A novel double-negative feedback loop between miR-489 and the HER2-SHP2-MAPK signaling axis regulates breast cancer cell proliferation and tumor growth

Human epidermal growth factor receptor 2 (HER2 or ErBb2) is a receptor tyrosine kinase overexpressed in 20-30% of breast cancers and associated with poor prognosis and outcome. Dysregulation of several microRNAs (miRNAs) plays a key role in breast cancer progression and metastasis. In this study, we screened and identified miRNAs dysregualted in HER2-positive breast cancer cells. Our molecular study demonstrated that miR-489 was specifically downregulated by the HER2-downstream signaling, especially through the MAPK pathway. Restoration or overexpression of miR-489 in HER2-positive breast cancer cells significantly inhibited cell growth in vitro and decreased the tumorigenecity and tumor growth in xenograft mice. Mechanistically, we found that overexpression of miR-489 led to the decreased levels of HER2 and SHP2 and thus attenuated HER2-downstream signaling. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2-SHP2-MAPK and miR-489 signaling pathways form a mutually inhibitory loop. Using quantitative real-time PCR analysis and Fluorescent in situ hybridization technique (FISH), we found that miR-489 was expressed at significantly lower level in tumor tissues compared to the adjacent normal tissues. Downregulation of miR-489 in breast cancers was associated with aggressive tumor phenotypes. Overall, our results define a double-negative feedback loop involving miR-489 and the HER2-SHP2-MAPK signaling axis that can regulate breast cancer cell proliferation and tumor progression and might have therapeutic relevance for HER2-positive breast cancer.


Colony formation assay
We transfected mimic-miR-489 and inhibitor-miR-489 in MCF7 and MD-MBA-231 cells and incubated cells for 24 hr [1]. After incubation, we collected all cells. For MCF-7 10000cells/well and for MD-MBA-231 4000cell/well were plated in 6 well plates in triplicates in DMEM media containing 10% Fetal Bovine Serum (FBS). After one week of incubation in 5% CO 2 , cells were washed with 1x phosphate-buffered saline (PBS) and fixed using 100% methanol. Once fixed, cells were washed again with 1x PBS and stained with 0.5% crystal violet. All the visible colonies were photographed and manually counted, and the average with standard error of mean was plotted using graphpad prism software.

MTT assay
Cell viability was examined by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [1]. Cells were seeded at a density of 4,000 cells/well in triplicate in 96-well plates. Cells transfected with either 0.32μl/well scrambled, miR-489 mimics, or miR-489 Inhibitor by using 0.32μl/well Lipofectamine RNAi and incubated at 37 °C for 72 hr. Cells were seeded at a density of 13,000 cells/well in triplicate in 24-well plates. Cells transfected with either 1μl/well scrambled, miR-489 mimics, or miR-489 Inhibitor by using 1μl/ well Lipofectamine RNAi and incubated at 37 °C for 72 hr. MTT assay was performed according to mention above. At the end of the incubation media was removed and formazan precipitates were dissolved by adding 200 μl of DMSO in each well. OD values were measured at both 570 and 630 nm and final values were calculated by subtracting OD 630 from OD 570 . These values for each time point were used to plot the growth curve for each clone using graphpad prism.

Cell cycle analysis
Cells were harvested using Accutase, washed with cold PBS and fixed with 70% cold ethanol. Cell pellets were then treated with 100 μg/ml RNase A for 15 min at 37°C and stained using 50 μg/ml Propidium Iodide (PI) for 30 min on ice. Samples were analyzed using BD Accuri flow cytometer [3].

Luciferase assay
MCF-7 vect and HER2 cells were seeded at 1.25 x 10 5 cells/well 24 h prior to the transfection in a 24 well plate. Cells were transfected with 500 ng of pGL3 promoter vector containing 3′UTR of HER2 or mutated 3′UTR, 20 ng of pRL-CMV (Renilla luciferase vector-Promega) and 500 ng of miR-489 pcDNA 3.1 vector or empty pcDNA 3.1 vector. After 48 h post transfection, luciferase activity were measured using Dual-luciferase reporter assay system (Promega Cat# E1980) as per manufacturer′s instructions and normalized to the levels of Renilla luciferase activity [5].

miRNA-nanoparticles preparation
For preparing miRNA-nanoparticles, 50 μl of prepared cationic liposomes 28.6 mM (composed of DOTAP/cholesterol) and 16.6 μl of 2 mg/ml protamine were mixed in a final volume of 100 μl nuclease free water (suspension A). The miRNA liposomes were obtained by thoroughly mixing suspension A with 100 μl of 46 μM miRNA (suspension B). The miRNA-liposomes were allowed to stand at room temperature for 10 min. For HA (Hyaluronic Acid) receptors targeting, miRNA-nanoparticles were decorated with sodium hyaluronate by adding 27.7 μl of 2 mg/ml sodium hyaluronate solution to suspension B and kept at room temperature for another 10 min before further application. The resulting miRNAliposomes were used within 20 min for the following experiments or were lyophilized in 30% sucrose for long term storage. The particle size and Zeta potential were measured by dynamic light scattering (DLS) (Malvern Nano-Zs). The sizes of targeted and scramble miRNA liposome were 132.1 ±0.91 and 123.8 ±0.07 nm, respectively (Supplementary Figure S5).

Xenograft experiments
Five to six weeks old athymic female nude mice were purchased from Jackson Laboratories. All mice were handled and maintained under supervision of veterinarian in accordance with institutional guidelines and under a University of South Carolina Institutional Animal Care and Use Committee (IACUC) approved protocol. All mice were subcutaneously injected with 2 x 10 6 HCC1954 in both right and left flank of each mice (n=5/group). The mice tumors were palpated starting day 9 post injection. Tumor volumes were calculated by measuring length and width twice a week for 4 weeks. After 4 weeks of tumor palpation, all mice in the control group were photographed and tumors were extracted after euthanizing all animals. Tumor volumes of each tumor was calculated by modified ellipsoidal formula (1/2(lxw 2 )). Tumor volumes were plotted using graphpad prism software [1,4].
Similarly, for nano-particle delivery experiment five to six week old athymic nude female mice were subcutaneously injected with 2 million wt-HCC1954 cells. The day tumors start palpating, each mice were given intratumoral injection of nano-particle containing either scramble or mimic-miR-489 every 3 days for one month (25 μM/treatment). Tumor volumes were measured, calculated and plotted as mentioned above.