Basal but not Luminal Mammary Epithelial Cells Require PI3K/mTOR Signaling for Ras-Driven Overgrowth

Abstract

The mammary ducts of humans and mice are comprised of two main mammary epithelial cell (MEC) subtypes: a surrounding layer of basal MECs and an inner layer of luminal MECs. Breast cancer subtypes show divergent clinical behavior that may reflect properties inherent in their MEC compartment-of-origin. How the response to a cancer-initiating genetic event is shaped by MEC subtype remains largely unexplored. Using the mouse mammary gland, we designed organotypic 3D culture models that permit challenge of discrete MEC compartments with the same oncogenic insult. Mammary organoids were prepared from mice engineered for compartment-restricted co-expression of oncogenic H-RASG12V together with a nuclear fluorescent reporter. Monitoring of H-RASG12V-expressing MECs during extended live cell imaging permitted visualization of Ras-driven phenotypes via video microscopy. Challenging either basal or luminal MECs with H-RASG12V drove MEC proliferation and survival, culminating in aberrant organoid overgrowth. In each compartment, Ras activation triggered modes of collective MEC migration and invasion that contrasted with physiological modes used during growth factor-initiated branching morphogenesis. Although basal and luminal Ras activation produced similar overgrowth phenotypes, inhibitor studies revealed divergent use of Ras effector pathways. Blocking either the phosphoinositide 3-kinase (PI3K) or the mammalian target of rapamycin (mTOR) pathway completely suppressed Ras-driven invasion and overgrowth of basal MECs, but only modestly attenuated Ras-driven phenotypes in luminal MECs. We show that MEC subtype defines signaling pathway dependencies downstream of Ras. Thus, cells-of-origin may critically determine the drug sensitivity profiles of mammary neoplasia.

Three-dimensional organotypic culture: experimental models of mammalian biology and disease

“…Mosaic, but not ubiquitous, expression of an activated form of the GTPase HRAS induces multicellular protrusions in MCF-10A aggregates 117 . In a separate study, activated HRAS was sufficient to drive tissue overgrowth when expressed in myoepithelial or luminal mammary epithelial cells 118 . However, inhibitor studies demonstrated the involvement of different RAS effectors, which showed that different pathways were used in the two cell types to induce proliferation downstream of the same oncogene 118 .…”

“…In a separate study, activated HRAS was sufficient to drive tissue overgrowth when expressed in myoepithelial or luminal mammary epithelial cells 118 . However, inhibitor studies demonstrated the involvement of different RAS effectors, which showed that different pathways were used in the two cell types to induce proliferation downstream of the same oncogene 118 . Taken together, these studies show that 3D culture enables elucidation of the cellular and molecular effects of gene activity in distinct cell populations within a tissue.…”

NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells

“…Circularity values are possible between 0 and 1, with a score of 1 indicating a perfect circle. The circularity scoring method was adapted for enteroids from a previously reported system in mammary epithelial cultures 51 .…”

Synthetic lethality by targeting the RUVBL1/2-TTT complex in mTORC1-hyperactive cancer cells

“…Meanwhile, mTORC2 is a major upstream regulator of Akt, protein kinase C (PKC), and serum/glucocorticoid-regulated kinase (SGK) kinases, which contribute to proliferation, migration, metabolism, and survival via separate and overlapping effector pathways (2). Because of its central role in these processes, aberrant mTOR signaling is frequently implicated in cancer pathogenesis, with oncogenes including PI3K and Ras demonstrated to up-regulate mTOR activation in proliferating cells (3,4). The frequent loss of tumor suppressors including p53, PTEN, TSC1/2, and LKB1 in malignant cells further promotes mTOR pathway activation (3,5,6).…”