ABT-263 induces apoptosis and synergizes with chemotherapy by targeting stemness pathways in esophageal cancer

Abstract

Activation of cancer stem cell signaling is central to acquired resistance to therapy in esophageal cancer (EC). ABT-263, a potent Bcl-2 family inhibitor, is active against many tumor types. However, effect of ABT-263 on EC cells and their resistant counterparts are unknown. Here we report that ABT-263 inhibited cell proliferation and induced apoptosis in human EC cells and their chemo-resistant counterparts. The combination of ABT-263 with 5-FU had synergistic lethal effects and amplified apoptosis that does not depend fully on its inhibition of BCL-2 family proteins in EC cells. To further explore the novel mechanisms of ABT-263, proteomic array (RPPAs) were performed and gene set enriched analysis demonstrated that ABT-263 suppresses the expression of many oncogenes including genes that govern stemness pathways. Immunoblotting and immunofluorescence further confirmed reduction in protein expression and transcription in Wnt/β-catenin and YAP/SOX9 axes. Furthermore, ABT263 strongly suppresses cancer stem cell properties in EC cells and the combination of ABT-263 and 5-FU significantly reduced tumor growth in vivo and suppresses the expression of stemness genes. Thus, our findings demonstrated a novel mechanism of ABT-263 antitumor effect in EC and indicating that combination of ABT-263 with cytotoxic drugs is worthy of pursuit in patients with EC.

Keywords: 5-fluorouracil; ABT-263; cancer stem cells; esophageal cancer; stemness pathways.

Figure 1. ABT-263 potently inhibit EC cell growth and synergizes with 5-FU on both sensitive and resistant EC cells

A. & B. Four EAC cell lines (left panel) and two ESCC cell lines (right panel) were treated with 0.1% DMSO (as control) or ABT-263 at different dosage as indicated for 5 days, cell growth inhibition was measured using MTS assay and calculated as percent of control. C. Four EC cell lines treated with 5-FU at different dosage and in combination with ABT263 at 0.1 μM and 1 μM for 3 days and cell growth inhibition was measured using MTS assay. D. SK4 cells and their resistant cells SK4-Rf were treated with 5-FU at 10 μM and ABT-263 at 1 μM either alone or in combination for 3 days, cell growth inhibition was measured using MTS assay. E. YES-6 cells and their resistant cells YES-6-Rf were treated with 5-FU at 10 μM and ABT-263 at 1 μM either alone or in combination for three days, cell growth inhibition was measured using MTS assay. **p < 0.01.

Figure 2. ABT-263 propels the arrested S-phase cells induced by 5-FU into apoptosis

A. The SKGT-4, KATO-TN and YES-6 cells were seeded onto 6-well plates and treated with 0.1% DMSO (as control) or with ABT-263 1 μM or 5-FU 10 μM or in combination for 48 hours and then fixed and stained for DNA with propidium iodide and then analyzed for DNA histograms and cell cycle phase distribution by flow-cytometry using a FACSCalibur instrument, which showed that 5-FU induced the cells arrested in S-phase and ABT-263 induced the cells arrested in the sub-G1-phase, but the combination resulted in significantly increasing cells in sub-G1 phase. B. The cell cycle distribution of SKGT-4, YES-6 and KATO-TN cells were demonstrated in bar graphs according to the proportion of their Sub-G1, G0G1, S and G2M phase after the treatment.

Figure 3. ABT-263 strongly induce apoptosis especially in combination with 5-FU in EC cells

A. SKGT-4, Yes-6 and KATO-TN cells were treated with 0.1% DMSO (as control) or ABT-263 1 μM or 5-FU 10 μM or in combination and determined the apoptosis index by flow cytometry, which indicated that the apoptosis index were increased, especially in combination treatment cells. B. and C. Apoptosis associated proteins-PARP, Cleaved PARP and antiapoptotic proteins MCL-1, BCL-2 and BCL-XL were detected using immunoblotting at SKGT-4 and JHESO EC cells treated with 5-FU and ABT263 or in their combination as dosage indicated (Top panel); quantification of BCL-2 expression in SKGT-4 and JHESO EC cells treated with 5-FU and ABT263 or in their combination as dosage indicated was performed using Image J (Lower panel).

Figure 4. Gene set enriched analysis of RPPA proteomic data on ABT-263 treated JHESO cells and the effects of ABT-263 on cell survival and stemness pathways

A. Gene set enriched analysis (GSEA) conducted by a specialized bioinformatist (B.L) demonstrated that many genes involving oncogenic (EGFR, PI3K/mTOR) and cancer stemness signaling are down-regulated in ABT-263 (1 μM for 48 hours) treated JHESO cells (Figure 4A). B. Down-regulation of genes in PI3K/mTOR and survival and stem cell signaling after normalization by RPPA analysis. C. Up-regulation of Genes by ABT-263 in pro-apoptosis and tumor suppression after normalization by RPPA analysis. D. Significantly down-regulation of YAP1, β-catenin, c-MYC and MCL-1 by ABT-263 was confirmed using quantitative real-time-PCR. **p < 0.01.

Figure 5. ABT-263 strongly inhibits expression and activation of Wnt/β-catenin and YAP1/SOX9 axes in EC cells

A. Protein levels of YAP, SOX9, β-catenin and its target Cyclin D1 were determined by immunoblotting in EC cells treated with different dosage of ABT263 for 48 hours. B. Protein levels of YAP, β-catenin and its target Cyclin D1 were detected by immunoblotting in SKGT-4 EC cells treated with different dosage of ABT263 and or in combination with 5-FU for 48 hours. C. expression level of YAP1, β-catenin and Cyclin D1 was detected by immunofluorescence as described in materials & Methods. D. Transient transfection of Super-TOP luciferase (represent Wnt/β-catenin activity) or YAP1 or SOX9 luciferase promoters into SKGT-4 EC cells and treated with ABT263 for 48 hours in different dosage as indicated; Luciferase reporter activities were detected after 48 hours. For all experiments, values shown represent the mean and SD of at least triplicate assays (**p < 0.01).

Figure 6. ABT-263 strongly inhibits tumor-sphere formation in both ALDH1+ and ALDH1- EA cells and ABT-263 in combination with 5-FU significantly inhibit ALDH1 positive and induced YAP1 high cell growth

A. & B. ALDH1 positive or negative cells were sorted from JHESO EC cells and tumor sphere assays were done in the sorted cells and add ABT-263 at 1 μM at the beginning of the tumor sphere culture. After 8–10 days of culture, the tumor sphere numbers formed were counted under microscope. Representative fields (A) and the bar graph (B) are demonstrated that ALDH1+ cells formed larger and more tumor spheres than ALDH1- cells, and ABT-263 inhibited the tumor sphere formation in both ALDH1+ and ALDH- cells, but preferentially in the former. C. & D. JHESO cells were treated with ABT-263 at 1 μM or control for 48 hours and then labeling with ALDH1 antibody that showed ABT-263 reduced the fraction of ALDH1+ cells in the JHESO cells. E. ALDH1 positive or negative cells were sorted from JHESO EC cells were treated with 5-FU and ABT-263 either alone or in combination at the concentration indicated for six days, cell growth inhibition was measured using MTS assay. **p < 0.01. F. SKGT-4 (PIN20YAP) cells with (DOX+) or without (DOX-) YAP induction by doxycycline and treated with 5-FU and ABT-263 either alone or in combination at the concentration indicated for six days, cell growth inhibition was measured using MTS assay. **p < 0.01.

Figure 7. ABT263 in combination with 5-FU strongly inhibit EC tumor growth and suppress expression of stemness genes (YAP1/SOX9) in vivo

JHESO cells (1.5 × 10⁶) were injected subcutaneously in nude mice, each mouse have two sites (left, right) injections; 5 mice/group and treated with either ABT263 alone, 5-FU alone or in combination as described in Materials & Methods. Tumor Volume A. tumor weight B. and mouse body weight in each group D. were measured and calculated as described in Materials & Methods. Representative tumors C. from each group after 4 weeks are shown. Each point represents mean tumor volume/weight and SD from five mice. E. Immunohistochemistry for YAP1, SOX9 and Ki67 was performed in mouse tumor tissues derived from JHESO xenograft nude mice. F. Proposed model by which effects of ABT263 on EC cell growth and resistance by targeting stemness pathways and oncogenic signaling in addition to its canonical function on BCL-2 inhibition.