miR-200c/141 Regulates Breast Cancer Stem Cell Heterogeneity via Targeting HIPK1/β-Catenin Axis

Abstract

Increasing evidence demonstrates the existence of two inter-convertible states of breast cancer stem cells (BCSCs) with distinct behaviors in proliferation and mobility, and the BCSC heterogeneity is accurately regulated by sophisticated mechanisms including microRNAs. The microRNA-200 family including miR-200c/141 cluster was reported to affect cancer cell invasion and metastasis by regulating epithelial to mesenchymal transition (EMT). However, the effect of miR-200 family on BCSC heterogeneity is uncertain. Thus, we investigated whether the miR-200c/141 cluster had different effects on breast tumor growth and metastasis by switching the two states of BCSC. Methods: The spontaneous mammary tumor mouse model with miR-200c/141 conditional knockout was utilized for analyzing the role of miR-200c/141 cluster in vivo. The effect of miR-200c/141 cluster on BCSCs was performed by CD24/CD29 staining and ALDEFLUOR assay. miR-200c/141 target expression and EMT-related marker expression were verified in tumor sections, primary cells and breast cancer cell lines by qRT-PCR or western blotting. Statistical analysis was determined using two-way ANOVA and Student's t-test. All values were presented as the mean ± s.e.m. Results: The deletion of miR-200c/141 cluster regulated BCSC heterogeneity and promoted the EMT-like BCSC generation, which resulted in increased tumor metastasis and inhibited tumor growth by directly upregulating the target gene homeodomain-interacting protein kinase 1 (HIPK1) and sequential β-catenin activation. Conclusions: Our results indicated that miR-200c/141 played biphasic roles in breast tumor progression via affecting the BCSC heterogeneity, suggesting targeting BCSC heterogeneity to simultaneously restrict breast cancer initiation and metastasis could be a promising therapeutic strategy for breast cancer.

Keywords: HIPK1; breast cancer; cancer stem cell; heterogeneity; miR-200c/141.

Figure 1

Deletion of miR-200c/141 cluster inhibits mammary tumor formation. (A) Schematic view of cross-breeding among MMTV-PyMT, MMTV-Cre transgenic mice and mice carrying loxP sites flanking the miR-200c/141fl/fl, and the generation of spontaneous breast tumor mice with mammary-specific deletion of miR-200c/141 cluster (miR-200c/141-/-; PyMT). (B) Kinetics of mammary tumor onset in MMTV-PyMT female mice of indicated genotypes. (log-rank test; WT: n=40,cKO:n=41) *p < 0.05. (C) The percentage of tumor-free mammary glands at indicated ages and indicated genotypes of mice. (D) Total tumor burden in WT and cKO cohorts evaluated at 15 weeks in the same mice as in (C). *p < 0.05. (E) The representative images for Ki67 IHC staining in tumor sections and quantification of Ki67 positive cells per tumor section in the same cohort of mice from (C). ***p < 0.001

Figure 2

Deletion of miR-200c/141 cluster increases mammary tumor lung metastasis and induces EMT. (A) Images of lungs from the same cohort of mice as shown in Figure 1B. Red arrows represent metastatic nodules in lungs. (B) HE staining of lung sections from (A) and the numbers of metastatic lesions per lung section were counted. ***p < 0.001. (C) The representative images for Ki67 IHC staining in lung sections from WT or cKO mice and quantification of Ki67 positive cells. ***p < 0.001. (D) FACS was performed to detect the effect of miR-200c/141 cluster on breast cancer stem cells (BCSCs) with CD24/CD29 and ALDEFLUOR assay. (E) E-cadherin (E-cad) and Vimentin (Vim) expression was verified by Immuno- fluorescence staining in tumor sections with indicated genotypes. (F) E-cadherin (E-cad), Vimentin (Vim) and Zeb1 protein immunoblotting in freshly lytic tumors and primary cells from the mice with indicated genotypes.

Figure 3

Inhibition of miR-200c and miR-141 results in reduced cell proliferation and increased cell invasion in vitro by regulating BCSC plasticity. SUM149 and T47D cells were treated with 200nM miR-200c and miR-141 inhibitors (miR200c- 141inh) or negative control inhibitor (NC) for 48h. (A) Treated SUM149 or T47D cells were accessed for proliferation ability which was performed with MTT assay as described in methods. **p<0.01. (B-C) The plate colony formation assay was carried out with 500 treated SUM149 (B) or T47D(C) colonies were counted in the whole field for statistics. **p<0.01. (D-E) The invasive ability of treated SUM149 (D) or T47D (E) cells was investigated with the transwell assay according to the manufacturer's protocol. Quantitative analysis was accessed for the total invasive cells from three independent experiments. *p<0.05, **p<0.01. (F-G) Flow cytometry analysis of BCSCs was carried out for the CD24/CD44 and ALDEFLUOR assay in treated SUM149 (F) and T47D (G) cells. (H) After being treated for 48 hours, cells were harvested to detect the protein expression levels of E-cadherin (E-cad), Snail, Vimentin (VIM) and ZEB1 by Western Blotting.

Figure 4

HIPK1 is a direct and functional target of miR-200c and miR-141, and is correlated with breast cancer malignancy. (A) Schematic diagram of genes targeted by both of miR-200c and miR-141 were analyzed based on miRBase and targetScan databases. (B) Candidate target genes were confirmed with qRT-PCR assay in both tumor and primary cells from miR-200c/141 cKO mice. (C) The protein expression levels of HIPK1 and YWHAG were detected by Western Blotting. (D) mRNA expression levels of HIPK1 and YWHAG were confirmed with qRT-PCR assay in SUM149 treated with miR200c and miR141 inhibitors or negative control. **p<0.01. (E) Protein expression levels of HIPK1 and YWHAG were confirmed by Western Blotting in SUM149 and T47D cells treated with indicated inhibitors. (F) Predicted miR-200c and miR-141 binding region sequence in 3'UTR of HIPK1. (G) The potential miR-200c and miR-141 binding regions in 3'UTR of HIPK1 were confirmed with luciferase reporter assay. The relative luciferase activity is defined as the value of activity of luciferase gene folded over internal control. **p<0.01. (H) The Kaplan-Meier survival curve of breast cancer patients (log-rank test, ***p < 0.001), data from TCGA. (I) The HIPK1 protein level in clinical breast tumor tissues and paratumor tissues was shown by immunohistochemistry (IHC) (Brown: HIPK1). Bar, 200um. (J) Quantification of HIPK1-positive cells ratio in IHC staining sections of breast cancer patient tissues from (I). ***p<0.001

Figure 5

HIPK1 knockdown rescues the effects of miR-200c/141 inhibition on breast cancer cells. SUM149 and T47D cells were transfected with HIPK1-shRNA lentivirus and then treated with miR-200c/141 inhibitors. (A) Protein expression level of HIPK1, E-Cadherin (E-cad), Snail and Vimentin (VIM) were detected by Western Blotting. Samples were harvested after cells had been treated for 48 hours. (B-C) MTT assay was performed with treated SUM149 (B) and T47D (C) cells for cell proliferation ability. *p<0.05, **p<0.01. (D-E) The invasive ability was investigated with treated SUM149 (D) or T47D (E) cells by transwell assay. Quantitative analysis of the total invasive cells from three independent experiments. ***p<0.001 (F-G) The heterogeneity of BCSCs was analyzed by Flow cytometry with two sets of markers (CD24-CD44+ and ALDH+) in treated SUM149 (F) and T47D (G) cells.

Figure 6

miR-200c/141 cluster regulates breast cancer metastasis and BCSCS states through β-catenin activity. (A) The downregulated genes from primary tumors of cKO PyMT mice compared to WT PyMT mice were applied to Gene Set Enrichment Analysis (GSEA). (B) The transcriptional activity of β-catenin was measured in both miR-200c/141-inhibited and HIPK1-overexpressed cells by TOP/FOP luciferase assay. (C) The activated β-catenin (non-phosphorylation at ser33/37/thr41 sites) and Phosphorylation of β-catenin in ser552 site in both miR-200c/141-inhibited and HIPK1- overexpressed cells as well as in the tumors from miR-200c/141 knockout mice were measured by Western Blotting. (D) Rescue experiment was performed in miR-200c/141 and HIPK1 double knockdown cell lines by Western Blotting. (E-G) The cell proliferation ability was measured for HIPK1-overexpressing SUM149 cells in the absence or presence of ICG-001 (10uM, 24h) by the MTT assay (E) and plate colony formation assay (F) and the cell invasive ability was tested by transwell assay (G). Quantitative analysis of the cells was done from three independent experiments. ICG- 001: Wnt/β-catenin pathway inhibitor. (H-I) Flow cytometry analysis of BCSCs was carried out for the CD24/CD44 analysis and ALDEFLUOR assay in SUM149 cells.