| BackgroundAmong all breast cancers,estrogen receptor α(ERα)-positive tumors constitute the largest proportion(～70%).The proliferation of ERα+breast cancer cells mainly depends on the activation of ERα signaling pathway.Therefore,endocrine therapy targeting ERαsignaling pathway has been widely used as a first line adjuvant therapy for these tumors.Tamoxifen is the earliest and most widely used endocrine therapy drug,which can significantly reduce the recurrence rate and prolong the overall survival of breast cancer patients.However,more than 30%of the patients eventually develop resistance to tamoxifen,which is associated with metastatic recurrence.Therefore,tamoxifen resistance presents a tremendous clinical challenge.Studies have shown that the deregulation of ERa and the activation of the PI3K-Akt signaling pathway are the main causes of tamoxifen resistance.However,the mechanism of ERa downregulation and PI3K-Akt signaling pathway activation remains unclear.Furthermore,there are few clinically available strategies that can effectively reverse tamoxifen resistance.Realistically,how tumor cells respond to therapy depends not solely on the genomic aberrations they harbor but also on the characteristics of the tumor microenvironment.As the indispensable "soil" for tumor development,tumor microenvironment can support tumor cell growth,metastasis,angiogenesis,immune escape and drug resistance through dynamic information interaction.It was reported that tumor microenvironment plays an essential role in breast cancer chemotherapy and immunotherapy resistance,but its role in tamoxifen resistance is unclear.Therefore,from a therapeutic perspective,it is urgent to elucidate the mechanism of tamoxifen resistance from the perspective of the whole tumor microenvironment.AimsThe purpose of this study is to elucidate the causes and molecular mechanisms of tamoxifen resistance in breast cancer from the perspective of tumor microenvironment,so as to provide potential drug targets and strategies for overcoming tamoxifen resistance in breast cancer.Methods1.The dynamic changes of ERα expression and the response to tamoxifen treatment were observed in MMTV-PyMT spontaneous breast cancer mouse model.2.Single cell RNA sequencing was used to chart the microenvironment landscape of tamoxifen resistant and sensitive breast cancer tissues.CD63+CAFs and CD63-CAFs were then isolated and co-cultured with ERα+breast cancer cells to verify the effect of CD63+CAFs on tamoxifen sensitivity.Then,the relationship between CD63+CAF and prognosis of breast cancer was verified in breast cancer clinical samples.3.The ERα+breast cancer cells were treated with exosomes form CD63+CAFs or CD63CAFs.Then the expression of ERα and viability of breast cancer cells were measured by Western blot,CCK8 and colony formation assay.The key enzyme mediating microRNA maturation(Dicer)was knocked-down and the effect of CAFs derived exosomes on breast cancer cells was measured to explore whether CD63+CAFs play their role via exosomal microRNA.4.microRNA sequencing was performed on exosomes from CD63+and CD63-CAFs,and the microRNA with high expression in exosomes from CD63+CAFs was screened and confirmed by real-time PCR.5.Co-culture and confocal assays were used to explore whether CD63+CAFs could deliver miR-22 to breast cancer cells through exosomes and thus induce breast cancer cell ERα downregulation and tamoxifen resistance.6.The effects of miR-22 on tamoxifen resistance in breast cancer were detected by in vitro co-culture assay and in vivo mouse model experiments.The relationship between miR22 and prognosis of breast cancer patients was confirmed through clinical sample analysis.7.The molecular and signaling pathways related to the high expression of CD63 and miR22 in CAFs were screened by bioinformatic analysis and transcription factor screening assay.ChIP,luciferase reporter assay and Western blot were applied to further clarify the mechanism underling the high expression of CD63 and miR-22 in CD63+CAFs.8.Bioinformatic analysis,luciferase reporter assay and Western blot were performed to further elucidate the molecular mechanism of miR-22 in breast cancer tamoxifen resistance.9.Combined treatment with CD63 neutralizing antibody and tamoxifen was performed on breast cancer mouse model to explore the effects of CD63 neutralizing antibody on tamoxifen sensitivity.The infiltration of CD63+CAFs in microenvironment and ERαexpression in breast cancer cells were investigated by immunofluorescence and We stern blot experiments.10.cRGD-decorated nanoparticles encapsulating the miR-22 sponge were prepared and their effects on tamoxifen sensitivity were tested in breast cancer mouse model.Results1.With the development of breast cancer in MMTV-PyMT mice,the expression of ERαin breast cancer cells gradually decreased,and the sensitivity of mice to tamoxifen treatment gradually decreased as well.2.Single-cell sequencing revealed that there are a large number of CD63+CAFs in tamoxifen resistant breast cancer microenvironment,which were validated to induce ERα downregulation and tamoxifen resistance in breast cancer.Further clinical sample analysis confirmed that CD63+CAFs were closely related to decreased ERα expression and poor prognosis of breast cancer.3.CD63+CAFs derived exosomes could induce breast cancer ERα downregulation and tamoxifen resistance;When Dicer(a key enzyme that mediates microRNA maturation)was knocked down,CD63+CAFs-derived exosomes could no longer induce ERαdownregulation and tamoxifen resistance in breast cancer.4.Exosome microRNA sequencing showed that miR-22,miR-148a and miR-152-3p were highly expressed in exosomes derived from CD63+CAFs.Further real-time PCR assay confirmed that miR-22 was highly expressed in exosomes derived from CD63+CAFs.5.CD63+CAFs could deliver miR-22 to breast cancer cells via exosome and thus induce ERα downregulation and tamoxifen resistance.Conversely,the above effect of exosomes from CD63+CAFs was abolished when miR-22 was sequestrated by miR-22 sponge.6.Overexpression of miR-22 inhibited the expression of ERα in breast cancer cells and induced tamoxifen resistance.In vivo tumor suppression experiments confirmed that miR-22 KO mice were more sensitive to tamoxifen treatment;Further public database analysis showed that miR-22 was associated with poor prognosis of breast cancer.7.TIMP1,the classical ligand of CD63,was highly expressed in CD63+CAFs,which could activate the transcription factor STAT3.More importantly,the activated STAT3 could bind with the promoter regions of CD63 and miR-22,enhance the expression of CD63 and miR-22,thereby maintaining the phenotype and function of CD63+CAFs.8.miR-22 could bind to the mRNA 3’ UTR region of ESR1 and PTEN and inhibit the expression of ESR1 and PTEN,thereby inducing tamoxifen resistance in breast cancer.9.Anti-CD63 neutralizing antibody could significantly enhance the therapeutic sensitivity of tamoxifen.Immunofluorescence assay and Western blot showed that anti-CD63 neutralizing antibody could effectively remove CD63+CAFs in breast cancer microenvironment and restore breast cancer cell ERα Expression.10.miR-22 sponge nanoparticles could reverse CD63+CAFs exosome mediated ERα and PTEN downregulation and enhance tamoxifen sensitivity in breast cancer.Conclusion1.In this study,we identify a new subtype of CAFs in the tumor microenvironment—CD63+CAFs,which suppresse ERa and PTEN expression in breast cancer via exosomal miR-22,thus confer tamoxifen resistance on breast cancer.2.The binding of TIMP1 to CD63 on the cell surface sustains the expression of CD63 and miR-22 in CD63+CAFs through the Jak-STAT3 signaling pathway.3.miR-22 can bind to the mRNA 3 ’UTR region of ESR1 and PTEN and inhibit the expression of ESR1 and PTEN,thereby inducing tamoxifen resistance in breast cancer.4.Blocking CD63+CAFs—exosomal miR-22—ESR1/PTEN signal axis through CD63 neutralizing antibody or miR-22 sponge nanoparticles can effectively enhance the sensitivity of breast cancer to tamoxifen treatment. |
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