Selective KGFR Antagonists for the Prevention of Cancer Metastasis

背景：成年雌性动物的乳腺对KGF非常敏感。KGF作用于KGFR，对乳腺癌细胞增殖和运动产生快速而深刻的刺激。此外，kgf诱导的乳腺癌细胞运动是通过Erk1/2信号通路介导的。因此，KGF/KGFR信号转导的增强可能是乳腺癌转移进展的早期步骤。KGFR受体模型用于鉴定具有高受体亲和力的KGFR TKI分子库。已经合成了40个KGFR TKI，可用于研究KGFR信号在乳腺癌转移中的作用。假设：KGFR是乳腺癌的重要治疗靶点；因此，选择性抑制kgfr介导的信号传导将减少或消除乳腺癌细胞的增殖、运动和转移。特异性目的：第一个特异性目的将测量40 KGFR TKI对人乳腺癌细胞中kgf介导的KGFR信号转导的相对活性和特异性。第二个特定目标将检查这40种抑制剂在体外减少kgf诱导的乳腺癌细胞增殖和运动的能力。第三个特异性目标将确定最有选择性和最有效的KGFR TKI（在体外特异性目标#1和2中确定）在小鼠异种移植模型中对kgf介导的人乳腺癌细胞生长和转移的抑制作用。方法：在特异性目标#1中，MCF-7乳腺癌细胞将接受KGF （50 ng/ml）或KGFR TKI（剂量范围为10至200 μ M）或KGF (50 ng/ml) + KGFR TKI（每种抑制剂剂量）或易瑞沙（表皮生长因子受体选择性TK抑制剂；用作阴性对照）在相同剂量范围内或KGF +易瑞沙（相同剂量）或载体对照。在KGF给药后1至48小时的不同时间，收集细胞，通过Western blotting检测KGFR、phospho-KGFR、Erk1/2、phospho-Erk1/2和¿-actin的蛋白表达。在特异性目标#2中，MCF-7细胞的增殖和迁移将在1至3天的时间内通过延时视频显微镜和培养损伤试验进行评估，使用与第一个特异性目标中描述的实验组和剂量相同。在特异性目的#3中，将确定抑制KGFR对肿瘤移植生长和转移的影响。异种移植研究将通过将MCF-7细胞植入裸鼠乳腺脂肪垫进行。实验结束后，取肿瘤、肺、肝切片冷冻，荧光显微镜观察肿瘤的生长及肺、肝微转移的发展情况。意义：KGFR作为重要的治疗靶点的确立，以及特异性强效KGFR TKI的发现，应该会导致一类新的高选择性治疗药物的快速发展，以防止癌症的转移进展。KGFR作为一个重要的治疗靶点的确立，以及特异性和强效的KGFR TKI的鉴定，应该会导致一类新的高选择性治疗药物的快速发展，旨在防止癌症的转移进展。该项目涉及国家癌症研究所的几个重点领域；也就是说，癌症转移的生物学和高选择性和分子靶向治疗剂的发展。

DESCRIPTION (provided by applicant): Background: The mammary glands of adult female animals are remarkably sensitive to KGF. KGF acts at the KGFR to produce a rapid and profound stimulation of breast cancer cell proliferation and motility. Further, KGF-induced motility in breast cancer cells is mediated via the Erk1/2 signaling pathway. Thus, enhancement of KGF/KGFR signal transduction may be an early step in the metastatic progression of breast cancer. Receptor modeling of KGFR was used to identify a library of selective KGFR TKI molecules with high receptor affinity. Forty of the KGFR TKI have been synthesized and are available to study the involvement of KGFR signaling in breast cancer metastasis. Hypothesis: KGFR is an important therapeutic target in breast cancer; and thus, selective inhibition of KGFR-mediated signaling will reduce or eliminate breast cancer cell proliferation, motility and metastasis. Specific Aims: The First Specific Aim will measure the relative activity and specificity of the 40 KGFR TKI on KGF-mediated KGFR signal transduction in human breast cancer cells. The Second Specific Aim will examine the ability of these 40 inhibitors to reduce KGF-induced breast cancer cell proliferation and motility in vitro. The Third Specific Aim will determine the inhibitory effect of the most selective and potent KGFR TKI (as determined in vitro in Specific Aims #1 and 2) on KGF-mediated growth and metastasis of human breast cancer cells in a mouse xenograft model. Methods: In Specific Aim #1, MCF-7 breast cancer cells will receive either a motility stimulating dose of KGF (50 ng/ml) or KGFR TKI at doses ranging from 10 to 200 ¿M or KGF (50 ng/ml) + KGFR TKI at each inhibitor dose or Iressa (epidermal growth factor receptor selective TK inhibitor; used as a negative control) over the same dosage range or KGF + Iressa (at the same doses) or vehicle control. At various times, from 1 to 48 hrs following KGF administration, the cells will be harvested to determine the protein expression of KGFR, phospho-KGFR, Erk1/2, phospho-Erk1/2 and ¿-actin by Western blotting. In Specific Aim #2 proliferation and migration of MCF-7 cells will be evaluated with both time-lapse videomicroscopy and a culture wounding assay over a period of 1 to 3 days using the same experimental groups and doses described in the first specific aim. In Specific Aim #3 the influence of KGFR inhibition on the growth and metastasis of tumor xenografts will be determined. The xenograft studies will be conducted by implanting MCF-7 cells into mammary fat pads of nude mice. At the end of the experiment, tumor, lung and liver will be removed and frozen sections examined by fluorescent microscopy to quantify tumor growth and development of lung and liver micro-metastasis. Significance: The establishment of KGFR as an important therapeutic target, together with the identification of specific and potent KGFR TKI, should result in the rapid development of a new class of highly selective therapeutic agents to prevent the metastatic progression of cancer. The establishment of KGFR as an important therapeutic target, together with the identification of specific and potent KGFR TKI, should result in the rapid development of a new class of highly selective therapeutic agents designed to prevent the metastatic progression of cancer. This project addresses several focus areas of the National Cancer Institute; namely, the biology of cancer metastasis and the development of highly selective and molecularly targeted therapeutic agents.