Article Figures & Data
Figures
- Figure 1.
BCLs display two drug-response profiles to abexinostat. Cell viability was measured with an MTS assay after 72 hours of treatment for 16 BCLs (n = 12). Dotted line allows determination of IC50 for each BCL. BCLs sensitive to low doses of abexinostat are represented with green curves; BCLs sensitive to high doses of abexinostat are represented with red curves. Error bars represent mean ± SD.
- Figure 2.
Cell-cycle progression was differentially altered by abexinostat according to drug-response profile. A, measurement of apoptosis induction by measuring caspase-3/7 activation in BCL panel (excluding MCF7) treated by abexinostat (n = 3; *, P < 0.01). B, cell proliferation was estimated using MTS viability test, and proliferation rate was calculated in 16 BCLs treated by abexinostat (high-dose sensitive BCLs: red curve; low-dose sensitive BCLs: green curve; n = 3). C, flow charts representing cell-cycle distribution of a low-dose sensitive BCL (SK-BR-7) and high-dose sensitive BCL (MDA-MB-436) along 72 hours of treatment. Quantifications of cells in each cell-cycle phase are represented in D (n = 3; *, P < 0.01). Similar results are reported for MDA-MB-231 and HCC1954 BCLs (Supplementary Fig. S6). Error bars represent mean ± SD.
- Figure 3.
The CSC population is differentially modulated according to abexinostat-response profile. A–D, the effect of abexinostat on the CSC population was assessed using ALDEFLUOR assay (A and B) and tumorsphere formation (C and D). Representative flow charts for ALDEFLUOR assay (A) and pictures of tumorspheres (C) are presented. (n = 6; *, P < 0.05.) Error bars represent mean ± SD.
- Figure 4.
Abexinostat induces CSC differentiation in low-dose sensitive BCLs. A, optical microscopy showed that morphology of cells of four representative low-dose sensitive BCLs changed after 72 hours of treatment and cells formed large cell clusters. B, cell differentiation was monitored by measuring expression of differentiation markers using immunofluorescent staining (green staining); nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; blue staining). Abexinostat treatment induces a modification of phenotypic profile. Scale bar = 10 μm.
- Figure 5.
lncRNA Xist predicts response to abexinostat. A, transcriptomic analysis of low-dose sensitive BCLs versus high-dose sensitive BCLs. Results are plotted according to their gene differential expression between both BCL groups (y axis) and their corresponding statistical significance (x axis). Xist lncRNA (red arrow) was the most differentially expressed gene between the two BCL populations with an overxepression up to 139-fold (P < 0.00001; FDR q-val: 0.03) in high-dose sensitive BCLs. B, BCLs are classified according to increasing Xist expression level measured by cDNA microarrays; opposite the cDNA microarray measurements is a histogram presenting Xist expression level measured by qRT-PCR, R = 0.84 [(0.58–0.95), P = 8.7E-5; n = 3]. C, box plots represent Xist expression level in low-dose and high-dose sensitive BCLs measured by qRT-PCR. High-dose sensitive BCLs are significantly enriched in Xisthigh BCLs compared with low-dose sensitive BCLs (n = 3; *, P = 0.055). D, repartition of X chromosomes per cell in both BCL groups (*, P = 0.0023). Error bars represent mean ± SD.
- Figure 6.
Abexinostat targets the CSC population in PDX with low Xist expression. A, tumor growth kinetic of PDX treated with docetaxel, abexinostat, or placebo (arrow indicates beginning of treatment) is presented for CRCM311X (Xistlow), CRCM226X (Xistlow), CRCM392X (Xistmed), and CRCM389X (Xisthigh; n = 4). B, evaluation of the ALDEFLUOR-positive population in all four PDXs after 3 weeks of treatment with abexinostat or docetaxel. Results are represented normalized with the proportion of ALDEFLUOR-positive cells in the placebo-treated tumors (n = 4; *, P < 0.05). C, three-week treated PDXs were reimplanted into new mice and tumor growth was monitored. Tumor cells isolated from abexinostat-treated tumors were unable to regenerate a tumor for CRCM311X (n = 8; *, P = 0.02) and CRCM226X (n = 4; *, P = 0.02) PDX compared with the cells isolated from placebo- or docetaxel-treated PDXs. Error bars represent mean ± SD.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supporting Information - PDF file 68K, Supplementary materials and methods
- Supplementary Figure 8 - PDF file 35K, Expression level of Xist in patient-derived xenograft
- Supplementary Table 1 - PDF file 44K, Clinical and pathological features of the breast cancer cell lines (BCLs)
- Supplementary Table 2 - PDF file 43K, Phenotype of BCLs after abexinostat treatment
- Supplementary Table 3 - PDF file 54K, Karyotypes of BCLs
- Supplementary Figure Legend - PDF file 86K, supplementary figure legend
- Supplementary Figure 1 - PDF file 166K, Identification of two drug-response groups using a Gaussian mixture model (GMM)
- Supplementary Figure 2 - PDF file 192K, HDACi treatments define two drug-response profiles in BCLs
- Supplementary Figure 3 - PDF file 109K, Conventional molecular parameters do not predict drug-response to abexinostat
- Supplementary Figure 4 - PDF file 123K, HDAC activity is impaired by treatment independently of drugresponse profiles
- Supplementary Figure 5 - PDF file 164K, Cell cycle progression was differentially altered by abexinostat according to drug-response profile
- Supplementary Figure 6 - PDF file 85K, Histone deacetylase inhibitors target the CSC population in lowdose sensitive BCLs
- Supplementary Figure 7 - PDF file 126K, The effect of abexinostat on CSC is achieved through the specific inhibition of HDACs
Volume 19, Issue 23
