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An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers

View Article: PubMed Central - PubMed

ABSTRACT

Epithelial–mesenchymal transition (EMT), a crucial mechanism in development, mediates aggressiveness during carcinoma progression and therapeutic refractoriness. The reversibility of EMT makes it an attractive strategy in designing novel therapeutic approaches. Therefore, drug discovery pipelines for EMT reversal are in need to discover emerging classes of compounds. Here, we outline a pre-clinical drug screening platform for EMT reversal that consists of three phases of drug discovery and validation. From the Phase 1 epithelial marker promoter induction (EpI) screen on a library consisting of compounds being approved by Food and Drug Administration (FDA), Vorinostat (SAHA), a histone deacetylase inhibitor (HDACi), is identified to exert EMT reversal effects by restoring the expression of an epithelial marker, E-cadherin. An expanded screen on 41 HDACi further identifies 28 compounds, such as class I-specific HDACi Mocetinosat, Entinostat and CI994, to restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells. Mocetinostat is the most potent HDACi to restore epithelial differentiation with the lowest concentration required for 50% induction of epithelial promoter activity (EpIC-50).The HDACi exerts paradoxical effects on EMT transcriptional factors such as SNAI and ZEB family and the effects are context-dependent in epithelial- and mesenchymal-like cells. In vitro functional studies further show that HDACi induced significant increase in anoikis and decrease in spheroid formation in ovarian and bladder carcinoma cells with mesenchymal features. This study demonstrates a robust drug screening pipeline for the discovery of compounds capable of restoring epithelial differentiation that lead to significant functional lethality.

No MeSH data available.


Effects of HDACi on in vitro anchorage independence growth. (a) Graph represents the percentage of SKOV3 (dark gray) and T24 (light gray) Annexin V-positive (% Annexin V positive) cells (y-axis) in suspension culture treated with 500 nM and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994) after 48 h of incubation. (b) Graph represents the percentage of OVCA429_shLuc (dark gray) and OVCA429_shGRHL2 (light gray) Annexin V-positive (% Annexin V positive) cells (y-axis) in suspension culture treated with 500 nM and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994) after 48 h of incubation. Representative flow cytometry scatter plots of Annexin V (x-axis) and PI (y-axis) channels. (c) Number of spheroids formed per 1000 cells (y-axis) in SKOV3 (dark gray) and T24 cells (light gray) after day 7 treated with 500 nm and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994). (d) Phase contrast image of SKOV3 and T24 cells treated with 5 μM concentration of HDACi in suspension culture at day 7. All the experiments were performed as three independent experiments, *P<0.05, **P<0.01, ***P<0.001.
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fig6: Effects of HDACi on in vitro anchorage independence growth. (a) Graph represents the percentage of SKOV3 (dark gray) and T24 (light gray) Annexin V-positive (% Annexin V positive) cells (y-axis) in suspension culture treated with 500 nM and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994) after 48 h of incubation. (b) Graph represents the percentage of OVCA429_shLuc (dark gray) and OVCA429_shGRHL2 (light gray) Annexin V-positive (% Annexin V positive) cells (y-axis) in suspension culture treated with 500 nM and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994) after 48 h of incubation. Representative flow cytometry scatter plots of Annexin V (x-axis) and PI (y-axis) channels. (c) Number of spheroids formed per 1000 cells (y-axis) in SKOV3 (dark gray) and T24 cells (light gray) after day 7 treated with 500 nm and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994). (d) Phase contrast image of SKOV3 and T24 cells treated with 5 μM concentration of HDACi in suspension culture at day 7. All the experiments were performed as three independent experiments, *P<0.05, **P<0.01, ***P<0.001.

Mentions: EMT reversal and restoration of E-cadherin expression are associated with increased anoikis.8,9,23 We subsequently utilized the ovarian carcinoma SKOV3 and bladder carcinoma T24 lines for functional studies to test if these class I HDACi affect the anoikis resistance in vitro. The percentage of the Annexin V-positive populations at 48 h in ultra-low attachment cultures was applied as the indication of cells entering apoptosis. The HDACi-treated T24 cells showed significant increase in the cell fractions entering the early anoikis phase (Annexin Vhigh/PIlow populations; Figure 6a). This anoikis-inducing effect followed dose dependency of HDACi whereas increased percentage of Annexin V-positive cells was found in the 5 μM compared with the 500 nM treated group (Figure 6a; Supplementary Figure 3). In the isogenic EMT model, similar trends were also evident in the mesenchymal OVCA429 shGRHL2 cells that all three class I HDACi significantly enhanced anoikis (Figure 6b). Of note, the epithelial OVCA429 shLUC line consistently showed higher anoikis fraction compared with the mesenchymal OVCA429 shGRHL2 line. In addition, the HDACi-treated cancer cells showed decreased spheroid forming efficiencies. Consistent with the anoikis-inducing effect, Mocetinostat-treated cells showed a significant reduction of spheroid formation at 500 nM in both SKOV3 and T24 (Figure 6c). The effect was even more prominent at 5 μM of Mocetinostat and Entinostat (Figures 6c and d). In conclusion, the HDACi abolished the anchorage independence growth and overcome the anoikis resistance of mesenchymal-like cancer cells while restoring the epithelial differentiation.


An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers
Effects of HDACi on in vitro anchorage independence growth. (a) Graph represents the percentage of SKOV3 (dark gray) and T24 (light gray) Annexin V-positive (% Annexin V positive) cells (y-axis) in suspension culture treated with 500 nM and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994) after 48 h of incubation. (b) Graph represents the percentage of OVCA429_shLuc (dark gray) and OVCA429_shGRHL2 (light gray) Annexin V-positive (% Annexin V positive) cells (y-axis) in suspension culture treated with 500 nM and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994) after 48 h of incubation. Representative flow cytometry scatter plots of Annexin V (x-axis) and PI (y-axis) channels. (c) Number of spheroids formed per 1000 cells (y-axis) in SKOV3 (dark gray) and T24 cells (light gray) after day 7 treated with 500 nm and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994). (d) Phase contrast image of SKOV3 and T24 cells treated with 5 μM concentration of HDACi in suspension culture at day 7. All the experiments were performed as three independent experiments, *P<0.05, **P<0.01, ***P<0.001.
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Related In: Results  -  Collection

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fig6: Effects of HDACi on in vitro anchorage independence growth. (a) Graph represents the percentage of SKOV3 (dark gray) and T24 (light gray) Annexin V-positive (% Annexin V positive) cells (y-axis) in suspension culture treated with 500 nM and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994) after 48 h of incubation. (b) Graph represents the percentage of OVCA429_shLuc (dark gray) and OVCA429_shGRHL2 (light gray) Annexin V-positive (% Annexin V positive) cells (y-axis) in suspension culture treated with 500 nM and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994) after 48 h of incubation. Representative flow cytometry scatter plots of Annexin V (x-axis) and PI (y-axis) channels. (c) Number of spheroids formed per 1000 cells (y-axis) in SKOV3 (dark gray) and T24 cells (light gray) after day 7 treated with 500 nm and 5 μM concentration of HDACi (x-axis, SAHA, Mocetinostat, Entinostat and CI994). (d) Phase contrast image of SKOV3 and T24 cells treated with 5 μM concentration of HDACi in suspension culture at day 7. All the experiments were performed as three independent experiments, *P<0.05, **P<0.01, ***P<0.001.
Mentions: EMT reversal and restoration of E-cadherin expression are associated with increased anoikis.8,9,23 We subsequently utilized the ovarian carcinoma SKOV3 and bladder carcinoma T24 lines for functional studies to test if these class I HDACi affect the anoikis resistance in vitro. The percentage of the Annexin V-positive populations at 48 h in ultra-low attachment cultures was applied as the indication of cells entering apoptosis. The HDACi-treated T24 cells showed significant increase in the cell fractions entering the early anoikis phase (Annexin Vhigh/PIlow populations; Figure 6a). This anoikis-inducing effect followed dose dependency of HDACi whereas increased percentage of Annexin V-positive cells was found in the 5 μM compared with the 500 nM treated group (Figure 6a; Supplementary Figure 3). In the isogenic EMT model, similar trends were also evident in the mesenchymal OVCA429 shGRHL2 cells that all three class I HDACi significantly enhanced anoikis (Figure 6b). Of note, the epithelial OVCA429 shLUC line consistently showed higher anoikis fraction compared with the mesenchymal OVCA429 shGRHL2 line. In addition, the HDACi-treated cancer cells showed decreased spheroid forming efficiencies. Consistent with the anoikis-inducing effect, Mocetinostat-treated cells showed a significant reduction of spheroid formation at 500 nM in both SKOV3 and T24 (Figure 6c). The effect was even more prominent at 5 μM of Mocetinostat and Entinostat (Figures 6c and d). In conclusion, the HDACi abolished the anchorage independence growth and overcome the anoikis resistance of mesenchymal-like cancer cells while restoring the epithelial differentiation.

View Article: PubMed Central - PubMed

ABSTRACT

Epithelial&ndash;mesenchymal transition (EMT), a crucial mechanism in development, mediates aggressiveness during carcinoma progression and therapeutic refractoriness. The reversibility of EMT makes it an attractive strategy in designing novel therapeutic approaches. Therefore, drug discovery pipelines for EMT reversal are in need to discover emerging classes of compounds. Here, we outline a pre-clinical drug screening platform for EMT reversal that consists of three phases of drug discovery and validation. From the Phase 1 epithelial marker promoter induction (EpI) screen on a library consisting of compounds being approved by Food and Drug Administration (FDA), Vorinostat (SAHA), a histone deacetylase inhibitor (HDACi), is identified to exert EMT reversal effects by restoring the expression of an epithelial marker, E-cadherin. An expanded screen on 41 HDACi further identifies 28 compounds, such as class I-specific HDACi Mocetinosat, Entinostat and CI994, to restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells. Mocetinostat is the most potent HDACi to restore epithelial differentiation with the lowest concentration required for 50% induction of epithelial promoter activity (EpIC-50).The HDACi exerts paradoxical effects on EMT transcriptional factors such as SNAI and ZEB family and the effects are context-dependent in epithelial- and mesenchymal-like cells. In vitro functional studies further show that HDACi induced significant increase in anoikis and decrease in spheroid formation in ovarian and bladder carcinoma cells with mesenchymal features. This study demonstrates a robust drug screening pipeline for the discovery of compounds capable of restoring epithelial differentiation that lead to significant functional lethality.

No MeSH data available.