Abstract
Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.
Highlights
Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional mammary epithelial cells (MECs) culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths
Leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis
Loss of apical and basolateral polarity is an invariant feature of tumors arising from epithelial cells, known as carcinomas, which account for most human cancers [3]
Summary
Antibodies and Other Reagents—Rabbit anti-Vav peptide sera were generated against Vav amino acids 208 –222 (QETEAKYYRTLEDIE) through a commercial vendor (Animal Pharma Inc.). The monoclonal anti-EGFR (528, ATCC) and anti-E-cadherin (clone E4.6) [49] (provided by Drs Michael Brenner and Jonathan Higgins, Brigham and Women’s Hospital, Boston, MA) antibodies were purified from mouse hybridoma supernatants. Purified anti-phosphotyrosine antibody 4G10 [50] was provided by Dr Brian Druker (Oregon Health Sciences University, Portland, OR). The following antibodies were commercially obtained: monoclonal anti-RhoA, antiRac, and anti-GM130 (BD Biosciences); anti-Rac (Cytoskeleton); anti--actin (Sigma); anti-␣6 integrin. AGTTGGCTTTG (sequence 1) and CGACAGCCCTGATAGTTTA (sequence 2); for Rac, GACACGATC-. Vav cDNA was obtained from the Signaling Consortium (The Signaling Gateway) and subcloned into the pRevTRE vector (Invitrogen) through an engineered SalI site
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
