Loss of p14 reduces immunogenicity in melanoma by affecting the peptide surface density via non-canonical Wnt signaling

Immunotherapy has achieved tremendous success in melanoma. However, only around 50 % of advanced melanoma patients benefit from immunotherapy with immunogenicity of melanomas playing a key role. CDKN2A encoding the two tumor-suppressor proteins p14ARF and p16INK4a belongs to the most frequently inactivated gene loci in advanced melanoma where decreased T cell infiltration is accompanied. While the role of p16INK4a has been aberrantly investigated, knowledge about the function of p14ARF in melanoma therapy is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished the recognition and killing by melanoma differentiation antigen (MDA)-specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. While antigen presentation via HLA-I molecules was enhanced upon p14ARF downregulation in general, absolute and relative expression of cognate peptides was decreased. Limiting WNT5A signaling reverted this phenotype suggesting an involvement of non-canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA-specific T cell responses by decreasing both absolute and relative MDA-peptide presentation in melanoma. To investigate the influence of p14 and p16, melanoma cell line MaMel51 was knocked down for p14, p16 or both of them with respective doxycyclin-inducible shRNAs or a control scramble (scr) shRNA. Three biological triplicates were analyzed for each condition.

免疫治疗 (immunotherapy) 在黑色素瘤 (melanoma) 领域已取得显著进展。然而仅约50%的晚期黑色素瘤患者可从免疫治疗中获益，而黑色素瘤的免疫原性 (immunogenicity) 在此过程中发挥核心调控作用。CDKN2A编码p14ARF与p16INK4a两种抑癌蛋白，是晚期黑色素瘤中最易发生失活的基因位点之一，此类失活常伴随肿瘤微环境中T细胞浸润水平降低。尽管针对p16INK4a的功能已有大量研究，但关于p14ARF在黑色素瘤治疗中的作用，目前相关认知仍较为匮乏。本研究旨在阐明p14ARF表达下调对黑色素瘤免疫原性的影响。实验结果显示，在黑色素瘤细胞系中敲低p14ARF的表达，会削弱黑色素瘤分化抗原 (melanoma differentiation antigen, MDA) 特异性T细胞对肿瘤细胞的识别与杀伤效应。该耐药表型的产生，源于肿瘤细胞表面呈递的MDA肽段密度降低。尽管总体而言，p14ARF表达下调会增强HLA-I (human leukocyte antigen class I) 分子介导的抗原呈递过程，但同源肽段的绝对与相对表达水平均出现显著下降。抑制WNT5A信号通路可逆转该表型，提示非经典Wnt信号通路参与了这一调控过程。综上，本研究数据揭示了一种全新的免疫逃逸机制：通过降低黑色素瘤中MDA肽段呈递的绝对与相对水平，从而抑制MDA特异性T细胞的抗肿瘤应答。为探究p14与p16的功能影响，本研究使用分别靶向p14、p16或同时靶向二者的多西环素 (doxycyclin) 诱导型短发卡RNA (short hairpin RNA, shRNA)，以及乱序对照 (scramble, scr) shRNA，对黑色素瘤细胞系MaMel51进行基因敲低处理。每种处理条件均设置3次生物学重复，并完成相关检测分析。