27-hydroxycholesterol induces multidrug resistance in estrogen receptor- positive breast cancer cells via elevation of HER2

doi:10.21203/rs.3.rs-4861893/v1

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Research Article

27-hydroxycholesterol induces multidrug resistance in estrogen receptor- positive breast cancer cells via elevation of HER2

https://doi.org/10.21203/rs.3.rs-4861893/v1

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Background

Estrogen receptor-positive breast cancer is prone to drug resistance during endocrine therapy and chemotherapy, a complex phenomenon known as multidrug resistance (MDR). 27-Hydroxycholesterol (27HC), a main metabolite of cholesterol in the body, is conformed to be is an independent risk factor for estrogen receptor-positive breast cancer. However, the potential association of 27HC accumulation in vivo with endocrine therapy or chemotherapy resistance remains to be determined. We recently demonstrated that human epidermal growth factor receptor 2 (HER2) upregulation represents a novel mechanism underlying endocrine resistance in breast cancer. The potential role of 27HC in HER2 expression and MDR of breast cancer is currently unknown.

Methods

In this study, human ER-positive breast cancer cell lines with low HER2 expression, T47D and MCF-7, were used to study the effects of exposure to 27HC on MDR in breast cancer in vitro.

Results

Long-term exposure to 27HC clearly induced MDR in ERα-positive breast cancer cells. In terms of the underlying mechanism, 27HC-induced reactive oxygen species (ROS) promoted HER2 expression, which is an important causative factor of MDR. Based on the data, we infer that ROS activate the IL-6/STAT3 pathway through phosphorylation of ERK1/2, enhancing HER2 expression and further promoting a HER2-ERK1/2-STAT3 positive feedback loop, which ultimately leads to the development of MDR.

Conclusion

Our collective data indicate that 27HC interferes with endocrine therapy and chemotherapy in breast cancer, representing a novel mechanism of MDR. Accordingly, we propose that hypercholesterolemia or accumulation of 27HC in the body is a potential health risk for breast cancer patients. Moreover, HER2 may have clinical utility as an intervention target to reduce the occurrence of MDR in patients and ultimately improve the efficacy of endocrine therapy and/or chemotherapy.

27-Hydroxycholesterol

Breast cancer

HER2

Multidrug resistance

Breast cancer is the most common tumor type and a leading cause of cancer-related death among women worldwide [1]. Nearly 70% of diagnosed breast cancers are hormone receptor-positive and predominantly express the estrogen receptor (ER) [2]. Consequently, endocrine therapy represents the first and most efficacious targeted treatment for patients with estrogen receptor-positive (ER+) breast cancer. Endocrine therapy includes the use of selective estrogen receptor modulators (SERMs, e.g., tamoxifen), selective estrogen receptor downregulators (SERDs, e.g., fulvestrant) and aromatase inhibitors (AI, e.g., letrozole) [3]. However, intrinsic and acquired resistance occurs in a significant proportion of patients, limiting the efficacy of treatment [4]. Chemotherapy is recommended for adjuvant treatment of breast cancer, but resistance is commonly reported during the course of treatment [5]. The current clinical guidelines usually list the following categories of chemotherapy: anthracycline-based, anthracycline + taxane, and taxane-based [6]. Multidrug resistance (MDR) of tumor cells remains an important cause of clinical treatment failure in patients with breast cancer.

Hypercholesterolemia is an independent risk factor for ER-positive breast cancer [7], and is associated with decreased responsiveness to endocrine therapy [8, 9]. 27-Hydroxycholesterol (27HC) is the main metabolite of cholesterol in the body that is converted from cholesterol by cytochrome oxidase CYP27A1 [10]. 27HC, as an endogenous selective estrogen receptor modulator (SERM), has been shown to promote the ER-dependent growth of breast cancer in mouse [11, 12]. Nelson et al. have further reported that the 27HC metabolite, and not cholesterol itself, is related to the occurrence and development of breast cancer and upregulation of tamoxifen (TAM) resistance-related genes [8].

The complex mechanism of MDR has been a significant focus of breast cancer research. Overexpression of human epidermal growth factor receptor 2 (HER2) is related to not only endocrine resistance but also chemotherapy resistance. MAP kinase shows markedly increased activity in breast cancer cell lines overexpressing HER2, negating the inhibitory effects of TAM [13]. Wang et al. have reported that HER2 suppresses the sensitivity of ovarian cancer cells to chemotherapy drugs through inducing stem cell-like properties [14]. Earlier, our group demonstrated that HER2 upregulation caused by long-term exposure to genistein promotes endocrine resistance in breast cancer [15].

The precise role of 27HC in enhancement of HER2 expression and MDR of breast cancer remains to be confirmed. The inflammatory response induced by 27HC is indispensable in the occurrence and development of tumors [16]. Upregulation of interleukin-6 (IL-6) has been shown to promote MDR through activation of Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathways [17, 18]. STAT3 binds to the HER2 promoter to upregulate HER2 expression in MCF-7 cells [19]. However, it is unclear whether the IL-6/STAT3 pathway is associated with increased HER2 expression and MDR potentially induced by 27HC.

To clarify the mechanisms by which 27HC induces MDR in breast cancer, we selected HER2-low/ER-positive MCF-7 and T47D breast cancer cells as a model to investigate whether prolonged exposure to 27HC could induce transformation of sensitive breast cancer cells to resistant cells through elevation of HER2 expression. Furthermore, associations of inflammatory cytokine levels and STAT3 phosphorylation with HER2 and MDR were explored.

2.1 Cell culture and reagents. Human breast cancer cells (T47D, MCF-7) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). T47D and MCF-7 cells were maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin and cultured in an incubator under 5% CO₂ at 37°C. 27HC (purity ≥ 99.9%) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in absolute ethanol at a stock concentration of 20 mM, and stored at -80°C. NAC was purchased from MedChemExpress (Princeton, NJ, USA), dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM, and stored at -80°C. Stattic, a STAT3 inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA), U0126, a ERK1/2 inhibitor, from Beyotime Biotechnology (Nanjing, Jiangsu, China), 17β-E₂, TAM, and fulvestrant (ICI 182780) from Sigma (St. Louis, MO, USA), and doxorubicin (DOX) and paclitaxel (PTX) from MedChemExpress.

2.2 Western blot. Total protein was extracted using Radio-Immunoprecipitation Assay buffer (Beyotime Biotechnology) and concentrations measured with a bicinchoninic acid (BCA) kit (Beyotime Biotechnology). Proteins (20 µg) were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk for 1 h, membranes were incubated with primary antibodies for HER2/ErbB2, p-STAT3^Tyr705, p-ERK (CST Boston, USA, 1:500 dilution) and β-actin (Beyotime Biotechnology, 1:500 dilution) overnight at 4°C. After washing off the primary antibody with TBST, samples were incubated with horseradish peroxidase-conjugated secondary antibody (Beyotime Biotechnology, 1:2000 dilution) at 37°C for 1 h. Immune complexes were detected using an enhanced chemiluminescence kit (Millipore, Billerica, USA) and densitometric quantification performed using Image Pro-Plus 6.0 software. Following normalization of the internal reference, the gray ratio of the control group was added to the western blot band for objective analysis of the trend of molecular changes.

2.3 Quantitative real-time polymerase chain reaction (qRT-PCR). TRIzol reagent (Yifeixue Bio Tech, Nanjing, Jiangsu, China) was used to extract total cellular RNA. Primers used for PCR (Table 1) were synthesized by Tsingke (Nanjing, Jiangsu, China). Total RNA (2 µg) was reverse-transcribed into cDNA with AMV reverse transcriptase (ABM, Zhenjiang, Jiangsu, China) and qRT-PCR performed using Light Cycler 96 SYBR Green I MasterMix (Roche, Basel, Switzerland).

2.4 Cell viability assay. Breast cancer cells were seeded into 96-well plates at a density of 4000 per well for 24 h of incubation, followed by treatment with different concentrations of TAM, ICI, DOX, and PTX for 72 h. The medium containing the drug was replaced with 100 µL medium containing 10% Cell Counting Kit-8 reagent (Beyotime Biotechnology), followed by incubation in the dark for 4 h. Absorbance was measured at 450 nm with a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA).

2.5 Cell transfection. Breast cancer cells were seeded in cell culture dishes at a density of 5×10⁵ cells per dish for 24 h and transiently transfected with a mixture of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and Si-Con or specific small interfering RNA (siRNA) for 6 h (Table 2). The medium was removed and transfected cells subsequently cultured in fresh medium (10% FBS) without penicillin-streptomycin for 48 h.

2.6 Measurement of intracellular ROS production. For detection of intracellular ROS, cells were incubated in serum-free culture medium containing 10 µM DCFH-DA (Beyotime Biotechnology) for 20 min at 37°C, followed by two washes with serum-free culture medium to remove extracellular DCFH-DA. Next, cells were resuspended in PBS and subjected to flow cytometry. All experiments were performed on the FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) and data analyzed using FlowJo software (Treestar, Ashland, OR, USA).

2.7 Statistical Analysis. Data are expressed as mean ± standard deviation (mean ± SD) and analysis of variance (ANOVA) and t-tests used to determine the statistical differences between groups. Data sets were compared, analyzed and graphed using GraphPad 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Differences were considered statistically significant at P < 0.05.

3.1 Long-term exposure to 27HC induces MDR in ER-positive breast cancer cells

We initially examined whether 27HC induces MDR in ER-positive breast cancer cells. Circulating levels of 27HC were determined as about 200 nM, with a maximum value of 600 nM, in a cohort study of 2,282 women diagnosed with breast cancer [20]. Accordingly, we selected 0.5 µM as the dose of 27HC, which is close to the physiological concentration, for our experiments. ERα-positive breast cancer cells (T47D, MCF-7) were cultured with or without 27HC for 3 months (3 m). After this time, culture medium containing 27HC was discarded and further culture continued for 48 h. Both groups of cells were treated with a range of concentrations of the endocrine therapy drugs TAM (0, 5, 10, 20 µM) and ICI (0, 20, 100, 200 nM) and chemotherapeutic drugs DOX (0, 1, 5, 10 nM) and PTX (0, 1, 5, 10 nM) for 72 h, respectively, and viability measured. Compared with the corresponding control cells, the sensitivities of T47D (Fig. 1A) and MCF-7 (Fig. 1B) to TAM, ICI, DOX, and PTX were significantly decreased after exposure to 27HC for 3 m. Our results suggest that continuous exposure to 27HC induces resistance to endocrine therapy and chemotherapy in ER-positive breast cancer cells.

3.2 Elevated HER2 is associated with 27HC-induced MDR

High expression of HER2 is related to endocrine therapy and chemotherapy resistance [13, 14]. We further focused on whether elevated HER2 is associated with 27HC-induced MDR in ER-positive breast cancer cells. Notably, both mRNA and protein levels of HER2 in T47D cells were elevated following long-term exposure to 0.5 µM 27HC for 1, 2, and 3 months, respectively (Fig. 2A). Conversely, E₂ induced a decrease in expression of HER2. Although 27HC is an endogenous SERM, it does not affect HER2 expression in the same manner as E₂. Similar results were obtained with MCF-7 cells (Fig. 2B). Overall, expression of HER2 was elevated during long-term exposure to 27HC. To further explore the role of HER2 in 27HC-induced MDR, we overexpressed HER2 in ER-positive breast cancer cells, followed by treatment with TAM (10 µM), ICI (100 nM), DOX (1 nM) or PTX (1 nM) for 72 h. Compared with the corresponding control cells, HER2-overexpressing T47D (Fig. 2C) and MCF-7 cells (Supplementary Figure. 1A) showed resistance to both endocrine and chemotherapeutic agents. For two-way validation, we performed HER2 knockdown with siRNA in cells with long-term 27HC exposure (T47D-3m, MCF-7-3m), followed by separate treatments with TAM (10 µM), ICI (100 nM), DOX (1 nM) and PTX (1 nM) for 72 h. Upon treatment with HER2 siRNA, T47D-3m (Fig. 2D) and MCF-7-3m cells (Supplementary Figure. 1B) recovered their sensitivity. Our collective data indicate that HER2 confers endocrine and chemotherapy resistance induced by 27HC to ER-positive breast cancer cells.

3.3 IL-6/STAT3 signaling is involved in MDR and HER2 elevation induced by 27HC

Considerable evidence indicates crosstalk between IL-6/STAT3 and MDR in cancer [17, 18]. Accordingly, we explored whether the IL-6/STAT3 pathway is involved in the MDR-promoting effect of 27HC. Upon long-term exposure of T47D and MCF-7 cells to 0.5 µM 27HC, mRNA levels of IL-6 were increased at the three time-points of 1, 2, and 3 months (Fig. 3A). STAT3 phosphorylation was elevated in T47D cells, while in MCF-7 cells, STAT3 phosphorylation was initially decreased and showed a subsequent increase at 3 months (Fig. 3B). Overall, our data suggest that long-term exposure to 27-HC activates IL-6/STAT3 signaling. To explore whether STAT3 signaling induced by 27HC is involved in MDR, cells with long-term 27HC exposure (T47D-3m, MCF-7-3m) were pretreated with the STAT3 inhibitor Stattic, followed by treatment with 10 µM TAM, 100 nM ICI, 1 nM DOX, and 1 nM PTX for 72 h. Notably, compared with the corresponding control cells, T47D-3m (Fig. 3C) and MCF-7-3m (Supplementary Figure. 2A) cells pretreated with Stattic showed greater sensitivity to TAM, ICI, DOX, and PTX. Upon treatment of T47D-3m cells with Stattic, inhibition of STAT3 phosphorylation was observed, along with downregulation of HER2 (Fig. 3D). Similar results were obtained upon transfection with STAT3 siRNA (Fig. 3E). Based on the results, we suggest that IL-6/STAT3 contributes to 27HC-induced resistance to endocrine therapy and chemotherapy through elevation of HER2.

3.4 ROS increases expression of HER2 through activation of IL-6/STAT3 signaling

Previous studies by our group showed that 27-HC induces oxidative stress in breast cancer cells [21]. Crosstalk between reactive oxygen species (ROS) and HER2 has been reported [22]. Accordingly, we further examined the hypothesis that elevated HER2 expression mediated by IL-6/STAT3 signaling is associated with 27-HC-induced oxidative stress. Our experiments showed greater ROS production in cells exposed to 27HC (T47D-3m, MCF-7-3m) than control cells. Upon treatment of T47D-3m or MCF-7-3m cells with the antioxidant N-acetyl-L-cysteine (NAC), ROS production was decreased (Fig. 4A). Inhibition of oxidative stress led to reduced IL-6 mRNA (Fig. 4B) and STAT3 phosphorylation (Fig. 4C) as well as HER2 levels (Fig. 4D) in T47D-3m cells. Compared with the corresponding control cells, NAC-pretreated T47D-3m (Fig. 4E) and MCF-7-3m (Supplementary Figure. 2B) cells showed greater sensitivity to TAM, ICI, DOX, and PTX treatments. Accordingly, we propose that ROS serve as a contributory factor to 27HC-mediated increase in HER2 expression, which is driven through IL-6/STAT3 signaling.

3.5 Modulation of ERK1/2 phosphorylation via ROS contributes to activation of IL-6/STAT3 signaling pathway and MDR

ROS elicited by 27HC can activate downstream ERK signaling pathways [23]. Earlier studies have shown that 27HC stimulates NF-κB activity via ERK1/2-dependent IκBα degradation and upregulates proinflammatory genes [24]. ERK1/2 phosphorylation was elevated at 1, 2, and 3 months in T47D cells continuously exposed to 0.5 µM 27HC. In MCF-7 cells, ERK1/2 phosphorylation showed a significant increase at 1 and 3 months (Fig. 5A). To establish the involvement of ROS in ERK1/2 activation induced by 27HC, T47D-3m cells were pretreated with NAC. Inhibition of oxidative stress clearly led to reduced ERK1/2 phosphorylation (Fig. 5B). Next, we evaluated whether phosphorylated ERK1/2 affects IL-6/STAT3 signaling and HER2 levels. Following treatment of T47D-3m cells with the MEK inhibitor, U0126, inhibition of ERK1/2 phosphorylation downregulated 27HC-induced IL-6 mRNA (Fig. 5C) and STAT3 phosphorylation as well as HER2 levels (Fig. 5D) and restored the sensitivity of cells to endocrine therapy and chemotherapy drugs (Fig. 5E). Taken together, the results suggest that modulation of ERK1/2 phosphorylation via ROS contributes to activation of IL-6/STAT3 signaling and MDR.

3.6 Feedback effects of HER2 signals on ERK1/2 and STAT3

Previous studies have shown that HER2 acts upstream of ERK1/2 and STAT3 signaling [25, 26]. In T47D and MCF-7 cells overexpressing HER2, phosphorylation of ERK1/2 and STAT3 was elevated (Fig. 6A), along with IL-6 mRNA (Fig. 6C). For two-way validation, we conducted HER2 knockdown in T47D-3m and MCF-7-3m cells, which led to the opposite results, whereby phosphorylation of ERK1/2 and STAT3 (Fig. 6B) and IL-6 mRNA levels were decreased (Fig. 6C). Therefore, as downstream signals, activation of STAT3 and ERK1/2 signaling appear to depend on high expression of HER2. Based on the above results, we propose that 27HC activates STAT3 through ERK1/2 and promotes HER2 expression, thereby maintaining activity of the HER2-ERK1/2-STAT3 positive feedback loop, further enhancing expression of HER2, and ultimately, leading to the development of MDR in ERα-positive breast cancer cells.

Overexpression or mutation of the proto-oncogene, HER2, poses a serious problem in the treatment of breast cancer [27]. HER2 is highly expressed in a variety of tumor cells and associated with malignant phenotypes, such as cancer cell survival, proliferation, and drug resistance [28]. Earlier studies have reported conversion to positive serum HER-2/neu status in approximately 25% of patients receiving first-line hormone therapy [29]. HER2 inhibition is reported to markedly increase the sensitivity of ovarian cancer cells to DOX or PTX [14]. In our study, long-term treatment of ERα-positive breast cancer cells (T47D, MCF-7) with 27HC induced an increase in HER2 expression. In addition, both types of cells were insensitive to endocrine therapy drugs, TAM and ICI, and chemotherapy drugs, DOX and PTX following 27HC treatment, resulting in a MDR phenotype. Under conditions of high expression of HER2, cells showed a MDR phenotype and sensitivity was restored after knockdown of the gene. The collective findings suggest that HER2 is a key molecule in 27HC-induced acquired MDR.

We further explored the potential mechanisms underlying the increase in HER2 expression induced by long-term exposure to 27HC. As STAT3 is a key upstream regulator of HER2 [19, 30], we examined whether 27HC-induced HER2 elevation is associated with STAT3 activation in ER-positive breast cancer cells. In the present study, STAT3 signaling was activated in T47D and MCF-7 cells after long-term exposure to 27HC, consistent with our previous data on short-term exposure to 27HC [21]. Moreover, when STAT3 signals were blocked with specific inhibitors in drug-resistant cells, HER2 expression decreased and the sensitivity of drug-resistant cells to endocrine therapy and chemotherapy drugs was restored to some extent.

27HC stimulates the activation of NF-κB and promotes release of the inflammatory cytokine IL-6 in macrophages [24]. IL-6 is a major mediator of inflammation and activator of STAT3. The IL-6/STAT3 signaling axis is considered an essential intrinsic pathway of cancer inflammation [31]. The mRNA levels of IL-6 were also elevated in ER-positive breast cancer cells after long-term exposure to 27HC. Accordingly, we propose that 27HC activates inflammatory and IL-6/STAT3 signals in breast cancer cells that are involved in regulation of HER2 expression by 27HC, which, in turn, contributes to the development of MDR in breast cancer cells.

ROS play important roles in tumorigenesis and disease progression [32]. Several studies have shown that a high-fat/high-cholesterol diet can trigger increased ROS levels [33]. Previous research by our group showed that 27HC, a metabolite of cholesterol, promotes production of ROS in breast cancer cells [34]. Considerable crosstalk between ROS and HER2 has been reported [22], which can participate in the progression, migration, invasion and drug resistance of breast cancer cells [35] by promoting the expression or activity of HER2 [36–38]. In this study, 27HC induced ROS generation in ER-positive breast cancer cells. In cells pretreated with the ROS scavenger NAC, HER2 was downregulated, concomitant with suppression of 27HC-induced resistance and blockage of IL-6/STAT3 signaling in cells. Accordingly, we suggest that elevated HER2 expression and MDR mediated by IL-6/STAT3 signaling are associated with 27HC-induced oxidative stress.

Interestingly, upregulation of ERK1/2 kinases was shown to be closely dependent on early 27HC-induced intracellular increase in ROS [23]. ROS induce structural changes in serine/threoninase and the altered protein conformations promote upregulation of the MAPK signaling cascade, leading to activation of NF-kB transcription factors [39]. Our experiments revealed that 27HC continuously activates ERK1/2 signaling through ROS. Earlier studies have reported that 27HC induces IκBα phosphorylation through phosphorylation of ERK1/2, leading to ubiquitination, to activate NF-κB and promote expression of macrophage inflammatory factor genes [24]. We found that upon blockage of ERK1/2 signaling with specific inhibitors, IL-6/STAT3 signaling was blocked, HER2 expression was downregulated, and drug sensitivity of cells exposed to 27HC was restored. Our findings suggest that 27HC-induced ROS promote HER2 expression through ERK1/2 activation. There is abundant evidence that HER2 acts upstream of STAT3 and ERK1/2 [25, 26]. In our study, phosphorylation levels of ERK1/2 and STAT3 proteins were reduced upon knockdown of highly expressed HER2 in drug-resistant cells. Following overexpression of HER2 in sensitive cells, phosphorylation levels of ERK1/2 and STAT3 proteins were restored. At the same time, crosstalk occurred between STAT3 and ERK1/2 signals. In view of the collective findings, we believe that a positive ERK1/2-STAT3-HER2 feedback loop exists, which plays a role in maintaining high expression of HER2 and thus promoting MDR.

This study shows that long-term exposure to 27HC promotes the development of MDR in ER-positive breast cancer cells. High expression of HER2 may serve as an important factor underlying 27HC-induced resistance to endocrine therapy and chemotherapy. 27HC-induced ROS play a critical role in upregulation of HER2 and development of MDR in breast cancer cells. Mechanistically, ROS activate the IL-6/STAT3 pathway through stimulation of ERK1/2 and enhance HER2 expression, further promoting the HER2-ERK1/2-STAT3 positive feedback loop. This pathway provides additional survival signals to breast cancer cells, which counteract the effects of endocrine and chemotherapy drugs, resulting in a drug-resistant phenotype (Fig. 7). Our results suggest that 27HC induces transformation of ER-positive breast cancer cells into drug-resistant cells, which interferes with endocrine therapy and chemotherapy, representing a novel mechanism of MDR. Based on the collective findings, we propose that hypercholesterolemia or accumulation of 27HC in the body is a potential health risk for breast cancer patients. Moreover, HER2 may provide an effective intervention target to reduce the occurrence of MDR, and ultimately improve the efficacy of endocrine therapy and/or chemotherapy.

MDR

Multidrug resistance

27HC

27-Hydroxycholesterol

HER2

Human epidermal growth factor receptor 2

Estrogen receptor

TAM

Tamoxifen

ICI 182780

Fulvestrant

DOX

Doxorubicin

PTX

Paclitaxel

IL-6

Interleukin-6

STAT3

Signal transducer and activator of transcription 3

ROS

Reactive oxygen species

NAC

N-acetyl-L-cysteine.

Ethics approval and consent to participate

This study does not include human or animal studies.

Consent for publication

All of the authors agree for publication.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Declaration of competing interest

All the authors declare that they have no conflicts of interest.

Author Contributions Statement

ZL, CH and QZ designed the study. QZ and MW prepared Figs. 1, 2, 3 and 4. GT, YY and YF prepared Figs. 5 and 6. YM and YH prepared Figs. 7. MZ provided technical support. ZL and CH wrote and revised the manuscript. All the authors contributed to the study and approved the final manuscript.

Acknowledgments

The authors gratefully acknowledge study staff for the cooperation during the study, and thank International Science Editing for editing this manuscript.

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27-hydroxycholesterol induces multidrug resistance in estrogen receptor- positive breast cancer cells via elevation of HER2

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Abstract

Background

Methods

Results

Conclusion

Figures

1 Introduction

2 Materials and Methods

3 Results

3.1 Long-term exposure to 27HC induces MDR in ER-positive breast cancer cells

3.2 Elevated HER2 is associated with 27HC-induced MDR

3.3 IL-6/STAT3 signaling is involved in MDR and HER2 elevation induced by 27HC

3.4 ROS increases expression of HER2 through activation of IL-6/STAT3 signaling

3.5 Modulation of ERK1/2 phosphorylation via ROS contributes to activation of IL-6/STAT3 signaling pathway and MDR

3.6 Feedback effects of HER2 signals on ERK1/2 and STAT3

4 Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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