Signaling amplitude molds the Ras mutation tropism of urethane
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Curated by eLife
Evaluation Summary:
This work helps explain some enduring mysteries about why certain activating mutations appear in the KRAS gene more frequently than others. This paper provides experimental support for an emerging concept within the Ras field that there is a sweet-spot of Ras signal strength that promotes tumorigenesis and that this explains why different mutations are observed in different contexts. The experiments are sound and the conclusions are fair. Given that certain KRAS mutations may be more amenable to therapeutic interventions than others, it is important to understand the basis for mutational tropism, and this work provides strong in vivo evidence that addresses this issue.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
RAS genes are commonly mutated in cancers yet despite many possible mutations, cancers have a ‘tropism’ towards a specific subset. As driver mutations, these patterns ostensibly originate from normal cells. High oncogenic RAS activity causes oncogenic stress and different oncogenic mutations can impart different levels of activity. Here we show that changing rare codons to common in the murine Kras gene to increase translation shifts tumors induced by the carcinogen urethane from arising from canonical Q 61 to biochemically less active G 12 Kras driver mutations, despite the carcinogen still being biased towards generating Q 61 mutations. Loss of p53 to blunt oncogenic stress partially reversed this effect, restoring Q 61 mutations. Finally, transcriptional analysis revealed similar signaling amongst tumors driven by different mutations and Kras alleles. These finding suggest that the RAS mutation tropism of urethane is largely product of selection in normal cells for mutations promoting proliferation without causing oncogenic stress.
Impact statement
The bias towards specific Kras driver mutations during urethane carcinogenesis appears to arise predominantly from the selection of a narrow window of oncogenic signaling in normal cells.
Article activity feed
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Author Response to Public Reviews
Public comment #1: “The mRNA data are interpreted as evidence for changes in protein expression and Ras signalling activity - there is no formal evidence that this is the case.
Response: As requested, while increase mRNA is a strong indication of elevated expression, we completely agree with this comment and added the caveat that protein analysis was not performed.
Public comment #2: “It is also intriguing how there wasn't a more complete switch to Q61 in the high KRAS tumours when p53 was deleted.”
Response: As requested, we now bring up this point in the discussion.
Public comment #3: “Whilst the Ras signalling dosing/oncogenic stress nexus are a reasonable explanation, the model/methods are a snapshot in time and don't have the resolution to fully understand the detail of what is going on here.”
Author Response to Public Reviews
Public comment #1: “The mRNA data are interpreted as evidence for changes in protein expression and Ras signalling activity - there is no formal evidence that this is the case.
Response: As requested, while increase mRNA is a strong indication of elevated expression, we completely agree with this comment and added the caveat that protein analysis was not performed.
Public comment #2: “It is also intriguing how there wasn't a more complete switch to Q61 in the high KRAS tumours when p53 was deleted.”
Response: As requested, we now bring up this point in the discussion.
Public comment #3: “Whilst the Ras signalling dosing/oncogenic stress nexus are a reasonable explanation, the model/methods are a snapshot in time and don't have the resolution to fully understand the detail of what is going on here.”
Response: As requested, while we have a different take on the degree by which this study informs RAS mutation tropism, we appreciate the position of this reviewer, particularly the point that using genetics to modulate oncoprotein levels and the stress response thereof at the endogenous levels in vivo with Krasex3op and Trp53fl alleles coupled with measuring the expression of three established downstream RAS target genes is no substitute for following signaling at the protein level at the moment Ras mutations are induced and thereafter throughout tumorigenesis, and hence note this caveat in the discussion.
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Reviewer #2 (Public Review):
This manuscript builds upon some important thought-leading work within the Ras field that the authors have published in recent years. They have previously demonstrated how changing the protein expression levels of KRAS can modulate the number of Ras-driven tumours that are observed and posited that this suggests an optimal level of Ras signalling that is neither too stressing nor too insufficient to promote tumourigenesis.
In this manuscript they use urethane to induce lung tumours in mouse models that have either normal or high levels of KRAS expression (also higher oncogenic stress). They are also able to modulate the associated oncogenic stress levels by the presence (higher stress) or deletion (lower stress) of p53. Urethane normally generates Q61 KRAS mutations, biochemical analysis by other groups has …
Reviewer #2 (Public Review):
This manuscript builds upon some important thought-leading work within the Ras field that the authors have published in recent years. They have previously demonstrated how changing the protein expression levels of KRAS can modulate the number of Ras-driven tumours that are observed and posited that this suggests an optimal level of Ras signalling that is neither too stressing nor too insufficient to promote tumourigenesis.
In this manuscript they use urethane to induce lung tumours in mouse models that have either normal or high levels of KRAS expression (also higher oncogenic stress). They are also able to modulate the associated oncogenic stress levels by the presence (higher stress) or deletion (lower stress) of p53. Urethane normally generates Q61 KRAS mutations, biochemical analysis by other groups has previously shown that these mutations are more active than G12 mutations. Following urethane induction, they observe an improved competence to support tumorigenesis in the high KRAS model when p53 is removed. They also observe a shift towards G12 mutants under genetic conditions where oncogenic stress is higher (higher KRAS expression, presence of p53). ie. stress compensators (p53 loss or weaker activating mutation) permit promotion of tumourigenesis in the high KRAS model. The converse was also observed. Loss of p53 (lower stress) resulted in higher mRNA levels of G12 mutants - suggesting that the weaker mutant increases protein expression/cancer signalling to occupy the new oncogenic stress headroom that has been created. Some support for the hypothesis that these effects are mediated by differences in Ras signalling amplitude between the different mutants was provided by analysing the expression of three key Ras gene targets. As predicted, higher expression (signalling output) was seen in Q61 vs Q12 mutants and when p53 was deleted.
Strengths:
The mouse model conditions provide a suitable range of options to allow the hypothesis to be tested. The data are all internally consistent and broadly support the general conclusions.
Weaknesses:
The mRNA data are interpreted as evidence for changes in protein expression and Ras signalling activity - there is no formal evidence that this is the case.
The similarity in G12/13 mutations between the KRAS normal and high KRAS mice in Figure 2C is unexpected. The authors focussed on the potential for higher G12/13 mutant expression in the KRAS normal mice to explain this. It is also intriguing how there wasn't a more complete switch to Q61 in the high KRAS tumours when p53 was deleted. Whilst the Ras signalling dosing/oncogenic stress nexus are a reasonable explanation, the model/methods are a snapshot in time and don't have the resolution to fully understand the detail of what is going on here.
This study represents a solid contribution supporting an important model and will stimulate future work to understand Ras variant cancer contributions.
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Reviewer #1 (Public Review):
The centrality of RAS proteins in human malignancies has long been established, but many issues regarding their regulation and functions remain unresolved. The results of this paper provide strong supporting evidence for an emerging model that posits that activated KRAS can only be tolerated by cells up to a certain point, after which the stress it imposes outweigh its transforming potential. These restrictions impose limits on the amount of KRAS expressed in tumor cells and are also consistent with the frequent coupling of KRAS mutations with loss of the tumor suppressor p53, as the latter relieves the stress signals induced by KRAS.
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Evaluation Summary:
This work helps explain some enduring mysteries about why certain activating mutations appear in the KRAS gene more frequently than others. This paper provides experimental support for an emerging concept within the Ras field that there is a sweet-spot of Ras signal strength that promotes tumorigenesis and that this explains why different mutations are observed in different contexts. The experiments are sound and the conclusions are fair. Given that certain KRAS mutations may be more amenable to therapeutic interventions than others, it is important to understand the basis for mutational tropism, and this work provides strong in vivo evidence that addresses this issue.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
Evaluation Summary:
This work helps explain some enduring mysteries about why certain activating mutations appear in the KRAS gene more frequently than others. This paper provides experimental support for an emerging concept within the Ras field that there is a sweet-spot of Ras signal strength that promotes tumorigenesis and that this explains why different mutations are observed in different contexts. The experiments are sound and the conclusions are fair. Given that certain KRAS mutations may be more amenable to therapeutic interventions than others, it is important to understand the basis for mutational tropism, and this work provides strong in vivo evidence that addresses this issue.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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