APLP2 regulates the expression of MHC class I molecules on irradiated Ewing's sarcoma cells

Abstract

Ewing's sarcoma (EWS) is a pediatric cancer that is conventionally treated by surgery, chemotherapy, and radiation therapy. Innovative immunotherapies to treat EWS are currently under development. Unfortunately for EWS patients, when the disease is found to be resistant to current therapeutic approaches, the prognosis is predictably grim. Radiation therapy and immunotherapy could potentially synergize in the eradication of EWS, as some studies have previously shown that irradiation increases the presence of immune receptors, including MHC class I molecules, on the surface of tumor cells. However, EWS cells have been reported to express low levels of MHC class I molecules, a phenotype that would inhibit T-cell mediated lysis. We have previously demonstrated that the transgene-driven overexpression of amyloid β (A4) precursor-like protein 2 (APLP2) reduces the expression of MHC class I molecules on the surface of human cervical carcinoma HeLa cells. We thus examined whether endogenously expressed APLP2 downregulates MHC class I expression on EWS cells, particularly upon irradiation. We found that irradiation induces the relocalization of APLP2 and MHC class I molecules on the surface of EWS cells, redistributing cells from subpopulations with relatively low APLP2 and high MHC class I into subpopulations with relatively high APLP2 and low MHC class I surface expression. Consistent with these findings, the transfection of an APLP2-targeting siRNA into EWS cells increased MHC class I expression on the cell surface. Furthermore, APLP2 was found by co-immunoprecipitation to bind to MHC class I molecules. Taken together, these findings suggest that APLP2 inhibits MHC class I expression on the surface of irradiated EWS cells by a mechanism that involves APLP2/MHC class I interactions. Thus, therapeutic strategies that limit APLP2 expression may boost the ability of T cells to recognize and eradicate EWS in patients.

Keywords: Ewing’s sarcoma; HLA; MHC class I molecule; amyloid β (A4) precursor-like protein 2; immune evasion; immunotherapy; pediatric cancer; radiation therapy.

Figure 1. APLP2 is associated with MHC class I molecules in EWS cells. The W6/32 antibody was used to immunoprecipitate folded, MHC class I molecules from TC71 and A673 cell lysates. As a control, the procedure was also performed with no immunoprecipitating antibody (lanes labeled as “Protein A-Sepharose”) and with an irrelevant IgG (isotype control) antibody (anti-CD3 antibody, lanes labeled as “Control Ab”). Immunoprecipitates were then probed by immunoblotting with the HC10 antibody against the MHC class I heavy chain (MHC-I HC) and APLP2. The results shown are representative of 6 separate experiments.

Figure 2. APLP2 is expressed on the surface of EWS cells and can be redistributed by ionizing radiation. (A-E) Radiation shifts EWS cells toward subpopulations with high levels of surface-exposed APLP2, but does not increase total APLP2 expression. EWS cells were irradiated with the indicated dose of γ rays and cultured for 24 h prior to analysis. (A) Representative histograms of surface APLP2 expression on TC71 (left) and A673 (right) cells 24 h post-irradiation as determined by flow cytometry. (B) The mean frequencies of EWS cells with APLP2^low (box), APLP2^int (triangle) and APLP2^high (diamond) phenotypes are shown with respect to radiation intensity. Error bars denote SD (n = 6 experimental replicates within 1 experiment that yielded results representative of several experiments (2 experiments including both TC71 and A673, 2 with A673 but not TC71, and 3 with TC71 but not A673). (C–E) Irradiated TC71 and A673 cells were lysed, and the lysate supernatants were subjected to immunoblotting to determine total expression levels of APLP2 (C and D). Actin levels were monitored to ensure equal lane loading in C and D. Separate experiments were performed to demonstrate that there is no decrease in the expression of another loading control, HSC70, in TC71 and A673 cells exposed to 25 Gy (E). Immunoblotting data are representative of n = 2-3 independent experiments yielding similar results.

Figure 3. The APLP2^high phenotype identifies EWS cells with consistently relatively low MHC class I surface expression upon irradiation. (A-B) EWS cells exposed to the indicated doses of γ rays were examined by flow cytometry for the co-expression of APLP2 and folded MHC class I molecules (using the W6/32 antibody) on their surface. Three cell subpopulations were identified: APLP2^highMHCI^low, APLP2^intMHCI^high, and APLP2^lowMHCI^high cells. MHC class I-dependent fluorescence (in terms of mean fluorescence units, MFU) is depicted for each subpopulation of TC71 (A) and A673 (B) cells. Error bars denote SD (n = 6 experimental replicates within 1 representative experiment out of 3 independent ones performed that yielded similar results, with 1 experiment including both TC71 and A673, and 2 experiments including 1 of the 2 cell lines). Representative dot plots can be found in Figure S3.

Figure 4. APLP2 downregulation increases MHC class I surface expression on EWS cells. (A-B) TC71 and A673 EWS cells were transfected with a siRNA pool targeting APLP2 or a non-targeting siRNA pool (as a negative control) for 48 h. (A) APLP2 downregulation upon transfection with APLP2-specific siRNA was confirmed by immunoblotting. Actin levels were monitored to ensure proper lane loading. (B) Surface-exposed MHC class I molecules were detected by flow cytometry on TC71 (black bars) and A673 (white bars) cells transfected with APLP2-specific or control siRNAs. The pan-human MHC-reactive W6/32 antibody and the allotype-specific BB7.2 antibody were used to detect MHC class I molecules and HLA-A2 molecules, respectively. The bar graphs depict the percent change (means ± SD) in MHC class I-dependent fluorescence (in terms of mean fluorescence units, MFU) elicited with APLP2-specific siRNAs (with cells transfected with control siRNA represented by 100%, as indicated by the dashed line). Individual MFU_siAPLP2/MFU_siControl results were obtained from paired samples, and data from 3 independent experiments were pooled. For each cell line and each antibody, siAPLP2 transfection (compared to siControl transfection) resulted in a significantly higher surface MHC class I level. Statistical significance was determined by means of unpaired Student t-test (*p < 0.05). The values used for derivation of this graph are presented in Table S1.