Long non-coding RNA GRASLND affects melanoma phenotypic switch and modulates immunogenicity by inhibition of the IFN? signaling pathway (GRASLND KD).

Melanoma is a highly malignant tumor, that stands as the most lethal form of skin cancer and is characterized by notable phenotypic plasticity and intratumoral heterogeneity. Melanoma plasticity is involved in tumor growth, metastasis and therapy resistance. Long non-coding RNAs (lncRNAs) could influence plasticity due to their regulatory function. However, their role and mode of action are poorly studied. Here, we show a relevance of lncRNA GRASLND in melanoma phenotypic switch, IFN? signaling and immunogenicity. GRASLND knockdown revealed switching towards a dedifferentiated, slow-proliferating and highly-invasive cell state. Interestingly, GRASLND is overexpressed in differentiated melanomas and associated with poor prognosis. Accordingly, we found GRASLND expressed in immunological “cold” tumors and it negatively correlates with gene signatures of immune response activation. In line, silencing of GRASLND under IFN? enhanced the expression of IFN?-stimulated genes, including HLA-I antigen presentation, demonstrating suppressive activity of GRASLND on IFN? signaling. Based on our findings, we hypothesize an adaptive resistance mechanism of melanoma cells evading the immune system via inhibition of IFN? signaling by GRASLND overexpression and highlight its value as a negative prognostic factor for melanoma. Overall design: To investigate effects of GRASLND knockdown on 501Mel melanoma cells, we generated dox-inducible shRNA knockdown cell lines with one control shRNA (lacZ) and 2 GRASLND-targeting shRNAs (sh4 and sh8)

黑色素瘤（Melanoma）是一类高度恶性肿瘤，为皮肤癌中致死性最强的亚型，其核心特征为表型可塑性（phenotypic plasticity）与肿瘤内异质性（intratumoral heterogeneity）。黑色素瘤可塑性与肿瘤生长、转移及治疗耐药紧密相关。长链非编码RNA（long non-coding RNAs, lncRNAs）可通过其调控功能影响肿瘤可塑性，但目前对其在黑色素瘤中的功能与作用机制研究尚不充分。
本研究揭示了长链非编码RNA GRASLND与黑色素瘤表型转换、干扰素γ（IFN-γ）信号通路及免疫原性的关联。敲低GRASLND可促使细胞向去分化、低增殖且高侵袭的细胞状态转化。值得注意的是，GRASLND在分化型黑色素瘤中呈高表达，且与患者不良预后显著相关。进一步研究发现，GRASLND在免疫"冷"肿瘤中高表达，并与免疫应答激活的基因特征呈负相关。此外，在干扰素γ处理条件下敲低GRASLND可上调干扰素刺激基因的表达，包括HLA-I抗原提呈相关基因，证实GRASLND对干扰素γ信号通路具有抑制活性。
基于上述研究结果，我们提出假说：黑色素瘤细胞可通过过表达GRASLND抑制干扰素γ信号通路，从而逃逸免疫系统，这是一种适应性耐药机制；同时本研究也阐明了GRASLND可作为黑色素瘤的不良预后标志物。
实验整体设计：为探究敲低GRASLND对501Mel黑色素瘤细胞的影响，我们构建了多西环素（dox）诱导的shRNA敲低细胞系，设置1个对照shRNA（lacZ）与2条靶向GRASLND的shRNA（sh4与sh8）。