S6K1调控的蛋白表达图谱及其对乳腺癌细胞恶性表型的影响

doi:10.3969/j.issn.1004-616x.2024.03.003

摘要/Abstract

摘要： 目的：探究核糖体蛋白S6激酶β-1(S6K1)所调控的蛋白表达图谱及其对乳腺癌细胞恶性表型的影响。方法：通过cBioPortal和GEPIA数据库分析S6K1基因在乳腺癌组织中的扩增及表达，并评估其表达对乳腺癌患者临床特征的影响；利用小干扰RNA在乳腺癌MCF7细胞中敲降S6K1，通过质谱技术检测S6K1所调控的蛋白表达图谱；采用细胞成球实验探究S6K1表达水平改变对乳腺癌细胞干性的影响；通过细胞迁移实验探究S6K1对乳腺癌细胞迁移能力的影响。结果：S6K1基因在乳腺癌组织中存在高频扩增现象，在浸润性乳腺癌中的扩增率高于非浸润性乳腺癌(P<0.05)，且S6K1的高频扩增与患者的不良预后相关(P<0.01)；S6K1在乳腺癌组织中的表达水平较癌旁组织高(P<0.01)，而S6K1在乳腺癌不同临床分期中表达水平无明显差异(P>0.05)。质谱检测表明敲降S6K1后，乳腺癌MCF7细胞中有251个蛋白表达上调，224个蛋白表达下调；GO通路富集分析表明，差异表达蛋白参与了细胞质翻译、蛋白质稳定、干细胞群体维持、细胞迁移等多个生物学过程相关通路；与对照组相比，成球实验和迁移实验表明敲降S6K1可显著抑制乳腺癌细胞干性表型和迁移能力(P<0.01)。结论：S6K1在乳腺癌细胞中调控多个生物学过程相关蛋白的表达，敲降S6K1明显抑制了乳腺癌细胞的干性和迁移能力，表明S6K1可能成为乳腺癌治疗的新靶点。

关键词: 核糖体蛋白S6激酶β-1, 乳腺癌, 细胞干性, 迁移, 蛋白组学

Abstract: OBJECTIVE：To investigate the expression and regulation of the ribosomal protein S6 kinase β-1(S6K1) on the malignant phenotype of breast cancer cells. METHODS：Amplification and expression of S6K1 in breast cancer tissues were analyzed by cBioPortal and GEPIA databases，and their relationships with clinical characteristics of the breast cancers were also analyzed. Small interfering RNA was employed to knock down S6K1 in the breast cancer MCF7 cells in vitro to investigate its impact on protein expression profile using mass spectrometry，stemness of breast cancer cells using the sphere formation assay，and migration ability using the migration assay. RESULTS：S6K1 gene exhibited frequent amplification in breast cancer tissues，with a higher amplification rate in invasive compared with non-invasive breast cancers (P<0.05)，and with significant relationship to poor prognosis of patients (P<0.01). In addition，the expression was higher in breast cancer tissues compared with adjacent tissues (P<0.01)，while the expression levels did not differ significantly among different stages of breast cancer (P>0.05). Mass spectrometry revealed 251 upregulated and 224 downregulated proteins following the S6K1 knockdown in the MCF7 cells. GO pathway enrichment analysis indicated that S6K1 regulated the expression of multiple proteins involved in biological processes such as cytoplasmic translation，protein stabilization，stem cell population maintenance，and cell migration. Additional assays demonstrated that S6K1 knockdown significantly inhibited the stemness phenotype and migration ability of breast cancer cells (P<0.05). CONCLUSION：S6K1 regulated the expression of proteins involved in multiple biological processes in breast cancer cells in vivo，and the knockdown of S6K1 significantly inhibited the stemness and migration ability of breast cancer cells in vitro，indicating a crucial regulatory role for S6K1 and highlighting its potential as a novel therapeutic target for breast cancer therapy.

Key words: S6K1, breast cancer, stemness of cells, migration, proteomics

中图分类号:

R737.9

舒杏梅, 石小倩, 刘燕, 李丹. S6K1调控的蛋白表达图谱及其对乳腺癌细胞恶性表型的影响[J]. 癌变·畸变·突变, 2024, 36(3): 179-186,223.

SHU Xingmei, SHI Xiaoqian, LIU Yan, LI Dan. Expression of ribosomal protein S6K1 and its regulation on malignant phenotypes in breast cancer cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2024, 36(3): 179-186,223.

参考文献

[1] BRAY F, LAVERSANNE M, SUNG H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2024. doi: 10.3322/caac.21834.
[2] HAN B F, ZHENG R S, ZENG H M, et al. Cancer incidence and mortality in China, 2022[J]. J Natl Cancer Cent, 2024, 4(1): 47-53.
[3] BURSTEIN H J. Systemic therapy for estrogen receptorpositive, HER2-negative breast cancer[J]. N Engl J Med, 2020, 383(26): 2557-2570.
[4] MO H N, LIU X F, XUE Y, et al. S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer[J]. Mol Cancer, 2022, 21(1): 171.
[5] CSIBI A, LEE G, YOON S O, et al. The mTORC1/S6K1 pathway regulates glutamine metabolism through the eIF4Bdependent control of c-Myc translation[J]. Curr Biol, 2014, 24(19): 2274-2280.
[6] WU X J, XIE W, XIE W X, et al. Beyond controlling cell size: functional analyses of S6K in tumorigenesis[J]. Cell Death Dis, 2022, 13(7): 646.
[7] RAUGHT B, PEIRETTI F, GINGRAS A C, et al. Phosphorylation of eucaryotic translation initiation factor 4B Ser422 is modulated by S6 kinases[J]. EMBO J, 2004, 23(8): 1761-1769.
[8] HARMS U, ANDREOU A Z, GUBAEV A, et al. eIF4B, eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle[J]. Nucleic Acids Res, 2014, 42(12): 7911-7922.
[9] BROWNE G J, PROUD C G. Regulation of peptide-chain elongation in mammalian cells[J]. Eur J Biochem, 2002, 269(22): 5360-5368.
[10] WANG X, LI W, WILLIAMS M, et al. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase[J]. EMBO J, 2001, 20(16): 4370-4379.
[11] LI P D, ZHANG W J, ZHANG M Y, et al. Overexpression of RPS₆KB₁ predicts worse prognosis in primary HCC patients[J]. Med Oncol, 2012, 29(5): 3070-3076.
[12] ISMAIL H M S. Overexpression of S6 kinase 1 in brain tumours is associated with induction of hypoxia-responsive genes and predicts patients survival[J]. J Oncol, 2012, 2012: 416927.
[13] AMARAL C L, FREITAS L B, TAMURA R E, et al. S6Ks isoforms contribute to viability, migration, docetaxel resistance and tumor formation of prostate cancer cells[J]. BMC Cancer, 2016, 16: 602.
[14] HSIAO Y H, HUANG Y T, HUNG C Y, et al. PYK2 via S6K1 regulates the function of androgen receptors and the growth of prostate cancer cells[J]. Endocr Relat Cancer, 2016, 23(8): 651-663.
[15] SHEN H, WANG G C, LI X, et al. S6K1 blockade overcomes acquired resistance to EGFR-TKIs in non-small cell lung cancer[J]. Oncogene, 2020, 39(49): 7181-7195.
[16] LU Q J, WANG J S, YU G, et al. Expression and clinical significance of mammalian target of rapamycin/P70 ribosomal protein S6 kinase signaling pathway in human colorectal carcinoma tissue[J]. Oncol Lett, 2015, 10(1): 277-282.
[17] IP C K, YUNG S, CHAN T M, et al. p70 S6 kinase drives ovarian cancer metastasis through multicellular spheroidperitoneum interaction and P-cadherin/b1 integrin signaling activation[J]. Oncotarget, 2014, 5(19): 9133-9149.
[18] SHAO X Y, CHENG Z W, XU M L, et al. Prognosis, significance and positive correlation of Rab1A and p-S6K/Gli1 expression in gastric cancer[J]. Anticancer Agents Med Chem, 2019, 19(11): 1359-1367.
[19] NAJAFI M, MORTEZAEE K, MAJIDPOOR J. Cancer stem cell (CSC) resistance drivers[J]. Life Sci, 2019, 234: 116781.
[20] BROWN H K, TELLEZ-GABRIEL M, HEYMANN D. Cancer stem cells in osteosarcoma[J]. Cancer Lett, 2017, 386: 189-195.
[21] NAKANO T, WARNER K A, OKLEJAS A E, et al. mTOR inhibition ablates cisplatin-resistant salivary gland cancer stem cells[J]. J Dent Res, 2021, 100(4): 377-386.
[22] ANDRADE N P, WARNER K A, ZHANG Z C, et al. Survival of salivary gland cancer stem cells requires mTOR signaling[J]. Cell Death Dis, 2021, 12(1): 108.
[23] FANFONE D, WU Z C, MAMMI J, et al. Confined migration promotes cancer metastasis through resistance to anoikis and increased invasiveness[J]. Elife, 2022, 11: e73150.
[24] TREPAT X, CHEN Z Z, JACOBSON K. Cell migration[J]. Compr Physiol, 2012, 2(4): 2369-2392.
[25] HANAHAN D. Hallmarks of cancer: new dimensions[J]. Cancer Discov, 2022, 12(1): 31-46.
[26] TALIAFERRO-SMITH L, NAGALINGAM A, ZHONG D, et al. LKB1 is required for adiponectin-mediated modulation of AMPK-S6K axis and inhibition of migration and invasion of breast cancer cells[J]. Oncogene, 2009, 28(29): 2621-2633.
[27] ZHOU H Y, WONG A S T. Activation of p70S6K induces expression of matrix metalloproteinase 9 associated with hepatocyte growth factor-mediated invasion in human ovarian cancer cells[J]. Endocrinology, 2006, 147(5): 2557-2566.