J Breast Cancer.  2019 Sep;22(3):375-386. 10.4048/jbc.2019.22.e43.

In Vitro and In Vivo Study on the Effect of Lysosome-associated Protein Transmembrane 4 Beta on the Progression of Breast Cancer

• Tao D
• Liang J
• Pan Y
• Zhou Y
• Feng Y
• Zhang L
• Xu J
• Wang H
• He P
• Yao J
• Zhao Y
• Ning Q
• Wang W
• Jiang W
• Zheng J
• Wu X

• KMID: 2458859
• DOI: http://doi.org/10.4048/jbc.2019.22.e43

Abstract

PURPOSE
Although the effect of lysosome-associated protein transmembrane 4 beta (LAPTM4B) on the proliferation, migration, and invasion of breast cancer (BC) cells has already been studied, its specific role in BC progression is still elusive. Here, we evaluated the effect of different levels of LAPTM4B expression on the proliferation, invasion, adhesion, and tumor formation abilities of BC cells in vitro, as well as on breast tumor progression in vivo.
METHODS
We investigated the influence of LAPTM4B expression on MCF-7 cell proliferation, invasion, adhesion, and tube formation abilities in vitro through its overexpression or knockdown and on breast tumor progression in vivo.
RESULTS
Cell growth curves and colony formation assays showed that LAPTM4B promoted the proliferation of breast tumor cells. Cell cycle analysis results revealed that LAPTM4B promoted the entry of cells from the G1 into the S phase. Transwell invasion and cell extracellular matrix adhesion assays showed that LAPTM4B overexpression increased the invasion and adhesion capabilities of MCF-7 cells. More branches were observed in MCF-7 cells overexpressing LAPTM4B under an electron microscope. In comparison with LAPTM4B overexpression, LAPTM4B knockdown decreased the expression of vascular endothelial growth factor-A and significantly inhibited the vasculogenic tube formation ability of tumors. These results were also verified with western blot analysis.
CONCLUSION
LAPTM4B promoted the proliferation of MCF-7 cells through the downregulation of p21 (WAF1/CIP1) and caspase-3, and induced cell invasion, adhesion, and angiogenesis through the upregulation of hypoxia-inducible factor 1 alpha, matrix metalloproteinase 2 (MMP2), and MMP9 expression. This specific role deems LAPTM4B as a potential therapeutic target for BC treatment.

Keyword

Breast neoplasms; Disease progression; LAPTM4B protein, human; MCF-7 cells

MeSH Terms

Blotting, Western
Breast Neoplasms*
Breast*
Caspase 3
Cell Cycle
Disease Progression
Down-Regulation
Extracellular Matrix
Hypoxia-Inducible Factor 1
In Vitro Techniques*
Matrix Metalloproteinase 2
MCF-7 Cells
S Phase
Up-Regulation
Vascular Endothelial Growth Factor A
Caspase 3
Hypoxia-Inducible Factor 1
Matrix Metalloproteinase 2
Vascular Endothelial Growth Factor A

Figure

• 

  Figure 1 LAPTM4B promotes the proliferation of breast tumor cells. (A) Relative levels of LAPTM4B in MCF-7 cells treated as indicated. Glyceraldehyde 3-phosphate dehydrogenase was used as the loading control. (B) Proliferation rates of MCF-7 cells treated as indicated. (C) Colony formation ability of MCF-7 cells treated as indicated. (D) Cycle distribution of MCF-7 cells treated as indicated. LAPTM4B = lysosome-associated protein transmembrane 4 beta; NC = negative control; shRNA = short-hairpin RNA; OD = optical density. *p < 0.05, †p < 0.01, ‡p <0.001.

• 

  Figure 2 LAPTM4B promotes the invasion and adhesion of breast tumor cells. (A) Invasion ability of MCF-7 cells treated as indicated (scale bar = 100 μm); (B) Adhesion ability of MCF-7 cells treated as indicated (scale bar = 100 μm). LAPTM4B = lysosome-associated protein transmembrane 4 beta; NC = negative control; shRNA = short-hairpin RNA; OD = optical density. *p < 0.05, †p < 0.01, ‡p <0.001.

• 

  Figure 3 LAPTM4B promotes vasculogenic tube formation in breast tumor cells. (A) Representative images of vasculogenic tubes formed in MCF-7 cells treated as indicated (scale bar = 100 μM). (B) Number of branch points formed in MCF-7 cells treated as indicated. (C) Concentration of VEGF secreted by MCF-7 cells treated as indicated. (D and E) Expression levels of various important regulators in MCF-7 cells treated as indicated. LAPTM4B = lysosome-associated protein transmembrane 4 beta; NC = negative control; shRNA = short-hairpin RNA; VEGF = vascular endothelial growth factor; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; Bcl = B cell lymphoma; CDK = cyclin-dependent kinase; HIF = hypoxia-inducible factor; MMP = matrix metalloproteinase; p = phosphorylated; AKT = protein kinase B; mTOR = mammalian target of rapamycin. *p < 0.05, †p < 0.01.

• 

  Figure 4 LAPTM4B promotes the progression of breast tumors in vivo. (A) The size of tumors in different treatment groups. (B) Growth curves of tumors in different treatment groups. (C) Expression of various important proteins in tumors induced as indicated. LAPTM4B = lysosome-associated protein transmembrane 4 beta; NC = negative control; shRNA = short-hairpin RNA; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; p = phosphorylated; AKT = protein kinase B; mTOR = mammalian target of rapamycin; Bcl = B cell lymphoma; CDK = cyclin-dependent kinase; HIF = hypoxia-inducible factor. *p <0.001.

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