AMPK对TGFbeta信号转导和乳腺癌转移的抑制作用

Breast cancer is the most common cancer in women and the second cause for cancer deaths, for which the major cause is metastasis at a late stages. Some cancer cells undergo epithelial cell to messenchymal transition (EMT) and acquire features of cancer stem cells (CSC), such that they are able to invade, metastasize, grow and repopulate at distant sites. During this course, it has been well documented that TGFbeta plays a pivotal role. Recent studies have shown that the tumor suppressor LKB1 and its downstream effector, AMPK, are implicated in tumor onset, progression and metastasis; loss of function mutation of LKB1 is frequently found in human cancer and the mutation promotes tumor progression and metastasis; in breast cancer samples, it has been reported that AMPK activity is inhibited, the extent of which correlates to disease progression, whereas the TGFbeta signal pathway is upregulated. However, the relationship between AMPK and TGFbeta in cancer has never been reported. Our preliminary study in breast cancer cells has revealed that the activation of AMPK induces an inhibition of TGFbea signaling, whereas it is enhanced by suppression of AMPK and its downstream target LITAF, concurrent with changes in markers of EMT/CSC. Therefore, we hypothesize that during progression of breast cancer, AMPK is suppressed, which induces upregation of TGFbeta signaling, leading to EMT/CSC, a prerequisite condition for breast cancer metastasis. To test this hypothesis, we will expand our study by establishing more breast cancer cell lines expressing AMPK shRNA and overexpressing LKB1 and examining the effects of AMPK on EMT/CSC and TGFbeta signaling in association with malignant behaviors in vitro. We will also establish animal tumor models including othotopic Xenograft and transgenic mouse expressing the dominant negative AMPK mutant to demonstrate if inhibition of AMPK will lead to activation of TGFbeta and breast cancer metastasis. Altogether, our study will provide a novel view on the mechanism underlying AMPK regulation of tumorigenesis and progression. Therefore, completion of this study will provide us important insights into the mechanism of tumor metastasis and rationale for developing new drugs targeting AMPK for cancer therapy.

乳腺癌在女性癌症患者位居第一，死亡率居第二。死亡的主要原因是晚期转移。癌细胞在发生上皮细胞至间充质细胞转化（EMT）、获得干细胞CSC特性后，发生侵润、转移，在远位第二次生长, TGFbeta被证明在此过程中起关键作用。近年的研究显示肿瘤抑制因子LKB/AMPK与肿瘤的发生、发展和转移有关，但是，LKB1/AMPK和TGFbeta之间的关系从未有过研究报道。我们的初步研究结果表明，在乳腺癌细胞中，AMPK的激活可以抑制TGFbeta信号转导导通路，而抑制其下游蛋白LITAF的表达导致后者的激活，并上调EMT／CSC成分。因此，在本课题中，我们将进一步证实AMPK对EMT/CSC和TGFbeta信号通道的抑制作用，检测细胞学效应。并用乳腺原位异体移植肿瘤和转基因鼠动物模型确认AMPK对肿瘤转移抑制作用。本课题对于肿瘤转移的机制研究可以提供重要的信息，并为其防治提供新的依据和靶点。

乳腺癌在女性癌症患者位居第一，死亡率居第二。死亡的主要原因是晚期转移。TGF-beta在肿瘤发生的早期阶段发挥抑制作用，在晚期却促进肿瘤进展和转移。单磷酸腺苷激活蛋白激酶（AMPK）是细胞能量感受器，在正常和癌细胞中维持能量稳态，是肿瘤代谢抑制因子。近年的研究显示LKB/AMPK 与肿瘤的发生、发展和转移有关，但是，LKB1/AMPK 和TGF-beta 之间的关系从未有过研究报道。因此本课题中描述AMPK和TGF-beta两者之间的关系，阐明AMPK的肿瘤抑制作用。通过本课题我们研究发现：1、在乳腺癌细胞中表达AMPK 上游激酶LKB1和 AMPK阻止TGF-beta诱导的Smad磷酸化及其转录活性，而通过转入shRNA敲除LKB1或AMPK表达时却增强TGF-beta的效应;2、激活AMPK降低了TGF-beta1的启动子活性。与那些没有服用二甲双胍的2型糖尿病患者相比，服用二甲双胍患者的血清显示TGF-beta1减少的趋势。此外小鼠服用二甲双胍后其血清中的TGF-beta1显著减少。这些结果表明AMPK抑制TGF-beta1的转录，导致其在血清中的浓度降低；3、二甲双胍抑制乳腺上皮细胞的上皮向间质转化（EMT）以及TGF-beta诱导的细胞迁移和侵袭；4、乳腺癌组织中TGF-beta关键成分Smad2的磷酸化（pSmad2）和下游靶基因β-catenin表达高于邻近组织，可能作为治疗和预后的乳腺癌潜在的生物标记物；5、为了进一步证实AMPK在肿瘤发生发展中的作用，建立AMPK DN转基因小鼠，发现AMPK活性降低小鼠乳腺上皮AMPK 活性降低促进青春期小鼠导管上皮细胞的增殖，促进TEBs 的发育，对妊娠期发育无明显变化，同时，二甲双胍抑制妊娠期初期导管发育，显示AMPK 活性变化能影响小鼠乳腺的阶段发育，但未能观察AMPK DN小鼠成瘤，提示AMPK的抑制不足以引起肿瘤，但是可以为肿瘤的进展创造条件。这些研究成果表明AMPK通过多种途径抑制TGF-beta信号传导，明确AMPK对肿瘤发生发展的作用，发现AMPK突变可能通过乳腺上皮增生，从而影响肿瘤形成。总之，我们的研究支持AMPK可以作为治疗乳腺癌进展以及其他由TGF-beta1升高引起的疾病的药物靶点，也有助于调整当前临床试验把二甲双胍用于预防癌症转移。