miR-630 targets IGF1R to regulate response to HER-targeting drugs and overall cancer cell progression in HER2 over-expressing breast cancer

Abstract

Background: While the treatment of HER2 over-expressing breast cancer with recent HER-targeted drugs has been highly effective for some patients, primary (also known as innate) or acquired resistance limits the success of these drugs. microRNAs have potential as diagnostic, prognostic and predictive biomarkers, as well as replacement therapies. Here we investigated the role of microRNA-630 (miR-630) in breast cancer progression and as a predictive biomarker for response to HER-targeting drugs, ultimately yielding potential as a therapeutic approach to add value to these drugs.

Methods: We investigated the levels of intra- and extracellular miR-630 in cells and conditioned media from breast cancer cell lines with either innate- or acquired- resistance to HER-targeting lapatinib and neratinib, compared to their corresponding drug sensitive cell lines, using qPCR. To support the role of miR-630 in breast cancer, we examined the clinical relevance of this miRNA in breast cancer tumours versus matched peritumours. Transfection of miR-630 mimics and inhibitors was used to manipulate the expression of miR-630 to assess effects on response to HER-targeting drugs (lapatinib, neratinib and afatinib). Other phenotypic changes associated with cellular aggressiveness were evaluated by motility, invasion and anoikis assays. TargetScan prediction software, qPCR, immunoblotting and ELISAs, were used to assess miR-630's regulation of mRNA, proteins and their phosphorylated forms.

Results: We established that introducing miR-630 into cells with innate- or acquired- resistance to HER-drugs significantly restored the efficacy of lapatinib, neratinib and afatinib; through a mechanism which we have determined to, at least partly, involve miR-630's regulation of IGF1R. Conversely, we demonstrated that blocking miR-630 induced resistance/insensitivity to these drugs. Cellular motility, invasion, and anoikis were also observed as significantly altered by miR-630 manipulation, whereby introducing miR-630 into cells reduced cellular aggression while inhibition of miR-630 induced a more aggressive cellular phenotype.

Conclusions: Taken together, our findings suggest miR-630 as a key regulator of cancer cell progression in HER2 over-expressing breast cancer, through targeting of IGF1R. This study supports miR-630 as a diagnostic and a predictive biomarker for response to HER-targeted drugs and indicates that the therapeutic addition of miR-630 may enhance and improve patients' response to HER-targeting drugs.

Figure 1

miR-630 expression decreases with both acquired- and innate resistance. (A) miR-630 expression was significantly decreased in cells with acquired lapatinib resistance ((i) HCC1954-LR and (ii) SKBR3-LR) and acquired neratinib-resistance ((iii) HCC1954-NR) compared to their age-matched control cells (HCC1954-Ag, SKBR3-Ag). miR-630 expression was also significantly decreased in cells with innate resistance to lapatinib ((iv) MDA-MB-453) compared to cells with innate sensitivity (SKBR3). (B) Extracellular expression of miR-630 was also assessed in the corresponding conditioned medium of these cells and trends followed that of the cells. All results represent biological repeats n = 3 ± SEM, where *p <0.05, **p <0.01, ***p <0.001.

Figure 2

Clinical relevance of miR-630 in breast cancer. Using a publically available data set (GSE40525) from Gene Expression Omnibus (GEO) the expression of miR-630 was determined in breast cancer tumour tissues compared to matched peritumour tissue. (A) miR-630 was significantly decreased in all breast tumours (n = 56) and also in (B) HER2+ breast cancer tissue (n = 6) compared to matched peritumours. *p <0.05, ***p <0.001

Figure 3

Inhibition of miR-630 increased cell motility, migration, invasion and resistance to anoikis. Following transfection with miR-630 inhibitor or a negative control (NC) inhibitor in (i) HCC1954-Ag and (ii) SKBR3-Ag (A) motility, assessed by wound-healing assay (B) migration, through transwells; (C) invasion, through ECM-coated transwells; and (D)anoikis, on poly(hydroxyethyl methacrylic) acid coated plates; were found to be significantly changed as a consequence of miR-630 inhibition. Results represent n = 3 ± SEM, where *p <0.05, **p <0.01.

Figure 4

Over-expression of miR-630 in lapatinib resistant cells decreases cell motility, migration, invasion and anoikis. Following transfection with miR-630 mimic or a negative control (NC) mimic in HCC1954-LR (i) and SKBR3-LR (ii) (A) motility, assessed by wound-healing assay; (B) migration, through transwells; (C) invasion, through ECM-coated transwells; (D)anoikis, on Poly(hydroxyethyl methacrylic) acid coated plates; were found to be significantly altered as a consequence of miR-630 induced expression. Results represent n = 3 ± SEM, where *p <0.05, **p <0.01, ***p<0.001.

Figure 5

miR-630’s mechanism of action. IGF1R was predicted as a direct target of miR-630. (A) (i) Initially the expression of IGF1R, HER2 and EGFR was assessed in our acquired lapatinib-resistant cells (HCC1954-LR and SKBR3-LR) and was found to be increased compared to levels in their age-matched parent cells. (ii) Inhibition of miR-630 induced an increase in IGF1R, HER2 and EGFR expression in both HCC1954-Ag and SKBR3-Ag cells compared to levels in the negative control transfected cells. (iii) Conversely, induced expression of miR-630 in lapatinib-resistant HCC1954-LR and SKBR3-LR cells caused a decrease in IGF1R, HER2 and EGFR levels. (B) The phosphorylated forms of IGF1R, HER2 and EGFR were assessed using ELISAs, indicating that (i) inhibition of miR-630 induced an increase in pIGF1R, pHER2 and pEGFR, while (ii) over-expression of miR-630 decreased the pIGF1R, pHER2 and pEGFR levels. Representative immunoblots of n = 3 biological repeats. n = 3 ± SEM, where *p <0.05, **p <0.01, ***p < 0.001.

Figure 6

Proposed model for manipulation of miR-630 in breast cancer cells. Elevated IGF1R, HER2 and EGFR have been associated with drug resistance and cancer cell aggression. (A) Introducing miR-630 into acquired or innately resistant breast cancer cells can (1) directly inhibit the mRNA translation of IGF1R. (2) Crosstalk of IGF1R to other receptor tyrosine kinases (HER2 and EGFR) can lead to their subsequent down regulation and so (3) cause cells to become more sensitive to HER-targeting agents as well as decreasing their aggressive phenotype (in terms of motility, invasion and resistance to anoikis). (B) Conversely, inhibiting the expression of miR-630 in sensitive breast cancer cells can (1) prevent its binding to and translational suppression of target mRNA IGF1R. This causes an increase in IGF1R protein expression which in turn can (2) induce elevated HER2 and EGFR expression and as a result, (3) cells exhibit a more aggressive phenotype.