MYC directs transcription of MCL1 and eIF4E genes to control sensitivity of gastric cancer cells toward HDAC inhibitors

Figures & data

Table 1. Response of gastric cancer cell lines to SAHA

Cell line	IC50 (48 h) (μM SAHA)	CI (95%)
MGC8	1.0	0.7–1.3
ST23123	1.4	1.1–1.7
MGC4	1.5	1.2–1.8
HSC45-M2	2.3	1.9–2.8
KatoIII	2.4	1.8–3.3
ST2957	2.8	2.1–3.7
NuGC4	2.9	2.4–3.6
MKN45	3.2	2.6–3.9

Figure 1. BCL_XL restricts SAHA efficacy of gastric cancer cells. (A) western blot analysis of MCL1 and BCL_XL expression of the indicated gastric cancer cell lines. β-actin served as loading control. (B) ST23132 and MKN45 cells were treated with increasing doses SAHA for 24 h as indicated or were left as vehicle treated controls. Western blots detected expression of BCL_XL and β-actin (loading control). (C) ST2957 and MKN45 cells were transfected with the indicated siRNAs. 48 h after the transfection whole-cell lysates were prepared and western blots detected expression of BCL_XL, MCL1 and β-actin (loading control). (D) ST2957 and MKN45 were transfected with the indicated siRNAs. 48 h after the transfection cells were treated with SAHA as indicated for additional 24 h. Viability of cells was measured in MTT assays and viability vehicle treated controls were arbitrary set to 100% in order to compare the SAHA responses within each siRNA condition. Data are presented as mean and standard error of the mean (S.E.M). (Student’s t-test: * p < 0.05 vs. controls).

Figure 2. MCL1 restricts SAHA efficacy of gastric cancer cells. (A) ST23132 and MKN45 cells were treated for 24 h with increasing doses SAHA as indicated or left as vehicle treated controls. Western blots detected expression of MCL1 and β-actin (loading control). (B) ST2957 and MKN45 cells were transfected with the indicated siRNAs. Forty-eight hours after transfection whole-cell lysates were prepared and western blots detected expression of MCL1, BCL_XL and β-actin (loading control). (C) ST2957 and MKN45 cells were transfected with the indicated siRNAs. 48 h after the transfection cells were treated with SAHA as indicated for additional 24 h. Viability of cells was measured in MTT assays and viability of vehicle treated controls were arbitrary set to 100% in order to compare the SAHA responses within each siRNA condition. Data are presented as mean and standard error of the mean (S.E.M). (D) ST2957 and MKN45 cells were transfected with the indicated siRNAs. For MCL1, the MCL1–1 siRNA was used. siRNA amount was kept constant using control siRNA. Forty-eight hours after transfection cells were treated with SAHA for additional 24 h as indicated or were left as untreated controls. Western blots detected expression of MCL1, BCL_XL and β-actin (loading control). (E) ST2957 and MKN45 cells were transfected with the indicated siRNAs. For MCL1, the MCL1–1 siRNA was used. siRNA amount was kept constant using control siRNA. Forty-eight hours after transfection cells were treated with SAHA for additional 24 h as indicated or were left as untreated controls. Cells were stained with Propidium iodide (PI) and FITC-labeled anti-Annexin V. Depicted is the Annexin V positive fraction (early apoptosis = Annexin V⁺/PI^- and late apoptosis = Annexin V⁺/PI⁺). (Student’s t-test: * p < 0.05 vs. controls).

Figure 3. c-MYC controls MCL1 and BCL_XL expression of gastric cancer cells. (A) ST2957 and MKN45 cells were transfected with the indicated siRNAs. 48 h after the transfection whole-cell lysates were prepared and western blots detected expression of c-MYC, BCL_XL, MCL1 and β-actin (loading control). (B) ST2957 and MKN45 cells were transfected with the indicated siRNAs. 48 h after the expression of c-MYC, MCL1 and BCL_XL mRNAs were determined by quantitative RT-PCR using cyclophilinA mRNA as reference. (C) ST2957 and MKN45 cells were transfected with the indicated siRNAs. 48 h after the transfection cells were treated with SAHA as indicated for additional 24 h. Viability of cells was measured in MTT assays and viability of vehicle treated controls were arbitrary set to 100% in order to compare the SAHA responses within each siRNA condition. Data are presented as mean and standard error of the mean (S.E.M). (Student’s t-test: * p < 0.05 vs. controls).

Figure 4. MCL1 is a direct c-MYC target in gastric cancer cells. (A) ST2957 and MKN45 cells were treated with the MYC inhibitor (myc-I) for 24 h. Western blots detected the expression of BCL_XL, MCL1 and β-actin (loading control). (B) ST2957 and MKN45 cells were treated with the MYC inhibitor (myc-I), SAHA or the combination of both for 24 h as indicated or were left as an untreated control. Cells were stained with Propidium iodide (PI) and FITC-labeled anti-Annexin V. Depicted is the Annexin V positive fraction (early apoptosis = Annexin V⁺/PI^- and late apoptosis = Annexin V⁺/PI⁺). (Student’s t-test: * p < 0.05). (C) and (D) ST2957 and MKN45 cells were treated with the MYC inhibitor for 24 h or were left as an vehicle treated control. ChIP analysis revealing the binding of c-MYC (C) or the RNA Polymerase II (D) to the E-box of the MCL1 promoter or an 13 kb 3′ control.

Figure 5. MYC controls eIF4E transcription to regulate BCL_XL expression in gastric cancer cells. (A) ST2957 and MKN45 cells were transfected with the indicated siRNAs. 48 h after the transfection whole-cell lysates were prepared and western blots detected expression of c-MYC, eIF4E and β-actin (loading control). (B) ST2957 and MKN45 cells were transfected with the indicated siRNAs. 48 h after the expression of the eIF4E mRNA were determined by quantitative RT-PCR using cyclophilinA mRNA as reference. (C) ST2957 and MKN45 cells were treated with the MYC inhibitor for 24 h. Western blots detected the expression of eIF4E and β-actin (loading control). (D) and (E) ST2957 and MKN45 cells were treated with the MYC inhibitor for 24 h or were left as an vehicle treated control. ChIP analysis revealing the binding of c-MYC (D) or the RNA Polymerase II (E) to the E-box of the eIF4E promoter or an cyclophilin A 3′ control. (F) ST2957 and MKN45 cells were transfected with the indicated siRNAs. 48 h after the transfection whole-cell lysates were prepared and western blots detected expression of eIF4E, BCL_XL and β-actin (loading control).

Figure 6. MYC expression is regulated by HDACi in gastric cancer cells. (A) ST2957 and MKN45 cells were treated with increasing doses SAHA for 24 h as indicated or were left as vehicle treated controls. Western blots detected expression of c-MYC, MCL1, eIF4E and β-actin (loading control). (B) ST2957 and MKN45 cells were pre-treated with myc-I as indicated or were left as an untreated control. After 24 h cells were again treated with myc-I or with the combination of the myc-I and SAHA for additional 24 h. Untreated cells remained untreated. Viability of cells was measured in MTT assays and viability of vehicle treated controls were arbitrary set to 100%. Data are presented as mean and standard error of the mean (S.E.M). (Student’s t-test: * p < 0.05 vs. controls). (C) Schematic presentation of how gene expression signatures induced by c-MYC restrain the efficacy of HDACi against gastric cancer cells.