Department of Oncology, Wayne State University, Detroit, Michigan, USA.
J Cell Physiol. 2011 Oct;226(10):2691-701. doi: 10.1002/jcp.22620.
We have previously shown that SUM-149 human breast cancer cells require an amphiregulin (AREG) autocrine loop for cell proliferation. We also demonstrated that AREG can increase epidermal growth factor receptor (EGFR) stability and promote EGFR localization to the plasma membrane. In the present studies we successfully knocked-down AREG expression in SUM-149 cells by lentiviral infection of AREG shRNA. In the absence of AREG expression, SUM-149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by Western blot. Immunofluorescence and confocal microscopy showed that following AREG knock-down, EGFR continued to localize to the cell surface. Soft agar assays demonstrated that AREG knock-down cells retain anchorage-independent growth capacity. Additionally mammosphere forming assays and Adefluor staining analysis showed that knock-down of AREG expression did not affect the expression of stem cell phenotypes. However, following AREG knock-down, SUM-149 cells demonstrated a dramatic decrease in their ability to invade a Matrigel matrix. Consistent with this observation, microarray analysis comparing cells infected with a non-silencing vector to the AREG knock-down cells, identified genes associated with the invasive phenotype such as RHOB and DKK1, and networks associated with cell motility such as integrin-linked kinase signaling, and focal adhesion kinase signaling. AREG was also found to modulate WNT and Notch signaling in these cells. Thus, AREG functions in regulating the invasive phenotype, and we propose that this regulation may be through altered signaling that occurs when AREG activates plasma membrane localized EGFR.
我们之前已经证明 SUM-149 人乳腺癌细胞增殖需要一个表皮生长因子受体（EGFR）自分泌循环。我们还证明了 AREG 可以增加 EGFR 的稳定性并促进 EGFR 向质膜的定位。在本研究中，我们通过慢病毒感染 AREG shRNA 成功敲低了 SUM-149 细胞中的 AREG 表达。在没有 AREG 表达的情况下，SUM-149 细胞的生长速度减慢，但并未完全受到抑制。此外，用 AREG shRNA 构建物感染的细胞通过 Western blot 显示 EGFR 蛋白表达增加。免疫荧光和共聚焦显微镜显示，在 AREG 敲低后，EGFR 继续定位到细胞膜。软琼脂测定表明 AREG 敲低细胞保持锚定独立生长能力。此外，类器官形成测定和 Adefluor 染色分析表明，敲低 AREG 表达不会影响干细胞表型的表达。然而，在 AREG 敲低后，SUM-149 细胞表现出侵袭 Matrigel 基质的能力急剧下降。与这一观察结果一致，比较感染非沉默载体的细胞与 AREG 敲低细胞的微阵列分析确定了与侵袭表型相关的基因，如 RHOB 和 DKK1，以及与细胞迁移相关的网络，如整合素连接激酶信号和粘着斑激酶信号。还发现 AREG 可以调节这些细胞中的 WNT 和 Notch 信号。因此，AREG 在调节侵袭表型中起作用，我们提出这种调节可能是通过 AREG 激活质膜定位的 EGFR 时发生的改变信号。
