miR‐200c suppresses stemness and increases cellular sensitivity to trastuzumab in HER2+ breast cancer

Abstract Resistance to trastuzumab remains a major obstacle in HER2‐overexpressing breast cancer treatment. miR‐200c is important for many functions in cancer stem cells (CSCs), including tumour recurrence, metastasis and resistance. We hypothesized that miR‐200c contributes to trastuzumab resistance and stemness maintenance in HER2‐overexpressing breast cancer. In this study, we used HER2‐positive SKBR3, HER2‐negative MCF‐7, and their CD44+CD24− phenotype mammospheres SKBR3‐S and MCF‐7‐S to verify. Our results demonstrated that miR‐200c was weakly expressed in breast cancer cell lines and cell line stem cells. Overexpression of miR‐200c resulted in a significant reduction in the number of tumour spheres formed and the population of CD44+CD24− phenotype mammospheres in SKBR3‐S. Combining miR‐200c with trastuzumab can significantly reduce proliferation and increase apoptosis of SKBR3 and SKBR3‐S. Overexpression of miR‐200c also eliminated its downstream target genes. These genes were highly expressed and positively related in breast cancer patients. Overexpression of miR‐200c also improved the malignant progression of SKBR3‐S and SKBR3 in vivo. miR‐200c plays an important role in the maintenance of the CSC‐like phenotype and increases drug sensitivity to trastuzumab in HER2+ cells and stem cells.

families of breast cancer patients. 8 To overcome resistance and improve the efficacy of trastuzumab treatment in HER2-overexpressing breast cancer patients, we must perform a thorough inquiry into the mechanisms of trastuzumab resistance and develop new effective treatment programmes.
A variety of potential molecular mechanisms of resistance to trastuzumab have previously been published, [9][10][11][12] and the vast majority involve the biological functions of breast cancer stem cells (BCSCs). 12,13 Cancer stem cells (CSCs) are a rare fraction of tumour cells that have the abilities of self-renewal, unlimited proliferation and multi-potent differentiation. 14 Like normal tissues, tumour tissues are composed of a variety of heterogeneous tumour cells and originate from corresponding stem cells. CSCs have been isolated from a variety of tumours, such as leukaemia, lung cancer and breast cancer. [15][16][17] There are two recognized methods of separating BCSCs from breast cancer patients and cell lines: one is surface phenotypic marker screening, and the other is Hoechst 33 342 dye exclusion. CD44 + CD24 − is a well-known surface maker for the isolation and identification of BCSCs in breast cancer tissues and cell lines. 17,18 There is mounting evidence that CSCs are responsible for tumour formation, infinite growth, recurrence and metastasis. CSCs have congenital resistance features. Conventional drug therapy, including chemotherapy drugs, radiotherapy drugs and targeted therapy drugs, can kill only the active non-stem cells, whereas residual CSCs can eventually lead to tumour recurrence and metastasis. CSCs are the root cause of drug resistance and treatment failure. 19,20 The mechanisms of drug resistance in CSCs include overexpression of ATP-binding cassette transporters, over-activation of cell detoxification enzymes, abnormal activation of cell survival and apoptosis-related signal transduction pathways, the protective effect of tumour niches on tumour stem cells, and that most CSCs are in a quiescent phase. [21][22][23] By intervening in these processes, we may reverse resistance to trastuzumab and improve the survival and prognosis of breast cancer patients.
MicroRNAs (miRNAs) are a class of endogenous small RNAs that regulate many crucial biological processes in cancer pathogenesis and progression. miRNAs are highly conserved and specific. By binding to the 3′-untranslated region (UTR) of target messenger RNA (mRNAs), miRNAs can regulate gene expression by inhibiting translation and inducing degradation of mRNAs. 24 miRNAs act as either oncogenes or tumour suppressors in cancer management. 25  Several studies associated miR-200c and its target mRNAs with the establishment, maintenance and regulation of the CSC phenotype.
Down-regulation of miR-200c is relevant for stem cell functions in cancer, including self-renewal, clonal expansion and differentiation. 27 miR-200c inhibits tumour growth, differentiation and selfreplication of CSCs by targeting TUBB3 and restoring sensitivity to microtubule-targeting drugs. 28 Members of the miR-200 family control the EMT process and sensitivity to EGFR therapy in bladder cancer. 29 Down-regulation of miR-200c expression is a marker of tumour invasion and drug resistance in female genital tumours, such as ovarian cancer, cervical cancer and breast cancer. 30 Therefore, exploring the role of miR-200c in BCSCs may help us understand the mechanism of drug resistance.
Increasing evidence suggests that the Notch, Wnt and Hedgehog signal transduction pathways are the most important pathways in stemness maintenance and drug resistance of CSCs. 31,32 The Notch family consists of transmembrane signal proteins, and their main function is to determine the direction of cell development. Notch is highly expressed in tissue stem cells and precursor cells. Notch promotes stem-like mammosphere formation and increases the recurrence of dormant tumour cells following HER2/neu-targeted therapy of BCSCs. 33,34 Abnormal activation of the Wnt pathway interferes with the survival, selfrenewal, proliferation and drug insensitivity of BCSCs, causes an increase in the proportion of stem cells, induces a conversion from mammary stem cells into BCSCs, and ultimately results in the recurrence and malignant progression of breast cancer. 35,36 The Hedgehog signalling pathway is highly activated in BCSCs, and inhibition of the Hedgehog pathway significantly inhibits the proliferation of CSCs and enhances their sensitivity to drugs. 37,38 In addition, Jagged1, ZEB1 and Bmi1 are highly critical genes in these three signalling pathways. They are the direct target genes of the miR-200 family in many cancers, including breast cancer. [39][40][41] Based on the above, BCSCs are the key factor responsible for trastuzumab resistance in HER2-overexpressing breast cancer. miR-200c may suppress stemness and increase the sensitivity to trastuzumab in HER2+ breast cancer cells and stem cells.
The study of the mechanism may be helpful for targeting BCSCs that are not successfully killed by trastuzumab and may improve the survival and prognosis of HER2-overexpressing breast cancer patients.  The spheres were counted and collected 5 days after being planted.

| Clinical samples
They were mechanically dissociated with a 25 G needle (BD) and reseeded in fresh medium.

| Mammosphere formation assay
Cells were plated in 6-well plates for 24 hour and transfected. After incubation for 48 hour, cells were harvested and plated at 10 4 cells/ well in ultra-low attachment six-well plates in serum-free DMEM/ F12 supplemented with B27 (1:50), EGF (20 ng/mL), bFGF (10 ng/ mL) and insulin (10 mg/mL). Mammospheres were counted after 5 days of culture using a Nikon Eclipse TE2000-S microscope (Japan) and photographed with MetaMorph.

| RNA extract and qRT-PCR
Total RNA from cells was extracted with TRIzol reagent (Invitrogen) following the manufacturer's protocol. Reverse transcription and quantitative real-time PCR analysis (qRT-PCR) reactions were performed using a qSYBR-Green-containing PCR kit (Qiagen) and a Bio-Rad IQTM5 Multicolor Real-Time PCR Detection System. The primers for Jagged1, ZEB1 and Bmi1 mRNA for qRT-PCR detection were synthesized by Invitrogen. Each experiment was performed three times in triplicate.

| Protein isolation and western blotting
Protein was extracted from cells lines using RIPA lysis buffer with proteinase inhibitors (Thermo). The Protein BCA Assay Kit (Thermo) was used to measure the concentration of protein in the lysates.
Each lane was loaded with 20 µg protein mixed with 2× SDS loading buffer (Thermo). The protein was separated by 12% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were incubated with 5% skim milk powder at room temperature for 1 hour to block non-specific binding. Subsequently, the membranes were incubated with antiserum containing antibodies against Jagged1, ZEB1 and Bmi1 (CST), at 4°C for 12 hour. To visualize the target proteins, a 1:5000 dilution of peroxidase-conjugated secondary antibody and ECL Western blotting detection reagents (GE) were used. A Bio Image Intelligent Quantifier 1-D was used to quantify the proteins (Version 2.2.1, Nihon-Bio Image Ltd). An anti-GAPDH antibody was used as a protein loading control (Boster).

| Apoptosis rate determined by flow cytometry
SKBR3 and SKBR3-S were divided into five groups as in the MTT assay. Cells at 5 × 10 5 cells/mL were inoculated into 6-well culture plates and incubated at 37ºC. After culture for 72 hour, cells were collected after digestion with 0.25% trypsin, washed with PBS three times and resuspended in 500 mL binding buffer (10 mmol/L HEPES/ NaOH, 140 mmol/L NaCl, 2.5 mmol/L CaCl 2 , PH 7.4). FITC-labelled Annexin V (50 mg/mL, 5 mL) and PI (50 mg/mL, 5 mL) were added, followed by incubation at room temperature in the dark for 30 minutes.
The apoptosis rate was immediately measured by flow cytometry.

| Sort BCSCs by flow cytometry
Serum-free suspension culture stem cell spheres and cells were collected and rinsed twice with PBS. Then, cells were stained with phycoerythrin-conjugated anti-human CD24 antibody (Invitrogen) and FITC-conjugated anti-human CD44 antibody (Invitrogen) for 30 minutes at room temperature according to the manufacturer's instructions. The labelled cells were then analysed using a FACSCalibur flow cytometer and Cell Quest software (BD).

| Statistical analysis
Each experiment was repeated three times, and the results are presented as the mean ± SE. The statistical analysis was performed using Student's t test and Pearson correlation analysis. Fisher's exact test was used to analyse the significance of in vivo experiment results. The significance level was set at P < .05. All statistical analyses were performed using SPSS 17.0 software.  Figure 1C,D). The expression of miR-200c was much lower in CD44 + CD24 − phenotype SKBR3-S than in the suspension microspheres SKBR3-SM ( Figure 1C). However, there were no significant differences between the expression of miR-200c in MCF-7-S and MCF-7-SM ( Figure 1D).

| Overexpression of miR-200c resulted in a decrease in the number of tumour spheres and proportion of CD44 + CD24 − phenotype cells in HER2+ BCSCs
To   Figure 3A). Next, we assessed the proportion of CD44 + CD24 − phenotype cells and found that the proportion was also noticeably decreased in SKBR3-S but not MCF-7-S cells after treatment with 10 μg/mL trastuzumab ( Figure 3B). Images of tumour spheres

| miR-200c significantly inhibited tumour growth and metastasis of HER2+ breast cancer cells and stem cells in vivo
To explore the role of miR-200c for BCSC function in vivo, we per-

| Jagged1, ZEB1 and Bmi1 were highly expressed and positively related with each other in clinical specimens from breast cancer patients
All tissue samples in this study were collected from 50 breast cancer patients. The breast cancer tissues (Breast cancer) and their matched adjacent tissues (Normal) were removed during surgery and immediately stored in RNA later for RNA extraction and subsequent qRT-PCR assay. qRT-PCR results revealed that Jagged1, ZEB1 and Bmi1 mRNAs were up-regulated in breast cancer tissues compared with matched adjacent tissues ( Figure 5A).
Then, we explored the correlation between Jagged1, ZEB1 and Bmi1 expression using Pearson correlation analysis. The correlation coefficient between Jagged1 and ZEB1 was .731 (linear R 2 = .534), between Jagged1 and Bmi1 was .362 (linear R 2 = .131), and between ZEB1 and Bmi1 was .397 (linear R 2 = .157), indicating that the expression levels of Jagged1 and ZEB1 were more strongly related than those of Jagged1 and Bmi1 or ZEB1 and Bmi1 (2-tailed, Figure 5B,C).

| D ISCUSS I ON
Trastuzumab has significantly improved the overall survival of HER2overexpressing breast cancer patients. However, more than 70% of trastuzumab-treated patients have experienced disease progression or relapse in past years. Inherent and acquired resistance to chemotherapy, radiotherapy or targeted therapy among the CSC population can be used to explain why conventional therapies fail and tumour relapses occur. Because of insufficient targeting to BCSCs, trastuzumab treatment finally fails. 43 can control the EMT process and sensitivity to EGFR therapy in bladder cancer. 29 We hypothesized that miR-200c can suppress stemness and increase the cellular sensitivity to trastuzumab in HER2+ breast cancer cells and stem cells via Jagged1, ZEB1 and Bmi1. in the Notch, Wnt and Hedgehog pathways that induce pluripotent F I G U R E 3 miR-200c reduced the stemness and increased the sensitivity to trastuzumab in HER2+ breast cancer cells and stem cells. A, The number and diameter of tumour spheres formed per 100 cells from SKBR3-S and MCF-7-S cells. SKBR3-S and MCF-7-S cells were treated with trastuzumab at 0 or 10 μg/mL. Trastuzumab was administered for 3 d and refreshed trastuzumab every 24 h. The spheres were counted for 10 fields in each group. Trastuzumab treatment increased the number and diameter of tumour spheres formed per 100 cells from SKBR3-S but not MCF-7-S 72 h after trastuzumab treatment (n = 3, *P < .05). B, Flow cytometric profiles of CD44 + CD24 − phenotype cells for SKBR3-S and MCF-7-S cells after 0 or 10 μg/mL trastuzumab treatment. The proportion was noticeably decreased in the 10 μg/mL trastuzumab treatment group of SKBR3-S but not MCF-7-S cells (n = 3, *P < .05). C, Representative images showing tumour spheres formed from SKBR3-S and MCF-7-S cells with 0 or 10 μg/mL trastuzumab treatment (magnification of 100x). D, qRT-PCR was used to evaluate the expression of miR-200c in SKBR3, SKBR3-S, MCF-7 and MCF-7-S after 0 or 10 μg/mL trastuzumab treatment (n = 3, *P < .05). E, MTT assay was used to assess the proliferation of SKBR3 and SKBR3-S in different groups. SKBR3 and SKBR3-S cells were divided into five groups: Mock (blank control), Trastuzumab ( Then, we treated SKBR3-S and MCF-7-S cells with trastuzumab at 0 or 10 μg/mL. The results showed that the number and diameter of tumour spheres formed and the proportion of CD44 + CD24 − phenotype cells from SKBR3-S were noticeably decreased. The expression of miR-200c was noticeably increased in the 10 μg/ F I G U R E 4 miR-200c significantly inhibited tumour growth and metastasis of HER2+ breast cancer cells and stem cells in vivo. A, Images of subcutaneous tumours from SKBR3-S and MCF-7-S cells that were stably transfected with lentiviral vector coding miR-200c or control. B, Tumour volume in mouse xenograft models. SKBR3-S and MCF-7-S cells expressing the indicated constructs were injected subcutaneously into BALC/c mice (five in each group). The tumour volume was measured every four days. miR-200c significantly reduced the volume of tumours from SKBR3-S cells (*P < .05 vs Control), whereas there was almost no decrease in the volume of tumours from MCF-7-S cells. C, Tumour weight in mouse xenograft models. After 28 d, the mice were killed, necropsies were performed, and the tumours were weighed. miR-200c significantly reduced the weight of tumours from SKBR3-S cells (*P < .05 vs Control), whereas there was almost no decrease in the weight of tumours from MCF-7-S cells. D, Lung tissues and their HE staining images from SKBR3, SKBR3-S, MCF-7 and MCF-7-S cells that were stably transfected with lentiviral vector coding miR-200c or control(*P < .05 vs Control) F I G U R E 5 Jagged1, ZEB1 and Bmi1 were highly expressed and positively related with each other in clinical specimens from breast cancer patients. A, qRT-PCR was used to detect the expression of Jagged1, ZEB1 and Bmi1 mRNAs in breast cancer patients (*P < .05 vs Normal). B, Simple scatter plot of the Pearson correction analysis between the expression of Jagged1, ZEB1 and Bmi1 mRNAs in breast cancer patients. C, The expression levels of Jagged1 and ZEB1 were more strongly related than those of Jagged1 and Bmi1 or ZEB1 and Bmi1 (2-tailed) In conclusion, these results are consistent with our hypothesis that miR-200c reduces stemness and increases sensitivity to trastuzumab in HER2+ breast cancer cells and stem cells via inhibition of Jagged1, ZEB1 and Bmi1, which are three important genes in the stemness-related Notch, Wnt and Hedgehog pathways. However, many molecular mechanisms remain unclear. We will continue to further explore the detailed molecular mechanisms and biological and pathological functions of miR-200c in increasing drug sensitivity to trastuzumab via these three pathways.

ACK N OWLED G EM ENTS
We are grateful to all the SYSUCC colleagues, especially Dr Bo Chen and Xiaojia Huang, for their permanent support, inspiration, constructive discussions and critical reading of the manuscript. F I G U R E 6 Mechanisms of trastuzumab resistance in HER2-overexpressing breast cancer. By directly targeting Jagged1, ZEB1 and Bmi1, miR-200c co-ordinates the stemness-related Notch, Wnt and Hedgehog signalling pathways. Finally, miR-200c affects stem cell properties and drug resistance in HER2-overexpressing breast cancer