Distinct requirements for Ras oncogenesis in human versus mouse cells

Abstract

The spectrum of tumors associated with oncogenic Ras in humans often differs from those in mice either treated with carcinogens or engineered to sporadically express oncogenic Ras, suggesting that the mechanism of Ras transformation may be different in humans. Ras stimulates primarily three main classes of effector proteins, Rafs, PI3-kinase, and RalGEFs, with Raf generally being the most potent at transforming murine cells. Using oncogenic Ras mutants that activate single effectors as well as constitutively active effectors, we find that the RalGEF, and not the Raf or PI3-kinase pathway, is sufficient for Ras transformation in human cells. Thus, oncogenic Ras may transform murine and human cells by distinct mechanisms, and the RalGEF pathway-previously deemed to play a secondary role in Ras transformation-could represent a new target for anti-cancer therapy. Extracellular signals detected by cell-surface receptors can be transmitted to the cell by stimulating the conversion of the Ras oncoprotein from an inactive GDP-bound to an active GTP-bound state. In the GTP-bound state, Ras stimulates downstream targets, or effectors, which, in turn, affect numerous activities of the cell, such as proliferation, apoptosis, and differentiation (Shields et al. 2000). Mutated and constitutively activated forms of Ras are found in 30% of all human cancers (Bos 1989). Significant experimental and epidemiological evidence supports that such aberrant Ras activation plays a critical role in human oncogenesis. Consequently, there is considerable effort to develop inhibitors of the Ras-signaling pathway for use as novel anticancer drugs (Kloog and Cox 2000;Shields et al. 2000).Ras transformation is mediated by interactions with multiple downstream effectors whose contribution to Ras transformation has been evaluated primarily in rodent fibroblast model cell systems. Substantial evidence from these studies supports the role of Raf serine/threonine kinases (c-Raf-1, A-Raf, and B-Raf) as key effectors and mediators of Ras-transforming activity. Raf kinases phosphorylate and activate the MEK1 and MEK2 dual specificity kinases, which, in turn, phosphorylate and activate the ERK1/p44 and ERK2/p42 mitogen-activated protein kinases (MAPKs) (Kyriakis et al. 1992). The critical contribution of the Raf/MEK/ERK pathway to Ras transformation is shown by the ability of constitutively activated mutants of Raf or MEK to cause tumorigenic transformation of NIH 3T3 cells (Bonner et al. 1985;Stanton et al. 1989;Leevers et al. 1994;Stokoe et al. 1994). Additionally, dominant-negative mutants of Raf-1, MEK, and ERK, as well as pharmacologic inhibitors of MEK, have been shown to effectively block Ras transformation in vitro or suppress tumorigenic growth of cancer cell lines in vivo (Kolch et al. 1991;Schaap et al. 1993;Cowley et al. 1994;Westwick et al. 1994;Khosravi-Far et al. 1995;Qiu et al. 1995;Monia et al. 1996;Sebolt-Leopold et al. 1999).Perhaps the second best-characterized effector important for Ras transformation is phosphatidylinositol 3-kina...

TRF2 inhibition promotes anchorage-independent growth of telomerase-positive human fibroblasts

“…Indeed, the immortalized cell line utilized in this study can form tumors in vivo only when expressing high levels of the Ha-RasV12 oncogene (Rangarajan et al, 2004). Such a dissociation between the ability to grow in soft agar and to form tumor is not unprecedented (Hamad et al, 2002) and is consistent with the fact that the transforming activity of M-TRF2 DBDM is not as effective as Ha-Ras V12 (Figure 3a and b). Nevertheless, TRF2 inhibition appears to be more potent at conferring an anchorage-independent growth phenotype than an exposure to g-radiation, even at doses exhibiting a marked impact on cell growth and chromosome stability.…”

Response to MLN8237 in Pancreatic Cancer Is Not Dependent on RalA Phosphorylation

“…2–4 × 10 3 cells were seeded in a 12-well culture dish as described previously (32, 33). DMSO or MLN8237 were included in the agar and in growth medium throughout the assay.…”

Immortalized cells as experimental models to study cancer

“…Multiple effector arms of RAS signaling are involved in transformation, yet the specific requirements for activation of these pathways may differ between cell types. Indeed, the results of Hamad et al and Rangarajan et al indicate that different RAS effector pathways participate in human and murine cell transformation (Hamad et al 2002;Rangarajan et al 2004). It is additionally unclear why the RAS expression levels required for tumorigenesis in these types of experiments is higher than those found in human tumors.…”

“…Since many molecules have been implicated in RAS signaling, it is difficult to identify the particular effectors that mediate the transforming capacity of RAS. However, Hamad et al and Rangarajan et al used individual HRas V12 mutant alleles (T35S, Y40C, E37G) that each activate one of these downstream pathways (Raf, PI3K, RalGEF, respectively) to delineate the roles of RAS-effector pathways in tumorigenesis (Hamad et al 2002;Rangarajan et al 2004). In HEK cells and human astrocytes, expression of RAS E37G cooperates with hTERT, LT, and ST to induce growth in soft agar, yet expression of RAS T35S or RAS Y40C fails to do so (Hamad et al 2002) (Figure 1).…”

“…However, HEK cells transformed by the co-expression of hTERT, LT, ST, and RAS E37G fail to form tumors in nude mice. The additional co-expression of RAS T35S and RAS Y40C renders such cells tumorigenic (Hamad et al 2002), suggesting that the combined effects of the Raf, PI3K and RalGEF pathways together mediate RAS-induced tumorigenesis in vivo. Indeed, different pathways downstream of RAS mediate the transformation of different human cell types (Rangarajan et al 2004).…”