Epigenetic targeting drugs potentiate chemotherapeutic effects in solid tumor therapy

Abstract

Epigenetic therapy is a novel tumor therapeutic method and refers to the targeting of the aberrant epigenetic modifications presumably at cancer-related genes by chemicals which are epigenetic targeting drugs (ETDs). Not like in treating hematopoietic cancer, the clinical trials investigating the potential use of ETDs in the solid tumor is not encouraging. Instead, the curative effects of ETD delivered together with DNA targeting chemo drugs (DTDs) are quite promising according to our meta-analysis. To investigate the synergistic mechanism of ETD and DTD drug combination, the therapeutic effect was studied using both cell lines and mouse engrafted tumors. Mechanically we show that HDAC inhibitors and DNMT inhibitors are capable of increasing the chromatin accessibility to cisplatin (CP) and doxorubicin (Dox) through chromatin decompaction globally. Consequently, the combination of ETD and DTD enhances the DTD induced DNA damage and cell death. Engrafted tumors in SCID mice also show increased sensitivity to irradiation (IR) or CP when the tumors were pretreated by ETDs. Given the limited therapeutic effect of ETD alone, these results strongly suggest that the combination of DTD, including irradiation, and ETD treatment is a very promising choice in clinical solid tumor therapy.

Figure 1

Epigenetic targeting drugs application in clinical. (A) Global gene expression from 18 primary AML samples in response to DEC treatment were collected (GSE40442) and the portion of consensus upregulated TSGs or Oncogenes, relative to total TSGs or oncogenes are plotted. P value is based on Chi-square test. (B) IC 50 of hematopoietic (HM) versus other cell lines to HDAC inhibitors. Each dot represents the IC 50 of one cell line to one drug as labeled on the top. The left group is the HM cell lines and the right group is the collection of all the other cell lines except HM cell lines. (C) The response rate of ETD alone applied to 1110 patients with solid tumors. CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. Response identification based on Response Evaluation Criteria in Solid Tumors (RECIST). (D) The response rate of classic chemical drugs applied to solid tumors according to TCGA. (E) Forest plot of objective response rate (ORR) related to DTD combined with ETD. The overall effect was statistically significant (P < 0.0001). Vertical line, “no-difference” point between two regimens; Horizontal line, 99% CI; Square, mean difference; Diamond, pooled mean difference for all studies. M-H fixed = Mantel-Haenszel fixed model. An odds ratio less than 1 means that combined therapy has successfully inhibited tumor progression.

Figure 2

ETD Increases the sensitivity to DTD induced apoptosis. (A,B and C), Skov3 cell viability when cells were exposed to 30 uM CP combined with 0 uM, 4 uM, 8 uM or 12 uM SAHA for 24 h, 0 uM, 1 uM, 2 uM or 4 uM DEC for 24 h, or 0 uM, 0.2 uM, 2 uM or 20 uM 2-DG for 36 h. (D and E) Skov3 cell viability when cells were exposed to 5 ug/ml DOX combined with 0 nM, 2.5 nM, 5 nM or 10 nM Rom for 24 h or 0 uM, 1 uM, 2 uM or 4 uM DEC for 24 h. (F) A549 cell viability when cells were exposed to 30 uM or 3 uM CP combined with 0 uM, 0.5 uM, 1 uM DEC for 24 h. (G) MCF7 cell viability when cells were exposed to 30 uM or 3 uM CP combined with 0 nM, 5 nM or 10 nM Rom for 24 h. (H) A549 cell viability when cells were exposed to 30 uM or 3 uM CP combined with 0 nM, 5 nM or 10 nM Rom for 24 h. (I and J), MCF7 cell viability when cells were exposed to 30 uM CP combined with 0 uM, 1 uM, 2 uM or 4 uM DEC for 24 h or 0 mM, 0.2 mM, 2 mM or 20 mM 2-DG for 36 h. (* means P < 0.05, ** means P < 0.001, n ≥ 3.)

Figure 3

ETD increases DNA damage induced by DTD. (A) γH2AX immunofluorescence staining when cells were exposed to 30 uM CP alone or combining with 2.5 nM Rom, 1 uM Dec or 2 mM 2-DG in SKOV3 cells. (B) γH2AX immunofluorescence staining when SKOV3 cells were exposed to 10 Gy IR alone or combining with 2.5 nM Rom, 1 uM Dec or 2 mM 2-DG. (C and D) Cropped western blots show γH2AX expression level when SKOV3 cells or MCF-7 cells were treated with 30 uM CP alone or combing with 2 mM 2-DG, 1 uM Dec, or 2.5 nM Rom. Uncropped images are in Supplementary information. (E) γH2AX immunofluorescence staining when MCF-7 cells were exposed to 30 uM CP alone or combining with 2.5 nM Rom, 1 uM Dec or 2 mM 2-DG. (F) γH2AX immunofluorescence staining when MCF-7 cells exposed to 10 Gy irradiation alone or combining with 2.5 nM Rom, 1 uM Dec or 2 mM 2-DG. (G and H) Western blotting detection of phosphorylated p53 (p-p53) and phosphorylated ATM upon treatment of MCF-7 (G) and SKOV3 (H) by Rom (left) or DEC (right). GAPDH serves as an endogenous control. 2 time scale (12 H, 12 hours treatment; 24 H, 24 hours treatment) and 2 dosage (Rom, 4 nM and 8 nM; DEC, 2 uM and 4 uM) were applied.

Figure 4

ETD increases DTD accessibilities in chromosome. (A) Dot blot analysis of CP-DNA adduct when SKOV3 cells were treated with 2-DG, SAHA, Rom or DEC with indicated concentration for 6 h, or SKOV3 cells were treated with a combination of CP 30 uM 4 h and 2-DG, SAHA, Rom or Dec with indicated concentration for another 6 h. (B) Flow cytometer detection of DOX autofluorescence in nuclei from cells treated with Dox alone or combined with 2-DG, DEC, SAHA or Rom. X axis indicates the 488/580 Dox fluorescence value, Y axis indicates SSC.

Figure 5

Epigenetic drugs affect global chromosome compaction. (A) Nuclear chromatin compaction heterogeneity by DAPI staining (top) in SKOV3 cells treated with ethanol, 2 mM 2-DG, 1 uM DEC or 2.5 nm Rom for 12 h. The bottom chart is the quantitation of the chromatin compaction heterogeneity. (B) Cropped western blots show acetyl-H3 expression and chromatin bound HP1α level when SKOV3 cells were treated with ethanol, 2 mM 2-DG, 4 uM SAHA, 2.5 nM Rom or 1 uM DEC. Uncropped western blots are in Supplementary information. (C) SKOV3 cells were stably transfected with GFP-H2B plasmid, then cells were treated with ethanol, 10 uM 2-DG, 1 uM DEC or 2.5 nM Rom for 12 h. 3D-reconstruction shows the H2B distributions in nucleus representing the chromatin 3D conformation. (D) MNase assay shows mononucleosome bound DNA quantity when cells treated with ethanol, 2-DG, SAHA, Rom and DEC for 0 min, 5 min, 10 min, and 15 min.

Figure 6

ETD promotes DTD therapeutic effect in engrafted tumors. (A) Growth curves of xenograft tumors in mice administrated with solvent (control), IR and SAHA combined with IR, (N = 12). (B) Growth curves of xenograft tumors in mice treated with solvent (control), IR and DEC combined with IR (N = 12). (C) Tumors are shown after being dissociated from mice with indicated conditions. (D) Tumor growth curves of xenograft tumors in mice treated with solvent (control), CP and DEC combined with CP, N = 12. (E) Growth curves of xenograft tumors in mice treated with solvent (control), CP and SAHA combined with CP (N = 12). (F) Tumors are shown after being dissociated from mice with indicated conditions. (G) The box plots show the mean tumor weights ± SEM (N ≥ 10) with indicated treatments including CP, CP combined with DEC or SAHA. (H) Representative caspase-3 histochemical staining of xenograft tumors treated with solvent (control), CP, SAHA combined with CP and DEC combined with CP. (I) Western blotting detection of acetylated H3, gH2AX, BAX, PARP and cleaved-PARP upon the cells are treated by CP/IR, or CP/IR plus SAHA/DEC, using tubulin as endogenous control, in mouse engrafted tumors.