肿瘤蛋白TPD52调控AMPK激酶活性及肿瘤发生的功能与分子机制研究

Breast cancer is the most common cancer and the second leading cause of cancer death in women. Better understanding breast cancer etiology would help prevent and treat this disease. The tumor protein D52 (TPD52) gene maps to a region on chromosome 8q21 that is commonly amplified in many tumors, and it is also associated with poor prognosis in cancers such as breast cancer. It remains unclear whether TPD52 amplification passively reflects its gene location or actively contributes to tumorigenesis. In addition, TPD52 is commonly overexpressed in breast cancer,but how TPD52 is regulated at the transcriptional level remains unclear. In preliminary studies, we found that TPD52 expression can be regulated by NFkB pathway. We also found that TPD52, as previously reported, is overexpressed in multiple breast tumor cell lines as well as clinical tumor samples and that alteration of TPD52 expression levels changes cell proliferation rate. To further explore its function, we performed an in vitro pulldown assay followed by mass spectrometry, and we found that AMPK interacts with TPD52, which was confirmed by Co-Immunoprecipitation (Co-IP). We further demonstrated that TPD52 negatively regulates AMPK activity, possibly through a phosphorylation-dependent process. The LKB1-AMPK pathway has been identified as the central regulator of energy metabolism and growth control in tumor suppression. AMPK activators such as metformin, a drug used to treat type II diabetes, have shown beneficial effect in cancer prevention and treatment. In the current application, we propose the following Specific Aims : 1）To study how TPD52 levels are regulated and how TPD52 regulates AMPK activity and downstream pathways. 2）To study how the interaction between TPD52 and AMPK is regulated upon activation of AMPK. 3）To determine the role of TPD52 in breast cancer tumorigenesis using clinical samples and transgenic mouse models. These studies will reveal a novel role of TPD52 in the regulation of an extremely important pathway, the AMPK pathway, which is involved in energy metabolism,autophagy and cell proliferation.

TPD52基因的拷贝数在乳腺癌中会扩增，其高表达与乳腺癌的不良预后有关。但TPD52的扩增是否有助于肿瘤发生尚不清楚。我们前期研究证实TPD52在多种乳腺癌细胞系及临床肿瘤样本中高表达，并且其表达可以被NFκB信号转导通路调控；进一步研究发现TPD52能负调控AMPK激酶的活性。LKB1-AMPK途径已被发现在抑制肿瘤细胞生长和能量代谢中起重要作用。迄今为止，TPD52是第一个与AMPK复合物直接相互作用的调控蛋白。基于以上研究结果，我们设定本研究目标为：1）用乳腺癌细胞作为研究模型来探索TPD52在AMPK通路中的调控功能及其分子机制；2）以临床样本及转基因小鼠模型分析探讨其在肿瘤发生和治疗中的作用。本课题的开展将为TPD52在乳腺癌中高表达和AMPK信号通路调控提供新的重要见解，弥补我们对于在多种肿瘤中扩增并过表达基因的认知不足，并针对TPD52高表达肿瘤的治疗提供思路。

AMP激活蛋白激酶（AMPK）在细胞能量代谢中是中枢调控蛋白，AMPK通路失调是代谢性疾病及肿瘤发生的重要原因。在能量压力条件下，AMPK被激活进而抑制合成代谢促进分解代谢。本项目中，我们着重研究了AMPK通路的一个新的调控因子。我们发现肿瘤蛋白D52（tumor protein D52，TPD52）与AMPKα亚基间存在相互作用。TPD52在多种肿瘤中过表达，在肿瘤细胞中与AMPKα形成稳定的复合体，其N端61个氨基酸对于两者结合起关键作用；在代谢压力条件下，TPD52与AMPK的相互作用减弱；通过调节LKB1-AMPK间的相互作用以及通过直接抑制AMPK对其底物的磷酸化，TPD52在AMPK激活过程中起较关键的负调控作用。通过构建TPD52转基因小鼠，我们发现过表达TPD52导致多组织内的AMPK活性抑制，使小鼠出现多种代谢缺陷表型。通过此研究，我们对于AMPK活性调控机制给出了新的见解，对于TPD52肿瘤蛋白的作用机制有了更深一步的了解，即TPD52通过调控AMPK激酶活性影响肿瘤细胞的糖酵解、脂类合成等生理过程。因TPD52在多种肿瘤中均有过表达，本项目对于TPD52过表达的肿瘤的诊断、治疗也有一定的意义，如在此类肿瘤中人为激活AMPK通路从而达到调节代谢异常的效果。.本项目还证实去泛素化酶USP10能与AMPKα亚基相互作用。USP10去泛素化酶可以特异去除AMPKα的K63链式泛素化，促进LKB1对于AMPKα的磷酸化。在能量压力下，在AMPK激活的过程中，AMPK可以磷酸化修饰USP10的Ser76位点，从而促进USP10的去泛素化酶活性，USP10-AMPK的相互调控形成一个能量压力下有利于AMPK激活的有效正反馈循环。在USP10肝脏特异性敲除小鼠模型中，敲除USP10导致AMPK通路活性异常，从而导致血脂、血糖升高等多重代谢缺陷。