一种新型磷酸脂酶PTP-PEST遗传变异促进结直肠癌发病的生物学机制研究

PTP-PEST，which belongs to a subfamily of protein tyrosine phosphatases，plays a critical role in tumor growth,death and invasion via dephosphorylating its substrates (including p130Cas, c-Src and Shc protein). However, diverse studies indicate a contraditory role of PTP-PEST in carcinogenesis. To clarify it ,we investigated the association between polymorphisms of PTP-PEST and colorectal cancer (CRC) risk. Our findings suggested that a genetic variant T573 (rs3750050), located within a exon of PTP-PEST gene, was associated with high risk of CRC and increased viability and mobility of CRC cells. As previous studies indicate that PTP-PEST negative regulates Ras/Raf/MEK/ERK signaling to suppress tumor growth and invasion by interacting with ShcA protein, we hypothesize that the variant fails to bind to ShcA protein, loses its ability to suppress the signaling of Ras/Raf/MEK/ERK pathway, increases CRC cell viability and mobility, and ultimately contributes to the development of CRCs. In the following study, we will perform several in vitro and in vivo experiments (including establishment of stable cell lines, tumor bearing nude mice，mouse model of colorectal cancer metastasis and so on) to verify the hypothesis. We believe that our research would reveal the role of PTPs in CRC carcinogenesis comprehensively and might contribute to the development of interventions and therapies for CRCs.

PTP-PEST是经典蛋白酪氨酸磷酸脂酶亚家族中的一员，其在肿瘤发生中的角色，不同研究观点迥异。为了厘清这点，本项目的前期工作采用了病例对照研究分析PTP-PEST的遗传变异与中国人群结直肠癌易感性的关联性，发现位于外显子区的错义遗传变异T573A（rs3750050）显著增加结直肠癌的风险，该变异能促进结直肠癌细胞增殖、侵袭，但相关分子机制未明。因该酯酶能通过去磷酸化Shc蛋白负调控下游的Ras/Raf/MEK/ERK通路抑制肿瘤增殖、侵袭，本研究推测该变异通过干扰PTP-PEST与Shc蛋白的互作，削弱了对Ras/ERK通路的抑制，促进了肿瘤增殖和侵袭，最终增加了罹患结直肠癌的风险。本研究拟采用稳定株筛选、裸鼠成瘤、转移模型构建等技术分别从体内/体外两层次证实该假设。该项目的开展有助于系统地阐述PTP-PEST遗传变异、结直肠癌发病间的机制联系，有助于开发结直肠癌诊疗新手段。

新型磷酸酯PTPN12作为一种肿瘤抑制分子近年来受到研究者的关注，但相关生物学机制尚未完全清晰。本研究的前期工作采用病例对照研究分析了PTPN12的遗传变异与中国人群结直肠癌（CRC）易感性的关联，发现位于外显子区错义变异T573A显著增加罹患CRC的风险。为了厘清PTPN12及其变异T573A影响CRC发生、发展的生物学机制，本研究首先分析了36对CRC及癌旁组织中PTPN12表达，发现该基因在肿瘤组织的表达显著低于对应的癌旁组织。其次，裸鼠成瘤实验分析发现PTPN12诱导表达能显著缩小HCT116细胞移植瘤体积、降低肿瘤细胞的增殖及减少瘤内血管数量，T573A突变体丧失了对肿瘤增殖及血管发生的抑制。同时，体外实验也得到了相同的结果：PTPN12能显著抑制ERK通路，将细胞阻滞在G0/G1期，显著削弱结直肠癌细胞HCT-116、HT-29及SW480细胞增殖，而T573A突变体丧失了抑制作用。此外，本研究发现使用PTPN12诱导表达的HCT-116及HT-29细胞条件培养基处理内皮细胞Ea.hy926，能有效抑制该细胞迁移及体外成管能力，而PTPN12 T573A诱导表达的CRC细胞条件培养基则对内皮细胞迁移及成管无明显影响。进一步分析发现，PTPN12能通过抑制EGF激活的MEK-ERK通路信号转导，负调控血管生成因子VEGFA、MMP-2、MMP-9及IL-8的表达，最终抑制肿瘤血管发生；而T573A突变体不具备抑制能力。本项目研究结果系统揭示了PTPN12及T573A变异参与CRC发病的生物学机制，有望为该疾病防治提供干预靶点。