γ干扰素通过c-Abl/HDAC2信号通路调节乳腺上皮细胞恶性生长（英文）
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摘要
目的:在之前的研究中我们发现γ干扰素(IFN-γ)通过营养感受器GCN2诱导牛乳腺上皮细胞(BMEC)的恶性转化。在恶性转化的表型中,包括细胞周期缩短、细胞增殖增加、细胞迁移和侵袭的发生,而细胞生长的异常调节是细胞恶性转化的第一步。因此,本研究旨在探讨IFN-γ诱导细胞恶性生长的分子机制。创新点:实验对象γ-BMEC能够更好的用于乳腺癌发生的基础研究,是一种新的研究工具。我们选择恶性细胞生长来详细研究IFN-γ的作用机制,为IFN-γ、恶性细胞生长甚至乳腺癌之间的密切关系提供直接证据。方法:通过MTT实验检测IFN-γ长期刺激下细胞的增殖能力;蛋白质印迹(Westernblot)检测cyclin D1/CDK4、p21、HDAC2、c-Abl蛋白的表达;免疫荧光观察c-Abl入核。结论:IFN-γ通过c-Abl/HDAC2信号通路促进恶性BMEC的生长。
Interferon-γ(IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells(BMECs) in vitro. In this study, we investigated the mechanisms underlying pthe malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ(10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4(CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene(c-Abl) and histone deacetylase 2(HDAC2). The HDAC2 inhibitor, valproate(VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.
Interferon-γ(IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells(BMECs) in vitro. In this study, we investigated the mechanisms underlying pthe malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ(10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4(CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene(c-Abl) and histone deacetylase 2(HDAC2). The HDAC2 inhibitor, valproate(VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.
引文
Allington TM,Galliher-Beckley AJ,Schiemann WP,2009.Activated Abl kinase inhibits oncogenic transforming growth factor-βsignaling and tumorigenesis in mammary tumors.Faseb J,23(12):4231-4243.https://doi.org/10.1096/fj.09-138412
Barneda-Zahonero B,Parra M,2012.Histone deacetylases and cancer.Mol Oncol,6(6):579-589.https://doi.org/10.1016/j.molonc.2012.07.003
Bougarn S,Cunha P,Gilbert FB,et al.,2011.Technical note:validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli.J Dairy Sci,94(5):2425-2430.https://doi.org/10.3168/jds.2010-3859
Bradley WD,Koleske AJ,2009.Regulation of cell migration and morphogenesis by Abl-family kinases:emerging mechanisms and physiological contexts.J Cell Sci,122(19):3441-3454.https://doi.org/10.1242/jcs.039859
Brightbill H,Schlissel MS,2009.The effects of c-Abl mutation on developing B cell differentiation and survival.Int Immunol,21(5):575-585.https://doi.org/10.1093/intimm/dxp027
Carpi A,Nicolini A,Antonelli A,et al.,2009.Cytokines in the management of high risk or advanced breast cancer:an update and expectation.Curr Cancer Drug Tar,9(8):9888-9903.https://doi.org/10.2174/156800909790192392
Creagan ET,Schaid DJ,Ahmann DL,et al.,1990.Disseminated malignant melanoma and recombinant interferon:analysis of seven consecutive phase II investigations.JInvest Dermatol,95(S6):S188-S192.https://doi.org/10.1111/1523-1747.ep12875512
Gonzalez-Zu?iga M,Contreras PS,Estrada LD,et al.,2014.c-Abl stabilizes HDAC2 levels by tyrosine phosphorylation repressing neuronal gene expression in Alzheimer’s disease.Mol Cell,56(1):163-173.https://doi.org/10.1016/j.molcel.2014.08.013
Hildmann C,Riester D,Schwienhorst A,2007.Histone deacetylases-an important class of cellular regulators with a variety of functions.Appl Microbiol Biotechnol,75(3):487-497.https://doi.org/10.1007/s00253-007-0911-2
Horikoshi T,Fukuzawa KF,Hanada N,et al.,1995.In vitro comparative study of the antitumor effects of human interferon-α,βandγon the growth and invasive potential of human melanoma cells.J Dermatol,22(9):631-636.https://doi.org/10.1111/j.1346-8138.1995.tb03889
Kaplan DH,Shankaran V,Dighe AS,et al.,1998.Demonstration of an interferonγ-dependent tumor surveillance system in immunocompetent mice.Proc Natl Acad Sci USA,95(13):7556-7561.https://doi.org/10.1073/pnas.95.13.7556
Kr?mer OH,2009.HDAC2:a critical factor in health and disease.Trends Pharmacol Sci,30(12):647-655.https://doi.org/10.1016/j.tips.2009.09.007
Kr?mer OH,Zhu P,Ostendorff HP,et al.,2003.The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2.EMBO J,22(13):3411-3420.https://doi.org/10.1093/emboj/cdg315
Matsuzaki J,Tsuji T,Luescher IF,et al.,2015.Direct tumor recognition by a human CD4+T-cell subset potently mediates tumor growth inhibition and orchestrates antitumor immune responses.Sci Rep,5:14896.https://doi.org/10.1038/srep14896
Mojic M,Takeda K,Hayakawa Y,2018.The dark side of IFN-γ:its role in promoting cancer immunoeevasion.Int J Mol Sci,19(1):89.https://doi.org/10.3390/ijms19010089
Montgomery RL,Davis CA,Potthoff MJ,et al.,2007.Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis,growth,and contractility.Gene Dev,21(14):1790-1802.https://doi.org/10.1101/gad.1563807
Müller BM,Jana L,Kasajima A,et al.,2013.Differential expression of histone deacetylases HDAC1,2 and 3 in human breast cancer-overexpression of HDAC2 and HDAC3 is associated with clinicopathological indicators of disease progression.BMC Cancer,13:215.https://doi.org/10.1186/1471-2407-13-215
Muss HB,Caponera M,Zekan PJ,et al.,1986.Recombinant gamma interferon in advanced breast cancer:a phase IItrial.Invest New Drugs,4(4):377-381.https://doi.org/10.1007/BF00173511
Nam SW,Park JY,Ramasamy A,et al.,2005.Molecular changes from dysplastic nodule to hepatocellular carcinoma through gene expression profiling.Hepatology,42(4):809-818.https://doi.org/10.1002/hep.20878
Noh JH,Jung KH,Kim JK,et al.,2011.Aberrant regulation of HDAC2 mediates proliferation of hepatocellular carcinoma cells by deregulating expression of G1/S cell cycle proteins.PLo S ONE,6(11):e28103.https://doi.org/10.1371/journal.pone.0028103
Ren WB,Li Y,Xia XJ,et al.,2018.Arginine inhibits the malignant transformation induced by interferon-gamma through the NF-κB-GCN2/eIF2αsignaling pathway in mammary epithelial cells in vitro and in vivo.Exp Cell Res,368(2):236-247.https://doi.org/10.1016/j.yexcr.2018.05.003
Sarhan D,D'Arcy P,Wennerberg E,et al.,2013.Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ.Euro J Immunol,43(1):249-257.https://doi.org/10.1002/eji.201242735
Schiller JH,Pugh M,Kirkwood JM,et al.,1996.Eastern cooperative group trial of interferon gamma in metastatic melanoma:an innovative study design.Clin Cancer Res,2(1):29-36.
Trivedi CM,Zhu WT,Wang QH,et al.,2010.Hopx and Hdac2interact to modulate Gata4 acetylation and embryonic cardiac Myocyte proliferation.Dev Cell,19(3):450-459.https://doi.org/10.1016/j.devcel.2010.08.012
Wang JYJ,2014.The capable ABL:what is its biological function?Mol Cell Biol,34(7):1188-1197.https://doi.org/10.1128/MCB.01454-13
Weichert W,R?ske A,Gekeler V,et al.,2008.Histone deacetylases 1,2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy.Br J Cancer,98(3):604-610.https://doi.org/10.1038/sj.bjc.6604199
Xia XJ,Che YY,Gao YY,et al.,2016a.Arginine supplementation recovered the IFN-γ-mediated decrease in milk protein and fat synthesis by inhibiting the GCN2/eIF2αpathway,which induces autophagy in primary bovine mammary epithelial cells.Mol Cell,39(5):410-417.https://doi.org/10.14348/molcells.2016.2358
Xia XJ,Gao YY,Zhang J,et al.,2016b.Autophagy mediated by arginine depletion activation of the nutrient sensor GCN2 contributes to interferon-γ-induced malignant transformation of primary bovine mammary epithelial cells.Cell Death Discov,2:15065.https://doi.org/10.1038/cddiscovery.2015.65
Xia XJ,Che YY,Zhang J,et al.,2016c.Diet-driven interferon-γenhances malignant transformation of primary bovine mammary epithelial cells through nutrient sensor GCN2-activated autophagy.Cell Death Dis,7(3):e2138.https://doi.org/10.1038/cddis.2016.48
Yamaguchi T,Cubizolles F,Zhang Y,et al.,2010.Histone deacetylases 1 and 2 act in concert to promote the G1-to-Sprogression.Genes Dev,24(5):455-469.https://doi.org/10.1101/gad.552310
Zaidi MR,Merlino G,2011.The two faces of interferon-γin cancer.Clin Cancer Res,17(19):6118-6124.https://doi.org/10.1158/1078-0432.CCR-11-0482
Zhao HJ,Ho PC,Lo YH,et al.,2012.Interaction of proliferation cell nuclear antigen(PCNA)with c-Abl in cell proliferation and response to DNA damages in breast cancer.PLo S ONE,7(1):e29416.https://doi.org/10.1371/journal.pone.0029416
Zhao HJ,Chen MS,Lo YH,et al.,2013.The Ron receptor tyrosine kinase activates c-Abl to promote cell proliferation through tyrosine phosphorylation of PCNA in breast cancer.Oncogene,33(11):1429-1437.https://doi.org/10.1038/onc.2013.84
Zhou HY,Cai Y,Liu DN,et al.,2018.Pharmacological or transcriptional inhibition of both HDAC1 and 2 leads to cell cycle blockage and apoptosis via p21Waf1/Cip1 and p19INK4d upregulation in hepatocellular carcinoma.Cell Proliferat,51(3):e12447.https://doi.org/10.1111/cpr.12447
Zhu JF,Yamane H,Paul WE,2010.Differentiation of effector CD4 T cell populations.Annu Rev Immunol,28:445-489.https://doi.org/10.1146/annurev-immunol-030409-101212
Zuo H,Tell GS,Vollset SE,et al.,2014.Interferon-γ-induced inflammatory markers and the risk of cancer:the hordaland health study.Cancer,120(21):3370-3377.https://doi.org/10.1002/cncr.28869
Barneda-Zahonero B,Parra M,2012.Histone deacetylases and cancer.Mol Oncol,6(6):579-589.https://doi.org/10.1016/j.molonc.2012.07.003
Bougarn S,Cunha P,Gilbert FB,et al.,2011.Technical note:validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli.J Dairy Sci,94(5):2425-2430.https://doi.org/10.3168/jds.2010-3859
Bradley WD,Koleske AJ,2009.Regulation of cell migration and morphogenesis by Abl-family kinases:emerging mechanisms and physiological contexts.J Cell Sci,122(19):3441-3454.https://doi.org/10.1242/jcs.039859
Brightbill H,Schlissel MS,2009.The effects of c-Abl mutation on developing B cell differentiation and survival.Int Immunol,21(5):575-585.https://doi.org/10.1093/intimm/dxp027
Carpi A,Nicolini A,Antonelli A,et al.,2009.Cytokines in the management of high risk or advanced breast cancer:an update and expectation.Curr Cancer Drug Tar,9(8):9888-9903.https://doi.org/10.2174/156800909790192392
Creagan ET,Schaid DJ,Ahmann DL,et al.,1990.Disseminated malignant melanoma and recombinant interferon:analysis of seven consecutive phase II investigations.JInvest Dermatol,95(S6):S188-S192.https://doi.org/10.1111/1523-1747.ep12875512
Gonzalez-Zu?iga M,Contreras PS,Estrada LD,et al.,2014.c-Abl stabilizes HDAC2 levels by tyrosine phosphorylation repressing neuronal gene expression in Alzheimer’s disease.Mol Cell,56(1):163-173.https://doi.org/10.1016/j.molcel.2014.08.013
Hildmann C,Riester D,Schwienhorst A,2007.Histone deacetylases-an important class of cellular regulators with a variety of functions.Appl Microbiol Biotechnol,75(3):487-497.https://doi.org/10.1007/s00253-007-0911-2
Horikoshi T,Fukuzawa KF,Hanada N,et al.,1995.In vitro comparative study of the antitumor effects of human interferon-α,βandγon the growth and invasive potential of human melanoma cells.J Dermatol,22(9):631-636.https://doi.org/10.1111/j.1346-8138.1995.tb03889
Kaplan DH,Shankaran V,Dighe AS,et al.,1998.Demonstration of an interferonγ-dependent tumor surveillance system in immunocompetent mice.Proc Natl Acad Sci USA,95(13):7556-7561.https://doi.org/10.1073/pnas.95.13.7556
Kr?mer OH,2009.HDAC2:a critical factor in health and disease.Trends Pharmacol Sci,30(12):647-655.https://doi.org/10.1016/j.tips.2009.09.007
Kr?mer OH,Zhu P,Ostendorff HP,et al.,2003.The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2.EMBO J,22(13):3411-3420.https://doi.org/10.1093/emboj/cdg315
Matsuzaki J,Tsuji T,Luescher IF,et al.,2015.Direct tumor recognition by a human CD4+T-cell subset potently mediates tumor growth inhibition and orchestrates antitumor immune responses.Sci Rep,5:14896.https://doi.org/10.1038/srep14896
Mojic M,Takeda K,Hayakawa Y,2018.The dark side of IFN-γ:its role in promoting cancer immunoeevasion.Int J Mol Sci,19(1):89.https://doi.org/10.3390/ijms19010089
Montgomery RL,Davis CA,Potthoff MJ,et al.,2007.Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis,growth,and contractility.Gene Dev,21(14):1790-1802.https://doi.org/10.1101/gad.1563807
Müller BM,Jana L,Kasajima A,et al.,2013.Differential expression of histone deacetylases HDAC1,2 and 3 in human breast cancer-overexpression of HDAC2 and HDAC3 is associated with clinicopathological indicators of disease progression.BMC Cancer,13:215.https://doi.org/10.1186/1471-2407-13-215
Muss HB,Caponera M,Zekan PJ,et al.,1986.Recombinant gamma interferon in advanced breast cancer:a phase IItrial.Invest New Drugs,4(4):377-381.https://doi.org/10.1007/BF00173511
Nam SW,Park JY,Ramasamy A,et al.,2005.Molecular changes from dysplastic nodule to hepatocellular carcinoma through gene expression profiling.Hepatology,42(4):809-818.https://doi.org/10.1002/hep.20878
Noh JH,Jung KH,Kim JK,et al.,2011.Aberrant regulation of HDAC2 mediates proliferation of hepatocellular carcinoma cells by deregulating expression of G1/S cell cycle proteins.PLo S ONE,6(11):e28103.https://doi.org/10.1371/journal.pone.0028103
Ren WB,Li Y,Xia XJ,et al.,2018.Arginine inhibits the malignant transformation induced by interferon-gamma through the NF-κB-GCN2/eIF2αsignaling pathway in mammary epithelial cells in vitro and in vivo.Exp Cell Res,368(2):236-247.https://doi.org/10.1016/j.yexcr.2018.05.003
Sarhan D,D'Arcy P,Wennerberg E,et al.,2013.Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ.Euro J Immunol,43(1):249-257.https://doi.org/10.1002/eji.201242735
Schiller JH,Pugh M,Kirkwood JM,et al.,1996.Eastern cooperative group trial of interferon gamma in metastatic melanoma:an innovative study design.Clin Cancer Res,2(1):29-36.
Trivedi CM,Zhu WT,Wang QH,et al.,2010.Hopx and Hdac2interact to modulate Gata4 acetylation and embryonic cardiac Myocyte proliferation.Dev Cell,19(3):450-459.https://doi.org/10.1016/j.devcel.2010.08.012
Wang JYJ,2014.The capable ABL:what is its biological function?Mol Cell Biol,34(7):1188-1197.https://doi.org/10.1128/MCB.01454-13
Weichert W,R?ske A,Gekeler V,et al.,2008.Histone deacetylases 1,2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy.Br J Cancer,98(3):604-610.https://doi.org/10.1038/sj.bjc.6604199
Xia XJ,Che YY,Gao YY,et al.,2016a.Arginine supplementation recovered the IFN-γ-mediated decrease in milk protein and fat synthesis by inhibiting the GCN2/eIF2αpathway,which induces autophagy in primary bovine mammary epithelial cells.Mol Cell,39(5):410-417.https://doi.org/10.14348/molcells.2016.2358
Xia XJ,Gao YY,Zhang J,et al.,2016b.Autophagy mediated by arginine depletion activation of the nutrient sensor GCN2 contributes to interferon-γ-induced malignant transformation of primary bovine mammary epithelial cells.Cell Death Discov,2:15065.https://doi.org/10.1038/cddiscovery.2015.65
Xia XJ,Che YY,Zhang J,et al.,2016c.Diet-driven interferon-γenhances malignant transformation of primary bovine mammary epithelial cells through nutrient sensor GCN2-activated autophagy.Cell Death Dis,7(3):e2138.https://doi.org/10.1038/cddis.2016.48
Yamaguchi T,Cubizolles F,Zhang Y,et al.,2010.Histone deacetylases 1 and 2 act in concert to promote the G1-to-Sprogression.Genes Dev,24(5):455-469.https://doi.org/10.1101/gad.552310
Zaidi MR,Merlino G,2011.The two faces of interferon-γin cancer.Clin Cancer Res,17(19):6118-6124.https://doi.org/10.1158/1078-0432.CCR-11-0482
Zhao HJ,Ho PC,Lo YH,et al.,2012.Interaction of proliferation cell nuclear antigen(PCNA)with c-Abl in cell proliferation and response to DNA damages in breast cancer.PLo S ONE,7(1):e29416.https://doi.org/10.1371/journal.pone.0029416
Zhao HJ,Chen MS,Lo YH,et al.,2013.The Ron receptor tyrosine kinase activates c-Abl to promote cell proliferation through tyrosine phosphorylation of PCNA in breast cancer.Oncogene,33(11):1429-1437.https://doi.org/10.1038/onc.2013.84
Zhou HY,Cai Y,Liu DN,et al.,2018.Pharmacological or transcriptional inhibition of both HDAC1 and 2 leads to cell cycle blockage and apoptosis via p21Waf1/Cip1 and p19INK4d upregulation in hepatocellular carcinoma.Cell Proliferat,51(3):e12447.https://doi.org/10.1111/cpr.12447
Zhu JF,Yamane H,Paul WE,2010.Differentiation of effector CD4 T cell populations.Annu Rev Immunol,28:445-489.https://doi.org/10.1146/annurev-immunol-030409-101212
Zuo H,Tell GS,Vollset SE,et al.,2014.Interferon-γ-induced inflammatory markers and the risk of cancer:the hordaland health study.Cancer,120(21):3370-3377.https://doi.org/10.1002/cncr.28869
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