Entinostat Enhances the Efficacy of Chemotherapy in Small Cell Lung Cancer Through S-phase Arrest and Decreased Base Excision Repair

Abstract

Purpose: Acquired chemoresistance is a frequent event in small cell lung cancer (SCLC), one of the deadliest human malignancies. Histone deacetylase inhibitors (HDACi) have been shown to synergize with different chemotherapeutic agents including cisplatin. Accordingly, we aimed to investigate the dual targeting of HDAC inhibition and chemotherapy in SCLC.

Experimental design: The efficacy of HDACi and chemotherapy in SCLC was investigated both in vitro and in vivo. Synergistic drug interactions were calculated based on the HSA model (Combenefit software). Results from the proteomic analysis were confirmed via ICP-MS, cell-cycle analysis, and comet assays.

Results: Single entinostat- or chemotherapy significantly reduced cell viability in human neuroendocrine SCLC cells. The combination of entinostat with either cisplatin, carboplatin, irinotecan, epirubicin, or etoposide led to strong synergy in a subset of resistant SCLC cells. Combination treatment with entinostat and cisplatin significantly decreased tumor growth in vivo. Proteomic analysis comparing the groups of SCLC cell lines with synergistic and additive response patterns indicated alterations in cell-cycle regulation and DNA damage repair. Cell-cycle analysis revealed that cells exhibiting synergistic drug responses displayed a shift from G1 to S-phase compared with cells showing additive features upon dual treatment. Comet assays demonstrated more DNA damage and decreased base excision repair in SCLC cells more responsive to combination therapy.

Conclusions: In this study, we decipher the molecular processes behind synergistic interactions between chemotherapy and HDAC inhibition. Moreover, we report novel mechanisms to overcome drug resistance in SCLC, which may be relevant to increasing therapeutic success.

Figure 1.

Sensitivity to HDACi is associated with neuroendocrine features and cisplatin responsiveness in SCLC. A, Heat map depicting the percentage of viable cells (right Y-axis) after 72-hour treatment with entinostat at the indicated doses (left Y-axis). A: SCLC-A, P: SCLC-P, N: SCLC-N, Y: SCLC-Y. B, IC₅₀ values for entinostat with respect to molecular subtypes. Each dot represents one cell line. NE: neuroendocrine, NNE: nonneuroendocrine, A: SCLC-A, P: SCLC-P, N: SCLC-N, Y: SCLC-Y. t test, *, P < 0.05. C, Significantly upregulated proteins in Enti^sens (red) and Enti^res (blue) cells. The dotted line indicates P = 0.05. D, Results from 1D annotation enrichment analysis show significantly overrepresented KEGG pathways in Enti^sens (red) and Enti^res (blue) cell lines. E, IC₅₀ values of entinostat and cisplatin. A: SCLC-A, P: SCLC-P, N: SCLC-N, Y: SCLC-Y. F, Spearman correlation of entinostat and cisplatin IC₅₀ values (r = 0.8079, P = 0.0003). Each dot represents one cell line. G, IC₅₀ values were calculated from dose–response curves from cisplatin treatment for 72 hours. A: SCLC-A, P: SCLC-P, N: SCLC-N, Y: SCLC-Y. ANOVA with Dunn multiple comparisons tests; *, P ≤ 0.05.

Figure 2.

Entinostat synergizes with chemotherapy in the chemoresistant subset of SCLC cell lines in vitro. A, Dose–response curves after 72 hours of treatment with entinostat and cisplatin at indicated doses showed synergistic (H841 and H372) and additive (CRL-2177 and COR-L311) effects. Data, mean ± SEM of at least three experiments performed in triplicates. B, Corresponding HSA synergy maps, generated with the Combenefit software. Green areas indicate additive, blue areas highlight synergistic, and red areas show antagonistic drug interactions. C, Maximum synergism score derived from HSA analysis ranking all 16 cell lines from highly synergistic to additive. The red line (max. synergism score: 35) indicates the cutoff between SYN and ADD cells. D, Spearman correlation analysis of the maximum synergism score with entinostat and cisplatin, respectively. *, P ≤ 0.05. E, Pooled results of intracellular Pt measurements via ICP-MS of 2 cell lines per group (SYN: H841, H372; ADD: CRL-2177, H1694). Cells were treated with either cisplatin for 3 hours or pretreated with entinostat for 24 hours followed by 3 hours of cisplatin exposure. Data are shown as mean ± SEM of two experiments performed in triplicates. ANOVA and Sidak's multiple comparisons tests; *, P ≤ 0.05; **, P < 0.01; ***, P< 0.001. F, Pooled maximum synergism scores of entinostat in combination with carboplatin, etoposide, epirubicin, and irinotecan in SYN (blue = H841, H372) and ADD (green = CRL-2177, H1694) cells. Representative synergy maps of two representative cell lines treated with entinostat (Enti) and etoposide (Eto) were created with Combenefit.

Figure 3.

Entinostat and cisplatin synergize in vivo in a double-resistant SCLC cell line. A, Calculated tumor volumes over 30 days of treatment with vehicle (Co), entinostat (Enti), cisplatin (Cis), or a combination of both (Enti + Cis). Green and red arrows indicate days of cisplatin and entinostat treatment, respectively. Data are shown as mean ± SEM of 6–8 mice per group. Multiple t tests with Holm–Sidak correction, ***, P < 0.001 vs. Co, ^###P < 0.001 vs. Enti. B, Tumor weight and (C) amount of intratumoral Pt measured by ICP-MS. ANOVA and Kruskal–Wallis multiple comparisons test in B, t test in C. *, P ≤ 0.05. D, Quantification of TUNEL (green, nuclei: blue), cPARP, and Ki-67 (brown, nuclei: blue) in xenografts. Between 6 and 20 representative images were evaluated. ANOVA and Dunn multiple comparisons test; *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001. E, Representative images of xenografts used for evaluation. Scale bars, TUNEL: 2 mm, cPARP: 50 μm, Ki-67: 25 μm.

Figure 4.

Pathway analysis reveals alteration in cell-cycle and DNA damage repair. A, ToppCluster analysis of the differentially expressed proteins between the top six synergistic and bottom six additive cell lines according to the maximum synergism score shows associated hallmark gene sets within each group (blue: SYN; green: ADD). Gene sets with a P value cutoff of 0.05 using Bonferroni correction are depicted. B, 1D annotation enrichment analysis using KEGG pathways highlighting dysregulated pathways in SYN (blue) or ADD (green) cell lines. This analysis included a strict grouping of the top four synergistic and bottom four additive cell lines according to the maximum synergism score. The dotted line indicates the significance cutoff (FDR = 0.05). C, GSEA based on data from the strict grouping of SYN and ADD groups shows enrichment in DNA repair (ADD), EMT, and apoptosis (SYN). The Y-axis indicates the enrichment score (ES) and the x-axis shows identified genes (vertical black lines) represented in each pathway.

Figure 5.

Combination therapy of entinostat and cisplatin results in enhanced apoptosis in synergistic SCLC cell lines. A, Representative immunoblots of SYN/ADD cell line panels after 24 hours of treatment with entinostat (Enti) or cisplatin (Cis) at indicated doses. Densitometric quantification was performed using ImageJ of at least two repeats. AcH3 was normalized to H3 and the results are shown as the mean ± SEM. B, Flow cytometry-based apoptosis assay using Annexin V and propidium iodide (PI) staining after 48 hours of treatment with 2.5 μmol/L entinostat, 5 μmol/L cisplatin, a combination of both or solvent. Data, mean ± SEM of three repeats performed in triplicate of pooled results of three cell lines per group, respectively (blue, synergistic; green, additive). ANOVA and Sidak's multiple comparisons tests, *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001. C, Representative immunoblots of SYN/ADD cell line panels after 24 hours of treatment with 2.5 μmol/L entinostat or 5 μmol/L cisplatin alone or in combination. cPARP was normalized to PARP, and the results shown are mean ± SEM. The same GAPDH lane is used in A and C; the same lysates were used to study different protein expressions and at least one of the proteins shown in each figure was detected on the same membrane.

Figure 6.

Cell-cycle analysis indicates S-phase arrest and less BER capacity in SCLC cell lines related to synergistic responses upon combination therapy with entinostat and cisplatin. A, Cell-cycle distribution was analyzed via flow cytometry after 24 hours of treatment with respective IC₅₀ values of each cell line (calculated after 72 hours). Experiments were performed in triplicate and data are shown as mean ± SEM of pooled results of three cell lines per group, respectively (blue, synergistic; green, additive). ANOVA and Sidak's multiple comparisons tests; *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001. B, Representative immunoblots of SYN/ADD cell line panels after 24 hours of treatment with 2.5 μmol/L entinostat (Enti) or 5 μmol/L cisplatin (Cis) alone or in combination. Densitometric quantification was performed using ImageJ of at least 2 repeats. γH2AX was normalized to GAPDH and is shown as the mean ± SEM. The GAPDH lane is the same as in Fig. 5A and 5C; the same lysates were used to study different protein expressions and at least one of the proteins shown in each figure was detected on the same membrane. C, Standard comet assay of SYN and ADD cell lines (N = 3, respectively) determined baseline DNA-damage levels, and data, mean ± SEM of at least two independent experiments. Mann–Whitney test; *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001. D, Repair enzyme comet assay of pooled results (ADD or SYN cell lines, N = 3, respectively) elucidating the NER or BER capacity. Data, mean ± SEM of at least two independent experiments. Mann–Whitney test; *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001.