1 contributes to EMT and metastatic potential in triple-negative breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Patients with triple-negative breast cancer (TNBC), which lacks estrogen receptor and progesterone receptor and has low HER2 expression, have a very poor prognosis because the disease often metastasizes. Preventing metastasis, as well as inhibiting tumor growth, is crucial to improving the prognosis of patients with TNBC. We previously showed using reverse-phase protein array analysis that patients with ERK2-overexpressing TNBC had a higher risk of death than those with low-ERK2-expressing TNBC. TNBC is characterized by an epithelial-to-mesenchymal transition (EMT) phenotype, which has features in common with those of cancer stem cells. Cells undergoing EMT may contribute to the metastatic process and have increased ability to form mammospheres in vitro. The roles of the 2 ERK isoforms, ERK1 and ERK2, in TNBC are not well defined. We hypothesized that ERK2, but not ERK1promotes EMT and metastasis in TNBC. Methods & Results: We compared parental SUM-149 TNBC cells and stable clones that constitutively expressed shERK1 or shERK2 and observed no differences in cell proliferation. However, reducing the expression of ERK2, but not reducing the expression of ERK1, changed the mesenchymal phenotype of TNBC to an epithelial phenotype, reduced formation of mammospheres in a dose-dependent manner, and reduced the stem cell subpopulation (CD44+/CD24-), which correlated with a reduction in migration and invasion. In addition, ERK2 knockdown inhibited anchorage-independent growth, an indicator of in vivo tumorigenicity. To determine which genes are involved in ERK2-induced EMT and metastasis, we performed transcriptional profiling using real-time PCR-based EMT and metastasis arrays. After normalization, we observed that mRNA levels of the epithelial markers KRT7 and KRT14 were increased by 4 and 1.8 fold, respectively, in ERK2-silenced cells as in control cells; mRNA levels of the mesenchymal markers Twist1, vimentin, and ZEB1 were reduced by 0.6, 0.1 and 0.2 fold respectively, in ERK2-silenced cells as in control cells; and mRNA levels of the metastasis-related genes MMP-2, MMP-7, MMP-13, IL1B, and COL4A2 were reduced by 0.3, 0.3, 0.6, 0.6 and 0.3 fold respectively, in ERK2-silenced cells as in control cells. Further, using an Affymetrix gene chip and the Inflammatory Breast Cancer World Consortium patient data set (n=389), we did a class comparison test in which the P value was calculated by Wilcoxon test. We observed significantly lower ERK1 mRNA expression in the TNBC group than the non-TNBC group (P < 0.0001) but significantly higher ERK2 mRNA expression in the TNBC group than in the non-TNBC group (P < 0.005). Conclusions: ERK2 but not ERK1 may promote tumorigenesis of the TNBC subgroup by enriching the cancer stem cell population and inducing EMT and metastasis. Our long-term goal is to confirm these findings in xenograft studies and develop ERK2-targeted therapy for TNBC. Citation Format: Chandra Bartholomeusz, Hitomi Saso, Ali Dadbin, Hiroko Masuda, Takahiro Kogawa, Steven Van Laere, Francois Bertucci, Gabriel N. Hortobagyi, Naoto T. Ueno. ERK2 rather than ERK1 contributes to EMT and metastatic potential in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 858. doi:10.1158/1538-7445.AM2013-858

Abstract LB-362: The MEK inhibitor selumetinib (AZD6244 - ARRY-142886) prevents lung metastasis in a triple-negative breast cancer (TNBC) xenograft model

Patients with TNBC are negative for estrogen receptor, progesterone receptor, and HER2 expression, and have a very poor prognoses because tumors often metastasize, leading to death. Preventing metastasis as well as inhibiting the tumor growth, is crucial to improving the prognosis of TNBC. We previously showed that patients with ERK2-overexpressing TNBC were at higher risk of death than those with low-ERK2-expressing tumors. Interestingly, when we treated the TNBC cells lines MDA-MB-231 and SUM-149 with the MEK1/2 ATP uncompetitive kinase inhibitor (selumetinib) at concentrations of 0.1 and 1.0 μM, it did not reduce cell viability in a two-dimensional (2D) cell culture. However, in a three-dimensional (3D) cell culture model, selumetinib changed the mesenchymal phenotype of TNBC to an epithelial phenotype. Cells undergoing epithelial-mesenchymal transition are well known to potentially contribute to the metastatic process and have increased ability to form mammospheres. Thus, we hypothesized that targeted inhibition of the MEK-ERK pathway by selumetinib may prevent lung metastasis in TNBC. In the present study, we observed that SUM-149 cells treated with selumetinib (0.1, 1.0 μM) exhibited cell-cycle arrest at G1 but did not exhibit apoptosis indicated by an increased proportion of cells at sub-G1 phase. Both SUM-149 and MDA-MB-231 cells treated with selumetinib exhibited significant inhibition of anchorage-independent growth, an indicator of in vivo tumorigenicity (P 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 LB-362. doi:1538-7445.AM2012-LB-362

Abstract 3368: MEK inhibitor selumetinib (AZD6244 – ARRY-142886) promotes mesenchymal to epithelial transition in triple-negative breast cancer

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 Selumetinib9s 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

PD08-06: ERK2 Promotes Stem Cell-Like Characteristics in Triple-Negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) is resistant to targeted therapies such as hormonal therapy and HER2−targeted therapies. Thus, no specific targeted therapy is currently available for TNBC. ERK2, a component of the MAPK pathway, plays an important role in epithelial-mesenchymal transition (EMT) in MCF-7-10A, a nontransformed human mammary epithelial cell line and is required for full acquisition of stem cell-like characteristics. Inhibition of ERK2 dramatically reduces cell growth, whereas inhibition of ERK1 significantly facilitates proliferation. ERK1 mutant mice have a strikingly milder phenotype than ERK2 mutant mice, which die early in development. In addition, we have shown that the upstream target of ERK, MEK, may be a target for treating TNBC with the MEK inhibitor selumetinib. Our reverse-phase protein array analysis showed that of patients with TNBC, those with ERK2-overexpressing tumors were at higher risk of death than those with low-ERK2-expressing tumors. Therefore, we hypothesized that ERK2 promotes the tumorigenesis and metastasis of TNBC. We sought to determine the role of ERK2 in the tumorigenic and metastatic activities in TNBC and whether ERK2 is required for acquisition of stem cell-like characteristics in TNBC. Methods: We studied the role of ERK1 and ERK2 in the TNBC cell line SUM-149. We used shRNA to specifically knockdown ERK1 and ERK2 (shERK1 and shERK2) and examined whether knocking down ERK1 and ERK2 correlated with EMT regulation, migration, and tumorigenicity in TNBC cells. Results: Compared with parental SUM-149 cells, stable clones that constitutively expressed shERK1 or shERK2 showed no difference in growth rate. However, knocking down ERK2 significantly inhibited migration and the acquisition of stem cell-like characteristics (CD44+/CD24-) in SUM-149 cells in vitro. In addition, knockdown of ERK2 also inhibited anchorage-independent growth, an indicator of in vivo tumorigenicity, whereas knockdown of ERK1 did not inhibit anchorage-independent growth. However, inhibition of ERK1 or ERK2 in 2D or 3D cell culture did not correlate with changes in epithelial and mesenchymal markers by western blot analysis. Conclusion: Our data demonstrate that ERK2 may promote the tumorigenesis of TNBC via enriching cancer stem cells. Our long-term goal is to develop ERK-targeted therapy for TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-06.

P2-02-01: A Novel Inflammatory Breast Cancer-Specific Oncogene, Tazarotene-Induced Gene 1, Promotes Tumorigenicity and Invasiveness through the Receptor Tyrosine Kinase Axl.

Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and is highly metastatic. The prognosis of patients with IBC is poor, and effective standard therapies for IBC are limited because the molecular mechanisms underlying the pathogenesis of IBC remain unknown. We recently found that tazarotene-induced gene 1 (TIG1) expression is significantly higher in IBC cell lines than in non-IBC cell lines. In both IBC and non-IBC data sets, estrogen receptor-negative/HER2−negative samples had significantly higher expression of TIG1 than did other clinical subtypes (estrogen receptor-positive/HER2−negative and HER2−positive). Therefore, we hypothesized that TIG1 plays an important role in the malignant process of IBC. In these studies, we determined the biological function of TIG1 in IBC cells and elucidated the molecular mechanism by which TIG1 regulates the invasiveness of IBC cells. Methods: TIG1 expression in SUM149 and KPL-4 IBC cells was stably knocked down, and the effects of this knockdown on in vitro cell proliferation, migration, and invasion were analyzed. The effects of restoring TIG1 expression on TIG1-silencing IBC cells were also examined. To determine the tumorigenic activity of TIG1 in vivo, TIG1 stable-knockdown SUM149 cells and control shRNA-transfected cells were implanted into the mammary fat pads of athymic nude mice, and tumor growth was monitored. The receptor tyrosine kinase Axl, a potential functional partner of TIG1, was identified using DNA microarray analysis. The interaction between TIG1 and Axl in IBC cells was examined using immunoprecipitation and confocal microscopy assays. The signaling pathway in IBC cells in which TIG1 participates was also investigated. Results: Knockdown of TIG1 expression in IBC cells reduced their proliferation, migration, and invasion in vitro. Also, silencing of TIG1 dramatically inhibited IBC tumor growth in a xenograft model. Moreover, restoring TIG1 expression rescued the proliferation, motility, and invasiveness of TIG1-silenced IBC cells. Most importantly, we identified Axl as a functional partner of TIG1 by showing that TIG1 interacted with and stabilized Axl in IBC cells. TIG1 regulated the invasiveness of IBC cells through mediation of the Axl signaling pathway. In SUM149 cells, TIG1 depletion decreased Axl expression, which led to downregulation of expression of matrix metalloproteinase-9, a molecule required for Axl-mediated invasion, and inactivation of nuclear factor-kB, ultimately leading to decreased invasiveness of IBC cells. Conclusion: Our results identified TIG1 as an oncogenic gene that contributes to the tumorigenic and metastatic properties of IBC. Our data also linked TIG1 with the key tumorigenic gene Axl in IBC cells. Further studies designed to establish TIG1 as a therapeutic target in the treatment of patients with IBC are under way. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-01.