Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity - PubMed (original) (raw)

. 2015 Mar;14(3):779-787.

doi: 10.1158/1535-7163.MCT-14-0228. Epub 2015 Feb 11.

Maria Ouzounova #, April Davis, Daejin Choi 1, Stevie M Tchuenkam 1, Gwangil Kim, Tahra Luther, Ahmed A Quraishi, Yasin Senbabaoglu, Sarah J Conley, Shawn G Clouthier, Khaled A Hassan, Max S Wicha, Hasan Korkaya

Affiliations

Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity

Rosemarie C D'Angelo et al. Mol Cancer Ther. 2015 Mar.

Abstract

Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch(+)) or reduced activity (Notch(-)) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch(+) cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch(+) cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch(-) cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch(+) cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics.

©2015 American Association for Cancer Research.

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Figures

Figure 1

Figure 1. Identification of Notch activity in multiple breast cancer cell lines is determined by the Notch reporter system

Breast cancer cell lines infected with the lentiviral Notch reporter that expresses GFP and Luciferase under the minimal CMV promoter driven by Notch transcription response element (GTGGGAACGGCATTGTAGCG). (TRE: transcription response element, NICD Notch intracellular domain, TC: transcription complex). (A) A schematic illustration of the lentiviral constructs used in studies presented here (B, C) Basal breast cancer cell lines, MDA-MB-231, Sum159, MDA-MB-436 display a higher Notch activity compared to luminal breast cancer cell lines, ZR-75-1, MCF7, ZR-75-30, T47D. Means ± SD (n=3),*p≤0.05, ** p≤0.005.

Figure 2

Figure 2. Notch reporter positive (Notch+) cells display CSC features

(A) FACS sorted Notch+ MCF7 cells showed a higher ALDH1 expression compared to Notch- cells. (B) Notch activity and CD44+CD24- phenotype in MCF7 cells are not correlated with statistical significance. (C) A higher sphere forming capacity was observed in Notch+ cells compared to the Notch- cells. (D) Notch+ MCF7 cells show reduced ER expression compared to the Notch- cells (E, F) Notch agonist Delta-Serrate-Lag (DSL) peptide or recombinant DLL4 ligand induces Notch reporter activity in MCF7 cells as demonstrated by GFP expression. (G) Notch activity is increased when MCF7 cells co-cultured with the human endothelial HUVEC cells. **(**Means ± SD (n=3),*p≤0.05, ** p≤0.005.

Figure 3

Figure 3. Notch reporter positive cells express higher Notch1, Notch2 and Notch4 receptors and downstream effectors compared to the negative cells

(A, B) qPCR analyses show increased expressions of Notch1, 2 and 4, Notch ligands Jagged 1 and DLL1 and downstream effector Hey1 in Notch+ cells compared to Notch- cell populations. (C) Notch+ cells compared to the Notch- cells display higher Hey1 expression and (D) increased nuclear β-catenin localization as assessed by IF. (E, F) Analyses of TCGA breast cancer data set (cBioPortal) revealed that elevated Notch4/Hey1 expressions but bot Notch2/Hey1 expressions correlated with poor patient survival. Means ± SD (n=3),*p≤0.05, ** p≤0.005.

Figure 4

Figure 4. Notch- cells fails to generate Notch+ cells in vitro

(A) Notch reporter lentivirus infected Sum159 cells were presorted by FACS and Notch+ cells were kept in culture for two weeks during which Notch+ cells generated Notch+ as well as Notch- cells. (B) Using the presorted Notch+ cells, single Notch+ or Notch- cells were FACS sorted and independent clones were grown. We analyzed these independent clones that are generated from single Notch- (n=13) or Notch+ (n=12) cells. None of the clones of single Notch- cells was able to generate the Notch+ cell population while the clones generated from single Notch+ cells were able to generate both Notch+ as well as Notch- cell populations. Means ± SD (n=3),*p≤0.05, ** p≤0.005.

Figure 5

Figure 5. Notch+ cells possess tumor initiating capacity and show lineage differentiation in mouse xenografts

(A, C) Notch+ Sum159 and MCF7 cells compared to Notch- cells demonstrated a higher tumor forming capacity in mice xenografts. (B, D) FACS analyses of primary tumor cells showed that Notch+ tumors reconstituted the initial heterogeneity in vivo as evidenced by the presence of both Notch+ and Notch- cells (two independent FACS data presented).

Figure 6

Figure 6. Gamma-secretase inhibitor (GSI) reduces the Notch activity and targets breast CSCs in mouse xenografts

Mice were implanted with Notch reporter expressing Sum159 cells and treated with two cycles of GSI or Docetaxel alone or in combination starting at three weeks post implantation (Supplementary Figure 2). (A) Bioluminescent imaging of mice during the course of treatment showed that the Notch activity was significantly reduced in GSI alone or GSI plus Docetaxel treated animals. (B) Reduced Notch activity in tumors was also confirmed by reduced GFP expression in GSI treated animals. (C) Secondary re-implantation of residual tumors from GSI treated mice revealed a reduced tumor initiating capacity. Means ± SD (n=3),*p≤0.05, ** p≤0.005.

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