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

Affiliations
  • 1Department of Oncological Surgery, Enze Hospital of Taizhou Enze Medical Group, Luqiao, Zhejiang, China.
  • 2The Affiliated People's Hospital of Inner Mongolia Medical University, Hohhot, Inner Mongolia, China.
  • 3Gynecology of Taizhou Enze Medical Center (Group) Enze Hospital, Taizhou, Zhejiang, China.
  • 4Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China. wuxia_imu@163.com

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.


Reference

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011; 61:69–90.
Article
2. Youlden DR, Cramb SM, Yip CH, Baade PD. Incidence and mortality of female breast cancer in the Asia-Pacific region. Cancer Biol Med. 2014; 11:101–115.
3. Ginsburg O, Bray F, Coleman MP, Vanderpuye V, Eniu A, Kotha SR, et al. The global burden of women's cancers: a grand challenge in global health. Lancet. 2017; 389:847–860.
Article
4. Bertos NR, Park M. Breast cancer - one term, many entities? J Clin Invest. 2011; 121:3789–3796.
Article
5. Curigliano G, Burstein HJ, Winer EP, Gnant M, Dubsky P, Loibl S, et al. De-escalating and escalating treatments for early-stage breast cancer: the St. Gallen International Expert Consensus Conference on the Primary Therapy of Early Breast Cancer 2017. Ann Oncol. 2017; 28:1700–1712.
Article
6. Tono Y, Ishihara M, Miyahara Y, Tamaru S, Oda H, Yamashita Y, et al. Pertuzumab, trastuzumab and eribulin mesylate therapy for previously treated advanced HER2-positive breast cancer: a feasibility study with analysis of biomarkers. Oncotarget. 2018; 9:14909–14921.
Article
7. Li F, Dou J, Wei L, Li S, Liu J. The selective estrogen receptor modulators in breast cancer prevention. Cancer Chemother Pharmacol. 2016; 77:895–903.
Article
8. Chen Y, Wang L, Zhu Y, Chen Z, Qi X, Jin L, et al. Breast cancer resistance protein (BCRP)-containing circulating microvesicles contribute to chemoresistance in breast cancer. Oncol Lett. 2015; 10:3742–3748.
Article
9. Meng F, Song H, Luo C, Yin M, Xu Y, Liu H, et al. Correlation of LAPTM4B polymorphisms with cervical carcinoma. Cancer. 2011; 117:2652–2658.
Article
10. Shao GZ, Zhou RL, Zhang QY, Zhang Y, Liu JJ, Rui JA, et al. Molecular cloning and characterization of LAPTM4B, a novel gene upregulated in hepatocellular carcinoma. Oncogene. 2003; 22:5060–5069.
Article
11. Liu X, Zhou R, Zhang Q, Zhang Y, Shao G, Jin Y, et al. Identification and characterization of LAPTM4B encoded by a human hepatocellular carcinoma-associated novel gene. Beijing Da Xue Xue Bao. 2003; 35:340–347.
12. Li Y, Zou L, Li Q, Haibe-Kains B, Tian R, Li Y, et al. Amplification of LAPTM4B and YWHAZ contributes to chemotherapy resistance and recurrence of breast cancer. Nat Med. 2010; 16:214–218.
Article
13. Xiao M, Jia S, Wang H, Wang J, Huang Y, Li Z. Overexpression of LAPTM4B: an independent prognostic marker in breast cancer. J Cancer Res Clin Oncol. 2013; 139:661–667.
Article
14. Fan M, Liu Y, Zhou R, Zhang Q. Association of LAPTM4B gene polymorphism with breast cancer susceptibility. Cancer Epidemiol. 2012; 36:364–368.
Article
15. Zhou L, He XD, Yu JC, Zhou RL, Shan Y, Rui JA. Overexpression of LAPTM4B-35 attenuates epirubucin-induced apoptosis of gallbladder carcinoma GBC-SD cells. Surgery. 2011; 150:25–31.
Article
16. Kang Y, Yin M, Jiang W, Zhang H, Xia B, Xue Y, et al. Overexpression of LAPTM4B-35 is associated with poor prognosis in colorectal carcinoma. Am J Surg. 2012; 204:677–683.
Article
17. Gomez-Pinillos A, Ferrari AC. mTOR signaling pathway and mTOR inhibitors in cancer therapy. Hematol Oncol Clin North Am. 2012; 26:483–505.
Article
18. Cordes N, Rödel F, Rodemann HP. Molecular signaling pathways. Mechanisms and clinical use. Strahlenther Onkol. 2012; 188:Suppl 3. 308–311.
19. Chen PS, Shih YW, Huang HC, Cheng HW. Diosgenin, a steroidal saponin, inhibits migration and invasion of human prostate cancer PC-3 cells by reducing matrix metalloproteinases expression. PLoS One. 2011; 6:e20164.
Article
20. González-Arriaga P, Pascual T, García-Alvarez A, Fernández-Somoano A, López-Cima MF, Tardón A. Genetic polymorphisms in MMP 2, 9 and 3 genes modify lung cancer risk and survival. BMC Cancer. 2012; 12:121.
Article
21. Akhtar S, Mahjabeen I, Akram Z, Kayani MA. CYP1A1 and GSTP1 gene variations in breast cancer: a systematic review and case-control study. Fam Cancer. 2016; 15:201–214.
Article
22. Baldwin RM, Bejide M, Trinkle-Mulcahy L, Côté J. Identification of the PRMT1v1 and PRMT1v2 specific interactomes by quantitative mass spectrometry in breast cancer cells. Proteomics. 2015; 15:2187–2197.
Article
23. Xiao M, Yang S, Meng F, Qin Y, Yang Y, Jia S, et al. LAPTM4B predicts axillary lymph node metastasis in breast cancer and promotes breast cancer cell aggressiveness in vitro. Cell Physiol Biochem. 2017; 41:1072–1082.
Article
24. Abeyrathna P, Su Y. The critical role of Akt in cardiovascular function. Vascul Pharmacol. 2015; 74:38–48.
Article
25. Raina D, Kharbanda S, Kufe D. The MUC1 oncoprotein activates the anti-apoptotic phosphoinositide 3-kinase/Akt and Bcl-xL pathways in rat 3Y1 fibroblasts. J Biol Chem. 2004; 279:20607–20612.
Article
26. Wang CD, Yuan CF, Bu YQ, Wu XM, Wan JY, Zhang L, et al. Fangchinoline inhibits cell proliferation via Akt/GSK-3beta/ cyclin D1 signaling and induces apoptosis in MDA-MB-231 breast cancer cells. Asian Pac J Cancer Prev. 2014; 15:769–773.
Article
27. Tang C, Lu YH, Xie JH, Wang F, Zou JN, Yang JS, et al. Downregulation of survivin and activation of caspase-3 through the PI3K/Akt pathway in ursolic acid-induced HepG2 cell apoptosis. Anticancer Drugs. 2009; 20:249–258.
Article
28. Wang XX, Cheng Q, Zhang SN, Qian HY, Wu JX, Tian H, et al. PAK5-Egr1-MMP2 signaling controls the migration and invasion in breast cancer cell. Tumour Biol. 2013; 34:2721–2729.
Article
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