Numb PRRL剪切异构体亚型在肝细胞癌复发转移中的作用机制研究

Hepatocellular carcinoma (HCC) disproportionately affects China due to hepatitis B virus infection, and contributes to more than 50% of the total worldwide annual HCC incidence and mortality.Currently,there are no effective systemic therapies for HCC.This situation highlights the importance and urgency of further research investment in deciphering the underlying molecular mechanisms of HCC's uncontrollable progression and recurrence, and identifying novel biomarkers and therapeutic targets. Cancer stem cells (CSCs) have been demonstrated in a growing range of epithelial and other solid malignancies, suggesting that the majority of malignancies are dependent on such a critical compartment. Liver cancer stem cells (LCSCs) have been associated with HCC's therapeutic resistance, recurrence and poor prognoses. LCSCs present a very difficult therapeutic challenge because existing treatment approaches cannot effectively eliminate them. Numb (a cell fate regulator) is a key factor for cell asymmetric division. Numb's isoform (PRRL) protien is associated with the proliferation of many kinds of cancer cells and the fate of CSCs . Our preliminary studies have shown: 1) an increased expression of Numb PRRL isoform in HCC tumor tissue and Epithelial Cell Adhesion Molecule (EpCAM) positive LCSCs; 2) Numb PRRL isoform promotes the proliferation of liver cancer cells. Based on these findings, we hypothesize that the level of Numb PRRL isoform expressed may correlate with HCC's aggressive progression patterns and poor clinical prognoses. This correlation is tentatively linked to liver cancer cells including LCSCs, and may impact on their functions. This proposal has three aims: Aim 1. Examine the relationship between Numb PRRL isoform expression and liver cancer cells' proliferation, differentiation, apoptosis and chemo-resistance. Aim 2. Examine the impact of Numb PRRL isoform expression on LCSCs' self-renewal, differentiation，invasiveness, and tumor-initiation and growth ability in NOD/SCID mice. Aim 3. Stratify and correlate the level of Numb PRRL isoform expression with LCSCs and HCC's recurrence, pathological grades, and clinical stages and prognoses. Summary: We propose to test whether Numb PRRL isoform is implicated in influencing liver cancer cells and LCSCs' functions that may impact HCC's recurrence, progression and prognosis. If our hypothesis is confirmed, Numb PRRL could serve as a novel biomarker for developing improved diagnosis and treatment methods for HCC.

复发转移是肝细胞癌（HCC）根治性治疗的主要障碍, 肝癌干细胞（LCSCs）在此过程中的作用备受关注；决定肿瘤干细胞（CSCs）产生非对称分裂的细胞命运决定子Numb成为研究热点，其PRRL剪切异构体亚型（Isoform）被认为是调控多种肿瘤细胞增殖和CSCs命运的关键因子。我们前期研究发现Numb PRRL亚型在HCC组织及上皮粘附分子（EpCAM）阳性的LCSCs中高表达，有促进肝癌细胞增殖和调控LCSCs生物学功能的作用，可能是HCC复发转移的特异性标志。本项目拟研究Numb PRRL亚型过表达或抑制表达对肝癌细胞和LCSCs增殖、分化、凋亡、侵袭、成瘤能力的影响；分析Numb PRRL亚型在HCC切除复发前、后肝癌组织、LCSCs中表达变化与病理分级、临床分期、预后的相关性；阐明Numb PRRL亚型参与调控LCSCs驱动HCC复发转移的机制，为寻找HCC特异性肿瘤标志提供理论依据

细胞命运决定子Numb 在癌症中异常表达。Numb 作为选择性剪接子包括PRRL和PRRS两个亚基。最近研究发现，PRRL亚基能增强乳腺癌和肺癌细胞增殖。然而，Numb的选择性剪接在原发性肝癌中的重要性仍然不是很清楚。在这里，我们发现肝癌中Numb PRRL表达升高与术后复发和生存率降低是相关的。通过对肝癌细胞系的研究表明，PRRL促进细胞增殖、迁移、侵袭和克隆形成，而PRRS则起到抑制效应。敲除PRRS导致一种蛋白激酶Akt磷酸化和原癌基因c-Myc表达增加，抑制Akt和沉默c-Myc抑制Numb PRRS基因敲除引起的细胞增殖的影响。在细胞模型研究中结果显示，选择性剪接因子Rbfox2和SRPK2协同调节Numb PRR亚基，敲除前者会引起PRRL积累，而敲除SRPK2会引起PRRS的积累。SRPK2的亚细胞定位是由分子伴侣蛋白热休克蛋白90调节的，抑制热休克蛋白90或者敲除SRPK2促进Numb PRRS亚基的积累。最后，主要表达PRRL的肝癌细胞系热休克蛋白90抑制剂具有显著敏感性。结论：Numb可变剪接在原发性肝癌中可能提供一个有用的预后标志物。