Role of NKX2-5 on tamoxifen resistance in breast cancer patients and its regulation mechanism

doi:10.3969/j.issn.1673-8640.2020.01.011

Abstract

Abstract:

Objective To investigate the role of NKX2-5 on tamoxifen（TAM） resistance in breast cancer and its regulation mechanism. Methods The expressions of NKX2-5 in breast cancer tissues and adjacent tissues were determined by immunohistochemical staining assay. Using Gene Expression Omnibus（GEO） database,the relative data of breast cancer patients were analyzed,and the correlation between NKX2-5 and the survival rate of breast cancer patients was analyzed. The TAM-resistant cell lines（MCF-7/TAM） were constructed,and cell viability was determined by CCK-8 kit after the treatment with different concentrations of TAM. The expressions of the related proteins were determined by western blotting,and NKX2-5 mRNA expression was determined by reverse transcription polymerase chain reaction（RT-PCR）. Results The expression of NKX2-5 was higher in adjacent tissues than that in cancer tissues（P<0.05）,and NKX2-5 expression in TAM-sensitive tissues was higher than that in TAM-resistant tissues（P<0.05）. Low expression of NKX2-5 was associated with poor overall survival rate and relapse free survival rate in breast cancer patients（P<0.05）,and high NKX2-5 expression level also predicted better outcome for patients with TAM endocrine therapy（P<0.05）. Compared to the MCF-7 cells,the expression of NKX2-5 protein was decreased in MCF-7/TAM cells（P<0.05）. Overexpression NKX2-5 plasmid（Ad-NKX2-5） and empty plasmid（Ad-NC） were transfected in MCF-7/TAM cell（Ad-NKX2-5 group and Ad-NC group） by transient transfection technology. NKX2-5-small interfering RNA plasmid（NKX2-5-siRNA） and blank control（NC-siRNA） were transfected in MCF-7 cell（NKX2-5-siRNA group and NC-siRNA group）. After treatment with 50 and 100 μmol/L TAM,the cell proliferation inhibition rates were increased in Ad-NKX2-5 group compared to Ad-NC group（P<0.05）. The cell proliferation inhibition rates were decreased in NKX2-5-siRNA group compared to NC-siRNA group（P<0.05）. The expressions of phosphorylated-P38 mitogen-activated protein kinase（p-P38MAPK）,phosphorylated-c-Jun N-terminal kinase（p-JNK）,phosphorylated-extracellular regulated protein kinase（p-ERK）proteins were increased in MCF-7/TAM cell compared to MCF-7 cell（P<0.05）. The expressions of p-P38,p-JNK and p-ERK proteins were increased in Ad-NKX2-5 group compared to Ad-NC group（P<0.05）. Meanwhile,the expression of p-P38,p-JNK and p-ERK proteins were decreased in NKX2-5-siRNA group compared to NC-siRNA group（P<0.05）. Conclusions NKX2-5 is down-regulated in TAM-resistant breast cancer cell,and overexpressive NKX2-5 overcomes TAM resistance in breast cancer via suppressing mitogen-activated protein kinase（MAPK） signaling pathway.

Key words: Transcriptional factor NKX2-5, Breast cancer, Tamoxifen, Mitogen-activated protein kinase signaling pathway

CLC Number:

R446.1

ZHAO Zheng, LI Yi, YANG Feixiang. Role of NKX2-5 on tamoxifen resistance in breast cancer patients and its regulation mechanism[J]. Laboratory Medicine, 2020, 35(1): 47-55.

Figures/Tables 12

References 21

[1]	ZENG H,CHEN W,ZHENG R,et al.Changing cancer survival in China during 2003-15:a pooled analysis of 17 population-based cancer registries[J]. Lancet Glob Health,2018,6(5):e555-e567.
[2]	BLOK E J,DERKS M G,VAN DER HOEVEN J J,et al. Extended adjuvant endocrine therapy in hormone-receptor positive early breast cancer:current and future evidence[J]. Cancer Treat Rev,2015,41(3):271-276.
[3]	PRASHANTH KUMAR B N,RAJPUT S,BHARTI R,et al. BI2536-A PLK inhibitor augments paclitaxel efficacy in suppressing tamoxifen induced senescence and resistance in breast cancer cells[J]. Biomed Pharmacother,2015,74:124-132.
[4]	GEORGE V,COLOMBO S,TARGOFF K L.An early requirement for nkx2.5 ensures the first and second heart field ventricular identity and cardiac function into adulthood[J]. Dev Biol,2015,400(1):10-22.
[5]	KWABI-ADDO B,WANG S,CHUNG W,et al.Identification of differentially methylated genes in normal prostate tissues from African American and Caucasian men[J]. Clin Cancer Res,2010,16(14):3539-3547.
[6]	GRAZIANI G,ARTUSO S,DE LUCA A,et al.A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib[J]. Biochem Pharmacol,2015,95(1):16-27.
[7]	LI G,ZHANG J,JIN K,et al.Estrogen receptor-α36 is involved in development of acquired tamoxifen resistance via regulating the growth status switch in breast cancer cells[J]. Mol Oncol,2013,7(3):611-624.
[8]	KOOL M,FONTEIN D B,MEERSHOEK-KLEIN KRANENBARG E,et al. Long term effects of extended adjuvant endocrine therapy on quality of life in breast cancer patients[J]. Breast,2015,24(3):224-229.
[9]	YE W,WANG J,SONG Y,et al.A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaker cell fate in the pulmonary vein myocardium and sinoatrial node[J]. Development,2015,142(14):2521-2532.
[10]	JEONG H S,JUNG E S,SIM Y J,et al.Fbxo25 controls Tbx5 and Nkx2-5 transcriptional activity to regulate cardiomyocyte development[J]. Biochim Biophys Acta,2015,1849(6):709-721.
[11]	KOJIC S,NESTOROVIC A,RAKICEVIC L,et al.Cardiac transcription factor Nkx2.5 interacts with p53 and modulates its activity[J]. Arch Biochem Biophys,2015,569:45-53.
[12]	DAREMIPOURAN M,WANG S P,ELLIS A,et al.NKX2-5,a potential tumor suppressor gene in prostate cancer[J]. Cancer Res,2011,71(8):3033.
[13]	杨莉,阮文静,陈恩国,等. 血清NKX2-1蛋白在原发性肺癌患者中的诊断价值[J]. 浙江大学学报(医学版),2012,41(5):535-539.
[14]	杨莉. NKX2-1在肺癌细胞中的生物学效应及与抗癌药物耐药性关系的研究[D]. 杭州:浙江大学,2011.
[15]	ALCÁNTARA-ORTIGOZA M A,DE RUBENS-FIGUEROA J,REYAN-FABIAN M E,et al. Germline mutations in NKX2-5,GATA4,and CRELD1 are rare in a Mexican sample of Down syndrome patients with endocardial cushion and septal heart defects[J]. Pediatr Cardiol,2015,36(4):802-808.
[16]	LI C M,GOCHEVA V,OUDIN M J,et al.Foxa2 and Cdx2 cooperate with Nkx2-1 to inhibit lung adenocarcinoma metastasis[J]. Genes Dev,2015,29(17):1850-1862.
[17]	CHANG N J,WENG W H,CHANG K H,et al.Genome-wide gene expression profiling of ischemia-reperfusion injury in rat kidney,intestine and skeletal muscle implicate a common involvement of MAPK signaling pathway[J]. Mol Med Rep,2015,11(5):3786-3793.
[18]	DANG N,PANG S,SONG H,et al.Knockdown of filaggrin influences the epidermal terminal differentiation via MAPK pathway in normal human epidermal keratinocytes[J]. Mol Biol Rep,2015,42(2):337-343.
[19]	XU C,SUN X,QIN S,et al.Let-7a regulates mammosphere formation capacity through Ras/NF-κ B and Ras/MAPK/ERK pathway in breast cancer stem cells[J]. Cell Cycle,2015,14(11):1686-1697.
[20]	QI X,YIN N,MA S,et al.p38γ MAPK is a therapeutic target for triple-negative breast cancer by stimulation of cancer stem-like cell expansion[J]. Stem Cells,2015,33(9):2738-2747.
[21]	张闻龙,卢仁泉,江铭磊,等. 血浆热休克蛋白90α在乳腺癌诊疗中的应用价值[J]. 检验医学,2017,32(5):374-377.

患者来源	例数	年龄/岁	TNM分期				ER表达			PR表达			HER-2表达
患者来源	例数	年龄/岁	Ⅰ期	Ⅱ期	Ⅲ期	Ⅳ期	阳性	阴性	不详	阳性	阴性	不详	阳性	阴性	不详
本研究	50	44.5±9.4	8	23	17	2	37	13	0	32	18	0	21	29	0
GEO	104	45.1±8.7	18	52	27	7	67	30	7	60	24	20	30	62	12

患者来源	例数	年龄/岁	TNM分期				ER表达			PR表达			HER-2表达
患者来源	例数	年龄/岁	Ⅰ期	Ⅱ期	Ⅲ期	Ⅳ期	阳性	阴性	不详	阳性	阴性	不详	阳性	阴性	不详
本研究	50	44.5±9.4	8	23	17	2	37	13	0	32	18	0	21	29	0
GEO	104	45.1±8.7	18	52	27	7	67	30	7	60	24	20	30	62	12

细胞株	TAM/（μmol/L）
细胞株	5	20	50	100
MCF-7	26.3±3.7	89.4±5.8	91.4±6.7	82.4±6.5
T47D	28.2±2.5	47.8±7.1	63.5±10.4	63.4±5.3
BCAP37	21.7±4.3	31.2±4.7	68.1±6.4	60.7±5.2
SKBR3	25.2±3.1	38.5±7.3	58.6±5.4	54.6±6.6
MCF-7/TAM	24.5±3.7	31.6±5.3	53.1±6.1	50.4±4.8

细胞株	TAM/（μmol/L）
细胞株	5	20	50	100
MCF-7	26.3±3.7	89.4±5.8	91.4±6.7	82.4±6.5
T47D	28.2±2.5	47.8±7.1	63.5±10.4	63.4±5.3
BCAP37	21.7±4.3	31.2±4.7	68.1±6.4	60.7±5.2
SKBR3	25.2±3.1	38.5±7.3	58.6±5.4	54.6±6.6
MCF-7/TAM	24.5±3.7	31.6±5.3	53.1±6.1	50.4±4.8

组别	TAM/（μmol/L）
组别	5	20	50	100
Ad-NKX2-5组	8.7±0.5	21.4±5.7	56.9±10.1^*	67.9±7.8^*
Ad-NC组	4.3±0.4	19.7±4.5	28.9±7.3	49.7±7.1
NKX2-5- siRNA组	19.8±5.4	38.9±6.2^*#	40.9±5.4^*#	40.5±5.6^*#
NC-siRNA组	21.2±6.5	59.4±8.4	60.1±8.9	67.7±7.6