Sorafenib对肝癌侵袭转移潜能的影响及其干预—实验研究

【作者】 张伟；

【导师】 汤钊猷；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 手术切除是原发性肝癌最有效的治疗方法,但即使根治术后5年转移复发率仍高达60-70%。对不能切除的肝癌和肝癌术后转移复发的预防,抗血管生成是一条可探索的途径,但新近的报道,抗血管生成也可促进转移。分子靶向药物sorafenib是目前唯一经Ⅲ期临床试验证实提高晚期肝癌病人生存期的药物,但仅延长3个月生存期；在东方晚期肝癌病人的Ⅲ期临床试验中,sorafenib虽延长生存期,但仅推迟肿瘤进展。作为包括抗血管生成作用的sorafenib是否也有促进癌转移的作用,也是需要弄清的问题。本文用不同转移潜能的人肝癌裸鼠模型,研究sorafenib治疗过程中肿瘤进展的原因,以及对肿瘤侵袭转移潜能的影响。鉴于肿瘤转移与肿瘤细胞及其所处微环境有关,在肝癌中也发现微环境中的炎症因子、巨噬细胞可预测肝癌患者术后复发转移。为此,本文拟从肿瘤细胞和肿瘤相关巨噬细胞两个方面进行研究,并探索进一步提高sorafenib疗效的途径。1.低剂量sorafenib诱导肝癌侵袭转移潜能增强本部分工作的主要目的是在不同转移潜能的人肝癌裸鼠模型中,探索不同剂量sorafenib对肿瘤侵袭转移潜能的影响。首先在我所高转移人肝癌裸鼠模型(LCI-D20)中,发现sorafenib低剂量(30mg/kg)和高剂量(100mg/kg)均明显抑制肿瘤生长,并延长荷瘤鼠生存期。Sorafenib虽抑制肺转移灶数量,但并未降低肺转移率；而考虑到sorafenib治疗组肿瘤体积更小,以标化肺转移率(肺转移灶数目/肿瘤体积)计算,sorafenib治疗组有促进肺转移趋势(sorafenib低剂量组与对照组比较,P=0.069)。研究还发现sorafenib低剂量(30mg/kg)明显促进肝内播散。尽管sorafenib大剂量(100mg/kg)组在用药期间未观察到促转移,但治疗一周后停药,总体生存更差,在对照组小鼠全部死亡之前,大剂量组小鼠都已死亡。为了进一步验证此现象,我们以低剂量sorafenib (30mg/kg)在荧光标记的不同转移潜能的人肝癌裸鼠原位模型LM3-RFP(高转移潜能,简称LM3)和HepG2-GFP(低转移潜能,简称HepG2)中重复此试验,发现低剂量sorafenib (30mg/kg)明显促进肝内播散,且不降低LM3模型腹腔转移率和肺转移率。通过流式细胞仪检测外周血肿瘤细胞(circulating tumor cells, CTC),发现低剂量sorafenib (30mg/kg)明显促进CTC增多。2.乏氧导致上皮-间质转化是sorafenib促侵袭转移的重要机理我们通过免疫组化染色内源性乏氧标记物乏氧诱导因子(hypoxia inducible factor-1α, HIF-1α)和外源性乏氧标记物哌莫硝唑(Pimodazole)发现sorafenib治疗组肿瘤组织明显乏氧。乏氧可通过诱导上皮-间质转化(EMT)促进侵袭转移,因此我们在LM3和]HepG2模型中,分别以免疫组化染色EMT相关指标E-cadherin、N-cadherin和Vimentin,发现sorafenib显著下调E-cadherin,同时上调N-cadherin和Vimentin,提示sorafenib治疗后肿瘤发生上皮-间质转化,且侵袭性增强。3. Sorafenib治疗下调HTATIP2等抑癌基因导致凋亡逃脱是重要分子基础我们发现sorafenib治疗后残余肿瘤对凋亡发生抵抗,“肿瘤信号通路发现者PCR芯片”提示6个抑癌基因HTATIP2、SERPINB5、SYK、Rb1、FAS、P53等和1个促凋亡基因肿瘤坏死因子(TNF)均下调,这些下调的基因均为促凋亡基因,且多数与P53有关。逃脱凋亡是恶性肿瘤细胞的6大特征之一,诱导或促进凋亡可抑制肿瘤细胞发生转移。为此我们推测,sorafenib治疗后,抗凋亡基因SERPINB5、SYK、Rb1、FAS、HTATIP2、P53、TNF等的下调,导致肿瘤逃脱凋亡。4.阿司匹林上调HTATIP2等,抑制sorafenib的促侵袭转移作用上述研究提示sorafenib治疗后,抑癌基因HTATIP2、SERPINB5、Rb1、FAS、P53、TNF等均下调,这些抗凋亡基因均与P53有关,且促进侵袭转移。而aspirin具有上调P53和促凋亡作用,为此探索合用aspirin是否可通过P53作为sorafenib的增敏剂。研究结果表明,合用aspirin后显著抑制肿瘤生长、转移,肝内播散灶消失,生存期延长。基因水平检测几种抑癌基因,发现aspirin仅显著上调HTATIP2-mRNA,但对P53-mRNA无影响。蛋白水平检测证实aspirin上调HTATIP2蛋白。我们推测sorafenib诱导的肝内播散灶增多和aspirin逆转sorafenib促肝内播散增多可能主要通过HTATIP2发挥作用。为此选择慢病毒下调的方法,得到慢病毒稳定转染shHTATIP2的细胞株LM3-LV-shHTATIP2和转染相应空载体对照细胞株LM3-LV-shNon,发现LM3-LV-shHTATIP2肿瘤相比LM3-LV-shNon肿瘤边界不清,提示局部侵袭性更强,LM3-LV-shHTATIP2肿瘤相比LM3-LV-shNon肿瘤,在sorafenib治疗下促进肝内播散的作用较弱,而合用aspirin后对sorafenib诱导的肝内播散的抑制作用也较弱(合用aspirin+sorafenib相比单用sorafenib,在LM3-LV-shHTATIP2肿瘤降低50%的肝内播散；而在LM3-LV-shNon肿瘤中降低100%肝内播散)。另外合用aspirin+sorafenib对肺转移的抑制作用,在LM3-LV-shHTATIP2肿瘤中也较LM3-LV-shNon肿瘤中更弱。5.体外用aspirin与sorafenib合用观察促凋亡作用体外实验以sorafenib 5μM作用于LM3细胞,选择不同时间点(0h、3h、6h、12h、24h)观察sorafenib对HTATIP2-mRNA的作用。发现sorafenib 5μM明显下调HTATIP2-mRNA;选择aspirin 0.1mM/0.5mM作用于细胞株LM3-LV-shHTATIP2、LM3-LV-shNon和LM3-wt,选择不同时间点观察,发现aspirin 0.1 mM/0.5 mM均明显上调HTATIP2-mRNA和HTATIP2蛋白；合用sorafenib 5μM+aspirin 0.1mM/0.5mM明显逆转sorafenib 5μM对HTATIP2的抑制作用。Annexin V/PI检测细胞凋亡发现,sorafenib+aspirin明显增强sorafenib对LM3-wt和LM3-LV-shHTATIP2的促凋亡作用。因此证明aspirin主要通过上调HTATIP2增强sorafenib的促凋亡作用。6.巨噬细胞抑制剂减少sorafenib诱导的巨噬细胞浸润而增效在血浆和肿瘤组织中发现sorafenib治疗组巨噬细胞趋化因子VEGF、SDF-1α和CSF-1明显升高,伴随肿瘤组织中F4/80和CD11b阳性巨噬细胞浸润明显增多,以及外周血中F4/80和CD11b阳性细胞数目增多。合用巨噬细胞抑制剂唑来磷酸和氯磷酸盐脂质体通过减少巨噬细胞的浸润进一步抑制肿瘤生长,并减少肺转移。本部分工作提示,肿瘤在sorafenib治疗过程中导致肿瘤相关巨噬细胞通过分泌VEGF对抗sorafenib治疗作用,可用抑制骨转移药物唑来磷酸增强sorafenib对肝癌的抑制作用。结论1. Sorafenib低剂量在不同转移潜能人肝癌原位裸鼠模型中促进肝癌侵袭转移潜能。2. Sorafenib的促侵袭转移作用与下调抑癌基因HTATIP2有关。3. Aspirin上调HTATIP2促肝癌细胞凋亡,有逆转sorafenib的促侵袭转移潜能作用。4.巨噬细胞抑制剂唑来磷酸通过清除sorafenib诱导的巨噬细胞而增效。应用价值1.发现低剂量sorafenib促进肝癌侵袭转移潜能增强,合用aspirin和sorafenib通过上调HTATIP2促进肿瘤细胞凋亡和抑制肿瘤侵袭转移,为提高sorafenib临床疗效提供线索。2.合用巨噬细胞抑制剂唑来磷酸可提高sorafenib对肝癌的抑制作用。3.为今后针对凋亡通路开发抗肿瘤药物,或为寻找sorafenib的合用药物提供依据。创新点1.在人肝癌原位裸鼠模型发现sorafenib通过下调HTATIP2促进肝癌侵袭转移潜能。2.发现合用aspirin通过上调HTATIP2促进凋亡和抑制肿瘤侵袭转移,增强sorafenib对肝癌生长和转移的抑制作用。3.发现巨噬细胞浸润增多为sorafenib治疗逃脱机制之一,清除巨噬细胞可增强sorafenib抑制肝癌作用。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Asia and Africa. Despite endeavors have been made to improve its prognosis, the overall survival, however, is still unsatisfied. The high rate of metastasis and recurrence after curative resection was responsible for the poor prognosis of resectable HCC. Anti-angiogenesis is an attractive treatment for HCC, because HCC is a typical hypervascular cancer, its growth, metastasis or even hepatocarcinogenesis depend on angiogenesis. Angiogenesis inhibitors have been hailed as opening a new era in cancer therapy. But a flurry of animal studies suggests that such drugs may in certain situations actually accelerate the spread of cancer.The phase III sorafenib SHARP trial as well as oriental sorafenib trial finally demonstrated a significant survival benefit, making sorafenib the new reference standard for systemic therapy of patients with advanced HCC. However, the survival benefit and a retardation of tumor progression were only about three months. As an angiogenesis inhibitor, it should be clarified whether sorafenib promote metastasis.Using different orthotopic human hepatocelluar carcinoma nude mice model with deferent metastatic potential, we investigated the mechanism of tumor progression under sorafenib treatment, and the effect of sorafenib on metastatic potential of HCC. As tumor metastasis can be also affected by tumor microenvironment, including the inflammatory cytokines or macrophages. Therefore, we investigated both tumor cell and tumor associated macrophages, and to explore clinical available approach to improve the effect of sorafenib in HCC.1. Low-dose sorafenib promote local invasion and metastatic potential of HCC.The purpose of this part is to investigated different dosage of sorafenib on invasion and metastatic potential of sorafenib with human HCC orthotopic nude mouse model with different metastatic potential. In a patient-like model LCI-D20, we found low-dose sorafenib and high-dose sorafenib significantly inhibited tumor growth and prolonged survival of mice bearing tumors. Sorafenib reduced number of metastatic lesion in lung but did not decrease lung metastatic rate. Because sorafenib-treated tumor was much smaller than that of control, when applying standardized lung metastatic rate (number of lung metastasis/tumor volume) we found sorafenib significantly promoted metastasis potential to lung. Low-dose sorafenib also induced intrahepatic metastasis, but this phenomenon was not observed in high-dose sorafenib group.To confirm this effect, we reconducted sorafenib treatment using red-or green-fluoresce protein infected tumor cells established in our institute LM3-RFP (high metastatic potential) and HepG2-GFP (low metastatic potential) and found that low-dose sorafenib did significantly promoted intrahepatic metastasis with no reduction of abdomen metastatic rate or lung metastatic rate. Low-dose sorafenib also induced significant increased circulating tumor cells as measured by flow cytometry.2. Hypoxia induced EMT is one mechanism of sorafenib to promote invasion and metastatic potential of HCC.Immunohistochemistry staining for HIF-la and Pimodazole revealed significant hypoxia induced by sorafenib. Hypoxia promotes metastasis by inducing EMT. In LM3-RFP and HepG2 model, sorafenib induce significant change in expression of a panel of EMT markers, including decrease of E-cadherin and elevation of N-cadherin and Vimentin, indicating enhanced invasion and metastatic potential of HCC undergoing sorafenib treatment.3. Down regulation of HTATIP2 and evasion of apoptosis induced by sorafenib as important molecular basis.Tumor Pathway Finder PCR Array was applied to screen possible key genes responsible for sorafenib induced invasion and metastatic potential. Twelve genes was significantly changed by sorafenib, and 6 of them were antioncogene HTATIP2、SERPINB5、SYK、Rb1、FAS、P53 and 1 was pro-apoptosis gene TNF. Since most of the antioncogenes were related to P53 and function as pro-apoptosis genes, we postulated that antioncogene down-regulation may help tumor cells evading apoptosis. 4. Aspirin up-regulated HTATIP2 to revert the metastatic promotion effect of sorafenib.Aspirin has been revealed to induce apoptosis by up-regulating P53 and is clinical available, so we combined aspirin with sorafenib. Combination of aspirin and sorafenib significantly suppressed tumor growth and reduced lung metastasis in HCC model, and intrahepatic metastasis induced by sorafenib disappeared after combined with aspirin. RT-PCR was applied to evaluate gene level change of antioncogene and found aspirin only up-regulated HTATIP2-mRNA, but P53 and other antioncogenes were unaffected by aspirin. Down-regulation of HTATIP2 was also confirmed by western blotting at protein level. So we postulated that HTATIP2 was responsible for anti-metastatic potential effect of aspirin when combined with sorafenib.We constructed LM3 cells infected with LV-sh HTATIP2, LV-shNon, and started treatment with sorafenib or combination of sorafenib and aspirin after orthotopically inoculation. We found LM3-LV-shHTATIP2 model had more invasive tumor compared with LM3-wt tumor, and sorafenib did not induce more intrahepatic metastasis compared to control. And after treat by combination of aspirin, intrahepatic metastasis was reduced to a less extent compared with that in the LM3-wt model (50% vs.100). Aspirin also reduced lung metastasis in combination with sorafenib, but to a less extent compared with that in the LM3-wt model.5. In vitro experiments demonstrated apoptosis induction effect of combination of aspirin and sorafenib.We treated LM3 cells with sorafenib 5μM by a time course of 0h,3h,6h,9h and 12h, and revealed a reduction of HTATIP2 was most prominent in 3-6h. LM3-LV-shHTATIP2, LM3-LV-shNon and LM3-wt cell lines were exposes to 5μM sorafenib or 0.1 mM/0.5 mM aspirin, HTATIP2-mRNA was measured by RT-PCR and apoptosis were evaluated by Annexin V-PI using flow cytometry. Aspirin reverted down-regulation of HTATIP2 by sorafenib, enhanced apoptosis and reduced invasion cells when combined with sorafenib compared to sorafenib alone. 6. Macrophage depletion enhanced effect of sorafenib by antiangiogenic and antimetastatic effect.Although sorafenib significantly inhibited tumor growth, it induced elevation of F4/80-and CD11b-positive cells in peripheral blood and increased infiltration of intratumoral macrophages, which was associated with elevation of plasma VEGF, SDF-la and CSF-1, suggesting that macrophages played a role in the progression of tumor undergoing sorafenib treatment. With depletion of macrophages by clodrolip or zoledronic acid, tumor progression, tumor angiogenesis, and metastasis were significantly inhibited compared with that of mice treated by sorafenib alone. Zoledronic acid is clinical available and is promising to be combined with sorafenib for HCC patients.Conclusions:1. Low-dose sorafenib promoted invasion and metastatic potential in human HCC nude mouse models with different metastatic potential.2. HTATIP2 down-regulation contributed to enhanced metastatic potential of HCC induced by sorafenib.3. Combination of aspirin with sorafenib promoted apoptosis and reverted sorafenib’s pro-invasion and pro-metastasis effect by down-regulating HTATIP2.4. Zoledronic acid enhanced antitumor effect of sorafenib by macrophage depletion.The novelties of this work:1. Sorafenib promoted metastatic potential by down-regulating HTATIP2 in two orthotopic human HCC nude mouse models.2. Combination of aspirin enhanced antitumor effect of sorafenib by promoting apoptosis and inhibiting invasion and metastasis through up-regulating HTATIP2.3. Macrophage recruitment and infiltration contributed to evasive resistance in mice undergoing sorafenib treatment. Macrophage depletion enhanced antitumor effect of sorafenib.更多还原