Tenascin-C蛋白选择性自噬降解异常促进三阴乳腺癌逃避T细胞免疫攻击及其分子机制

Compared with other forms of breast cancer, triple negative breast cancer (TNBC) is associated with advanced stage at diagnosis, increased risk of poorer outcome and lack of specific targets. Accumulating evidence suggests that TNBC may be the most regulated by intratumoral T cells, and thus the most responsive to immunotherapies. Immune-checkpoint inhibitors have achieved certain effect in the clinical trials, there is still much to do to improve the effect. Although autophagy is a catabolic process that widely involves in the whole process of human cancer, the role of autophagy in cells stimulating or limiting the T cell immune system’s attack on tumor cells has not been well understood.In our preliminary study, we found that protein levels of autophagy-related genes decreased in TNBC breast cancer tissues. Animal experiments showed that autophagy-deficient 4T1 tumors inhibited the number and activity of tumor infiltrating T cells, and decreased the sensitivity to anti-PD1 antibody treatment. Inhibition of autophagic activity in MEF and MDA-MB-231 cells resulted in impaired susceptibility to cytotoxic T-cell-mediated lysis. Through SILAC proteomics analysis, we identified the extracellular matrix protein Tenascin-C (TNC), upregulated in autophagy-deficient cells as a potential immunosuppressive molecule. In this proposal, we will establish the autophagy-deficient TNBC cell models and animal models. Within these models, we will explore the inhibitory effect（including T cell mediated cytotoxicity and proliferation）of activated T cells on tumor cells with different autophagic activity , to further confirm that cancer cells with low autophagic activity is prone to escape T cell immune attack. We will investigate the mechanism that extracellular matrix protein TNC is highly expressed in autophagy-deficient TNBC cells because of abnormality of selective autophagy degradation. We will also explore the mechanisms that how TNC interacts with integrin on the cell surface of T cells and inhibits T-cell activation. More importantly, we will evaluate the synergistic effect of anti-TNC antibody and T cell immune-checkpoint inhibitors for providing new strategies for TNBC treatment.

三阴乳腺癌恶性度大预后差，尽管其免疫原性相对强，但免疫检查点抑制剂临床试验疗效未达到预期。肿瘤细胞自噬如何影响T细胞对其攻击的能力及分子机制尚不清楚。我们发现TNBC中自噬基因低表达且与预后差相关；动物实验显示自噬缺陷的4T1细胞抑制肿瘤浸润性T细胞的数量及活性，且对PD1抗体治疗敏感性降低；细胞实验显示自噬缺陷的TNBC细胞可能更容易逃避T细胞攻击，其分子机制与细胞外基质TNC蛋白相关。本课题拟进一步在自噬缺陷的模型上研究T细胞对TNBC细胞的杀伤作用，对其增殖成瘤的抑制作用，明确自噬能力低是TNBC患者对T细胞免疫治疗耐受的重要原因；通过从选择性自噬降解异常的角度阐明TNC蛋白在自噬缺陷细胞中高表达的分子机制，明确其通过与T细胞表面整合素受体结合来抑制T细胞信号通路，探讨其能否成为TNBC患者免疫治疗的疗效预测指标；明确TNC单抗与免疫检查点抑制剂的联合用药作用，为患者提供新的治疗策略

三阴乳腺癌（TNBC）恶性度大预后差，尽管其免疫原性相对强，但免疫检查点抑制剂临床试验疗效未达到预期。肿瘤细胞自噬如何影响T细胞对其攻击的能力及分子机制尚不清楚。在本项目中我们重点探讨了自噬缺陷对TNBC肿瘤免疫耐受的影响及机制。重要结论及关键数据如下：①明确自噬缺陷的TNBC细胞对T细胞免疫杀伤的耐受现象：我们发现自噬缺陷的TNBC细胞能逃逸T细胞的免疫攻击自噬缺；发现自噬缺陷的4T1肿瘤组织成瘤能力显著增强，浸润性T细胞的数量和活性显著降低，且对anti-PD1 和anti-PDL1治疗耐受；②获得TNC是自噬缺陷介导的肿瘤免疫耐受关键分子的证据：结合SILAC蛋白标记技术筛选出可能受自噬调控的关键免疫抑制分子Tenascin-C(TNC)；发现自噬缺陷细胞的TNC蛋白水平显著上调；敲低TNC回复自噬缺陷细胞对T细胞杀伤的敏感性，且对T细胞的增殖抑制减弱；TNC敲除后，自噬缺陷细胞移植瘤中浸润性T细胞的数量和活性显著降低，肿瘤生长速度显著减缓；③ 阐明E3连接酶Skp2催化TNC发生K63型泛素化，随后在自噬受体p62介导下发生自噬性降解：发现自噬受体蛋白p62通过与TNC蛋白结合，且结合依赖于p62的UBA结构域；证实TNC能发生K63型泛素化修饰，且受自噬调控；干扰Skp2后能使TNC蛋白泛素化水平降低及蛋白水平升高；发现饥饿诱导自噬使野生型TNBC细胞的TNC蛋白水平明显下降，但自噬缺陷的TNBC细胞中TNC降解被阻断,明确Tenascin-C 蛋白在自噬缺陷的三阴乳腺癌细胞中高表达的原因；④ 明确TNC的表达与TNBC患者病程进展和预后的影响：发现TNBC肿瘤组织中TNC高表达是患者不良的独立预后因素；TNC表达与TNBC患者中CD8+ T细胞肿瘤浸润呈显著负相关，与LC3B表达呈负相关；⑤ 明确靶向TNC能增强免疫检查点抑制剂对自噬缺陷肿瘤的疗效：发现TNC单抗与免疫检查点抑制剂联用比单用药能显著增强T细胞对自噬缺陷细胞的杀伤作用；发现诱导性敲低TNC能够增强PD1单抗对自噬缺陷移植瘤的治疗效果。本研究首次阐明TNC是自噬缺陷细胞的关键免疫抑制分子，并创造性地提出通过联合靶向TNC与免疫治疗的治疗以增强自噬缺陷肿瘤的疗效，为TNBC患者提供了具有应用前景的治疗策略。