ADAMTS8在结直肠癌中的抑癌作用及其负调控MAPK/ERK通路的机制研究

Some members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) metalloproteinase family have recently been reported to function as candidate tumor suppressor. However, there are still no systemic study carried out in the colorectal cancer (CRC) for this family by now. In our previous screening study for the 19 human ADAMTS genes, ADAMTS8 was revealed to be silenced by promoter CpG methylation in multiple CRC cell lines. Re-expression of ADAMTS8 could inhibit the colony formation ability of the HT29 CRC cell and meanwhile suppress its MAPK/ERK activity, suggesting that ADAMTS8 could be a functional tumor suppressor candidate in CRC. However, the biological role of ADAMTS8 and related molecular mechanism are still unknown. Based on our preliminary data, we presume that ADAMTS8 is a novel candidate tumor suppressor gene with multiple tumor-suppressing roles which is silenced by promoter methylation in CRC. ADAMTS8 may play its role through suppressing the MAPK/ERK signaling and it may mediate the MAPK/ERK inhibition via the EGF, VEGF or Integrin pathways. To prove above hypotheses, we plan to study the role of ADAMTS8 in the regulation of the proliferation, apoptosis and angiogenesis of CRC using in vitro and in vivo assays. Besides, we will also analyze if ADAMTS8 play its anti-tumorigenesis role via inhibiting MAPK/ERK signaling and try to reveal the mechanism underlying ADAMTS8-mediated MAPK/ERK suppression using a series of over-expression, gene knockdown and protein mutant assays. Finally, we will evaluate the feasibility of using the ADAMTS8 expression level and promoter methylation status as molecular markers for CRC. Results of this project may help to reveal the role of ADAMTS8 as an epigenetically silenced novel tumor suppressor candidate, to explore the underlying tumor suppressing mechanism of ADAMTS8 and to discover novel CRC molecular markers. Thus, this study is of prominent basic scientific significance and may potentially contribute to better clinical treatment for CRC.

新型金属蛋白酶ADAMTS家族某些成员近期被发现具有抑癌作用，但在结直肠癌中仍缺乏针对该家族的系统研究。前期在结直肠癌细胞系中对该家族进行的筛选研究发现成员ADAMTS8因启动子甲基化在癌细胞系中频繁沉默，恢复其表达可抑制肿瘤克隆形成并下调MAPK/ERK活性，提示它在结直肠癌中可能具有抑癌作用，但具体功能及机制不清。我们推测ADAMTS8可能通过负调控EGF、VEGF或Integrin通路下调MAPK/ERK活性而发挥抗增殖、促凋亡或抗血管生成等抑癌功能，并在原发结直肠癌组织中因甲基化沉默。为验证这一假设，拟利用癌细胞系及裸鼠模型开展体内、体外实验以研究ADAMTS8在结直肠癌细胞增殖、凋亡及血管生成中的抑癌功能及关键分子机制，并分析它在原发结直肠癌中的表达和甲基化状态及与肿瘤分期、预后等的相关性。结果将为研发新型结直肠癌治疗靶点和策略提供理论依据，并有可能发现新型结直肠癌标志物。

新型金属蛋白酶ADAMTS家族某些成员近期被发现具有抑癌作用，但在结直肠癌中仍缺乏针对该家族的系统研究。前期在结直肠癌细胞系中对该家族进行的筛选研究发现成员ADAMTS8因启动子甲基化在癌细胞系中频繁沉默，恢复其表达可抑制肿瘤克隆形成并下调MAPK/ERK活性，提示它在结直肠癌中可能具有抑癌作用，但具体功能及机制不清。我们推测ADAMTS8可能通过负调控EGF、VEGF或Integrin通路下调MAPK/ERK活性而发挥抗增殖、促凋亡或抗血管生成等抑癌功能，并在原发结直肠癌组织中因甲基化沉默。但本研究进行过程中发现ADAMTS8基因在原发结直肠癌组织中甲基化比率低且癌及癌旁组织中蛋白表达无统计学差异，可能不是结直肠癌中起重要作用的抑癌基因。进而我们将研究扩展到了其他恶性肿瘤中，发现ADAMTS8基因在鼻咽癌、食管癌及胃癌细胞系中因甲基化表达下调，重要的是该基因在这些癌种的原发肿瘤组织中发生频繁甲基化。在HONE1及KYSE150细胞系中的功能及分子机制研究发现，ADAMTS8基因过表达可以抑制肿瘤细胞克隆形成、抑制肿瘤细胞迁移、诱导肿瘤细胞凋亡，并下调MAPK/ERK通路的活性。针对上游通路的研究发现ADAMTS8可以通过下调磷酸化EGFR蛋白水平抑制EGFR/MAPK/ERK通路活性，ADAMT8作为一个分泌蛋白酶导致磷酸化EGFR水平下调的具体分子机制仍有待探讨。本研究发现ADAMTS8可以通过抑制EGFR/MAPK/ERK通路活性在恶性肿瘤中发挥抑癌基因作用，目前靶向EGFR蛋白的单抗药物在实体瘤中获得了很好的疗效，因此继续对ADAMTS8抑制EGFR/MAPK/ERK通路的分子机制进行深入研究，具有重要的科学意义及潜在的临床应用价值。