Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes’ ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes’ ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes’ ability to transfer their cargo into the heterologous cells.

No abstract

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.

Brassinosteroids (BS), a class of plant‐specific steroid hormones, are considered as new potential anticancer agents for the treatment of tumors of different origin, including hormone‐dependent cancers. Effects of a synthetic brassinosteroid BS4 ((22R,23R,24R)‐22,23‐dihydroxy‐24‐methyl‐B‐homo‐7‐oxa‐5α‐cholest‐2‐en‐6‐one ((3aS,7aR,7bS,9aS,10R,12aS,12bS)‐10‐[(2S,3R,4R,5R)‐3,4‐dihydroxy‐5,6‐dimethylheptan‐2‐yl]‐7a,9a‐dimethyl‐1,3a,4,7,7a,7b,8,9,9a,10,11,12,12a,12b‐tetradecahydro‐3H‐benzo[c]indeno[5,4‐e]oxepin‐3‐one)) on hormone‐dependent breast cancer cells and normal epithelial cells and its impact on the estrogen receptor signaling were evaluated. Cytotoxicity was assessed by MTT‐test; expression of estrogen receptor α and survivin was measured by immunoblotting. Transactivation analysis of luciferase reporter gene was performed for ERα and AP‐1 factors after the brassinosteroid treatment. Dock6 and Autodock Vina were used for molecular docking. BS4 revealed a significant antiproliferative effect towards the hormone‐dependent breast cancer cells and was not active against normal epithelial cells. BS4 action on MCF‐7 breast cancer cells was found to be complex: a decrease in ERα expression as well as in its transcription activity was accompanied by inhibition of ERα‐related signaling pathways (AP‐1 complex and survivin). BS4 binding mode to ERα ligand‐binding domain was analyzed by molecular docking. The obtained results show that antiproliferative and antiestrogenic properties of the brassinosteroid BS4, as well as its ability to inhibit the anti‐apoptotic protein survivin may be of interest for further development of anticancer agents.

The phenomenon of the primary or acquired resistance of cancer cells to antitumor drugs is among the key problems of oncology. For breast cancer, the phenomenon of the resistance to hormonal or target therapy may be based on the numerous mechanisms including the loss or mutation of estrogen receptor, alterations of antiapoptotic pathways, overexpression of growth-related signaling proteins, etc. The perspective approaches for overcoming the resistance may be based on the usage of compounds such as inhibitors of the cell energetic metabolism. Among the latter, the antidiabetic drug metformin exerts antitumor activity via the activation of AMPK and the subsequent inhibition of mTOR signaling. The experiments were performed on the ERα-positive MCF-7 breast cancer cells, the MCF-7 sublines resistant to tamoxifen (MCF-7/T) and rapamycin (MCF-7/Rap), and on triple-negative MDA-MB-231 breast cancer cells. We have demonstrated metformin’s ability to enhance the cytostatic activity of the tamoxifen and rapamycin on both parent MCF-7 cells and MCF-7-resistant derivates mediated via the suppression of mTOR signaling and growth-related transcriptional factors. The cooperative effect of metformin and tested drugs was realized in an estrogen-independent manner, and, in the case of tamoxifen, was associated with the activation of apoptotic cell death. Similarly, the stimulation of apoptosis under metformin/tamoxifen co-treatment was shown to occur in the MCF-7 cells after steroid depletion as well as in the ERα-negative MDA-MB-231 cells. We conclude that metformin co-treatment may be used for the increase and partial restoration of the cancer cell sensitivity to hormonal and target drugs. Moreover, the combination of metformin with tamoxifen induces the apoptotic death in the ERα-negative breast cancer cells opening the additional perspectives in the treatment of estrogen-independent breast tumors.