HPI/AMF inhibition halts the development of the aggressive phenotype of breast cancer stem cells.

ABSTRACT

Cancer stem cells are responsible for tumor recurrence and metastasis. A new highly reproducible procedure for human breast cancer MCF-7 stem cells (BCSC) isolation and selection was developed by using a combination of hypoxia/hypoglycemia plus taxol and adriamycin for 24h. The BCSC enriched fraction (i) expressed (2-15 times) the typical stemness protein markers CD44+, ALDH1A3 and Oct 3/4; (ii) increased its clonogenicity index (20-times), invasiveness profile (>70%), migration capacity (100%) and ability to form mammospheres, compared to its non-metastatic MCF-7 counterpart. This isolation and selection protocol was successful to obtain stem cell enriched fractions from A549, SiHa and medulloblastoma cells. Since the secretion of HPI/AMF cytokine seems involved in metastasis, the effects of erytrose-4-phosphate (E4P) and 6-phosphogluconate (6PG), potent HPI inhibitors, on the acquisition of the breast stem cell-like phenotype were also evaluated. The presence of E4P during the BCSC selection deterred the development of the stemness phenotype, whereas both extracellular E4P (5-250nM) and 6PG (1µM) as well as siRNA HPI/AMF depressed the BCSC invasiveness ability (>90%), clonogenicity index (>90%) and contents (50-96%) of stemness (CD44, ALDH1A), pluripotency (p38 MAPK, Oct3/4, wnt/ß-catenin) and EMT (SNAIL, MMP-1, vimentin) markers. The cytokine inhibitor repertaxin (10nM) or the anti-IL-8 or anti-TGF-ß monoclonal antibodies (10µg/mL) did not significantly affect the BCSC metastatic phenotype. E4P also diminished (75%) the formation and growth of MCF-7 stem cell mammospheres. These results suggested that E4P by directly interacting with extracellular HPI/AMF may be an effective strategy to deter BCSC growth and progression.