Identification of FDA-approved drugs targeting breast cancer stem cells along with biomarkers of sensitivity

Abstract

Recently developed genomics-based tools are allowing repositioning of Food and Drug Administration (FDA)-approved drugs as cancer treatments, which were employed to identify drugs that target cancer stem cells (CSCs) of breast cancer. Gene expression datasets of CSCs from six studies were subjected to connectivity map to identify drugs that may ameliorate gene expression patterns unique to CSCs. All-trans retinoic acid (ATRA) was negatively connected with gene expression in CSCs. ATRA reduced mammosphere-forming ability of a subset of breast cancer cells, which correlated with induction of apoptosis, reduced expression of SOX2 but elevated expression of its antagonist CDX2. SOX2/CDX2 ratio had prognostic relevance in CSC-enriched breast cancers. K-ras mutant breast cancer cell line enriched for CSCs was resistant to ATRA, which was reversed by MAP kinase inhibitors. Thus, ATRA alone or in combination can be tested for efficacy using SOX2, CDX2, and K-ras mutation/MAPK activation status as biomarkers of response.

Figure 1. ATRA reverses CD44+/CD24+ phenotype of Ful-R cells.

(A) Parental MCF-7, tamoxifen-resistant (OHTR), and Fulvestrant-resistant (Ful-R) variants were treated with ethanol or ATRA (1 μM) for 72 hours and subjected to flow cytometry as indicated. Percentage of CD44+/CD24+ Ful-R cells changed from 46 ± 3 to 13 ± 0.5 upon ATRA treatment (p = 0.003). (B) Morphological changes in Ful-R cells upon long-term exposure to ATRA. Cells were passaged for one month with or without ATRA treatment and photographed. (C) Cell surface CD44 and CD24 expression status of long-term ATRA treated Ful-R cells. (D) The effects of ATRA on CD271+ CSCs. MCF-7, OHTR, and Ful-R cells were treated with vehicle or ATRA (1 μM) for 72 hours and cell surface expression of CD271 was measured by flow cytometry. Representative isotype control (purple), untreated (green), and ATRA treated cell (pink) histograms from three experiments are shown.

Figure 2. The effects of ATRA on mammospheres.

(A) MCF-7, OHTR, and Ful-R cells were treated with ethanol or ATRA for 72 hours under adherent growth conditions and then subjected to mammosphere assay with or without ATRA. Mammospheres were photographed after 7 days. Size of mammospheres is indicated (n > 3). (B) Mammosphere formation in presence of methylcellulose. (C) The effect of ATRA on growth of Ful-R cells in 2D culture. Cells were treated with ATRA for 72 hours and BrdU-incorporation ELISA was used to measure cell proliferation. (D) Differences in secondary mammosphere formation by Ful-R cells with or without ATRA. Mammospheres were filtered through 40-micron filter and stained with Wright-Giemsa. (E) Cell cycle analysis of Ful-R cells-derived mammospheres grown with or without ATRA for 7 days. (F) Apoptotic cells in mammospheres with or without ATRA for 7 days (n = 3, Average ± SD, p < 0.002, untreated versus ATRA treated). (G) MDA-MB-231, TMD-231, and MDA-MB-436 cells-derived primary/secondary mammospheres with or without ATRA treatment. (H) Secondary and tertiary mammospheres from MDA-MB-436 cells were visualized using Wright-Giemsa stain. ATRA substantially reduced the size of secondary and tertiary mammospheres. (I) The effect of ATRA on proliferation of MDA-MB-436 cells in 2D culture. Cell proliferation was measured using BrDU-incorporation ELISA.

Figure 3. The effects of ATRA on the expression of CSC-associated genes.

(A) ATRA reduced EGFR protein in all but MDA-MB-231 cells. Experiments were conducted in identical condition and cropped blots are shown. Full-length gels are shown in supplementary Figure 1 (Figure S1). (B) The effect of ATRA on mRNA levels of EGFR, SERPINE1, SOX2, and SLUG in parental MCF-7, OHTR, and Ful-R cells. Differences in the basal expression of these genes amongst three cell types are shown (top left). The effect of ATRA on gene expression is presented after normalizing basal expression to one in each cell type. Cells were treated with ATRA (1 μM) for indicated time and mRNA levels were measured by qRT-PCR (n = 3). *p < 0.001, untreated versus ATRA treated. (C) The effects of ATRA on SERPINE1, SOX2, and SLUG expression in MDA-MB-231 and MDA-MB-436 cells. ATRA reduced SOX2 expression but not other genes in MDA-MB-436 cells. *p < 0.01 untreated versus treated. (D) MDA-MB-231 cells express lower levels of SOX2 compared to MDA-MB-436 cells.

Figure 4. Prognostic relevance of combined expression of ATRA-repressible genes EGFR, SERPINE1, SLUG, and SOX2.

Recurrence-free and metastasis-free survival of patients with ER-negative (A and B) or basal type (C and D) breast cancer expressing high (red) or low (black) levels of EGFR, SERPINE1, SLUG and SOX2.

Figure 5. SOX2/CDX2 ratio has prognostic implications.

(A) ATRA induced CDX2 expression in Ful-R cells. qRT-PCR was used to measure CDX2 levels. (B) SOX2 and CDX2 mRNA levels in adjoining normal breast, ER-positive and ER-negative breast cancer. Oncomine database was used to analyze the TCGA dataset. (C–E) Prognostic relevance of SOX2/CDX2 ratio in breast cancer. NKI dataset was analyzed for SOX2/CDX2 ratio.

Figure 6. PD03925901 (Selumetinib) increased sensitivity of MDA-MB-231 cells to ATRA.

(A) K-ras mutation confers resistance to ATRA. ATRA IC50 values for K-ras mutant and wild type cell lines are indicated. (B) Drug sensitivity of K-ras mutant cell lines. K-ras mutant cell lines are sensitive to drugs shown in green, which include AZ628 and PD03925901. These K-ras mutant cell lines are resistant to several other drugs, which are shown in red. (C) PD03925901 with or without ATRA has minimum effect on proliferation of MDA-MB-231 cells in 2D culture. (D) PD03925901 (0.5 μM) with ATRA (1 μM) inhibited mammosphere formation.