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

Corcoran C, Rani S, Breslin S, Gogarty M, Ghobrial IM, Crown J, O'Driscoll L - Mol. Cancer (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Pharmacy and Pharmaceutical Sciences & Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. lodrisc@tcd.ie.

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.

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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.
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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.

Mentions: Through the use of target prediction software (TargetScanHuman Release 6.2), IGF1R was predicted to be regulated by miR-630. Initially we assessed the levels of IGF1R in our acquired lapatinib-resistant cells (HCC1954-LR and SKBR3-LR) and found a significant increase of this protein compared to the corresponding parent cells (Figure 5A (i)). We also observed that IGF1R to be increased in our neratinib-resistant cells (HCC1954-NR) compared to its corresponding parent cells (not shown).


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

Corcoran C, Rani S, Breslin S, Gogarty M, Ghobrial IM, Crown J, O'Driscoll L - Mol. Cancer (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234346&req=5

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.
Mentions: Through the use of target prediction software (TargetScanHuman Release 6.2), IGF1R was predicted to be regulated by miR-630. Initially we assessed the levels of IGF1R in our acquired lapatinib-resistant cells (HCC1954-LR and SKBR3-LR) and found a significant increase of this protein compared to the corresponding parent cells (Figure 5A (i)). We also observed that IGF1R to be increased in our neratinib-resistant cells (HCC1954-NR) compared to its corresponding parent cells (not shown).

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Pharmacy and Pharmaceutical Sciences & Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. lodrisc@tcd.ie.

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.

Show MeSH
Related in: MedlinePlus