Oncogenic K-Ras requires activation for enhanced activity

Abstract

Oncogenic Ras mutations are widely considered to be locked in a permanent 'On' state and 'constitutively active'. Yet, many healthy people have cells possessing mutant Ras without apparent harm, and in animal models mutant Ras causes transformation only after upregulation of Ras activity. Here, we demonstrate that oncogenic K-Ras is not constitutively active but can be readily activated by upstream stimulants to lead to prolonged strong Ras activity. These data indicate that in addition to targeting K-Ras downstream effectors, interventions to reduce K-Ras activation may have important cancer-preventive value, especially in patients with oncogenic Ras mutations. As other small G proteins are regulated in a similar manner, this concept is likely to apply broadly to the entire Ras family of molecules.

Figure 1

Oncogenic Ras requires activation for enhanced activity. (a) Lysates from HEK293 cells transfected with K-, N- and H-Ras cDNA were used to determine the specificity of corresponding antibodies by western blotting. (b) Active K-, N- and H-Ras were measured by a Raf pull-down assay from pancreas lysates of 2-month-old control and Panc-Ras mice (lanes a). For comparison with total corresponding Ras isoform proteins, 5% of total lysates used for pull-down assay were loaded to adjacent wells (lanes t) and probed with isoform-specific antibodies. Densitometry was performed using ImageJ software. (c) Active K-Ras was measured in isolated pancreatic acinar cells from control and Panc-Ras mice after EGF (50 ng/ml) stimulation. (d) Oncogenic Ras is partially active in cancer cells. Active K-, N- and H-Ras (lanes a) were compared with 10% of the corresponding Ras isoform levels used for pull-down assay (lanes t) in human pancreatic cancer cells (Bxpc3, Panc1 and Mpanc96) and pancreatic cancer cells derived from Panc-Ras mouse (K8484). Note all cells except BxPC-3 possess oncogenic K-Ras mutations.

Figure 2

Targeting both upstream and downstream of Ras signaling for cancer prevention. Multiple factors are able to act as Ras activators through interactions with upstream receptors (e.g., EGF receptor). The effect on wild-type Ras is a transient elevation of downsteam signaling pathways including those regulated by RALGDS, RAF1 and PI3K (not shown). However, when oncogenic Ras is present, the downstream signaling after stimulation is prolonged at a high level. Elevated Ras activity generates inflammatory mediators that further activate Ras, and thus sustain Ras activity at high levels through a feed-forward loop. Previously emphasis has been focused on targeting downstream pathways activated by Ras, as oncogenic Ras was considered to be constitutively active. However, endogenous expression of mutant Ras gives rise only low level of Ras activity, which does not cause pathological effects unless Ras activity is upregulated. On the basis of the evidence that oncogenic Ras is not active without input from upstream activators, we propose that targeting the upstream mechanisms of Ras activation would be an alternative approach for cancer prevention, especially in patients with Ras mutations.