Comparative analysis of the exosomal cargo of the estrogen-resistant breast cancer cells

Exosomes are 30-100 nm-sized microvesicles that are generated in the cells and released into the extracellular space accumulating in many biological fluids, including urine, milk, semen, cerebrospinal fluid, lymph, saliva, etc. [1]. It is noteworthy that tumor cells produce much more exosomes than normal cells [2]. The exosomes involvement in the pathogenesis of tumors is based on their property to incorporate into the recipient cells resulting in the both genomic and epigenomic changes [3-8]. Recently, the ability of the exosomes secreted by the drug- or hormone-resistant tumor cells to transfer the resistant properties to recipient cells has been demonstrated in different cell models [9, 10].

The main goal of the present study was to analyse the features of the exosomes of the estrogen-resistant breast cancer cells and to identify the exosomal factors respondent for transferring of the resistant phenotype to the donor cells.

Earlier, using the estrogen-dependent MCF-7 breast cancer cells and estrogen-independent MCF-7/T cells we have demonstrated the ability of the resistant cells-derived exosomes to initiate the estrogen-independent growth of the parent MCF-7 cells. The parallel experiments were performed on the MCF-7/М resistant subline developed under long-term cultivation of the parent MCF-7 cells with biguanide metformin and characterized by the cross-resistance to metformin and tamoxifen. The treatment of the parent MCF-7 cells with the MCF-7/M exosomes resulted in the cell cross-resistance to metformin and tamoxifen. Both types of resistant exosomes, MCF-7/T and MCF-7/M, induced the similar changes in the cell signaling: inhibition of the estrogen signaling and stimulation of the Akt protein kinase and transcription factors AP-1and NF-kB [11].

Here, to elucidate the common features of the both types of resistant exosomes, the proteome and microRNA cargo of the control and both types of the resistant exosomes were analyzed. Exosomes were prepared from the MCF-7, MCF-7/T and MCF-7/M’ conditioned medium by the differential ultracentrifugation, and exosome imaging was carried out by transmission electron microscope. For proteome study, an AB Sciex 5800 MALDI TOF/TOF mass spectrometer (Sciex, Germany) was used. Analysis of MS and MS/MS spectra was done with Protein Pilot software using the UniProtKB/SwissProt/NCBI international protein databases. Then the differentially expressed proteins in the exosomes of MCF-7, MCF-7/T and MCF-7/M cells were detected. Totally, more than 400 proteins were identified in the exosome samples. Among them, 131 differentially expressed proteins (DEPs) were found in the exosomes of MCF-7/T cells versus MCF-7 exosomes and 97 DEPs were found in the MCF-7/M exosomes.

To find the common changes in the proteome of the resistant exosomes, DEPs in the exosomes from MCF-7/T and MCF-7/M cells were compared. As revealed, only two proteins, DMBT1 (Deleted in Malignant Brain Tumors 1) and THBS1 (Thrombospondin-1), were commonly expressed in the both resistant exosomes (less than 5% from total DEPs) demonstrating the unique protein composition of each type of the resistant exosomes. Noteworthy, the bioinformatical analysis showed correlation between expression of two identified proteins and breast cancer risk. Namely, single-nucleotide polymorphisms (SNPs) and overexpression of DMB1 were found to be associated with the breast cancer [12, 13]. Several studies demonstrated that high level of THBS1 mediates chemotherapy resistance through the integrin β1/mTOR pathway [14] and promotes aggressive phenotype via epithelial-mesenchymal transition (EMT) [15].

The analysis of exosomal microRNAs was performed by HiSeq2500 and at least 5 million reads per samples were obtained. Library preparation

Table 1

Table 2

Differentially expressed miRNAs in the exosomes of the resistant MCF-7/T and MCF-7/M cells

Differentially expressed exosomal miRNAs associated with hormonal resistance

and sequencing was done by ZAO Genoanalytica (Moscow, Russia) as follow: microRNA was extracted from by PureLink RNA Micro Kit (#12183-016) according to manual. Library preparation was carried out with NEBNext® Small RNA Library Prep Set for Illumina® (E7330S) according to manual. More than 2500 miRNAs were identified in the exosomal samples. The comparison of miRNA profile of MCF-7 and MCF-7/T exosomes revealed 877 miRNA differentially expressed in MCF-7/T exosomes, among them 459 miRNA were up-regulated, and 418 miRNA were down-regulated. Study of miRNA of MCF-7/M exosomes showed 751 differentially expressed miRNA including 388 up-regulated and 363 down-regulated miRNAs. The comparative analysis of the miRNA differentially expressed in the both MCF-7/T and MCF-7/M resistant exosomes revealed 180 up-regulated and 202 down-regulated miRNAs (Table 1).

The following bioinformatical analysis of the common differentially expressed miRNAs revealed 4 up-regulated and 8 down-regulated miRNAs associated with progression of hormonal resistance of breast tumors. Importantly, we revealed the strong correlation between change vector of miRNA expression in the resistant exosomes and type of miRNA activity. Namely, all of 4 up-regulated miRNAs were described as resistance-associated mitogenic factors, whereas 8 down-regulated miRNAs were considered as the pro-apoptotic or hormone-sensitive- associated factors (Table 2).

As mentioned above, the parent cells response to the resistant exosomes involves the activation of Akt – one of the key signaling supporting the growth of the hormone-resistant cells [30]. The key role of the Akt signaling in the transferring of the resistant phenotype was substantiated in our experiments, showing the full block of the exosome-induced resistance of MCF-7 cells in the presence of PI3K inhibitor wortmannin [11]. Here, the analysis of 4 resistance-associated exosomal miRNAs revealed 2 miRNAs, mir-101, mir-181b, which up-regulated PI3K signaling. Both of miRNAs exert their effect via the suppression of PTEN phosphatase which is main physiological antagonist of PI3K [16].

Totally, we demonstrated the unique protein and miRNA composition of the exosomes of the resistant cells, identified the possible intercellular targets of exosomes and revealed the key exosomal miRNAs associated with hormonal resistance. Further studies are required to explore the role of the each of the identified miRNAs in the progression of the exosome-induced hormonal resistance.