Fu Yong, Ding Liang, Yang Xihu, Ding Zhuang, Huang Xiaofeng, Zhang Lei, Chen Sheng, Hu Qingang, Ni Yanhong
Central Laboratory of Stomatology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China.
Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China.
Front Oncol. 2021 Mar 10;11:637226. doi: 10.3389/fonc.2021.637226. eCollection 2021.
Dysregulated amino acids metabolism reciprocally interplays with evolutionary phenotypic characteristics of cancer cells to enhance metastasis. The high metastasis potential of oral squamous cell carcinoma (OSCC) can manifest with perineural invasion (PNI). We here aimed to determine the role of amino acids metabolism in OSCCs with different PNI statuses. Targeted metabolomics was used to quantify 48 amino acids in 20 fresh OSCC samples and 25 amino acids were successfully detected, within which 9 were significantly up-regulated in PNI positive (PNI) samples. As its highest area under the curve value (0.9063), l-asparagine was selected as the biomarker to distinguish PNI from PNI negative (PNI). Then, the key enzyme of l-asparagine, asparagine synthetase (ASNS), was investigated using immunohistochemistry with 86 OSCC patients. The results showed that ASNS mainly expressed in tumor epitheliums and positively correlated with lymph node metastasis and PNI. Moreover, subgroup survival analysis revealed that ASNS expression combined with PNI status significantly improved their prognostic value, which was confirmed by the TCGA OSCC cohort (n = 279). To validate whether ASNS promotes PNI, we determined ASNS expression levels in five OSCC cell lines and one normal oral keratinocyte, and HSC3 showed the lowest ASNS level but CAL33 had the highest. Therefore, HSC3 and CAL33 (or PBS as control) were selected and injected separately into sciatic nerves to construct the PNI mouse models. Although both models eventually developed the hind-limb paralysis, nerve dysfunction in the CAL33 model progressed significantly earlier than HSC3 (Day 9 vs. Day 24). Besides, CAL33 migrated significantly farther than HSC3 in the nerve microenvironment ( = 0.0003), indicating high ASNS expression is indispensable for OSCC progression, especially PNI formation, through l-asparagine metabolism alteration. This study provides novel insights into how amino acids metabolism disorders alter tumor neurotropism which helps cancer metastasis.
失调的氨基酸代谢与癌细胞的进化表型特征相互作用，以增强转移能力。口腔鳞状细胞癌（OSCC）的高转移潜能可表现为神经周围浸润（PNI）。我们在此旨在确定氨基酸代谢在具有不同PNI状态的OSCC中的作用。采用靶向代谢组学方法对20个新鲜OSCC样本中的48种氨基酸进行定量，成功检测到25种氨基酸，其中9种在PNI阳性（PNI）样本中显著上调。由于其曲线下面积值最高（0.9063），l-天冬酰胺被选为区分PNI与PNI阴性（PNI）的生物标志物。然后，使用免疫组织化学方法对86例OSCC患者研究了l-天冬酰胺的关键酶天冬酰胺合成酶（ASNS）。结果显示，ASNS主要在肿瘤上皮中表达，与淋巴结转移和PNI呈正相关。此外，亚组生存分析显示，ASNS表达与PNI状态相结合显著提高了其预后价值，这在TCGA OSCC队列（n = 279）中得到了证实。为了验证ASNS是否促进PNI，我们测定了五种OSCC细胞系和一种正常口腔角质形成细胞中的ASNS表达水平，HSC3显示出最低的ASNS水平，而CAL33具有最高水平。因此，选择HSC3和CAL33（或PBS作为对照）分别注射到坐骨神经中构建PNI小鼠模型。虽然两种模型最终都出现了后肢麻痹，但CAL33模型中的神经功能障碍比HSC3显著更早进展（第9天对第24天）。此外，CAL33在神经微环境中的迁移明显比HSC3更远（P = 0.0003），表明高ASNS表达对于OSCC进展，尤其是通过l-天冬酰胺代谢改变形成PNI是不可或缺的。本研究为氨基酸代谢紊乱如何改变肿瘤嗜神经性从而促进癌症转移提供了新的见解。
