Abstract
BackgroundResistance to humanized monoclonal erbB2/HER2 antibody, trastuzumab (Herceptin), has become a pivotal obstacle for targeted therapy of HER2-positive breast cancers. The activation of alternative growth factor receptors, in particular, the insulin-like growth factor 1 receptor (IGF1R), represents a common feature of trastuzumab-refractory cells; however, the underlying mechanism remains elusive.MethodsTrastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3 cells in the presence of trastuzumab. Among the differentially expressed microRNAs (miRNAs) screened by microarray analysis, candidate miRNA(s) predicted to target IGF1R was studied for its role in conferring trastuzumab resistance. The mechanism underlying decreased expression of IGF1R-targeted miRNA in refractory cells was also addressed.ResultsmiR-375, which was downregulated and predicted to target IGF1R in trastuzumab-resistant HER2-positive breast cancer cells, could indeed inhibit the cellular luciferase activity in a reporter construct containing the 3′-UTR of IGF1R. Overexpression of miR-375 restored the sensitivity of cells to trastuzumab, while inhibition of miR-375 conferred trastuzumab resistance on HER2-positive breast cancer cells. Blockade of DNA methylation and histone deacetylation restored the expression of miR-375 in trastuzumab-resistant cells. A reverse correlation between the levels of miR-375 and IGF1R was validated in clinical breast cancers.ConclusionsEpigenetic silencing of miR-375 causes the upregulation of IGF1R, which at least partially underlies trastuzumab resistance of breast cancer cells. Our study has implications for miR-375 as a potential target in combination with trastuzumab for treating HER2-positive breast cancers.
Highlights
Resistance to humanized monoclonal erbB2/Human epidermal growth factor receptor 2 (HER2) antibody, trastuzumab (Herceptin), has become a pivotal obstacle for targeted therapy of HER2-positive breast cancers
Compared with the parental cells, the resistant SKBr-3 cells displayed dramatically increased colony formation on the agar plates (Figure 1A) and had a significantly higher viability or proliferative capacity in an MTT assay (p < 0.05, Figure 1B). These results suggest that trastuzumab-resistant HER2-positive breast cancer cells exhibit anchorage-independent growth and proliferation advantages in vitro over non-resistant cells
Among the differentially expressed miRNAs, we focused on miR-375, which showed the second largest absolute fold change in the microarray analysis (p < 0.001), because this miRNA was predicted to target insulin-like growth factor 1 receptor (IGF1R), a receptor tyrosine kinase dominantly upregulated in trastuzumabresistant cells (Figure 1D) [13]
Summary
Resistance to humanized monoclonal erbB2/HER2 antibody, trastuzumab (Herceptin), has become a pivotal obstacle for targeted therapy of HER2-positive breast cancers. Most patients that respond to the initial trastuzumab treatment develop resistance within a year [5]; clarifying the mechanisms underlying trastuzumab resistance will provide great impetus for the development of novel strategies for breast cancer therapy [7]. Various mechanisms have been reported to cause resistance of breast cancers to trastuzumab, including reduced HER2 expression or antibody affinity, increased pro-survival signaling through alternative receptor tyrosine kinases, and altered intracellular signaling such as the loss of PTEN expression, reduced activity of cell cycle regulator p27kip, or increased Akt activity, which result in the over-proliferation of cells [8,9]. Insulin-like growth factor-1 receptor (IGF1R) is thought to play a key role in the acquisition of cancer resistance to trastuzumab and other targeted pharmaceuticals [10,11]; little is currently known regarding the regulation of IGF1R in these cells during the development of resistance to trastuzumab
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