Partial Truncation of the C-Terminal Domain of PTCH1 in Cancer Enhances Autophagy and Metabolic Adaptability.
Partial Truncation of the C-Terminal Domain of PTCH1 in Cancer Enhances Autophagy and Metabolic Adaptability.
复制标题
PTCH1中C末端结构域的部分截断可增强自噬和代谢适应性。
作者：
我们最近报道了肿瘤抑制因子PTCH 1（刺猬蛋白受体）的胞浆C末端结构域（CTD）与自噬相关蛋白ATG 101相互作用并损害自噬通量。在这项研究中，我们确定了相互作用区域，并发现它在结直肠癌，胃癌和子宫内膜癌的子集中不存在。我们证明，截短突变体缺乏约束自噬的能力，导致增殖优势和降低的敏感性，自噬诱导剂或糖酵解抑制剂的细胞系携带内源性PTCH 1 CTD突变相比，表达野生型PTCH 1的同基因细胞。总之，这项研究强调了PTCH 1-ATG 101相互作用在调节癌细胞的基础和刺激自噬以及代谢灵活性中的重要性。Hedgehog受体Patched 1（PTCH 1）是一种众所周知的肿瘤抑制因子。虽然肿瘤抑制因子的活性主要归因于其作为经典Smoothened/Gli途径的阻遏物的功能，但据报道其C-末端结构域（CTD）具有额外的非经典功能。其中之一是通过与Unc-51直接相互作用减少自噬通量，如自噬激活激酶（ULK）复合物亚基自噬相关蛋白-101（ATG 101）。为了研究PTCH 1的这种功能在癌细胞适应性中是否重要，我们首先在癌症数据库中鉴定了PTCH 1 CTD中的移码突变。我们证明了这些突变破坏了PTCH 1与ATG 101的相互作用，并增加了自噬通量。在免疫共沉淀研究中使用PTCH 1 CTD的缺失突变体，我们确定了1309-1447区域对于与ATG 101的相互作用是必要且充分的。我们接下来发现，在子宫内膜、胃和结肠腺癌中导致S1203、R1308和Y1316移码的三种最常见的PTCH 1 CTD突变缺乏与ATG 101相互作用并限制自噬通量的能力，这是由自噬标志物LC 3BII和p62的巴弗洛霉素A1敏感性积累确定的。我们接下来通过CRISPR/Cas9在SW 620结肠癌细胞中在S1223处工程化PTCH 1插入缺失突变。比较两个独立的克隆窝藏PTCH 1 S1223 fs突变的同基因亲本细胞系表达野生型PTCH 1显示了显着增加的基础和雷帕霉素刺激的自噬通量，预测损失的ATG 101相互作用。此外，PTCH 1 CTD突变体细胞在雷帕霉素存在下表现出增殖增加，对糖酵解抑制剂的敏感性降低。我们的研究结果表明，PTCH 1 CTD突变导致的PTCH 1-ATG 101相互作用的丧失可能通过刺激自噬和促进对营养剥夺条件的适应而赋予选择性优势。
We have recently reported that the cytosolic C-terminal domain (CTD) of the tumour suppressor PTCH1, the Hedgehog proteins receptor, interacts with the autophagy-related protein ATG101 and impairs autophagic flux. In this study, we identified the interaction region and found that it is absent in a subset of colorectal, stomach and endometrial cancers. We demonstrated that truncation mutants lack the ability to constraint autophagy, resulting in a proliferative advantage and reduced sensitivity to autophagy inducers or glycolysis inhibitors in cell lines harbouring endogenous PTCH1 CTD mutations compared to isogenic cells expressing wild-type PTCH1. In summary, this study highlights the importance of the PTCH1-ATG101 interaction in the regulation of basal and stimulated autophagy and the metabolic flexibility in cancer cells. The Hedgehog receptor, Patched1 (PTCH1), is a well-known tumour suppressor. While the tumour suppressor’s activity is mostly ascribed to its function as a repressor of the canonical Smoothened/Gli pathway, its C-terminal domain (CTD) was reported to have additional non-canonical functions. One of them is the reduction of autophagic flux through direct interaction with the Unc-51, like the autophagy activating kinase (ULK) complex subunit autophagy-related protein-101 (ATG101). With the aim of investigating whether this function of PTCH1 is important in cancer cell fitness, we first identified frameshift mutations in the CTD of PTCH1 in cancer databases. We demonstrated that those mutations disrupt PTCH1 interaction with ATG101 and increase autophagic flux. Using deletion mutants of the PTCH1 CTD in co-immunoprecipitation studies, we established that the 1309–1447 region is necessary and sufficient for interaction with ATG101. We next showed that the three most common PTCH1 CTD mutations in endometrial, stomach and colon adenocarcinomas that cause frameshifts at S1203, R1308 and Y1316 lack the ability to interact with ATG101 and limit autophagic flux, determined by bafilomycin A1-sensitive accumulation of the autophagy markers LC3BII and p62. We next engineered PTCH1 indel mutations at S1223 by CRISPR/Cas9 in SW620 colon cancer cells. Comparison of two independent clones harbouring PTCH1 S1223fs mutations to their isogenic parental cell lines expressing wild-type PTCH1 showed a significant increase in basal and rapamycin-stimulated autophagic flux, as predicted by loss of ATG101 interaction. Furthermore, the PTCH1 CTD mutant cells displayed increased proliferation in the presence of rapamycin and reduced sensitivity to glycolysis inhibitors. Our findings suggest that loss of the PTCH1-ATG101 interaction by mutations in the CTD of PTCH1 in cancer might confer a selective advantage by stimulating autophagy and facilitating adaptation to nutrient deprivation conditions.
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影响因子：
5.6
作者：
Yoshii SR;Mizushima N
通讯作者：
Mizushima N
DOI：
10.1038/nrc3262
发表时间：
2012-04-26
期刊：
Nature reviews. Cancer
影响因子：
--
作者：
通讯作者：
--
影响因子：
5.3
作者：
Chen, Xiaole L.;Chinchilla, Pilar;Riobo, Natalia A.
通讯作者：
Riobo, Natalia A.
影响因子：
64.8
作者：
Taipale, J;Beachy, PA
通讯作者：
Beachy, PA
影响因子：
--
作者：
Brennan, Donna;Chen, Xiaole;Cheng, Lan;Mahoney, My;Riobo, Natalia A.
通讯作者：
Riobo, Natalia A.