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A. we treated a panel of breast cancer cells MDA-MB-468, SUM190, KPL-4, and MDA-IBC-3 with different concentrations of selumetinib and assessed cell viability by WST-1 assay 72 hours later. We observed that in MDA-MB-468, SUM190, KPL-4, and MDA-IBC-3 cells, the 50% inhibitory concentration (IC50) was above 20 µM. B. MDA-MB-231 and SUM149 cells were treated with selumetinib for 72 hours, and FACS analysis was performed to determine cell cycle distribution using propidium iodide.

Supplementary Figures S1-S3 from Effects of mTOR Inhibitor Everolimus (RAD001) on Bladder Cancer Cells

Purpose: We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder cancer (BC) cells in vitro and in vivo.Experimental Design: The effect of RAD001 on the growth of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 BC cells were assessed by crystal violet and [3H]thymidine incorporation assays. Flow cytometric cell-cycle analyses were done to measure the apoptotic cell fraction. Protein synthesis was measured using tritium-labeled leucine incorporation assays. The effects of RAD001 on the mTOR pathway were analyzed by Western blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, and UM-UC-9 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with RAD001 or placebo. Tumors were harvested for immunohistochemical analysis.Results:In vitro, RAD001 transiently inhibited BC cell growth in a dose-dependent manner. This effect was augmented by re-treatment of cells after 3 days. UM-UC-14 cells were the most sensitive to RAD001, whereas UM-UC-9 cells were the least sensitive. After re-treatment with RAD001, only sensitive cell lines showed G1-phase arrest, with no evidence of apoptosis. RAD001 significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of protein synthesis through the S6K and 4EBP1 pathways seems to be the main mechanism for the RAD001-induced growth inhibition. However, inhibition of angiogenesis was the predominant mechanism of the effect of RAD001 on UM-UC-9 cells.Conclusions: The mTOR inhibitor RAD001 inhibits growth of BC cells in vitro. RAD001 is effective in treating BC tumors in an in vivo nude mouse model despite the heterogeneity of in vitro responses. Clin Cancer Res; 17(9); 2863–73. ©2011 AACR.

Objective Oral squamous cell carcinoma (OSCC) represents 3%-4% of all cancers. Despite the increasing incidence of OSCC distant metastasis and poor prognosis, few animal models of OSCC distant metastasis have been reported. In this study, we established mouse models of OSCC lung metastasis by orthotopic and tail vein injection of new OSCC cell lines. Methods For the tail vein model, we used a novel cell line isolated from lung metastases reproduced in vivo after intravenous injection of HSC-3 GFP/luciferase cells and sorted for GFP expression (HSC-3 M1 GFP/luciferase). Lung metastases were assessed by imaging techniques and further confirmed by histology. For the orthotopic model, HSC-3 GFP/luciferase cells were injected into the tongue of athymic nude mice. The primary tumor and metastases were assessed by in vivo imaging, histology, and immunohistochemistry. Results The orthotopic model presented spontaneous lung metastases in 50% of the animals and lymph node metastases were present in 83% of cases. In the tail vein model, a lung metastasis rate of 60% was observed. Conclusions Lung metastases were successfully reproduced by orthotopic and tail vein injection. Since lymph node metastases were present, the orthotopic model with HSC-3 GFP/luciferase cells may be suitable to investigate metastatic dissemination in OSCC.

Adipose stromal cells (ASCs) have been identified as a mesenchymal cell population recruited from white adipose tissue (WAT) by tumors and supporting cancer progression. We have previously reported the existence of a non-glycanated decorin isoform (ngDCN) marking mouse ASCs. We identified a peptide CSWKYWFGEC that binds to ngDCN and hence can serve as a vehicle for ASC-directed therapy delivery. We used hunter-killer peptides composed of CSWKYWFGEC and a pro-apoptotic moiety to deplete ASCs and suppress growth of mouse tumors. Here, we report the discovery of the human non-glycanated decorin isoform. We show that CSWKYWFGEC can be used as a probe to identify ASCs in human WAT and tumors. We demonstrate that human ngDCN is expressed on ASC surface. Finally, we validate ngDCN as a molecular target for pharmacological depletion of human ASCs with hunter-killer peptides. We propose that ngDCN-targeting agents could be developed for obesity and cancer treatment.

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.

Purpose: We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder cancer (BC) cells in vitro and in vivo. Experimental Design: The effect of RAD001 on the growth of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 BC cells were assessed by crystal violet and [3H]thymidine incorporation assays. Flow cytometric cell-cycle analyses were done to measure the apoptotic cell fraction. Protein synthesis was measured using tritium-labeled leucine incorporation assays. The effects of RAD001 on the mTOR pathway were analyzed by Western blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, and UM-UC-9 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with RAD001 or placebo. Tumors were harvested for immunohistochemical analysis. Results: In vitro, RAD001 transiently inhibited BC cell growth in a dose-dependent manner. This effect was augmented by re-treatment of cells after 3 days. UM-UC-14 cells were the most sensitive to RAD001, whereas UM-UC-9 cells were the least sensitive. After re-treatment with RAD001, only sensitive cell lines showed G1-phase arrest, with no evidence of apoptosis. RAD001 significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of protein synthesis through the S6K and 4EBP1 pathways seems to be the main mechanism for the RAD001-induced growth inhibition. However, inhibition of angiogenesis was the predominant mechanism of the effect of RAD001 on UM-UC-9 cells. Conclusions: The mTOR inhibitor RAD001 inhibits growth of BC cells in vitro. RAD001 is effective in treating BC tumors in an in vivo nude mouse model despite the heterogeneity of in vitro responses. Clin Cancer Res; 17(9); 2863–73. ©2011 AACR.

Progression of many cancers is associated with tumor infiltration by mesenchymal stromal cells (MSC). Adipose stromal cells (ASC) are MSC that serve as adipocyte progenitors and endothelium-supporting cells in white adipose tissue (WAT). Clinical and animal model studies indicate that ASC mobilized from WAT are recruited by tumors. Direct evidence for ASC function in tumor microenvironment has been lacking due to unavailability of approaches to specifically inactivate these cells. Here, we investigate the effects of a proteolysis-resistant targeted hunter-killer peptide D-WAT composed of a cyclic domain CSWKYWFGEC homing to ASC and of a proapoptotic domain KLAKLAK2. Using mouse bone marrow transplantation models, we show that D-WAT treatment specifically depletes tumor stromal and perivascular cells without directly killing malignant cells or tumor-infiltrating leukocytes. In several mouse carcinoma models, targeted ASC cytoablation reduced tumor vascularity and cell proliferation resulting in hemorrhaging, necrosis, and suppressed tumor growth. We also validated a D-WAT derivative with a proapoptotic domain KFAKFAK2 that was found to have an improved cytoablative activity. Our results for the first time demonstrate that ASC, recruited as a component of tumor microenvironment, support cancer progression. We propose that drugs targeting ASC can be developed as a combination therapy complementing conventional cancer treatments.

Cross-contamination of cell lines is a serious but often unrecognized problem. We describe the authentication of a panel of transitional cell carcinoma cell lines using the short tandem repeat profiling technique to detect cross-contamination.Genomic DNA was isolated from UM-UC-1, UM-UC-2, UM-UC-3 (ATCC), UM-UC-6, UM-UC-9, UM-UC-10, UM-UC-11, UM-UC-13, UM-UC-14, UM-UC-16, T24 and KU7 cell lines. Short tandem repeat loci (D3S1358, D16S539, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820) and a segment of the X-Y homologous gene amelogenin were co-amplified by polymerase chain reaction. Profiling was done using POP-4TM performance optimized polymer 4 (Applied Biosystems) with an ABI Prism 310 genetic analyzer. DNA sequencing of TP53 and immunohistochemistry for p53 were performed in UM-UC-3 and UM-UC-3-GFP.All cell lines had a unique short tandem repeat profile except UM-UC-2 and T24, which were virtually identical. T24 short tandem repeat profiles matched those of early passage number UM-UC-2. Stable transfection of the green fluorescence protein marker gene did not alter UM-UC-6, UM-UC-14 or KU7 profiles. However, the short tandem repeat profile for UM-UC-3-GFP was different from that of UM-UC-3. DNA sequencing showed a difference in TP53 between UM-UC-3 and UM-UC-3-GFP, confirming that UM-UC-3-GFP is not derived from UM-UC-3.Short tandem repeat profiling provides a unique genetic signature of human cell lines that does not significantly change with passage or green fluorescence protein transduction. Using short tandem repeat profiling we noted that the cell line UM-UC-2 is T24. DNA fingerprinting using short tandem repeat profiling is an easy and reliable tool that can be used to verify cell lines.

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, François 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

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.

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