Curcumin targets breast cancer stem-like cells with microtentacles that persist in mammospheres and promote reattachment

Abstract

Cancer stem-like cells (CSC) and circulating tumor cells (CTC) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTN), a type of tubulin-based protrusion of the plasma cell membrane that forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the human mammary epithelial (HMLE) cell line presents increased McTNs compared with its isogenic non-stem-like subpopulation. This increase was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live-cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. Although exposed to the environment, they also function as extensions between adjacent cells along cell-cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTN in breast CSC, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an antimetastatic strategy.

Figure 1. Stem-like mammary epithelial cells have increased microtentacles

A) HMLE cells have distinct subpopulations of stem-like (CD44^hi/CD24^lo) and non-stem-like (CD44^lo/CD24^hi) cells. B) Flow sorted stem-like subpopulations of HMLEs display significantly higher microtentacle frequencies than non-stem-like subpopulations. Columns, mean for three blinded experiments where at least 100 CellMask-stained cells were counted, representative of three independent experiments; bars, SD (P ≤ 0.00005, t test, black asterisk). C) Phase-contrast images of detached HMLE subpopulations where stem-like HMLEs display increased microtentacles (black arrows) compared to non-stem-like HMLEs.

Figure 2. Stem-like mammary epithelial cells have microtentacle-promoting cytoskeletal alterations

A) Representative western blot analysis of HMLE subpopulations shows that stem-like HMLEs have increased vimentin and detyrosinated tubulin (Glu) whereas total α-tubulin and GAPDH are unchanged (cropped). B) Attached non-stem-like HMLE cells show weak, diffuse staining for Glu-tubulin (b) and a loss of vimentin protein and organization (c), whereas stem-like HMLE cells display bundling of filamentous Glu-tubulin (f) and a robust network of vimentin filaments (g). Hoechst was used to visualize the nuclei (d, h). C) Suspended immunofluorescence where detached HMLE subpopulations were spun down onto glass coverslips and fixed. Immunostaining reveals the presence of Glu-tubulin (f) and vimentin (g) in McTN protrusions (white arrows) in the stem-like HMLE subpopulation but not in the non-stem-like subpopulation (b,c). Hoescht was used to visualize the nuclei (a,e).

Figure 3. Mammary stem-like cells have increased tubulin-dependent initial reattachment from suspension

A) Stem-like HMLE cells attach at significantly faster rates than non-stem-like HMLE cells as determined by electrical impedance expressed as Cell Index. Stem-like HMLE cells and non-stem-like HMLE cells both have significantly reduced attachment when treated with the microtubule polymerization inhibitor colchicine (50μM). For all reattachment assays: lines=mean for quadruplicate wells; bars=SD; representative graph from three independent experiments is shown. B) Phase contrast images of HMLE stem-like (a-d) and non-stem-like (e-h) subpopulations reattaching from suspension. Panels, 10x magnification; Insets, 60x magnification.

Figure 4. Microtentacles persist in mammospheres

A) Live cell confocal imaging of a HMLE mammosphere stained with CellMask Orange revealing dynamic microtentacles (a, black arrows) that disappear upon treatment with microtubule polymerization inhibitor Colchicine (b, 50μM, 10 minutes). B) Live cell confocal imaging of mammospheres stained with CellMask Orange (red) and transduced with GFP-membrane (green) suspended over a glass surface reveals McTNs from the GFP-membrane cell encircling other cells in the mammosphere. Three-dimensional computer-rendered image of HMLE (a) and BT-549 (d) mammospheres viewed from the bottom of the spheres. GFP-membrane transduced cells reveal long, flexible protrusions (b, e) that encircle other cells in the mammospheres (c, f).

Figure 5. Stem-cell targeting agent Curcumin reduces microtentacles and reattachment

A) Cellular viability assay using CellTiter 96 AQ_ueous One Solution (Promega) reveals no significant differences in viability between BT-549 cells treated with doses of curcumin (5, 10, 25, 40, and 50μM) compared to vehicle (DMSO) for 6 hours (P ≥ 0.05 for each time point). B) Western blot analysis reveals no significant cleavage of poly ADP-ribose polymerase (ΔPARP) with up to 50 μM Curcumin exposure for 6 hrs BT-549s, while treatment with 1 μM staurosporine for 16 hrs positively induced PARP cleavage (far right lane). C) Treatment with 50 μM Curcumin for 6hr significantly reduces the proportion of cells in stem-like population as assessed by surface marker phenotype. Columns=mean for three independent experiments where at least 10,000 BT-549 cells per treatment group were analyzed; bars=SD (P ≤ 0.005, t test, black asterisk). D) Vehicle-treated BT-549s display significantly higher microtentacle frequencies than BT-549s treated with 50μM curcumin for 6 hours. Columns=mean for three blinded experiments where at least 100 CellMask-stained cells were counted, representative of three independent experiments; bars=SD (P ≤ 0.0005, t test, black asterisk). E) Vehicle-treated BT-549s attach at significantly faster rates than BT-549s pretreated with 50μM curcumin for 6 hours and then seeded into E-plates containing vehicle or drug, respectively, as determined by electrical impedance expressed as Cell Index. Lines=mean for quadruplicate wells; bars=SD; representative graph from three independent experiments is shown.