| The overexpression of human epidermal growth factor(HER2)is related to the development,drug resistance and poor prognosis of human breast cancer cells.HER2 forms dimers with other members of epidermal growth factor receptor(EGFR)family to activate downstream signaling pathways,such as mitogen-activated protein kinase(MAPK),phosphatidylinositol 3-kinase(PI3K),signal transducer and activator of transcription 3(STAT3),to mediates the proliferation of breast cancer cells and resistance to anticancer drugs.Endocrine therapy and chemotherapy are less effective in obese breast cancer patients.Hypercholesterolemia,an important comorbidity of obesity,has been proved to be an independent risk factor for ER positive breast cancer,and is related to the decreased responsiveness to endocrine therapy of breast cancer cells.It is not cholesterol,but its main metabolite27-hydroxycholesterol(27-HC)plays an important role in it.27-HC can activate inflammatory signals that are indispensable in the occurrence and development of tumors,and can also cause a sharp increase in reactive oxygen species(ROS),then promote oxidative sress that regulates the occurrence,development and metastasis of cancer.Cancer cells show high levels of ROS,which can activate downstream growth factor signals and induce the expression of resistance-related membrane proteins,ultimately promoting the invasion,metastasis,proliferation,angiogenesis and multidrug resistance(MDR)of breast cancer cells.Some studies have shown that there is crosstalk between oxidative stress and HER2,ROS can promote the expression and/or activity of HER2.Therefore,we speculate that if ER positive breast cancer cells are exposed to27-HC for a long period of time,wether the relevant growth facter signals will contine to be activated,and wether these signals will affect the expression of HER2 and eventually induce multidrug resistance?Objective: In this study,breast cancer cells with positive ER alpha and low HER2 expression were used to observe the changes in the protein levels of HER2,p-STAT3 and p-ERK1/2 under long-term low-dose 27-HC exposure,and to explore the relationship between STAT3,ERK1/2 signals and HER2,to clarify the mechanism that may lead to multidrug resistance,and to explore possible strategies for the treatment of breast cancer patients who is obesity,hypercholesterolemia.Methods: We used Western Blot assay to detect the protein expression of HER2,p-STAT3 and p-ERK1/2.Real-time Quantitative PCR was adopted to detect the m RNA level of HER2,IL-6 and IL-8.The cell proliferation activity under different treatments was detected by CCK-8 assay.Results:一、Long-term low-dose exposure of 27-HC induces multidrug resistance in ER alpha positive breast cancer cellsTwo ER alpha positive breast cancer cells(T47D,MCF-7)were treated with27-HC at the concentration of 0.5,1μM for 3 months.It was found that both of them had a higher survival rate at 0.5μM concentration,and their sensitivity to anticancer drugs was reduced,such as tamoxifen(TAM)and fulvestrant(ICI),which were used in endocrine therapy,and doxorubicin(DOX)and paclitaxel(PTX),which were used in chemotherapy.Western Blot results showed that the expression of proliferating cell nuclear antigen(PCNA)increased.二、The role of HER2 in 27-HC-induced multidrug resistance of ER alpha positive breast cancer cells1、Long-term low-dose exposure of 27-HC affects the expression of HER2T47D and MCF-7 cells were treated with 0.5μM 27-HC for 3 months.The expression and m RNA of HER2 increased in both of two cells.2、HER2 affects the sensitivity of ER alpha positive breast cancer cells to commonly used anticancer drugsKnockdown of HER2 with si RNA in the 27-HC-induced drug-resistant cells(T47D-R,MCF-7-R)restored the sensitivity to TAM,ICI,DOX and PTX,and reduced the expression of PCNA.Overexpression of HER2 in the sensitive cells(T47D-S,MCF-7-S)reduced its sensitivity to these anticancer drugs,and increased the expression of PCNA.三、HER2 forms a feedback loop with STAT3 and ERK1/2 to participate in the multidrug resistance of ER alpha positive breast cancer cells induced by 27-HC1、Long-term low-dose exposure of 27-HC activated STAT3 and ERK1/2signaling pathwaysWestern Blot results showed that the phosphorylation of STAT3 and ERK1/2protein increased in T47D-R and MCF-7-R.The q RT-PCR results showed that m RNA of IL-6 and IL-8 also increased accordingly.2、STAT3 and ERK1/2 signals affects the sensitivity of ER alpha positive breast cancer cells to commonly used anticancer drugsPretreatment of T47D-R and MCF-7-R with Stattic,the STAT3 pathway inhibitor,restored the sensitivity of these induced drug-resistant cells to TAM,ICI,DOX and PTX.At the same time,the expression of PCNA reduced.Treating T47D-R and MCF-7-R with U0126,the inhibitor of ERK1/2 signal,also restored the sensitivity to these anticancer drugs,and the expression of PCNA also reduced.3、The activation of STAT3 and ERK1/2 signals depends on the high expression of HER2HER2 can act as an upstream signal to activate the STAT3 and ERK1/2pathways.Knockdown of HER2 with si RNA in T47D-R and MCF-7-R decreased the phosphorylation of STAT3 and ERK1/2 protein,and the m RNA of IL-6 and IL-8also decreased.Overexpression of HER2 in T47D-S and MCF-7-S promoted phosphorylation of STAT3 and ERK1/2,and the m RNA level of IL-6 and IL-8 also rised.4、Feedback effects of STAT3 and ERK1/2 signals on HER2HER2 can activate the downstream signals,such as STAT3 and ERK1/2pathways.The activation of STAT3 and ERK1/2 signals can also promote the expression of HER2.T47D-R and MCF-7-R were treated with Stattic,the protein expression and m RNA of HER2 decreased in them.The same phenomena were obtained by knocking down STAT3 with si RNA.Treatment of T47D-R and MCF-7-R with U0126 for 48 h also reduced HER2 expression.At the same time,pretreatment of T47D-R and MCF-7-R with Stattic can reduce the phosphorylation of ERK1/2 protein.Using U0126 to treat T47D-R and MCF-7-R can also decrease the phosphorylation of STAT3 protein.There is crosstalk between STAT3 and ERK1/2 signals.Therefore,a positive feedback loop is formed between HER2,STAT3 and ERK1/2.四、The effect of ROS on HER2 expression and 27-HC-induced multidrug resistance of ER alpha positive breast cancer cellsTreatment of T47D-R with antioxidant NAC,it was found that the expression of HER2,P-STAT3 and P-ERK1/2 protein decreased.The m RNA of HER2,IL-6 and IL-8 also reduced.CCK-8 results showed that both of these drug-resistant cells(T47D-R,MCF-7-R)restored the sensitivity to TAM,ICI,DOX and PTX after operation of NAC,and the expression of PCNA also reduced.Conclusion: Long-term low-dose treatment with 27-HC induces oxidative stress in ER alpha positive breast cancer cells(T47D and MCF-7).Excessive ROS can result in high expression of HER2 and eventually lead to multidrug resistance.In this process,HER2 activates the downstream STAT3 and ERK1/2 signals,and the activation of STAT3 and ERK1/2 pathways feedbackly regulate the expression of HER2.At the same time,there is crosstalk between STAT3 and ERK1/2 signals.A positive feedback loop is formed to maintain the high expression of HER2.This positive feedback loop provides extra survival signals for ER alpha positive breast cancer cells to counteract cell death caused by anticancer drugs.Therefore,the high circulating level of 27-HC and its induced ROS,HER2,and downstream STAT3 and ERK1/2 signals can serve as intervention targets to reduce drug resistance in the treatment of obese or hypercholesterolemia breast cancer patients,and to improve the efficacy of endocrine therapy and chemotherapy. |
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