Study of the effects of miR-200 overexpression and LOXL4 downregulation on triple-negative breast cancer progression : miR-200의 과발현과 LOXL4 발현 억제가 삼중음성 유방암 진행에 미치는 영향에 관한 연구

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의과대학 의과학과
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서울대학교 대학원
Triple-negative breast cancer (TNBC)microRNA-200 (miR-200)vascular endothelial growth factor (VEGF)Lysyl oxidase-like 4 (LOXL4)CollagenTumor progressionOverall survival
학위논문 (박사)-- 서울대학교 대학원 의과대학 의과학과, 2017. 8. 문우경.
Introduction: The microRNA-200 (miR-200) family is known to have a pivotal role in regulating epithelial to mesenchymal transition through the suppression of transcription factors of E-Cadherin, ZEB1, and ZEB2, but the role of miR-200 family in the migration and invasion of breast cancer cells is controversial. The lysyl oxidase-like 4 (LOXL4), a member of the lysyl oxidase (LOX) family, is involved in extracellular matrix (ECM) modulation and signaling pathway related to cancer cell growth and survival. However, the role of LOXL4 in breast cancer progression and metastasis is still unclarified. This study investigated the mechanisms by which the miR-200 family modulated the migratory and invasive abilities of aggressive triple-negative breast cancer (TNBC) cells, which have the worst prognosis among breast cancer subtypes. In addition, the role of LOXL4 in tumor formation and metastasis in TNBC was investigated and overall survival (OS) was analyzed to evaluate its clinical significance as a prognostic factor in TNBC patients.

Methods: The miR-200 family (miR-200b/200a/429 and miR-141/200c clusters) coupled with green fluorescence protein (GFP), the LOXL4 shRNA (shLOXL4) coupled with red fluorescence protein (RFP) and the firefly luciferase (Luc) coupled with GFP were transduced into MDA-MB-231 cells, TNBC cells, using a lentiviral system. Gene expression was evaluated using real-time polymerase chain reaction (PCR) or reverse transcriptase-PCR (RT-PCR). The migratory and invasive abilities were assessed using trans-well or wound-healing assays. The secreted cytokines and growth factors from cells were quantified using a Bio-Plex 200 multiplex array system. Western blot and immunofluorescence staining were conducted to investigate the signaling pathways. The xenograft tumor models were produced by injection with breast cancer cells into the mammary gland or tail vein of 6-week old female Balb/c nude mice for an orthotopic or lung metastatic model, respectively. Primary tumor volumes were measured by a digital caliper. The primary tumor in mammary gland and metastatic lung burden were obtained using IVIS and Maestro imaging system. Histological analysis was assessed by H&E, Picrosirius red, Massons trichrome and immunostaining. Second harmonic generation (SHG) imaging was conducted to quantify the collagen fiber lengths, straightness, and widths. A public database (BreastMark) for overall survival (OS) was used to examine the prognostic value of the LOXL4, collagen I, and collagen IV genes in breast cancer patients.

Results: The overexpression of the miR-200b/200a/429 or miR-141/200c cluster suppressed cell growth, but significantly increased migration and invasion of MDA-MB-231 cells, and led to an increase in the phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT). Chemical inhibitors of FAK and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT suppressed the migration and invasion of MDA-MB-231 cells which was enhanced by the overexpression of the miR-200b/200a/429 or miR-141/200c cluster. Compared to the miR-200b/200a/429 cluster-transduced MDA-MB-231 cells, the miR-141/200c cluster-transduced MDA-MB-231 cells exhibited a significant increase in vascular endothelial growth factor (VEGF)-A secretion and integrin-alphaV (integrin-αV) expression. Treatment with an anti-VEGF-A-neutralizing antibody inhibited the increase in migration and invasion and significantly reduced the phosphorylation of FAK and AKT in the miR-200b/200a/429- or miR-141/200c-transduced MDA-MB-231 cells. In the mouse xenograft models, LOXL4 knockdown increased the primary tumor growth and lung colonization in MDA-MB-231 cells. LOXL4 knockdown caused a significant increase in the levels of collagen I and IV, as well as lysine hydroxylase (PLOD)1, PLOD2, prolyl 4-hydroxylase subunit alpha (P4HA)1, and P4HA2, which are the key enzymes in collagen biogenesis. The collagen bundle thickening was observed in LOXL4 knockdown tumors as compared with that of control tumors. The OS analysis of the breast cancer patients (n = 584) revealed that low LOXL4 expression and combination of low LOXL4 and high collagen I and IV expression were significantly correlated with a poor OS, particularly, TNBC patients (n = 101).

Conclusions: This study demonstrates the miR-141/200c cluster promotes the migratory and invasive abilities of MDA-MB-231 cells through FAK- and PI3K/AKT-mediated signaling by means of increased VEGF-A secretion. In addition, the low LOXL4 expression triggered the ECM remodeling by inducing the collagen synthesis, deposition, and structural changes, which resulted in a significant promotion of tumor growth and metastasis. LOXL4 might be used as a prognostic marker in TNBC patients.
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Biomedical Sciences (대학원 의과학과)Theses (Ph.D. / Sc.D._의과학과)
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