Bray Kristi, Gillette Melissa, Young Jeanette, Loughran Elizabeth, Hwang Melissa, Sears James Cooper, Vargo-Gogola Tracy
Breast Cancer Res. 2013;15(5):R91. doi: 10.1186/bcr3487.
The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast tumors compared to normal breast tissue. Cdc42 regulates key processes that are critical for mammary gland morphogenesis and become disrupted during the development, progression, and metastasis of breast cancer. However, the contribution of Cdc42 to normal and neoplastic mammary gland development in vivo remains poorly understood. We were therefore interested in investigating the effects of Cdc42 overexpression on mammary gland morphogenesis as a first step toward understanding how its overexpression may contribute to mammary tumorigenesis.
We developed a tetracycline-regulatable Cdc42 overexpression mouse model in which Cdc42 can be inducibly overexpressed in the developing mammary gland. The effects of Cdc42 overexpression during postnatal mammary gland development were investigated using in vivo and in vitro approaches, including morphometric analysis of wholemounted mammary glands, quantification of histological markers, and primary mammary epithelial cell (MEC) functional and biochemical assays.
Analysis of Cdc42-overexpressing mammary glands revealed abnormal terminal end bud (TEB) morphologies, characterized by hyperbudding and trifurcation, and increased side branching within the ductal tree. Quantification of markers of proliferation and apoptosis suggested that these phenotypes were not due to increased cell proliferation or survival. Rather, Cdc42 overexpressing MECs were more migratory and contractile and formed dysmorphic, invasive acini in three-dimensional cultures. Cdc42 and RhoA activities, phosphorylated myosin light chain, and MAPK signaling, which contribute to migration and invasion, were markedly elevated in Cdc42 overexpressing MECs. Interestingly, Cdc42 overexpressing mammary glands displayed several features associated with altered epithelial-stromal interactions, which are known to regulate branching morphogenesis. These included increased stromal thickness and collagen deposition, and stromal cells isolated from Cdc42 overexpressing mammary glands exhibited elevated mRNA expression of extracellular matrix proteins and remodeling enzymes.
These data suggest that Cdc42 overexpression disrupts mammary gland branching morphogenesis by altering Rho GTPase and MAPK signaling, leading to increased MEC contractility and migration in association with stromal alterations. Our studies provide insight into how aberrant Cdc42 expression may contribute to mammary tumorigenesis.
与正常乳腺组织相比，Rho GTP酶Cdc42在乳腺肿瘤中过表达且过度激活。Cdc42调节对乳腺形态发生至关重要的关键过程，这些过程在乳腺癌的发生、发展和转移过程中会受到破坏。然而，Cdc42在体内对正常和肿瘤性乳腺发育的作用仍知之甚少。因此，我们有兴趣研究Cdc42过表达对乳腺形态发生的影响，作为理解其过表达如何导致乳腺肿瘤发生的第一步。
我们构建了一种四环素调控的Cdc42过表达小鼠模型，其中Cdc42可在发育中的乳腺中诱导性过表达。使用体内和体外方法研究了产后乳腺发育过程中Cdc42过表达的影响，包括对整个乳腺的形态计量分析、组织学标记物的定量分析以及原代乳腺上皮细胞（MEC）的功能和生化分析。
对过表达Cdc42的乳腺的分析显示，终末芽（TEB）形态异常，表现为过度出芽和三叉分支，并且导管树内的侧支分支增加。增殖和凋亡标记物的定量分析表明，这些表型并非由于细胞增殖或存活增加所致。相反，过表达Cdc42的MEC迁移性和收缩性更强，并且在三维培养中形成了畸形的侵袭性腺泡。在过表达Cdc42的MEC中，有助于迁移和侵袭的Cdc42和RhoA活性、磷酸化肌球蛋白轻链以及MAPK信号传导均明显升高。有趣的是，过表达Cdc42的乳腺表现出一些与上皮-基质相互作用改变相关的特征，而上皮-基质相互作用已知可调节分支形态发生。这些特征包括基质厚度增加和胶原蛋白沉积增加，并且从过表达Cdc42的乳腺中分离出的基质细胞表现出细胞外基质蛋白和重塑酶的mRNA表达升高。
这些数据表明，Cdc42过表达通过改变Rho GTP酶和MAPK信号传导破坏乳腺分支形态发生，导致MEC收缩性和迁移增加，并伴有基质改变。我们的研究为异常的Cdc42表达如何导致乳腺肿瘤发生提供了见解。
