Ling Ling, Tan Si Kee, Goh Ting Hwee, Cheung Edwin, Nurcombe Victor, van Wijnen Andre J, Cool Simon M
Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore.
Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, #02-01 Genome, Singapore, 138672, Singapore.
Mol Cancer. 2015 Jul 23;14:136. doi: 10.1186/s12943-015-0391-4.
Aberrant activation of fibroblast growth factor receptors (FGFRs) deregulates cell proliferation and promotes cell survival, and may predispose to tumorigenesis. Therefore, selective inactivation of FGFRs is an important strategy for cancer therapy. Here as a proof-of-concept study, we developed a FGFR1 neutralizing antisera, IMB-R1, employing a novel strategy aimed at preventing the access of essential heparan sulfate (HS) co-receptors to the heparin-binding domain on FGFR1.
The mRNA and protein expression level of FGFR1 and other FGFRs were examined in several lines of breast cancer and osteosarcoma cells and corresponding normal cells using Taqman real-time quantitative PCR and Western blot analysis. The specificity of IMB-R1 against FGFR1 was assessed with various ELISA-based approaches and Receptor Tyrosine Kinase array. Proliferation assay and apoptosis analysis were performed to assess the effect of IMB-R1 on cancer cell growth and apoptosis, respectively, in comparison with known FGFR1 inhibitors. The IMB-R1 induced alteration of intracellular signaling and gene expression were analysed using Western blot and microarray approaches. Immunohistochemical staining of FGFR1 using IMB-R1 were carried out in different cancer tissues from clinical patients. Throughout the study, statistical differences were determined by Student's t test where appropriate and reported when a p value was less than 0.05.
We demonstrate that IMB-R1 is minimally cross-reactive for other FGFRs, and that it potently and specifically inhibits binding of heparin to FGFR1. Furthermore, IMB-R1 blocks the interaction of FGF2 with FGFR1, the kinase activity of FGFR1 and activation of intracellular FGFR signaling. Cancer cells treated with IMB-R1 displayed impaired FGF2 signaling, were unable to grow and instead underwent apoptosis. IMB-R1-induced cell death correlated with a disruption of antioxidative defense networks and increased expression of several tumor suppressors and apoptotic proteins, including p53. Immunostaining with IMB-R1 was stronger in human cancer tissues in which the FGFR1 gene is amplified.
Our study suggests that blocking HS interaction with the heparin-binding domains of FGFR1 inhibited cancer cell growth, which can be an attractive strategy to inactivate cancer-related heparin-binding proteins.
成纤维细胞生长因子受体（FGFRs）的异常激活会使细胞增殖失调并促进细胞存活，可能易引发肿瘤发生。因此，选择性失活FGFRs是癌症治疗的一项重要策略。在此，作为一项概念验证研究，我们采用了一种旨在阻止必需的硫酸乙酰肝素（HS）共受体与FGFR1上的肝素结合域结合的新策略，研发出了一种FGFR1中和抗血清IMB-R1。
使用Taqman实时定量PCR和蛋白质印迹分析，检测了几种乳腺癌和骨肉瘤细胞系以及相应正常细胞中FGFR1和其他FGFRs的mRNA和蛋白质表达水平。采用各种基于酶联免疫吸附测定（ELISA）的方法和受体酪氨酸激酶芯片评估IMB-R1对FGFR1的特异性。与已知的FGFR1抑制剂相比，分别进行增殖测定和凋亡分析，以评估IMB-R1对癌细胞生长和凋亡的影响。使用蛋白质印迹和基因芯片方法分析IMB-R1诱导的细胞内信号传导和基因表达变化。在临床患者的不同癌组织中，使用IMB-R1进行FGFR1的免疫组织化学染色。在整个研究中，适当情况下通过学生t检验确定统计学差异，当p值小于0.05时进行报告。
我们证明IMB-R1对其他FGFRs的交叉反应性极小，并且它能有效且特异性地抑制肝素与FGFR1的结合。此外，IMB-R1阻断FGF2与FGFR1的相互作用、FGFR1的激酶活性以及细胞内FGFR信号传导的激活。用IMB-R1处理的癌细胞显示FGF2信号传导受损，无法生长，而是发生凋亡。IMB-R1诱导的细胞死亡与抗氧化防御网络的破坏以及包括p53在内的几种肿瘤抑制因子和凋亡蛋白的表达增加相关。在FGFR1基因扩增的人类癌组织中，用IMB-R1进行的免疫染色更强。
我们的研究表明，阻断HS与FGFR1的肝素结合域的相互作用可抑制癌细胞生长，这可能是使癌症相关肝素结合蛋白失活的一种有吸引力的策略。
