OBJECTIVE: We examined the expression status of p21^{Waf1/Cip1} and p57^{Kip2} in breast cancer as well as their relationship with clinicopathological factors. Moreover, the diagnostic value of gene promoter methylation of p21^Waf1/Cip1 and p57^Kip2 was assessed in breast cancer patients.
METHODS: This study involved 85 patients diagnosed with breast cancer and 36 patients with benign breast lesions. The expression of p21^{Waf1/Cip1} and p57^{Kip2} in cell lysates was analyzed by ELISA and Western blot, respectively. The gene promoter methylation of p21^Waf1/Cip1 and p57^Kip2 was examined in cell lysates by methylation specific PCR.
RESULTS: p21^{Waf1/Cip1} expression was higher while p57^{Kip2} level was lower in breast cancer patients compared to patients with benign breast lesions. The combined use of p21^{Waf1/Cip1} and p57^{Kip2} provided sensitivity and specificity of 82.35% and 86.11%, respectively. None of the malignant and benign breast tumors were found to be hypermethylated at p21^Waf1/Cip1 gene promoter. However, aberrant methylation of p57^Kip2 gene promoter was detected in 49 of 85 (57.65%) of breast cancer tumors. High p21^{Waf1/Cip1} level was associated with high grade, late stages and lymph node involvement, whereas low p57^{Kip2} level was correlated with high grade and HER2 overexpressing breast cancer. Moreover, hypermethylatedp57^Kip2 gene promoter was associated with high grade.
CONCLUSION: Our findings show that the overexpression of p21^{Waf1/Cip1}, down-expression of p57^{Kip2} and gene promoter methylation of p57^Kip2 could be considered as promising diagnostic markers for breast cancer.
LimS. and KaldisP., Cdks, cyclins and CKIs: Roles beyond cell cycle regulation, Development140 (2013), 3079-3093.
2.
LuZ. and HunterT., Ubiquitylation and proteasomal degradation of the p21^{Cip1}, p27^{Kip1} and p57^{Kip2} CDK inhibitors, Cell Cycle9 (2010), 2342-2352.
3.
GomezV. and HergovichA., Cell-cycle control and DNA-damage signaling, in: Genome Stability from Virus to Human Application (Translational Epigenetics Series), KovalchukI. and KovalchukO., eds, Chapter 14, 1^{ed}., Academic Press, London, (2016); pp. 227-242.
4.
El-DeiryW.S., TokinoT., VelculescuV.E. et al., WAF1, a potential mediator of p53 tumor suppression, Cell75 (1993), 817-825.
5.
XiongY., HannonG.J., ZhangH. et al., P21 is a universal inhibitor of cyclin kinases, Nature366 (1993), 701-704.
6.
WagaS., HannonG.J., BeachD. et al., The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA, Nature369 (1994), 574-578.
7.
LiR., WagaS., HannonG.J. et al., Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair, Nature371 (1994), 534-537.
8.
BunzF., DutriauxA., LengauerC. et al., Requirement for p53 and p21 to sustain G_2 arrest after DNA damage, Science282 (1998), 1497-1501.
9.
MichieliP., ChedidM., LinD. et al., Induction of WAF1/CIP1 by a p53-independent pathway, Cancer Res54 (1994), 3391-3395.
10.
DattoM.B., LiY., PanusJ.F. et al., Transforming growth factor β induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism, Proc Natl Acad Sci USA92 (1995), 5545-5549.
11.
LiX.S., RishiA.K., ShaoZ.M. et al., Posttranscriptional regulation of p21WAF1/CIP1 expression in human breast carcinoma cells, Cancer Res56 (1996), 5055-5062.
12.
JiangM., ShaoZ.M., WuJ. et al., P21/waf1/cip1 and mdm-2 expression in breast carcinoma patients as related to prognosis, Int J cancer74 (1997), 529-534.
13.
PellikainenM.J., PekolaT.T., RopponenK.M. et al., P21^{WAF1} expression in invasive breast cancer and its association with p53, AP-2, cell proliferation, and prognosis, J Clin Pathol56 (2003), 214-220.
14.
BarbareschiM., CaffoO., DoglioniC. et al., P21^{WAF1} immunohistochemical expression in breast carcinoma: Correlations with clinicopathological data, oestrogen receptor status, MIB1 expression, p53 gene and protein alterations and relapse-free survival, Br J Cancer74 (1996), 208-215.
15.
CaffoO., DoglioniC., VeroneseS. et al., Prognostic value of p21^{WAF1} and p53 expression in breast carcinoma: An immunohistochemical study in 261 patients with long-term follow-up, Clin Cancer Res2 (1996), 1591-1599.
16.
BánkfalviA., ToryK., KemperM. et al., Clinical relevance of immunohistochemical expression of p53-targeted gene products mdm-2, p21 and bcl-2 in breast carcinoma, Pathol Res Pract196 (2000), 489-501.
17.
WintersZ.E., HuntN.C., BradburnM.J. et al., Subcellular localization of cyclin B, Cdc2 and p21^{WAF1/CIP1} in breast cancer: Association with prognosis, Eur J Cancer37 (2001), 2405-2412.
18.
BorrielloA., CaldarelliI., BencivengaD. et al., P57^{Kip2} and cancer: Time for a critical appraisal, Mol Cancer Res9 (2011), 1269-1284.
19.
WatanabeH., PanZ.Q., Schreiber-AgusN. et al., Suppression of cell transformation by the cyclin-dependent kinase inhibitor p57^{KIP2} requires binding to proliferating cell nuclear antigen, Proc Natl Acad Sci USA95 (1998), 1392-1397.
20.
MerloP., FulcoM., CostanzoA. et al., A role of p73 in mitotic exit, J Biol Chem280 (2005), 30354-30360.
21.
LarsonP.S., SchlechterB.L., KingC.L. et al., CDKN1C/ p57^{kip2} is a candidate tumor suppressor gene in human breast cancer, BMC Cancer8 (2008), 68.
22.
XuX.Y., WangW.Q., ZhangL. et al., Clinical implications of p57^KIP2 expression in breast cancer, Asian Pac J Cancer Prev13 (2012), 5033-5036.
23.
YangC., NanH., MaJ. et al., High Skp2/low p57^{Kip2} expression is associated with poor prognosis in human breast carcinoma, Breast Cancer (Auckl)9 (2015), 13-21.
24.
EdgeS.B., ByrdD.R., ComptonC.C. et al., Breast, in: AJCC Cancer Staging Manual, eds, 7^{ed.}, Springer, New York, NY, (2010), 345-377.
25.
TavassoliF.A. and DevileeP., World health organization classification of tumours, in: Pathology and Genetics, Tumours of the Breast and Female Genital Organs, Lyon: IARC Press, 2003, 19-23.
26.
ZohnyS.F. and El-ShinawiM., Significance of survivin and Bcl-2 homologous antagonist/killer mRNA in detection of bone metastasis in patients with breast cancer, Med Oncol28 (2011), S108-S114.
27.
BradfordN.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem72 (1976), 248-254.
28.
SambrookJ., FritschE.F. and ManiatisT., Molecular Cloning. A Laboratory Manual, 2^{ed.}, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press, USA, 1989, 42-59.
29.
FrommerM., McDonaldL.E., MillarD.S. et al., A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands, Proc Natl Acad Sci USA89 (1992), 1827-1831.
30.
StaalesenV., LeirvaagB., LillehaugJ.R. et al., Genetic and epigenetic changes in p21 and p21B do not correlate with resistance to doxorubicin or mitomycin and 5-fluorouracil in locally advanced breast cancer, Clin Cancer Res10 (2004), 3438-3443.
31.
ChimC.S., LiangR., FungT.K. et al., Infrequent epigenetic dysregulation of CIP/KIP family of cyclin-dependent kinase inhibitors in multiple myeloma, Leukemia19 (2005), 2352-2355.
32.
WeiC.Y., TanQ.X., ZhuX. et al., Expression of CDKN1A/p21 and TGFBR2 in breast cancer and their prognostic significance, Int J Clin Exp Pathol8 (2015), 14619-14629.
33.
BalbínM., HannonG.J., PendásA.M. et al., Functional analysis of a p21^{WAF1}, CIP1, SDI1 mutant (Arg^{94} \to Trp) identified in a human breast carcinoma. Evidence that the mutation impairs the ability of p21 to inhibit cyclin-dependent kinases, J Biol Chem271 (1996), 15782-15786.
34.
AsadaM., YamadaT., IchijoH. et al., Apoptosis inhibitory activity of cytoplasmic p21^{Cip1/WAF1} in monocytic differentiation, EMBO J18 (1999), 1223-1234.
35.
ZhouB.P., LiaoY., XiaW. et al., Cytoplasmic localization of p21^{Cip1/WAF1} by Akt-induced phosphorylation in HER-2/neu-overexpressing cells, Nat Cell biol3 (2001), 245-252.
36.
WintersZ.E., LeekR.D., BradburnM.J. et al., Cytoplasmic p21^{WAF1/CIP1} expression is correlated with HER-2/neu in breast cancer and is an independent predictor of prognosis, Breast Cancer Res5 (2003), R242-R249.
37.
XiaW., ChenJ.S., ZhouX. et al., Phosphorylation/cytoplasmic localization of p21^{Cip1/WAF1} is associated with HER2/neu overexpression and provides a novel combination predictor for poor prognosis in breast cancer patients, Clin Cancer Res10 (2004), 3815-3824.
38.
ChengX., XiaW., YangJ.Y. et al., Activation of p21(CIP1/WAF1) in mammary epithelium accelerates mammary tumorigenesis and promotes lung metastasis, Biochem Biophys Res Commun403 (2010), 103-107.
39.
RössigL., JadidiA.S., UrbichC. et al., Akt-dependent phosphorylation of p21^{Cip1} regulates PCNA and proliferation of endothelial cells, Mol Cell Biol21 (2001), 5644-5657.
40.
LiY., DowbenkoD. and LaskyL.A., AKT/PKB phosphorylation of p21^{Cip/WAF1} enhances protein stability of p21^{Cip/WAF1} and promotes cell survival, J Biol Chem277 (2002), 11352-11361.
41.
WeissR.H., MarshallD., HowardL. et al., Suppression of breast cancer growth and angiogenesis by an antisense oligodeoxynucleotide to p21^{Waf1/Cip1, Cancer Lett189 (2003), 39-48.
42.
FanY., BorowskyA.D. and WeissR.H., An antisense oligodeoxynucleotide to p21^{Waf1/Cip1} causes apoptosis in human cancer cells, Mol Cancer Ther2 (2003), 773-782.
43.
KobatakeT., YanoM., ToyookaS. et al., Aberrant methylation of p57KIP2 gene in lung and breast cancers and malignant mesotheliomas, Oncol Rep12 (2004), 1087-1092.
44.
ZhaoR., YangH.Y., ShinJ. et al., CDK inhibitor p57^{Kip2} is downregulated by Akt during HER2-mediated tumorigenicity, Cell Cycle12 (2013), 935-943.
