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High Throughput Kinase Inhibitor Screens Reveal TRB3 and MAPK-ERK/Tgfβ Pathways As Fundamental Notch Regulators in Breast Cancer

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High Throughput Kinase Inhibitor Screens Reveal TRB3 and MAPK-ERK/Tgfβ Pathways As Fundamental Notch Regulators in Breast Cancer High throughput kinase inhibitor screens reveal TRB3 and MAPK-ERK/TGFβ pathways as fundamental Notch regulators in breast cancer Julia Izrailita,b, Hal K. Bermana,c, Alessandro Dattid,e, Jeffrey L. Wranad, and Michael Reedijka,b,f,1 aCampbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, Toronto, ON, Canada M5G 2M9; bDepartment of Medical Biophysics, University of Toronto, Ontario Cancer Institute, Princess Margaret Hospital, Toronto, ON, Canada M5G 2M9; cDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada M5S 1A8; dCenter for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; eDepartment of Experimental Medicine and Biochemical Sciences, University of Perugia, 06100 Perugia, Italy; and fDepartment of Surgical Oncology, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada M5G 2M9 Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, and approved December 12, 2012 (received for review August 20, 2012) Expression of the Notch ligand Jagged 1 (JAG1) and Notch acti- TGFβ signaling pathways. These findings are consistent with vation promote poor-prognosis in breast cancer. We used high a previously reported association between TRB3 expression and throughput screens to identify elements responsible for Notch acti- poor overall survival in breast cancer (16) and establishes TRB3 vation in this context. Chemical kinase inhibitor and kinase-specific as a potential therapeutic target in this malignancy. small interfering RNA libraries were screened in a breast cancer cell line engineered to report Notch. Pathway analyses revealed MAPK- Results ERK signaling to be the predominant JAG1/Notch regulator and this MAPK Regulates Notch in Breast Cancer. To perform a functional was supported by gene set enrichment analyses in 51 breast cancer genetic screen for regulators of Notch signaling in breast cancer, cell lines. In accordance with the chemical screen, kinome small in- a dual-reporter cell line was created by transducing human mam- terfering RNA high throughput screens identified Tribbles homolog 3 mary carcinoma cells (MDA MB231) that expressed Renilla lucif- (TRB3), a known regulator of MAPK-ERK, among the most significant erase under the control of the thymidine kinase (TK) promoter, with hits. We demonstrate that TRB3 is a master regulator of Notch an adenovirus expressing a Notch-regulatable Hes1 promoter/firefly through the MAPK-ERK and TGFβ pathways. Complementary in vitro luciferase cassette (Fig. 1A). MDA MB231 cells were selected as and in vivo studies underscore the importance of TRB3 for tumor a suitable cell background because they express high levels of key growth. These data demonstrate a dominant role for TRB3 and Notch signaling pathway constituents, and they are Notch signaling MAPK-ERK/TGFβ pathways as Notch regulators in breast cancer, competent (5). We subjected the Notch dual-reporter line to both establishing TRB3 as a potential therapeutic target. γ-secretase inhibitor (GSI) and siRNA-mediated knockdown of JAG1 or NOTCH1 to confirm its reliability and power to identify compounds and siRNAs that inhibit Notch (Fig. S1A). reast cancer ranks second among causes of cancer death in Kinases such as c-Src, c-Abl, mitogen-activated protein kinases North American women and elevated expression of the B (MAPK), phosphotidylinositol-3-kinase (PI3K), and the epider- Notch ligand Jagged 1 (JAG1) is a marker of relapse and poor mal growth factor (EGF) receptor are commonly activated in outcome in this malignancy (1). Consistent with this, activated cancer cells and are known to contribute to tumorigenesis. To Notch signaling (2–4) and up-regulation of growth-promoting Notch target genes (5–9), are observed in breast cancer. Notch is comprehensively identify cross-talk between Notch and kinase- a highly conserved intercellular signaling system present in mul- regulated signaling pathways, a kinase inhibitor library that con- tains 240 kinase inhibitors was screened using our dual-reporter ticellular organisms (10). Mammals have four Notch proteins fl (NOTCH1–4) that function as receptors for five Notch ligands system to identify pathways that disproportionally in uenced – Hes1-firefly luciferase expression compared with TK-Renilla lu- [Delta-like (Dll)1, -3, -4, and JAG1 and -2]. Notch ligand receptor firefly interaction on neighboring cells leads to presenilin-protease ciferase. Of these compounds, 26 reduced the normalized / γ Renilla luciferase ratio greater than two standard deviations from ( -secretase) complex-mediated cleavage of the receptor, resulting B fi TK-Renilla in release of the cytoplasmic domain fragment, intracellular Notch the mean (Fig. 1 ) without signi cantly affecting the IC IC luciferase level (Fig. S1B). Control wells in the screen confirmed (N ). N enters the nucleus and modulates the expression of firefly Renilla C target genes, predominantly by converting the recombination low variability of the / luciferase ratio (Fig. S1 ). The fi A signal sequence-binding protein J kappa (RBPJκ) transcription ndings of the screen were validated separately (Fig. S2 )and fi factor from a repressor to an activator of transcription (11). a toxicity assay identi ed only one of 26 compounds as toxic under Knowledge of the events that promote Notch activation in breast the experimental conditions (Fig. S2B). When these 26 com- cancer is limited. Hypoxia, through up-regulation of p66shc or IL-6 pounds were analyzed according to major signaling pathways tar- can induce NOTCH3 and ERK-dependent JAG1 expression in geted, MAPK-ERK was found to be the predominant pathway breast cancer cells (12, 13). JAG1 expression is regulated by TGFβ promoting Notch activation in MDA MB231 cells. Strikingly, 6 of in a SMAD-dependent fashion in a mouse breast cancer bone me- 26 compounds targeted MEK1/2 and this comprised 66% of all tastasis model (14). Numb loss (2), Ras-induced γ-secretase stabi- MEK1/2 inhibitors included in the library; this was statistically lization (15) and Pin1-potentiated NOTCH1 cleavage by γ-secretase significant (P < 0.001). Upon repeat screen of the entire library, (8) promote Notch activation and have been implicated in the progression of breast cancer. Protein kinases and pseudokinases participate extensively in Author contributions: J.I. and M.R. designed research; J.I. performed research; A.D. and cellular signaling pathways and are key regulators of cell function. J.L.W. contributed new reagents/analytic tools; J.I., H.K.B., A.D., and M.R. analyzed data; Here we describe the use of large-scale chemical kinase inhibitor- and J.I. and M.R. wrote the paper. and kinase-specific small interfering (si)RNA-based screens to The authors declare no conflict of interest. gain insight into the mechanisms that promote Notch activation in This article is a PNAS Direct Submission. breast cancer. We show that the pseudokinase Tribbles homolog 3 1To whom correspondence should be addressed. E-mail: [email protected]. (TRB3), functions as a master regulator of JAG1-induced Notch This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activation and tumor growth through its control of MAPK and 1073/pnas.1214014110/-/DCSupplemental. 1714–1719 | PNAS | January 29, 2013 | vol. 110 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1214014110 Downloaded by guest on September 23, 2021 AB C MDA MB231 TK-R cells Chemical kinase U0126 - 1μM 10μM inhibitors 49 p-ERK1/2 FFFF TK TKR LuciferaseR Luciferase 49 RR ERK1/2 Activity (FF/R) Activity 180 Kinome siRNA siRNA JAG1 librarylibrary Adenoviral Luciferase 115 transduction NIC 64 uPA Hes1 FF Luciferase Normalized U0126 49 β-actin Kinase Inhibitors DE MDA MB231 HCC1143 MDA MB231 HCC1143 U0126 - 1μM 10μM - 1μM 10μM EGF --++++--++++ 180 JAG1 U0126 -+-+-+-+-+-+ 49 hr 7 7 7 7 24 24 77772424 β-actin 180 JAG1 49 p-ERK1/2 49 ERK1/2 Fig. 1. MEK1/2 inhibitors down-regulate JAG1 expression in breast cancer cell lines. (A) Dual-reporter MDA MB231 cell line was created by transducing cells that expressed Renilla (R) luciferase under the control of the thymidine kinase (TK) promoter (MB 231 TK-R) with adenovirus containing a Notch-regulatable Hes1 promoter/firefly (FF) luciferase cassette. In a robotic high throughput screen format, cells were cultured on microtiter plates, treated with chemical kinase inhibitor or kinome siRNA libraries, and FF and R luciferase quantified. (B) Kinase inhibitor screen scatter plot: FF/R ratios are plotted on the y axis against 240 corresponding kinase inhibitors (OICR-L100) on the x axis. The dashed lines represent two standard deviations (SD) away from the mean (solid line) and the circled dots represent the MEK1/2 inhibitors in OICR-L100. (C) Western blot analysis of p-ERK1/2, ERK1/2, JAG1, NIC, and uPA in MDA MB231 cells treated with U0126 (1 μMor10μM) for 24 h. (D) Expression of JAG1 in MDA MB231 or HCC1143 cells treated with U0126 (1 μMor10μM) for 24 h. (E) Western blot analysis of JAG1, p-ERK1/2, and ERK1/2 protein in MDA MB231 and HCC1143 cells grown in serum-free conditioned media for 24 h prior to stimulation with EGF for 4 h, followed by treatment with U0126 (10 μM) for the indicated time periods. β-Actin expression is included as a loading control. Molecular CELL BIOLOGY weight (MW) markers are shown in kilodaltons. there was an 87% overlap in the significant hits identified and this subtype, also associated significantly with JAG1 overexpression included all 6 of the aforementioned MEK1/2 inhibitors. (5, 9) (Fig. 2A). To a lesser degree, the MAPK-ERK activation To validate MAPK-ERK signaling as a regulator of Notch signature was present in human epidermal growth factor receptor activation, the dual-reporter line was treated with the six MEK1/2 2 positive (HER2+) cell lines (Fig. S3). To determine if MAPK- inhibitors identified in the screen, and Notch reporter activity was ERK activation genes showed a statistically significant, concor- found to be reduced in a dose-dependent manner in all cases dant difference as a function of continuous JAG1 gene ex- (Fig.
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