Homeoprotein SIX1 compromises anti-tumor immunity through TGF-β-mediated regulation of collagens. Homeoprotein SIX1 compromises anti-tumor immunity through TGF-β-mediated regulation of collagens

Purpose: The tumor microenvironment (TME), such as non-immune-infiltrated “cold” tumors, has been associated with immune suppression. However, the complex mechanisms regulating tumor immunogenicity have not been clearly elucidated. In this study, we investigated whether Transcription factor sine oculis homeobox 1 (SIX1) expression in the cancer cells regulated anti-tumor immunity by reshaping TME. Methods: The expression of SIX1 in human tumor tissues was analyzed based on TCGA data. Six1 knockout murine cell lines were generated to analyze the SIX1-associated tumor progression and anti-tumor immune response in tumor xenograft models. Immune cells and cytokines in TME were quantified by immunofluorescence, flow cytometry, quantitative reverse transcription-PCR, and ELISPOT assay. SIX1-associated differentially expressed genes were investigated in cancer cells and tissues with RNA-Seq. At the end, Correlation of SIX1 with TGFBR2 expression was assessed by Western blot and Dual-luciferase reporter assay. Results:SIX1 was highly upregulated in human tumor tissues, while rarely expressed in matched normal tissues. Deletion of the Six1 in cancer cells inhibited tumor growth, depending on adaptive immunity. Moreover, Six1 deficiency significantly enhanced CD8+ T cells infiltration, leading to eradication of poorly immunogenic tumors and maintaining a long-term protection from tumor re-challenge. Mechanistically, SIX1 as a transcription factor upregulated TGFBR2 expression, which induced collagen genes expression via the Smad2/3 phosphorylation, increased collagens deposition in TME and hampered CD8+ T cells infiltration. Conclusions: Six1 deletion in cancer cells improved tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncovered a crucial role of SIX1 on remodulating tumor immunogenicity and demonstrated a proof of concept for targeting SIX1 in cancer immunotherapy. Overall design: mRNA profiles of MCA205 wild type (WT) and SIX1-/- cell lines or tumor tissue.

研究背景与目的：肿瘤微环境（tumor microenvironment, TME），例如非免疫浸润的“冷”肿瘤，已被证实与免疫抑制密切相关。然而，调控肿瘤免疫原性的复杂机制尚未得到清晰阐明。本研究旨在探讨肿瘤细胞中的转录因子sine oculis homeobox 1（SIX1）是否可通过重塑肿瘤微环境来调控抗肿瘤免疫。
研究方法：基于癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据，分析人类肿瘤组织中SIX1的表达水平。构建Six1基因敲除的小鼠细胞系，以在肿瘤异种移植模型中分析与SIX1相关的肿瘤进展及抗肿瘤免疫应答。采用免疫荧光法、流式细胞术、定量反转录PCR及酶联免疫斑点（ELISPOT）检测，对肿瘤微环境中的免疫细胞与细胞因子进行定量分析。通过RNA测序（RNA-Seq）探究肿瘤细胞与组织中与SIX1相关的差异表达基因。最后，通过蛋白质印迹（Western blot）与双荧光素酶报告基因检测，评估SIX1与转化生长因子β受体2（TGFBR2）表达的相关性。
研究结果：SIX1在人类肿瘤组织中呈高表达，而在配对的正常组织中几乎不表达。肿瘤细胞中Six1基因敲除可抑制肿瘤生长，且该过程依赖于适应性免疫。此外，Six1基因缺失可显著增强CD8+ T细胞浸润，从而清除免疫原性较弱的肿瘤，并维持长期的抗肿瘤复发保护作用。从机制上而言，作为转录因子的SIX1可上调TGFBR2的表达，后者通过Smad2/3磷酸化诱导胶原基因表达，增加肿瘤微环境中的胶原沉积，并阻碍CD8+ T细胞浸润。
研究结论：肿瘤细胞中Six1基因缺失可提升肿瘤免疫原性，通过增强抗肿瘤免疫应答实现肿瘤清除。本研究结果揭示了SIX1在重塑肿瘤免疫原性中的关键作用，并为癌症免疫治疗中靶向SIX1的策略提供了概念验证。
整体实验设计：MCA205野生型（WT）与SIX1基因敲除（SIX1-/-）细胞系或肿瘤组织的mRNA表达谱。