BCSC are considered to be involved in the recurrence of breast cancer and its resistance to the systemic therapies. Their detection and targeting remain challenging.
Patients and methods:
The study was conducted in vitro, in xenografted immunecompromised mice and samples of 38 patients with early stage BC having biopsies and blood samples before and after anthraycycline + taxane-based neoadjuvant chemotherapy (NAC). All patients gave written informed consent before inclusion. 1) MCF7 cells grown as mammospheres (MS) for BCSC enrichment were treated with 5FU or paclitaxel (Pac) to select chemo-resistant BCSC. miRNA microarray was performed to identify specific miRNAs for chemo-resistant BCSC. The results were compared to miRNAs found in immortalized non-tumorigenic MCF10A cells to exclude miRNAs related to normal stem cells. 2) The correlation between the most highly expressed miRNA and the BCSC was confirmed by RT-qPCR in ALDH+ and ALDH- cells sorted from MCF7 and MDA-MB-231 cells by flow cytometry. 3) The impact of the miRNA on MS and colony development was assessed by up- and down-regulating its expression. MCF7 cells transfected with ectopic expression of miRNA, anti-miRNA or miRNA control were grown in MS before being injected in mice using a mouse INtraDuctal xenograft Model (MIND). In vivo tumor growth was assessed by luciferase imaging, then measured and quantified with human GAPDH ex vivo at 6 weeks. 4) The miRNA was quantified by RT-qPCR in tumor samples and sera of the patients before and after treatment, and its levels were correlated with pathological complete response and patients' outcomes.
379 miRNAs out of 2006 were altered in chemo-resistant versus untreated MCF7 MS. Thirteen were specific for 5FU and 5 for Pac. Three were common for both drugs. Of these, miR-363-3p was overexpressed specifically in BCSC-enriched chemo-resistant MCF7 cells and all the other tested BC cell lines, but not in non-tumorigenic MCF10A cells. Compared to adherent MCF7 cells, miR-363-3p was 12-, 60-, and 10-folds more expressed in BCSC-enriched MS treated with 5FU, Pac, or without treatment, respectively. miR-363-3p was 20- and 100-folds higher in ALDH+ compared to ALDH- in MCF7 and MDA-MB-231 cells. Anti-miR-363-3p reduced MS size and decreased their number 50%. A significant decrease of the number of colonies was also observed in soft agar. Consistently, miR-363-3p downregulation decreased tumor growth and metastasis by MCF7 cells transplanted in mice. In patients' sera with lower baseline level (n=15), miR-363-3p appeared decreased upon NAC. Patients with high miR-363-3p serum levels (n=22) had more risk to maintain higher level after chemotherapy. Triple-negative and HER2+ BC were more frequent in this second group. No significant difference was observed in term of pCR between the 2 groups. However 3 patients relapsed with distant metastases and all were in the second group with high baseline level and no decrease after NAC.
miR363-3p appeared to be a mediator of chemo-resistant BCSC. Its measurement in the serum of BC patients may predict resistance to neo-/adjuvant chemotherapy and higher risk of distant recurrence. Further investigations are warranted to confirm its role as biomarker and potential therapeutic target against BCSC.
Citation Format: Renaud S, Fiche M, Stravodimou A, Scabia V, Dormoy VM, Galmiche Rindisbacher M, Brisken C, Mermod N, Zaman K. miR363-3p mediates maintenance and resistance of breast cancer stem cells (BCSC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-11.