Homeobox protein MSX1 inhibits the growth and metastasis of breast cancer cells and is frequently silenced by promoter methylation

Yue,Yujuan; Yuan,Ying; Li,Lili; Fan,Jiangxia; Li,Chen; Peng,Weiyan; Ren,Guosheng

doi:10.3892/ijmm.2018.3468

Homeobox protein MSX1 inhibits the growth and metastasis of breast cancer cells and is frequently silenced by promoter methylation

Authors:
- Yujuan Yue
- Ying Yuan
- Lili Li
- Jiangxia Fan
- Chen Li
- Weiyan Peng
- Guosheng Ren
View Affiliations

Affiliations: Chongqing Key Laboratory of Molecular Oncology and Epigenetics, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China, Cancer Epigenetics Laboratory, Department of Clinical Oncology, State Key Laboratory of Oncology in South China, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong and CUHK Shenzhen Research Institute, Hong Kong, SAR 999077, P.R. China
Published online on: February 7, 2018 https://doi.org/10.3892/ijmm.2018.3468
Pages: 2986-2996

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Deregulation of msh homeobox 1 (MSX1) has been identified to be associated with multiple human malignant neoplasms. However, the association of the expression and biological function of MSX1 with breast tumorigenesis, and the underlying mechanism remain largely unknown. Therefore, the present study examined the expression and promoter methylation of MSX1 in breast tumor cell lines, primary breast tumors and normal breast tissues using semi-quantitative, quantitative and methylation-specific reverse transcription‑polymerase chain reaction. Colony formation assays, flow cytometric analysis, and wound healing and Transwell assays were used to assess various functions of MSX1. Western blot analyses were also conducted to explore the mechanism of MSX1. The results revealed that MSX1 was broadly expressed in normal human tissues, including breast tissues, but was frequently downregulated or silenced in breast cancer cell lines and primary tumors by promoter methylation. Methylation of the MSX1 promoter was observed in 7/9 (77.8%) breast cancer cell lines and 47/99 (47.5%) primary tumors, but not in normal breast tissues or surgical margin tissues, suggesting that tumor-specific methylation of MSX1 occurs in breast cancer. Pharmacological demethylation reduced MSX1 promoter methylation levels and restored the expression of MSX1. The ectopic expression of MSX1, induced by transfection with a lentiviral vector, significantly inhibited the clonogenicity, proliferation, migration and invasion of breast tumor cells by inducing G1/S cell cycle arrest and apoptosis. Ectopic MSX1 expression also inhibited the expression of active β-catenin and its downstream targets c-Myc and cyclin D1, and also increased the cleavage of caspase-3 and poly (ADP-ribose) polymerase. In conclusion, MSX1 exerts tumor-suppressive functions by inducing G1/S cell cycle arrest and apoptosis in breast tumorigenesis. Its methylation may be used as an epigenetic biomarker for the early detection and diagnosis of breast cancer.

View Figures

View References

1	Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Jemal A: Global cancer statistics, 2012. CA Cancer J Clin. 65:87–108. 2015. View Article : Google Scholar : PubMed/NCBI
2	Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar : PubMed/NCBI
3	Taby R and Issa JP: Cancer epigenetics. CA Cancer J Clin. 60:376–392. 2010. View Article : Google Scholar : PubMed/NCBI
4	Esteller M: Epigenetics in cancer. N Engl J Med. 358:1148–1159. 2008. View Article : Google Scholar : PubMed/NCBI
5	Hoque MO, Prencipe M, Poeta ML, Barbano R, Valori VM, Copetti M, Gallo AP, Brait M, Maiello E, Apicella A, et al: Changes in CpG islands promoter methylation patterns during ductal breast carcinoma progression. Cancer Epidemiol Biomarkers Prev. 18:2694–2700. 2009. View Article : Google Scholar : PubMed/NCBI
6	Xiang TX, Yuan Y, Li LL, Wang ZH, Dan LY, Chen Y, Ren GS and Tao Q: Aberrant promoter CpG methylation and its translational applications in breast cancer. Chin J Cancer. 32:12–20. 2013. View Article : Google Scholar :
7	Revet I, Huizenga G, Koster J, Volckmann R, van Sluis P, Versteeg R and Geerts D: MSX1 induces the Wnt pathway antagonist genes DKK1, DKK2, DKK3, and SFRP1 in neuroblastoma cells, but does not block Wnt3 and Wnt5A signalling to DVL3. Cancer Lett. 289:195–207. 2010. View Article : Google Scholar
8	Shah N and Sukumar S: The Hox genes and their roles in oncogenesis. Nat Rev Cancer. 10:361–371. 2010. View Article : Google Scholar : PubMed/NCBI
9	Bhatlekar S, Fields JZ and Boman BM: HOX genes and their role in the development of human cancers. J Mol Med (Berl). 92:811–823. 2014. View Article : Google Scholar
10	McGinnis W and Krumlauf R: Homeobox genes and axial patterning. Cell. 68:283–302. 1992. View Article : Google Scholar : PubMed/NCBI
11	Sliwinski T, Synowiec E, Czarny P, Gomulak P, Forma E, Morawiec Z, Morawiec J, Dziki L, Wasylecka M and Blasiak J: The c.469+46_56del mutation in the homeobox MSX1 gene - a novel risk factor in breast cancer? Cancer Epidemiol. 34:652–655. 2010. View Article : Google Scholar : PubMed/NCBI
12	Saadi I, Das P, Zhao M, Raj L, Ruspita I, Xia Y, Papaioannou VE and Bei M: Msx1 and Tbx2 antagonistically regulate Bmp4 expression during the bud-to-cap stage transition in tooth development. Development. 140:2697–2702. 2013. View Article : Google Scholar : PubMed/NCBI
13	Nassif A, Senussi I, Meary F, Loiodice S, Hotton D, Robert B, Bensidhoum M, Berdal A and Babajko S: Msx1 role in craniofacial bone morphogenesis. Bone. 66:96–104. 2014. View Article : Google Scholar : PubMed/NCBI
14	Bendall AJ and Abate-Shen C: Roles for Msx and Dlx homeoproteins in vertebrate development. Gene. 247:17–31. 2000. View Article : Google Scholar : PubMed/NCBI
15	Nakatomi M, Wang XP, Key D, Lund JJ, Turbe-Doan A, Kist R, Aw A, Chen Y, Maas RL and Peters H: Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis. Dev Biol. 340:438–449. 2010. View Article : Google Scholar : PubMed/NCBI
16	Ceyhan D, Kirzioglu Z and Calapoglu NS: Mutations in the MSX1 gene in Turkish children with non-syndromic tooth agenesis and other dental anomalies. Indian J Dent. 5:172–182. 2014. View Article : Google Scholar :
17	Dunwell TL, Hesson LB, Pavlova T, Zabarovska V, Kashuba V, Catchpoole D, Chiaramonte R, Brini AT, Griffiths M, Maher ER, et al: Epigenetic analysis of childhood acute lymphoblastic leukemia. Epigenetics. 4:185–193. 2009. View Article : Google Scholar : PubMed/NCBI
18	Park K, Kim K, Rho SB, Choi K, Kim D, Oh SH, Park J, Lee SH and Lee JH: Homeobox Msx1 interacts with p53 tumor suppressor and inhibits tumor growth by inducing apoptosis. Cancer Res. 65:749–757. 2005.PubMed/NCBI
19	Yamashita S, Tsujino Y, Moriguchi K, Tatematsu M and Ushijima T: Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2′-deoxycytidine treatment and oligonucleotide microarray. Cancer Sci. 97:64–71. 2006. View Article : Google Scholar
20	Bonds J, Pollan-White S, Xiang L, Mues G and D' Souza R: Is there a link between ovarian cancer and tooth agenesis? Eur J Med Genet. 57:235–239. 2014. View Article : Google Scholar : PubMed/NCBI
21	Baylin SB and Ohm JE: Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nat Rev Cancer. 6:107–116. 2006. View Article : Google Scholar : PubMed/NCBI
22	Park J, Park K, Kim S and Lee JH: Msx1 gene overexpression induces G1 phase cell arrest in human ovarian cancer cell line OVCAR3. Biochem Biophys Res Commun. 281:1234–1240. 2001. View Article : Google Scholar : PubMed/NCBI
23	Ringner M, Fredlund E, Hakkinen J, Borg A and Staaf J: GOBO: gene expression-based outcome for breast cancer online. PLoS One. 6:e179112011. View Article : Google Scholar : PubMed/NCBI
24	Xiang T, Li L, Yin X, Yuan C, Tan C, Su X, Xiong L, Putti TC, Oberst M, Kelly K, et al: The ubiquitin peptidase UCHL1 induces G0/G1 cell cycle arrest and apoptosis through stabilizing p53 and is frequently silenced in breast cancer. PLoS One. 7:e297832012. View Article : Google Scholar : PubMed/NCBI
25	Klaus A and Birchmeier W: Wnt signalling and its impact on development and cancer. Nat Rev Cancer. 8:387–398. 2008. View Article : Google Scholar : PubMed/NCBI
26	Giusti AF, O' Neill FJ, Yamasu K, Foltz KR and Jaffe LA: Function of a sea urchin egg Src family kinase in initiating Ca²⁺ release at fertilization. Dev Biol. 256:367–378. 2003. View Article : Google Scholar : PubMed/NCBI
27	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar
28	Chesi M and Bergsagel PL: Epigenetics and microRNAs combine to modulate the MDM2/p53 axis in myeloma. Cancer Cell. 18:299–300. 2010. View Article : Google Scholar : PubMed/NCBI
29	Clevers H: Wnt/beta-catenin signaling in development and disease. Cell. 127:469–480. 2006. View Article : Google Scholar : PubMed/NCBI
30	Veeman MT, Axelrod JD and Moon RT: A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell. 5:367–377. 2003. View Article : Google Scholar : PubMed/NCBI
31	Seidensticker MJ and Behrens J: Biochemical interactions in the wnt pathway. Biochim Biophys Acta. 1495:168–182. 2000. View Article : Google Scholar : PubMed/NCBI
32	Moon RT, Kohn AD, De Ferrari GV and Kaykas A: WNT and beta-catenin signalling: Diseases and therapies. Nat Rev Genet. 5:691–701. 2004. View Article : Google Scholar : PubMed/NCBI
33	Tao H, Guo L, Chen L, Qiao G, Meng X, Xu B and Ye W: MSX1 inhibits cell migration and invasion through regulating the Wnt/β-catenin pathway in glioblastoma. Tumour Biol. 37:1097–1104. 2016. View Article : Google Scholar
34	Hu Z, Fan C, Oh DS, Marron JS, He X, Qaqish BF, Livasy C, Carey LA, Reynolds E, Dressler L, et al: The molecular portraits of breast tumors are conserved across microarray platforms. BMC Genomics. 7:962006. View Article : Google Scholar : PubMed/NCBI