Division of Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA.
Cancer Rep (Hoboken). 2022 Sep;5(9):e1566. doi: 10.1002/cnr2.1566. Epub 2021 Nov 17.
CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types. Previous studies have shown that CD148 dephosphorylates growth factor receptors and their signaling molecules, including EGFR and ERK1/2, and negatively regulates cancer cell growth. Furthermore, research of clinical patients has shown that highly linked CD148 gene polymorphisms, Gln276Pro (Q276P) and Arg326Gln (R326Q), are associated with an increased risk of several types of cancer. However, the biological effects of these missense mutations have not been studied.
We aimed to determine the biological effects of CD148 Q276P/R326Q mutations in cancer cell proliferation and growth factor signaling, with emphasis on EGFR signaling.
CD148 forms, wild-type (WT) or Q276P/R326Q, were retrovirally introduced into A431D epidermoid carcinoma cells that lacks CD148 expression. The stable cells that express comparable levels of CD148 were sorted by flow cytometry. A431D cells infected with empty retrovirus was used as a control. CD148 localization, cell proliferation rate, EGFR signaling, and the response to thrombospondin-1 (TSP1), a CD148 ligand, were assessed by immunostaining, cell proliferation assay, enzyme-linked immunosorbent assay, and Western blotting.
Both CD148 forms (WT, Q276P/R326Q) were distributed to cell surface and all three cell lines expressed same level of EGFR. Compared to control cells, the A431D cells that express CD148 forms showed significantly lower cell proliferation rates. EGF-induced EGFR and ERK1/2 phosphorylation as well as cell proliferation were also significantly reduced in these cells. Furthermore, TSP1 inhibited cell proliferation in CD148 (WT, Q276P/R326Q)-expressing A431D cells, while it showed no effects in control cells. However, significant differences were not observed between CD148 WT and Q276P/R326Q cells.
Our data demonstrates that Q276P/R326Q mutations do not have major effects on TSP1-CD148 interaction as well as on CD148's cellular localization and activity to inhibit EGFR signaling and cell proliferation.
CD148 是一种跨膜蛋白酪氨酸磷酸酶，在多种细胞类型中表达。先前的研究表明，CD148 去磷酸化生长因子受体及其信号分子，包括 EGFR 和 ERK1/2，并负调控癌细胞生长。此外，对临床患者的研究表明，高度相关的 CD148 基因多态性，Gln276Pro（Q276P）和 Arg326Gln（R326Q），与多种类型的癌症风险增加有关。然而，这些错义突变的生物学效应尚未研究。
我们旨在确定 CD148 Q276P/R326Q 突变对癌细胞增殖和生长因子信号转导的生物学影响，重点是 EGFR 信号转导。
用逆转录病毒将 CD148 形式（野生型（WT）或 Q276P/R326Q）引入缺乏 CD148 表达的 A431D 表皮样癌细胞中。通过流式细胞术对表达相当水平 CD148 的稳定细胞进行分选。用空逆转录病毒感染的 A431D 细胞作为对照。通过免疫染色、细胞增殖测定、酶联免疫吸附测定和 Western blot 评估 CD148 定位、细胞增殖率、EGFR 信号转导以及对 CD148 配体血小板反应蛋白-1（TSP1）的反应。
两种 CD148 形式（WT、Q276P/R326Q）均分布于细胞膜表面，三种细胞系均表达相同水平的 EGFR。与对照细胞相比，表达 CD148 形式的 A431D 细胞的增殖率明显较低。EGF 诱导的 EGFR 和 ERK1/2 磷酸化以及细胞增殖也显著降低。此外，TSP1 抑制 CD148（WT、Q276P/R326Q）表达的 A431D 细胞的增殖，而在对照细胞中没有影响。然而，在 CD148 WT 和 Q276P/R326Q 细胞之间未观察到显著差异。
我们的数据表明，Q276P/R326Q 突变对 TSP1-CD148 相互作用以及 CD148 的细胞定位和抑制 EGFR 信号转导和细胞增殖的活性没有重大影响。
