ERK3介导TNF-α调控头颈鳞癌淋巴管生成的作用及机制

Lymph node metastasis is an important risk factor for the poor prognosis of patients with squasmous cell carcinoma of the head and neck (SCCHN), and its mechanism has not been fully clarified. The VEGF-C／VEGFR3 pathway plays a critical role in the lymph node metastasis of the majority cancer types, however, it was found that there was no association between VEGF-C／VEGFR3 expression and lymphatic metastasis of SCCHN, indicating that other pathways might be involved in this process. TNF-α, an important cytokine, plays a critical role in the initiation and regulation of immune and inflammatory responses. It has been shown that TNF-α could induce lymphangiogenesis under physical and pathological conditions, suggesting that it may play a pivotal role in the lymphatic metastasis of cancer. In our previous study, we found that TNF-α was associated with lymphatic metastasis of SCCHN, but the mechanism on TNF-α and lymphatic metastasis of SCCHN has not been fully clarified. ERK3, another member of MAPK subfamily, has been reported to play an important role in angiogenesis, a process similar to lymphangiogenesis, indicating that it may also play a vital role in TNF-α induced lymphangiogenesis. Thus, we conducted a pilot study and found that the expression of ERK3 was significantly increased in SCCHN tumor tissue, and its expression was associated with lymphangiogenesis and lymphatic metastasis of SCCHN. Furthermore, we also found that TNF-α could regulate the expression of ERK3 in human lymphatic endothelial cells. Thus, we hypothesized that TNF-α stimulates the lymphangiogenesis and lymphatic metastasis of SCCHN via the ERK3 pathway. To test the hypothesis, we will conduct a series of in vitro, in vivo and cohort studies to explore the roles of ERK3 in TNF-α-induced lymphangiogenesis and lymphatic metastasis of SCCHN, as well as the molecular mechanism underlying the observed findings. Therefore, this study will provide important theoretic basis for the lymphangiogenesis and lymphatic metastasis of SCCHN.

淋巴管生成是淋巴结转移的重要前提，而淋巴结转移大大影响头颈鳞癌的治疗效果，但淋巴管生成的机制尚不甚清楚。我们先前报道及预实验发现TNF-α与头颈鳞癌淋巴结转移密切相关，体外可以诱导淋巴管的生成，我们进一步预实验明确了ERK3介导TNF-α调控头颈鳞癌淋巴管生成的作用，结合文献在血管和淋巴管生成中ERK3信号通路及其家族成员与VEGF-C／VEGFR3信号通路的密切关系，我们推测ERK3介导TNF-α调控头颈鳞癌淋巴管生成通过VEGF-C／VEGFR3信号通路来实现。本课题通过离体、在体及队列研究，确认ERK3介导TNF-α对头颈鳞癌淋巴管生成的作用，明确ERK3介导TNF-α通过VEGF-C／VEGFR3信号通路对淋巴管生成的作用，从而阐明ERK3及其介导的TNF-α调控头颈鳞癌淋巴管生成的作用及分子机制。阐述了头颈鳞癌淋巴管生成的具体机制，有望筛选出对头颈鳞癌淋巴结转移有预测作用的靶标。

头颈部鳞状细胞癌（简称头颈鳞癌）作为全球第六大恶性肿瘤，由于其易在早期发生淋巴结转移，总体治疗效果欠佳，预后较差。淋巴管的生成是恶性肿瘤发生淋巴结转移的重要前提，在恶性肿瘤的发生发展过程中，也往往伴随着淋巴管的生成，但淋巴管的生成机制尚不清楚。在本研究中，我们首先向人淋巴内皮细胞hLEC的培养基中加入TNF-α和anti-TNF-α，并通过qRT-PCR和western blot观察hLEC细胞中ERK相关基因、蛋白的表达水平，发现VEGF3的表达水平在TNF-α加入后显著升高，而anti-TNF-α处理12h后ERK和VEGFC水平均降低。进一步用TNF-α和anti-TNF-α处理头颈鳞癌细胞系Hep-2，发现细胞上清中的ERK3, p38, Efe2, MMP2, MMP9, TNF-α, SRC3和SP1表达均受TNF-α诱导上调，获取细胞上清，加入到hLEC的培养基中，通过CCK-8实验检测hLEC的增殖能力变化，发现TNF-α组的Hep-2条件培养基能够抑制hLEC细胞的增殖，另一方面，利用条件培养基处理细胞后，通过细胞划痕实验发现hLEC细胞的迁移能力受到抑制。进一步在hLEC细胞中过表达ERK3，发现过表达的细胞的增殖和迁移能力都显著强于对照组。之后，通过在裸鼠背部注射Hep-2细胞，我们构建了异位瘤模型，分别使用生理盐水和TNF-α进行瘤内注射，我们发现TNF-α组的肿瘤组织中VEGF3和LYVE-1（内皮细胞透明质酸受体）表达水平升高，可能与新生血管和淋巴管的生成有关。将VEGF3和ERK3作为干预的靶点可能对抑制头颈鳞癌的发展和淋巴结的转移具有较有效的作用，而其中的具体分子机制仍需要进一步的探索研究。