Research ArticleExperimental Studies

Etoposide-induced MicroRNA-205-5p Suppresses Proliferation and Migration by Targeting ERBB4 in MCF-7 Cells

SEUNG WAN SON, SEUNG HYEOK HEO, SANG SU OH, CHAN HEE LEE and JONG KOOK PARK
Anticancer Research September 2022, 42 (9) 4265-4272; DOI: https://doi.org/10.21873/anticanres.15926

Abstract

Background/Aim: Patients with breast cancer frequently encounter a dismal prognosis due to the lack of effective and curative therapies. MicroRNAs (miRNAs) are aberrantly regulated in many types of cancer and have been recognized to play crucial roles in cancer progression. We performed a preclinical investigation of the anti-cancer effect of etoposide and microRNA-205-5p (miRNA-205-5p) and their relationship in MCF-7 cells. Materials and Methods: Two cell culture systems, namely monolayers and spheroids, were employed for evaluating the effect of etoposide and miRNA-205-5p on cell proliferation and migration. Real time quantitative polymerase chain reaction was used for the measurement of mRNA and miRNA levels. Luciferase and western blot assays were utilized for the validation of the target gene of miRNA-205-5p. Results: Treatment with etoposide, suppressed both cell proliferation and migration in MCF-7 monolayers. Also, the growth of MCF-7 spheroids as demonstrated by size measurements was inhibited by etoposide treatment. Furthermore, etoposide was found to upregulate the level of miRNA-205-5p. Over-expression of miRNA-205-5p inhibits cell proliferation and migration by directly targeting Erb-B2 receptor tyrosine kinase 4 (ERBB4). Conclusion: miRNA-205-5p may act as an important mediator of the anti-cancer effect of etoposide and miRNA-205-5p-based therapy may expand the therapeutic opportunities for breast cancer.

Key Words:

Breast cancer is widely regarded as one of the most significant causes of cancer-related mortality and is anticipated to affect 2,261,419 women worldwide in 2020, with 684,996 people dying from this disease (1). Analysis of the expression level of estrogen (ER) receptors, progesterone (PR) receptors, and Erb-B2 receptor tyrosine kinase 2 (ERBB2, also known as human epidermal growth factor receptor 2, HER2) provides the basis for the evaluation of breast cancer subtypes (2, 3). The occurrence of p53 mutations differs according to the subtype. For example, while basal-like breast cancer, which generally lacks ER, PR, and ERBB2 expression, has a high rate of p53 mutation, luminal A breast cancer (ER+/PR+/ERBB2–) is more likely to have wild-type p53 (4). While ER- and PR-positive breast cancer patients can be treated with hormonal therapies such as Tamoxifen, drug resistance can be developed, thus becoming one of the major issues in achieving efficient cancer treatment (5-7). These findings suggest the requirement of developing new treatment strategies for ER- and PR-positive breast cancer.

Etoposide is a semi-synthetic derivative of podophyllotoxin and induces DNA damage via targeting topoisomerase II (8, 9). Etoposide can augment and reduce the expression of B-cell CLL/lymphoma 2 (BCL2) and BCL2-associated X protein (BAX) via activating p53, inducing apoptotic cell death (10-12). It was also demonstrated that the sensitivity of cells to etoposide can be regulated by CtBP-interacting protein (CTIP) and E2F transcription factor 1 (E2F1) in a p53-independent manner (13, 14). However, it is still needed to uncover the molecular mechanism of etoposide to enhance its anti-cancer efficacy.

A number of extensive profiling studies have revealed that numerous microRNAs (miRNAs) are aberrantly expressed in several types of cancer (15-18). Comprehensive inhibition of miRNA biogenesis by targeting Drosha, Dicer1, or DGCR8 is known to enhance cellular transformation and tumorigenesis, distinctly highlighting the role of miRNAs in regulating the various signaling pathways involved in tumor development (19). Additionally, over-expressed miRNAs directly control the level of tumor-suppressive genes, thus acting as oncogenic miRNAs in cancer. In contrast, miRNAs can also serve as tumor-suppressive miRNAs via negatively modulating oncogene expression (20-23). For example, we previously identified that miRNA-205-5p impedes both proliferation and invasion of breast cancer cells by targeting high mobility group box 3 (HMGB3) (24).

In our present study, we observed that etoposide not only represses both cell proliferation and migration, but also positively regulates the level of miRNA-205-5p in MCF-7 cells, a luminal A subtype cell line. Moreover, we further identified ERBB4 (also called HER4) as a target of miRNA-205-5p.

Materials and Methods

Cell culture and transfection. MCF-7, a human breast cancer cell line, was acquired from the Korea Cell Line Bank (Seoul, Republic of Korea). MCF-7 cells were grown in a humidified incubator at 37°C with 5% (v/v) CO2 using Dulbecco’s Modified Eagle’s medium (DMEM) containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). For miRNA transfection, cells were transfected with 100 nM of miRNA mimics, namely miRNA-control or miRNA-205-5p (GE Healthcare, Chicago, IL, USA) using Opti-MEM medium (Invitrogen, Carlsbad, CA, USA) and RNAiMAX, a transfection reagent (Invitrogen) as previously described (24). A liquid overlay technique was used to generate and culture multicellular spheroids as described previously (25). The size of spheroids was evaluated by Eclipse Ts2R inverted microscope (Nikon, Japan).

Cell viability assay. Cells were transfected with miRNA mimics (miRNA-control or miRNA-205-5p) or exposed to etoposide at several different concentrations for 48 h or 96 h. Following treatments, 10% trichloroacetic acid and 0.4% sulforhodamine B (SRB) were used for cell fixation (for 1 h) and cell staining (for 30 mins), respectively. Then, 10 mM Tris base solution was used for the extraction of protein-bound dye, and the optical absorbance measurement of each well was evaluated at 565 nm as described previously (26).

Migration assay. MCF-7 cells were grown on 6-well plates until reaching 80% confluency and treated with etoposide or transfected with miRNA mimics (miRNA-control or miRNA-205-5p). At 48 h or 96 h post-treatment, scratch wounds were created with a pipette tip, and images were taken using a Zeiss AxioCam MR digital camera mounted on an Eclipse Ts2R inverted microscope (Nikon, Japan). The percentage of wound closure was calculated using ImageJ software as described previously (27).

Protein extraction and immunoblotting. Total proteins were isolated using RIPA cell lysis buffer containing phosphatase/protease inhibitor cocktails (Thermo Fisher Scientific, Waltham, MA, USA). The protein separation was conducted using 4-20% Tris-HCl gradient gels (Bio-Rad Laboratories, Hercules, CA, USA). Blotting was performed for ERBB4 and p85α (Cell Signaling Technology, Danvers, MA, USA) as well as GAPDH (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Real time quantitative polymerase chain reaction. Based on the manufacturer’s instructions, the miRNeasy kit (Qiagen, Hilden, Germany) was utilized to extract total RNA. To detect mRNA levels, Superscript III reverse transcription kit (Invitrogen) was used to synthesize cDNA. Real time qPCR (RT-qPCR) analysis was performed on the AriaMx real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) using the Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Primer sequences, which were utilized in this study, were designed by GENOTECH (Daejeon, Republic of Korea). All primer sequences were indicated as follows: cyclin dependent kinase inhibitor 1A (CDKN1A, also called p21Cip1), forward 5′-CAG AGG AAG ACC ATG TGG-3′ and reverse 5′-TTT CGA CCC TGA GAG TCT-3′ primary miRNA-205 transcript, forward 5′-CTA CAG GCT GAG GTT GAC-3′ and reverse 5′-AGA AGC ACA TGG ATT GTC TGA-3′ ERBB4, forward 5′-ACC AGC ATT GAG CAC AAC-3′ and reverse 5′-TAG CCT GTG ACT TCT CGA-3′ glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5′-GAA GGT GAA GGT CGG AGT C-3′ and reverse 5′-GAA GAT GGT GAT GGG ATT TC-3′. For the detection of mature miRNAs, one hundred nanograms (100 ng) of total RNA was primed using gene-specific looped primers in a 20 μl reaction volume, and TaqMan miRNA assays (Applied Biosystems, Foster City, CA, USA) were used for the quantification of cDNA as previously described (28).

Luciferase reporter constructs and assay. PCR products of the three prime untranslated regions (3′ UTR) of ERBB4 were generated using human genomic DNA as a template and a LongAmp™ TaqDNA Polymerase (New England BioLabs, Ipswich, MA, USA). The PCR amplicons were then ligated into the psiCHECK-2 vector (Promega, Madison, WI, USA). The mutant reporter constructs, which contain mutations of the first four nucleotides of the seed-match sequence, were generated using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA, USA). The interaction of the target gene with miRNA-205-5p was evaluated using the psiCHECK-2 vector, which harbors a wild type or mutant 3′UTR. Constructs were co-transfected with either miRNA-control or miRNA-205-5p mimic into cells. Twenty-four h after transfection, cell lysates were harvested and used to measure both firefly and renilla luciferase activities using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer’s instructions. For p53 promoter-based reporter assay, MCF-7 cells transiently transfected with the pGL4.38, a luciferase reporter vector containing a p53 response element, were treated with etoposide at indicated concentrations. The cell lysates were then prepared for reporter assays. The luciferase activity of each sample in relation to the control was calculated.

Data analysis. The IC50 was defined as the drug concentration which exhibited 50% cell viability of the untreated control and calculated using Eq. 1 and Eq. 2:Embedded Image Eq. 1 Embedded Image Eq. 2

where D is the drug concentration, m is the Hill-type coefficient, R is the residual unaffected fraction (the resistance fraction), and Kd is the concentration of drug that produces a 50% reduction of the drug’s maximum effect (100−R) (29, 30).

Statistical analysis. An unpaired t-test and analysis of variance were used to verify statistical significance. The data are demonstrated as mean±SD. The differences were considered significant at p-values < 0.05. All experiments were replicated in triplicate.

Results

Etoposide inhibits the proliferation and migration of MCF-7 cells. To explore the anti-cancer activity of etoposide, we evaluated the effect of etoposide on cell viability and migration in MCF-7 cells. A time- and dose-dependent reduction in cell proliferation was significantly observed following treatment with etoposide ranged up to 200 μM for 48 h and 96 h in monolayers (Figure 1A and Table I). MCF-7 cells were relatively resistant after 48 h exposure etoposide (R value is 68.5±7.9%). However, etoposide produced an improved anti-cancer activity with an IC50 value of 45.4±3.7 μM, an IC70 value of 96.2±5.5 μM, and a R value of 18.7±4.9 at 96 h (Figure 1A and Table I). In addition, we found that the migration of MCF-7 cells was suppressed when exposed to etoposide at approximate IC50/96 h (50 μM) and IC70/96 h (100 μM) concentrations (Figure 1B). Next, we sought to investigate the anti-proliferative effect of etoposide in MCF-7 spheroids. It was noted that the proliferative state of MCF-7 cells in spheroid culture system was also inhibited after treating with etoposide at 50 μM and 100 μM (Figure 1C), demonstrating the effectiveness of etoposide toward breast cancer.

Figure 1.
Figure 1.

Etoposide inhibits cell proliferation and migration. A) Representative dose-response curves of MCF-7 cells treated with various concentrations of etoposide. Cell viability was assessed by SRB assay following 48 h or 96 h continuous exposure. B) Scratch wound lines were generated following treatment with etoposide and the recovery rates were observed until 96 h of treatment. Bar graph shows the percentage of wound closure. C) MCF-7 spheroids were treated with etoposide and a reduction in size was observed at 48 and 96 h. A dot plot graph shows the relative size of spheroids after treatment with vehicle and etoposide (n=9). Significantly different at **p<0.01. Data are mean±SD.

View this table:
Table I.

Antiproliferative activity parameters of etoposide in MCF-7 cells. The dose-response curves were analyzed using the Emax model (Eq. 1 and Eq. 2).

Etoposide upregulates miRNA-205-5p expression. We previously demonstrated that miRNA-205-5p serves as a tumor-suppressive miRNA in breast cancer cells (24). Also, this miRNA is known to be induced by p53, which can be activated by etoposide (11, 31). In order to elucidate the contribution of miRNA-205-5p to etoposide-mediated suppression of cell proliferation and migration, we measured the etoposide-induced p53 activity and the expression level of cyclin dependent kinase inhibitor 1A (CDKN1A, also known as P21CIP1) and miRNA-205-5p in etoposide-treated MCF-7 cells (Figure 2). It was observed that etoposide activated p53 and that the level of CDKN1A, a well-known downstream target of p53, was increased in both monolayers and spheroids of MCF-7 cells (Figure 2A and B). Next, we observed whether etoposide has any effects on the level of miRNA-205-5p. Real-time qPCR assay showed that the levels of both primary transcripts of miRNA-205 and mature miRNA-205-5p were remarkably upregulated by etoposide in both monolayer and spheroid culture systems (Figure 2C).

Figure 2.
Figure 2.

Etoposide induces miRNA-205-5p in MCF-7 cells. A) Luciferase reporter vectors containing a p53 response element were transfected to MCF-7 cells and then cells were treated with etoposide for 24 h. Luciferase activity was measured, and relative activity was calculated. B) Real time quantitative polymerase chain reaction analyses of P21CIP1 levels in MCF-7 cells treated with either vehicle or etoposide for 48 h in monolayers and spheroids. C) The levels of both primary transcripts of miRNA-205 and mature miRNA-205-5p in MCF-7 cells were analysed using real time quantitative polymerase chain reaction. Monolayers and spheroids were treated with etoposide for 48 h and then total RNA was procured for cDNA synthesis. Expression levels were related to GAPDH, which serves as an internal control. The comparative CT method was utilized to calculate the data. The fold changes in miRNA levels are shown relative to vehicle treatment. Significantly different at **p<0.01. Data are mean±SD.

MiRNA-205-5p inhibits proliferation and migration in MCF-7 cells. Following the identification of etoposide-induced upregulation of miRNA-205-5p, we further investigated the anti-cancer effect of this miRNA on the proliferation and migration of MCF-7 cells. We found that miRNA-205-5p noticeably suppressed both cell proliferation and migration in MCF-7 monolayers compared to the negative control (Figure 3A and B). Besides, in spheroid culture system, the enforced expression of miRNA-205-5p was seen to cause a significant inhibition in the proliferative state (Figure 3C). Therefore, these results obviously indicated the tumor-suppressive function of miRNA-205-5p in MCF-7 cells.

Figure 3.
Figure 3.

MiRNA-205-5p inhibits both cell proliferation and migration in MCF-7 cells. A) MCF-7 cells were transfected with either miRNA-control or miRNA-205-5p mimic at 50 nM concentration for 48 h in 96 well plates. At 48 h after transfection, survival rates of cells were measured using SRB assay. B) Representative photographs showing in vitro migration assay of MCF-7 cells after transfection with miRNA-control or miRNA-205-5p mimic at 50 nM prior to the generation of scratch wound lines. The recovery rates were monitored until 96 h and the percentage of wound closure was indicated in a bar graph. C) Representative photographs showing the size of spheroids at 96 h after transfection of miRNA-control or miRNA-205-5p mimic (50 nM). A dot plot graph showing the relative size of spheroids in miRNA-control or miRNA-205-5p over-expressing spheroids (n=12). Significantly different at **p<0.01. Data are mean±SD. The bar indicates 50 μm.

MiRNA-205-5p targets ERBB4 in MCF-7 cells. We next sought to identify the bona fide target gene of miRNA-205-5p in order to understand the mechanisms by which miRNA-205-5p exhibited its anti-cancer effects on MCF-7 cells. There are predicted miRNA:mRNA binding sites of miRNA-205-5p in the 3′ untranslated region (UTR) of ERBB4 mRNA (Figure 4A). Luciferase reporter assay was carried out to experimentally validate whether miRNA-205-5p could directly interact with the 3′UTR of ERBB4. MiRNA-control or miRNA-205-5p was transfected into cells in combination with a reporter construct containing the 3′UTR of ERBB4. The ectopic expression of miRNA-205-5p resulted in a notable reduction in Renilla luciferase activities. In addition, mutation of the seed-matching sequences resulted in no significant difference of Renilla luciferase activities between miRNA-control- and miRNA-205-5p-over-expressing cells (Figure 4B). Moreover, both mRNA and protein levels of ERBB4 were remarkably repressed by miRNA-205-5p (Figure 4C and D), indicating that miRNA-205-5p is responsible for ERBB4 down-regulation through mRNA degradation and translational repression. Further, ERBB4 mRNA and protein levels were noticeably suppressed following etoposide treatment (Figure 4E and F), demonstrating that an increase in miRNA-205-5p levels can partly contribute to etoposide-mediated suppression of ERBB4 expression.

Figure 4.
Figure 4.

MiRNA-205-5p targets ERBB4 in MCF-7 cells. A) A schematic diagram of miRNA-205-5p binding sites in the 3′UTR region of human ERBB4 mRNA. Mutant reporter constructs (MU) were generated at the first four nucleotides of the seed-match sequences. B) Luciferase reporter plasmids carrying wild-type (WT) or mutant (MU) binding sequences in 3′UTR of ERBB4 were co-transfected in MCF-7 cells with miRNA-control or miRNA-205-5p mimic at 50 nM concentration. Dual-luciferase assays were conducted to measure the luciferase activity at 24 h after transfection. C) Real time quantitative polymerase chain reaction analyses of ERBB4 mRNA levels in MCF-7 cells transfected with miRNA-control or miRNA-205-5p mimic at 50 nM concentration for 48 h. D) MCF-7 cells were transfected with miRNA-control or miRNA-205-5p mimic at 50 nM concentration for 48 h, and protein lysates were isolated and subjected to western blot analysis of ERBB4. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control and relative gene expression levels are indicated. E-F) MCF-7 cells were treated with either vehicle, etoposide at 50 μM, or etoposide at 100 μM for 48 h. The level of ERBB4 mRNA (E) and protein (F) was measured by real time quantitative polymerase chain reaction and western blot, respectively. Significantly different at **p<0.01. Data are mean±SD.

Discussion

In this study, we found that miRNA-205-5p levels were induced by etoposide and that over-expression of miRNA-205-5p showed anti-proliferative and anti-migratory effects on MCF-7 cells, indicating the tumor-suppressive role of miRNA-205-5p. Moreover, we identified that ERBB4 is a direct target of miRNA-205-5p.

Accumulating evidence has shown that numerous oncogenes, namely zinc finger E-Box binding homeobox 1 (ZEB1), vascular endothelial growth factor A (VEGFA), and ERBB3, can be regulated by miRNA-205-5p (32-34). Moreover, we previously discovered that epigenetic modifying agents, 5-aza-2′-deoxycytidine and Trichostatin A, are able to derepress the expression of miRNA-205-5p and that high mobility group box 3 (HMGB3) is directly inhibited by miRNA-205-5p in breast cancer cells. Therefore, there is a possibility that miRNA-205-5p could be considered as an important mediator of tumor suppressive signaling pathways that are regulated by epigenetic modifying drugs (24). Since our data clearly indicate that etoposide also increases miRNA-205-5p levels, the combined treatment of etoposide with epigenetic modifying reagents can synergistically induce miRNA-205-5p, eventually leading to improved treatment outcomes.

Among all members of the ERBB subfamily of tyrosine kinase receptors, ERBB4 is highly considered as an essential and functional member and its over-expression is found in several types of cancer (35-37). ERBB4 has been known not only to facilitate the proliferation and migration of cancer cells, but also to function as a marker of poor prognosis in breast cancer (38-40). Indeed, it was reported that the pharmacological inhibition of ERBB4 using ibrutinib successfully suppresses the activity of MEK and ERK, eventually inhibiting the growth of cancer cells harboring high levels of ERBB4 (41). In addition, ERBB4 is known to impede ER-Tamoxifen interactions, reducing the efficacy of Tamoxifen in ER-positive breast cancer (42). Furthermore, it was interestingly noticed that the activation of ERBB4 upon the binding of neuregulin-1 enhances the self-renewal of cancer stem cells (CSCs) (43), thus suggesting that miRNA-205-5p over-expression may decrease CSC properties and reverse cancer therapeutic resistance since CSCs are generally insensitive to cancer treatments (44, 45). Further research is required to investigate the role of miRNA-205-5p in sensitizing cancer cells to anti-cancer therapies.

Collectively, our findings provide new insights into a molecular mechanism underlying etoposide-induced antiproliferation and anti-migration and how miRNA-205-5p acts as a tumor suppressive miRNA in breast cancer cells. Our data also pave the way for developing a new perspective that the modulation of miRNA-205-5p levels can provide a promising opportunity for developing novel strategies to treat breast cancer.

Acknowledgements

This research was supported by Hallym University Research Fund 2019 (HRF-201904-006).

Footnotes

  • ↵* These Authors contributed equally to this work.

  • Authors’ Contributions

    Conceptualization, S.W.S., S.H.H., and J.K.P.; Data acquisition, analysis, interpretation, and visualization, S.W.S., S.H.H., S.S.O., C.H.L., and J.K.P.; writing – original draft preparation, S.W.S., S.H.H., J.K.P.; writing – review and editing, J.K.P. All Authors have read and agreed to the published version of the manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Received April 20, 2022.
  • Revision received July 19, 2022.
  • Accepted July 20, 2022.

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Etoposide-induced MicroRNA-205-5p Suppresses Proliferation and Migration by Targeting ERBB4 in MCF-7 Cells
SEUNG WAN SON, SEUNG HYEOK HEO, SANG SU OH, CHAN HEE LEE, JONG KOOK PARK
Anticancer Research Sep 2022, 42 (9) 4265-4272; DOI: 10.21873/anticanres.15926
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