Expression of activated M-Ras in a murine mammary epithelial cell line induces epithelial–mesenchymal transition and tumorigenesis

Abstract

The expression of activated mutants of M-Ras (G22V or Q71L), but not wild-type M-Ras, in a murine mammary epithelial cell line, scp2, resulted in epithelial-mesenchymal transition (EMT) and oncogenic transformation. Cells expressing constitutively active M-Ras continued to grow in the absence of serum and exhibited a loss of the epithelial markers cytokeratin, E-cadherin and b-catenin, together with a gain of the mesenchymal marker vimentin, a loss of contact inhibition in monolayer growth and a gain of the capacity for anchorage-independent growth. Moreover, unlike the parental cells, they failed to form differentiated mammospheres on Matrigel and instead formed branched networks of cells that grew and invaded the Matrigel. The expression of activated p21 Ras (G12V H-Ras or Q61K N-Ras) also resulted in EMT and tumorigenesis, although there was evidence that expression of higher levels was toxic. Tumors derived from scp2 cells expressing activated M-Ras exhibited activation of Akt and of ERK. The levels of expression of Q71L M-Ras and G12V H-Ras required for tumorigenesis were comparable, although higher levels of the weaker G22V M-Ras mutant were selected for in vivo. These data indicate that the expression of activated mutants of MRas was sufficient for oncogenic transformation of a murine mammary epithelial cell line.

Cyclooxygenase‐2 overexpression in MCF‐10F human breast epithelial cells inhibits proliferation, apoptosis and differentiation, and causes partial transformation

“…30 Therefore, to investigate the effect of COX-2 expression on differentiation, we cultured the cells on Matrigel-coated plates, a method that has been used by other investigators to study epithelial differentiation. 23,24 Under these conditions, 10F-P and 10F-AS cells aggregated to form smooth spheroids (Fig. 4a), structures previously observed to form from well-differentiated mammary epithelial cells.…”

“…4a), structures previously observed to form from well-differentiated mammary epithelial cells. 23,24 The 10F-S cells, however, formed irregular 3-dimensional structures with many fibroblast-like cells showing invasion into the Matrigel (Fig. 4b), suggesting differentiation of these cells was inhibited.…”

“…Cells were suspended in complete medium and seeded at a density of 1 3 10 6 per well and incubated at 37°C for 24-72 hr, after which time morphology was assessed by light microscopy. 23,24 Cell transformation assays Cells were passaged until there were obvious morphological changes. At this time, proteins from whole cell lysates were separated on 10% SDS-polyacrylamide gels and probed with mouse monoclonal anti-E-cadherin (catalog number 13-5700, Zymed Laboratories, Inc., San Francisco, CA), mouse monoclonal antikeratin (catalog number MS-343-P, Lab Vision Corporation, Fremont, CA) or mouse monoclonal anti-vimentin (catalog number MS-129-P, Lab Vision Corporation).…”

Fra‐1 regulates vimentin during Ha‐RAS‐induced epithelial mesenchymal transition in human colon carcinoma cells

“…16 It has been reported that in mammary epithelial cells another activated mutant member of the RAS subfamily, M-Ras, increased vimentin expression and resulted in EMT. 24 It appears that Ha-RASV12 has a higher transforming potential as opposed to its isoform Ki-RASV12. The unique gene expression profiles generated by each of the 2 RAS isoforms probably are responsible for these differences.…”

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate

“…Activated forms of MRAS, such as the MRAS-G22V mutant which is constitutively in the GTP-bound form, mimic inducers (NGF or BMP2) and trigger neuronal or osteoblast differentiation in PC12 or C2C12 models, respectively (Kimmelman et al, 2002;Sun et al, 2006;WatanabeTakano et al, 2010), induce formation of peripheral microspikes in fibroblasts and participate in the reorganisation of the actin cytoskeleton (Matsumoto et al, 1997). In addition, these activated forms are oncogenic in murine mammary epithelial cell lines (Ward et al, 2004). MRAS function is potentially regulated by plexin B1, a dual-function GTPase-activating protein (GAP) for RRAS and MRAS, which remodels axon and dendrite morphology, respectively (Saito et al, 2009).…”