The MAPK Pathway Is a Predominant Regulator of HLA-A Expression in Esophageal and Gastric Cancer

Inhibition of MAPK signaling upregulates HLA-A expression in ESCC. Eight ESCC cell lines were treated with PD98059, wortmannin, lapatinib, or DMSO as a negative control for 48 h. (A and B) The expression of total HLA class I (W6/32 or G46-26 mAbs) and HLA-A02 or HLA-A24 was assessed by flow cytometry. Representative graphs are shown. (C) TE4, TE1, and KYSE30 cells were treated with wortmannin, PD98059, lapatinib, or DMSO as a negative control for 48 h. The expression of HLA-A02 or HLA-A24 was assessed by flow cytometry. *p < 0.05, **p < 0.01, PD98059 versus DMSO.

Total HLA class I and HLA-A expression of gastric cancer cell lines is regulated by the MAPK inhibitor. Six gastric cancer cell lines were treated with PD98059 (50 μM) or DMSO for 48 h. The expression of total HLA class I and HLA-A molecules after PD98059 treatment was assessed by flow cytometry. Clone W6/32 and clone G46-2.6 were used for measuring the expression of total HLA class I. *p < 0.05, **p < 0.01, PD98059 versus DMSO.

Inhibition of MAPK signaling upregulates total HLA class I and HLA-A expression in gastric cancer cell lines. MKN7 and OE19 cells were treated with the indicated doses of PD98059 and lapatinib for 48 h. (A and B) The effects of PD98059 and lapatinib on HER2-related signals were assessed by Western blot. (C and D) The effects of PD98059 and lapatinib on the expression of total HLA class I and HLA-A were assessed by flow cytometry. Clone W6/32 and clone G46-2.6 were used for measuring the expression of total HLA class I. *p < 0.05, **p < 0.01, PD98059 versus DMSO.

Expression of APM components upon MAPK signal inhibition. (A) KYSE30 and MKN7 cells were treated with 10 μM (PD-10) or 50 μM (PD-50) of PD98059 or DMSO for 48 h, and mRNA levels of APM components were evaluated by quantitative PCR. The results are expressed as fold change in expression relative to the DMSO-treated cells as negative controls. Relative quantification was done by normalizing to the average of β-actin, GAPDH, PPIA, and HPRT levels. (B) KYSE30 and MKN7 cells were treated with 10 μM (PD-10) or 50 μM (PD-50) of PD98059 or DMSO (PD-0) for 48 h and protein levels of APM components were evaluated by Western blot. *p < 0.05, **p < 0.01, PD98059 versus DMSO. β2-M, β2-microglobulin; HC, HLA class I H chains; LMP, low m.w. proteins; PA28, proteasome activators-28; PSX, the constitutive proteasome subunit X, PSY, the constitutive proteasome subunit Y; PSZ, the constitutive proteasome subunit Z; TAP, transporter associated with Ag processing.

Erk1/2-silencing and HLA-A expression. KYSE30 cells were treated with Erk1/2 siRNA in combination with the MAPK inhibitors PD98059 and PD0325901. (A) The levels of total Erk1/2 (Erk) and p-Erk1/2 (p-Erk) were assessed by Western blot after treatment with different combinations of Erk1/2 siRNA (siErk1/2), control siRNA (siCTR), MAPK inhibitors, and DMSO. Western blot data show one of three independent experiments with comparable results. (B) The expression of HLA-A24, HLA-A02, and total HLA-class I (W6/32 and G46-2.6 mAbs) was analyzed by flow cytometry and expressed as relative MFI (rMFI) compared with cells treated with control siRNA + DMSO (siCTR + DMSO). *p < 0.05, **p < 0.01, Erk1/2 siRNA and/or MAPK inhibitors versus siCTR + DMSO.

Inhibition of MAPK signaling results in an enhancement of tumor-specific CTL activity. HLA-A24–restricted, LY6K peptide–specific CTL lines and CTL clones were generated as described in Materials and Methods. (A) TE1 and KYSE30 targets were pretreated with PD98059 (50 or 100 μM) or DMSO as a negative control and cocultured with HLA-A24–restricted, LY6K peptide–specific CTL lines in an ELISPOT assay. (B) The CTL clone against LY6K peptide, rather than the cell line, was used as a responder under experimental conditions identical to those described in (A). (C) TE1 and MKN7 targets were pretreated with PD98059 (50 μM) or DMSO as negative control and cocultured with an HLA-A24–restricted, LY6K peptide–specific CTL clone in a cytotoxic assay, using the indicated E:T ratios, as described in Materials and Methods. (D) MKN7 targets were pretreated with PD98059 (50 μM), lapatinib (1.0 μM), or DMSO as negative control and cocultured with an HLA-A24–restricted, LY6K peptide–specific CTL clone in a cytotoxic assay. *p < 0.05, PD98059 versus DMSO, **p < 0.01, PD98059 versus DMSO.

HLA-A expression in EGFR- and HER3-expressing tumors treated with MAPK inhibitor and ligands. (A and B) The EGFR-overexpressing ESCC cell line KYSE30 was treated with a combination of EGF and PD98059 at the indicated doses. After incubation with EGF, treated cells were exposed to 20 μM (PD-20) or 50 μM (PD-50) of PD98059 or DMSO (PD-0). (A) The status of HER2-signaling molecules was assessed by Western blot. (B) The relative MFI (rMFI) of HLA-A02 and HLA-A24 was assessed by flow cytometry. (C and D) The HER3-expressing ESCC cell line KYSE30 was treated with a combination of NRG-1-β1 and PD98059 at the indicated doses. After incubation with NRG-1-β1, treated cells were exposed to 20 μM (PD-20) or 50 μM (PD-50) of PD98059 or DMSO control. (C) The status of phosphorylation of p44/42 Erk and Akt was assessed in treated KYSE30 cells by Western blot. (D) The rMFI of HLA-A02 and HLA-A24 in treated KYSE30 cells was assessed by flow cytometry. Data from Western blot show one of three independent experiments with comparable results. *p < 0.01, **p < 0.05, PD98059 versus DMSO.