Estrogen receptor α confers Nab-paclitaxel resistance in breast cancer by promoting miR199a-5p maturation to inhibit Caveolin 1 translation

Estrogen receptor α confers Nab-paclitaxel resistance in breast cancer by promoting miR199a-5p maturation to inhibit Caveolin 1 translation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Estrogen receptor α confers Nab-paclitaxel resistance in breast cancer by promoting miR199a-5p maturation to inhibit Caveolin 1 translation Jianping Zhang, Zuo Wang, Liyuan Zhu, Chaoqun Wang, Yiming Zhong, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2838943/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Estrogen receptor positive (ER+) breast cancer patients are poorly responsive to Nab-paclitaxel compared to ER negative (ER-) breast cancer patients. Herein, we conducted an investigation regarding the mechanism for ERα confers Nab-paclitaxel resistance in breast cancer. Methods Retrospectively reviewed 116 cases of breast cancer treated with nab-paclitaxel between Jan 2008 and May 2022 in Sir Run Run Shaw Hospital. StataSE 16 software was used to analyze the basic conditions and therapeutic effects. Protein-RNA interactions were validated through RNA immunoprecipitation and RNA pull-down assays. In vitro and in vivo experiments were carried out to testify the effect of ERα on Nab-paclitaxel resistance. Results We show that ERα limits the efficacy of nab-paclitaxel in breast cancer while genetic or pharmacological inhibition of ERα has a synergistic effect with Nab-paclitaxel. Meanwhile, CAV1 expression is negatively correlated to ERα and relevant to the better clinical benefits of Nab-paclitaxel treatment. Importantly, ERα stimulates miR199a-5p maturation to antagonize m6A modification of CAV1 mRNA, thus inhibiting its translation. Conclusions Our results define a novel role of ERα miR199a-5p/CAV1 axis responsible for nab-paclitaxel resistance and propose combining ER antagonist with nab-paclitaxel as a perspective strategy for ER + breast cancer patients. Estrogen receptor α (ERα) Nab-Paclitaxel Caveolin 1 (CAV1) translation N6-methyladenosine (m6A) miR199a-5p Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Estrogen receptor α (ERα) is the major driver of ∼75% of breast cancers (BC) with routine endocrine treatment ( 1 , 2 ). However, many ER + BC patients still relapse after endocrine treatment and require chemotherapy in the recurrent or metastatic phase ( 3 ). Nab-paclitaxel (Abraxane), the first clinically successful albumin-bound chemotherapeutic agent (paclitaxel bound to human albumin), is commonly used in the treatment of pancreatic cancer, non–small cell lung cancer (NSCLC), and breast cancer ( 4 – 6 ). However, some clinical trials and retrospective analyses have observed that nab-paclitaxel is less effective in ER + BC than in ER-BC ( 7 ), indicating that ER status may play an important role in Nab-paclitaxel response, despite the lack of understanding of the underlying mechanisms. Caveolin 1(CAV1) is the principal structural protein of Caveolae, which are 50–100 nm flask-shaped invaginations of the plasma membrane functioning in endocytosis, cholesterol homeostasis, signal transduction, and macromolecule transport ( 8 – 10 ). CAV1 is reported to be upregulated in pancreatic cancer, NSCLC, and breast cancer, and is related to increased invasion, metastasis, resistance to radiation or chemotherapy, and poor prognosis ( 11 – 13 ). In addition, a previous study claimed that CAV1 is an albumin transporter that facilitates nab-paclitaxel uptake to enhance its efficacy in pancreatic cancer and NSCLC ( 10 ). However, it remains unknown whether CAV1 is relevant to nab-paclitaxel resistance in ER + BC. In this study, we demonstrated that ERa mediates nab-paclitaxel resistance by inhibiting CAV1 translation. Mechanistically, ERa promotes miR199a-5p maturation, which binds to CAV1 mRNA, thus competitively suppressing m6A modification-dependent mRNA translation. Our study provides a rationale for targeting ERa to overcome nab-paclitaxel resistance in ER + breast cancer. Methods and Materials Patients and Retrospective study design A cohort of 116 breast cancer patients who received nab-paclitaxel at Sir Run Run Show Hospital, Zhejiang University, between January 2008 and May 2022 were retrospectively reviewed. All eligible patients received at least one dose of nab-paclitaxel. We analyzed the baseline ER status by IHC and evaluated disease progression using the RECIST guidelines ( 14 ). Cell lines, antibodies and reagents The breast cancer cell lines MCF-7, T-47D, MB231, and BT549 were purchased from the American Type Culture Collection (ATCC). Both were authenticated using short tandem repeat multi-amplification and tested negative for Mycoplasma. MCF-7 and T-47D cells were cultured in DMEM (Gibco, USA), and MB231 and BT549 cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Hyclone, USA),100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies/Gibco, Shanghai, China). Cells were grown at 37°C in a humidified incubator with 5% CO2 and 95% humidity. The following antibodies were used for western blotting or immunohistochemistry: ERα(Vector Laboratories, VP-E613), CAV1 (ABclonal, A1555), METTL3 (ABclonal, A8370), β-Actin (Cell Signaling Technology, 4970), Cleaved PARP1 (C-PARP1) (Cell Signaling Technology, 9541), Cleaved Caspase-3 (Cell Signaling Technology, 9661), 4-OH-Tamoxifen (579002), and β-estradiol (E8875) were purchased from Merck and Sigma Aldrich. Fulvestrant (HY-13636) was purchased from MedChemExpress (Shanghai, China). Nab-PTX was supported by the Celgene Corporation. DQ™ Green BSA( Invitrogen™,1014496) SiRNA, miRNA mimics/inhibitors and plasmid transfections Small interfering RNA (siRNA) targeting ERα, CAV1, METTL3, and microRNAs were synthesized by GenePharma (Shanghai, China) and RiboBio (Guangzhou, China). The sequences of the siRNAs and miRNA mimics/inhibitors are listed in Table S1 . The plasmids Flag-CAV1-pcDNA3.1 and Flag- ERα- pcDNA3.1 were purchased from Genechem (Shanghai, China). SiRNAs and miRNA mimics/inhibitors were transfected into cells seeded overnight using lipo2000 (Invitrogen, USA) or Lipofectamine RNAiMax transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA extraction, reverse transcription and qPCR Total RNA was extracted using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer's instructions, and RNA concentration was quantified using a NanoDrop 2000 (Nanodrop, Wilmington, DE, USA). RNA (1–2 µg) was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368813). Real-time PCR (qPCR) was conducted using a SYBR Green Master Mix Kit (ComWin Biotech, CW0659s, Beijing, China). The qPCR data were normalized to the control group, and the relative expression of the indicated genes shown in the histograms is expressed as mean ± SD. The primers used in this study are listed in Table S1 . Luciferase activity assay The fragment of the CAV1 3'-UTR containing the miR-199a-5p binding site was amplified by PCR. and was inserted into the pMIR-REPORTER vector (Promega). For the luciferase assay, HEK-293T in 6-well plates. The CAV1-3’UTR plasmids were co-transfected using lipo2000 with miR199a-5p mimics and pRL Renilla as an internal control (Invitrogen, USA). After 48 h, the luciferase activity was measured using the Dual-GLO Luciferase Assay System (Promega Corporation, USA). RNA immunoprecipitation (RIP) assay The RIP assay was conducted following the manufacturer’s instructions for the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore, No.17–700). Cells (> 2 × 10 7 ) were collected and lysed in 100 µl RIP lysis buffer, immunoprecipitated with the indicated antibody-linked beads or protein G magnetic beads overnight at 4°C, washed six times in Washing Buffer, and the protein was denatured at 55°C. Total RNA was isolated and quantified using RT-PCR analysis. Puromycin-labelling 5X 10 6 cells were plated in a 10 cm dish and incubated with 1:1000 biotin-dC-puromycin (NU-925-BIO-S, Jena Bioscience) for 24 h. Cells were collected and lysed in 1% NP40 buffer with protease inhibitor cocktail. After 15000rpm x30min centrifugation at 4°C, the supernatant was collected and incubated with 80ul streptavidin sepharose beads (GE17–5113-01, Sigma) by rotating at 4°C overnight. The mixture was washed five times with 1% NP40 buffer for 5 times and test CAV1 expression was tested by western blotting. Biotin pull down assay Cells were transfected with biotinylated miR199a-5p probes for 48 h and resuspended in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2, 60 U/mL SUPERase-In, 1 mM DTT, 0.05% Igepal, protease inhibitors). Lysates were incubated with prepared streptavidin beads (GE Healthcare). Yeast tRNA (Sigma-Aldrich) was used to block the lysates at 4°C for 3 h. The cells were washed five times with binding and wash buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl). Finally, the bound RNAs was extracted and purified for qPCR. In vitro pri-miRNA processing assays Probe biotin-pri-miR-199 was transcribed in vitro using a T7 based MEGA shortscript kit (Life Technologies). Biotin RNA-labeling Mix (Roche,11685597910) was used for the in vitro transcription reaction. For pri-miR-199 processing assays, biotin-pri-miR-199 was incubated with whole cell lysates of 293T cells at 37°C for 90 min. The Lysates were then incubated with streptavidin beads (GE Healthcare). Yeast tRNA (Sigma) was used to block lysates at 4°C for 3 h, and RNA purified from the reaction products was analyzed by qRT-PCR. The primers used for in vitro pri-miR-199 transcription are listed in Supplementary Table S1 . Animal experiments Six-week-old female BALB/c nude mice from the Center of Experimentation of Zhejiang University were used following the Institutional Animal Care and Use Committee and National Institute of Health (NIH) guidelines. Tumors were established by subcutaneous injection (1× 10 6 E0771 cells in 0.1 ml saline) into the flanks of the mice. After 12 days, tumors reached > 150 mm 3 in size, and the mice were randomly allocated into four groups and treated with Nab-PTX (22.3 mg/kg body weight; i.g, every 3 days), Fulvestrant (2.5 mg/kg body weight; i.p, every 3 days), Nab-PTX combined with fulvestrant, or vehicle PBS as control. Tumor volume was measured every 3 days. Mice were euthanized when the tumor size reached approximately 2000 mm 3 . Tumor volume was calculated using the following formula: length × width 2 ×0 .5. Statistical Analysis All data are presented as mean ± SD. The statistical approach used in every experiment to compare the differences between the groups is provided in the figure legends. Statistical significance was set at P < 0.05. Results ER + BC patients show worse response to nab-paclitaxel than ER- BC patients To test whether ER status dictates responsiveness to nab-paclitaxel, we retrospectively reviewed a cohort of 116 breast cancer patients who received nab-paclitaxel at Sir Run Run Show Hospital, Zhejiang University, between January 2008 and May 2022. All eligible patients received at least one dose of nab-paclitaxel (Fig. 1 A). We analyzed the baseline ER status to determine its ability to predict disease progression, as evaluated by the RECIST guidelines. 55 patients (64.7%) were classified as the ER-positive subtype, and 30 patients (35.2%) were classified as the ER-negative subtype (Supplementary Fig. 1A). In the ER + subgroup, four patients (7.3%) achieved partial response (PR), 17 patients (30.9%) had stable disease (SD), and 34 patients (61.8%) experienced disease progression (PD). In the ER − group, 16 patients (53.3%) achieved PR, 9 patients (30%) had SD, and 5 patients (16.7%) experienced PD. There was also a significant discrepancy between these two subgroups with regard to the objective response rate (ORR) and disease control rate (DCR) (Fig. 1 B-C), which is consistent with previous reports ( 7 ). In vitro experiments also verified that ERα-BC cells (MB231 and BT549) were more sensitive to nab-paclitaxel treatment than ERα + cells (MCF7 and T47D) (Fig. 1 D- 1 E and Supplementary Fig. 1B ). Taken together, we concluded that ER + BC patients were less responsive to nab-paclitaxel than ER-BC patients. ERα inhibit the sensitivity of breast cancer cells to Nab-paclitaxel Given the distinct response to Nab-paclitaxel in ER + and ER- breast cancers, we questioned whether ERα inhibits the anticancer activity of nab-paclitaxel. Indeed, genetic knockdown of ERα in MCF7 cells conferred sensitivity to nab-paclitaxel (Fig. 2 A- 2 C and Supplementary Fig. 2A). Similarly, chemical inhibition of ERα in MCF7 and T47D cells greatly enhanced the growth-inhibitory effects of Nab-paclitaxel (Fig. 2 D- 2 I and Supplementary Fig. 2B-2E). Furthermore, β-estradiol(β-E2) deprivation increased nab-paclitaxel-induced growth inhibition and apoptosis activation, which was reversed by β-E2 supplementation (Fig. 2 J- 2 L and Supplementary Fig. 2F). Taken together, ERα decreased the sensitivity of breast cancer cells to nab-paclitaxel. CAV1 is relevant to the sensitivity of breast cancer cells to Nab-paclitaxel As CAV1 is critical for the response to albumin-bound chemotherapeutics in NSLC and pancreatic cancer by affecting albumin endocytosis ( 10 ), we assessed the effect of CAV1 on nab-paclitaxel sensitivity in breast cancer cells. After CAV1 expression was knocked down by siRNA, MB231 and BT549 cells displayed resistance to nab-paclitaxel (Fig. 3 A- 3 C, Supplementary Fig. 3A-3E). In contrast, exogenous CAV1 expression conferred MCF7 and T47D cells increased sensitivity to Nab-paclitaxel (Fig. 3 D- 3 E, Supplementary Fig. 3F-3G). However, their sensitivity to paclitaxel was not altered (Fig. 3 F), suggesting a role of CAV1 in mediating the internalization of albumin-bound drugs. Indeed, upon encapsulation with DQ-BSA for 30 min in culture medium ( 15 ), MB231 cells accumulated high levels of DQ-BSA in the cytoplasm, which was abrogated after CAV1 knockdown (Fig. 3 G). Importantly, patients with higher CAV1 expression had a better benefit from nab-paclitaxel treatment (Fig. 3 H and 3 I), revealing a positive correlation between CAV1 expression and the disease control rate in breast cancer after nab-paclitaxel treatment. In conclusion, CAV1 expression is important for determining the nab-paclitaxel response. ERα downregulates CAV1 protein expression by inhibiting its translation To explore the potential correlation between ERα and CAV1, we analyzed their expression based on reverse-phase protein arrays (RPPA) (n = 627) from The Cancer Proteome Atlas (TCPA) database ( 16 ). CAV1 protein levels were significantly negatively correlated with ERα protein levels (Fig. 4 A). However, there was no such correlation between CAV1 and ESR1 mRNA levels based on results from The Cancer Genome Atlas (TCGA) (Supplementary Fig. 4A). In addition, CAV1 protein expression was much lower in ERα-positive breast cancer tissues than in ERα-negative breast cancer tissues (Fig. 4 B and 4 C). Moreover, CAV1 protein levels were notably higher in ERα-negative breast cancer cell lines than in ERα-positive breast cancer cell lines (Fig. 4 D). After knockdown of ERα expression by siRNA or treatment with ERα inhibitors including 4-OH-Tamoxifen and fulvestrant, the CAV1 protein was upregulated (Fig. 4 E- 4 F and Supplementary Fig. 4B-4D) while CAV1 mRNA remained unchanged (Supplementary Fig. 4E-4G). In contrast, exogenous ERα overexpression or ER activation by 17β-E2 inhibited the expression of CAV1 protein, but not mRNA (Fig. 4 F-G and Supplementary Fig. 4H). In addition, either inhibition or activation of ERα failed to affect the expression of exogenous CAV1 (Supplementary Fig. 4I-4J), since exogenous CAV1 doesn’t have a 3’UTR. Therefore, ERα appears to inhibit the translation of CAV1. In fact, the synthesis of nascent CAV1 protein increased upon ERα depletion by siRNA or chemical inhibition by fulvestrant (Fig. 4 H-I). In summary, ERα downregulated CAV1 expression by inhibiting its translation. ERα stimulates miR199a-5p maturation to inhibit CAV1 translation MicroRNAs (miRNAs) are widely recognized as negative regulators of protein translation through partial base-pairing with the 3’-UTR of mRNA to block formation of the translation initiation complex ( 17 – 20 ). We queried potential miRNAs that could be upregulated upon E2 stimulation (data from GSE78167) and predicted target CAV1 using TargetScan, miRDB, and StarBase, which revealed an overlap of seven miRNAs, including hsa-miR-493-3p, hsa-miR-520a*, hsa-miR-384, hsa-miR-124-3p, hsa-miR-512-3p, hsa-miR-199a-5p, and hsa-miR-302b* (Fig. 5 A). However, inhibition of miR-199a-5p, miR-512-3p, or miR-520a, but not of other miRNAs, upregulated CAV1 protein levels (Fig. 5 B), whereas knockdown of ERα downregulated the expression of only two miRNAs, miR-124-3p and miR-199a-5p (Fig. 5 C). Therefore, we postulated that miR-199a-5p may be involved in the ERα-mediated regulation of CAV1 protein translation. In fact, the miR-199a-5p mimic reduced while its inhibitor increased CAV1 protein level (Fig. 5 D). In addition, luciferase activity driven by the CAV1 mRNA 3’-UTR was significantly inhibited by miR199a-5p (Fig. 5 E). The interaction of miR199a-5p with CAV1 mRNA was further confirmed by a biotin pulldown assay (Fig. 5 F). Importantly, the miR-199a-5p mimic succeeded in rescuing CAV1 upregulation induced by fulvestrant (Figure.5G), highlighting the relevance of miR199a-5p upregulation to ERα-mediated CAV1 downregulation. Next, we found that miR199a-5p was highly expressed in ERα + MCF7 cells compared to ERα- cell MB231 (Fig. 5 H). Besides, the expression of miR199a-5p has positive correlated with ERα expression in breast cancer tissues (Fig. 5 I). Inhibition of ERα by Fulvestrant decreased mature miR-199a-5p and its precursor miR-199a, but increased the level of primary miR-199a (Fig. 5 J), suggesting that ERα stimulates miR-199a-5p maturation. To verify the role of ERα in miR-199a-5p maturation, we performed an in vitro RNA processing assay using in vitro-transcribed pri-miR199 incubated with whole cell lysates before and after fulvestrant treatment. Inhibition of ERα led to reduced generation of pre-miR199 from pri-miR199 (Fig. 5 K). In summary, ERα stimulates miR199a-5p maturation to inhibit CAV1 translation. miR199a-5p antagonizes m6A modification to inhibit CAV1 translation Recently, N6-methyladenosine (m6A) modification has been found to play a critical role in regulating protein translation ( 21 – 25 ). Analysis of the meRIP-Seq results revealed a hypermethylated peak in the 3’-UTR of CAV1 mRNA (Fig. 6 A). Indeed, m6A modification of CAV1 mRNA was confirmed by RIP-qPCR (Fig. 6 B). The m6A writer METTL3 was also found to bind to CAV1 mRNA (Fig. 6 C). METTL3 knockdown effectively reduced the m6A modification of CAV1 mRNA (Fig. 6 D), accompanied by reduced synthesis of nascent CAV1 protein (Fig. 6 E). Taken together, METTL3-mediated m6A modification of CAV1 mRNA is important for efficient translation. Importantly, inhibition of ERα by fulvestrant or knockdown by siRNA increased not only m6A modification but also METTL3 binding to CAV1 mRNA (Fig. 6 F- 6 G and Supplementary Fig. 5A-5B). Furthermore, upregulation of CAV1 protein induced by ERα inhibition was reversed by METTL3 knockdown (Fig. 6 H and Supplementary Fig. 5C), highlighting the dependence of ERα-regulated CAV1 protein translation on METTL3-mediated m6A modification. Interestingly, the seed sequence of the miR-199a-5p interaction contained a classical RRACH motif potential for m6A modification (AGACA) (Fig. 6 I), indicating that miR-199a-5p might antagonize m6A modification to inhibit CAV1 translation. Indeed, the miR-199a-5p inhibitor increased CAV1 mRNA m6A levels, while the miR-199a-5p mimic rescued the fulvestrant-induced increase in m6A modification (Fig. 6 J& 6 L). Interestingly, miR-199a-5p was bound to CAV1 the 3’UTR upon knockdown of METTL3 (Fig. 6 K), thus representing a competitive interplay between the miR-199a-5p interaction and m6A modification of CAV1 mRNA. Taken together, upregulated miR199a-5p antagonizes m6A modification to inhibit CAV1 translation. Fulvestrant has a synergistic effect with Nab-Paclitaxel in ER + breast cancer Based on the mechanism identified above, we explored the clinical prospects of ERα inhibitors combined with Nab-paclitaxel in ER + breast cancer patients. We first tested this hypothesis in a xenograft model using the ER + murine breast cancer cell line, E0771( 26 , 27 ) (Fig. 7 A and Supplementary Fig. 6A-6C). Twelve days after the subcutaneous injection, nude mice were randomized to treatment with vehicle, fulvestrant alone (2.5 mg/kg), nab-paclitaxel alone (22.3 mg/kg), or fulvestrant plus nab-paclitaxel. The combination treatment significantly delayed tumor growth, as evidenced by tumor volume and weight (Figure.7B-C and Supplementary Fig. 6D), accompanied by increased apoptosis and decreased Ki67 expression (Fig. 7 D- 7 E). Notably, CAV1 expression was negatively correlated with ERα expression in xenograft tumor tissues (Supplementary Fig. 6E). More importantly, ERα + breast cancer patients receiving fulvestrant plus nab-paclitaxel showed a Partial Response with significantly decreased target lesion sizes (Fig. 7 F- 7 G). Taken together, these results indicate that fulvestrant has a synergistic effect with Nab-Paclitaxel in ER + breast cancer. Discussion Estrogen receptors (ER) play an important role in the development and progression of breast cancers; However, ER + breast cancers have low sensitivity to chemotherapy, and the survival benefit of chemotherapy is limited ( 28 – 31 ). Overcoming chemoresistance remains a pressing need for ER + breast cancer. However, the combination of endocrine therapy with chemotherapy is controversial because tamoxifen may antagonize chemotherapeutic agents, partly because of its estrogen-like agonist activity ( 32 – 35 ). In contrast, fulvestrant, an ER-selective inhibitor, overcomes the disadvantages of tamoxifen with regard to estrogen-like agonist activity and downregulates ER expression ( 36 , 37 ). Previous studies have reported that ERα mediates chemotherapy resistance by inhibiting cell apoptosis and that fulvestrant has a synergistic effect with cytotoxic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) in breast cancer ( 28 , 38 ). Nevertheless, whether ERα inhibition enhances the effects of nab-paclitaxel has not yet been reported. In our study, we found that targeting ERα has the therapeutic potential to boost nab-paclitaxel efficacy in ER + breast cancer, extending the choice of chemotherapeutic agents combined with fulvestrant-based endocrine therapy. CAV1 is a membrane invagination protein involved in endocytosis of albumin-bound or conjugated chemotherapeutics. Gemcitabine upregulates CAV1 expression and imparts survival advantages to nab-paclitaxel ( 39 , 40 ). Similarly, we reported that fulvestrant synergizes with nab-paclitaxel by restoring CAV1 expression. Therefore, CAV1 serves as a biomarker for predicting nab-paclitaxel response and tailoring treatments to the appropriate patient subset. Protein translation is a complicated process influenced by multiple factors. For example, m6A modification facilitates the translation of heat shock factor 1 (HSF1) mRNA ( 24 ), while promoting the degradation of LncRNA AS-ARHGAP5 ( 41 ). Our current study shows that ERα also restricts m6A modification and translation efficiency of CAV1 mRNA, adding a layer of complexity to m6A-regulated RNA fates. We also found that miR199a-5p targets CAV1 mRNA and inhibits translation, which is consistent with previous reports ( 42 , 43 ). Thus, we propose ERa suppresses CAV1 translation by affecting the competitive interplay between the miR199a-5p interaction and m6A modification of CAV1 mRNA. Regulation of miRNA maturation has recently attracted wide attention, since miRNAs have been found to be functional in many diseases ( 44 ). Generally, miRNA biogenesis involves three steps. First, long primary miRNA (pri-miRNA) transcripts with a stem loop hairpin structure are encoded by miRNA genes, which are transcribed by RNA Polymerase II or III. In this process, transcriptional regulation mainly involves the interplay of genomic cis-regulatory elements with trans-factors, including transcription factors, co-activators, co-repressor complexes, and chromatin modifications ( 45 , 46 ). Second, pri-miRNAs are cropped to hairpin intermediates (pre-miRNAs) by the microprocessor complex, which is comprised of RNase III Drosha and its obligate RNA-binding protein partner, DiGeorge syndrome critical region gene (DGCR8). Pre-miRNAs are then exported from the nucleus to the cytoplasm by exportin 5. Third, pre-miRNAs are processed into short-lived double-stranded duplexes by cytoplasmic RNase III Dicer, which also employs an RNA binding cofactor, TAR RNA-binding protein (TRBP). These duplexes were separated, and one strand was selected as the mature miRNA, whereas the other strand was rapidly degraded. Regulators of miRNA processing bind to the stem or loop regions of miRNA precursors and influence their processing via Drosha and/or Dicer ( 47 ). For example, hnRNPA1 binds to a conserved region of the pri-miR-18a loop and promotes its cleavage by Drosha ( 48 ). Similarly, KSRP binds to sequences in the loop region of several miRNA precursors and recruits Drosha and Dicer to the pri- and pre-miRNA, respectively, enhancing their processing ( 49 ). In our study, we found that ERα promotes pri-miR199 maturation, which emphasizes an overlooked function of estrogen receptors, besides transcription factors, in the regulation of miRNA processing. However, it remains unknown how ER modulates the pri-miR199 splice, which might be controlled by RNA-binding proteins such as Drosha and DGCR8. If so, how ER influences Drosha/DGCR8 affinity for pri-miR199 is also a good question to discover. Conclusion In Summary, our study delineates a previously unappreciated nab-paclitaxel resistance mechanism in ER + breast cancer, where ERα stimulates CAV1-targeting miR199a-5p maturation to antagonize CAV1 mRNA m6A modification and compromise CAV1 translation (Fig. 8 ). Most importantly, we provide compelling preclinical and clinical evidence that combining the ERα inhibitor fulvestrant with nab-paclitaxel represents a synergistic therapeutic strategy for ER-positive breast cancer. Abbreviations BC: Breast Cancer; ERα: Estrogen receptor α; Nab-PTX: nano albumin-paclitaxel CAV1: Caveolin1; PR: partial response; SD: stable disease; PD: disease progression ORR: objective response rate; DCR: disease control rate; β-E2: β-estradiol 4-OH-TAM:4-OH-Tamoxifen; UTR: Untranslated Regions; miRNA: microRNA m6A: N6-methyladenosine; meRIP: methylated RNA Immunoprecipitation METTL3: Methyltransferase-like 3; pri-miR199: primary microRNA 199 pre-miR199: precursor microRNA 199 Declarations Ethics approval and consent to participate: The study was approved by the ethical Review Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. All the patients had signed the informed consent Consent for publication: All authors give consent for the publication Availability of data and materials: The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests ： All authors declare that they have no competing interests Funding: this study was supported by the grant from Zhejiang Natural Science Foundation to Dr. Xian Wang (LD22H160003) Authors' contributions: ZJP performed vivo and vitro experiment and visualization. WZ and ZLY carried out the data curation, formal analysis, validation and investigation. WCQ carried out the tissue processing, ZYM and YPT performed the TCGA, TCPA, GEO and other computational biology analysis. WHY performed the writing review and editing. LQL and FLF gave suggestions on the project. WX provided the funding, JHC supervised the project, writing-original draft, writing review and editing. All authors read and approved the final manuscript. Acknowledgements ： Not applicable Authors' information (optional): 1 Biomedical Research Center, Sir Runrun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China. 2 Department of Medical Oncology, Cancer Institute of Zhejiang University, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China. 3 Department of pathology, Dongyang People’s Hospital, Zhejiang, China 4 The Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou, China References Siersbæk R, Kumar S, Carroll JS. Signaling pathways and steroid receptors modulating estrogen receptor α function in breast cancer. Genes Dev. 2018;32(17-18):1141-54. Davies C, Godwin J, Gray R, Clarke M, Cutter D, Darby S, et al. Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet. 2011;378(9793):771-84. Musgrove EA, Sutherland RL. 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Zhu L, Zhu Y, Han S, Chen M, Song P, Dai D, et al. Impaired autophagic degradation of lncRNA ARHGAP5-AS1 promotes chemoresistance in gastric cancer. Cell Death Dis. 2019;10(6):383. Du F, Zhang Y, Xu Q, Teng Y, Tao M, Chen AF, et al. Preeclampsia serum increases CAV1 expression and cell permeability of human renal glomerular endothelial cells via down-regulating miR-199a-5p, miR-199b-5p, miR-204. Placenta. 2020;99:141-51. Peng W, He D, Shan B, Wang J, Shi W, Zhao W, et al. LINC81507 act as a competing endogenous RNA of miR-199b-5p to facilitate NSCLC proliferation and metastasis via regulating the CAV1/STAT3 pathway. Cell Death Dis. 2019;10(7):533. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(7):2257-61. Chang TC, Zeitels LR, Hwang HW, Chivukula RR, Wentzel EA, Dews M, et al. Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(9):3384-9. Schanen BC, Li X. Transcriptional regulation of mammalian miRNA genes. Genomics. 2011;97(1):1-6. Bronevetsky Y, Ansel KM. Regulation of miRNA biogenesis and turnover in the immune system. Immunological reviews. 2013;253(1):304-16. Guil S, Cáceres JF. The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a. Nature structural & molecular biology. 2007;14(7):591-6. Trabucchi M, Briata P, Garcia-Mayoral M, Haase AD, Filipowicz W, Ramos A, et al. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature. 2009;459(7249):1010-4. Additional Declarations No competing interests reported. Supplementary Files supplementarymaterial.pdf Supplementary Information The online version contains supplementary material available. Supplementary Table S1. RNA oligonucleotide sequences. Supplementary Figure 1. Lower response to Nab-paclitaxel in ER+BC than ER-BC. Supplementary Figure 2. inhibition of ERα enhance the sensitivity to Nab-paclitaxel in vitro. Supplementary Figure 3. CAV1 is relevant to the sensitivity of breast cancer cells to Nab-paclitaxel. Supplementary Figure 4. ERα downregulates CAV1 protein expression by inhibiting its translation. Supplementary Figure 5. m6A modification interplay with miR199a-5p in CAV1 translation. Supplementary Figure 6. Fulvestrant has a synthetic lethal with Nab-Paclitaxel in vivo. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2838943","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":195972552,"identity":"a7fc30db-8a1e-4b37-bd81-ea399a7f7ce6","order_by":0,"name":"Jianping Zhang","email":"","orcid":"","institution":"Sir Run Run Show Hospital of Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Zhang","suffix":""},{"id":195972556,"identity":"713fd593-018a-4bdb-8c17-ae823cf7c12d","order_by":1,"name":"Zuo Wang","email":"","orcid":"","institution":"Sir Run Run Show Hospital of Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zuo","middleName":"","lastName":"Wang","suffix":""},{"id":195972557,"identity":"2cc7a647-4572-419c-980b-5054e415abcb","order_by":2,"name":"Liyuan Zhu","email":"","orcid":"","institution":"Sir Run Run Show Hospital of Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liyuan","middleName":"","lastName":"Zhu","suffix":""},{"id":195972558,"identity":"3b1d4ac2-9c6d-4e34-bf6e-69316dd60b74","order_by":3,"name":"Chaoqun Wang","email":"","orcid":"","institution":"Dongyang People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chaoqun","middleName":"","lastName":"Wang","suffix":""},{"id":195972559,"identity":"b0c97715-2232-447e-a781-f51636196dff","order_by":4,"name":"Yiming Zhong","email":"","orcid":"","institution":"Sir Run Run Show Hospital of Zhejiang 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Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACxmYGNgaGCgZmEEeCBC1nSNECBGwMjG0QFnFamNt5jz34Oa+O3eAA88HbPAx2eUQ4jC/dsHcbG7PBAbZkax6G5GIitPCYSTNu4wFqATJ4GA4kNhCnZY4EUAv/N1K0NBiAbGEjXotkz7EEZsnDbMaWcwySCWsx7D9jJvGjpi6Z73jzwxtvKuyI0AJVkQyJTANC6oFAHkrbEaF2FIyCUTAKRioAAD+4L2+lGtRMAAAAAElFTkSuQmCC","orcid":"","institution":"Sir Run Run Show Hospital of Zhejiang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hongchuan","middleName":"","lastName":"Jin","suffix":""}],"badges":[],"createdAt":"2023-04-20 02:44:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2838943/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2838943/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36591181,"identity":"81bb3158-b353-44a7-b316-0193c5396f67","added_by":"auto","created_at":"2023-05-03 21:24:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":483636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLower response to Nab-paclitaxel in ER+BC than ER-BC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Work flow of Patient Enrollment and Responses. (B) Evaluate of Disease progression in ER+ and ER- BC patients by the RECIST guideline. (C) Therapeutic Evaluation in ER+ and ER- BC patients. (D) Analysis of cell viability in ER+BC (MCF7 and T47D) and ER-BC (MB231 and BT549) with Nab-PTX treatment by MTS assays. (E) Analysis of apoptosis in ER+BC (MCF7 and T47D) and ER-BC (MB231 and BT549) with or without Nab-PTX treatment by western blot. T test was performed for significant analysis, significant difference is indicated with * for p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/9f2f9c30879255b884f7236e.png"},{"id":36591182,"identity":"4affd33a-cef3-4a4d-8397-e2407fb783fc","added_by":"auto","created_at":"2023-05-03 21:24:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1351264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003einhibition of ERα enhance the sensitivity to Nab-paclitaxel in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Analysis of cell viability after siESR1 with Nab-PTX in MCF7 cells using MTS assay. (B) and (C) Analysis of apoptosis after siESR1 with Nab-PTX (10ng/ml) in MCF7 cells by flow cytometry and western blotting.(D) Analysis of cell viability after combining 4-OH-TAM (5uM) with Nab-PTX in MCF7 cells using MTS assays.(E) and (F) Analysis of apoptosis after combining 4-OH-TAM (5uM) with Nab-PTX (10ng/ml) in MCF7 cells by flow cytometry and western blotting.(G) Analysis of cell viability after combining Fulvestrant (1uM) with Nab-PTX in MCF7 cells using MTS assays.\u003c/p\u003e\n\u003cp\u003e(H) and (I) Analysis of apoptosis after combining Fulvestrant (1uM) with Nab-PTX (10ng/ml) in MCF7 cells by flow cytometry and western blotting.(J) Analysis of cell viability before and after β-E2 treatment with Nab-PTX in MCF7 cells by MTS assays. (K) Colony formation assay before and after β-E2 treatment with Nab-PTX (10ng/ml) in MCF7cells. (L) Analysis of apoptosis before and after β-E2 treatment with Nab-PTX (10ng/ml) by western blotting.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/175e643d3d6a457ecfcbdcdd.png"},{"id":36592150,"identity":"2453f3f8-4118-4158-8a3e-3ad75d4e94f4","added_by":"auto","created_at":"2023-05-03 21:40:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8625555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAV1 is relevant to the sensitivity of breast cancer cells to Nab-paclitaxel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Analysis of cell viability after siCAV1 with Nab-PTX in MB231 cells using MTS assay. (B) and (C) Analysis of apoptosis after siCAV1 with Nab-PTX (10ng/ml) in MB231 cells by flow cytometry and western blotting.(D) Analysis of cell viability after overexpression of CAV1 with Nab-PTX in MCF7 cells by the MTS assay. (E) Analysis of apoptosis after overexpression of CAV1 with Nab-PTX in MCF7 cells using western blotting.(F) Analysis of cell viability after siCAV1 with PTX in MB231 cells by the MTS assay.(G) Immunofluorescence after siCAV1 with DQ-BSA encapsulated for 30 min in MB231 cells.(H) and (I) Immunohistochemical analysis of CAV1 protein expression in Nab-PTX-treated patients.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/263cb921aafba999f538f69e.png"},{"id":36591796,"identity":"2e836c92-7ac3-4189-bb29-cc8c97d057b5","added_by":"auto","created_at":"2023-05-03 21:32:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9906565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eERα downregulates CAV1 protein expression by inhibiting its translation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Correlation between ERa and CAV1 mRNA levels in BC tissues from TCPA. (B) and (C) IHC analysis of the association between CAV1 and ERa protein expression in BC tissues. (D) Western blot analysis of CAV1 and ERa expression in different BC cell lines. (E) Western blot analysis of CAV1 protein expression after siESR1 treatment in MCF7 cells. (F) Western blot analysis of CAV1 protein expression after treatment with 4-OH-TAM,fulvestrant,or β-E2 in MCF7 cells. (G) Western blot analysis of CAV1 protein expression after ERa overexpresses in MB231. (H)\u0026amp;(I) Puromycin-labelling test for CAV1 translation after siESR1 or fulvestrant treatment in MCF7 cells.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/a9d9699d945c41b4102c83a2.png"},{"id":36591795,"identity":"cacc5618-c368-4e04-998e-d8a1b2159778","added_by":"auto","created_at":"2023-05-03 21:32:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1925886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eERα stimulates miR199a-5p maturation to inhibit CAV1 translation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Venn diagram: miRNAs upregulated by E2 stimulate VS predicted CAV1-targeting miRNAs. (B) Western blot analysis of CAV1 protein expression after treatment with the miRNA inhibitor. (C) Real-time PCR analysis of CAV1 mRNA levels after siESR1. (D) Western blot analysis of CAV1 protein expression after mimic miR199a-5p and inhibitor miR199a-5p. (E) Luciferase assay analysis of miR199a-5p binding ability to CAV1. (F) RNA pull-down analysis miR199a-5p binding ability to CAV1. (G) Western blot analysis of CAV1 protein expression after mimic miR199a-5p was rescued by fulvestrant. (H) Real-time PCR analysis of CAV1 mRNA levels in MB231 and MCF7cells. (I) Correlation between miR199a-5p and ERa protein. (J) Real-time PCR analysis of Pri-miR199, Pre-miR199, and mature miR199a-5p expression after treatment with fulvestrant.\u003c/p\u003e\n\u003cp\u003e(K) In vitro RNA processing assay analysis of Pri-miR199 splice efficiency after fulvestrant treatment.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/228a1e1ffd655dcf15e2a152.png"},{"id":36591185,"identity":"4f97b848-86cd-4233-8846-3dec1a59028b","added_by":"auto","created_at":"2023-05-03 21:24:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1291197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003em6A modification interplay with miR199a-5p in CAV1 translation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) meRIP-Seq analysis of CAV1 3’UTR m6A modification. (B) RIP analysis of CAV1 m6A modification. (C) RIP analysis of METTL3 binding to the CAV1 mRNA. (D) RIP analysis of CAV1 m6A level after siMETTL3 transfection. (E) Puromycin-labelling test for CAV1 translation after siMETTL3. (F) RIP analysis CAV1 m6A level after fulvestrant treatment.(G) RIP analysis showing that METTL3 binds to CAV1 mRNA after fulvestranttreatment. (H) Western blot analysis of CAV1 protein expression after siMETTL3 was rescued by fulvestrant.(I) miR199a-5p seed region overlap CAV1 3’UTR m6A motif “AGACA”. (J) RIP analysis of CAV1 m6A modification after miR199a-5p inhibition. (K) RNA pull-down analysis of miR199a-5p bind to the CAV1 3’UTR after siMETTL3 transfection. (L) RIP analysis of CAV1 m6A modification after miR199a-5p inhibition and rescue by fulvestrant.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/32247c4e856cc74ea5f6e424.png"},{"id":36591189,"identity":"d27760ac-be8e-484c-8e3a-a03df475b5b6","added_by":"auto","created_at":"2023-05-03 21:24:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9402012,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFulvestrant has a synthetic lethal with Nab-Paclitaxel in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Animal Experiment schematic diagram: nude mice were randomized to treatment with control, fulvestrant alone (2.5 mg/kg), nab-paclitaxel alone (22.3 mg/kg) or fulvestrant plus nab-paclitaxel\u003c/p\u003e\n\u003cp\u003e(B) Dynamic tumor growth. (C) Weight of tumors. (D) Western blot analysis of c-parp and c-caspase3 protein expression. (E) IHC analysis of Ki-67 protein expression. (F) Clinical patient therapeutic schedule.(G) Radiology showing a decrease in the target lesion diameter after treatment\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/7252fca980e8c2c7be6e5ed4.png"},{"id":36591187,"identity":"3c324466-4030-4a25-9ef3-84fe41255ed8","added_by":"auto","created_at":"2023-05-03 21:24:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5671839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eIn Estrogen receptor (ER)+ breast cancer, ERα stimulates miR199a-5p maturation to antagonize Caveolin 1(CAV1) mRNA m6A modification and compromise CAV1 translation, which leads to resistance to nab-paclitaxel.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/9a1e4771cb3a8f9a09a4d351.png"},{"id":37500201,"identity":"10ec9cd3-5012-4e97-ae98-ba9c40ae8aaf","added_by":"auto","created_at":"2023-05-25 15:59:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4139594,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/338153ef-edb5-44d2-9551-839e6252a4dd.pdf"},{"id":36591184,"identity":"af0e04c9-e998-42ea-9a96-3c6d8b21eeb8","added_by":"auto","created_at":"2023-05-03 21:24:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":405318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available.\u003c/p\u003e\n\u003cp\u003eSupplementary Table S1. RNA oligonucleotide sequences.\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 1. Lower response to Nab-paclitaxel in ER+BC than ER-BC.\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 2. inhibition of ERα enhance the sensitivity to Nab-paclitaxel in vitro.\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 3. CAV1 is relevant to the sensitivity of breast cancer cells to Nab-paclitaxel.\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 4. ERα downregulates CAV1 protein expression by inhibiting its translation.\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 5. m6A modification interplay with miR199a-5p in CAV1 translation.\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 6. Fulvestrant has a synthetic lethal with Nab-Paclitaxel in vivo.\u003c/p\u003e","description":"","filename":"supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2838943/v1/ab75864166479f527f58edfd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Estrogen receptor α confers Nab-paclitaxel resistance in breast cancer by promoting miR199a-5p maturation to inhibit Caveolin 1 translation","fulltext":[{"header":"Background","content":"\u003cp\u003eEstrogen receptor α (ERα) is the major driver of \u0026sim;75% of breast cancers (BC) with routine endocrine treatment (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, many ER\u0026thinsp;+\u0026thinsp;BC patients still relapse after endocrine treatment and require chemotherapy in the recurrent or metastatic phase (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Nab-paclitaxel (Abraxane), the first clinically successful albumin-bound chemotherapeutic agent (paclitaxel bound to human albumin), is commonly used in the treatment of pancreatic cancer, non\u0026ndash;small cell lung cancer (NSCLC), and breast cancer (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, some clinical trials and retrospective analyses have observed that nab-paclitaxel is less effective in ER\u0026thinsp;+\u0026thinsp;BC than in ER-BC (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), indicating that ER status may play an important role in Nab-paclitaxel response, despite the lack of understanding of the underlying mechanisms.\u003c/p\u003e \u003cp\u003eCaveolin 1(CAV1) is the principal structural protein of Caveolae, which are 50\u0026ndash;100 nm flask-shaped invaginations of the plasma membrane functioning in endocytosis, cholesterol homeostasis, signal transduction, and macromolecule transport (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). CAV1 is reported to be upregulated in pancreatic cancer, NSCLC, and breast cancer, and is related to increased invasion, metastasis, resistance to radiation or chemotherapy, and poor prognosis (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition, a previous study claimed that CAV1 is an albumin transporter that facilitates nab-paclitaxel uptake to enhance its efficacy in pancreatic cancer and NSCLC (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, it remains unknown whether CAV1 is relevant to nab-paclitaxel resistance in ER\u0026thinsp;+\u0026thinsp;BC.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that ERa mediates nab-paclitaxel resistance by inhibiting CAV1 translation. Mechanistically, ERa promotes miR199a-5p maturation, which binds to CAV1 mRNA, thus competitively suppressing m6A modification-dependent mRNA translation. Our study provides a rationale for targeting ERa to overcome nab-paclitaxel resistance in ER\u0026thinsp;+\u0026thinsp;breast cancer.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Retrospective study design\u003c/h2\u003e \u003cp\u003eA cohort of 116 breast cancer patients who received nab-paclitaxel at Sir Run Run Show Hospital, Zhejiang University, between January 2008 and May 2022 were retrospectively reviewed. All eligible patients received at least one dose of nab-paclitaxel. We analyzed the baseline ER status by IHC and evaluated disease progression using the RECIST guidelines (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell lines, antibodies and reagents\u003c/h2\u003e \u003cp\u003eThe breast cancer cell lines MCF-7, T-47D, MB231, and BT549 were purchased from the American Type Culture Collection (ATCC). Both were authenticated using short tandem repeat multi-amplification and tested negative for Mycoplasma. MCF-7 and T-47D cells were cultured in DMEM (Gibco, USA), and MB231 and BT549 cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Hyclone, USA),100 U/ml penicillin, and 100 \u0026micro;g/ml streptomycin (Life Technologies/Gibco, Shanghai, China). Cells were grown at 37\u0026deg;C in a humidified incubator with 5% CO2 and 95% humidity.\u003c/p\u003e \u003cp\u003eThe following antibodies were used for western blotting or immunohistochemistry: ERα(Vector Laboratories, VP-E613), CAV1 (ABclonal, A1555), METTL3 (ABclonal, A8370), β-Actin (Cell Signaling Technology, 4970), Cleaved PARP1 (C-PARP1) (Cell Signaling Technology, 9541), Cleaved Caspase-3 (Cell Signaling Technology, 9661), 4-OH-Tamoxifen (579002), and β-estradiol (E8875) were purchased from Merck and Sigma Aldrich. Fulvestrant (HY-13636) was purchased from MedChemExpress (Shanghai, China). Nab-PTX was supported by the Celgene Corporation. DQ\u0026trade; Green BSA( Invitrogen\u0026trade;,1014496)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSiRNA, miRNA mimics/inhibitors and plasmid transfections\u003c/h2\u003e \u003cp\u003eSmall interfering RNA (siRNA) targeting ERα, CAV1, METTL3, and microRNAs were synthesized by GenePharma (Shanghai, China) and RiboBio (Guangzhou, China). The sequences of the siRNAs and miRNA mimics/inhibitors are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The plasmids Flag-CAV1-pcDNA3.1 and Flag- ERα- pcDNA3.1 were purchased from Genechem (Shanghai, China). SiRNAs and miRNA mimics/inhibitors were transfected into cells seeded overnight using lipo2000 (Invitrogen, USA) or Lipofectamine RNAiMax transfection reagent (Invitrogen, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, reverse transcription and qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer's instructions, and RNA concentration was quantified using a NanoDrop 2000 (Nanodrop, Wilmington, DE, USA). RNA (1\u0026ndash;2 \u0026micro;g) was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368813). Real-time PCR (qPCR) was conducted using a SYBR Green Master Mix Kit (ComWin Biotech, CW0659s, Beijing, China). The qPCR data were normalized to the control group, and the relative expression of the indicated genes shown in the histograms is expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The primers used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase activity assay\u003c/h2\u003e \u003cp\u003eThe fragment of the CAV1 3'-UTR containing the miR-199a-5p binding site was amplified by PCR. and was inserted into the pMIR-REPORTER vector (Promega). For the luciferase assay, HEK-293T in 6-well plates. The CAV1-3\u0026rsquo;UTR plasmids were co-transfected using lipo2000 with miR199a-5p mimics and pRL Renilla as an internal control (Invitrogen, USA). After 48 h, the luciferase activity was measured using the Dual-GLO Luciferase Assay System (Promega Corporation, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation (RIP) assay\u003c/h2\u003e \u003cp\u003eThe RIP assay was conducted following the manufacturer\u0026rsquo;s instructions for the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore, No.17\u0026ndash;700). Cells (\u0026gt;\u0026thinsp;2 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e) were collected and lysed in 100 \u0026micro;l RIP lysis buffer, immunoprecipitated with the indicated antibody-linked beads or protein G magnetic beads overnight at 4\u0026deg;C, washed six times in Washing Buffer, and the protein was denatured at 55\u0026deg;C. Total RNA was isolated and quantified using RT-PCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePuromycin-labelling\u003c/h2\u003e \u003cp\u003e5X 10\u003csup\u003e6\u003c/sup\u003e cells were plated in a 10 cm dish and incubated with 1:1000 biotin-dC-puromycin (NU-925-BIO-S, Jena Bioscience) for 24 h. Cells were collected and lysed in 1% NP40 buffer with protease inhibitor cocktail. After 15000rpm x30min centrifugation at 4\u0026deg;C, the supernatant was collected and incubated with 80ul streptavidin sepharose beads (GE17\u0026ndash;5113-01, Sigma) by rotating at 4\u0026deg;C overnight. The mixture was washed five times with 1% NP40 buffer for 5 times and test CAV1 expression was tested by western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBiotin pull down assay\u003c/h2\u003e \u003cp\u003eCells were transfected with biotinylated miR199a-5p probes for 48 h and resuspended in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2, 60 U/mL SUPERase-In, 1 mM DTT, 0.05% Igepal, protease inhibitors). Lysates were incubated with prepared streptavidin beads (GE Healthcare). Yeast tRNA (Sigma-Aldrich) was used to block the lysates at 4\u0026deg;C for 3 h. The cells were washed five times with binding and wash buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl). Finally, the bound RNAs was extracted and purified for qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro pri-miRNA processing assays\u003c/h2\u003e \u003cp\u003eProbe biotin-pri-miR-199 was transcribed in vitro using a T7 based MEGA shortscript kit (Life Technologies). Biotin RNA-labeling Mix (Roche,11685597910) was used for the in vitro transcription reaction. For pri-miR-199 processing assays, biotin-pri-miR-199 was incubated with whole cell lysates of 293T cells at 37\u0026deg;C for 90 min. The Lysates were then incubated with streptavidin beads (GE Healthcare). Yeast tRNA (Sigma) was used to block lysates at 4\u0026deg;C for 3 h, and RNA purified from the reaction products was analyzed by qRT-PCR. The primers used for in vitro pri-miR-199 transcription are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eSix-week-old female BALB/c nude mice from the Center of Experimentation of Zhejiang University were used following the Institutional Animal Care and Use Committee and National Institute of Health (NIH) guidelines. Tumors were established by subcutaneous injection (1\u0026times; 10\u003csup\u003e6\u003c/sup\u003e E0771 cells in 0.1 ml saline) into the flanks of the mice. After 12 days, tumors reached\u0026thinsp;\u0026gt;\u0026thinsp;150 mm\u003csup\u003e3\u003c/sup\u003e in size, and the mice were randomly allocated into four groups and treated with Nab-PTX (22.3 mg/kg body weight; i.g, every 3 days), Fulvestrant (2.5 mg/kg body weight; i.p, every 3 days), Nab-PTX combined with fulvestrant, or vehicle PBS as control. Tumor volume was measured every 3 days. Mice were euthanized when the tumor size reached approximately 2000 mm\u003csup\u003e3\u003c/sup\u003e. Tumor volume was calculated using the following formula: length \u0026times; width\u003csup\u003e2\u003c/sup\u003e \u0026times;0 .5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical approach used in every experiment to compare the differences between the groups is provided in the figure legends. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eER\u0026thinsp;+\u0026thinsp;BC patients show worse response to nab-paclitaxel than ER- BC patients\u003c/h2\u003e \u003cp\u003eTo test whether ER status dictates responsiveness to nab-paclitaxel, we retrospectively reviewed a cohort of 116 breast cancer patients who received nab-paclitaxel at Sir Run Run Show Hospital, Zhejiang University, between January 2008 and May 2022. All eligible patients received at least one dose of nab-paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We analyzed the baseline ER status to determine its ability to predict disease progression, as evaluated by the RECIST guidelines. 55 patients (64.7%) were classified as the ER-positive subtype, and 30 patients (35.2%) were classified as the ER-negative subtype (Supplementary Fig.\u0026nbsp;1A). In the ER\u0026thinsp;+\u0026thinsp;subgroup, four patients (7.3%) achieved partial response (PR), 17 patients (30.9%) had stable disease (SD), and 34 patients (61.8%) experienced disease progression (PD). In the ER\u0026thinsp;\u0026minus;\u0026thinsp;group, 16 patients (53.3%) achieved PR, 9 patients (30%) had SD, and 5 patients (16.7%) experienced PD. There was also a significant discrepancy between these two subgroups with regard to the objective response rate (ORR) and disease control rate (DCR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C), which is consistent with previous reports (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In vitro experiments also verified that ERα-BC cells (MB231 and BT549) were more sensitive to nab-paclitaxel treatment than ERα\u0026thinsp;+\u0026thinsp;cells (MCF7 and T47D) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;1B ). Taken together, we concluded that ER\u0026thinsp;+\u0026thinsp;BC patients were less responsive to nab-paclitaxel than ER-BC patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eERα inhibit the sensitivity of breast cancer cells to Nab-paclitaxel\u003c/h2\u003e \u003cp\u003eGiven the distinct response to Nab-paclitaxel in ER\u0026thinsp;+\u0026thinsp;and ER- breast cancers, we questioned whether ERα inhibits the anticancer activity of nab-paclitaxel. Indeed, genetic knockdown of ERα in MCF7 cells conferred sensitivity to nab-paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;2A). Similarly, chemical inhibition of ERα in MCF7 and T47D cells greatly enhanced the growth-inhibitory effects of Nab-paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI and Supplementary Fig.\u0026nbsp;2B-2E). Furthermore, β-estradiol(β-E2) deprivation increased nab-paclitaxel-induced growth inhibition and apoptosis activation, which was reversed by β-E2 supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL and Supplementary Fig.\u0026nbsp;2F). Taken together, ERα decreased the sensitivity of breast cancer cells to nab-paclitaxel.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCAV1 is relevant to the sensitivity of breast cancer cells to Nab-paclitaxel\u003c/h2\u003e \u003cp\u003eAs CAV1 is critical for the response to albumin-bound chemotherapeutics in NSLC and pancreatic cancer by affecting albumin endocytosis (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), we assessed the effect of CAV1 on nab-paclitaxel sensitivity in breast cancer cells. After CAV1 expression was knocked down by siRNA, MB231 and BT549 cells displayed resistance to nab-paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;3A-3E). In contrast, exogenous CAV1 expression conferred MCF7 and T47D cells increased sensitivity to Nab-paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, Supplementary Fig.\u0026nbsp;3F-3G). However, their sensitivity to paclitaxel was not altered (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), suggesting a role of CAV1 in mediating the internalization of albumin-bound drugs. Indeed, upon encapsulation with DQ-BSA for 30 min in culture medium (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), MB231 cells accumulated high levels of DQ-BSA in the cytoplasm, which was abrogated after CAV1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Importantly, patients with higher CAV1 expression had a better benefit from nab-paclitaxel treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), revealing a positive correlation between CAV1 expression and the disease control rate in breast cancer after nab-paclitaxel treatment. In conclusion, CAV1 expression is important for determining the nab-paclitaxel response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eERα downregulates CAV1 protein expression by inhibiting its translation\u003c/h2\u003e \u003cp\u003eTo explore the potential correlation between ERα and CAV1, we analyzed their expression based on reverse-phase protein arrays (RPPA) (n\u0026thinsp;=\u0026thinsp;627) from The Cancer Proteome Atlas (TCPA) database (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). CAV1 protein levels were significantly negatively correlated with ERα protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, there was no such correlation between CAV1 and ESR1 mRNA levels based on results from The Cancer Genome Atlas (TCGA) (Supplementary Fig.\u0026nbsp;4A). In addition, CAV1 protein expression was much lower in ERα-positive breast cancer tissues than in ERα-negative breast cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Moreover, CAV1 protein levels were notably higher in ERα-negative breast cancer cell lines than in ERα-positive breast cancer cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). After knockdown of ERα expression by siRNA or treatment with ERα inhibitors including 4-OH-Tamoxifen and fulvestrant, the CAV1 protein was upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and Supplementary Fig.\u0026nbsp;4B-4D) while CAV1 mRNA remained unchanged (Supplementary Fig.\u0026nbsp;4E-4G). In contrast, exogenous ERα overexpression or ER activation by 17β-E2 inhibited the expression of CAV1 protein, but not mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G and Supplementary Fig.\u0026nbsp;4H). In addition, either inhibition or activation of ERα failed to affect the expression of exogenous CAV1 (Supplementary Fig.\u0026nbsp;4I-4J), since exogenous CAV1 doesn\u0026rsquo;t have a 3\u0026rsquo;UTR. Therefore, ERα appears to inhibit the translation of CAV1. In fact, the synthesis of nascent CAV1 protein increased upon ERα depletion by siRNA or chemical inhibition by fulvestrant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). In summary, ERα downregulated CAV1 expression by inhibiting its translation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eERα stimulates miR199a-5p maturation to inhibit CAV1 translation\u003c/h2\u003e \u003cp\u003eMicroRNAs (miRNAs) are widely recognized as negative regulators of protein translation through partial base-pairing with the 3\u0026rsquo;-UTR of mRNA to block formation of the translation initiation complex (\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). We queried potential miRNAs that could be upregulated upon E2 stimulation (data from GSE78167) and predicted target CAV1 using TargetScan, miRDB, and StarBase, which revealed an overlap of seven miRNAs, including hsa-miR-493-3p, hsa-miR-520a*, hsa-miR-384, hsa-miR-124-3p, hsa-miR-512-3p, hsa-miR-199a-5p, and hsa-miR-302b* (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, inhibition of miR-199a-5p, miR-512-3p, or miR-520a, but not of other miRNAs, upregulated CAV1 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), whereas knockdown of ERα downregulated the expression of only two miRNAs, miR-124-3p and miR-199a-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Therefore, we postulated that miR-199a-5p may be involved in the ERα-mediated regulation of CAV1 protein translation. In fact, the miR-199a-5p mimic reduced while its inhibitor increased CAV1 protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In addition, luciferase activity driven by the CAV1 mRNA 3\u0026rsquo;-UTR was significantly inhibited by miR199a-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The interaction of miR199a-5p with CAV1 mRNA was further confirmed by a biotin pulldown assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Importantly, the miR-199a-5p mimic succeeded in rescuing CAV1 upregulation induced by fulvestrant (Figure.5G), highlighting the relevance of miR199a-5p upregulation to ERα-mediated CAV1 downregulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we found that miR199a-5p was highly expressed in ERα\u0026thinsp;+\u0026thinsp;MCF7 cells compared to ERα- cell MB231 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Besides, the expression of miR199a-5p has positive correlated with ERα expression in breast cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Inhibition of ERα by Fulvestrant decreased mature miR-199a-5p and its precursor miR-199a, but increased the level of primary miR-199a (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ), suggesting that ERα stimulates miR-199a-5p maturation. To verify the role of ERα in miR-199a-5p maturation, we performed an in vitro RNA processing assay using in vitro-transcribed pri-miR199 incubated with whole cell lysates before and after fulvestrant treatment. Inhibition of ERα led to reduced generation of pre-miR199 from pri-miR199 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). In summary, ERα stimulates miR199a-5p maturation to inhibit CAV1 translation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003emiR199a-5p antagonizes m6A modification to inhibit CAV1 translation\u003c/h2\u003e \u003cp\u003eRecently, N6-methyladenosine (m6A) modification has been found to play a critical role in regulating protein translation (\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Analysis of the meRIP-Seq results revealed a hypermethylated peak in the 3\u0026rsquo;-UTR of CAV1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Indeed, m6A modification of CAV1 mRNA was confirmed by RIP-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The m6A writer METTL3 was also found to bind to CAV1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). METTL3 knockdown effectively reduced the m6A modification of CAV1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), accompanied by reduced synthesis of nascent CAV1 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Taken together, METTL3-mediated m6A modification of CAV1 mRNA is important for efficient translation. Importantly, inhibition of ERα by fulvestrant or knockdown by siRNA increased not only m6A modification but also METTL3 binding to CAV1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and Supplementary Fig.\u0026nbsp;5A-5B). Furthermore, upregulation of CAV1 protein induced by ERα inhibition was reversed by METTL3 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH and Supplementary Fig.\u0026nbsp;5C), highlighting the dependence of ERα-regulated CAV1 protein translation on METTL3-mediated m6A modification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, the seed sequence of the miR-199a-5p interaction contained a classical RRACH motif potential for m6A modification (AGACA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), indicating that miR-199a-5p might antagonize m6A modification to inhibit CAV1 translation. Indeed, the miR-199a-5p inhibitor increased CAV1 mRNA m6A levels, while the miR-199a-5p mimic rescued the fulvestrant-induced increase in m6A modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ\u0026amp; \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Interestingly, miR-199a-5p was bound to CAV1 the 3\u0026rsquo;UTR upon knockdown of METTL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK), thus representing a competitive interplay between the miR-199a-5p interaction and m6A modification of CAV1 mRNA. Taken together, upregulated miR199a-5p antagonizes m6A modification to inhibit CAV1 translation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFulvestrant has a synergistic effect with Nab-Paclitaxel in ER\u0026thinsp;+\u0026thinsp;breast cancer\u003c/h2\u003e \u003cp\u003eBased on the mechanism identified above, we explored the clinical prospects of ERα inhibitors combined with Nab-paclitaxel in ER\u0026thinsp;+\u0026thinsp;breast cancer patients. We first tested this hypothesis in a xenograft model using the ER\u0026thinsp;+\u0026thinsp;murine breast cancer cell line, E0771(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;6A-6C). Twelve days after the subcutaneous injection, nude mice were randomized to treatment with vehicle, fulvestrant alone (2.5 mg/kg), nab-paclitaxel alone (22.3 mg/kg), or fulvestrant plus nab-paclitaxel.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe combination treatment significantly delayed tumor growth, as evidenced by tumor volume and weight (Figure.7B-C and Supplementary Fig.\u0026nbsp;6D), accompanied by increased apoptosis and decreased Ki67 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Notably, CAV1 expression was negatively correlated with ERα expression in xenograft tumor tissues (Supplementary Fig.\u0026nbsp;6E). More importantly, ERα\u0026thinsp;+\u0026thinsp;breast cancer patients receiving fulvestrant plus nab-paclitaxel showed a Partial Response with significantly decreased target lesion sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Taken together, these results indicate that fulvestrant has a synergistic effect with Nab-Paclitaxel in ER\u0026thinsp;+\u0026thinsp;breast cancer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEstrogen receptors (ER) play an important role in the development and progression of breast cancers; However, ER\u0026thinsp;+\u0026thinsp;breast cancers have low sensitivity to chemotherapy, and the survival benefit of chemotherapy is limited (\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Overcoming chemoresistance remains a pressing need for ER\u0026thinsp;+\u0026thinsp;breast cancer. However, the combination of endocrine therapy with chemotherapy is controversial because tamoxifen may antagonize chemotherapeutic agents, partly because of its estrogen-like agonist activity (\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In contrast, fulvestrant, an ER-selective inhibitor, overcomes the disadvantages of tamoxifen with regard to estrogen-like agonist activity and downregulates ER expression (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Previous studies have reported that ERα mediates chemotherapy resistance by inhibiting cell apoptosis and that fulvestrant has a synergistic effect with cytotoxic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) in breast cancer (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Nevertheless, whether ERα inhibition enhances the effects of nab-paclitaxel has not yet been reported. In our study, we found that targeting ERα has the therapeutic potential to boost nab-paclitaxel efficacy in ER\u0026thinsp;+\u0026thinsp;breast cancer, extending the choice of chemotherapeutic agents combined with fulvestrant-based endocrine therapy.\u003c/p\u003e \u003cp\u003eCAV1 is a membrane invagination protein involved in endocytosis of albumin-bound or conjugated chemotherapeutics. Gemcitabine upregulates CAV1 expression and imparts survival advantages to nab-paclitaxel (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Similarly, we reported that fulvestrant synergizes with nab-paclitaxel by restoring CAV1 expression. Therefore, CAV1 serves as a biomarker for predicting nab-paclitaxel response and tailoring treatments to the appropriate patient subset.\u003c/p\u003e \u003cp\u003eProtein translation is a complicated process influenced by multiple factors. For example, m6A modification facilitates the translation of heat shock factor 1 (HSF1) mRNA (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), while promoting the degradation of LncRNA AS-ARHGAP5 (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Our current study shows that ERα also restricts m6A modification and translation efficiency of CAV1 mRNA, adding a layer of complexity to m6A-regulated RNA fates. We also found that miR199a-5p targets CAV1 mRNA and inhibits translation, which is consistent with previous reports (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Thus, we propose ERa suppresses CAV1 translation by affecting the competitive interplay between the miR199a-5p interaction and m6A modification of CAV1 mRNA.\u003c/p\u003e \u003cp\u003eRegulation of miRNA maturation has recently attracted wide attention, since miRNAs have been found to be functional in many diseases (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Generally, miRNA biogenesis involves three steps. First, long primary miRNA (pri-miRNA) transcripts with a stem loop hairpin structure are encoded by miRNA genes, which are transcribed by RNA Polymerase II or III. In this process, transcriptional regulation mainly involves the interplay of genomic cis-regulatory elements with trans-factors, including transcription factors, co-activators, co-repressor complexes, and chromatin modifications (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Second, pri-miRNAs are cropped to hairpin intermediates (pre-miRNAs) by the microprocessor complex, which is comprised of RNase III Drosha and its obligate RNA-binding protein partner, DiGeorge syndrome critical region gene (DGCR8). Pre-miRNAs are then exported from the nucleus to the cytoplasm by exportin 5. Third, pre-miRNAs are processed into short-lived double-stranded duplexes by cytoplasmic RNase III Dicer, which also employs an RNA binding cofactor, TAR RNA-binding protein (TRBP). These duplexes were separated, and one strand was selected as the mature miRNA, whereas the other strand was rapidly degraded. Regulators of miRNA processing bind to the stem or loop regions of miRNA precursors and influence their processing via Drosha and/or Dicer (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). For example, hnRNPA1 binds to a conserved region of the pri-miR-18a loop and promotes its cleavage by Drosha (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Similarly, KSRP binds to sequences in the loop region of several miRNA precursors and recruits Drosha and Dicer to the pri- and pre-miRNA, respectively, enhancing their processing (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). In our study, we found that ERα promotes pri-miR199 maturation, which emphasizes an overlooked function of estrogen receptors, besides transcription factors, in the regulation of miRNA processing. However, it remains unknown how ER modulates the pri-miR199 splice, which might be controlled by RNA-binding proteins such as Drosha and DGCR8. If so, how ER influences Drosha/DGCR8 affinity for pri-miR199 is also a good question to discover.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn Summary, our study delineates a previously unappreciated nab-paclitaxel resistance mechanism in ER\u0026thinsp;+\u0026thinsp;breast cancer, where ERα stimulates CAV1-targeting miR199a-5p maturation to antagonize CAV1 mRNA m6A modification and compromise CAV1 translation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Most importantly, we provide compelling preclinical and clinical evidence that combining the ERα inhibitor fulvestrant with nab-paclitaxel represents a synergistic therapeutic strategy for ER-positive breast cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBC: Breast Cancer; ER\u0026alpha;: Estrogen receptor \u0026alpha;; Nab-PTX: nano albumin-paclitaxel\u003c/p\u003e\n\u003cp\u003eCAV1: Caveolin1; PR: partial response; SD: stable disease; PD: disease progression ORR: objective response rate; DCR: disease control rate; \u0026beta;-E2: \u0026beta;-estradiol \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4-OH-TAM:4-OH-Tamoxifen; UTR: Untranslated Regions; miRNA: microRNA \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003em6A: N6-methyladenosine; meRIP: methylated RNA Immunoprecipitation \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMETTL3: Methyltransferase-like 3; pri-miR199: primary microRNA 199 \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003epre-miR199: precursor microRNA 199\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThe study was approved by the ethical Review Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. All the patients had signed the informed consent\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eAll authors give consent for the publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eAll authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e this study was supported by the grant from Zhejiang Natural Science Foundation to Dr. Xian Wang (LD22H160003)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e ZJP performed vivo and vitro experiment and visualization. WZ and ZLY carried out the data curation, formal analysis, validation and investigation. WCQ carried out the tissue processing, ZYM and YPT performed the TCGA, TCPA, GEO and other computational biology analysis. WHY performed the writing review and editing. LQL and FLF gave suggestions on the project. WX provided the funding, JHC supervised the project, writing-original draft, writing review and editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information (optional):\u0026nbsp;\u003c/strong\u003e1 Biomedical Research Center, Sir Runrun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2 Department of Medical Oncology, Cancer Institute of Zhejiang University, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.\u003c/p\u003e\n\u003cp\u003e3 Department of pathology, Dongyang People\u0026rsquo;s Hospital, Zhejiang, China\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4 The Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiersb\u0026aelig;k R, Kumar S, Carroll JS. 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Nature. 2009;459(7249):1010-4.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Estrogen receptor α (ERα), Nab-Paclitaxel, Caveolin 1 (CAV1), translation, N6-methyladenosine (m6A), miR199a-5p","lastPublishedDoi":"10.21203/rs.3.rs-2838943/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2838943/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEstrogen receptor positive (ER+) breast cancer patients are poorly responsive to Nab-paclitaxel compared to ER negative (ER-) breast cancer patients. Herein, we conducted an investigation regarding the mechanism for ERα confers Nab-paclitaxel resistance in breast cancer.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRetrospectively reviewed 116 cases of breast cancer treated with nab-paclitaxel between Jan 2008 and May 2022 in Sir Run Run Shaw Hospital. StataSE 16 software was used to analyze the basic conditions and therapeutic effects. Protein-RNA interactions were validated through RNA immunoprecipitation and RNA pull-down assays. In vitro and in vivo experiments were carried out to testify the effect of ERα on Nab-paclitaxel resistance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe show that ERα limits the efficacy of nab-paclitaxel in breast cancer while genetic or pharmacological inhibition of ERα has a synergistic effect with Nab-paclitaxel. Meanwhile, CAV1 expression is negatively correlated to ERα and relevant to the better clinical benefits of Nab-paclitaxel treatment. Importantly, ERα stimulates miR199a-5p maturation to antagonize m6A modification of CAV1 mRNA, thus inhibiting its translation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results define a novel role of ERα miR199a-5p/CAV1 axis responsible for nab-paclitaxel resistance and propose combining ER antagonist with nab-paclitaxel as a perspective strategy for ER\u0026thinsp;+\u0026thinsp;breast cancer patients.\u003c/p\u003e","manuscriptTitle":"Estrogen receptor α confers Nab-paclitaxel resistance in breast cancer by promoting miR199a-5p maturation to inhibit Caveolin 1 translation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-03 21:24:00","doi":"10.21203/rs.3.rs-2838943/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e10989d1-0965-4130-9d49-f5a3791a54ac","owner":[],"postedDate":"May 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-05-25T15:59:19+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-03 21:24:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2838943","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2838943","identity":"rs-2838943","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}