Exosomes derived from bone marrow mesenchymal stem cells promote proliferation and migration via upregulation yes-associated protein/transcriptional coactivator with PDZ binding motif expression in breast cancer cells

Wanming Wu; Renfeng Huang; Linyang Ou; Ruiwen Lei

doi:10.4103/0304-4920.359800

ORIGINAL ARTICLE

Year : 2022 | Volume : 65 | Issue : 5 | Page : 233-240

Exosomes derived from bone marrow mesenchymal stem cells promote proliferation and migration via upregulation yes-associated protein/transcriptional coactivator with PDZ binding motif expression in breast cancer cells

Wanming Wu, Renfeng Huang, Linyang Ou, Ruiwen Lei
Department of Head, Neck and Breast Surgery, Yue Bei People's Hospital, Shaoguan, Guangdong, China

Date of Submission	30-May-2022
Date of Decision	01-Jul-2022
Date of Acceptance	31-Jul-2022
Date of Web Publication	27-Oct-2022

Correspondence Address:
Prof. Ruiwen Lei
Department of Head, Neck and Breast Surgery, Yue Bei People's Hospital, Shaoguan, 512000, Guangdong
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0304-4920.359800

Abstract

Bone marrow mesenchymal stem cells (BM-MSCs), with the properties of self-renewal and pluripotency, can migrate to the tumor sites and exert complex effects on tumor progression and communications by releasing exosomes. However, to our knowledge, only a few studies have reported the effects of BM-MSCs exosomes on breast cancer cells development. Here, utilizing exosomes isolated from in vitro BM-MSCs, we systematically investigated this issue in a breast cancer cell line. In this study, we found that BM-MSCs exosomes are actively incorporated by breast cancer cell MDA-MB-231 cells and subsequently promote MDA-MB-231 cells proliferation and migration. Mechanistically, we further found Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) which are Hippo signaling components were involved in this promoting progress. Consistently, YAP and TAZ knockdown could significantly reverse breast cancer cells proliferation and migration improved by BM-MSCs exosomes. Taken together, our findings demonstrated a new mechanism through which BM-MSCs-derived exosomes may contribute to breast cancer cells proliferation and migration, which might provide an evidence for novel drug discovery based on exosomes and Hippo signaling.

Keywords: Breast cancer cells, exosome bone marrow mesenchymal stem cells, migration, proliferation, Yes-associated protein, transcriptional coactivator with PDZ binding motif

How to cite this article:
Wu W, Huang R, Ou L, Lei R. Exosomes derived from bone marrow mesenchymal stem cells promote proliferation and migration via upregulation yes-associated protein/transcriptional coactivator with PDZ binding motif expression in breast cancer cells. Chin J Physiol 2022;65:233-40

How to cite this URL:
Wu W, Huang R, Ou L, Lei R. Exosomes derived from bone marrow mesenchymal stem cells promote proliferation and migration via upregulation yes-associated protein/transcriptional coactivator with PDZ binding motif expression in breast cancer cells. Chin J Physiol [serial online] 2022 [cited 2023 Nov 30];65:233-40. Available from: https://www.cjphysiology.org/text.asp?2022/65/5/233/359800

Authors Wanming Wu and Renfeng Huang contributed equally to this work.

Introduction

Breast cancer is one of the most common cancers in the world.^[1] The best treatment for breast cancer is surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, and endocrine therapy. However, patients with breast cancer usually metastasize after several years of resection of the primary tumor.^[2] This phenomenon indicates that breast cancer cells could survive in the body in a state of cancer dormancy for a long time. In other words, the cells stop dividing and stay in a quiescent state. When in an appropriate environmental conditions, the cancer cells will proliferate again. Studies reported that the breast cancer cells can be detected in bone marrow at the early stage,^[3] which might form micrometastases and invade to other distant organs.^[4]

Bone marrow mesenchymal stem cells (BM-MSCs), with the properties of self-renewal and pluripotency, belong to one type of hematopoietic stem cells. It has been reported that coculture BM-MSCs with breast cancer cells can greatly promote cancer cells metastatic potency.^[5] These studies indicate that there might be an intimate interaction between breast cancer cells and BM-MSCs. However, the underlying mechanism governing BM-MSCs crosstalk with breast cancer cells is not fully understood.

BM-MSCs can migrate to the tumor sites and exert complex effects on tumor progression and communications by releasing exosomes, which is a novel way of cell-cell communication and still plays an important role in various biological processes. Researchers found that exosomes also participate in tumor development by transporting intracellular contents, such as protein, mRNA, and microRNAs (miRNAs).^{[6],[7],[8],[9],[10]} Moreover, exosomes secreted by cancer cells act a critical role in cancer progression by the education of bone marrow-derived cells, neutrophil infiltration and promoting angiogenesis.^{[11],[12],[13]} Conversely, in the tumor microenvironment, exosomes secreted by stromal cells can transmit their cargo to cancer cells, which may contribute to cancer progression.^[14],[15] It has been validated that exosomes secreted by mesenchymal stem cell-derived adipocytes promote breast cancer cell growth via activation of Hippo signaling pathway.^[16]

The Hippo signaling pathway regulates cell growth and fate decision, organ size and tissue homeostasis, and its dysregulation contributes to tumorigenesis.^[17],[18] Hippo component Yes-associated protein (YAP) has been widely reported to be involved in many aspects of tumor biology, and it promotes focal adhesion and tumor aggressiveness via transcriptionally activating THBS1/FAK signaling in breast cancer.^[19] Recently, a novel study from Guan KL's group confirmed that Hippo signaling maintains ER expression and ER⁺ breast cancer growth, which provided an insight to the functions of YAP and transcriptional coactivator with PDZ binding motif (TAZ) in promoting the growth of ERα⁺ breast cancer cells.^[20] Moreover, researchers found that exosomes secreted by mesenchymal stem cell-derived adipocytes promoted breast cancer cell growth, and mechanistically, the hippo signaling pathway was demonstrated to be partially responsible for the tumor-promoting effects of MSC-differentiated adipocyte exosomes.^[16]

To our knowledge, only a few studies have reported the effects of BM-MSCs exosomes and Hippo signaling on breast cancer cells development. Therefore, utilizing exosomes isolated from in vitro BM-MSC, we found that exosomes secreted by BM-MSCs could promote breast cancer cell proliferation and migration. Mechanistically, Hippo signaling components YAP and TAZ were demonstrated to be involved in the tumor-promoting effects of exosomes isolated from BM-MSC. Consistently, YAP and TAZ knockdown could significantly reverse breast cancer cells proliferation and migration improved by BM-MSCs exosomes.

Materials and Methods

Cell line and culture

BM-MSCs were purchased from Ningbo MINGZHUBIO Co., Ltd and cultured in α-modified Eagle's medium (12561056, Gibco) with 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin at 37°C in 5% CO₂. MDA-MB-231 cells were cultured in RPMI 1640 (R8758, Sigma-Aldrich) containing 10% heat-inactivated FBS (12106C, Sigma-Aldrich) and the 1% antibiotic-antimycotic at 37°C in 5% CO₂. BM-MSCs markers including positive ones CD29 (ab30394, Abcam) and CD90 (ab225, Abcam) and negative maker CD45 (ab40763, Abcam) were detected using flow cytometer (Aria II, BD).

Exosome isolation and analysis

Exosomes were isolated as previously described.^[21] Briefly, cells used to isolate exosomes were cultured in serum free medium, and then, exosomes were isolated from conditioned media at 48 h by serial centrifugation. After that, exosome pellets were resuspended in phosphate buffered saline (PBS). Morphology of the exosomes was examined by electron microscopy using negative staining. The sizes of exosomes were identified by nanoparticle tracking analysis with ZETA VIEW (Particle Metrix). Moreover, the protein concentrations of exosomes lysates were quantified by BCA protein quantification, and then, exosomes markers were examined by Western blot. The Exosomes markers we used including Calnexin (ab22595, Abcam), TSG101 (ab125011, Abcam), CD9 (ab92726, Abcam), CD63 (A5271, Abclonal), and CD81 (ab109201, Abcam). Exosomes taking-up was evaluated by labeling exosomes with PKH26 (UR53202, Umibio). The whole cell and nucleus were labeled with β-actin (ab8226, Abcam) and DAPI (ab228549, Abcam), respectively. Dye transfer was visualized by confocal (SP8, Leica). The coculture of exosomes pellets and MDA-MB-231 cells were performed at a concentration of 200 μg/ml.

Proliferation assay

Cells were plated in 96-well plates at a density of 2 × 10³ cells per well. To reduce differences within the group, each group of cells samples was set five parallel holes. Then, the cells were incubated with a cell counting kit (ab228554, Abcam) according to the manufacturer's instructions. The absorbance was measured by a microplate reader at a wavelength of 450 nm.

Colony formation assay

MDA-MB-231 cells were plated at 100 cells/well in 6-well plates. After 24 h of PBS or BM-MSCs exosomes coculture, plates were rinsed with PBS, and fixed with 4% paraformaldehyde for 20 min at room temperature, washed twice with PBS, and then colonies were imaged and quantified using an inverted Olympus IX81 fluorescence microscope with a CellR image system.

Wound-healing assay

Wound-healing assays were performed as previously described.^[22] Briefly, cells were grown to 70% confluence and monolayers were wounded by scratching with a sterile plastic 200 μl micropipette tip, washed, and incubated in the presence of PBS or BM-MSC exosomes. After 24 h, cells were fixed with 4% paraformaldehyde in PBS for 5 min at room temperature and photographed using a phase-contrast microscope.

Cell cycle analyses

Cell cycle distribution was analyzed using flow cytometer (Aria II, BD). Briefly, cells were seeded into 6-well plates and grown for 24 h and further grew in the presence of PBS or BM-MSC exosomes 24 h. The cells were harvested, washed twice with PBS, and fixed in 70% ethanol on 4°C for 1 h and centrifuged. The pellet was treated with RNase (20 μg/ml) at room temperature for 30 min and then incubated with PI (10 μg/ml) for 30 min. DNA content and cell cycle distribution were analyzed using flow cytometer.

Real-time polymerase chain reaction

Total mRNA of cells was extracted using Trizol. The quantitative real-time polymerase chain reaction (PCR) using AceQ quantitative PCR SYBR Green Master Mix was performed in ABI PRISM® 7500 Sequence Detection System according to the manufacturer's instructions. The primers for detecting YAP, TAZ, and GAPDH mRNAs are listed in [Supplementary Table 1]. Relative gene expression was normalized to GAPDH expression.

Western blot analysis

MDA-MB-231 cells were grown in six-well plates at 1 × 10⁶ cells/well. The cells were lysed in cell lysis buffer containing protease inhibitor and phosphatase inhibitor cocktails. Protein content in the cell lysates was quantified with the BCA protein assay Kit (BCA1-1KT, Thermo) and then equal amounts (5–10 μg) of total proteins were separated on 4%–12% Bis-Tris NuPAGE gels, followed by transfer to a PVDF membrane (1620177, Bio-Rad) for immunoblotting. The following antibodies were used for detection YAP (1:1000, ab56701, Abcam) and TAZ (1:1000, ab242313, Abcam). Blots were developed with the SuperSignal West Femto Maximum Sensitivity Substrate Kit (34095, Thermo Fisher Scientific) prior to be visualized by the Gel Doc XR+ (Bio-Rad). Densitometric measurement of band intensity was obtained using Quantity One software (Bio-Rad).

Small interfering RNA transfection

MDA-MB-231 cells were transfected using Lipofectamine RNAiMAX (13778-075, Thermo Fisher Scientific) with a small interfering RNA (siRNA) (designed by siDirect version 2.0) or YAP or TAZ or a non-targeting control siRNA (D-001206-13-20, Thermo Fisher Scientific). Cells were seeded at a density of 1 × 10⁶ cells/well in 6-well plates using DMEM with 0.5% FBS, as described above, and exposed to 40 nM of each targeting siRNA or equivalent amounts of non-targeting siRNA for at least 16 h. Knockdown of each siRNA target was confirmed by reverse transcription PCR and Western blot 72 h after transfection. The targeted position and sequence of YAP is 1514-1536, CTCAGGAATTGAGAACAATGACG. The targeted position and sequence of TAZ is 1116-1138, GACATGAGATCCATCACTAATAA.

Statistical analysis

The results were presented as mean ± standard error of mean (SEM). Student's t-test was performed to determine if there is a significant difference between two groups. * indicates statistically significant with P < 0.05, ** indicates statistically significant with P < 0.01, and *** indicates statistically significant with P < 0.001.

Results

Identification and characterization of exosomes derived from bone marrow mesenchymal stem cells

BM-MSCs have a characteristic morphology of fibroblast-like cells [Figure 1]a. They express high levels of CD29 and CD90, but are persistently negative for CD45 [Figure 1]b, which was consistently with previous report.^[23] Exosomes were isolated from the culture supernatants of BM-MSCs by differential centrifugation. The exosome concentration in BM-MSCs culture medium was 2.77E + 10 particles/ml. Moreover, under transmission electron microscopy, the average diameter of exosomes was around 78.9 nm [Figure 1]c. In addition, after evaluating the protein concentration of exosomes protein lysates (1.046074 μg/μl), the exosome markers including TSG101, CD9, CD63, and CD81 were confirmed by the Western blot [Figure 1]d. All these data indicated that the BM-MSCs exosomes were successfully isolated.

Figure 1: Characterization of exosomes secreted by BM-MSCs. (a) The morphology of BM-MSCs. (b) FACS analysis of BM-MSCs immunophenotype. (c) Electron micrograph of exosomes from BM-MSCs. (d) Detection protein markers in BM-MSCs exosomes by Western blot. BM-MSCs: Bone marrow mesenchymal stem cells, FACS: Fluorescence activating cell sorter.

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Bone marrow mesenchymal stem cells exosomes promote breast cancer cell proliferation and migration

We then evaluated exosomes effects on breast cancer cell proliferation and migration. First, we investigated whether BM-MSC-exosomes could be transferred into MDA-MB-231 cells. The exosomes were fluorescently labeled with PHK26 and incubated with MDA-MB-231 cells for 6 h. After treatment, over 90% of the MDA-MB-231 cells exhibited red fluorescence [Figure 2]a. In the following, the result of CCK8 assay showed that the proliferation rate of MDA-MB-231 cells treated with exosomes was significantly increased compared with that of control cells treated by PBS [Figure 2]b. Not surprisingly, the proportion of the G2/M cells were dramatically increased at 24 h after exosomes incubation, which indicates the cell cycle arrest [Figure 2]c. Consistently, the colony formation assay showed that BM-MSCs exosomes promote MDA-MB-231 cells colony formation [Figure 2]d and [Figure 2]e. Next, the wound healing assay demonstrated that MDA-MB-231 cells treated with BM-MSCs exosomes showed a higher migration rate than control cells treated with PBS, as manifested by more numbers of migrated cells [Figure 2]f and [Figure 2]g. Collectively, these results suggest that BM-MSCs exosomes are an important participant in promoting breast cancer cell proliferation and migration.

Figure 2: BM-MSCs-exosomes promoted proliferation and migration of MDA-MB-231 cells. (a) PKH26-labeled BM-MSCs-exosomes were detected in MDA-MB-231 breast cancer cells. Scale bar = 10 μm. (b) CCK8 analysis of MB-231 breast cancer cells treated with BM-MSCs-exosomes or PBS. (c) Cell cycle analysis of MDA-MB-231 cells treated with BM-MSCs-exosomes or PBS. (d and e) Colony formation assay of MDA-MB-231 cells treated with BM-MSCs-exosomes or PBS. (f and g) Migratory ability of MDA-MB-231 cells in exosome added culture medium or PBS added culture medium was determined by wound healing assay. BM-MSCs: Bone marrow mesenchymal stem cells.

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Bone marrow mesenchymal stem cells exosomes upregulate yes-associated protein and transcriptional coactivator with PDZ binding motif expression

It has been reported that YAP and TAZ are transcription coactivators enhancing the transcription of specific genes related to cell proliferation.^[24] The abnormal regulation of YAP/TAZ protein will affect cell proliferation, apoptosis, migration, and differentiation, resulting in a variety of diseases, including cancer.^[25] With the aim of identifying how exosomes regulate cell proliferation and migration, we investigated the Hippo signaling pathway in BM-MSCs exosomes treated cells. As revealed by quantitative real-time PCR, the results showed that the mRNA levels of both YAP and TAZ in BM-MSCs exosome-treated cells were significantly upregulated [Figure 3]a and [Figure 3]b. Consistently, the Western blot result showed that the protein levels of both YAP and TAZ were remarkably increased in BM-MSCs exosome-treated cells compared with PBS-treated control cells [Figure 3]c,[Figure 3]d,[Figure 3]e. These results indicate that Hippo signaling pathway might be involved in breast cancer cell proliferation and migration regulated by BM-MSCs exosomes.

Figure 3: BM-MSCs-exosomes activated the Hippo signaling pathway in MDA-MB-231 cells. (a and b) YAP and TAZ mRNA expression levels of MDA-MB-231 cells treated with BM-MSCs-exosomes or PBS were detected by real-time PCR. GAPDH was used as the internal control. (c) YAP and TAZ protein levels of MDA-MB-231 cells treated with BM-MSCs-exosomes or PBS were determined by Western blot analysis. GAPDH was used as the internal control. (d and e) The quantification of Western blot results with an image analyzer. BM-MSCs: Bone marrow mesenchymal stem cells, YAP: Yes-associated protein, TAZ: Transcriptional coactivator with PDZ binding motif, PBS: Phosphate buffered saline.

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Yes-associated protein and transcriptional coactivator with PDZ binding motif knockdown lead to impaired breast cancer cell proliferation and migration improved by bone marrow mesenchymal stem cells exosomes

To further confirm that YAP and TAZ are bona fide modulator of BM-MSCs exosomes in breast cancer cells, we next conducted a siRNA transfection study in MDA-MB-231 cells. As we expected, we found that YAP and TAZ knockdown markedly inhibited the proliferation induced by BM-MSCs exosomes of breast cancer cells [Figure 4]a, and the proportion of the G2/M cells were dramatically decreased in BM-MSCs exosomes with YAP and TAZ knockdown condition compared to BM-MSCs exosomes treated group [Figure 4]b and [Figure 4]c. Consistently, the colony formation assay showed that YAP and TAZ knockdown markedly inhibited MDA-MB-231 cells colony formation induced by BM-MSCs exosomes [Figure 4]d and [Figure 4]e. Next, the wound healing assay demonstrated that the higher migration rate of MDA-MB-231 cells induced by BM-MSCs exosomes was also blocked by YAP and TAZ knockdown, respectively [Figure 4]f and [Figure 4]g. These results provide evidence suggesting that Hippo signaling pathway is involved in regulating BM-MSCs exosomes in breast cancer cells proliferation and migration.

Figure 4: Inhibition of Hippo signaling impaired breast cancer cells proliferation and migration induced by BM-MSCs exosomes. MDA-MB-231 cells were transfected with non-targeting control siRNA, YAP siRNA or TAZ siRNA and incubated with BM-MSCs-exosomes. (a) CCK8 analysis for MB-231 breast cancer cells. (b and c) Cell cycle analysis for MDA-MB-231 cells. (d and e) Proliferative ability was detected by clone formation assay. (f and g) Migratory ability of MDA-MB-231 cells was detected by wound healing assay. BM-MSCs: Bone marrow mesenchymal stem cells, YAP: Yes-associated protein, TAZ: Transcriptional coactivator with PDZ binding motif, siRNA: Small interfering RNA.

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Discussion

Cell-to-cell communication, a dynamic mechanism, maintains tissue homeostasis and enables normal cellular activities. It has been reported that exosomes released by different cell types may act as a mediator of cell-to-cell communication. Genetic materials mRNAs and microRNAs contained in exosomes allow the communication exchange between cells.^[26] The neighboring cells of tumor cells can be epigenetically reprogramed by tumor cells, which via releasing exosomes.^[27],[28]

The microenvironment of tumors is composed of various types of cells, including vascular endothelial cells, immune cells, as well as mesenchymal cells. Hence, the communications and interactions between the cancer cells and the microenvironment have been extensively studied. Recently, the therapeutic potential of the mesenchymal stromal cell is intensively studied. Even though promising preclinical studies, most MSC secretome-based therapies have not been implemented in human medicine, because the complexity of bioactive factors secreted by MSCs is not completely understood. However, once MSCs are incorporated into tumors, they exert complex effects on tumors. Whether MSCs are pro- or anti-tumorigenic has been a subject of controversy. Interestingly, some of researchers showed that MSCs promote tumor growth, whereas other groups reported that MSCs suppress tumor progression.^{[2],[16],[29],[30]} Despite investigators proposed various mechanisms, the effects of MSC-derived exosomes on tumor cells remain to be explored.

The aim of this study was to investigate the effects of BM-MSCs-derived exosomes on cancer cells progression, especially in respect to breast cancer cells proliferation and migration. The exosomes were successfully isolated from the culture supernatants of BM-MSCs. The morphology and characterization of BM-MSCs-derived exosomes were consistent with other cells in terms of molecular contents.^[2],[16]

A recent study reported that exosomes from BM-MSCs contain a microRNA that promotes dormancy in metastatic breast cancer cells, resulting cancer cells maintaining a dormant state.^[2] Surprisingly, in this study, we demonstrated that BM-MSCs-derived exosomes greatly promoted cell proliferation and migration of breast cancer cells. These findings indicated that BM-MSCs-derived exosomes might indirectly promote tumor cells behavior.

To further study the molecular mechanisms underlying the exosomes regulating cells proliferation and migration, we paid attention to previous studies that exosomes secreted by mesenchymal stem cell-derived adipocytes promote breast cancer cell growth via activation of Hippo signaling pathway.^[16] Thus, we evaluated YAP and TAZ, the key molecules involved in Hippo signaling, expression levels in breast cancer cells treated with BM-MSCs-derived exosomes. Our data showed that both YAP and TAZ mRNA and protein levels were significantly increased in exosomes treated cancer cells. We next examined whether knockdown the YAP and TAZ can reverse the promoted proliferation and migration induced by exosomes. The results were concordant with our hypothesis that both YAP and TAZ knockdown can reverse the promoted proliferation and migration induced by BM-MSCs-derived exosomes.

It has been reported that asbestos exosomes significantly changed the expression of more than hundreds of genes expression in mesothelial cells;^[31] however, the underlying mechanisms on the capability of exosomes contents to affect mesothelial cell gene expression need further validation. However, researchers found that exosomes provide a unique mode of cell-to-cell communication in which microRNAs produced and released from one cell are taken up by cells at a distance where they can enact changes in gene expression.^{[9],[10],[32],[33]} Hence, our further research will dissect whether the microRNAs and which microRNAs in the exosome modulate the YAP and TAZ expression.

Exosome selection and uptake by recipient cells is highly intriguing. According to the results of past studies, signals are transferred from exosomes to recipient cells mainly by three methods: endocytosis/phagocytosis, direct membrane fusion, and receptor-ligand interactions. Some studies have also described the pathways of transmembrane signal transduction between exosomes and recipient cells.^[34] However, exosomes uptake by recipient cells is cell specific, and the mechanism of recipient cell selection is not yet clear. The interaction of surface molecules between specific cells and exosomes is critical for recipient cell targeting and adhesion. Hence, it is interesting to explore the target of the BM-MSCs exosomes in the breast cancer cells and investigate the subcellular localization of those exosomes.

Conclusion

This study demonstrated that BM-MSCs-derived exosomes promote breast cancer cells proliferation and migration via upregulating Hippo signaling pathway. Our study might provide a new insight into the role of BM-MSCs exosomes in the breast tumor microenvironment.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

RW performed the research conception and design. WW, RF, and LY performed the experiments. WW and RF analyzed and checked the data and drafted the manuscript. WW prepared figures. RW edited and revised manuscript. RW was primarily responsible for final content. WW and RF confirmed the authenticity of all the raw data. All authors read and approved the final manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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