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. 2013 Jan 29;110(5):1714-9.
doi: 10.1073/pnas.1214014110. Epub 2013 Jan 14.

High throughput kinase inhibitor screens reveal TRB3 and MAPK-ERK/TGFβ pathways as fundamental Notch regulators in breast cancer

Julia Izrailit  1 , Hal K Berman, Alessandro Datti, Jeffrey L Wrana, Michael Reedijk
Affiliations

High throughput kinase inhibitor screens reveal TRB3 and MAPK-ERK/TGFβ pathways as fundamental Notch regulators in breast cancer

Julia Izrailit et al. Proc Natl Acad Sci U S A. .

Abstract

Expression of the Notch ligand Jagged 1 (JAG1) and Notch activation promote poor-prognosis in breast cancer. We used high throughput screens to identify elements responsible for Notch activation in this context. Chemical kinase inhibitor and kinase-specific small interfering RNA libraries were screened in a breast cancer cell line engineered to report Notch. Pathway analyses revealed MAPK-ERK signaling to be the predominant JAG1/Notch regulator and this was supported by gene set enrichment analyses in 51 breast cancer cell lines. In accordance with the chemical screen, kinome small interfering RNA high throughput screens identified Tribbles homolog 3 (TRB3), a known regulator of MAPK-ERK, among the most significant hits. We demonstrate that TRB3 is a master regulator of Notch through the MAPK-ERK and TGFβ pathways. Complementary in vitro and in vivo studies underscore the importance of TRB3 for tumor growth. These data demonstrate a dominant role for TRB3 and MAPK-ERK/TGFβ pathways as Notch regulators in breast cancer, establishing TRB3 as a potential therapeutic target.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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 µM or 10 µM) for 24 h. (D) Expression of JAG1 in MDA MB231 or HCC1143 cells treated with U0126 (1 µM or 10 µM) 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 weight (MW) markers are shown in kilodaltons.
Fig. 2.
Fig. 2.
MAPK pathway activation in JAG1-expressing and basal-like tumor cells. (A) An ordered set of genes significantly up-regulated (yellow) or down-regulated (blue) following MAPK pathway activation in MCF7 cells, as previously defined (Materials and Methods), is comparatively visualized in a set of 51 breast tumor cell lines. Cell lines of the basal-like molecular subtype show the strongest MAPK signature pattern and JAG1 expression (red box). (B) Gene set enrichment analysis (GSEA) was used to determine if there are statistically significant, concordant differences in MAPK signature gene expression as a function of continuous JAG1 expression in 51 breast cancer cell lines (Genentech) versus a series of cancer cell lines of varied type (NCI-60). Shown are GSEA plots for a representative JAG1 probeset. ER+, estrogen receptor positive; ER-, estrogen receptor negative; HER2+, human epidermal growth factor receptor 2 positive; ES, enrichment score; NES, normalized enrichment score; p, nominal P value.
Fig. 3.
Fig. 3.
TRB3 is a regulator of JAG1. (A) Kinome siRNA library screen scatter plot: B-scores for two biological replicate screens are plotted on the x- and y axes. Dashed cross hairs separate hits that are 2 SD away from the mean B-scores. (B) Western blot analysis of JAG1 and NIC in the indicated cell lines 72 h after transfection with Scr or TRB3 siRNA. (C) Western blot analysis of p-ERK1/2, ERK1/2, and JAG1 in MDA MB231 cells treated with Scr or TRB3 siRNA. (D) Western blot analysis of TRB3, p-ERK1/2, ERK1/2, and JAG1 in HepG2 cells transduced either with adenoviral control or with adenovirus expressing TRB3 [multiplicity of infection (MOI) = 5], in the absence or presence of U0126. (E) Western blot analysis of JAG1, p-ERK1/2, and ERK1/2 in MDA MB231 cells treated with or without TGFβ and either Scr or TRB3 siRNA. (F) Western blot analysis of SMAD4 and JAG1 in MDA MB231 cells treated with Scr, TRB3, or SMAD4 siRNA. β-Actin expression is included as a loading control. MW markers are shown in kilodaltons.
Fig. 4.
Fig. 4.
JAG1 rescues the proliferation defect imposed by TRB3 knockdown. Proliferation of MDA MB231 (A) and JAG1 MDA MB231 (B) cell lines in monolayer culture after treatment with either Scr siRNA (dashed curves) or TRB3 siRNA (solid curves). Cells were counted at 24, 48, 72, and 96 h after transfection. Insets, Western blot analysis of JAG1 protein expression in MDA MB231 (A) or JAG1 MDA MB231 (B) cells treated with either Scr or TRB3 siRNA. β-Actin expression is included as a loading control. MW markers are shown in kilodaltons. Experiments were performed in triplicate; error bars represent SD. *P < 0.05 relative to Scr control.
Fig. 5.
Fig. 5.
TRB3 knockdown impairs xenograft growth of MDA MB231 cells. (A) Estimated cross-sectional area of tumor xenografts derived from Scr siRNA-treated (dashed curve; six NSG mice) or TRB3 siRNA-treated (solid curve; six NSG mice) MDA MB231 cells measured over 5.5 wk. (B) Weight of xenograft tumors derived from Scr siRNA-treated or TRB3 siRNA-treated MDA MB231 cells at 5.5 wk. Error bars represent SD.*P < 0.05 relative to Scr control.
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References

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