| Backgrounds:The incidence of breast cancer is the highest among all female cancers in the world.Surgery,chemotherapy,endocrine therapy and other methods can be used to treat breast cancer at present,but even for those patients who receive treatment at the early stage,there is still a poor prognosis.Therefore,researchers are working to develop new targets and treatment strategies to improve patient outcomes,among which the novel targeted medicine cyclin-dependent kinases 4/6（CDK4/6）inhibitor combined with endocrine therapy has become the first-line treatment for advanced estrogen receptor（ER）positive human epidermal growth factor receptor 2（HER2）negative breast cancer.Although the personalized treatment of breast cancer has made great progress,there are still some problems such as endocrine therapy resistance and lack of new therapeutic targets.The tumor suppressor p53 is a transcription factor which binds to the promoters of its target genes and regulates their expression.Upon DNA damage and other types of stresses,p53 blocks proliferation of pre-malignant and malignant cells or eliminates them by inducing apoptosis.p53 inactivation via mutations or enhanced degradation by MDM2 is the most frequent alteration in human cancers,which underscores the key role of p53 in combating cancer.Previous studies have shown that reinstatement of p53 by genetic means has demonstrated remarkable tumor suppression in animal models,including inhibition of aggressive metastatic lesions.This inspires the idea of developing small molecules reactivating p53 to fight cancer.Prof.Galina Selivanova’group from Karolinska Institute have screened and identified a small molecule RITA（Reactivation of p53 and Induction of Tumor cell Apoptosis）,which prevents p53/MDM2 interaction,induces p53 accumulation and transcriptional activity and triggers p53-dependent apoptosis in tumor cells of a different origin in vitro and in mice.And it is considered as a candidate p53-targeting compound.However,at levels similar to efficacious doses observed in mouse xenografts of human cancer cell lines,RITA caused pulmonary edema in rat,dog,and monkey,but not in mice,which are deficient for the expression of phenol sulfotransferase detoxification enzymes in lung.Further studies have showed that RITA can be modified as a substrate of sulfotransferase1A1（SULT1A1）to produce active carbocation,which can cause DNA damage.The development of therapeutics that selectively target cancer cells without extensive cytotoxicity is in line with the concept of modern precision medicine.With respect to RITA,we hypothesize that broad toxicity observed in multiple mammals was due to nonspecific biological activities of the SULT1A1-modified molecule.More target-selective analogs,recently designed by Developmental Therapeutics Program（DTP）at National Cancer Institute（NCI）,in collaboration with Dr Peter Wipf and colleagues,narrowed the cytotoxicity profile of RITA without elimination of in vivo antitumor activity.Using quantum mechanics,RITA analogs need higher transition state energy for formation of a carbocation from sulfonated versions compared to RITA,which was in line with our expectation that RITA could reduce the SULT1A1 dependence.Objectives:In this study,we investigated molecular mechanisms of RITA analogs NSC777196 and NSC782846 upon anti-cancer.We screened out the high response population of ER-positive breast cancer with NSC782846,which can be used in combination with endocrine therapy.This study aims to lay a theoretical foundation for the development of RITA analogs with high p53 specificity and low toxicity that can be clinically transformed in future,and to provide ideas for improving the therapeutic effect of ER-positive breast cancer patients.Methods:（1）CRISPR-Cas9 gene editing,correlation sensitivity analysis and short-term survival experiment were performed to compare the SULT1A1-dependent antitumor activity of RITA and its analogs.（2）CRISPR-Cas9 gene editing,q PCR and Western blotting were used to detect whether the inhibition of related oncogenes by RITA analogs was dependent on p53 expression,and to analyze the dose-dependence and different regulatory mechanisms of RITA analogs on the inhibition of related oncogenes.In addition,short-term and long-term survival experiments were performed to detect the dependence of the anti-cancer activity of RITA analogs on p53.The effect of RITA analogues on apoptosis of tumor cells was detected by flow cytometry.（3）q PCR and western blotting techniques were used to detect the effect of RITA analogs on representative oncogenes in SULT1A1-/-cell lines.Lentivirus-mediated p53knockdown was performed to detect the changes of oncogenes upon RITA analogs.Flow cytometry was performed to detect apoptosis of SULT1A1-/-cancer cells upon RITA analogs.（4）Western blotting and immunofluorescence were performed to detect DNA damage marker in SULT1A1-/-cells upon RITA analogs.Western blotting and 5-Ethynyluridine labeling were used to detect the effect of RITA analogs on transcription.（5）The amplification of MDM2 and MDM4 genes in breast cancer patients with different ER status was obtained from c Bio Portal website,and the correlation between their amplified mutations and TP53 gene mutations was analyzed.The correlation between the antitumor activity of RITA analog NSC782846 and MDM2 was analyzed by correlation sensitivity analysis.（6）The synergistic inhibitory effect of RITA analog NSC782846 combined with tamoxifen on estrogen receptor-positive breast cancer was detected by short-term and long-term survival experiments,and Calcu Syn software was used to calculate the combination index of the two compounds.Results:（1）Analysis of NCI-60 database,it was found that the correlation between SULT1A1expression and the growth inhibition of RITA,NSC777196 and NSC78284 on tumor cells was decreased.Seven tumor cell lines from different sources and stable SULT1A1 knockout and overexpression cell lines constructed by the research group were used.Western blotting and short-term survival experiment confirmed that the antitumor activity of RITA analog was less dependent on SULT1A1 than that of RITA.In particular,the RITA analog NSC782846.（2）q PCR and Western blotting showed that the two RITA analogs had inhibitory effects on related oncogenes,and this inhibitory effect was cancelled after p53 knockout.The inhibitory effects of RITA and its two analogs on oncogenes are dose-dependent and regulated in different ways.Short-term and long-term survival experiments proved that the proliferation inhibition of RITA analog NSC782746 was dependent on p53.Flow cytometry showed that two RITA analogs could induce strong apoptosis of tumor cells.（3）q PCR and Western blotting proved that RITA analogs NSC777196 and NSC782846had inhibitory effects on related oncogenes in SULT1A1-/-cell lines,and this inhibitory effect would be rescued after lentiviral transduction and knockdown of p53.Flow cytometry showed that RITA analogue NSC782846 induced apoptosis in SULT1A1-/-cells,which further demonstrated its non-SULT1A1 dependence.（4）Using MCF7 SULT1A1-/-cell line,Western blotting and immunofluorescence staining showed that the relative expression ofγH2AX protein in cells treated with RITA analog NSC782846 did not change significantly,while the relative expression of p53 protein was up-regulated.It is suggested that the activation mechanism of RITA analog NSC782846p53 is not related to DNA damage stress,that is,RITA analog NSC782846 activates p53 in a way without DNA damage to exert anti-tumor effect.Using MCF7 SULT1A1-/-cell line,Western blotting and EU labeling technology showed that RITA analogs NSC782846 and NSC777196 did not block the synthesis of new RNA and did not inhibit the expression of RNA pol II.These results suggest that the oncogene inhibition of RITA analogs in SULT1A1-/-cells is p53-mediated transcriptional inhibition rather than whole-gene transcriptional block,that is,RNA analogs NSC777196 and NSC782846 exert anti-tumor effects through non-transcriptional block.（5）From the information of 1897 breast cancer patients obtained from c Bio Portal website,it was found that the MDM2 gene of ER-positive breast cancer patients was more likely to be in the state of amplification,and the amplification of MDM2 and TP53 gene mutation were mutually exclusive,suggesting that ER-positive breast cancer patients may become the benefit population of RITA analog NSC782846 treatment.By analyzing the NCI-60 database,it was found that the correlation between the anti-cancer activity of RITA,RITA analogs NSC777196 and NSC782846 and the baseline m RNA expression of MDM2increased sequentially,suggesting that interfering with the binding of p53/MDM2 may have more important significance for the anti-tumor activity of NSC782846.（6）Short-term and long-term survival experiments showed that the combination of NSC782846 and tamoxifen could synergistically inhibit ER-positive breast cancer cells,and Calcu Syn software was used to calculate the combination index CI<1,which confirmed the synergistic inhibitory effect of NSC782846 and tamoxifen.Conclusions:（1）Decreased SULT1A1 dependence of RITA analogs along with improved p53specificity,especially NSC782846,which played an anti-tumor effect.（2）RITA analogs reactivate p53 to induce target genes,repress oncogenes exerting anti-tumor effects by directly activating p53.（3）Unlike RITA and its analog NSC777196,NSC782846 can activate p53 without causing DNA damage and transcription block.（4）RITA analog NSC782846 reduces SULT1A1 dependence and activates p53 without causing DNA damage and transcriptional blockade,suggesting that its cytotoxicity is lower than that of RITA.（5）NSC782846 synergized with 4OH-tamoxifen in killing ER-positive breast cancer cells... |
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