Phenotypic screening reveals topoisomerase I as a breast cancer stem cell therapeutic target

Abstract

Cancer stem cells (CSCs) are a subpopulation generally thought to be responsible for cancer initiation and progression. Because CSCs are often rare in the total tumor cell population and differentiate rapidly when grown in culture, it has been challenging to uncover compounds that selectively target CSCs. We previously described CSC-emulating cells derived from breast cancer cell lines that maintained a stable undifferentiated state. We optimized a phenotypic assay with these cells and screened 1,280-bioactive compounds, identifying five that preferentially inhibited CSC-like cell proliferation. Using a compound-guided target identification approach, we found high topoisomerase I (Topo I) expression levels in breast CSC-like cells and primary breast CSCs. Structurally unrelated small molecules targeting Topo I preferentially inhibited CSC-like cells. These results illustrate the substantial power of this CSC phenotypic screening platform and promote Topo I as a potential molecular therapeutic target for therapies aimed at expunging CSCs.

Figure 1. Concentration-dependent curves for the four compounds identified as selective against BC1A cells

(a) BC1A and BC1B cells were treated with rottlerin, A77636, β-lapachone, CGP74514A, doxorubicin, or etoposide at the indicated concentrations. CSC-like cells (●); non-CSC-like cells (□). Cell viability was accessed using alamar blue after 72 hr. The fluorescence readout (RFU) was normalized to in-plate control to calculate the percent control. Each concentration was tested in quadruplicate and the data are the mean ± SD; each drug panel is representative of three experiments with similar results. (b) Growth curves for the four CSC selective compounds. BC1A and BC1B cells were treated with rottlerin (0.8 μM), A77636 (4 μM), β-lapachone (1.5 μM), or CGP74514A (0.8 μM) for 4 or 6 days. BC2A and BC2B cells were treated with β-lapachone (1.5 μM) for 5 days. We used drug concentrations that yielded the maximum selective difference in growth inhibition from Figure 1a. Viable cells were counted using the Vi-Cell Cell Viability Analyzer and the number of viable cells on day 0 was used to normalize the relative cell number for the subsequent days. CSC-like cells (●); non-CSC-like cells (□). Experiments were performed in triplicate and the data were presented as the average relative cell number ± SD. Each growth curve was repeated three times with similar results and one representative curve is shown.

Figure 2. CSC selective growth inhibition by Topo I inhibitors

(a) BC1A and BC1B cells were treated with camptothecin and topotecan at indicated concentrations. Cell viability was accessed using the alamar blue after 72 hr. The fluorescence readout was normalized to in-plate control to calculate the percent of control. Each concentration was tested in quadruplicate and the data were presented as mean ± SD. Each curve was repeated three times with similar results and one representative curve is shown. CSC-like cells (●); non-CSC-like cells (□). (b) BC1A, BC1B, BC2A, BC2B, BC3A, and BC3B cells were treated with NSC 725776 or NSC 743400 at indicated concentrations and as described in panel A. CSC-like cells (●); non-CSC-like cells (□).

Figure 3. Elevated Topo I levels and activity in breast CSC-like cells

(A) Western blots for Topo I protein in CSC-like (S) and non-CSC-like (NS) cells from BC1, BC2 and BC3 cells. β-tubulin was used as a loading control. (B) Topo I mRNA levels were determined in the three CSC pairs by quantitative real-time RT-PCR. Human GAPDH was used as an internal control. Data were normalized to GAPDH mRNA level first. Topo I mRNA level in CSC-like cells were then normalized to non-CSC-like cell Topo I level to calculate the fold change. Each PCR reaction was performed in triplicate, and the data were presented as the average fold change ± SD. CSC-like cells = hatched bars; non-CSC-like cells = black bars. (C) Cell extracts from BC1A and BC1B cells were serial diluted 2-fold before the activity assay. Extracts were incubated with supercoiled plasmid substrate DNA at 37°C for 30 min. After incubation, DNA samples were separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. The supercoiled DNA (S.C, lane 1) and relaxed DNA (R, lane 2) samples are shown for reference. Lane 3 to 8 were DNA incubated with diluted extract, corresponding to 1:64, 1:32, 1:16, 1:8, 1:4, and 1:2 dilutions. Lane 9 was DNA incubated with undiluted extract.

Figure 4. Higher Topo I expression in primary breast CSCs

(A) CSCs and non-CSCs were isolated from primary tumors using CD49f as a marker and stained with anti-Topo I antibody (red) and DAPI (blue). Cells were directly examined by fluorescence microscopy and Topo I was found expressed exclusively in the nuclei. CSC (CD49f⁺ cells) had much higher level of Topo I staining compared to non-CSC (CD49f⁻ cells). Images showed 3 representative cells of CSC and non-CSC. (B) Topo I mean fluorescence intensity was higher in CSC-like cells. Fluorescence intensity of each cell was quantified using the Image J software. 41 CD49⁻ cells and 50 CD49f⁺ cells were measured and mean fluorescence intensities were calculated. (C) Co-localization of breast CSC marker ALDH1 and Topo I in frozen tumor sections. Frozen sections of 19 breast tumors were stained with DAPI (blue), anti-Topo I antibody (green), and anti-ALDH1 antibody (red). Only a small subset of tumor cells expressed ALDH1 and these cells formed clusters (a and b). Co-localization of ALDH1 and Topo I was evident in 15 out of the 19 tumors we examined. Panel c and d are enhanced magnifications.