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Breast Cancer

, Volume 26, Issue 5, pp 581–593 | Cite as

Breast cancer cell motility is promoted by 14-3-3γ

  • Emiko HiraokaEmail author
  • Takahiro Mimae
  • Masaoki Ito
  • Takayuki Kadoya
  • Yoshihiro Miyata
  • Akihiko Ito
  • Morihito Okada
Original Article
  • 201 Downloads

Abstract

Purpose

Pseudopodia are actin-rich ventral protrusions associated with cell motility and cancer cell invasion. We previously applied our established method of using excimer laser cell etching to isolate pseudopodial proteins from MDA-MB-231 breast cancer cells. We later identified 14-3-3γ as an oncogenic molecule among 46 candidate proteins that are specific to pseudopodia. The present study aimed to determine the function of 14-3-3γ in the motility of breast cancer cells.

Methods

MDA-MB-231 cells were cultured on 3-µm porous membranes and double stained to localize 14-3-3γ and phalloidin in pseudopodia using confocal imaging. We assessed pseudopodia numbers and length, as well as migration and wound healing in MDA-MB-231 cells with knockdown and forced expression of 14-3-3γ to determine 14-3-3γ involvement in cell motility. We also immunohistochemically analyzed 14-3-3γ in human breast cancer tissues with high-grade lymphatic invasion.

Results

We specifically located 14-3-3γ in pseudopodia of MDA-MB-231 cells. Knockdown and forced expression of 14-3-3γ, respectively, decreased and increased pseudopodial formation and elongation. Migration and wound healing assays also showed that 14-3-3γ knockdown and forced expression, respectively, decreased and increased the number of underside cells and acellular areas in MDA-MB-231 breast cancer cells. More 14-3-3γ was expressed in sites of lymphatic invasion, than in the center and periphery of human breast cancer tissues.

Conclusion

The role of 14-3-3γ in breast cancer invasiveness might be to promote cell motility. Inhibition of 14-3-3γ could, therefore, become a novel target of therapy to prevent invasion and metastasis in patients with breast cancers expressing 14-3-3γ.

Keywords

Pseudopodia Metastasis Triple-negativity Tumor cell migration Motility 

Notes

Acknowledgements

The authors thank Yusuke Motoi (Hiroshima University) for technical assistance. Part of this study was supported by the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to declare.

Ethical approval

The Institutional Review Board, Ethics Committee for Epidemiology at Hiroshima University approved this study.

Supplementary material

12282_2019_957_MOESM1_ESM.tif (13 mb)
Supplementary Fig. 1. Motility of MCF7, SKBR3 and BT474 breast cancer cells is promoted by 14-3-3γ. Cultured cells with wounds (a) were harvested at 0, 6, and 12 h after scratching. Bar graphs represent (b) acellular areas at 12 h divided by areas at 0 h as relative ratios (%) with standard error. *P < 0.05 and P < 0.01 by Mann–Whitney U test vs. control (si-Control) cells. Bars (a), 200 μm. (TIF 13272 KB)
12282_2019_957_MOESM2_ESM.tif (5.7 mb)
Supplementary Fig. 2. Breast cancer cell proliferation is not promoted by 14-3-3γ. Cultured cells were stained with Ki-67 (Alexa Fluor® 488) and DAPI at 48 h after seeding (a). Bar graphs (b) represent ratios of Ki-67-positive cells with standard error. (TIF 5793 KB)
12282_2019_957_MOESM3_ESM.tif (2.8 mb)
Supplementary Fig. 3. Promotion of EMT is not 14-3-3γ-dependent in breast cancer cell lines. Western blots of 14-3-3γ (1:250 dilution), RAB1A, fibronectin, vimentin, E-cadherin (1:100 dilution), and β-actin (1:5000 dilution) expression in breast cancer cell lines with or without 14-3-3γ knockdown. (TIF 2870 KB)

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Copyright information

© The Japanese Breast Cancer Society 2019

Authors and Affiliations

  1. 1.Department of Surgical Oncology, Division of Radiation Biology and Medicine, Graduate School of Biomedical and Health SciencesHiroshima UniversityHiroshima CityJapan
  2. 2.Department of Pathology, Faculty of MedicineKinki UniversityHigashiōsakaJapan

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