MiR-200c restoration inhibits FOXP3 and metastatic spread in breast cancer: evidence from in vitro and in vivo models

Abstract

Background: Metastatic breast cancer remains a leading cause of cancer-related mortality in women, often driven by molecular pathways that promote invasion and immune evasion. MicroRNA-200c (miR-200c) is a known tumor suppressor that inhibits epithelial-mesenchymal transition (EMT), while FOXP3, a transcription factor typically associated with regulatory T cells, is aberrantly expressed in breast cancer cells and may contribute to tumor progression. This study investigates whether targeting the miR-200c/FOXP3 axis can suppress metastasis in breast cancer.

Methods: Metastatic (MDA-MB-361, MDA-MB-468) and non-metastatic (MCF-7) breast cancer cell lines were transfected with miR-200c mimic or inhibitor. Cell proliferation, apoptosis, and invasion were assessed using MTT, Annexin V/PI staining, and transwell assays. FOXP3 mRNA and protein levels were quantified using qRT-PCR and immunohistochemistry. A metastatic mouse model was established via intracardiac injection of tumor cells, followed by treatment with miR-200c mimic, inhibitor, or Cisplatin.

Results: MiR-200c overexpression significantly suppressed proliferation and invasion and enhanced apoptosis in metastatic cells. FOXP3 mRNA and protein expression were downregulated in mimic-treated cells and tissues, while miR-200c inhibition led to increased FOXP3 expression. In vivo, miR-200c mimic treatment reduced tumor burden and metastatic infiltration in the brain and lungs. A strong inverse correlation between miR-200c and FOXP3 was observed (r = - 0.82, p < 0.01).

Conclusion: MiR-200c restoration inhibits FOXP3 and suppresses metastatic progression in breast cancer. Targeting the miR-200c/FOXP3 axis presents a novel and promising therapeutic approach for advanced breast cancer.

Keywords: Breast cancer; Cell invasion; FOXP3 transcription factor; Gene expression regulation; MicroRNA-200; Neoplasm metastasis; Neoplastic.

Fig. 1

Regulatory networks involving miR-200 family in breast cancer: (a) shows the chromosomal locations of the miR-200b/200a/429 cluster, which is located on chromosome 1p36.33. A schematic graph shows high levels of miR-200b/200a/429 in non-metastatic BC, which is attributed to the suppression of FOXP3 and the release of extracellular vesicles (ECVs) derived from tumor cells that are abundant in miR-200 clusters, which further promotes BC progression. b: shows the chromosomal locations of miR-200c/141 cluster is located on chromosome 12p13.31. A schematic graph showing low expression of miR-200c/141 in metastatic BC, which subsequently activates FOXP3. In addition, ECVs and CTC are released from tumor cells and localized in distant locations to induce metastases via EMT. Meanwhile, FOXP3 directly activates miR-200c, and in vitro stimulation of miR-200c inhibits EMT, progression, and metastases by reducing the expression of FOXP3. On the other hand, higher levels of miR-200c inhibit FOXP3 “TSG” and promote progression and EMT. Abbreviations: BC: breast cancer, Ch. Chromosome, TSG: tumor suppressor gene, CTC: circulating tumor cells, ECVs: extracellular vesicles, EMT: Epithelial to mesenchymal transition, green arrow: stimulation, red arrow: inhibition

Fig. 2

Restoration of miR200c inhibits non-metastatic and metastatic breast cancer cell proliferation and migration. a Effect of miR-200c modulation on viability of breast cancer cells, (b) Light microscope images of migrated cells, “cell migration assay,” stained with crystal violet, showed a significant reduction in the migration in BC cells transfected with miR-200c mimic. However, a significant increase in migrated cells was detected in BC cells after transfection with miR-200c inhibitor. c Effects of transfection of BC cells with miR-200c mimic and inhibitor on cell invasion measured by Boyden chamber invasion assay. The sample is performed in triplicate to ensure the accuracy of the test. Values represent the mean of three experiments ± Standard deviation, **, p-value < 0.01, *, p-value < 0.05. Comparative analysis was performed by One-Way ANOVA, followed by Tukey’s multicomparative test. Abbreviations: BC: breast cancer

Fig. 3

Effect of transfection with miR-200c mimic on apoptosis of MDA-MB361 and MCF7 cell lines (a) Images of apoptosis assay by Annexin V/propidium iodide following transfection of miR-200c mimic, measured by Flowcytometry. b Percent of viable, early apoptotic, and necrotic cells. Values represent the mean of three experiments ± Standard deviation of three independent measurements for each group. * indicates significant difference between compared groups (p < 0.05).Multiple group cmparisions was performed by One-Way ANOVA, followed by Tukey’s multicomparative test. Abbreviations: PI: propidium iodide, BC: breast cancer, PI: Propidium iodide

Fig. 4

Effect of transfection with miR-200c mimic on the expression of miR-200c-5p and its target gene “mRNA-FOXP3”, measured by Syber green-based Real-time PCR. Values represent the mean of three experiments ± Standard deviation in three tested BC cell lines. a and b: miR-200c-5p and mRNA-FOXP3 expression (FC) after transfection with miR-200c mimic or inhibitor, compared to untreated cells. Values represent the mean of three independent measurements for each experiment ± Standard deviation. *: denotes a significant difference between the compared groups (p < 0.05). Statistical significance was determined using a one-way ANOVA followed by Tukey’s post-hoc test. c Pearson’s correlation between the expression of miR-200c and FOXP3 in different groups. Abbreviations: BC: breast cancer, FC: fold change, r: correlation coefficient, CI: confidence interval

Fig. 5

Restoration of miR-200c decreases breast cancer xenograft tumor growth. a: present the tumor weight measured in tumors transfected with miR-200c mimic or inhibitors, compared to untreated tumor tissue. b: displays images stained with Hematoxylin and Eosin of breast cancer at both magnifications x100 and x400, along with breast cancer tumor size and immunohistochemical staining of forkhead box protein 3 (FOXP3) in breast cancer. c: FOXP3 expression is illustrated in representative images with nuclear and cytoplasmic localization at magnification x100, with the immunoreactive score (IRS) of each group presented on the respective image. Values represent the mean of three independent measurements for each experiment ± Standard deviation.*: denotes significance between the compared groups (p < 0.05). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test to determine differences among groups. Abbreviations: H&E: Hematoxylin and eosin, FOXP3: Forkhead box protein 3, IRS: immunoreactive score

Fig. 6

Restoration of miR-200c inhibits FOXP3 gene expression in breast cancer xenograft tumors. The expression level of miR-200c-5p (a) and FOXP3 (b) in tumor tissue was measured by RT-PCR. c: association between miR-200c-5p and mRNA-FOXP3 gene expression in tumor tissue. Values represent the mean of three experiments ± Standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test.*: Denotes significant difference between compared groups (p < 0.05)