Ras isoform abundance and signalling in human cancer cell lines

Abstract

The ubiquitously expressed major Ras isoforms: H-, K- and N-Ras, are highly conserved, yet exhibit different biological outputs. We have compared the relative efficiencies with which epidermal or hepatocyte growth factor activates Ras isoforms and the requirement for specific isoforms in the activation of downstream pathways. We find that the relative coupling efficiencies to each Ras isoform are conserved between stimuli. Furthermore, in both cases, inhibition of receptor endocytosis led to reduced N- and H-Ras activation, but K-Ras was unaffected. Acute knockdown of each isoform with siRNA allows endogenous Ras isoform function and abundance to be probed. This revealed that there is significant variation in the contribution of individual isoforms to total Ras across a panel of cancer cell lines although typically K> or =N>>H. Intriguingly, cancer cell lines where a significant fraction of endogenous Ras is oncogenically mutated showed attenuated activation of canonical Ras effector pathways. We profiled the contribution of each Ras isoform to the total Ras pool allowing interpretation of the effect of isoform-specific knockdown on signalling outcomes. In contrast to previous studies indicating preferential coupling of isoforms to Raf and PtdIns-3-kinase pathways, we find that endogenous Ras isoforms show no specific coupling to these major Ras pathways.

FIG. 1. Growth factor induced Ras isoform activation in normal and endocytosis-defective (K44A) HeLa cells

Serum-starved HeLa cells were stimulated with either 100ng/ml HGF or EGF for 5 min (A) or 5 and 30 min (B). Activated Ras was pulled-down using GST-RBD (K85A), 30μg of each lysate was immunoblotted with isoform-specific Ras antibodies to normalise for Ras content (B). (C) Equivalent Ras activation assays (5 minute stimulation) were performed in serum-starved K44A dynamin-expressing mutant HeLa cells. Only K-Ras activation is insensitive to inhibition of endocytosis (Tet-).

FIG. 2. H-Ras is a minor isoform in HeLa cells compared to K-Ras and N-Ras

(A) Isoform-specific Ras knock-down using RNAi; >90% knock-down of each isoform was achieved. Probing the knock-down lysates with the anti-pan-Ras antibody Y13-259 enables the relative abundance of each isoform to be determined. (B) Y13-259 as a true pan-Ras antibody. Lysates normalised for GFP expression from HeLa cells expressing GFP-tagged Ras isoforms were probed with a variety of anti-pan-Ras antibodies. Only Y13-259 exhibited no isoform preference.

FIG. 3. Quantitative analysis of Ras isoform abundance in cancer cell lines

(A) 10μg of each cell lysate was immunoblotted for Ras isoforms and total Ras (Y13-259). Cell lines harbouring oncogenic K-Ras (*) or N-Ras (#) are highlighted (B); for the leukaemia cells, the Ras mutational status is unknown. HeLa cells were used as a benchmark to determine comparative abundance of Ras isoforms in other cell lines. Primary lymphoid leukaemia samples were only available as lysates preventing determination of total Ras abundance per 1×10⁶ cells.

FIG. 4. Oncogenic Ras is largely uncoupled from effector activation

Phospho-Akt and phospho-ERK labelling is not correlated with the presence of oncogenic Ras isoforms (K-Ras,* or N-Ras, #) within cancer cell lines (A). Despite a lack of constitutive stimulation in starved conditions, HepG2 and A549 cells are still able to respond to acute serum stimulation (B). 1: normal 10% serum media overnight, 2: starved overnight, 3: starved overnight then 20% FBS treatment for 5 minutes. Other relevant mutations present in these cell lines include the potent B-Raf V600E (HT-29) and PtdIns-3-kinase p110α E545K (MCF-7 and NCI-H460) oncogenic mutations.

FIG. 5. Ras isoforms are redundant for MAP kinase activation

MAP kinase activation is preserved following Ras isoform-specific knock-down in HeLa cells stimulated with 100ng/ml EGF or HGF. 35μg of each cell lysate was probed with anti-phospho-p44/42 MAPK and anti-p44/42 MAPK antibodies.

FIG. 6. Ras isoforms are redundant for Akt activation

RNAi treated HeLa cells stimulated with 100ng/ml of HGF were analysed for activating Ser⁴⁷³ phosphorylation of Akt (A). Significant abrogation of Akt phosphorylation is seen with some Ras oligos although for H- and N-Ras this doesn't appear to correlate with the extent of Ras knockdown. (B) Recomplementation with RNAi resistant H- or N-Ras fails to fully restore Akt phosphorylation indicating non-specific RNAi effects on Akt signalling.