Sapi E, Flick M B, Rodov S, Gilmore-Hebert M, Kelley M, Rockwell S, Kacinski B M
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520-8040, USA.
Cancer Res. 1996 Dec 15;56(24):5704-12.
Invasion of tissue by macrophages and implantation into the uterine wall by placental trophoblasts are known to be regulated by the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R, the product of the c-fms proto-oncogene). Recently, the clinical importance of CSF-1 and CSF-1R in invasive breast carcinoma has been recognized, but the significance of coexpression of CSF-1 and CSF-1R in mammary epithelial cell invasion has not been explored. In the present study, we investigated the invasive potential of a noninvasive, CSF-1R-negative, mouse mammary epithelial cell line (HC11) expressing a high level of CSF-1, which was stably transfected with the mouse wild-type CSF-1R. Compared with parental cells, transfected cells expressing a wild-type CSF-1R invaded 100-fold more efficiently through a barrier of reconstituted basement membrane (Matrigel) and formed colonies in soft agar, whereas the cellular growth rate was only slightly increased. Analysis of cell-conditioned medium by zymography and quantitative enzyme activity assays showed that clones transfected with a wild-type CSF-1R expressed significantly higher levels of urokinase-type plasminogen activator than did untransfected clones. Furthermore, after injection into the tail veins of BALB/c mice, CSF-1R-expressing clones also produced a 10-fold higher incidence of lung tumors than the parental cell line. We also analyzed HC11 clones transfected with CSF-1R mutated at two major autophosphorylation sites (Tyr-->Phe807 and Tyr-->Phe721). Mutation at Tyr807 eradicated the stimulatory effect of Fms expression on the invasive ability of HC11 cells and substantially reduced the metastatic potential of the transfected clones but did not alter the Fms-induced anchorage-independent growth in soft agar. In contrast, mutation at Tyr721 of Fms had no effect on invasion as measured in the in vitro assay but markedly abolished Fms-induced colony formation in soft agar and eradicated the metastatic potential of the transfected clones. Our results suggest that expression of CSF-1R can facilitate cellular invasion and anchorage-independent growth in mammary epithelial cells, and these two processes are independently regulated by separate phosphotyrosine sites of CSF-1R.
巨噬细胞对组织的侵袭以及胎盘滋养层细胞对子宫壁的植入，已知受巨噬细胞集落刺激因子（CSF-1）及其受体（CSF-1R，即原癌基因c-fms的产物）调控。最近，CSF-1和CSF-1R在浸润性乳腺癌中的临床重要性已得到认可，但CSF-1和CSF-1R共表达在乳腺上皮细胞侵袭中的意义尚未得到探究。在本研究中，我们研究了一种非侵袭性、CSF-1R阴性、高水平表达CSF-1的小鼠乳腺上皮细胞系（HC11）的侵袭潜能，该细胞系用小鼠野生型CSF-1R进行了稳定转染。与亲代细胞相比，表达野生型CSF-1R的转染细胞通过重组基底膜（基质胶）屏障的侵袭效率提高了100倍，并在软琼脂中形成集落，而细胞生长速率仅略有增加。通过酶谱分析和定量酶活性测定对细胞条件培养基进行分析表明，用野生型CSF-1R转染的克隆表达的尿激酶型纤溶酶原激活剂水平明显高于未转染的克隆。此外，将表达CSF-1R的克隆注射到BALB/c小鼠尾静脉后，其肺肿瘤发生率也比亲代细胞系高10倍。我们还分析了在两个主要自磷酸化位点（酪氨酸突变为苯丙氨酸807和酪氨酸突变为苯丙氨酸721）发生突变的CSF-1R转染的HC11克隆。酪氨酸807位点的突变消除了Fms表达对HC11细胞侵袭能力的刺激作用，并大幅降低了转染克隆的转移潜能，但未改变Fms诱导的在软琼脂中不依赖贴壁的生长。相反，Fms的酪氨酸721位点突变对体外测定的侵袭没有影响，但明显消除了Fms诱导的在软琼脂中的集落形成，并消除了转染克隆的转移潜能。我们的结果表明，CSF-1R的表达可促进乳腺上皮细胞的细胞侵袭和不依赖贴壁的生长，并且这两个过程由CSF-1R的不同磷酸酪氨酸位点独立调控。
