研究背景:原发性肝癌是全球第六大常见癌症，也是第三大常见癌症死亡原因。肝癌具有发生隐匿、恶性程度高、发展迅速等特点。除手术切除病灶外，肝癌的治疗选择非常有限，且术后复发率高，对放化疗均不敏感，而免疫治疗和靶向药物也仅对少数患者有效。因此，需要深入研究肝癌的发病机制，发现治疗靶点和开发有效药物。CTNNB1(  编码β-catenin)(22%突变率)是肝癌中突变率最高的原癌基因。超过10%的肝细胞腺瘤和半数以上肝母细胞瘤患者携带CTNNB1突变。然而目前仍没有好的药物来针对这一通路进行肝癌的治疗。我们的研究旨在通过高通量药物筛选，探索能够选择性抑制 β-catenin活化突变细胞增殖和肿瘤生长的小分子化合物，为临床精准治疗β-catenin活化突变肝癌提供科研依据和实验基础。

研究方法:我们首先利用野生型和β-catenin活化突变的成纤维细胞系进行高通量药物筛选，发现能够选择性地抑制 β-catenin活化细胞增殖的药物。随后利用成纤维细胞系对候选药物进行验证。然后将肝癌细胞系根据β-catenin表达情况进行分组，并在β-catenin正常表达的肝癌细胞中过表达突变的β-catenin、在β-catenin活化的 肝癌细胞中敲低β-catenin，检测铁螯合剂处理后细胞增殖情况。然后，我们用铁螯合剂进行裸鼠成瘤实验，检测其抑制β-catenin活化肿瘤生长的能力。为了探讨铁螯合剂处理后，β-catenin 活化细胞增殖能力下降可能的机制，我们利用相差显微镜、细胞台盼蓝染色以及免疫荧光检测细胞活力、DNA复制和损伤情况。为进一步探讨铁螯合剂抑制细胞增殖的机制，我们用seahorse实验检测加药前后细胞线粒体代谢及ATP合成的变化。然后，我们用线粒体抑制剂S-Gboxin和Oligo处理β-catenin活化的细胞，检测线粒体抑制剂抑制β-catenin活化细胞增殖的能力。并用S-Gboxin处理裸鼠移植瘤，观察线粒体抑制剂对β-catenin活化移植瘤生长的影响。

研究结果:利用野生型和β-catenin活化突变的成纤维细胞系进行高通量药物筛选，我们发现二丙酸倍氯米松(Beclomethasone dipropionate)、地拉罗司(Deferasirox, DFX)、卡维地洛(Carvedilol)和维他命 B12(Vitamin B12)四种化合物能够选择性地抑制β-catenin活化突变的细胞。在成纤维细胞系中进行上述候选药物的验证，发现仅有铁螯合剂DFX的效果与高通量药物筛选结果一致。另外两种铁螯合剂去 铁胺甲磺酸酯(Deferoxamine, DFO)和VLX600的结果也与DFX一致。利用枸橼酸铁胺(Ferric ammonium citrate, FAC)外源性补充铁能够回补三种铁螯合剂对细胞增殖的抑制作用。细胞增殖实验表明，铁螯合剂能够选择性地抑制β-catenin突变表达的肝癌细胞系组的增殖。裸鼠成瘤实验也表明DFO能够抑制β-catenin活化突变的肿瘤生长(p<0.05)。利用相差显微镜、细胞台盼蓝染色以及免疫荧光实验，我们发现DFO处理后细胞活力没有明显下降，而DNA复制过程受阻、DNA损伤增加。我们用seahorse实验检测发现，DFO能够降低细胞线粒体能量代谢和ATP合成(p<0.05)。然后，我们发现线粒体抑制剂S-Gboxin和Oligo能够选择性抑制β-catenin活化突变的细胞增殖(p<0.05)。裸鼠移植瘤实验表明S-Gboxin能够有效抑 制β-catenin活化移植瘤的生长(p<0.05)。

研究结论:本研究首次发现铁螯合剂能够选择性地抑制β-catenin突变细胞增殖并抑制β-catenin活化移植瘤的生长。初步验证DFO能够抑制β-catenin活化突变细胞而非细胞毒的作用。这种抑制作用很可能是通过抑制线粒体能量代谢来实现的。线粒体抑制剂S-Gboxin也能够有效抑制β-catenin突变细胞增殖以及移植瘤的生长。

Background: Primary liver cancer is the sixth most common cancer and the third most common cause of cancer-related deaths worldwide. Liver cancer is characterized by its insidious onset, high malignancy, and rapid progression. Besides surgical resection of the lesion, treatment options for liver cancer are very limited and the postoperative recurrence rate is high. The cancer is not sensitive to radiotherapy or chemotherapy while immunotherapy and targeted drugs are only effective for a small number of patients. Therefore, it is necessary to conduct in-depth research on the pathogenesis of liver cancer, identify therapeutic targets and effective drugs. CTNNB1 (encoding β-catenin) has the highest mutation rate among oncogenes in liver cancer, with a 22% mutation rate. Morethan 10% of hepatocellular adenoma and more than half of hepatoblastoma patients carry CTNNB1 mutations. However, there are still no effective drugs targeting this pathway for the treatment of liver cancer. Our study aims to explore small molecule compounds that can selectively inhibit the proliferation and tumor growth of β-catenin-activated cells through high-throughput drug screening. This research will provide scientific evidence and experimental basis for the precise clinical treatment of β-catenin-activated liver cancer.

Methods: We first performed high-throughput drug screening using WT and β-catenin^Δ(ex3)/+ cells to identify drugs that selectively inhibit the proliferation of β-catenin-activated cells. Next, we verified the screened drugs using MEFs. The hepatocellular carcinoma cell lines were then grouped according to β-catenin expression, and cell proliferation was examined after iron chelator treatment by overexpressing mutant β-catenin in β-catenin-normal-expressing hepatocellular carcinoma cells and knocking down β-catenin in β-catenin-activated hepatocellular carcinoma cells. Subsequently, we conducted xenograft tumor experiments in nude mice using iron chelator to assess its ability to inhibit tumor growth. To explore the potential mechanisms underlying the decreased proliferative capacity of β-catenin-activated cells after treatment with iron chelators, we used phase-contrast microscopy, cell trypan blue staining, and immunofluorescence staining to detect cell viability, DNA replication and DNA damage. To further investigate the mechanism by which iron chelators inhibit cell proliferation, we used seahorse assay to detect changes in cellular mitochondrial metabolism and ATP synthesis before and after drug administration. Then, we treated β-catenin-activated cells with mitochondrial inhibitors S-Gboxin and Oligo to detect the ability of mitochondrial inhibitors to inhibit the proliferation of β-catenin-activated cells. Subsequently, we treated nude mice with S-Gboxin to observe the effect of mitochondrial inhibitors on the growth of xenograft tumors.

Results: Using WT and β-catenin^Δ(ex3)/+cells for high-throughput drug screening, we found that four compounds, namely beclomethasone dipropionate, deferasirox (DFX), carvedilol, and vitamin B12, could selectively inhibit the proliferation of β-catenin-activated cells. Manual drug validation in fibroblast cell lines showed that only the iron chelator DFX exhibited consistent effects with the high-throughput drug screening results. The other two iron chelators, deferoxamine (DFO) and VLX600, also showed results consistent with DFX. Exogenous supplementation of iron with ferric ammonium citrate (FAC) could reverse the inhibitory effect of the three iron chelators on cell proliferation. Cell proliferation assays showed that iron chelators could selectively inhibit the proliferation of β-catenin-mutated hepatocellular carcinoma cell lines group. Xenograft tumor experiments also showed that DFO could inhibit the growth of β-catenin-activated tumors (p<0.05). Using phase-contrast microscopy, cellular trypan blue staining, and immunofluorescence experiments, we found that cell viability did not decrease significantly after treatment with DFO, while DNA replication was inhibited and DNA damage increased. Seahorse experiments revealed that DFO could reduce mitochondrial energy metabolism and ATP synthesis in β-catenin^Δ(ex3)/+cells (p<0.05). We then found that mitochondrial inhibitors S-Gboxin and Oligo could selectively inhibit the proliferation of β-catenin^Δ(ex3)/+ cells (p<0.05). Xenograft tumor experiments showed that S-Gboxin could inhibit the growth of β-catenin-activated xenograft tumors (p<0.05).

Conclusion: In this study, we found that iron chelators can selectively inhibit the proliferation of β-catenin-activated cells and the growth of xenograft tumors for the first time. Preliminary verification showed that DFO could inhibit the proliferation of β-catenin-activated cells rather than being cytotoxic. This inhibitory effect is likely achieved by suppressing mitochondrial energy metabolism. Mitochondrial inhibitor S-Gboxin also selectively inhibited the proliferation of β-catenin-activated cells and the growth of xenograft tumors.