癌蛋白p28~(GANK)和酪氨酸激酶Pyk2对肝细胞癌复发转移的影响和机制研究
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摘要
第一部分癌蛋白p28GANK对肝细胞癌复发转移的影响及机制研究
研究背景和目的
原发性肝癌(primary liver cancer, PLC,以下简称肝癌)是临床上最常见的消化系统恶性肿瘤之一,居全球恶性肿瘤发病率第五位,居肿瘤相关死亡的第3位。90%的原发性肝癌为肝细胞性肝癌(hepacoeellular carcinoma, HCC)。我国是肝癌高发区之一在肿瘤相关死亡中仅次于肺癌,位居第二。近年来,以手术为主的综合治疗显著提高了肝癌的疗效。根治性的肝癌切除及肝脏移植术后5年生存率可达70%左右。但即使如此,术后5年复发率也在60%以上,小肝癌达40%-50%。高达90%以上的肝癌患者术后死亡因素与肿瘤复发转移有关。由此可见,肝癌的复发转移是阻碍肝癌患者获得远期生存的重要因素。肝癌的转移机制非常复杂,涉及到多种粘附分子、基质蛋白酶、细胞因子以及相应的信号转导机制和相关的基因改变。肿瘤转移的分子生物学机制研究表明,肝癌转移基因、转移相关基因的激活与转移抑制基因的失活可导致肝癌的侵袭转移表型。所以,从基因水平认识肝癌的发生、发展和转移的分子机制,并寻找干预策略是肝癌诊疗领域的重要课题。
2000年日本科学家Fujita等通过差减杂交法从人的肝癌组织cDNA文库中发现了一个在人肝癌组织中高表达的癌基因--p28GANK(又称gankyrin、PMSD10,简称p28)。我室于2001年克隆了该基因,同时发现和克隆了它在肝癌内的缺失突变体p28-Ⅱ(GenBank:AY057056,获国家发明专利：ZL 011 26927.8)；并通过大量的临床肝癌样本检测,确认其表达蛋白p28GANK为肝癌中相对特异性高表达的癌蛋白。p28GANK可能与甲胎蛋白AFP相似,在正常成人肝脏内呈沉默状态,而在肝硬化发生时开始启动,可能参与了由肝硬化向肝癌病理改变过程。我组前期研究发现利用RNA干扰技术下调肝癌细胞中p28的表达,可明显抑制细胞的增殖并可在体内外诱发肿瘤细胞凋亡,论文发表于国际胃肠病学领域最有影响力的期刊《Gastroenterology》。分子机制上研究表明,p28GANK主要参与了CDK4-Rb-E2F-1、MDM2-p53以及NF-κB等重要信号途径的调控,调控细胞周期进程、促进细胞增殖并抑制细胞凋亡,这些预示该基因在肝细胞增殖、肝再生以及肝癌发生发展中具有极为重要的作用。
随着研究的深入,新近研究发现p28的表达水平与食管癌及结肠癌患者的预后密切相关,这提示癌蛋白p28可能在消化道系统的肿瘤发生发展中发挥着重要作用,并与肿瘤的复发转移及预后密切相关。然而,p28对肝癌复发转移的影响目前尚未见报道。本课题将在原有研究结果的基础上结合体内外实验及临床样本资料,明确癌蛋白p28对肝癌复发转移的影响,探讨相关的调控机制,为肝癌防治提供新的思路和靶标。
研究方法
1.随机选取2001.9～2006.7年在我院行肝癌根治性切除术的原发性肝细胞癌患者标本201例,并收集完善病史及随访资料,制作组织芯片；
2.用免疫组化方法检测组织芯片中p28的表达,并统计分析p28的表达与肝癌患者各项临床特征(肿瘤大小、血管侵犯、肝内播散和远处转移等)及预后(无瘤生存率和总生存率)的关系；
3.选用不同的肝癌细胞系(SMMC-7721, MHCC-97L, HCC-LM3, HepG2等),以慢病毒过表达或下调肝癌细胞中p28的表达,体外检测p28对肝癌细胞迁移、粘附及侵袭能力的影响；
4.选用具有不同转移潜能的肝癌细胞系(SMMC-7721, MHCC-97L, HCC-LM3等)通过慢病毒介导的过表达或下调p28的表达,建立裸鼠肝脏原位荷瘤的肺转移模型,检测p28对肝癌细胞转移能力的影响；
5.p28调控肝癌复发转移分子机制研究：运用基因芯片筛选p28过表达或下调后复发转移相关分子的变化并以Western Blot及免疫组化方法进行验证；对验证得到的相关分子进行分析,探寻p28调控复发转移的机理。
结果
1.201例HCC组织芯片结果显示癌组织p28蛋白表达水平明显高于癌旁组织(P<0.001)；癌组织中p28表达水平与肿瘤大小、血管侵犯、肝内播散及术后远处转移密切相关。高表达p28病人的总体生存率和无瘤生存率明显低于低表达者(P=0.007,P=0.002)；
2.p28在伴有门脉癌栓及肝内血管侵犯的肝癌样本中具有更高的表达水平。划痕和Transwell等体外实验显示p28能促进肝癌细胞的迁移、粘附和侵袭能力；以慢病毒过表达p28处理的肝癌细胞进行裸鼠皮下荷瘤、肝内包埋,四周后肺内转移灶的数目增多,促血管生成趋势明显；
3.肝癌标本免疫组化结果提示p28与VEGF表达水平呈正相关(P<0.001)。SMMC-7721, MHCC-97L过表达p28后VEGF相应表达增强,提示p28可能通过调控VEGF表达影响了肿瘤的血管形成。
结论经201例病理样本显示,p28在HCC癌组织中的表达显著高于癌旁,且与血管侵犯、肝内播散及术后远处转移等因素密切相关。高表达p28的病人总体生存率和无瘤生存率均较低。体内外实验表明p28能增强肝癌细胞的迁移、粘附、侵袭和转移能力。此外,p28还可以增强肝癌细胞的血管生成能力,促进VEGF表达,这提示p28发挥促进肝癌复发转移可能与其调节VEGF的表达有关。
第二部分蛋白酪氨酸激酶Pyk2对肝细胞癌复发转移的影响和机制研究
研究背景和目的
Pyk2是一种非受体型蛋白酪氨酸激酶,也被称为蛋白酪氨酸激酶2/细胞粘附激酶β(proline—rich tyrosine kinase 2 /cellular adhesion kinaseβ, PYK2/CAKβ),是粘着斑激酶家族FAK (focal adhesion kinase)的新成员,主要在脑细胞,成纤维细胞、血小板及其它造血细胞的胞浆表达。Pyk2基因cDNA全长为4048bp,编码1009个氨基酸,分子量116kD,与FAK具有高度的同源序列(45%),它编码的蛋白与FAK具有相似的蛋白结构。FAK对肿瘤的增殖、生存、迁移、侵袭、转移和血管形成等行为起着重要的调控作用。目前已经证实,FAK在结肠癌、乳腺癌、膀胱癌等多种肿瘤组织中过度表达,FAK的高表达和酪氨酸磷酸化与肿瘤生成能力、侵袭和转移密切相关,这提示FAK可能是肿瘤发生发展中的一个重要环节。目前为止,有关Pyk2在肿瘤中的作用报道并不一致。一些研究表明,多种恶性肿瘤细胞的粘附、转移和浸润能力的变化与Pyk2的表达增加或改变有关。Pyk2能促进神经胶质瘤细胞的迁移和浸润,调节肺血管内皮细胞的增生；还能调节前列腺细胞的增生和分化,但其表达水平与前列腺癌的恶性程度呈负相关。过表达的Pyk2在乳腺癌、星形细胞瘤中有报道。相反,也有学者研究发现在晚期骨肉瘤、胃癌和高分化的前列腺癌中Pyk2相对正常组织低表达。然而,Pyk2在肝癌中的研究报道较少。信号网络调控方面,Pyk2调控JNK和Ras-MAPK通路。目前研究发现Pyk2是VEGF介导的P38活化的关键分子,P38的激活依赖Pyk2和Src的参与。在脑星形细胞瘤中FAK/Pyk2可能通过激活PI3K/AKT信号通路,进而诱导VEGF合成,促使肿瘤内血管密度增加。
本实验旨在通过组织芯片研究Pyk2在肝癌及癌旁组织中的表达特点及它对复发转移和预后的影响,并探讨相应的调控机制。
实验方法
1.随机选取2001.9～2006.7在我院行肝癌根治术的原发性肝细胞癌患者标本141例(包括癌及相应的癌旁肝组织),制作组织芯片,并收集完善病史及随访资料；
2.以免疫组化方法检测组织芯片标本中Pyk2的表达,统计分析肝癌及癌旁组织Pyk2的表达与肝癌患者各项临床特征(肿瘤大小、血管侵犯、肝内播散和远处转移等)及生存(无瘤生存率、总生存率和复发时间等)的关系；
3.免疫组化方法检测肝癌组织样本中VEGF与Pyk2表达的相关性；
4.运用正常肝细胞系HL-7702外转Pyk2或抑制型PRNK(Pyk2缺失了C端激酶区域的剪接体),检测转移相关分子的变化,探讨调节机制。
结果
1.141例HCC样本分析发现,癌和癌旁组织间Pyk2的表达无明显差异；高表达Pyk2的病人生存时间短,术后易出现远处转移(P<0.001)。但是,与癌组织相比,癌旁Pyk2的表达水平较癌组织对预测术后复发,尤其是晚期复发更有意义；
2.组织芯片结果显示癌或癌旁组织中相应的Pyk2和VEGF的表达水平呈正相关(P<0.001)；体外细胞实验发现Pyk2在蛋白和mRNA水平均能上调VEGF的表达；
3. HL-7702细胞过表达Pyk2后AKT磷酸化增加,VEGF表达增强。而转染PRNK后,AKT磷酸化减弱,VEGF表达减少。PI3K/AKT通路的抑制剂LY294002可以抑制Pyk2对VEGF的活化。
结论141例HCC样本经组织芯片分析发现,癌和癌旁之间Pyk2的表达无明显差异。癌
或癌旁组织中Pyk2表达水平的高低与病人术后是否出现远处转移及总体生存率密切
相关,但癌旁组织Pyk2的水平较癌组织对预测术后复发,尤其是晚期复发更有意义。
本实验还发现在肝癌组织中Pyk2与VEGF的表达密切相关；体外实验结果表明Pyk2通
过激活PI3K/AKT信号通路调控VEGF。Pyk2是否通过这一途径促进HCC的复发和转
移有待进一步研究证实。
PartⅠOncoprotein p28GANK Promotes Hepatocellular Carcinoma Progression and Metastasis
Primary liver cancer (PLC) is one of most prevalent malignant digestive cancers, the fifth most common cancer worldwide and the third most common cause of cancer-related death.90% PLC is hepacocellular carcinoma (HCC). PLC is most prevalent in our country and the second most common cause of cancer-related death, second only to lung cancer. The death cause of 90% HCC patients is related to tumor recurrence and metastasis after hepatectomy. Therefore, many biomarkers for prediction and intervention, mainly HCC metastasis-related chromosomes, genes, and proteins have been tried.
It has been previously demonstrated that oncoprotein p28GANK (Gankyrin, PSMD10 or p28), commonly amplified in HCC, plays an important role in hepatocarcinogenesis by destabilizing Rb and p53, but its functional role in HCC metastases has remained unexplored. Here we reported that increased p28GANK in clinical samples of human HCC tissues after hepatohectomy predicted marked poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that HCC patients with large tumors, vascular invasion, intrahepatic metastatic and distant metastatic tumor were prone to have higher p28GANK expression. Subsequent investigation revealed that tumors overexpressing p28GANK were more invasive, and exhibited increased angiogenesis associated with VEGF activation, whereas silencing endogenous p28GANK expression reduced motility/invasion. Taken together, it suggests that p28GANK plays an important role in recurrence and metastasis process.
PartⅡThe Role of Proline-rich Tyrosine Kinase 2 (Pyk2) in Hepatocellular Carcinoma Recurrence and Metastasis
Pyk2 (proline-rich tyrosine kinase-2), also known as RAFTK, CAK, and CADTK, is a non-receptor, proline-rich protein tyrosine kinase that is expressed in the cytoplasm of different cell types including brain cells, fibroblasts, platelets, and other hemopoietic cells. As a new member of focal adhesion kinase family, Pyk2 exhibits a high level (45% amino acid sequence) of sequence homology and similar structural organization to FAK. FAK has been reported to play a role on regulation of proliferation, survival, migration, invasion, metastasis and angiopoiesis in cancer cells. Pyk2 is an important signaling protein, serving as a upstream mediator of the JNK (c-Jun N-terminal kinase) signaling pathway as well as the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. Overexpression or altered activity of Pyk2 has been linked to changes in cell adhesion, as well as invasion and metastasis of various malignancies, such as glioma or prostate cancers. However, the role of Pyk2 in HCCs has been unclear.
In current study, we found that increased expression of Pyk2 in human HCC predicts poor survival and disease recurrence after surgery. HCC patients having large tumors, with vascular invasion and intrahepatic or distant metastasis, expressed high levels of Pyk2. Invasive tumors expressing Pyk2 exhibited increased vascular endothelia growth factor (VEGF) overexpression. Furthermore, Pyk2 activates AKT to promote VEGF promotion. Consistently, we detected a significant correlation between Pyk2 and VEGF levels in a cohort of HCC biopsie. Therefore, we proposed that Pyk2 plays a key role in HCC recurrence and metastasis via PI3K/AKT signaling pathway to induce VEGF expression.
研究背景和目的
原发性肝癌(primary liver cancer, PLC,以下简称肝癌)是临床上最常见的消化系统恶性肿瘤之一,居全球恶性肿瘤发病率第五位,居肿瘤相关死亡的第3位。90%的原发性肝癌为肝细胞性肝癌(hepacoeellular carcinoma, HCC)。我国是肝癌高发区之一在肿瘤相关死亡中仅次于肺癌,位居第二。近年来,以手术为主的综合治疗显著提高了肝癌的疗效。根治性的肝癌切除及肝脏移植术后5年生存率可达70%左右。但即使如此,术后5年复发率也在60%以上,小肝癌达40%-50%。高达90%以上的肝癌患者术后死亡因素与肿瘤复发转移有关。由此可见,肝癌的复发转移是阻碍肝癌患者获得远期生存的重要因素。肝癌的转移机制非常复杂,涉及到多种粘附分子、基质蛋白酶、细胞因子以及相应的信号转导机制和相关的基因改变。肿瘤转移的分子生物学机制研究表明,肝癌转移基因、转移相关基因的激活与转移抑制基因的失活可导致肝癌的侵袭转移表型。所以,从基因水平认识肝癌的发生、发展和转移的分子机制,并寻找干预策略是肝癌诊疗领域的重要课题。
2000年日本科学家Fujita等通过差减杂交法从人的肝癌组织cDNA文库中发现了一个在人肝癌组织中高表达的癌基因--p28GANK(又称gankyrin、PMSD10,简称p28)。我室于2001年克隆了该基因,同时发现和克隆了它在肝癌内的缺失突变体p28-Ⅱ(GenBank:AY057056,获国家发明专利：ZL 011 26927.8)；并通过大量的临床肝癌样本检测,确认其表达蛋白p28GANK为肝癌中相对特异性高表达的癌蛋白。p28GANK可能与甲胎蛋白AFP相似,在正常成人肝脏内呈沉默状态,而在肝硬化发生时开始启动,可能参与了由肝硬化向肝癌病理改变过程。我组前期研究发现利用RNA干扰技术下调肝癌细胞中p28的表达,可明显抑制细胞的增殖并可在体内外诱发肿瘤细胞凋亡,论文发表于国际胃肠病学领域最有影响力的期刊《Gastroenterology》。分子机制上研究表明,p28GANK主要参与了CDK4-Rb-E2F-1、MDM2-p53以及NF-κB等重要信号途径的调控,调控细胞周期进程、促进细胞增殖并抑制细胞凋亡,这些预示该基因在肝细胞增殖、肝再生以及肝癌发生发展中具有极为重要的作用。
随着研究的深入,新近研究发现p28的表达水平与食管癌及结肠癌患者的预后密切相关,这提示癌蛋白p28可能在消化道系统的肿瘤发生发展中发挥着重要作用,并与肿瘤的复发转移及预后密切相关。然而,p28对肝癌复发转移的影响目前尚未见报道。本课题将在原有研究结果的基础上结合体内外实验及临床样本资料,明确癌蛋白p28对肝癌复发转移的影响,探讨相关的调控机制,为肝癌防治提供新的思路和靶标。
研究方法
1.随机选取2001.9～2006.7年在我院行肝癌根治性切除术的原发性肝细胞癌患者标本201例,并收集完善病史及随访资料,制作组织芯片；
2.用免疫组化方法检测组织芯片中p28的表达,并统计分析p28的表达与肝癌患者各项临床特征(肿瘤大小、血管侵犯、肝内播散和远处转移等)及预后(无瘤生存率和总生存率)的关系；
3.选用不同的肝癌细胞系(SMMC-7721, MHCC-97L, HCC-LM3, HepG2等),以慢病毒过表达或下调肝癌细胞中p28的表达,体外检测p28对肝癌细胞迁移、粘附及侵袭能力的影响；
4.选用具有不同转移潜能的肝癌细胞系(SMMC-7721, MHCC-97L, HCC-LM3等)通过慢病毒介导的过表达或下调p28的表达,建立裸鼠肝脏原位荷瘤的肺转移模型,检测p28对肝癌细胞转移能力的影响；
5.p28调控肝癌复发转移分子机制研究：运用基因芯片筛选p28过表达或下调后复发转移相关分子的变化并以Western Blot及免疫组化方法进行验证；对验证得到的相关分子进行分析,探寻p28调控复发转移的机理。
结果
1.201例HCC组织芯片结果显示癌组织p28蛋白表达水平明显高于癌旁组织(P<0.001)；癌组织中p28表达水平与肿瘤大小、血管侵犯、肝内播散及术后远处转移密切相关。高表达p28病人的总体生存率和无瘤生存率明显低于低表达者(P=0.007,P=0.002)；
2.p28在伴有门脉癌栓及肝内血管侵犯的肝癌样本中具有更高的表达水平。划痕和Transwell等体外实验显示p28能促进肝癌细胞的迁移、粘附和侵袭能力；以慢病毒过表达p28处理的肝癌细胞进行裸鼠皮下荷瘤、肝内包埋,四周后肺内转移灶的数目增多,促血管生成趋势明显；
3.肝癌标本免疫组化结果提示p28与VEGF表达水平呈正相关(P<0.001)。SMMC-7721, MHCC-97L过表达p28后VEGF相应表达增强,提示p28可能通过调控VEGF表达影响了肿瘤的血管形成。
结论经201例病理样本显示,p28在HCC癌组织中的表达显著高于癌旁,且与血管侵犯、肝内播散及术后远处转移等因素密切相关。高表达p28的病人总体生存率和无瘤生存率均较低。体内外实验表明p28能增强肝癌细胞的迁移、粘附、侵袭和转移能力。此外,p28还可以增强肝癌细胞的血管生成能力,促进VEGF表达,这提示p28发挥促进肝癌复发转移可能与其调节VEGF的表达有关。
第二部分蛋白酪氨酸激酶Pyk2对肝细胞癌复发转移的影响和机制研究
研究背景和目的
Pyk2是一种非受体型蛋白酪氨酸激酶,也被称为蛋白酪氨酸激酶2/细胞粘附激酶β(proline—rich tyrosine kinase 2 /cellular adhesion kinaseβ, PYK2/CAKβ),是粘着斑激酶家族FAK (focal adhesion kinase)的新成员,主要在脑细胞,成纤维细胞、血小板及其它造血细胞的胞浆表达。Pyk2基因cDNA全长为4048bp,编码1009个氨基酸,分子量116kD,与FAK具有高度的同源序列(45%),它编码的蛋白与FAK具有相似的蛋白结构。FAK对肿瘤的增殖、生存、迁移、侵袭、转移和血管形成等行为起着重要的调控作用。目前已经证实,FAK在结肠癌、乳腺癌、膀胱癌等多种肿瘤组织中过度表达,FAK的高表达和酪氨酸磷酸化与肿瘤生成能力、侵袭和转移密切相关,这提示FAK可能是肿瘤发生发展中的一个重要环节。目前为止,有关Pyk2在肿瘤中的作用报道并不一致。一些研究表明,多种恶性肿瘤细胞的粘附、转移和浸润能力的变化与Pyk2的表达增加或改变有关。Pyk2能促进神经胶质瘤细胞的迁移和浸润,调节肺血管内皮细胞的增生；还能调节前列腺细胞的增生和分化,但其表达水平与前列腺癌的恶性程度呈负相关。过表达的Pyk2在乳腺癌、星形细胞瘤中有报道。相反,也有学者研究发现在晚期骨肉瘤、胃癌和高分化的前列腺癌中Pyk2相对正常组织低表达。然而,Pyk2在肝癌中的研究报道较少。信号网络调控方面,Pyk2调控JNK和Ras-MAPK通路。目前研究发现Pyk2是VEGF介导的P38活化的关键分子,P38的激活依赖Pyk2和Src的参与。在脑星形细胞瘤中FAK/Pyk2可能通过激活PI3K/AKT信号通路,进而诱导VEGF合成,促使肿瘤内血管密度增加。
本实验旨在通过组织芯片研究Pyk2在肝癌及癌旁组织中的表达特点及它对复发转移和预后的影响,并探讨相应的调控机制。
实验方法
1.随机选取2001.9～2006.7在我院行肝癌根治术的原发性肝细胞癌患者标本141例(包括癌及相应的癌旁肝组织),制作组织芯片,并收集完善病史及随访资料；
2.以免疫组化方法检测组织芯片标本中Pyk2的表达,统计分析肝癌及癌旁组织Pyk2的表达与肝癌患者各项临床特征(肿瘤大小、血管侵犯、肝内播散和远处转移等)及生存(无瘤生存率、总生存率和复发时间等)的关系；
3.免疫组化方法检测肝癌组织样本中VEGF与Pyk2表达的相关性；
4.运用正常肝细胞系HL-7702外转Pyk2或抑制型PRNK(Pyk2缺失了C端激酶区域的剪接体),检测转移相关分子的变化,探讨调节机制。
结果
1.141例HCC样本分析发现,癌和癌旁组织间Pyk2的表达无明显差异；高表达Pyk2的病人生存时间短,术后易出现远处转移(P<0.001)。但是,与癌组织相比,癌旁Pyk2的表达水平较癌组织对预测术后复发,尤其是晚期复发更有意义；
2.组织芯片结果显示癌或癌旁组织中相应的Pyk2和VEGF的表达水平呈正相关(P<0.001)；体外细胞实验发现Pyk2在蛋白和mRNA水平均能上调VEGF的表达；
3. HL-7702细胞过表达Pyk2后AKT磷酸化增加,VEGF表达增强。而转染PRNK后,AKT磷酸化减弱,VEGF表达减少。PI3K/AKT通路的抑制剂LY294002可以抑制Pyk2对VEGF的活化。
结论141例HCC样本经组织芯片分析发现,癌和癌旁之间Pyk2的表达无明显差异。癌
或癌旁组织中Pyk2表达水平的高低与病人术后是否出现远处转移及总体生存率密切
相关,但癌旁组织Pyk2的水平较癌组织对预测术后复发,尤其是晚期复发更有意义。
本实验还发现在肝癌组织中Pyk2与VEGF的表达密切相关；体外实验结果表明Pyk2通
过激活PI3K/AKT信号通路调控VEGF。Pyk2是否通过这一途径促进HCC的复发和转
移有待进一步研究证实。
PartⅠOncoprotein p28GANK Promotes Hepatocellular Carcinoma Progression and Metastasis
Primary liver cancer (PLC) is one of most prevalent malignant digestive cancers, the fifth most common cancer worldwide and the third most common cause of cancer-related death.90% PLC is hepacocellular carcinoma (HCC). PLC is most prevalent in our country and the second most common cause of cancer-related death, second only to lung cancer. The death cause of 90% HCC patients is related to tumor recurrence and metastasis after hepatectomy. Therefore, many biomarkers for prediction and intervention, mainly HCC metastasis-related chromosomes, genes, and proteins have been tried.
It has been previously demonstrated that oncoprotein p28GANK (Gankyrin, PSMD10 or p28), commonly amplified in HCC, plays an important role in hepatocarcinogenesis by destabilizing Rb and p53, but its functional role in HCC metastases has remained unexplored. Here we reported that increased p28GANK in clinical samples of human HCC tissues after hepatohectomy predicted marked poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that HCC patients with large tumors, vascular invasion, intrahepatic metastatic and distant metastatic tumor were prone to have higher p28GANK expression. Subsequent investigation revealed that tumors overexpressing p28GANK were more invasive, and exhibited increased angiogenesis associated with VEGF activation, whereas silencing endogenous p28GANK expression reduced motility/invasion. Taken together, it suggests that p28GANK plays an important role in recurrence and metastasis process.
PartⅡThe Role of Proline-rich Tyrosine Kinase 2 (Pyk2) in Hepatocellular Carcinoma Recurrence and Metastasis
Pyk2 (proline-rich tyrosine kinase-2), also known as RAFTK, CAK, and CADTK, is a non-receptor, proline-rich protein tyrosine kinase that is expressed in the cytoplasm of different cell types including brain cells, fibroblasts, platelets, and other hemopoietic cells. As a new member of focal adhesion kinase family, Pyk2 exhibits a high level (45% amino acid sequence) of sequence homology and similar structural organization to FAK. FAK has been reported to play a role on regulation of proliferation, survival, migration, invasion, metastasis and angiopoiesis in cancer cells. Pyk2 is an important signaling protein, serving as a upstream mediator of the JNK (c-Jun N-terminal kinase) signaling pathway as well as the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. Overexpression or altered activity of Pyk2 has been linked to changes in cell adhesion, as well as invasion and metastasis of various malignancies, such as glioma or prostate cancers. However, the role of Pyk2 in HCCs has been unclear.
In current study, we found that increased expression of Pyk2 in human HCC predicts poor survival and disease recurrence after surgery. HCC patients having large tumors, with vascular invasion and intrahepatic or distant metastasis, expressed high levels of Pyk2. Invasive tumors expressing Pyk2 exhibited increased vascular endothelia growth factor (VEGF) overexpression. Furthermore, Pyk2 activates AKT to promote VEGF promotion. Consistently, we detected a significant correlation between Pyk2 and VEGF levels in a cohort of HCC biopsie. Therefore, we proposed that Pyk2 plays a key role in HCC recurrence and metastasis via PI3K/AKT signaling pathway to induce VEGF expression.
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