Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migration.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Kristi
K
Bray
Melissa
M
Gillette
Jeanette
J
Young
Elizabeth
E
Loughran
Melissa
M
Hwang
James Cooper
JC
Sears
Tracy
T
Vargo-Gogola
D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D002460:Cell Line; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005260:Female; D015870:Gene Expression; D006801:Humans; D020935:MAP Kinase Signaling System; D008321:Mammary Glands, Animal / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D008822:Mice, Transgenic; D017154:Stromal Cells / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000235:genetics*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein