HDAC inhibitor entinostat restores responsiveness of letrozole-resistant MCF-7Ca xenografts to aromatase inhibitors through modulation of Her-2

Abstract

We previously showed that in innately resistant tumors, silencing of the estrogen receptor (ER) could be reversed by treatment with a histone deacetylase (HDAC) inhibitor, entinostat. Tumors were then responsive to aromatase inhibitor (AI) letrozole. Here, we investigated whether ER in the acquired letrozole-resistant tumors could be restored with entinostat. Ovariectomized athymic mice were inoculated with MCF-7Ca cells, supplemented with androstenedione (Δ(4)A), the aromatizable substrate. When the tumors reached about 300 mm(3), the mice were treated with letrozole. After initial response to letrozole, the tumors eventually became resistant (doubled their initial volume). The mice then were grouped to receive letrozole, exemestane (250 μg/d), entinostat (50 μg/d), or the combination of entinostat with letrozole or exemestane for 26 weeks. The growth rates of tumors of mice treated with the combination of entinostat with letrozole or exemestane were significantly slower than with the single agent (P < 0.05). Analysis of the letrozole-resistant tumors showed entinostat increased ERα expression and aromatase activity but downregulated Her-2, p-Her-2, p-MAPK, and p-Akt. However, the mechanism of action of entinostat in reversing acquired resistance did not involve epigenetic silencing but rather included posttranslational as well as transcriptional modulation of Her-2. Entinostat treatment reduced the association of the Her-2 protein with HSP-90, possibly by reducing the stability of Her-2 protein. In addition, entinostat also reduced Her-2 mRNA levels and its stability. Our results suggest that the HDAC inhibitor may reverse letrozole resistance in cells and tumors by modulating Her-2 expression and activity.

Figure 1

Figure 1A: Effect of ENT alone or in combination with letrozole or exemestane on the growth of letrozole resistant MCF-7Ca xenografts: Ovariectomized athymic nude mice were inoculated with MCF-7Ca cells. Tumors were allowed to form in the presence of androstenedione (Δ⁴A), aromatizable substrate for estrogen. When the tumors reached ~300mm³, mice were treated with letrozole (10μg/day) for total 15 weeks, during this time the tumors regressed, but eventually began to grow. When the tumors had reached double the initial volume, they were randomized again into 5 groups; letrozole continued, exemestane (250μg/day), ENT (50μg/day) or the combination of ENT with letrozole or exemestane. The mice were treated till week 26. The growth rates of tumors of mice treated with the combination of ENT with letrozole or exemestane was significantly better than single agent alone (p=0.0009 ENT vs ENT+letrozole; p=0.048 ENT vs ENT+exemestane; p<0.0001 ENT vs ENT+letrozole)). Figure 1B: Effect of ENT alone or in combination with letrozole or exemestane on the tumor protein expression in letrozole resistant MCF-7Ca xenografts: Protein expression in the tumors was examined by western immunoblotting as described in “materials and methods”. Blots were probed for β-actin to verify equal loading. The numbers below the blots show densitometric values that are corrected for loading. Figure 1C: Effect of ENT alone or in combination with letrozole or exemestane on the tumor aromatase activity in letrozole resistant MCF-7Ca xenografts: The aromatase activity in the homogenized tumors was analyzed as described in “materials and methods”. Tumors of mice treated with letrozole for 15 weeks followed by ENT (in presence of Δ⁴A) have significantly higher aromatase activity than the letrozole treated tumors (*_a p<0.01) and control tumors (*_b p<0.05).

Figure 2. ChIP analysis of LTLT-Ca cells to examine the activation of ERα, pS2 and aromatase (PI.3/II) promoter

Activation of ERα and aromatase promoter was measured by immunoprecipitating chromatin bound acetylated histone H₃. Blot shows conventional PCR analysis and the numbers below the blot show real-time qPCR analysis.

Figure 3

Western blotting analysis of (A) MCF-7Ca and (B) LTLT-Ca cells treated with entinostat: MCF-7Ca and LTLT-Ca cells were treated with ENT + letrozole. Cycloheximide (5μM) treatment was added to measure protein half-life. Figure 3C: RT-PCR analysis of MCF-7Ca and LTLT-Ca cells treated with entinostat in presence or absence of E₂, Δ⁴A and letrozole: MCF-7Ca and LTLT-Ca cells were treated with ENT (1μM) in presence or absence of E₂ (1nM), Δ⁴A (25nM) or Δ⁴A+ letrozole (1μM). Changes in the mRNA levels were measured with real-time qRT-PCR. Numbers are corrected for the expression of housekeeping gene (18s ribosomal RNA) and expressed as fold change over control (fixed as 1).

Figure 4

Figure 4A: Western blotting analysis of LTLT-Ca cells treated with entinostat alone or in presence of MG-132 (proteosomal inhibitor) or NH₄Cl (lysosomal inhibitor): LTLT-Ca cells were treated with ENT (1μM) alone or in presence of MG-132 (5μM) or NH₄Cl (100μM) or both. Protein expression in the cells was examined by western immunoblotting as described in “materials and methods”. Blots were probed for β-actin to verify equal loading. The numbers below the blots show densitometric values that are corrected for loading. Figure 4B: Western blotting analysis of SKBr3 and BT-474 cells treated with entinostat: Her-2 positive SKBr3 and BT-474 cells were treated with ENT (1μM). Protein expression in the cells was examined by western immunoblotting as described in “materials and methods”. Blots were probed for β-actin to verify equal loading. The numbers below the blots show densitometric values that are corrected for loading. Figure 4C: Western blotting analysis of AnR and ExR cells treated with entinostat: Her-2 positive anastrozole resistant (AnR) and exemestane resistant (ExR) cells were treated with ENT (1μM). Protein expression in the cells was examined by western immunoblotting as described in “materials and methods”. Blots were probed for β-actin to verify equal loading. The numbers below the blots show densitometric values that are corrected for loading.

Figure 5

Figure 5A: Co-immunoprecipitation of Her-2 and hsp-90 in MCF-7Ca xenografts: Tumor lysates (500μg) of MCF-7Ca xenografts (from fig 1) were immunoprecipitated with either anti-Her-2 or anti-hsp90 antibody. The blots were then probed for hsp90 or Her-2 to measure association between the two proteins. Blots were probed for IP antibody to verify equal loading. The numbers below the blots show densitometric values that are corrected for loading. Figure 5B: Co-immunoprecipitation of Her-2 and hsp-90 in MCF-7Ca and LTLT-Ca cells: Lysates (500μg) of MCF-7Ca and LTLT-Ca cells were immunoprecipitated with either anti-Her-2 or anti-hsp90 antibody. The blots were then probed for hsp90 or Her-2 to measure association between the two proteins. Blots were probed for IP antibody to verify equal loading. The numbers below the blots show densitometric values that are corrected for loading. Figure 5C: Western blotting analysis of MCF-7Ca and LTLT-Ca cells: Relative levels of Her-2 and hsp90 proteins in MCF-7Ca and LTLT-Ca cells were detected by western blotting and measured by densitometry. Blots were probed for β-actin to verify equal loading. The numbers below the blots show densitometric values that are corrected for loading. Figure 5D: Western blotting analysis of AnR and ExR cells: Relative levels of Her-2 and hsp90 proteins in AnR and ExR cells were detected by western blotting and measured by densitometry. Blots were probed for β-actin to verify equal loading. The numbers below the blots show densitometric values that are corrected for loading.

Figure 6

Figure 6A: RT-PCR analysis of Her-2 mRNA levels in LTLT-Ca cells: Relative levels of Her-2 mRNA after treatment with ENT were analyzed by real-time RT-qPCR. Cells were treated with ENT (1μM) alone or in presence of actinomycin D (5μM) and cells were collected at indicated time points. RNA was isolated, quantified and diluted to 0.08μg/μl. Total 0.64Mg of RNA was reverse transcribed and Her-2 was amplified using real-time qPCR as described in Materials and Methods. Graph represents relative levels (±SEM) of Her-2 mRNA compared to LTLT-Ca control (set at 1). Figure 6B, C: RT-PCR analysis of Her-2, HDAC1, ERα and 18s mRNA levels in LTLT-Ca cells: Relative levels of Her-2, HDAC1, ERα and 18s mRNA after treatment with ENT, or siRNA against HDAC1 were analyzed by real-time RT-qPCR. Cells were treated with ENT (1μM) or siRNAs against HDAC1 (5nM) or mock siRNA. The cells were collected after 48-hour incubation at 37°C. RNA was isolated, quantified and diluted to 0.08μg/μl. Total 0.64Mg of RNA was reverse transcribed and cDNA was amplified using real-time qPCR as described in Materials and Methods. Graph represents relative levels (±SEM) of (B) Her-2 and HDAC1 mRNA and (C) ERα and 18s mRNA. The relative mRNA levels (±SEM) are reported compared to LTLT-Ca control (set at 1). P-values: *p=0.0024, ‡p=0.0021, †p<0.0001 versus control. NS: not significant.

Figure 7. Effect of ENT alone or in combination with letrozole on the growth of MCF-7Ca xenografts

Ovariectomized athymic nude mice were inoculated with MCF-7Ca cells. Tumors were allowed to form in the presence of androstenedione (Δ⁴A), aromatizable substrate for estrogen. When the tumors reached ~300mm³, mice were treated with either letrozole (10μg/day), ENT (50μg/day) or the combination of letrozole and ENT for 20 weeks, The growth rates of tumors of mice treated with the combination of ENT plus letrozole was not significantly different than letrozole alone (p=0.28).