Abstract 4769: Targeting HER2 enriches Jagged1 high cancer stem cells in breast cancer
Authors: Shah Deep S. , Wyatt Debra , Baker Andrew , Green Andrew , Filipovic Aleksandra , Miele Lucio , Osipo Clodia 
Published: July 2017

The human epidermal growth factor receptor 2 (HER2) positive subtype of breast cancer is characterized by gene amplification and/or protein overexpression of HER2. It is driven by a subpopulation of cells possessing stem cell properties of self-renewal and differentiation, known as Cancer Stem cells (CSCs). CSCs are implicated in tumor growth as well as radiotherapy and chemotherapy associated resistance. Notch receptors promote breast CSCs survival and self-renewal, and overexpression of a Notch ligand Jagged1 mRNA predicts poor prognosis in women with breast cancer. Our lab has published that Jagged1 or Notch1 is a critical target in trastuzumab/lapatinib (LAP) resistant HER2+ breast cancer. The study aimed to determine whether anti-HER2 therapy selects for Jagged-1/Notch-dependent CSCs that are responsible for tumor initiation. Surface expression of Notch1 and Jagged1 upon HER2 blockade using LAP was measured in HER2+ breast cancer cell lines (MDA-MB-453 and HCC1954) using flow cytometry. LAP treatment increased the Jagged1-positive subpopulation compared to vehicle. Cells were sorted based on Jagged1 cell surface expression and assessed for CSC-like properties (i.e. mammosphere forming efficiency, Aldefluor activity, expression of CD44high /CD24low, Notch target transcripts expression, and limiting dilution tumor initiating potential in athymic, nude mice). In addition, immunohistochemistry was performed on 145 HER2+ breast tumor microarray to detect cytoplasmic, membrane, or nuclear Jagged1 protein expression. Kaplan-Meier analysis was performed to determine overall survival. The results showed that upon HER2 inhibition, Jagged1 cell surface expression increased and Notch1 cell surface expression was unchanged. The Jagged1high subpopulation of cells showed elevated levels of Aldehyde dehydrogenase activity, Notch target gene transcripts, and mammosphere formation efficiency compared to vehicle treated cells. The MRK-003 γ-secretase inhibitor (GSI) prevented mammosphere formation in the Jagged1high cells indicating that Notch activation drives Jagged1high CSC survival. Also, we confirmed that Jagged1 expression is required for the enrichment of CSCs using a Jagged1 siRNA. Combined knockdown of Notch1 and Notch3 receptors was necessary to reduce LAP-enriched mammospheres suggesting that targeting HER2 enriches for a Jagged1-Notch1+Notch3 driven CSC phenotype. Importantly, higher membrane expression of Jagged1 protein in 145 HER2+ breast tumor specimens correlated with significantly lower overall cumulative survival. These results reveal that HER2 blockade in breast cancer cells enriches for a Jagged1high subpopulation that has higher CSC potential and is resistant to HER2 inhibitors. The implications of this work are that dual blockade of Jagged-1/Notch1/Notch3 and HER2 could be more effective than either therapy alone to eliminate both HER2 and Jagged-1-dependent cancer cells.

Citation Format: Deep S. Shah, Debra Wyatt, Andrew Baker, Andrew Green, Aleksandra Filipovic, Lucio Miele, Clodia Osipo. Targeting HER2 enriches Jagged1 high cancer stem cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4769. doi:10.1158/1538-7445.AM2017-4769