BAsic ReseARch PAPeR
Autophagy 9:5, 1–16; May 2013; © 2013 Landes Bioscience
Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells interferes with their maintenance Wen Yue,1,2,† Ahmed hamaï,1,2,† Giovanni Tonelli,1,2 chantal Bauvy,1,2 Valérie Nicolas,2,3 hugo Tharinger,2,3 Patrice codogno1,2 and Maryam Mehrpour1,2,* 1
These authors contributed equally to this work.
Keywords: breast cancer stem-like/progenitor cell, salinomycin, autophagy, cell death, lysosome Abbreviations: ACTB, actin, beta; ATG12, autophagy-related 12; ATG5, autophagy-related 5; ATG16L1, autophagy-related ATG16L1; ALDH1, aldehyde dehydrogenase 1; Baf, bafilomycin A1; BECN1, Beclin 1, autophagy related; BCSCs, breast cancer stem-like /progenitor cells; CASP3, caspase 3; CASP8, caspase 8, apoptosis-related cysteine peptidase; CTNNB1, catenin (cadherin-associated protein), beta 1; CTS, cathepsin; EPCAM, epithelial cell adhesion molecule; LRP6, lipoprotein receptor related protein; LC3, microtubule-associated protein 1 light chain 3B; MTORC1, the mechanistic target of rapamycin complex 1; Nig, nigericin; PAS, phagophore assembly site; AO, acridine orange; RAB7A, member RAS oncogene family; ROS, reactive oxygen species; RPTOR, regulatory associated protein of MTOR, complex 1; Sal, salinomycin; TUBB, β-tubulin; WNT1, wingless-type MMTV integration site family
Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (cscs). We have recently shown that autophagy is essential for the tumorigenicity of these cscs. salinomycin (sal), a K+/h+ ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast cscs. however, its mechanisms of action are still unclear. We show here that sal blocked both autophagy flux and lysosomal proteolytic activity in both cscs and non-cscs derived from breast cancer cells. GFPLc3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/ lysosome fusion was not altered by sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in sal-treated cells. however, tandem mcherry-GFP-Lc3 assay indicated that the degradation of mcherry-GFP-Lc3 is blocked by sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BsA assay and measurement of cathepsin activity. Our data indicated that sal has a relatively greater suppressant effect on autophagic flux in the ALDh+ population in hMLeR cells than in the ALDh- population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDh+ population. ATG7 depletion accelerated the proapoptotic capacity of sal in the ALDh+ population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of sal to target cscs in vitro.
Introduction Cancer stem cells are a new target for drug discovery in cancer. The theory of cancer stem-like cells (CSCs) attempts to explain how cancer is initiated and how it recurs after treatment. The hypothesis postulates that tumors are organized in a cellular hierarchy that is maintained by a subpopulation of tumor cells endowed with self-renewal and multilineage differentiation capacities.1 CSCs have been identified in many different types of tumor, including breast cancer.2-5 In breast cancer, a *Correspondence to: Maryam Mehrpour; Email: firstname.lastname@example.org Submitted: 09/27/12; Revised: 02/12/13; Accepted: 02/13/13 http://dx.doi.org/10.4161/auto.23997 www.landesbioscience.com
population of CD44high /CD24−/low /EPCAM+ has been identified as being breast CSCs. As markers CD44, CD24 and EPCAM molecules were used to isolate progenitor/CSCs cells. A high...