Abstract
HER2 positive breast cancer accounts for approximately 20-25 % of breast cancers. Trastuzumab, a humanised monoclonal antibody, is approved for the treatment of HER2 positive breast cancer. However, the majority of metastatic HER2 positive breast cancers progress on trastuzumab treatment due to either innate or acquired resistance. The aim of this study is to investigate the role of microRNAs (miRNAs) in a cell line model of acquired trastuzumab resistance.
The trastuzumab resistant cell line, SKBR3-T was established by continuous exposure to trastuzumab (10 µg/ml) for 6 months. miRNA extracted from SKBR3 and SKBR3-T was profiled using Taqman low density arrays (TLDA). Differentially regulated miRNAs were selected using >2-fold change and a P-value of <0.05. Individual quantitative RT-PCR (qRT-PCR) was performed to confirm miRNA alterations. Proliferation assays were performed using the acid phosphatase method. Taqman gene expression assays for retinoic acid receptor alpha (RARA) were performed using GAPDH as an endogenous control. Immunodetection of RARA was performed using α-tubulin as a control.
Six differentially regulated miRNAs were identified in the SKBR3-T cells. qRT-PCR assays confirmed that four were significantly altered in SKBR3-T compared to SKBR3 cells including miR-9 which was 2.2 fold up-regulated (p=0.04).
Utilising miRWalk, we identified RARA as a target for miR-9. We confirmed that RARA mRNA and protein expression are reduced in SKBR3-T cells compared to SKBR3 cells. Treatment with all-trans retinoic acid (ATRA) (0.2 µM–0.025 µM) alone inhibited growth of the SKBR3 cell line in a dose dependent manner but not in the SKBR3-T cell line. Combined treatment with trastuzumab (10 µg/uL) and ATRA (0.2 µM) had a significantly greater growth inhibitory effect on the SKBR3 cell line (90.1±5.2 %) (p=0.0002) than either trastuzumab (39.1±4.0 %) (p= 0.0004) or ATRA alone (57.9±4.2 %) (p=0.001). Interestingly, despite relative insensitivity to ATRA in the SKBR3-T cells (23.7±5.0%) (p=0.86), combined treatment with trastuzumab produced significant growth inhibition in the SKBR3-T cells (74.2±5.1%) compared to either trastuzumab (24.1±4.4%) (p=0.0001) or ATRA (23.7±5.0%) (p=0.86) alone.
miR-9 is up-regulated in the acquired trastuzumab resistant SKBR3-T cell line, RARA expression is downregulated, and SKBR3-T cells are resistant to ATRA treatment compared to SKBR3 cells. However, SKBR3-T cells are sensitive to combined treatment with trastuzumab and ATRA. Thus combined treatment with ATRA and trastuzumab may overcome acquired resistance to trastuzumab in HER2 positive breast cancer.
Citation Format: Karen Howe, Brigid C Browne, Sinead Aherne, John Crown, Norma O'Donovan. miR-9 expression, retinoids and their potential role in trastuzumab resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-06.
Volume 75, Issue 9 Supplement
