The role of miR-26a and miR-30b in HER2+ breast cancer trastuzumab resistance and regulation of the CCNE2 gene

Eduardo Tormo,Joan Albanell,Ana Rovira,Pilar Eroles,Federico Rojo,Begoña Pineda,Ana Lluch,Sandra Ballester,Anna Adam-Artigues,Sandra Zazo,Paula González-Alonso

doi:10.1038/srep41309

Abstract

A subset of HER2+ breast cancer patients manifest clinical resistance to trastuzumab. Recently, miR-26a and miR-30b have been identified as trastuzumab response regulators, and their target gene CCNE2 seems to play an important role in resistance to trastuzumab therapy. Cell viability was evaluated in trastuzumab treated HER2+ BT474 wt (sensitive), BT474r (acquired resistance), HCC1954 (innate resistance), and MDA-MB-231 (HER2−) cell lines, and the expression of miR-26a, miR-30b, and their target genes was measured. BT474 wt cell viability decreased by 60% and miR-26a and miR-30b were significantly overexpressed (~3-fold, p = 0.003 and p = 0.002, respectively) after trastuzumab treatment, but no differences were observed in resistant and control cell lines. Overexpression of miR-30b sensitized BT474r cells to trastuzumab (p = 0.01) and CCNE2, was significantly overexpressed after trastuzumab treatment in BT474r cells (p = 0.032), but no significant changes were observed in sensitive cell line. When CCNE2 was silenced BT474r cell sensitivity to trastuzumab increased (p = 0.03). Thus, the molecular mechanism of trastuzumab action in BT474 cell line may be regulated by miR-26a and miR-30b and CCNE2 overexpression might play an important role in acquired trastuzumab resistance in HER2+ breast cancer given that resistance was diminished when CCNE2 was silenced.

Highlights

It has been previously described that trastuzumab blocks HER2 signaling by preventing HER2 homo/heterodimerization, inducing antibody-dependent cell-mediated cytotoxicity, promoting HER2 receptor internalization, and inhibiting its cleavage, which in turn blocks the MAPK, mTOR, and PI3K/Akt pathways[6]
First we examined the effect of trastuzumab on cell viability in our HER2-positive (BT474 wt, BT474r, and HCC1954) and HER2-negative (MDA-MB-231) breast cancer cell lines
Annexin-V assays showed that BT474 wt cell viability was reduced from 97.7% to 22.6% after trastuzumab treatment while BT474r cells were not affected by the same treatment

Summary

Introduction

It has been previously described that trastuzumab blocks HER2 signaling by preventing HER2 homo/heterodimerization, inducing antibody-dependent cell-mediated cytotoxicity, promoting HER2 receptor internalization, and inhibiting its cleavage, which in turn blocks the MAPK, mTOR, and PI3K/Akt pathways[6]. Following on from this work, our group focused on how miR-26a and miR-30b are implicated in this response to trastuzumab by examining the differences in miRNA expression in trastuzumab-sensitive, acquired-resistance, or innate-resistance HER2+breast cancer cell lines. We checked the regulation of CCNE2 (Cyclin E2) by these miRNAs and its expression in different HER2+breast cancer cell lines because this gene has been previously implicated in acquired trastuzumab resistance[10] and is a possible miR-30b target[9]. We describe possible miR-30b and miR-26a target genes related to the cell cycle and apoptosis that are likely implicated in trastuzumab response, and go on to suggest potential biomarkers which may be able to identify trastuzumab resistance in early treatment phases

Methods

Results

Conclusion