Kumbrink Joerg, de la Cueva Ana, Soni Shefali, Sailer Nadja, Kirsch Kathrin H
Department of Biochemistry, Boston University School of Medicine, 72 E. Concord Street, Boston, MA, 02118, USA.
Department of Medicine III, University Hospital Grosshadern, University of Munich, Munich, Germany.
Tumour Biol. 2016 Aug;37(8):10665-73. doi: 10.1007/s13277-016-4902-8. Epub 2016 Feb 11.
Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6-10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6-10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.
在侵袭性乳腺癌中发现p130Cas（Crk相关底物）水平升高，且其与患者预后不良及对标准治疗的耐药性相关。p130Cas主要通过其磷酸化的底物结构域（SD）发出信号，该结构域包含15个酪氨酸基序（YxxP），可募集效应分子。p130Cas的酪氨酸磷酸化对于介导迁移、侵袭、肿瘤促进和转移很重要。我们之前开发了一种Src*/SD融合分子方法，其中SD被组成性磷酸化。在多瘤病毒中间T抗原（PyMT）/Src*/SD双转基因小鼠模型中，Src*/SD加速PyMT诱导的肿瘤生长并促进更具侵袭性的表型。为了测试Src*/SD是否也驱动转移以及哪些YxxP基序参与此过程，将与Src融合的全长和截短SD分子在乳腺癌细胞中表达。在体外分析Src/SD片段的功能，并在原位小鼠模型中对活性蛋白进行体内测试。将表达全长SD和功能性较小SD片段（跨越SD基序6 - 10）的乳腺癌细胞注射到小鼠的乳腺脂肪垫中。通过生物发光成像和卡尺测量监测肿瘤进展。与对照动物相比，完整的SD促进原发性肿瘤生长并更早发生转移。重要的是，完整和截短的SD均显著增加了向多个器官转移的发生率。这些研究提供了强有力的证据，表明磷酸化的p130Cas SD基序6 - 10（Y236、Y249、Y267、Y287和Y306）对于驱动乳腺癌进展很重要。
