A Quinone Derivative ZSW Inhibits Triple Negative Breast Cancer Cell Proliferation By Targeting STAT3

Breast cancer originating from mammary epithelial tissue is the highest incidence of female malignant tumors in the world.In recent years,the incidence of breast cancer patients in our country has continued to increase.Due to the lack of estrogen receptor,progesterone receptor,and human epidermal growth factor 2 on the cell surface,triple negative breast cancer is insensitive to endocrine antagonistic therapy,prone to organ and bone metastasis,young age and poor prognosis,and lack of targeted therapeutic drugs,making it as the most aggressive and malignant subtype of all breast cancers.Breast cancer stem cells are a part of the cells with self-renewal potential and strong resistance to traditional radiotherapy or chemotherapy drugs,which is related to the drug resistance and relapse of patients with radiotherapy and chemotherapy.It is also an important factor causing metastasis and death of breast cancer patients.The development of inhibitory drugs targeting breast cancer stem cells is a new strategy for breast cancer treatment.Quinone compounds are a series of compounds with a quinone structure that exist widely in natural animals and plants.ZSW is a new naphthoquinone compound synthesized by our research group,which has the characteristics of high tumor inhibitory activity,good stability,and water solubility.Previous studies showed that ZSW has strong proliferation inhibition on breast cancer,colorectal cancer,and lung cancer cells and weak inhibition on immortalized epithelial cells.By exploring the molecular mechanism of ZSW inhibiting the proliferation and growth of triple negative breast cancer cells and breast cancer stem cells,this study provided drug candidates for the exploration and development of novel targeted inhibition of triple negative breast cancer and breast cancer stem cells.In this study,the efficacy evaluation of ZSW for breast cancer treatment was carried out at the cellular and animal levels.Through the synthesis of ZSW molecules labeled with Biotin probes,the target of ZSW in triple-negative breast cancer cells and the mechanism of inhibiting the proliferation of triple-negative breast cancer cells and breast cancer stem cells were explored,and reached the following conclusions:（1）It was shown that ZSW had a strong anti-proliferation ability to triple negative breast cancer cells and weak inhibition ability of immortalized breast epithelial cells 184B5.It was demonstrated that ZSW could significantly inhibit the growth of triple negative breast cancer cell line MDA-MB-231 in nude mice and showed inconspicuous toxic and side effects on body weight and vital organ lesions of nude mice.（2）Small molecule compound Biotin-ZSW with Biotin label and no significant change in anti-tumor activity was synthesized by organic chemistry,pull-down assay,and LC-MS/MS determined that STAT3 was the target of ZSW.（3）ZSW directly bound to STAT3 through Lys591,Glu594,Arg595,and Arg609 amino acids,which was provided by the MOE docking experiment.ITC showed that the binding strength of ZSW with STAT3was about 2.67×10^-7.In contrast,the binding strength of ZSW with mutated STAT3 was reduced by almost ten times.（4）ZSW down-regulated the expression levels of phosphorylated STAT3 and total STAT3 in triple negative breast cancer cells in a time and concentration-dependent manner,but had no significant inhibitory effect on its m RNA level.Immunoprecipitation experiments showed that ZSW could promote ubiquitination degradation of STAT3 and down-regulate its expression level.（5）As a transcription factor protein,STAT3 needs to be dimerized,transferred into the nucleus,binds to specific transcription factor binding sites,and starts the transcription of downstream genes.ZSW inhibited the dimerization process of STAT3 and down-regulated the STAT3 in the nucleus.Quantitative real-time PCR and luciferase assay proved that ZSW down-regulates the expression of the downstream oncogene of triple negative breast cancer cell line STAT3.The CRISPR-Cas9 system was used to construct the triple negative breast cancer cell line knocked out by STAT3.It was found that STAT3 knockout could significantly reduce the sensitivity of ZSW to triple negative breast cancer cells.（6）Acetaldehyde dehydrogenase1（ALDH1）is a marker of breast cancer stem cells.It was shown that ZSW could significantly down-regulate the proportion of ALDH1-positive cells in triple negative breast cancer,inhibit the proliferation of breast cancer stem cells,and reduce the volume of breast cancer stem cells.To restore drug sensitivity of clinical chemotherapy drug doxorubicin-resistant triple negative breast cancer cell line.In conclusion,ZSW can target Lys591,Glu594,Arg595,and Arg609amino acid residues that bind to STAT3,promote ubiquitination degradation of STAT3,inhibit the nuclear accumulation of STAT3,and reduce its expression in triple negative breast cancer cell lines.Down-regulating the transcription level of key downstream oncogenes of STAT3inhibits the proliferation of triple negative breast cancer cells,reduces the proportion of triple negative breast cancer stem cell-like cells,and restores the drug sensitivity of drug-resistant cell lines.We found ZSW,as a novel small quinone molecular compound that inhibits the proliferation of triple negative breast cancer,provides a new drug candidate structure for clinical therapy of breast cancer.