Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R

Abstract

Background: Resistance 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.

Methods: Trastuzumab-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.

Results: miR-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.

Conclusions: Epigenetic 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.

Figure 1

Trastuzumab-resistant and parental breast cancer SKBr-3 cells display distinct characteristics and miRNA expression profiles. A. SKBr-3 cells were cultured in the presence of trastuzumab (5 μg/ml) for 6 months to obtain resistant cells. A colony formation assay in soft agar was performed using parental and resistant cells. The images are representative of triplicate experiments on day 7 after cell seeding. B. MTT assays of parental and trastuzumab-resistant SKBr-3 cells in the absence of trastuzumab. C. Confirmation of the differential expression of the indicated miRNAs by qRT-PCR. Each assay was performed in triplicate and the expression levels of each miRNA were normalized to those of snRNA RNU6B (U6). D. The predicted human miR-375 binding site at nucleotides 2993-2999 of the 3′ UTR of wild-type (WT) IGF1R (sense). The sequence of the pGL3-IGF1R-mut construct containing mutated nucleotides at the miR-375 binding site within the 3′ UTR of IGF1R is also shown. All data are represented as the mean ± SD or are representative of n = 3 replicates. *P <0.05, **P <0.01 and ***P <0.001.

Figure 2

miR-375 modulates trastuzumab resistance in breast cancer cells. Trastuzumab-resistant SKBr-3 cells were infected with lentiviral vectors expressing pre-miR-control or pre-miR-375, and parental cells were transfected with a control RNA or a miR-375-specific inhibitor. A. qRT-PCR analyses of miR-375 expression. Data were normalized to mock-transfected cells and the expression levels of miR-375 were normalized to U6. B. MTT assays of modified cells after treatment with increasing concentrations of trastuzumab. C. Plate colony formation assays of modified trastuzumab-resistant cells in the presence of trastuzumab (5 μg/ml). D. Flow cytometry analyses of modified cells. Resistant and parental cells were treated with 10 μg/ml or 5 μg/ml trastuzumab, respectively, for 24 h, and then stained with FITC-conjugated Annexin V and PI. E. Microscopy images of modified trastuzumab-resistant SKBr-3 cells. Cells were treated with the indicated concentrations of trastuzumab for 24 h and images were captured using a phase contrast microscope. The arrow indicates autophagosome-like bodies. F. Upper panel: qRT-PCR assays were performed to identify the expression levels of miR-375 in human breast cancer BT474 and MDA-MB-453 cells. Lower panel: MTT assay of cells transfected with control or miR-375 antisense RNA and treated with various concentrations of trastuzumab for 24 h. G. Flow cytometry analyses of apoptosis in cells transfected with control or miR-375 antisense RNA. Twenty-four hours post-transfection, cells were treated with trastuzumab (5 μg/mL) for another 24 h, and cells were stained with FITC-conjugated Annexin V and PI. All data are represented as the mean ± SD or are representative of n = 3 replicates. *P <0.05, **P <0.01 and ***P <0.001.

Figure 3

miR-375 restores trastuzumab sensitivity by directly targeting IGF1R in breast cancer cell. A. Western blot (left) and qRT-PCR (right) analyses of IGF1R expression in parental (P) and trastuzumab-resistant (R) SKBr-3 cells. The qRT-PCR data were normalized to GAPDH. B. Luciferase activity measured 24 h after co-transfection of trastuzumab-resistant SKBr-3 with pGL3 constructs containing the wild-type or mutant 3′UTR of IGF1R, an internal control vector (pGL4.73), and synthetic miR-375 mimics. Data were normalized to the luciferase activity of control (vehicle transfected) cells. C. qRT-PCR analyses of IGF1R expression in SKBr-3 cells as modified in Figure 2. Data were normalized to mock-transfected cells. D. Pearson’s correlation analysis of the relative expression level of miR-375 (normalized to U6) and IGF1R mRNA (normalized to GAPDH) as determined using qRT-PCR in 40 human breast cancer tissue samples. E. Western blot and qRT-PCR analyses of trastuzumab-resistant SKBr-3 cells infected with a lentivirus vector expressing GFP- (control) or IGF1R-specific shRNA. Data were normalized to those of GAPDH. F. MTT assays of cells described in (E) after treatment with the indicated concentration of trastuzumab for 24 h prior to analysis. G. Flow cytometry analyses of cells in (E). Cells were treated with trastuzumab (10 μg/ml or 5 μg/ml) for 24 h and then stained with Annexin V and PI. All data are represented as the mean ± SD of n = 3 replicates. *P <0.05 and **P <0.01.

Figure 4

miR-375 modulates trastuzumab resistance of HER2-positive breast cancer xenografts. Nude mice were inoculated in the mammary fat pad with trastuzumab-resistant SKBr-3 cells overexpressing pre-miR-375 or control pre-miRNA to allow tumor development. Mice were intravenously injected with 10 mg/kg trastuzumab twice a week. A. Tumor volume in the trastuzumab-treated mice. Data are represented as the mean ± SD of six mice. *P <0.05. B. Tumor weight at the end of the treatment period (45 days after first trastuzumab injection). The bars represent the mean ± SD of six mice. C. Kaplan-Meier survival curves of the trastuzumab-treated mice (n = 6).

Figure 5

Epigenetic mechanisms underlie miR-375 deregulation, and PI3K/Akt pathway is involved in miR-375/IGF1R-mediated trastuzumab resistance. A. The luciferase reporter construct of miR-375 promoter was introduced into parental and trastuzumab-resistant SKBr-3 cells, followed by assays of relative cellular luciferase activity. Data were normalized to the luciferase activity of control (vehicle transfected) cells. B. Cells were treated with 5-Aza-CdR (5 μmol/L) for 3 days and/or TSA (100 μmol/L) for 24 hrs. qRT-PCR was performed to quantify miR-375. NC, non-treated control. C. The levels of acetylated histone H3K9 in the miR-375 promoter region were determined by chromatin immunoprecipitation assay in indicated cells. NAC, nonspecific antibody control. Ac-H3, acetylated H3. D. Methylation-specific PCR analysis of miR-375 promoter region in parental (P) and trastuzumab-resistant (R) SKBr-3 cells. M, methylated allele; U, unmethylated allele. E. Western blot analyses of trastuzumab-resistant SKBr-3 cells expressing pre-miR-375 or control pre-miRNA, and parental SKBr-3 cells transfected with miR-375 antisense RNA or control RNA. GAPDH was used as the loading control. Data are represented as the mean ± SD of n = 3 replicates for A and B. **P <0.01 and ***P <0.001.