NOTCH-1 and NOTCH-4 are novel gene targets of PEA3 in breast cancer: novel therapeutic implications

Abstract

IntroductionWomen with triple-negative breast cancer have the worst prognosis, frequently present with metastatic tumors and have few targeted therapy options. Notch-1 and Notch-4 are potent breast oncogenes that are overexpressed in triple-negative and other subtypes of breast cancer. PEA3, an ETS transcription factor, is also overexpressed in triple-negative and other breast cancer subtypes. We investigated whether PEA3 could be the critical transcriptional activator of Notch receptors in MDA-MB-231 and other breast cancer cells.MethodsReal-time PCR and Western blot analysis were performed to detect Notch-1, Notch-2, Notch-3 and Notch-4 receptor expression in breast cancer cells when PEA3 was knocked down by siRNA. Chromatin immunoprecipitation was performed to identify promoter regions for Notch genes that recruited PEA3. TAM-67 and c-Jun siRNA were used to identify that c-Jun was necessary for PEA3 enrichment on the Notch-4 promoter. A Notch-4 luciferase reporter was used to confirm that endogenous PEA3 or AP-1 activated the Notch-4 promoter region. Cell cycle analysis, trypan blue exclusion, annexin V flow cytometry, colony formation assay and an in vivo xenograft study were performed to determine the biological significance of targeting PEA3 via siRNA, Notch signaling via a γ-secretase inhibitor, or both.ResultsHerein we provide new evidence for transcriptional regulation of Notch by PEA3 in breast cancer. PEA3 activates Notch-1 transcription in MCF-7, MDA-MB-231 and SKBr3 breast cancer cells. PEA3 activates Notch-4 transcription in MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474 breast cancer cells where PEA3 levels are low, overexpression of PEA3 increases Notch-4 transcripts. Chromatin immunoprecipitation confirmed the enrichment of PEA3 on Notch-1 and Notch-4 promoters in MDA-MB-231 cells. PEA3 recruitment to Notch-1 was AP-1-independent, whereas PEA3 recruitment to Notch-4 was c-JUN-dependent. Importantly, the combined inhibition of Notch signaling via a γ-secretase inhibitor (MRK-003 GSI) and knockdown of PEA3 arrested growth in the G1 phase, decreased both anchorage-dependent and anchorage-independent growth and significantly increased apoptotic cells in vitro. Moreover, either PEA3 knockdown or MRK-003 GSI treatment significantly reduced tumor growth of MDA-MB-231 xenografts in vivo.ConclusionsTaken together, the results from this study demonstrate for the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast cancer cells. Targeting of PEA3 and/or Notch pathways might provide a new therapeutic strategy for triple-negative and possibly other breast cancer subtypes.

Discovery of Biomarkers Predictive of GSI Response in Triple-Negative Breast Cancer and Adenoid Cystic Carcinoma

“…For example, MRK-003 treatment produced dose-dependent growth cessation or regression of HCC1599 and MB-157 xenografts (Figure 5 A and Table1), but had only modest anti-tumor activity in the MDA-MB-231 xenograft model with wild type Notch genes (Supplemental Figure S7C, Table 1). Although previous studies have demonstrated significant anti-tumor activity with MRK-003 in MDA-MB-231 mode we would like to note that the dosing scheme utilized in these studies were not identical to our study (20). In this study we utilized a clinically tolerated dosing schedule (once a week dosing).…”

EZH2 expands breast stem cells through activation of NOTCH1 signaling

“…Despite interest in the association between EZH2 functions, breast TICs, and TN breast cancer (13), the molecular mechanisms underlying the tumorigenic function of EZH2 in this cancer subtype and the relationship to NOTCH1 signaling have not yet been considered. Furthermore, whereas a role for NOTCH1 in breast tumorigenesis has been established in vivo (30), the factors regulating increased NOTCH1 expression and signaling in breast cancer cells are largely unknown (31,32). We show that EZH2 is a regulator of NOTCH1 expression and pathway activation in TN breast cancer and that NOTCH signaling activation is required for EZH2-dependent stem cell expansion.…”

γ-Secretase inhibitors and modulators

“…For example, in the GSI AD trial, many individuals noted changes in hair color, apparently due to inhibition of tyrosinase, another γ-secretase substrate [101]. In a few studies other Notch paralogs and VEGFR1 have been considered to be targets [70, 102-103]. Given that tools are not readily available to perform facile, detailed studies on the impact of γ-secretase cleavage on γ-secretase substrates other than APP and Notch 1; it will be important to develop these tools to better understand the biological consequences of GSI based therapies [4].…”