Identification of CCN1 transferred by extracellular vesicles derived from breast cancer cells responsible for doxorubicin-resistance.

Abstract

e12623 Background: The anthracycline drug, doxorubicin, is one of the most frequently used chemotherapeutic drugs for breast cancer. However, not all patients respond to doxorubicin-containing combination chemotherapy due to chemo-resistance (natural or acquired). Preceding researches have shown that extracellular vesicles (EVs) can be readily harvested from peripheral blood for further biological analysis and thus perform an attractive source of tumor derived materials for identification of chemo-resistance factors. This study aims to provide a theoretical basis to use CCN1-containing EVs as new biomarkers and therapeutic targets for doxorubicin-resistance in breast cancer. Methods: A strategy combining RNA-sequencing technique and bioinformatic analysis was used to determine the level of differential expressed mRNAs in EVs samples from cellular supernatant (n = 4) and blood serum (n = 8) from chemo-resistant and chemo-sensitive patients. Serial gene expression analysis in patients (n = 203) who treated with neoadjuvant (GSE32603) were also employed to identified drug-resistance-related proteins. The expression of CCN1 was detected in doxorubicin-resistant cell lines (MCF-7/DOX),MCF-7 and their EVs by RT-qPCR and western blot. Then, we used cell apoptosis and immunofluorescence stain to confirm the ability of CCN1-containing EVs in conferring drug resistance. Immunohistochemistry stain and RT-qPCR were used to prove the expression level of CCN1 in tumor tissues from breast cancer patients (n = 80) and in serum EVs from patients treated with the whole neoadjuvant chemotherapy. Results: We combined the differentially expressed mRNAs from three datasets and found identified CCN1 as a drug-resistance-related factor in breast cancer. The expression level of CCN1 is much higher in MCF-7/DOX cells and their EVs compared with MCF-7 cells and their EVs. The addition of CCN1-containing EVs has been found significantly increase the resistance to doxorubicin in sensitive cells. Moreover, a higher expression level of CCN1 was detected in the progressive disease (PD)/stable disease (SD) group than in the partial response (PR)/complete response (CR) group both in tissues and serum EVs. Conclusions: All These findings demonstrate that CCN1-containing EVs appear to be essential in conferring resistance to doxorubicin. More importantly, we exhibit the potential applications of CCN1-containing EVs for predicting the efficiency and outcome of patients treated with doxorubicin-based chemotherapy.