mTORC1 Activity Is Necessary and Sufficient To Inhibit Mammary Epithelial Cell Invasion in 3D Culture
Susmita Ghosh, Lidenys Varela, Akshay Sood, Andrew J Ewald, Tamara L Lotan. Johns Hopkins School of Medicine, Baltimore, MD
Background: PTEN (phosphatase and tensin homologue) loss occurs in nearly one third of breast cancers, resulting unopposed PI3K (phosphoinositide-3-kinase) and mTORC1/2 (mammalian target of rapamycin) signaling. Despite the well-known role of PTEN and mTORC2 signaling in the regulation of single cell motility and chemotaxis, it remains unclear whether these pathways modulate epithelial motility and invasion.
Design: Mice harboring an inducible Cre (ROSA[ER]-Cre) were crossed with PTEN loxp/loxp and TSC1 loxp/loxp strains enabling diffuse and rapid inactivation of these genes in vitro. Murine mammary ductal fragments were isolated using a combination of manual dissection, enzymatic digestion and differential centrifugation and embedded in a laminin-rich extracellular matrix (ECM) supplemented with a completely-defined cell culture media containing FGF2 to induce epithelial branching and 4-OHT to induce genetic recombination. Appropriate gene inactivation was confirmed by immunoblot and immunohistochemistry (IHC). Mammary organoids were assessed for epithelial branching and invasion using a combination of time-lapse microscopic imaging and immunofluorescence (IF) after 7 days of culture.
Results: In vitro PTEN deletion resulted in enlarged mammary organoids with luminal filling resembling atypical ductal hyperplasia (ADH). These changes were reflected by increased luminal cell proliferation (BrdU) and decreased luminal cell apoptosis (caspase 3). Similar to PTEN-null human and murine breast tumors, PTEN-null mammary organoids showed decreased ER and increased K14 expression in luminal cells. Surprisingly, however, PTEN-null luminal epithelial cells were markedly less efficient at invading beyond the surrounding myoepithelial cell layer into the ECM when compared with wildtype counterparts. This inability to invade was due to increased mTORC1 activity, as incubation with rapamycin, an mTORC1 inhibitor, reversed this finding and resulted in increased epithelial invasion relative to wildtype controls. Increased mTORC1 activity was sufficient to inhibit mammary epithelial invasion, as TSC1 inactivation also resulted in decreased invasion reversible by rapamycin.
Conclusions: PTEN deletion results in luminal filling resembling ADH, however mTORC1 inactivation is required to initiate luminal epithelial invasion beyond the surrounding myoepithelial layer. This data suggests that single agent rapalog therapy for PTEN-null mammary tumors is likely to be inadequate and may even increase invasive potential.
Monday, March 19, 2012 11:00 AM
Platform Session: Section G2, Monday Morning