High throughput 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 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 such as c-Src, c-Abl, mitogen-activated 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 (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 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. S2C). Similarly, treatment of wild-type MDA MB231 cells pression, gene set enrichment analysis (GSEA) was used (Fig. with U0126 reduced phospho (p)-ERK1/2, JAG1, Notch activa- B IC 2 ). JAG1 and MAPK-ERK activation were compared in breast tion as measured by N levels, and expression of the Notch target, cancer cell lines, and in the NCI-60 cell lines originating from C urokinase plasminogen activator (uPA) (9) (Fig. 1 ). cancer tissues of multiple types (20). Interestingly, a statistically MDA MB231 cells contain an activating mutation in the fi BRAF signi cant association between MAPK-ERK activation and JAG1 proto-oncogene so they may be uniquely dependent upon expression was specific to breast cancer cell lines [enrichment the RAF/MAPK-ERK pathway for Notch activation. Indeed, score (ES) = 0.60 and 0.39 and P = 0.007 and 0.035 for up-reg- HCC1143 breast cancer cells, which do not display constitutive ulated and down-regulated MAPK-ERK genes, respectively], but RAF/MAPK-ERK activation, demonstrated a marginal decrease D not to diverse human cancer cell lines (NCI-60, ES = 0.25 and 0.22 in JAG1 expression after 24 h of U0126 treatment (Fig. 1 ). P = However, HCC1143 cells could be rendered permissive for RAF/ and 0.63 and 0.57 respectively). MAPK-ERK activation through serum starvation and EGF β stimulation (Fig. 1E). Under these conditions, HCC1143 cells TRB3 Is a Master Regulator of MAPK and TGF Pathway Activation and JAG1 Expression. As a complementary approach to identify demonstrated a transient increase in p-ERK1/2 and exquisite fl MAPK-ERK–dependent JAG1 expression; this was evidenced kinases that in uence Notch activation, a kinome siRNA library by the ability of U0126 treatment to abrogate JAG1 expression was screened using the dual-reporter system. The kinome siRNA by 24 h of treatment. These data suggest that the MAPK-ERK library contains siRNA pools that target 720 genes encoding pathway regulates JAG1 expression in breast cancer cells. protein kinases. Two biological replicate screens were performed and the results of each screen were analyzed using the B-score Correlation of JAG1 and MAPK Pathway Activation in Breast Cancer method of high throughput screen normalization to correct for Cell Lines. To further explore the link between MAPK-ERK and systematic and positional variability across the samples (21). The JAG1/Notch we looked for an association between a gene ex- results obtained in the two screens were reproducible and iden- pression signature indicative of MAP kinase activation (17, 18) tified five siRNA pools that demonstrated a reduction in the B- and JAG1 expression in 51 breast cancer cell lines (19). We found score of at least two SDs from the mean (correlation coefficient = that MAPK-ERK was activated in cell lines of the basal-like 0.61; Fig. 3A). These hits were independently validated in the

Izrailit et al. PNAS | January 29, 2013 | vol. 110 | no. 5 | 1715 Downloaded by guest on September 23, 2021 HER2+ A HER2+ HER2+ Weak- HER2+ ER+ / Luminal ER-/Basal ER-/CD24low Basal luminal luminal ER- MCF7 - RAF MCF7 - MEK MCF7 - EGFR MCF7 - HER2

9 4 2 0 -2 -4 -9

JAG1

B Genentech 51 NCI - 60

ES = 0.60 ES = -0.39 ES = -0.25 ES = -0.22 NES = 1.8 NES = -1.5 NES = -0.85 NES = -0.92 p=0.007 p=0.035 p=0.63 p=0.57

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.

dual-reporter cell line (Fig. S4A), and it was confirmed that they resulted in up-regulation of p-ERK1/2 and JAG1; this effect could regulated JAG1, NOTCH1, and Hes1 expression (Fig. S4B). be reversed with U0126, demonstrating MAPK-ERK depen- Consistent with the results of the chemical kinase inhibitor dence. These effects were more difficult to illustrate in MDA screen, TRB3, a known regulator of MAPK-ERK (22), was among MB231 cells, likely due to an overactivated MAPK pathway (Fig. the top five hits. TRB3 is a member of a family of three pseudo- S6B). Taken together with previous reports that TRB3 promotes kinases (TRB1–3) and these are the mammalian orthologs of activation of MAPK-ERK, and the finding that MAP-ERK reg- Tribbles, a protein that inhibits mitosis in early Drosophila de- ulates JAG1 (Fig. 1), these data are consistent with a model where velopment. Despite its lack of kinase activity, TRB3 participates MAPK-ERK is a mediator of TRB3-regulated JAG1 expression. as a scaffold molecule during protein complex formation (23). Studies in mouse models of breast cancer bone metastases have The matched response of both TRB3 and JAG1 expression to all implicated TGFβ-SMAD4 signaling in the regulation of JAG1 four siRNA species that comprised the TRB3 siRNA pool, reduced expression (14). In nonmammary cell lines TRB3 has been shown the likelihood that JAG1 knockdown was due to an off-target effect to stabilize SMADs and potentiate SMAD-mediated transcrip- (Fig. S4C). To ensure that our findings were not unique to the MDA tional activity by triggering degradation of SMAD ubiquitination MB231 cell line, we explored additional basal-like subtype breast regulatory factors (SMURFs) (24, 25). Based on these findings we cancer cell lines and found that JAG1 expression and Notch acti- tested whether TRB3 regulates TGFβ/SMAD4-dependent JAG1 vation were consistently dependent on TRB3 (Fig. 3B). expression in breast cancer. TGFβ treatment of serum-starved To explore the regulation of JAG1 expression and MAPK-ERK MDA MB231 cells resulted in up-regulation of JAG1 and this signaling by TRB3 in breast cancer, p-ERK1/2 levels were de- effect was dependent upon TRB3 (Fig. 3E). Furthermore, TRB3 termined by Western blotting after treatment of MDA MB231 cells knockdown resulted in a decrease in SMAD4 and JAG1 proteins with TRB3 siRNA (Fig. 3C). TRB3 knockdown resulted in mark- (Fig. 3F) without affecting SMAD4 mRNA levels (Fig. S7A), edly reduced p-ERK1/2 and a corresponding reduction in JAG1 suggesting a posttranscriptional mechanism of SMAD4 regulation protein. To reconcile the findings of the chemical inhibitor screen by TRB3. To confirm the importance of SMAD4 in this sequence we explored the ability of individual components within the MAPK- we demonstrated that SMAD4 knockdown alone reduced JAG1 ERK pathway to regulate JAG1 expression. Although BRAF, expression (Fig. 3F). To determine whether TRB3-mediated ERK1, or ERK2 knockdown resulted in decreased JAG1 expres- degradation of SMURFs was required for SMAD4 protein ex- sion, combined ERK1 and ERK2 knockdown was required to pression, we performed knockdown of TRB3 and double knock- overcome redundancy within the pathway and to match the effect of down of TRB3 and SMURF1 or SMURF2 (Fig. S7 B and C). TRB3 knockdown (Fig. S5). Consistent with these findings, over- Knockdown of TRB3 had no effect on SMURF1 and double expression of TRB3 in HepG2 human hepatocellular carci- knockdown conditions did not rescue SMAD4 or JAG1 levels, noma (Fig. 3D) or MCF10A mammary epithelial (Fig. S6A) cells suggesting that SMURFs are not involved in TRB3 regulation of

1716 | www.pnas.org/cgi/doi/10.1073/pnas.1214014110 Izrailit et al. Downloaded by guest on September 23, 2021 A B Correlation MDA MB231 MDA MB157 HCC1143 HCC38 coefficient = 0.61 Scr-siRNA +-+-+-+-

TRB3-siRNA -+-+-+-+

180 JAG1

115 NIC B-score screen 2

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B-score screen 1 C D E F AD control AD TRB3 TGF-β --++ U0126 -+-+ Scr-siRNA +-+- 49 TRB3 TRB3-siRNA -+-+

49 49 p-ERK1/2 180 p-ERK1/2 JAG1 80 SMAD4 49 49 ERK1/2 49 p-ERK1/2 ERK1/2 180 JAG1 180 180 JAG1 JAG1 49 ERK1/2 49 β-actin 49 49 β-actin β-actin 49 β-actin

MDA MB231 HepG2

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. CELL BIOLOGY

SMAD4 and JAG1. These findings suggest that in addition to slower than those derived from control cells as evaluated by tumor its control of MAPK-ERK, TRB3 regulates JAG1 expression size measurements (Fig. 5A) and average tumor weight at sacrifice through SMURF-independent TGFβ/SMAD4 signaling. (Fig. 5B). Taken together, these results demonstrate that TRB3 plays a crucial role in breast cancer tumorigenesis and that down- JAG1 Rescues a Proliferation Defect Imposed by TRB3 Knockdown. regulation of TRB3 can reduce tumor growth in vivo. Based on the effects of TRB3 on MAPK and TGFβ pathways and on JAG1 expression, we predicted that knockdown of TRB3 Discussion should have a profound effect on breast cancer cell proliferation. We performed high throughput screens to identify kinases and We examined the effect of siRNA-mediated depletion of TRB3 associated signal transduction pathways that promote JAG1/ on the proliferation of wild-type MDA MB231 cells and on an MDA MB231 variant that was engineered to overexpress JAG1 (JAG1 MDA MB231). Compared with Scr siRNA-treated cells, A B TRB3 siRNA achieved a significant knockdown of JAG1 in MDA MDA MB231 JAG1 MDA MB231 MB231 cells but not in JAG1 MDA MB231 cells (Fig. 4 A and B, 900 ) ) 700 3 Insets). Corresponding with this, TRB3 knockdown resulted in 3 800 * JAG1 600 JAG1 a significant reduction in MDA MB231 proliferative capacity, 700 600 β-actin 500 β-actin whereas JAG1 overexpression rescued this effect (Fig. 4). The * data demonstrate a critical role for TRB3 in maintaining MDA 500 400 400 MB231 proliferation and implicate JAG1 in mediating this effect. 300 300 200 TRB3 Knockdown Results in a Growth Deficiency of MDA MB231 Mouse 200 Total number of cells (10 Xenografts. To confirm the importance of TRB3 to MDA MB231 Total number of cells (10 100 Scr 100 Scr TRB3 TRB3 cell proliferation and tumorigenesis we performed an orthotopic 0 0 MDA MB231 tumor xenograft study, where MDA MB231 cells 24hr 48hr 72hr 96hr 24hr 48hr 72hr 96hr underwent ex vivo transfection with either Scr or TRB3 siRNA (Fig. S8A) followed by injection into the mammary fat pad of se- Fig. 4. JAG1 rescues the proliferation defect imposed by TRB3 knockdown. Proliferation of MDA MB231 (A) and JAG1 MDA MB231 (B) cell lines in verely immunodeficient nonobese diabetic (NOD/SCID/IL2Rγ − − monolayer culture after treatment with either Scr siRNA (dashed curves) or [ / ]) (NSG) female mice (26). We ensured injection of equivalent TRB3 siRNA (solid curves). Cells were counted at 24, 48, 72, and 96 h after numbers of viable cells in all models through the Trypan blue dye transfection. Insets, Western blot analysis of JAG1 protein expression in MDA exclusion method and an anoikis assay (27) (Fig. S8B). Parallel in MB231 (A) or JAG1 MDA MB231 (B) cells treated with either Scr or TRB3

vitro assays demonstrated persistent knockdown of JAG1 for at siRNA. β-Actin expression is included as a loading control. MW markers are least 14 d posttransfection (Fig. S8C). Tumors derived from MDA shown in kilodaltons. Experiments were performed in triplicate; error bars MB231 cells transfected with TRB3 siRNA grew substantially represent SD. *P < 0.05 relative to Scr control.

Izrailit et al. PNAS | January 29, 2013 | vol. 110 | no. 5 | 1717 Downloaded by guest on September 23, 2021 Notch activation in breast cancer. A kinase inhibitor screen and also depend upon the seeding of metastatic sites by tumor cells, follow-up GSEA identified MAPK-ERK as the predominant a process that Notch and TGFβ pathways have been shown to regulator of JAG1 expression and Notch activation in this ma- facilitate (14); TGFβ released from sites of bone metastases lignancy. Accordingly TRB3, a regulator of MAPK signaling was stimulates JAG1 expression in tumor cells, enabling paracrine identified as a top hit in a complementary kinome siRNA screen. Notch activation in osteoblasts and preosteoclasts and bone in- Herein we show that through the control of MAPK-ERK and vasion. Collectively, these findings predict that TRB3 can po- β TGF pathways, TRB3 is a master regulator of JAG1 expression tentiate tumor initiation and the metastatic capacity of breast and is required for the growth of basal-like breast cancer. cancer cells through its regulation of MAPK-ERK– and TGFβ- TRB3 is a pseudokinase, a class of proteins that comprises 10% mediated JAG1/Notch activation. It remains to be shown whether of the kinase superfamily and that lacks one or more of the activation of these pathways and processes account for the re- conserved amino acid motifs that are essential for catalytic ac- duced survival associated with tumors with elevated TRB3. tivity (28). Despite lacking these characteristic motifs, pseudoki- Interestingly, TRB3 knockout mice are phenotypically identical nases are not impotent, vestigial remnants of functional kinases to their wild-type littermates (44), suggesting that under normal but rather, are crucial regulators of diverse cellular functions. In physiological conditions this gene is either unnecessary or is some instances pseudokinases function as scaffolding proteins, TRB bringing together catalytically active molecules and their sub- functionally redundant with the other paralogs. Thus, whereas TRB3 may have minimal effects on normal cells, it has a major strates. TRB3 plays such a role in normal cells during conditions fi of hypoxic/endoplasmic reticulum stress or nutrient deprivation impact on the survival and tness of stressed cells including hypoxic where it becomes up-regulated and promotes survival by blunting tumor cells. This establishes TRB3 as an ideal therapeutic target fi potentially deleterious stress signals (29, 30). Indeed, TRB3 is up- whose elimination may render a low side-effect pro le while being regulated in cancers as a response to hypoxia (16, 29) and is as- deleterious to tumor progression and metastasis. sociated with poor outcome (16, 31). However, an apparent dis- cordance between the influence of TRB3 mRNA (16) and Materials and Methods protein (32) on outcome in breast cancer remains to be explained. Cell Culture and Reagents. Cell lines were purchased from American Type We hypothesize that in response to cellular stress, TRB3 protein Culture Collection. The MB231 TK-R cell line was generated by transfecting influences outcome by promoting activation of key cancer sig- MDA MB231 cells with the pRL-TK vector (TK promoter-driven Renilla lucif- naling pathways such as MAPK-ERK, TGFβ, and JAG1/Notch. erase; Promega) together with pBABE-puro plasmid with selection of stable JAG1/Notch signaling is emerging as a mediator of breast transfectants in 1μg/mL puromycin. JAG1-pcDNA 3.1 and pcDNA 3.1 were cancer progression and metastasis and is associated with the transfected into MDA MB231 cells to generate control and JAG1 over- basal-like subtype (6, 33–35). Approximately 20% of breast expressing cell lines. MEK1/2 inhibitor U0126 was purchased from Cell Signaling cancer patients have basal-like disease, and despite an initial Technology. GSI (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t- response to systemic cytotoxic chemotherapy, their disease fol- butyl ester; Calbiochem) was used at 50 μM. Human epidermal growth factor lows an aggressive clinical course with early recurrence (36). (EGF) (Austral Biologicals) was used at 50 ng/mL and TGFβ1 (Perpotech) was Therefore, JAG1/Notch signaling and regulators of this pathway used at 1 ng/mL. Transfections of siRNA (50 nmol/L; Table S1) were per- are attractive therapeutic targets in this breast cancer subtype. formed using Lipofectamine RNAiMAX (Invitrogen). How TRB3 influences tumor cell biology may be inferred from knowledge about the pathways that it regulates. Tumor-initiating High Throughput Screens for Notch Signaling Regulators. High throughput cells (TICs) represent a small population of cells within some screens were performed at the Simple Modular Assay Robotic Technology tumors that possess the unique ability to self-renew and to pro- (SMART) facility at the Samuel Lunenfeld Research Institute. For the kinase duce derivatives that maintain the tumor. The TRB3 target inhibitor screen MB231 TK-R cells were plated at a density of 6,500 cells per pathways Notch, MAPK-ERK, and TGFβ (37–39) have all been well in 384-well plates together with HES1 promoter/firefly (Hes1-FF) ade- implicated in TIC maintenance, suggesting that through the novirus at a multiplicity of infection (MOI) of 100 and cultured for 18 h. control of these pathways TRB3 may regulate tumor initiation. Transduced cells were treated with aliquots from a collection of 240 kinase The metastatic potential of epithelial tumors likely depends on inhibitors (OICR-L100, Medicinal Chemistry Platform at the Ontario Institute fi μ a process known as epithelial-to-mesenchymal transition (EMT), for Cancer Research) at a nal concentration of 5 M in 0.1% DMSO for 48 h. where epithelial cells acquire a migratory mesenchymal pheno- After incubation, Notch activation was assessed by luciferase assay. The type (40). The Notch, TGFβ, and MAPK-ERK pathways interact siRNA screen was performed using the Human siGENOME siRNA Library with each other and have a synergistic effect on the production of Protein Kinases SMARTpool (Dharmacon) targeting 720 genes encoding all factors that promote EMT and metastasis (41–43). Metastases known human protein kinases and consisting of a smart pool of 4 siRNAs per gene. Dharmacon nontargeting siRNA was used as a negative control and JAG1 siRNA as a positive control. MB231 TK-R cells were infected with Hes1- FF adenovirus at MOI 100 and then plated at a density of 16,500 cells per A B well in 96-well plates and cultured for 18 h before transfection with the 2 1.8 siRNA library and controls at 34 nm concentration using (Lipofectamine ) 2 1.8 Scr * 1.6 RNAiMAX; Invitogen). Forty-eight hours posttransfection, cells were assayed for luciferase activity. 1.6 TRB3 * 1.4 1.4 * 1.2 1.2 1 Gene Expression Analysis of MAP Kinase Activation. A gene expression sig- 1 * 0.8 nature indicative of MAP kinase activation (17, 18) was mapped to the 0.8 * 0.6 Affymetrix U133 2.0 plus platform yielding 387 of a possible 395 genes and 0.6 to the Affymetrix 133A platform yielding 341 genes. For each gene, a single 0.4 Tumor weight (g) 0.4 probeset was chosen based upon the highest level of variability between 0.2 0.2 Cross sectional area (cm Cross sectional samples. To determine if MAPK kinase activation genes show statistically 0 0 fi 2.5 3 3.5 4 4.5 5 5.5 signi cant, concordant differences as a function of continuous JAG1 gene expression, the GSEA method was used with the dataset collapsed to gene Weeks symbols, 1,000 permutations and phenotype permutation type, and Pearson Fig. 5. TRB3 knockdown impairs xenograft growth of MDA MB231 cells. (A) metric for ranking genes as previously described (45, 46). JAG1 and MAPK Estimated cross-sectional area of tumor xenografts derived from Scr siRNA- activation was compared in two sets of breast cancer cell lines (19, 47), and in treated (dashed curve; six NSG mice) or TRB3 siRNA-treated (solid curve; six the National Cancer Institute (NCI)-60 cell lines originating from cancer tis- NSG mice) MDA MB231 cells measured over 5.5 wk. (B) Weight of xenograft sues of multiple types (20) prepared as previously described. Methodology of tumors derived from Scr siRNA-treated or TRB3 siRNA-treated MDA MB231 hierarchical clustering and classification of molecular subtypes of breast cells at 5.5 wk. Error bars represent SD.*P < 0.05 relative to Scr control. cancer cell lines was previously reported (9).

1718 | www.pnas.org/cgi/doi/10.1073/pnas.1214014110 Izrailit et al. Downloaded by guest on September 23, 2021 Western Blotting and Antibodies. Cells were lysed in RIPA buffer (25 mM Tris, generated by cloning the TRB3 cDNA (Origene) into the pShuttle CMV vector. pH 7.6, 150 mM NaCl, 1% NP40, 1% DOC, 0.1% SDS) and protein was re- AD-293 packaging cells were used for recombinant adenovirus generation and solved by SDS/PAGE and blotted on PVDF membranes (Bio-Rad). Anti-JAG1 amplification. For adenovirus purification and viral titer determination, the (sc-8303), anti-TRB3 (sc-271572), anti-Smad4 (sc-7966), anti-SMURF1 (sc- Adeno-X purification and qPCR titration kits (Clontech) were used, respectively. 25510), and horseradish peroxidase (HRP) (sc-1615 HRP) were purchased from Santa Cruz Biotechnology. Anticleaved NOTCH1-Val-1744 (no. 2421), Mouse Xenografts. MDA MB231 cells were transfected with Scr or TRB3 siRNA anti-ERK1/2 (no. 4695), and anti-p-ERK1/2 (no. 9101S) were purchased from followed by injection into the mammary fat pad of six female NSG mice to Cell Signaling Technology and anti-uPA was purchased from Chemicon. generate xenografts. Tumors were measured at constant intervals and growth curves plotted. Excised tumors were weighed. Proliferation Assays. Sixty thousand cells were transfected in 24-well tissue culture plates in triplicate. At each time point cells were trypsinized and counted in a Vi-Cell XR Coulter counter. Data Analysis. The screens were analyzed using two different high throughput screen normalization methods: The first method was based on Quantitative Real-Time (QRT) PCR. Total RNA was extracted using RNeasy plus the FF/R ratios that were calculated for each test compound and siRNA. The (Qiagen) following the manufacturer’s instructions. Reverse transcription of second method determined a “B-score” where FF/R ratios were normalized total RNA was carried out using iScript cDNA synthesis kit (Bio-Rad). PCR was and corrected for systematic and positional variability within the samples performed with Power Sybr Green (Applied Biosystems). See Table S2 for (21). Each screen was performed in duplicate. Inhibitors or siRNAs that re- primer sequences. producibly decreased the FF/R ratio or the B-score by more than 2 SD from the mean were considered as hits. Luciferase activity was assessed using the Adenovirus Construction, Amplification, and Purification. Adenoviruses were Dual-Glo luciferase reporter assay system (Promega). constructed using the AdEasy system (Stratagene). Notch reporter was gen- − − erated by inserting the human Hes1 promoter ( 106 to 4) upstream of the ACKNOWLEDGMENTS. This study was supported by funds (to M.R.) from the firefly luciferase gene in pGL3-Basic vector (Promega) using traditional cloning Canadian Institutes of Health Research and in part by the Ontario Ministry of methods. Hes1-FF was than cloned into the pShuttle vector. ADTRB3 was Health and Long Term Care.

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