Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer

Abstract

Experimental evidence suggest that breast tumors originate from breast cancer stem cells (BCSCs), and that mitochondrial biogenesis is essential for the anchorage-independent clonal expansion and survival of CSCs, thus rendering mitochondria a significant target for novel treatment approaches. One of the recognized side effects of the FDA-approved drug, doxycycline is the inhibition of mitochondrial biogenesis. Here we investigate the mechanism by which doxycycline exerts its inhibitory effects on the properties of breast cancer cells and BCSCs, such as mammosphere forming efficiency, invasion, migration, apoptosis, the expression of stem cell markers and epithelial-to-mesenchymal transition (EMT) related markers of breast cancer cells. In addition, we explored whether autophagy plays a role in the inhibitory effect of doxycycline on breast cancer cells. We find that doxycyline can inhibit the viability and proliferation of breast cancer cells and BCSCs, decrease mammosphere forming efficiency, migration and invasion, and EMT of breast cancer cells. Expression of stem cell factors Oct4, Sox2, Nanog and CD44 were also significantly downregulated after doxycycline treatment. Moreover, doxycycline could down-regulate the expression of the autophagy marker LC-3BI and LC-3BII, suggesting that inhibiting autophagy may be responsible in part for the observed effects on proliferation, EMT and stem cell markers. The potent inhibition of EMT and cancer stem-like characteristics in breast cancer cells by doxycycline treatment suggests that this drug can be repurposed as an anti-cancer drug in the treatment of breast cancer patients in the clinic.

Keywords: autophagy; breast cancer; cancer stem cells; doxycycline; epithelial-to-mesenchymal transition; mitochondria.

Figure 1.

Doxycycline inhibits cell viability of breast cancer cells. (A) Breast cancer cells propagated as monolayers (differentiated cultures, solid line) or mammospheres (enriched in BCSCs, dotted line) were treated with the indicated concentrations of doxycycline for 72h. The % viability of cells at each doxycycline dose at the end of treatment was measured using a Cell Counting Assay Kit. The horizontal dotted line represents the value of IC50 (MCF7 and MDA-MB-468 was 11.39 and 7.13uM, respectively). (B) Doxycycline significantly reduced the number of colonies in both breast cancer cell lines, MCF7 and MDA-MB-468. (C-D) Doxycycline treatment induced both early (Annexin-V_pos/P_Ineg) and late (Annexin-V_pos/P_Ipos) apoptotic cell death in both breast cancer lines.

Figure 2.

Doxycycline inhibits self-renewal of breast cancer cells. Doxycycline decreased the % of CD44⁺CD24^-/low BCSC population in MCF7 and MDA-MB-468 cells (A), as well as mammosphere formation efficiency in both lines tested (B). (C) Relative mRNA expression levels of self-renewal related genes are significantly downregulated in doxycycline treated breast cancer cells. The dotted line represents mRNA expression of non-treated breast cancer cells. (D) Western blot analysis of the stem cell related genes. (Data are reported as means ± standard deviation, *p < 0.05, **p < 0.01) MCF7 and MDA-MB-468 were treated with 11.39 μM and 7.13 μM doxycycline, respectively.

Figure 3.

Doxycycline inhibits migration and invasion in MDA-MB-468 breast cancer cells. Doxycycline inhibits migration (A) and invasion (B) in MDA-MB-468 breast cancer cells in a transwell assay. MDA-MB-468 cells were treated with doxycycline for 72 h with a single dose of IC₅₀.

Figure 4.

Doxycycline decreased the EMT phenotype in MDA-MB-468 breast cancer cells. (A) Relative mRNA expression levels of EMT-related genes in doxycycline treated breast cancer cells. The dotted line represents mRNA expression of non-treated breast cancer cells. (Data are reported as means ± standard deviation, *p < 0.05, **p < 0.01) (B) Western-blot analysis for EMT-related proteins. MDA-MB-468 cells were treated with doxycycline for 72 h with a single dose of IC₅₀.

Figure 5.

Doxycycline inhibits decreases autophagy-related protein levels. LC3BI and LC3BII protein levels were analyzed (A) and measured (B) in MCF-7 and MDA-MB-468 cells after doxycycline treatment. MCF7 and MDA-MB-468 were treated with 11.39 and 7.13 μM doxycycline for 72 h, respectively.