Role And Mechanism Of ROS In Breast Cancer Epithelial Cell Polarity Disruption And Monocyte Infiltration

BACKGROUND:Cell polarity is a fundamental feature of normal epithelial tissue,and loss of cell polarity is an early cellular event during breast cancer development.Disruption of cell polarity causes unnormal tissue organization,increases susceptibility to breast cancer and ultimately leads to formation of breast cancer.ROS and associated oxidative stress are considered to be the driving force of breast cancer development and progression.ROS induce oxidative damages in DNA,lipids,proteins and other cellular components and the damages are sufficient to induce malignant transformation.However,it is unclear whether ROS levels help to determine the maintenance of epithelial cell polarity,and whether help to determine the infiltration of monocyte.OBJECTIVE:To explore the association between the increased levels of ROS and the destruction of normal polarization acinar structure following by the infiltration of monocyte,to confirm that elevated levels of ROS is necessary and sufficient to disrupt mammary epithelial cell polarity and promote monocyte infiltration,ultimately define the molecular mechanism of ROS in disruption cell polarity and monocyte infiltration.METHODS:1.In this thesis project,a 3D culture system was optimized in vitro to simulate the physiological microenvironment in vivo,and the human breast cancer progression series HMT-3522(the non-polarized S1 cell line and its polarized counterpart T4-2)and the normal human mammary epithelial cells MCF-10 A were 3D cultured in vitro.The cell polarity was authenticated using the basal marker ?6-integrin.The cell proliferation was assessed by quantification of proportion of cells with Ki-67 positive staining.Cellular ROS levels were assessed and quantified by Cell ROX Deep Red staining.2.T4-2 cells were phenotypically reversed into T4 R cells by tyrphostin AG 1478(TYR),an EGFR inhibitor;GM6001(GM),a MMP inhibitor;or LY294002(LY),a PI3 K inhibitor.Non-polarized T4-2 and polarized S1 and T4 R cells were cultured in 3D Matrigel and stained with Cell ROX Deep Red,followed by live cell imaging and quantification to evaluate ROS levels.3.Disruption of the acinar structure during breast cancer development is accompanied by loss of tissue polarity and increased cell proliferation,so we constitutively active RAC1 L61 and dominantly active Myr-Akt are introduced into S1,T4-2 and MCF-10 A cells respectively,to decouple cell proliferation from polarity establishment/disruption,to investigate whether cell polarity damage caused by elevated levels of ROS depend on cell proliferation.4.To investigate whether increased ROS levels contribute to the disruption of polarized acinar structures in 3D culture,we lowered ROS levels in T4-2 cells using antioxidants NAC(N-Acetyl-L-cysteine,NAC)or GSH(L-glutathione Reduced,GSH),and increased ROS levels in S1 cells or MCF-10 A cells with GO(Glucose Oxidase,GO).5.To recapitulate the interaction of mammary epithelial cells with monocytes during breast tumorigenesis,we developed a 3D co-culture assay in vitro.We 3D co-cultured of HMT-3522/MCF-10 A cells with monocyte(GFP-expressing THP-1 cells,human CD14+ monocytes labeled with Cell Tracker Green CMFDA),and followed by fluorescence imaging,to investigate whether increased ROS levels contribute to monocyte infiltration.6.Luciferase assay and western blot analysis were used to determine NF-?B activity.Compared expression of cytokines in non-polarized T4-2 cells and with expression in polarized S1 and T4 R cells by q RT-PCR and ELISA,to investigate whether loss of tissue polarity enhances cytokine expression and subsequently promotes monocyte infiltration.7.Oxygen consumption rate(OCR)in S1 and T4-2 cells were performed with a Seahorse Bioscience XF96 extracellular flux analyzer,and q RT-PCR and ELISA were used to determine NDUFA11 expression level.RESULTS:1.The association between ROS levels and breast cancer epithelial cell polarization: Cell ROX Deep Red staining revealed that non-polarized breast cancer cell line,T4-2,generated significantly higher levels of ROS compared to the polarized S1 cells in 3D culture,approximately 1.96 times than that of S1 cells(p<0.05).The ROS levels decreased when T4-2 cells phenotypically reverted by TYR,GM or LY,respectively reduced to 40%,53% and 59%(p<0.05).2.Breast cancer epithelial cell polarity disruption and cell proliferation:(1)Introduction of RAC1 L61,a constitutively active RAC1,into T4-2 cells significantly elevated ROS levels in TYR treated T4-2 cells(p<0.05),inhibited restoration of polarity in response to TYR(p<0.001),but had little effect on cell proliferation(p=0.42).However,expression of the dominant-active Akt(Myr-Akt)only slightly increased ROS levels in TYR treated T4-2 cells(p=0.07),although cell proliferation was significantly increased(p<0.001).(2)Over-expression of RAC1 L61 significantly elevated ROS levels in polarized S1 cells(p<0.05),followed by polarity disruption.Over-expression of Myr-Akt in S1 cells only increased cell proliferation but not significantly affected the levels of ROS(p=0.09).(3)Over-expression of RAC1 L61 significantly elevated ROS levels in polarized MCF-10 A cells(p<0.001),followed by polarity disruption.Over-expression of Myr-Akt in MCF-10 A cells only increased cell proliferation but not significantly affected the levels of ROS(p=0.10).3.ROS levels and polarized acinar structure formation: Treatment with antioxidant NAC or GSH significantly reduced ROS levels in 3D cultured T4-2 cells(p<0.01),and reprogramed the cells to form polarized spheroids(p<0.001)and inhibited cell proliferation(p<0.001);GO treatment elevated ROS levels in 3D cultured S1 cells(p<0.001),and impaired polarized acinar formation(p<0.001);GO treatment elevated ROS levels in 3D cultured MCF-10 A cells(p<0.001),and impaired polarized acinar formation(p<0.05).4.Disruption of polarized acinar structure promotes monocyte infiltration: Non-polarized T4-2 cells recruitmented the infiltration of monocyte in the 3D co-culture system.Reduced ROS levels with NAC or GSH in T4-2 cells inhibited monocyte infiltration(p<0.001),whereas elevated ROS levels with GO in S1 cells promoted monocyte infiltration(p<0.001).The infiltration of monocyte is associated with the destruction polarity of breast cancer cells caused by ROS.5.The correlation between ROS levels and NF-?B activity and cytokine expression in mammary epithelial cells:(1)The results of luciferase activity assay showed that the transactivation activities of NF-?B were significantly reduced when T4-2 cells reverted by TYR(T4R cells).NAC treatment significantly inhibited NF-?B activity in 3D cultured T4-2 cells.(2)Quantification of immunoblotting results showed that,in non-polarized T4-2 cells Ser536 phosphorylation of p65(p-p65)was increased to 2.50 times(p<0.05)that of polarized S1 cells,nuclear translocation of p65 was increased to 1.43 times(p<0.05).The expression levels of p-p65 and nuclear p65 decreased to 60%,50% when T4-2 cells polarity were reverted by TYR(p<0.05).In 3D cultured T4-2 cells NAC significantly inhibited phosphorylation of NF-?B p65 at Ser536 to 50%(p<0.05),and also significantly inhibited the nuclear translocation of p65 to 40%(p<0.05).(3)q RT-PCR results demonstrated that the cytokines interleukin(IL)-24,CXCL1,CXCL3 and CXCL8 were significantly upregulated in non-polarized T4-2 cells compared to polarized S1 cells,respectively increased to 131 times,78 times,21 times that of S1 cells(p<0.05).IL-24,CXCL1,CXCL3 and CXCL8 m RNA expression levels decreased when T4-2 cells polarity were reverted by TYR.Treatment with the NF-?B inhibitor Wedelolactone in T4-2 cells significantly reduced m RNA levels of IL-6,IL-24,IL-32,CXCL1,CXCL2,CXCL3,CXCL6 and CXCL8 to 70%,51%,74%,50%,55%,37%,39% and 73%(p<0.05).NAC treatment significantly decreased m RNA levels of IL-6,IL-24,CXCL1,CXCL2,CXCL3 and CXCL8 to 19%,27%,15%,35%,18% and 14% in T4-2 cells(p<0.05).GO treatment in S1 cells significantly increased m RNA levels of IL-6,IL-24,IL-32,CXCL1,CXCL2,CXCL3 and CXCL8 to 6.87 times,7.27 times,3.43 times,4.46 times,2.79 times,4.28 times and 14.66 times(p<0.05).(4)ELISA assay showed that the secretion of IL-6 and CXCL-1 was significantly increased in the T4-2 cells compared to the S1 cells,respectively increased to 241 times,4 times that of S1 cells(p<0.05).And NAC treatment significantly inhibited the secretion of IL-6,CXCL-1 to 50%,49% in 3D cultured T4-2 cells(p<0.05).6.The correlation between ROS levels and mitochondrial oxidative phosphorylation: The seahorse experiment showed that T4-2 cells exhibited significantly up-regulated basal respiration,ATP turnover,Max respiration,and Spare respiration capacity compared to S1 cells,respectively increased to 1.27 times,1.46 times,1.41 times and 1.66 times that of S1 cells(p<0.05).q RT-PCR and western blot results demonstrated that NDUFA11 was significantly up-regulated in non-polarized T4-2 cells compared to polarized S1 cells.Oxidative phosphorylation is elevated in non-polarized T4-2 cells,which may contribute to ROS generation.CONCLUSIONS:1.RAC1 is a key regulator which integrates non-polarized tissue formation and ROS production,and RAC1 activation increases ROS production and disrupts polarized acinar formation.2.Increased ROS production disrupts breast cancer epithelial cell polarity,and disruption of the polarized acinar structure directly elevates ROS levels.Elevation of ROS is necessary and sufficient to disrupt polarized acinar structure formation,and cell polarity damage caused by elevated levels of ROS does not depend on cell proliferation.3.Elevation of ROS is necessary to disrupt polarized acinar formation,followed by activating NF-?B pathway to secrete a large number of cytokines,such as IL-6,IL-24,IL-32,CXCL1,CXCL2,CXCL3,CXCL6 and CXCL8.And these cytokines recruit monocytes/macrophages to reach the tumor site,promoting breast cancer development.4.NDUFA11 expression in mitochondria in the non-polarized breast cancer cells is increased,and oxidative phosphorylation is elevated,contributing to ROS generation in the non-polarized breast cancer cells.