When we think of how cancers develop, we usually imagine mutations that lead to proteins gaining aberrant, tumor-promoting functions, that differ from their normal functions. But does altering the levels of normal proteins also elicit changes that can lead to cancer? In a recent study published in Science Signaling by the Berger Lab, part of the Human Biology Division at Fred Hutch, researchers found that protein abundance of mutant RIT1, but also wild-type, may promote oncogenic signaling in lung adenocarcinoma. In this common form of lung cancer, roughly 30% of tumors harbor activating mutations in KRAS, part of the RAS family of guanine triphosphatases (GTPases). While oncogenic KRAS mutations are being extensively studied, mutations in a gene encoding another RAS family GTPase, RIT1, were discovered only recently and are poorly understood. This study led by graduate student April Lo, used multiomic approaches to explore the biological pathways regulated by RIT1 and how they relate to oncogenic KRAS signaling. Lo explains that as a recently discovered oncogene, there have been studies looking at RIT1’s “role in cancer and how it differs from the more infamous KRAS,” but their goal was to “generate an unbiased functional profile of RIT1's signaling network,” and shed light on its oncogenic mechanisms.
To understand how oncogenic RIT1 may perturb cell signaling in the context of lung cancer, and if it does so in a similar manner as KRAS, the authors ectopically expressed either wild-type or mutant forms for RIT1 and KRAS in human airway epithelial cells. They then looked at genome-wide changes in gene expression elicited by RIT1 and KRAS variants, finding that oncogenic KRAS, in addition to both wild-type and mutant RIT1, induced KRAS transcriptional signatures. Using mass spectrometry, the authors then sought to identify proteins that were differentially abundant and phosphorylated across all KRAS and RIT1 variant conditions. Through examining phosphorylation of abundant proteins, they were able to predict which kinases and signaling pathways were responsible for driving differences in protein phosphorylation in the different conditions. Consistent with their transcriptional analysis, the differential phosphoproteome revealed that mutant RIT1, like mutant KRAS, can activate the canonical RAS effector pathways.
Lo et., al next asked how the proteomes and phosphoproteomes of wild-type and mutant KRAS and RIT1 relate to each other. Curiously, they found high correlation among the proteome and phosphoproteome of all cell types, with the exception of wild-type KRAS cells. This finding meant that both wild-type and mutant RIT1 were highly similar to oncogenic KRAS expressing cells and also highlight a critical divergence between KRAS and RIT1. While expression of wild-type KRAS is not capable of activating downstream oncogenic RAS signaling, expression of wild-type RIT1 induces transcriptional and proteomic signatures similar to ones activated by RIT1 and KRAS mutants. So how is it that expression of both mutant and wild-type RIT1 can lead to the same activation of oncogenic signaling? Common mutations in RIT1 actually reside in part of the protein that target it for degradation, leaving the molecular switches of RIT1 in tact but promoting unregulated levels of this protein, which resembles wild-type RIT1 overexpression.
To further investigate the consequence of RIT1 expression, the researchers asked what type of genes were upregulated in KRAS and RIT1 expressing cells that might play a role in development of lung adenocarcinoma. They identified genes regulating epithelial to mesenchymal transition (EMT) as being highly enriched among up-regulated proteins across all KRAS and RIT1 expressing cells. EMT is an important process in carcinogenesis that can promote tumor growth and metastasis. While KRAS has previously been known to promote EMT, little had been known about whether RIT1 also played a role in EMT. To functionally determine whether EMT characteristics are present in RIT1-transformed airway epithelial cells, the researchers performed a scratch assay, a wound healing assay used to study cell migration as cells undergo EMT to close the wound. Consistent with a role for RIT1 in promoting EMT, wild-type and mutant RIT1 expressing cells showed enhanced migration, closing the wound up to 1.5x and 1.9x faster than control cells, respectively. Lo notes that this result was particularly exciting since they were able to use their “proteomics and transcriptomic data to hypothesize a role for RIT1 and then follow it up with a few directed experiments to strengthen the association.” Future work aims to better understand RIT1's function from different angles, particularly looking at how the RIT1 protein is regulated. “The long-term goal,” Lo adds, “is to enable the design and application of RIT1-specific therapies for lung adenocarcinomas to improve the lives and survival rates of patients with RIT1-mutant cancers.”
This work was funded by the National Institutes of Health, the National Cancer Institute, the National Institute of General Medical Sciences and the National Science Foundation.
UW/Fred Hutch Cancer Consortium member Alice Berger contributed to this work.
Lo A, Holmes K, Kamlapurkar S, Mundt F, Moorthi S, Fung I, Fereshetian S, Watson J, Carr SA, Mertins P, Berger AH. Multiomic characterization of oncogenic signaling mediated by wild-type and mutant RIT1. Sci Signal. 2021 Nov 30;14(711):eabc4520. doi: 10.1126/scisignal.abc4520. Epub 2021 Nov 30. PMID: 34846918.