Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.


Introduction
In recent years, many new classes of antitumour compounds with a significant effect on the signalling pathways in tumour cells have been developed [1][2][3][4][5][6]. Along with the new classes, antihormonal drugs-SERMs (selective oestrogen receptor modulators) and SERDs (selective oestrogen receptor degraders)-remain highly relevant as an antitumour therapy [7][8][9]. Hormone therapy [10][11][12][13][14] is one of the most common types of treatment of hormone-dependent tumours including breast cancer, ovarian cancer, endometrial and prostate tumours. Hormone therapy is based on the principle of creating an artificial deficiency of hormones necessary for the growth of hormone-dependent tumours, oestrogens (for tumours of the female reproductive system) and androgens (for prostate tumours). This effect is achieved mainly in two ways: by reducing the concentration of endogenous hormones, due to the inhibition of their synthesis (aromatase inhibitors), or replacing hormones with their inactive analogues (antioestrogens or antiandrogens).
According to the World Health Organization, there were more than 2.2 million women diagnosed with breast cancer and 685,000 deaths worldwide in 2020. There are about eight xenografts [64]. The suppression of miR-7, targeting ABC drug efflux pump MRP1 (multidrug resistance-associated protein 1), was found to promote multidrug resistance in breast cancer and other cancer types [65].
Recent data demonstrate the possibility of the horizontal transfer of hormonal resistance (cell-to-cell) [66][67][68][69]. Extracellular vesicles play a critical role in this process due to the exosomal miRNAs, among which miRNAs have been identified that are involved in the negative regulation of the oestrogen receptor: miR-342, Let7b / Let-7i, miR-1280 and others [70]. Delivered into the target cell, such miRNAs can induce a rearrangement or blockage of hormonal signalling and, as a consequence, a decrease in the sensitivity to hormone therapy. Since miRNAs are expressed and/or delivered to a cell with exosomes in the form of a "rich cocktail" containing hundreds of miRNAs, it is extremely important to identify exosomal miRNAs associated with the hormonal resistance and to determine their ability to induce the irreversible rearrangement of oestrogen signalling.

Results
Experiments were performed on the in-vitro-cultured oestrogen-dependent MCF7 cells and tamoxifen-resistant MCF7/T subline obtained by long-term tamoxifen treatment of the parent cells. Earlier we have shown that long-term treatment of the MCF7 cells with the exosomes from the resistant MCF7/T cells results in the development of tamoxifen resistance in the recipient cells [71]. To further the study of the mechanism of the exosome-induced resistance, the profile of exosomal miRNAs was analysed. We proposed that continuous incorporation of the miRNA-suppressors of the oestrogen receptor (ERα) into the cells results in the irreversible block of the oestrogen signalling and compensatory activation of the oestrogen-independent growth pathways forming the tamoxifen-resistant phenotype.
MiRNA profile of the MCF7 and MCF7/T exosomes was studied, and miRNAs overexpressed in the resistant cells and exosomes were identified (Supplementary data, Table S1). Furthermore, we have searched for associations between identified miRNAs and ESR1 using the integrative database of human miRNA target predictions mirDIP, and six miRNAs overexpressed in the exosomes of resistant cells were annotated as potentially targeting ERα and suppressors of oestrogen signalling (Table 1). Among them, miR-181a-2 was overexpressed in both resistant cells and respective exosomes (Table S1). Moreover, miR-181a-2 was found to be within 1% of top miRNAs annotated with ESR1 (very high confidence class) supporting the possible involvement of the latter in the acquisition of hormonal resistance.  [74,78] The comparative analysis of the identified miRNAs' influence on the ERα expression showed that miR-181a-2 demonstrates the maximal inhibitory activity (Figure 1a), and all following experiments were performed with the latter. The role of this miRNA in cancer progression is of great interest as its expression correlates with cell proliferation and with the survival of cancer patients [79][80][81].
Using PCR analysis of miR-181a-2 content in the exosome-treated cells we have revealed the constitutively increased level of miR-181a-2 in the MCF-7 cells treated with the MCF-7/T exosomes demonstrating the pivotal role of exosomal miR-181a-2 in the transferring of the resistance (Figure 1b). The full protocol of the exosome preparation and development of exosome-induced resistance was described previously [82].
following experiments were performed with the latter. The role of this miRNA in cancer progression is of great interest as its expression correlates with cell proliferation and with the survival of cancer patients [79][80][81].
Using PCR analysis of miR-181a-2 content in the exosome-treated cells we have revealed the constitutively increased level of miR-181a-2 in the MCF-7 cells treated with the MCF-7/T exosomes demonstrating the pivotal role of exosomal miR-181a-2 in the transferring of the resistance (Figure 1b). The full protocol of the exosome preparation and development of exosome-induced resistance was described previously [82]. The single transfection by the miRNAs mimetics was performed as described in Methods. Twenty-four hours after transfection the Western blot analysis of ERα expression in the cell lysates was performed. Protein loading was controlled by membrane hybridization with αtubulin antibodies. Densitometry for immunoblotting data (right diagram) was carried out using ImageJ software (Wayne Rasband, NIH) with the recommendations from the work [83]; * p < 0.05 versus scrambled (scr); (b) quantification of endogenous miR-181a expression (vertical axis) in the exosome-treated MCF-7 cells by qRT-PCR. The MCF-7 cells were cultured in the presence of the exosomes isolated from MCF-7 (exoC) and MCF-7/T (exoT) cells for 30 days with following cell cultivation within the next 30 days after exosomes withdrawal. Three separate measurements were performed for each sample. The expression of RNU6B was used as an internal control. Error bars indicate standard deviation; * p < 0.05 versus MCF-7 cells treated with exoC.
We have found that single transfection of miR-181a-2 into MCF7 cells results in temporary tamoxifen resistance being decreased in 5 days after transfection. On the contrary, multiple transfections (totally, 20 rounds of transfection) of miR-181a-2 induce the persistent tamoxifen resistance, and transfected cells retain the resistance to tamoxifen within at least 2 months of cultivation after the last transfection ( Figure 2). The single transfection by the miRNAs mimetics was performed as described in Methods. Twenty-four hours after transfection the Western blot analysis of ERα expression in the cell lysates was performed. Protein loading was controlled by membrane hybridization with α-tubulin antibodies. Densitometry for immunoblotting data (right diagram) was carried out using ImageJ software (Wayne Rasband, NIH) with the recommendations from the work [83]; * p < 0.05 versus scrambled (scr); (b) quantification of endogenous miR-181a expression (vertical axis) in the exosome-treated MCF-7 cells by qRT-PCR. The MCF-7 cells were cultured in the presence of the exosomes isolated from MCF-7 (exoC) and MCF-7/T (exoT) cells for 30 days with following cell cultivation within the next 30 days after exosomes withdrawal. Three separate measurements were performed for each sample. The expression of RNU6B was used as an internal control. Error bars indicate standard deviation; * p < 0.05 versus MCF-7 cells treated with exoC.
We have found that single transfection of miR-181a-2 into MCF7 cells results in temporary tamoxifen resistance being decreased in 5 days after transfection. On the contrary, multiple transfections (totally, 20 rounds of transfection) of miR-181a-2 induce the persistent tamoxifen resistance, and transfected cells retain the resistance to tamoxifen within at least 2 months of cultivation after the last transfection ( Figure 2). Furthermore, the transfected cells were characterized by the sustained suppression of the ERα level and its transcriptional activity accompanied by the constitutive activation of Akt ( Figure 3).
The analysis of the cell sensitivity to nonhormonal cytostatic drugs, cisplatin, docetaxel, fluorouracil and doxorubicin, revealed no difference in the sensitivity of the miR-181atransfected cells and control cells to these drugs demonstrating the high specificity of the acquired tamoxifen resistance in these cells (Figure 4).  Furthermore, the transfected cells were characterized by the sustained suppression of the ERα level and its transcriptional activity accompanied by the constitutive activation of Akt (Figure 3). diagram) was carried out using ImageJ software; * p < 0.05 versus scrambled (scr).
The analysis of the cell sensitivity to nonhormonal cytostatic drugs, cisplatin, docetaxel, fluorouracil and doxorubicin, revealed no difference in the sensitivity of the miR-181a-transfected cells and control cells to these drugs demonstrating the high specificity of the acquired tamoxifen resistance in these cells (Figure 4). As mentioned above, the exosomes of the resistant MCF-7/T cells are characterized with overexpression of miR-181a-2 (Table S1). To compare the effects of miR-181a-2 and exosomes produced by the parent or resistant cells the analysis of the oestrogen signalling in the exosome-treated cells was performed. Exosomes were isolated from the MCF7 and MCF7/T conditioned medium by the differential ultracentrifugation, and exosome imaging was carried out by transmission electron microscope as described in Methods ( Figure  5a). The recipient MCF7 cells were cultured in the presence of exosomes from MCF7 or resistant MCF7/T cells in the final concentration of 1.7 μg/mL for 1 month and oestrogen and Akt signalling was analysed. We have revealed the same tendency: suppression of ERα expression and activation of Akt phosphorylation in the cells treated with the exosomes from the MCF7/T-resistant cells (Figure 5b). As mentioned above, the exosomes of the resistant MCF-7/T cells are characterized with overexpression of miR-181a-2 (Table S1). To compare the effects of miR-181a-2 and exosomes produced by the parent or resistant cells the analysis of the oestrogen signalling in the exosome-treated cells was performed. Exosomes were isolated from the MCF7 and MCF7/T conditioned medium by the differential ultracentrifugation, and exosome imaging was carried out by transmission electron microscope as described in Methods (Figure 5a). The recipient MCF7 cells were cultured in the presence of exosomes from MCF7 or resistant MCF7/T cells in the final concentration of 1.7 µg/mL for 1 month and oestrogen and Akt signalling was analysed. We have revealed the same tendency: suppression of ERα expression and activation of Akt phosphorylation in the cells treated with the exosomes from the MCF7/T-resistant cells (Figure 5b). Exosomes were collected from the MCF7 and MCF7/T conditioned medium by the differential ultracentrifugation, labelled by the gold nanoparticles and imaged as described in Methods. (b) ERα and Akt expression. MCF7 cells were treated with exosomes from MCF7 and MCF7/T for 1 month and Western blot analysis of ERα and Akt was performed in the cell extracts. Protein loading was controlled by membrane hybridization with α-tubulin antibodies. Densitometry for immunoblotting data (right diagram) was carried out using ImageJ software; * p < 0.05 versus scrambled (scr).
Recently many miRNAs have been found to be associated with the progression of tamoxifen resistance of breast cancer cells [89][90][91], however, the miRNAs involvement in Figure 5. Exosome influence on the ERα and Akt level in MCF7 cells. (a) The transmission electron microscopy of the exosomes. Exosomes were collected from the MCF7 and MCF7/T conditioned medium by the differential ultracentrifugation, labelled by the gold nanoparticles and imaged as described in Methods. (b) ERα and Akt expression. MCF7 cells were treated with exosomes from MCF7 and MCF7/T for 1 month and Western blot analysis of ERα and Akt was performed in the cell extracts. Protein loading was controlled by membrane hybridization with α-tubulin antibodies. Densitometry for immunoblotting data (right diagram) was carried out using ImageJ software; * p < 0.05 versus scrambled (scr).
Recently many miRNAs have been found to be associated with the progression of tamoxifen resistance of breast cancer cells [89][90][91], however, the miRNAs involvement in the resistance development is still unclear. Here we have found that some miRNAssuppressors of oestrogen signalling are overexpressed in the exosomes of the tamoxifenresistant cells. Considering the possible involvement of exosomes in the transferring of hormonal resistance [32,92], the role of some of these miRNAs in the progression of acquired resistance was analysed.
Many researchers point to the important role of the miRNA axis in the progression of various cancers [93][94][95][96]. It is now clear that miRNAs could be evaluated as predictors of the response to therapy and as biomarkers. Aiko Sueta and colleagues revealed that a combined signature of four miRNAs (miR-4448, miR-2392, miR-2467-3p and miR-4800-3p) could be used to discriminate between pCR and non-pCR patients with triple-negative breast cancer [97]. It is interesting to note that not only intracellular miRNAs, but also circulating miRNAs, have great potential for use as potent biomarkers [98]. Intriguing new data suggest that miRNAs will be used to overcome resistance. Bernice Monchusi and Mandeep Kaur suggested that targeting the miRNAs hsa-miR-34a-5p and hsa-miR-373-3p could provide a way to alter the cell's response to drugs via modulating cholesterol pathways in cancer stem cells [99].
As revealed, the multiple (but not single) transfection of miR-181a-2 into oestrogendependent MCF7 cells induces the irreversible tamoxifen resistance demonstrating the important role of this miRNA in the formation of the resistant phenotype. We suppose that prolonged cell treatment with the miRs-ERα suppressors induces the continuous block of the oestrogen signalling resulting in the progression of the hormone resistance. Probably, the mechanism of such resistance may be similar to that induced by prolonged antioestrogen treatment-in both cases, the central event includes the prolonged suppression of oestrogen receptor with the following cell switch to oestrogen-independent growth. In agreement with the latter, we have revealed the constitutive activation of Akt in the cells after multiple transfections of miR-181a-2 showing the involvement of PI3K/Akt signalling in the growth regulation of the resistant cells. The miRs-ERα suppressors are overexpressed in the exosomes of the resistant cells, allowing us to consider such miRs as one of the key factors involved in the progression of exosome-induced resistance. The results obtained are in agreement with the recent publications demonstrating the ability of the exosomes of tamoxifen-resistant cells to induce resistance in the recipient cells [100,101]. Here we have revealed oestrogen receptor machinery as one of the possible targets of exosomal miRNAs transferring the resistant phenotype in breast cancer cells.

Conclusions
Presently, we have demonstrated that miR-181a-2 has the ability to downregulate ERα expression in the oestrogen-dependent MCF-7 breast cancer cells. Analysis of the profile of exosomal miRs in the MCF-7 cells and resistant MCF-7/T subline revealed the overexpression of miR-181a-2 in the exosomes of the resistant cells. We have shown that regular treatment of the MCF-7 cells with the resistant exosomes as well as the multiple (but not single) transfection of miR-181a-2 results in the progression of tamoxifen resistance in the treated cells accompanied with the constitutive activation of PI3K signalling. In general, we suggest that ERα-targeting miRNAs may be involved in the transferring of the hormonal resistance via the prolonged blockage of oestrogen signalling accompanied with the activation of oestrogen-independent pathways.

Cell Lines
The human breast cancer cell line MCF7 was purchased from ATCC. The cells were cultured in a standard DMEM medium (Gibco (Thermo Fisher Scientific, Waltham, MA, USA)) supplemented with 10% foetal bovine serum (FBS) (HyClone (Cytiva, Marlborough, MA, USA)) at 37 • C and 5% CO 2 . The tamoxifen-resistant MCF7/T subline was established from the parent MCF7 cells by long-term tamoxifen treatment, as described [66].

Exosome Isolation and Visualization
Exosomes were prepared from the MCF7 and MCF7/T conditioned medium by the differential ultracentrifugation and were characterized as described in our recent paper [102]. Transmission electron microscopy (TEM) with immunogold labelling was used to visualize the exosome samples. Imaging was carried out using a JEM-1011 (JEOL Ltd., Tokyo, Japan) transmission electron microscope at 80 kV. At least 30 images were obtained for the exosomes of each type.

MiRNA Analysis
The analysis of exosomal miRNAs was performed by HiSeq2500 (Illumina, San Diego, CA, USA) and at least 5 million reads per sample were obtained. Library preparation and sequencing was done by ZAO Genoanalytica as follows: miRNA was extracted from exosomes by PureLink RNA Micro Kit (12183-016 (Thermo Fisher Scientific, Waltham, MA, USA)) according to the manual. Library preparation was carried out with the NEBNext Small RNA Library Prep Set for Illumina (E7330S (New England Biolabs, Hitchin, UK)). The associations between miRNAs and ESR1 were searched by using the integrative database of human miRNA target predictions mirDIP [103], which aggregates the data from all known miRNA databases. The likelihood level was specified as medium. All studied miRNAs were found as potentially targeting ESR1. Some of them (miR-181a-2-3p and hsa-miR-874-3p) were found to be within 1% of top miRNAs annotated with ESR1 (very high confidence class). Moreover, these 6 miRNAs were selected concerning the literature indicated in Table 1. All hyperexpressed miRNAs are provided in Table S1 in the supporting file.

MiRNA Transfection
MiRNA constructs were purchased from Syntol. RNAs were dissolved in annealing buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA) at 100 µM concentration and annealed at room temperature following heating to 95 • C. Transient single or multiple transfections of miRNAs were performed using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) to result in the final RNA concentration of 50 nM. Multiple transfections were performed twenty times, once every three days.

Immunoblotting, Reporter Analysis, and MTT-Test
Protein expression was determined by immunoblotting [104], reporter analysis used to study the transcriptional activity of oestrogen receptor (ERα) was described in our work [105]. Cell viability was assessed by the MTT test as described in [106]. All in vitro experiments were performed in triplicates. The Student's t-test was used to evaluate the significance of differences in comparisons. The p-value of <0.05 was considered statistically significant.