ΔNp63调控Sonic Hedgehog通路在邻苯二甲酸酯类促进乳腺癌干细胞活性中的作用

The health effects of phthalates(PAEs) has attracted tremondeous attention scientifically and socially. Cancer stem cell (CSCs) play central role in the formation and development of cancer. The biological function of p63 has been recognized substantially in cancer research. Growing evidence suggests that PAEs can promote the growth, invasion, metastasis, and drug resistance of breast cancer cells. To date, no studies have been conducted yet to reveal the effects of PAEs on breast CSCs, as well as the involvement and function of ΔNp63 in PAEs-activated breast CSCs. In our preliminary study we found that PAEs (DEHP, DBP and BBP) promoted the sphere formation of breast cancer cells cultured in serum-free-medium, increased CSCs markers' expression, upregulated Sonic Hedgehog pathway signaling, and elevated ΔNp63 level. However, the role and regulation of Δ Np63/Sonic Hedgehog in PAEs-mediated breast CSCs stemness is totally unknown. Using serum-free-medium cultured breast CSCs and nude mouse xenograft models, the proposed research will for the first time to illustrate the effects of PAEs on breast CSCs. Meanwhile, The transcriptional regulation of ΔNp63 in PAEs-mediated Sonic Hedgehog pathway activation, target gene expression and breast CSCs activity will be further explored. Elucidating the regulatory role of ΔNp63/Sonic Hedgehog axis in PAEs-mediated breast CSCs will provide important new insights into the molecular mechanisms by which PAEs promote breast cancer and its target intervention as well.

邻苯二甲酸酯类（PAEs）的健康效应一直是关注重点。p63的作用近年越为人所重视。肿瘤干细胞研究已成为相关领域的前沿和热点。PAEs已证实可促进乳腺癌细胞增殖、侵袭转移及耐药；然其对乳腺癌干细胞的影响尚未见报道。我们前期研究发现，低浓度的PAEs（DEHP、DBP和BBP）可促进乳腺癌MCF-7和MDA-MB-231悬浮细胞球形成，上调乳腺癌干细胞标志物表达， 激活Sonic Hedgehog通路并升高ΔNp63水平。然ΔNp63/Sonic Hedgehog是否真正介导PAEs的促乳腺癌干细胞作用尚属未知。本课题拟采用体外无血清悬浮培养克隆成球模型和裸鼠移植瘤模型相结合的方法，创新性地探讨PAEs对乳腺癌干细胞活性的影响，并研究ΔNp63/Sonic Hedgehog在PAEs调控乳腺癌干细胞活性中的作用，可望为探索PAEs促乳腺癌的分子机制、并为以机制为导向的靶向干预研究提供科学依据。

乳腺癌是女性最常见的肿瘤。邻苯二甲酸酯类（PAEs）在环境中普遍存在，可增加乳腺癌发病风险，并促进乳腺癌细胞增殖、侵袭、转移和耐药。肿瘤干细胞在上述过程中发挥重要作用；而ΔNp63α 是维持干细胞特性的一个主要调节因子。本研究用低浓度（10-9M）的PAEs（DEHP、DBP和BBP）长期处理ER(+)的MCF-7和ER(-)的MDA-MB-231乳腺癌细胞（传代40代以上），发现经PAEs（10-9M）长期处理后的两种乳腺癌细胞的悬浮培养细胞球（富集了乳腺癌干细胞）体积增大、数量增多，乳腺癌干细胞标志物Oct-4、Nanog、CD133、CD44和ALDH1A1的mRNA和蛋白表达水平均升高， Sonic Hedgehog通路蛋白SHH、Smo、Gli1和Gli2被激活，同时ΔNp63的mRNA和蛋白表达水平也升高。使用Sonic Hedgehog通路抑制剂Gli1的siRNA与PAEs联合处理，发现单独PAEs处理引起的悬浮培养细胞球体积增大、上述干细胞标志物蛋白表达上调、上述通路蛋白表达上调的效应会被逆转（或部分逆转）。在乳腺癌细胞中转染ΔNp63α质粒同时给予PAEs处理，发现ΔNp63α可以进一步增强PAEs促进乳腺癌悬浮细胞球形成、上调干细胞分子标志物和Sonic Hedgehog通路蛋白的作用。相反，转染ΔNp63α的siRNA并联合处理PAEs，发现单独PAEs处理引起的促进乳腺癌悬浮细胞球形成、增强细胞干性标志物表达、上调Sonic Hedgehog通路蛋白的效应可以被ΔNp63α的siRNA逆转（或部分逆转）。文献报道环境中PAEs暴露的中位浓度为10-8M-10-6M，本研究中10-9M浓度的PAEs对ER(+)和ER(-)乳腺癌存在的共性效应当引起重视研究结果也有望为探索PAEs促乳腺癌的共性分子机制、并为以机制为导向的靶向干预研究提供科学依据。