白介素1受体相关激酶1在乳腺癌紫杉醇耐受患者治疗中的作用机制研究
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摘要
目的探讨白介素1受体相关激酶1(IRAK1)在乳腺癌紫杉醇耐受患者治疗中的作用机制。方法选取2013年5月—2015年9月赣南医学院第一附属医院肿瘤科收集的乳腺组织(对照)及乳腺癌组织,其中对照女性16例,Basal-like型乳腺癌患者38例,Luminal-A型乳腺癌患者25例,Luminal-B型乳腺癌患者31例,HER2过表达型乳腺癌患者31例,三阴乳腺癌患者58例。取对照乳腺组织及乳腺癌组织,采用实时荧光定量PCR(q PCR)检测IRAK1 mRNA的相对表达水平,采用酶联免疫吸附试验(ELISA)法检测炎性因子白介素(IL)-6、IL-8和CXC趋化因子配体1(CXCL-1)表达水平。培养乳腺癌细胞株HMEC、MCF7、MB415、MB436、MB468、BT549、SUM159,采用体外低浓度梯度递增联合大剂量间断冲击方法诱导乳腺癌细胞株产生紫杉醇耐受性,检测未经处理的细胞株和紫杉醇耐受细胞株IRAK1 mRNA相对表达水平。经shRNA或空白序列NC-shRNA转染紫杉醇耐受乳腺癌细胞株,采用q PCR检测增殖相关基因细胞周期蛋白(Cyclin)D1、增殖细胞核抗原(PCNA)和Cyclin A相对表达水平,凋亡相关基因p53、c-myc、Bcl-2、c-erb-2相对表达水平,迁移相关基因Cullin 1、Bcl-6和KLF6相对表达水平,采用ALDEFLUOR实验检测醛脱氢酶(ALDH)活性。结果各类型乳腺癌紫杉醇耐受患者IRAK1 mRNA相对表达水平均高于不耐受患者(P<0.05),三阴乳腺癌紫杉醇耐受患者IRAK1 mRNA相对表达水平均高于其他类型乳腺癌紫杉醇耐受患者(P<0.05)。对照者及各类型乳腺癌紫杉醇耐受患者IL-6、IL-8、CXCL-1表达水平比较,差异均有统计学意义(P<0.05)。紫杉醇耐受的各乳腺癌细胞株IRAK1 mRNA相对表达水平均高于未经紫杉醇处理的细胞株(P<0.05)。经shRNA转染的紫杉醇耐受的各乳腺癌细胞株Cyclin D1、PCNA、Cyclin A相对表达水平均低于经NC-shRNA转染的各乳腺癌细胞株,p53、c-myc、Bcl-2、c-erb-2相对表达水平均高于经NC-shRNA转染的各乳腺癌细胞株,Cullin 1、Bcl-6相对表达水平、ALDH活性均低于经NC-shRNA转染的各乳腺癌细胞株,KLF6相对表达水平高于经NC-shRNA转染的各乳腺癌细胞株(P<0.05)。结论 IRAK1在紫杉醇耐受的乳腺癌细胞,尤其是三阴乳腺癌细胞中发挥重要作用。抑制IRAK1表达可显著降低乳腺癌细胞的增殖和迁移能力,诱导癌细胞凋亡,IRAK1抑制剂可用于乳腺癌尤其是三阴乳腺癌的治疗。
Objective To investigate the mechanisms of interleukin-1 receptor-associated kinase 1( IRAK1) in the treatment for breast cancer patients with resistance to paclitaxel.Methods From May 2013 to September 2015,the Department of Oncology,the First Affiliated Hospital of Gannan Medical University,collected breast tissues of 16 control female( breast fibrous tumor) and those of patients with differential subtypes of breast cancer,including 38 Basal-like breast cancer,25Luminal-A breast cancer,31 Luminal-B breast cancer,31 HER2 overexpressed breast cancer,and 58 TNBC breast cancer.The relative expression level of IRAK1 mRNA in controls and different breast cancer patients were measured by real-time PCR.The level of inflammatory cytokines such as IL-6,IL-8,CXCL-1 were detected by ELISA.Breast cancer cell lines( HMEC,MCF7,MB415,MB436,MB468,BT549 and SUM159) were cultured.Through low concentration gradient increment combined with large dose intermittent shock,breast cancer cell lines obtained resistance to paclitaxel.The relative expression level of IRAK1 mRNA of various breast cancer cell lines with resistance to paclitaxel and without paclitaxel treatment were measured by real-time PCR.When breast cancer cell lines with resistance to paclitaxel were transfected by shRNA or blank sequence NC-shRNA,real-time PCR was performed to detect the relative expression level of proliferation-associated genes such as Cyclin D1,PCNA and Cyclin A,that of apoptosis-related genes such as p53,c-myc,Bcl-2 and c-erb-2,and that of migration-related genes such as Cullin 1,Bcl-6 and KLF6.ALDEFLUOR testing was used to detect the activity of aldehyde dehydrogenase( ALDH).Results The relative expression level of IRAK1 mRNA in various subtypes of paclitaxel-resistant breast cancer patients was much higher than that of the non-resistant patients( P < 0.05).The relative expression level of IRAK1 mRNA in paclitaxel-resistant TNBC was higher than that of other subtypes of paclitaxel-resistant patients( P < 0.05).The differences in the expression levels of IL-6,IL-8 and CXCL-1 between the controls and the various paclitaxel-resistant breast cancer patients showed statistical significance( P < 0.05).The relative expression level of IRAK1 mRNA in various subtypes of paclitaxel-resistant breast cancer cell lines was higher than that of the cell lines without paclitaxel treatment( P <0.05).Compared with the paclitaxel-resistant breast cancer cell lines with NC-shRNA transfection,the various paclitaxel-resistant breast cancer cell lines with shRNA tranfection had lower Cyclin D1,PCNA,Cyclin A,Cullin 1 and Bcl-6 relative expression levels and lower ALDH activity,but higher p53,c-myc,Bcl-2,c-erb-2 and KLF6 relative expression levels( P <0.05).Conclusion IRAK1 plays an essential role in paclitaxel-resistance breast cancer cells especially in TNBC.When the expression of IRAK1 was inhibited,the proliferation and migratory ability could be remarkably decreased inducing abundant breast cancer cell apoptosis,so IRAK1 inhibitors could be used in breast cancer therapy especially in TNBC.
Objective To investigate the mechanisms of interleukin-1 receptor-associated kinase 1( IRAK1) in the treatment for breast cancer patients with resistance to paclitaxel.Methods From May 2013 to September 2015,the Department of Oncology,the First Affiliated Hospital of Gannan Medical University,collected breast tissues of 16 control female( breast fibrous tumor) and those of patients with differential subtypes of breast cancer,including 38 Basal-like breast cancer,25Luminal-A breast cancer,31 Luminal-B breast cancer,31 HER2 overexpressed breast cancer,and 58 TNBC breast cancer.The relative expression level of IRAK1 mRNA in controls and different breast cancer patients were measured by real-time PCR.The level of inflammatory cytokines such as IL-6,IL-8,CXCL-1 were detected by ELISA.Breast cancer cell lines( HMEC,MCF7,MB415,MB436,MB468,BT549 and SUM159) were cultured.Through low concentration gradient increment combined with large dose intermittent shock,breast cancer cell lines obtained resistance to paclitaxel.The relative expression level of IRAK1 mRNA of various breast cancer cell lines with resistance to paclitaxel and without paclitaxel treatment were measured by real-time PCR.When breast cancer cell lines with resistance to paclitaxel were transfected by shRNA or blank sequence NC-shRNA,real-time PCR was performed to detect the relative expression level of proliferation-associated genes such as Cyclin D1,PCNA and Cyclin A,that of apoptosis-related genes such as p53,c-myc,Bcl-2 and c-erb-2,and that of migration-related genes such as Cullin 1,Bcl-6 and KLF6.ALDEFLUOR testing was used to detect the activity of aldehyde dehydrogenase( ALDH).Results The relative expression level of IRAK1 mRNA in various subtypes of paclitaxel-resistant breast cancer patients was much higher than that of the non-resistant patients( P < 0.05).The relative expression level of IRAK1 mRNA in paclitaxel-resistant TNBC was higher than that of other subtypes of paclitaxel-resistant patients( P < 0.05).The differences in the expression levels of IL-6,IL-8 and CXCL-1 between the controls and the various paclitaxel-resistant breast cancer patients showed statistical significance( P < 0.05).The relative expression level of IRAK1 mRNA in various subtypes of paclitaxel-resistant breast cancer cell lines was higher than that of the cell lines without paclitaxel treatment( P <0.05).Compared with the paclitaxel-resistant breast cancer cell lines with NC-shRNA transfection,the various paclitaxel-resistant breast cancer cell lines with shRNA tranfection had lower Cyclin D1,PCNA,Cyclin A,Cullin 1 and Bcl-6 relative expression levels and lower ALDH activity,but higher p53,c-myc,Bcl-2,c-erb-2 and KLF6 relative expression levels( P <0.05).Conclusion IRAK1 plays an essential role in paclitaxel-resistance breast cancer cells especially in TNBC.When the expression of IRAK1 was inhibited,the proliferation and migratory ability could be remarkably decreased inducing abundant breast cancer cell apoptosis,so IRAK1 inhibitors could be used in breast cancer therapy especially in TNBC.
引文
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[5]MIYATA M,LEE J Y,SUSUKI-MIYATA S,et al.Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M[J].Nat Commun,2015,6(3):6062.
[6]KIM J M,CHO H H,LEE S Y,et al.Role of IRAK1 on TNFinduced proliferation and NF-κB activation in human bone marrow mesenchymal stem cells[J].Cell Physiol Biochem,2012,30(1):49-60.
[7]RHYASEN G W,STARCZYNOWSKI D T.Starczynowski,IRAK signalling in cancer[J].Br J Cancer,2015,112(2):232-237.
[8]NIK-ZAINAL S,DAVIES H,STAAF J,et al.Landscape of somatic mutations in 560 breast cancer whole-genome sequences[J].Nature,2016,13(3):131-133.
[9]HURVITZ S A,ANDRE F,JIANG Z,et al.Combination of everolimus with trastuzumab plus paclitaxel as first-line treatment for patients with HER2-positive advanced breast cancer(BOLERO-1):a phase 3,randomised,double-blind,multicentre trial[J].Lancet Oncol,2015,16(7):816-829.
[10]CAMARDA R,ZHOU A Y,KOHNZ R A,et al.Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer[J].Nat Med,2016,22(4):427-432.
[11]SUN X,MAO Y,WANG J,et al.IL-6 secreted by cancerassociated fibroblasts induces tamoxifen resistance in luminal breast cancer[J].Oncogene,2014,15(3):124-132.
[12]LI Z,YOUNGER K,GARTENHAUS R,et al.Inhibition of IRAK1/4 sensitizes T cell acute lymphoblastic leukemia to chemotherapies[J].J Clin Invest,2015,125(3):1081-1097.
[13]ZHU T,LIU C L,ZHANG Y F,et al.A phaseⅡtrial of dosedense(biweekly)paclitaxel plus carboplatin as neoadjuvant chemotherapy for operable breast cancer[J].Breast Cancer Res Treat,2016,156(1):117-124.
[14]ANDERSON R A,WALLACE W H.Chemotherapy risks to fertility of childhood cancer survivors[J].Lancet Oncol,2016,11(3):121-126.
[15]DONG Y,YIN S,SONG X,et al.Involvement of ROS-p38-H2AX axis in novel curcumin analogues-induced apoptosis in breast cancer cells[J].Mol Carcinog,2016,55(4):323-334.
[16]KHORRAMI S,ZAVARAN HOSSEINI A,MOWLA S J,et al.Verification of ALDH activity as a biomarker in colon cancer stem cells-derived HT-29 cell line[J].Iran J Cancer Prev,2015,8(5):e3446.
[17]GORELIK M,ORLICKY S,SARTORI M A,et al.Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface[J].Proc Natl Acad Sci U S A,2016,113(13):3527-3532.
[18]CHIANG C,IHRIE R A.Controlling COR competence:Bcl-6regulates neurogenesis and tumor suppression[J].Cancer Cell,2014,26(6):773-774.
[19]MALLIPATTU S K,HE J C.KLF 6:a mitochondrial regulator in the kidney[J].Oncotarget,2015,6(18):15720-15721.
[2]SISTI J S,COLLINS L C,ELIASSEN A H,et al.Reproductive risk factors in relation to molecular subtypes of breast cancer:results from the nurses'health studies[J].Int J Cancer,2016,138(10):2346-2356.
[3]FERNANDES-ALNEMRI T,KANG S,ANDERSON E S,et al.Cutting edge:TLR signaling licenses IRAK1 for rapid activation of the NLRP3 inflammasome[J].J Immunol,2013,191(8):3995
[4]BURNS K,JANSSENS S,BRISSON B,et al.Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced,short form of My D88 is due to its failure to recruit IRAK-4[J].J Exp Med,2003,197(2):263-268.
[5]MIYATA M,LEE J Y,SUSUKI-MIYATA S,et al.Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M[J].Nat Commun,2015,6(3):6062.
[6]KIM J M,CHO H H,LEE S Y,et al.Role of IRAK1 on TNFinduced proliferation and NF-κB activation in human bone marrow mesenchymal stem cells[J].Cell Physiol Biochem,2012,30(1):49-60.
[7]RHYASEN G W,STARCZYNOWSKI D T.Starczynowski,IRAK signalling in cancer[J].Br J Cancer,2015,112(2):232-237.
[8]NIK-ZAINAL S,DAVIES H,STAAF J,et al.Landscape of somatic mutations in 560 breast cancer whole-genome sequences[J].Nature,2016,13(3):131-133.
[9]HURVITZ S A,ANDRE F,JIANG Z,et al.Combination of everolimus with trastuzumab plus paclitaxel as first-line treatment for patients with HER2-positive advanced breast cancer(BOLERO-1):a phase 3,randomised,double-blind,multicentre trial[J].Lancet Oncol,2015,16(7):816-829.
[10]CAMARDA R,ZHOU A Y,KOHNZ R A,et al.Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer[J].Nat Med,2016,22(4):427-432.
[11]SUN X,MAO Y,WANG J,et al.IL-6 secreted by cancerassociated fibroblasts induces tamoxifen resistance in luminal breast cancer[J].Oncogene,2014,15(3):124-132.
[12]LI Z,YOUNGER K,GARTENHAUS R,et al.Inhibition of IRAK1/4 sensitizes T cell acute lymphoblastic leukemia to chemotherapies[J].J Clin Invest,2015,125(3):1081-1097.
[13]ZHU T,LIU C L,ZHANG Y F,et al.A phaseⅡtrial of dosedense(biweekly)paclitaxel plus carboplatin as neoadjuvant chemotherapy for operable breast cancer[J].Breast Cancer Res Treat,2016,156(1):117-124.
[14]ANDERSON R A,WALLACE W H.Chemotherapy risks to fertility of childhood cancer survivors[J].Lancet Oncol,2016,11(3):121-126.
[15]DONG Y,YIN S,SONG X,et al.Involvement of ROS-p38-H2AX axis in novel curcumin analogues-induced apoptosis in breast cancer cells[J].Mol Carcinog,2016,55(4):323-334.
[16]KHORRAMI S,ZAVARAN HOSSEINI A,MOWLA S J,et al.Verification of ALDH activity as a biomarker in colon cancer stem cells-derived HT-29 cell line[J].Iran J Cancer Prev,2015,8(5):e3446.
[17]GORELIK M,ORLICKY S,SARTORI M A,et al.Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface[J].Proc Natl Acad Sci U S A,2016,113(13):3527-3532.
[18]CHIANG C,IHRIE R A.Controlling COR competence:Bcl-6regulates neurogenesis and tumor suppression[J].Cancer Cell,2014,26(6):773-774.
[19]MALLIPATTU S K,HE J C.KLF 6:a mitochondrial regulator in the kidney[J].Oncotarget,2015,6(18):15720-15721.