Ndfip1/Nedd4-1调控PTEN亚细胞定位参与乳腺癌化疗耐药的分子机制研究

Breast cancer is the most common malignancy in women and chemoresistance is a major reason for failure of chemotherapy-based treatment. Inactivation of PTEN is an important mechanism involving in chemotherapy resistance development of breast cancer. The loss of PTEN protein in breast cancer is 33% ~ 50%, the protein post-translational modification may be the main reason leading to its inactivation, but the mechanism is not clear. Recently, nuclear PTEN plays a more important role in chromosome stability, DNA repair, cell cycle arrest and cellular apoptosis. PTEN enters the nucleus by several mechanisms including monoubiquitylation-dependent import. However, cytoplasmic PTEN is instability and may be degradation easily by poly ubiquitination. .NEDD4-1 is a proto-oncogenic Ubiquitin E3 ligase regulates PTEN activity by ubiquitination. The newly discovered Ndfip1 (Nedd4 family–interacting protein 1), an adaptor protein for the Nedd4 family of ubiquitin ligases, can bind the WW domain of Nedd4-1 by its PY motif which is also necessary for uniquitination of pten. Recent studies have found that Ndfip1-mediated ubiquitination of PTEN can lead to either monoubiquitination promoting nuclear accumulation or polyubiquitination promoting PTEN degradation in the cytoplasm. However, there is no related research on development and resistance effect of Ndfip1/Nedd4-1/PTEN ubiquitin pathway in tumor especially in breast cancer. .Our previous studies showed that:.1. The expression of nuclear PTEN was observed to be down-regulated in human breast cancer cells.However,up-regulated Ndfip1 and Nedd4-1 were observed in tissues of non-responders to new neoadjuvant chemotherapy as compared to those of responders by immunohistochemistry. .2. The resistant MCF-7/ADM cells were observed to have a lower neclear PTEN and higher expression of p-Akt, Nedd4-1 and Ndfip1 than that of sensitive cells by western blot. PTEN and Akt inhibitor perifosine reversed MDR phenotype partially by downregulation of MDR-1 gene expression by the inhibition of PI3K/Akt/NF-κB pathway in the MCF-7/ADM cell line..3. Transfection of Nedd4-1 enhanced nuclear PTEN export and down-regulated PTEN expression..Taken together, our findings indicate that Ndfip1/Nedd4-1-PTEN pathway might be the main mechanism of drug resistance of breast cancer cells. And it may act as a promising therapeutic target for improvement of responsiveness to chemotherapy in breast cancer. However, the role of Ndfip1/Nedd4-1 in modulating proliferation and chemoresistance of breast cancer remains largely unexplored. In this study, we aimed to investigate the possible role of Ndfip1/Nedd4-1 in the development and chemoresistance of breast cancer cells using tissue microarray, confocal, immunoprecipitation and RNA interference technologies. This maybe helpful for finding an attractive target for reveral of chemoresistance of breast cancer and provide a theoretical basis for breast cancer treatment and reseach.

PTEN蛋白失活是乳腺癌发生、发展及化疗耐药产生的重要机制。Nedd4-1是新近发现的调控PTEN特异性泛素化的E3连接酶，可在衔接蛋白Ndfip1介导下导致PTEN亚细胞定位异常促进其泛素化降解，但在乳腺癌的发生和化疗耐药的关系未见报道。我们前期实验显示， Ndfip1/Nedd4-1在乳腺癌化疗耐药组肿瘤细胞及多药耐药细胞系MCF-7/ADM中表达显著升高，而核PTEN表达缺失。由此我们提出：Ndfip1/Nedd4-1可促进PTEN的泛素化从而改变其亚细胞定位可能促进乳腺癌的发生发展和化疗耐药。为此，本课题拟以细胞模型转染Ndfip1/Nedd4-1表达质粒和iRNA质粒，研究其对PTEN的泛素化影响及细胞生长和多药耐药逆转情况，并在动物模型和临床病理标本中验证，并深入研究Ndfip1/Nedd4-1促进PTEN的泛素化影响耐药的分子机制，有助于为临床逆转乳腺癌多药耐药提供新靶点。

NEDD4-1是新发现的E3泛素化连接酶，在衔接蛋白Ndfip1介导下促进PTEN泛素化及调节亚细胞定位，但目前与乳腺癌的发生发展和化疗耐药的关系未见详细报道。本课题以细胞模型转染Ndfip1/NEDD4-1表达质粒和iRNA，通过分子生物学技术研究对PTEN的泛素化状态、细胞生长和多药耐药表型的影响，并在动物模型和临床病理组织标本中验证，探讨Ndfip1/NEDD4-1影响PTEN泛素化诱导耐药的分子机制。本研究结果显示：1. 乳腺癌组织中NEDD4-1和Ndfip1表达均较周围乳腺组织上调，而PTEN表达缺失，三者均与乳腺癌的肿瘤进展和化疗耐受相关，多因素分析显示NEDD4-1高表达和PTEN低表达是提示乳腺癌患者不良预后的分子指标。2. 外源性NEDD4-1能诱导MCF-7细胞生长，抑制凋亡，促进细胞浸润和迁移能力；而NEDD4-1沉默有效的抑制细胞生长、促进凋亡以及降低浸润和迁移能力。3. NEDD4-1沉默后，MCF-7细胞胞质PTEN泛素化水平下降表达增加，下调p-Akt水平，抑制PI3K/Akt信号通路活性，通路下游靶蛋白BAD表达上调，mTOR磷酸化水平下降及细胞录因子NF-Kb p65表达下降。4.多药耐药乳腺癌细胞NEDD4-1转染沉默后，MCF-7/ADM细胞对阿霉素的敏感性均不同程度的增加，IC50值下降，并检测到多药耐药蛋白P-gp、BCRP和MRP表达均降低。总之，NEDD4-1在乳癌组织中高表达通过泛素化拮抗PTEN的活性，活化了PI3K/Akt细胞信号通路并作用于多种靶蛋白，从而促进了乳腺癌细胞的生长，浸润和迁移过程，同时还增加了乳腺癌细胞对于化疗药物的耐受性，并部分的促进了多药耐药相关耐药蛋白的表达，继而参与乳腺癌的肿瘤进展过程，并提示患者的不良预后。因此，我们认为NEDD4-1可能使乳腺癌治疗的一个新的分子靶点，可能成为逆转乳腺癌化疗耐药的分子靶蛋白，对于临床治疗提供了新的思路和策略。