The miR-124-3p/Neuropilin-1 Axis Contributes to the Proliferation and Metastasis of Triple-Negative Breast Cancer Cells and Co-Activates the TGF-β Pathway

Triple-negative breast cancer (TNBC) accounts for 90% of breast cancer-associated mortality. Neuropilin-1 (NRP-1) acts as a non-tyrosine kinase receptor for several cellular signaling pathways involved in the proliferation and metastasis of cancer cells. However, the miRNAs that regulate NRP-1 expression and the underlying mechanisms in TNBC cells remain unclear. In the present study, we found that TNBC cells expressed higher levels of NRP-1 than non-TNBC cells. Stable transfectants depleted of NRP-1 were generated from two TNBC cell lines, human MDA-MB-231 and mouse 4T1 cells. NRP-1 depletion significantly suppressed the proliferation of TNBC cells by arresting the cell cycle at phase G0/G1 by upregulating p27 and downregulating cyclin E and cyclin-dependent kinase 2. NRP-1 depletion also repressed cell migration and epithelial-mesenchymal transition (EMT) by inducing the upregulation of E-cadherin and the downregulation of N-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9, and reducing MMP-2 and MMP-9 activities as detected by gelatin zymography assay. By applying multiple miRNA-target prediction tools, we screened potential miRNAs with binding sites with the 3’-untranslated region of the NRP-1 gene and selected 12 miRNA candidates, among which miR-124-3p displayed the most vigorous activity to downregulate NRP-1 as validated by luciferase assay and miRNA transfection assay. By downregulating NRP-1, miR-124-3p mimics inhibited the proliferation, migration, and invasion of TNBC cells, and antagomiR-124-3p could partially abolish the effects of NRP-1 depletion. In the animal experiments, NRP-1 depletion inhibited tumorigenesis and liver metastasis of TNBC cells, while miR-124-3p mimics inhibited the growth of established TNBC tumors. In the mechanistic exploration, we revealed that NRP-1 co-interacted with transforming growth factor (TGF)-β to activate the TGF-β pathway, which regulates EMT-related molecules. In summary, the present results indicate that the miR-124-3p/NRP-1 axis contributes to the proliferation and metastasis of TNBC cells and co-activates the TGF-β pathway, suggesting that these molecules may present as potential therapeutic targets and valuable biomarkers for TNBC.

[1]  Ping Wang,et al.  miR-124-3p Regulates FGF2–EGFR Pathway to Overcome Pemetrexed Resistance in Lung Adenocarcinoma Cells by Targeting MGAT5 , 2020, Cancer management and research.

[2]  Muhammad Nawaz,et al.  miR-124-3p Suppresses the Invasiveness and Metastasis of Hepatocarcinoma Cells via Targeting CRKL , 2020, Frontiers in Molecular Biosciences.

[3]  Y. Zhang,et al.  Neuropilin1, a novel independent prognostic factor and therapeutic target in triple-negative breast cancer. , 2020, Neoplasma.

[4]  J. S. Lee,et al.  Neoadjuvant Treatment for Triple Negative Breast Cancer: Recent Progresses and Challenges , 2020, Cancers.

[5]  A. Jemal,et al.  Cancer statistics, 2020 , 2020, CA: a cancer journal for clinicians.

[6]  Stephen T. C. Wong,et al.  Differential contributions of pre- and post- EMT tumor cells in breast cancer metastasis. , 2019, Cancer research.

[7]  B. Zhai,et al.  The miR-141/neuropilin-1 axis is associated with the clinicopathology and contributes to the growth and metastasis of pancreatic cancer , 2019, Cancer Cell International.

[8]  L. Saal,et al.  Whole-genome-sequencing of triple negative breast cancers in a population-based clinical study , 2019, Nature Medicine.

[9]  Junjie Zhao,et al.  MicroRNA‐124: An emerging therapeutic target in cancer , 2019, Cancer medicine.

[10]  Yang Zhang,et al.  Neuropilin 1 modulates TGF-β1-induced epithelial-mesenchymal transition in non-small cell lung cancer , 2019, International journal of oncology.

[11]  Hui Wang,et al.  RNA binding protein PUM2 promotes the stemness of breast cancer cells via competitively binding to neuropilin-1 (NRP-1) mRNA with miR-376a. , 2019, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[12]  M. Al Riyami,et al.  Neoadjuvant Chemotherapy Alters Neuropilin-1, PlGF, and SNAI1 Expression Levels and Predicts Breast Cancer Patients Response , 2019, Front. Oncol..

[13]  Y. Bidet,et al.  New Insights into the Implication of Epigenetic Alterations in the EMT of Triple Negative Breast Cancer , 2019, Cancers.

[14]  F. Sato,et al.  Long isoform of VEGF stimulates cell migration of breast cancer by filopodia formation via NRP1/ARHGAP17/Cdc42 regulatory network , 2018, International journal of cancer.

[15]  A. Khan,et al.  miRNA‐124‐3p/neuropilin‐1(NRP‐1) axis plays an important role in mediating glioblastoma growth and angiogenesis , 2018, International journal of cancer.

[16]  M. Bandehpour,et al.  Neuropilin-1 expression is associated with lymph node metastasis in breast cancer tissues , 2018, Cancer management and research.

[17]  D. Mukhopadhyay,et al.  Genetic status of KRAS influences Transforming Growth Factor-beta (TGF-β) signaling: An insight into Neuropilin-1 (NRP1) mediated tumorigenesis. , 2018, Seminars in cancer biology.

[18]  Jun Lu,et al.  Neuropilin‐1 regulated by miR‐320 contributes to the growth and metastasis of cholangiocarcinoma cells , 2018, Liver international : official journal of the International Association for the Study of the Liver.

[19]  M. Al Riyami,et al.  Neuropilin-1 Associated Molecules in the Blood Distinguish Poor Prognosis Breast Cancer: A Cross-Sectional Study , 2017, Scientific Reports.

[20]  B. Zhai,et al.  Dual inhibition of Akt and c‐Met as a second‐line therapy following acquired resistance to sorafenib in hepatocellular carcinoma cells , 2017, Molecular oncology.

[21]  Xinhan Zhao,et al.  VEGF/NRP-1axis promotes progression of breast cancer via enhancement of epithelial-mesenchymal transition and activation of NF-κB and β-catenin. , 2016, Cancer letters.

[22]  Wassim Raffoul,et al.  A Preclinical Model for ERα-Positive Breast Cancer Points to the Epithelial Microenvironment as Determinant of Luminal Phenotype and Hormone Response. , 2016, Cancer cell.

[23]  Linhao Li,et al.  Neuropilin-1 is associated with clinicopathology of gastric cancer and contributes to cell proliferation and migration as multifunctional co-receptors , 2016, Journal of experimental & clinical cancer research : CR.

[24]  S. Estrem,et al.  Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway , 2015, Drug design, development and therapy.

[25]  Zhen Wang,et al.  miRNA-148b suppresses hepatic cancer stem cell by targeting neuropilin-1 , 2015, Bioscience reports.

[26]  C. Betsholtz,et al.  TGF-β1-induced EMT promotes targeted migration of breast cancer cells through the lymphatic system by the activation of CCR7/CCL21-mediated chemotaxis , 2015, Oncogene.

[27]  M. Kerin,et al.  Metastatic breast cancer: the potential of miRNA for diagnosis and treatment monitoring , 2015, Cancer and Metastasis Reviews.

[28]  Prahlad T. Ram,et al.  Functional proteomics identifies miRNAs to target a p27/Myc/phospho-Rb signature in breast and ovarian cancer , 2015, Oncogene.

[29]  Pete E. Pascuzzi,et al.  The Antitumorigenic Function of EGFR in Metastatic Breast Cancer is Regulated by Expression of Mig6 , 2015, Neoplasia.

[30]  B. Zhai,et al.  Inhibition of Akt Reverses the Acquired Resistance to Sorafenib by Switching Protective Autophagy to Autophagic Cell Death in Hepatocellular Carcinoma , 2014, Molecular Cancer Therapeutics.

[31]  Samy Lamouille,et al.  Molecular mechanisms of epithelial–mesenchymal transition , 2014, Nature Reviews Molecular Cell Biology.

[32]  Lianfeng Zhang,et al.  STAT3 interacts with Skp2/p27/p21 pathway to regulate the motility and invasion of gastric cancer cells. , 2013, Cellular signalling.

[33]  J. Balko,et al.  TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer. , 2013, The Journal of clinical investigation.

[34]  J. Norman,et al.  Neuropilin-1-dependent regulation of EGF-receptor signaling. , 2012, Cancer research.

[35]  C. Ling,et al.  MiR-124 suppresses cell proliferation in hepatocellular carcinoma by targeting PIK3CA. , 2012, Biochemical and biophysical research communications.

[36]  Jiri Bartek,et al.  Autocrine VEGF–VEGFR2–Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth , 2012, The Journal of experimental medicine.

[37]  Y. Glinka,et al.  Neuropilin-1 exerts co-receptor function for TGF-beta-1 on the membrane of cancer cells and enhances responses to both latent and active TGF-beta. , 2011, Carcinogenesis.

[38]  Pedagógia,et al.  Cross Sectional Study , 2019 .

[39]  J. Slingerland,et al.  p27: A Barometer of Signaling Deregulation and Potential Predictor of Response to Targeted Therapies , 2010, Clinical Cancer Research.

[40]  S. Dent The role of VEGF in triple-negative breast cancer: where do we go from here? , 2009, Annals of oncology : official journal of the European Society for Medical Oncology.

[41]  R. Crystal,et al.  A SNAIL1–SMAD3/4 transcriptional repressor complex promotes TGF-β mediated epithelial–mesenchymal transition , 2009, Nature Cell Biology.

[42]  Y. Glinka,et al.  Neuropilin-1 is a receptor for transforming growth factor β-1, activates its latent form, and promotes regulatory T cell activity , 2008, Journal of leukocyte biology.

[43]  R. Weinberg,et al.  Tumour invasion and metastasis initiated by microRNA-10b in breast cancer , 2007, Nature.

[44]  D. Bartel MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[45]  G. Watkins,et al.  The Hepatocyte Growth Factor Regulatory Factors in Human Breast Cancer , 2004, Clinical Cancer Research.

[46]  W. Birchmeier,et al.  Met, metastasis, motility and more , 2003, Nature Reviews Molecular Cell Biology.

[47]  J. Balko,et al.  TGF-b inhibition enhances chemotherapy action against triple-negative breast cancer , 2018 .

[48]  Xin-Yun Huang,et al.  Mouse models for tumor metastasis. , 2012, Methods in molecular biology.

[49]  N. Dubrawsky Cancer statistics , 1989, CA: a cancer journal for clinicians.