Upstream activators are dominated by GTP Exchange Factors (GEFs), which number almost 100 different genes in the human genome. GEFs are activated by cell surface receptors e.g. GPCRs, RTKs, integrins. In turn, GEFs activate small G-proteins by increasing GTP/GDP exchange rate at the nucleotide-binding site. e.g. Tiam, Vav1 and Vav2 are GEFs of Rac, and Dbl, Dbs, and ITSN are GEFs of Cdc42 and some, Tiam1, Vav1,2 activate both GTPases (Fig.1). Due to Mg2+:GTP having a higher binding affinity than GDP, and a ten-fold higher level than GDP in cells, the exchange of nucleotides favors GTP in a homeostatic cancer cell. GEFs can be upregulated by mutations that increase binding affinity to the small G-protein, or mutations that simulate its phosphorylated form, or simply increased expression via demethylation of their promoter regions (reviewed in 14).
Only in the last 10 years, has the prevalence of GEF oncogenes in metastatic cancer been found to be broad due to the advent of genomic databases15. Although GEFs are broadly represented as oncogenes, individually mutated GEF genes make up a small portion of the total pool of players. For example, in melanoma metastasis, Tiam GEF mutations have a 9.0% prevalence while Vav2 mutations have a 3.6% prevalence, whereas all mutated RC GEFs are present in 80.7% of melanomas14. Under these circumstances, many GEF targets are necessary for a comprehensive drug development effort, and tumor screening is necessary to delineate causative GEF genes in individual patients. There are many options for drug development in R&C GEFs, for example small molecules could bind the protein interaction site, or an allosteric site, or mask the phosphorylation site thus inhibiting its activity16.
Fewer players are currently known on the downstream side of the R&C axis. Most are kinases that are activated by GTP-bound forms of R&C. In particular, p21-activated kinase (PAK), activated Cdc42-associated kinase (ACK), and more recently myotonic dystrophy-related Cdc42 binding kinase (MRCK) have been the focus of drug development efforts17. For example, Unbekandt et al. (2018) present a tour de force of MRCK inhibitor development for melanoma18.
As a side note, many of the oncogenic GEFs’ promotors are demethylated during transition to metastasis14, which could conceivably be targeted with demethylase inhibitors, but the question of specificity comes into the picture when using those enzymes with a broad, sometimes undefined, substrate range.
Interdependence of the Ras and R&C axes in metastatic cancer
Critically, it has been reported that primary cancer oncogenes, such as K-Ras and N-Ras, work in concert with the R&C pathway when transitioning from primary to metastatic phenotype19,20, which makes primary cancer progression to metastasis highly dependent on R&C axis activation/dysfunction. Some GEFs, e.g. P-Rex, have been shown to contribute at a later stage metastatic cancer progression21,22, which opens the possibility to develop later stage-specific therapeutics. The multitude of RC axis modulators make it essential to develop diagnostics to determine which GEFs and effectors are differentially expressed or mutated, this information will allow oncologists to pick the matched drug for a particular patient.
Although metastatic cancer is destined to kill on average 58% of cancer patients5,6, this realization is not equated to the NIH & FDA’s focus to encourage therapeutics in this area. In fact, only 16% of active cancer clinical trials are for metastatic disease23. Clinical trials targeting prevention and treatment of metastasis are a difficult sell when evidence of metastasis is not apparent early in the disease. And hence, its imperative for scientists and clinical oncologists to communicate this aspect at every opportunity so the barriers are decreased as early as possible. Cytoskeleton is proud to provide highly dependable and accurate Pulldown and GLISA Activation Assay Kits which have been used by researchers in this field over the past 20 years.
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23 Clinicaltrials.gov as of 2021-09-16, and search for “cancer” and limit to “active, not recruiting”. Then calculate percent of titles containing the word “metastatic”. Result: 1271 active, not recruiting, cancer trials, wherein 203 contained the word “metastatic” in the title.