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Mutations
in the RAS oncogene have been found in about 30% of human cancers.
Among them, the mutation frequency of KRAS is much higher than that of HRAS and NRAS
.
For example, about 12-15% of patients with non-small cell lung cancer carry the KRAS G12C mutation, and more than 90% of patients with KRAS ductal adenocarcinoma (PDAC), especially the KRAS G12D and KRASG12V subtypes.
However, the development of RAS inhibitors has been so difficult that RAS is considered an "undruggable target.
"
Recently, small molecule compounds that directly target the KRASG12C mutation have been approved for therapeutics, but targeting other KRAS mutations
is still considered clinically impractical.
Recently, researchers from the First Affiliated Hospital of Sun Yat-sen University and Nanjing University have found that a protein RASON encoded by a long chain of non-coding RNA between genes (LINC00673) is a key regulator of the oncogenic RAS signaling pathway and is expected to become a therapeutic target for KRAS mutated
cancers.
The results were published Oct.
14 in
the journal Cell Research.
1.
Research materials and methods
In this study, the researchers used multiple pancreatic cancer cell lines and knocked out the rason gene
from multiple cell lines using CRISPR technology.
To study pancreatic tumor generation, they also used multiple mouse models and commissioned Saiye Biologics to construct Rason-/− mice
.
They determined RASON expression by western blotting and LC-MS analysis, and analyzed RASON-KRAS interactions
by experiments such as immunoprecipitation, surface plasmon resonance, and nuclear magnetic resonance.
2.
Research results
(1) RASON protein is associated with poor prognosis of PDAC
There are a large number of non-coding regions in the human genome that were previously thought to be non-coding proteins, but in recent years translationome studies have identified hundreds of functional peptides or proteins
from the non-coding regions.
The researchers set out to analyze
the noncoding regions of pancreatic cancer samples.
After comparing the three PDAC cell lines with normal pancreatic duct cell lines, they found differential translations of long non-coding RNA (lincRNAs) between 8 genes, with LINC00673 ranking first
.
Comparison of tumor samples from PDAC patients with normal tissues adjacent to cancer also showed that LINC00673 was differentially expressed
.
They confirmed by western blot analysis that LINC00673 encodes a 108-amino acid protein and named it RASON
.
In a separate cohort of 105 PDAC cancer samples and paired normal pancreas samples, the researchers found that LINC00673 was highly expressed in cancer samples and that its expression was positively correlated
with tumor grade.
In line with this, the expression of the rason protein in cancer tissues was also significantly increased and positively correlated with shorter tumor grade and overall survival (Figure 1).
These results suggest that RASON is an oncoprotein that acts as a prognostic marker for
PDACs.
Figure 1.
RASON is overexpressed in human pancreatic cancer
(2) RASON is essential for KRAS-driven tumorigenesis
To investigate RASON's potential function in PDAC, the researchers constructed two pancreatic cancer cell lines
that knocked out RASON.
They found that the RAS downstream signaling pathway was inhibited after rason knockout, and the downstream pathway
was enhanced after rason overexpression.
MEANWHILE, ACTIVE KRAS-GTP LEVELS WERE ALSO DOWNREGULATED
IN RASON KO CELLS.
In addition, they generated Rason-/− homologate knockout (Rason−/−) mice (which were provided by Saiye).
Both embryonic fibroblasts and pancreatic tissues of Rason−/− mice showed significant downregulation of RAS signaling pathway, indicating that rason was involved in the regulation
of oncogenic KRAS signaling pathway.
To explore whether RASON is involved in KRAS-driven tumorigenesis, they used RAS-less MEF cell lines stably transfected with KRAS G12D or KRASG12V and tested the effect of
Rason KO on KRAS-induced MEF cell transformation.
They generated two Rason KO clones and found that Rason KO almost completely reduced the tumorigenic potential
of KRAS G12D or KRASG12V MEF cells.
Subcutaneous tumorigenesis experiments in nude mice also confirmed the key role
of rason in mutation-induced MEF cell transformation.
Compared to control cells, Rason KO almost completely eliminates tumor formation
.
The researchers then crossed Rason-/− mice with KC mice, a mature model for studying the early stages of PDAC, to generate KCR mice
.
KCR mice had a significant reduction in all precancerous lesion formation at 6 and 9 months of age compared to KC mice, suggesting that RASON was involved in the early stages of KRAS-driven pancreatic tumor formation (Figure 2).
Studies on another classic PDAC model mouse (KPC mouse) confirmed this
.
These data suggest that RASON is required for
KRAS-driven pancreatic tumorigenesis in mice.
Figure 2.
RASON is required for KRAS-driven pancreatic tumorigenesis
Immunoprecipitation analysis showed a direct interaction
between RASON and KRAS.
Surface plasmon resonance analysis (SPR) showed that RASON binds
to KRAS G12D and KRASG12V with very high affinity.
The results of nuclear magnetic resonance (NMR) titration experiments also strongly support the direct binding of
rason to KRAS.
Subsequent analyses showed that rason inhibits GTPase-activated protein (GAP)-induced GTP hydrolysis, keeping KRAS in an activated state at all times (Figure 3).
Figure 3.
Schematic diagram of RASON's regulation of oncogenic RAS signaling pathways
(3) RASON is a potential therapeutic target for PDAC
Due to the presence of KRAS mutations, most patients with PDAC develop resistance
to EGFR inhibitors (cetuximab).
However, the combination of cetuximab and RASON shRNA minimized tumor growth in nude mice, suggesting that rason inhibition made KRAS-mutated cancer cells sensitive
to cetuximab.
The effect of this combination therapy has also been demonstrated on organoids derived from PDAC patients, indicating that RASON is a potential therapeutic target for PDAC patients with KRAS mutations
.
3.
Conclusion
Overall, the researchers identified a rason protein
that is essential for KRAS signaling.
This protein encoded by lincRNA binds directly to KRAS, allowing KRAS to stabilize in an over-activated state bound to GTP and is therefore necessary for
KRAS-driven tumorigenesis and tumor maintenance.
A decrease in RASON increases pancreatic cancer's sensitivity
to EGFR inhibitors.
They believe that for the "undruggable" KRAS signaling pathway in human cancer, the KRAS−rason interaction may be an attackable target
.
Original text search
Cheng, R.
, Li, F.
, Zhang, M.
et al.
A novel protein RASON encoded by a lncRNA controls oncogenic RAS signaling in KRAS mutant cancers.
Cell Res (2022).
https://doi.
org/10.
1038/s41422-022-00726-7
