H-Ras is a unique isoform of the Ras GTPase family, one of the most prominently mutated oncogene families across the cancer landscape. Relative to other isoforms, though, mutations of H-Ras account for the smallest proportion of mutant Ras cancers. Yet, in recent years, there have been renewed efforts to study this isoform, especially as certain H-Ras–driven cancers, like those of the head and neck, have become more prominent. Important advances have therefore been made not only in the understanding of H-Ras structural biology but also in approaches designed to inhibit and impair its signaling activity. In this review, we outline historic and present initiatives to elucidate the mechanisms of H-Ras–dependent tumorigenesis as well as highlight ongoing developments in the quest to target this critical oncogene.

This article is featured in Highlights of This Issue, p. 973

Despite incredible advances in targeted therapies and a vast, multipronged approach to its study, cancer grows increasingly prominent in a majority of the world. In the United States, as incidence rates continue to rise, cancer is even poised to overtake cardiovascular disease as the leading cause of death (1). A major unanswered question and persistent challenge facing present treatment options is the existence of various oncogenic mechanisms of intrinsic and acquired resistance, some of which have only recently been discovered.

A well-known and well-characterized point of origin for drug resistance is attributed to mutations within the Ras subfamily of small GTPases, of which mutations in three members—K-Ras, N-Ras, and H-Ras—hold clinical significance in humans (2). Cycling between active GTP-bound and inactive GDP-bound states, these proteins function as molecular on-and-off switches, playing vitally important roles in a host of different cell processes, ranging from motility to growth to senescence (3). Under normal conditions, Ras activation is under tight regulation by guanine exchange factors (GEF) and GTPase-activating proteins (GAP), which mediate GDP to GTP exchange and GTP hydrolysis, respectively (Fig. 1A; ref. 4). However, in about one-third of human cancers, this robust system of control is ablated by a single-point mutation occurring primarily at the G12, G13, or Q61 site of Ras (3). Without regular control, Ras preferentially locks into a GTP-bound active state, an end result that is ultimately oncogenic (5).

Figure 1.

Ras regulation cycle and isoform sequences. A, Ras GTPases exist under tight enzymatic regulation. GEFs, such as son-of-sevenless, effect Ras activation by exchanging a bound GDP for a GTP. GAPs will then bind to, stabilize, and accelerate enzyme activity. This terminates with the cleavage of the terminal phosphate, returning Ras to its inactive, GDP-bound state. B, Ras isoforms share a high degree of sequence homology (82–90%). In the G domain's effector lobe, which includes the vital Switch I and II domains, protein sequence is perfectly conserved across all isoforms. It is the HVR that is the primary source of sequence variation. Composed of the secondary signal domain and the CAAX motif, the HVR directly affects Ras prenylation and membrane localization. H-Ras, specifically, carries a unique dual-cysteine (Cys-181 and Cys-184) sequence in its SSD (denoted by stars).

Figure 1.

Ras regulation cycle and isoform sequences. A, Ras GTPases exist under tight enzymatic regulation. GEFs, such as son-of-sevenless, effect Ras activation by exchanging a bound GDP for a GTP. GAPs will then bind to, stabilize, and accelerate enzyme activity. This terminates with the cleavage of the terminal phosphate, returning Ras to its inactive, GDP-bound state. B, Ras isoforms share a high degree of sequence homology (82–90%). In the G domain's effector lobe, which includes the vital Switch I and II domains, protein sequence is perfectly conserved across all isoforms. It is the HVR that is the primary source of sequence variation. Composed of the secondary signal domain and the CAAX motif, the HVR directly affects Ras prenylation and membrane localization. H-Ras, specifically, carries a unique dual-cysteine (Cys-181 and Cys-184) sequence in its SSD (denoted by stars).

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Although first linked to cancer over 30 years ago, Ras mutations have continued to trouble both researchers and medical professionals alike (6). This is not only because Ras proteins are notoriously unsuited for direct inhibition, but also because Ras-mutant cancers exhibit an uncanny ability to render therapeutic agents ineffective, increasing tumor fitness (7, 8). Across this spectrum of mutant Ras-driven cancers, mutations of K-Ras are most frequent, occurring in about 25% of all cancers. This is followed by N-Ras and H-Ras mutations, which exist in about 10% and 4% of cancers, respectively. Interestingly, these mutations are not evenly spread across the cancer landscape; rather, mutant Ras isoforms seem to preferentially select for different cancer types. For example, pancreatic ductal, lung, and colorectal cancers are almost exclusively K-Ras mutated, whereas N-Ras mutations predominate in melanomas and leukemia (9). H-Ras mutants, despite their elevated expression in bladder–urothelial and head and neck cancers, remain infrequently observed. Accordingly, recent attention within the research community has largely been focused on the more abundantly expressed Ras isoforms. In this review, however, we specifically focus on H-Ras, discussing recent advances made in understanding its biology and highlighting novel therapeutic strategies against H-Ras-mutant cancers.

Early beginnings

A common misconception concerning the study of Ras holds that K-Ras is the most intensively studied and therefore best understood Ras family member. Given its role in driving a number of frequently diagnosed cancers, this seems a logical conclusion to draw (10). Yet, it is actually H-Ras that is, historically, the best studied; in fact, studies involving H-Ras were the earliest source for foundational knowledge related to Ras biochemistry, structure, and tumorigenesis (6, 11). This fact, however, was not necessarily an intentional choice. Harvey Ras, so named for the researcher who discovered it in 1964, was the first of the Ras family to be extensively isolated and studied (12). Coupled with the generally-held yet ultimately misleading belief in functional redundancy, whereby all Ras members overlapped in structure and mechanism, it took decades before experimental data revealed the existence of rather robust isoform specificities, including differential patterns of expression, variations in posttranslational modification, and slight disparities in subcellular localization (13, 14).

Structure and function

As previously stated, H-Ras structural studies served as the foundation of all information concerning Ras protein composition. This was due, in part, to the high degree of homology shared by Ras family members (82–90%; Fig. 1B). As such, introducing the structure of H-Ras is tantamount to introducing the structure of Ras in general. Briefly, Ras proteins consist of two “sections,” a highly conserved G domain (aa 1–165) and a hypervariable region (HVR; aa 166-188/189; ref. 15). The G domain contains the vital switch I and II domains within its “effector” lobe (residues 1–86), an area that is perfectly conserved across all Ras isoforms (16). This region is also responsible for allowing Ras to interact with effector protein pathways, primarily Raf-Mek-ERK and PI3K/AKT, to mediate such events as cell growth, survival, and proliferation among others (Fig. 2). It is also this domain that houses the most common sites (G12, G13, and Q61) of Ras oncogenic mutations (17).

Figure 2.

H-Ras signaling pathways. The major H-Ras signal transduction pathways are similar to those of other family members. Primarily, the Raf–Mek–ERK signal chain carries growth receptor signals into the nucleus, where transcription factors such as Myc initiate transcription of genes related to cell survival, proliferation, and epithelial-to-mesenchymal transition. On the other hand, H-Ras has also been linked to PI3K/AKT signaling. This pathway carries signals that eventually result in the activation of mTOR, which then facilitates protein synthesis, cell migration, and cell proliferation among others. PDK, phosphoinositide-dependent kinase-1; Raf, rapidly accelerated fibrosarcoma.

Figure 2.

H-Ras signaling pathways. The major H-Ras signal transduction pathways are similar to those of other family members. Primarily, the Raf–Mek–ERK signal chain carries growth receptor signals into the nucleus, where transcription factors such as Myc initiate transcription of genes related to cell survival, proliferation, and epithelial-to-mesenchymal transition. On the other hand, H-Ras has also been linked to PI3K/AKT signaling. This pathway carries signals that eventually result in the activation of mTOR, which then facilitates protein synthesis, cell migration, and cell proliferation among others. PDK, phosphoinositide-dependent kinase-1; Raf, rapidly accelerated fibrosarcoma.

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In contrast, the HVR displays little sequence similarity. Because this region contains the CAAX motif, which facilitates the posttranslational modifications requisite for proper Ras function, the end result is differential lipid attachment to individual Ras isoforms (Fig. 3; ref. 18). Broadly speaking, however, the canonical process of Ras modification begins with the farnesylation of the cysteine within the CAAX motif. CAAX prenyl protease 2, otherwise known as Ras-converting enzyme 1 (RCE1), then follows with the removal of the motif's remaining amino acids (AAX). Finally, protein-S-isoprenylcysteine O-methyltransferase (ICMT) attaches a methyl group to the carboxyl group of the now-farnesylated cysteine (19). Attempts to disrupt this process have met with varying degrees of success depending on Ras isoform, suggesting isoform-specific variations in the pathway (20). Accordingly, much of the identifiable distinctions between isoforms are attributed to differences within the HVR. As will be discussed later, however, recent experimental insights have provided newfound reason to believe such agents as farnesyltransferase inhibitors (FTI) might be particularly effective in combatting H-Ras–driven cancers.

Figure 3.

Posttranslational modification sequence of H-Ras. The modification sequence of the H-Ras CAAX motif generally identical to that of the other isoforms, with a few notable exceptions. FTase initiates the process by attaching a farnesyl moiety to the motif's cysteine residue. In other Ras isoforms, this step may be alternatively conducted by a geranylgeranyltransferase. Next, RCE1 cleaves the remaining motif residues (Val-Lys-Ser, for H-Ras). Immediately following, ICMT attaches a methyl to the carboxyl group of the now-farnesylated cysteine. Finally, in a step unique to H-Ras, PAT palmitoylates Cys-181 and Cys-184 directly upstream of the CAAX motif. These palmitoylated cysteines will aid in H-Ras membrane localization. FT, farnesyltransferase; PAT, palmitoyl acyltransferase.

Figure 3.

Posttranslational modification sequence of H-Ras. The modification sequence of the H-Ras CAAX motif generally identical to that of the other isoforms, with a few notable exceptions. FTase initiates the process by attaching a farnesyl moiety to the motif's cysteine residue. In other Ras isoforms, this step may be alternatively conducted by a geranylgeranyltransferase. Next, RCE1 cleaves the remaining motif residues (Val-Lys-Ser, for H-Ras). Immediately following, ICMT attaches a methyl to the carboxyl group of the now-farnesylated cysteine. Finally, in a step unique to H-Ras, PAT palmitoylates Cys-181 and Cys-184 directly upstream of the CAAX motif. These palmitoylated cysteines will aid in H-Ras membrane localization. FT, farnesyltransferase; PAT, palmitoyl acyltransferase.

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Membrane localization

Around the turn of the century, accumulating data began to chip away at the theory of Ras functional redundancy. Ras knockout experiments revealed H-Ras was not as crucial to mouse embryonic development as K-Ras, whereas other work suggested H-Ras operated through different signal transduction proteins than other isoforms (21, 22). In-depth studies of H-Ras structural idiosyncrasies, specifically those involving the HVR, served to explain some of these biological inconsistencies.

H-Ras is known to undergo unique palmitoylation at cysteines 181 and 184, directly upstream of the CAAX motif, as the final step of its activation prior to membrane localization (Figs. 1B and 3). Often, this region is considered a second signal domain, working in conjunction with the CAAX motif to effect Ras membrane targeting. Importantly, the H-Ras second signal sequence differs from that of other isoforms: the K-Ras sequence is a polybasic stretch of lysines (aa 175–180), whereas that of N-Ras includes a single, palmitoylated cysteine (aa 181; ref. 23). Functionally, this translates to a unique membrane localization scheme for H-Ras. Roy and colleagues found that, in depleting plasma membrane cholesterol, H-Ras–dependent Raf-1 signaling was drastically mitigated whereas K-Ras–dependent signaling was unaffected (24). This immediately suggested a unique tendency for H-Ras to target towards membrane lipid raft domains. Subsequent studies, however, suggested a slightly more complex process. It was found that GTP-bound H-Ras predominantly anchors to nonraft domains, whereas GDP-bound proteins exist more so in lipid regions. In terms of Raf signaling, this GTP-dependent liberation of H-Ras from lipid domains appears critical to its ability to bind and activate Raf protein (25). Whether this dependency exists in other H-Ras effector proteins is the subject of ongoing study.

Recent studies have furthered this avenue of H-Ras investigation. Pezzarossa and colleagues found, using techniques of superresolution imaging, that 60% of inactive, full-length H-Ras proteins group together in membrane clusters of 80 to 180 nm. This percentage increased significantly under conditions favoring the coalescence of cholesterol domains, furthering the theory of a strong H-Ras preference for raft domains (26). Interestingly, whether this clustering pattern applies similarly to mutant H-Ras proteins is yet uncertain, as emerging immunofluorescence data does not show significant colocalization between H-RasG12V clusters and raft biomarkers (27). What is more, the observed link between GDP-bound H-Ras and lipid domains seems to show a certain degree of context dependency. Specifically, although studies done in HeLa and other cells continue to find inactive H-Ras clustered on raft domains, data from human erythroleukemia (HEL) and colorectal adenocarcinoma (HT29) cells seem to show the opposite, with active, GTP-bound H-Ras migrating to membrane lipid domains (28). Thus, the model wherein GDP-bound H-Ras is sequestered to lipid-rich domains until GTP exchange occurs might not be as universally applicable as previously thought.

The functional relationship between H-Ras membrane localization and effector activity is still, as of yet, poorly understood. Although initial membrane attachment experiments looked at Raf signaling, it is perhaps the PI3K/AKT pathway that displays more H-Ras dependency (29). In one study, H-Ras silencing strongly disrupted phosphotidylinositol 3-phosphate levels in fibroblast cell (NIH3T3), which is a key secondary messenger molecule produced by PI3K enzyme to facilitate AKT activation. More specifically, H-Ras silencing affected asymmetric localization of PIP3 to the cell membrane during membrane extension and cell migration, despite the presence of global PIP3 levels. K-Ras and N-Ras knockdown, on the other hand, produced no such inhibitory response. This could suggest H-Ras to possess a critical role in cell migration, perhaps even in the context of tumor metastasis (30). What is more, the presence of membrane bound H-Ras appears critical to this activity, as soluble, GTP-loaded H-Ras demonstrated no effect on lipid kinase activity for any class I PI3K variants (31). At present, it is uncertain to what degree, if any, this interaction might be dependent on membrane lipid composition, although a relation between lipid rafts and PI3K/AKT signaling has been previously reported (32). The oncogenic implications of this relationship will be discussed later.

It is important to note, however, that debate continues about the formation of these Ras-filled microdomains. The currently prevailing theory holds that Ras proteins help to structure the domains in which they operate, thus helping to create an environment suited for their activity. This is in contrast to other theories which hold that Ras proteins are simply targeted to preformed microdomains.

Allosteric lobe effects

Although Ras sequence dissimilarities are generally introduced as residing almost exclusively in the HVR, there is another region of isoform divergence. Within residues 87 to 166 of the Ras protein sequence, also known as the “allosteric lobe,” sequence homology is roughly 80% to 90% (16). Accordingly, this region has emerged as an exploratory avenue of Ras research.

Broadly speaking, the allosteric lobe, as the name suggests, performs more of a supporting role in the proper function of Ras signaling. Such activities as membrane binding, ligand interactions, and nucleotide binding are all facilitated by, and to a certain degree dependent on, allosteric lobe interactions (16).

Structural analysis of H-Ras reveals a functionally important helix 4 (α4) region, which helps stabilize the H-RAS GTP structure as it binds to the membrane. What is more, residues R128 and R135, both part of the allosteric α4region, have been reported as “hotspots” of protein interactions, and disruptions or mutations to these sites seem to result in decreased Ras activity (16). As a result, the allosteric lobe has garnered some initial interest as a possible avenue of direct Ras inhibition. Recent experiments attempting to disrupt H-Ras allosteric lobe activity have reported promising outcomes for further study (33, 34). This strategy of Ras-targeting will be further discussed later.

Ubiquitination

Over the years, investigation into Ras isoform-specific trafficking, function, and localization patterns has led researchers to constantly seek previously unexplored areas of the Ras protein landscape. One of these areas which has begun to receive notable attention in the recent decade is that of reversible Ras ubiquitination, as there is increasing evidence that ubiquitination contributes to the differentiation of Ras isoforms.

Indeed, mono- and di-ubiquitin deficient H-Ras variants appear to be far more effective in recruiting Raf-1 to the plasma membrane, where activation occurs, and are therefore much more adept at mediating Erk activation (35, 36). This is attributed to linked ubiquitin chains at lysine-63, which has previously been reported to function in processes of membrane trafficking (37). Taken in context, ubiquitinated H-Ras is sequestered to endosomes, reducing not only the plasma membrane bound population of H-Ras but also its capacity to recruit Raf-1. Because Raf membrane recruitment is critical to activation, this observation would be consistent with data suggesting that K-Ras is a more potent Raf activator, as K-Ras seems either unaffected by or impervious to lysine-63 ubiquitination (21, 36, 38). Further investigation identified Rab GEF-1, otherwise known as Rabex-5, as a Ras E3 ligase, mediating the ubiquitination and thus endosomal compartmentalization of Ras (39, 40). These studies, however, focused primarily on the implications of ubiquitination on Ras localization and not on potential effects in Ras conformation or stability. Indeed, a literature search did not reveal many studies on the matter. In just one study done using only HEK293T cells, monoubiquitination of H-Ras at lysine-117 upregulates the process of GDP to GTP exchange, especially in HEK293T cells (41). Although this seems to directly challenge previous reports of Ras endosomal targeting, what is conceivably more likely is a complex model of ubiquitin regulation that is at once site, isoform, and cell line specific.

The tumorigenic capacity of Ras ubiquitination is no less controversial. OTUB1, an enzyme inhibiting Ras ubiquitination, promotes Ras activation, membrane sequestration, and tumorigenesis of wild-type (WT) Ras in non–small cell lung cancer cells. Elevated OTUB1 levels are also associated with increased ERK activation as well as worse patient outcomes (42). On the other hand, clinical data suggest that higher Rabex-5 expression, which was earlier deemed a Ras E3 ligase, results in poorer prognoses in a variety of cancer types (43). The degree to which these conflicting results are isoform- or cancer-specific remains to be seen. Increased research efforts are critical to furthering the existing knowledge base and evaluating ubiquitination as a viable target for Ras dysregulation.

One of the more commonly diagnosed cancers is squamous cell carcinomas of the head and neck (HNSCC), which results in over 650,000 new cases and 300,000 deaths annually (44). As previously mentioned, mutations in H-Ras are disproportionately expressed in this tumor type. Cancers of the thyroid, bladder, and prostate, among others, are also well known to carry H-Ras mutations, which almost exclusively occur at codons 12 and 61 (45). The detailed mechanisms by which H-Ras dysregulation functionally drives tumorigenesis, however, are still poorly understood, especially when viewed relative to the wealth of knowledge concerning K-Ras driver mutations. However, some exciting insights into H-Ras–driven tumorigenesis have recently emerged.

H-Ras and the PI3K/AKT pathway

Ras proteins operate through a large variety of effector pathways, and mutations that give rise to constitutively active Ras isoforms also tend to result in upregulated effector activity. There is mounting evidence to suggest the existence of differential effector pathway preferences among isoforms, although this is primarily related to Raf-pathway interactions and not fully understood in others (46). To that end, interactions between H-Ras and the PI3K/AKT pathway have been well-reported, and recent literature lends further credence to a previously hypothesized model whereby H-Ras mutations favor the PI3K–AKT pathway to drive oncogenic transformation (47).

In a recombinant study, an altered C-terminal targeting domain (tD) was used to fully sequester WT H-Ras to raft domains independent of GTP status. Although this action only minimally impacted PI3K activation, Erk phosphorylation noticeably diminished, consistent with previous reports (25). In addition, PI3K inhibition almost completely ablated H-Ras–driven proliferation, whereas MEK inhibition translated to only a modest inhibitory effect (29). These suggest, in H-Ras, an innate capacity to target PI3K, which is not wholly dependent on membrane microdomain location. Not only do these findings support an H-Ras preference for PI3K, they also furthers the notion that lipid rafts are more conducive to PI3K activation, thus explaining why K-Ras is more often linked to Raf signaling than is H-Ras (48).

In estrogen receptor–negative breast adenomyoepitheliomas, a rare form of cancer, recurrent H-Ras mutations at site Q61 were highly correlated with coordinate PIK3CA mutations. Forced expression of H-Ras Q61R in noncancerous breast epithelial cells resulted in the development of pronounced cancerous phenotypes followed by strong activation of the PI3K–AKT–mTOR pathway. Here, however, inhibition of either the MAPK or AKT pathway caused a comparable decline in cell proliferation, although this result was not unexpected (49). In H-Ras mutant HNSCC, H-Ras depletion fully inhibited PI3K signaling even in the presence of an activated EGF signal. Interestingly, EGF blockade by cetuximab treatment resulted in decreased phopho-AKT expression in WT BB49 (HNSCC cell line) with active H-Ras signaling. Together, these suggest the necessity of H-Ras and EGFR coexpression in allowing for the oncogenic effects of aberrant PI3K–AKT signaling to manifest. At the same time, this contradicts the notion that aberrant Ras activity may bypass the need for EGF signaling. Along those same lines, whereas treatment of Cal-33 (tongue SCC) cells with cetuximab did not reduce the number of colonies seen on colony-formation assay, treatment with cetuximab following H-Ras depletion fully inhibited colony growth (50).

These findings suggest the existence of a delicate balance between EGFR, H-Ras, and PI3K/AKT in determining oncogenic proliferation capacity. Clinically, the interconnected nature of these mutations could aid in developing more effective treatment strategies, especially in cetuximab-resistant cancer cells.

H-Ras–mediated drug resistance

Directly tied into Ras-driven tumorigenesis is the ability of these mutants to evade both endogenous and exogenous mechanisms of tumor suppression. Already, K-Ras mutant cancers, such as those of the colon, lung, and pancreas, have already demonstrated an ability to develop resistance against established methods of targeted therapy (51). Although less frequently encountered, H-Ras mutant tumors present similar degrees of treatment immunity.

In one study of WT HNSCC cell lines, introduction of H-RasG12V conveyed characteristics of cetuximab resistance; conversely, H-Ras silencing in resistant cell lines resensitizes them to treatment. The clinical impact mirrored these findings. In a clinical cohort of 55 patients with HNSCC, those with de novo H-Ras mutations were far less likely than those without H-Ras mutants to respond to cetuximab therapy (42.9% vs. 81.3%, respectively; ref. 50). In another case, Boidot and colleagues recently reported a patient presenting with metastatic colon adenocarcinoma that was nonresponsive to treatment with both panitumumab, an anti-EGFR antibody, and bevacizumab, a monoclonal antibody designed to impair angiogenesis by inhibition of VEGF-A (52). Intriguingly, sequencing analysis revealed no mutations in K-Ras, N-Ras, or B-Raf; however, a single G13D mutation was found in H-Ras. Subsequent in vitro studies revealed that introduction of H-RasG13D to WT Ras colorectal cell lines conferred resistance to cetuximab, similar to studies of HNSCC. This is quite possibly the first reported association of mutant H-Ras and EGFR therapy resistance in colorectal cancer, a cancer type wherein mutant K-Ras predominates (52). Clearly, this indicates a capacity for mutant H-Ras to mediate treatment resistance.

The ability of H-Ras mutations to elevate tumor fitness extends beyond that of EGFR therapy resistance. As the need to overcome cetuximab resistance rises, strategies of combinatory targeting have led to the development of many classes of inhibitors, including PI3K-pathway inhibitors. Although these have shown some promise in clinical trials, they are rendered ineffective in H-RASG12V HNSCC cell lines. Comparable to cetuximab, cell lines were only resensitized to PI3K inhibition upon H-Ras knockdown or silencing (53). Further investigation revealed constitutive activation of mTOR signaling, suggesting that MAPK pathway signaling might be bypassing PI3K signaling to continue mediating proliferative effects. Indeed, exposure to AZD8055, a mTOR inhibitor, dramatically ablated cell growth in both WT and mutant H-Ras cells.

Even endogenous mechanisms of tumor suppression seem to be disrupted by aberrant H-Ras activity. A major pathway of proliferation in animals is the Hippo (Hpo) pathway. Among the components of this signaling cascade, a variety of both oncogenes and tumor suppressors has been identified. Protein kinases Mst1 and Mst2 are one such group of suppressors, existing in an active homodimer formation as well as an inactive heterodimer form. Oncogenic H-Ras disrupts this regulation by shifting the conformational balance towards that of the inactive heterodimer, thus mitigating tumor suppressive activity and promoting tumor transformation (54).

Taken together, these discoveries reflect an incredibly diverse array of H-Ras–driven resistance mechanisms that drastically complicate efforts to combat mutant H-Ras cancers.

Novel implications of H-Ras in tumor biology

As Ras research continues to beget novel discoveries, the breadth of knowledge concerning H-Ras tumorigenesis has expanded as well. Increasingly, H-Ras mutations are being found in cancers beyond its usual landscape in cancers of the skin, head and neck, and bladder. A study of gastric cancer reported an ability for H-Ras to not only drive but also to enhance cancer aggressiveness. As stated by the authors of the study, “overexpression of H-Ras in gastric carcinoma cells accelerated proliferation, invasion, and angiogenesis.” In addition, this enhanced cancer phenotype was attributed to H-Ras–dependent activation of the PI3K/AKT and Raf-1 pathways (55). In a somewhat similar case, genetic screening of papillary thyroid carcinoma (PTC) revealed a novel H-Ras variant that expressed a rather unique 5′-UTR sequence. This variant was found more frequently in PTC tissues than in healthy samples, although the authors cautioned a requirement for larger studies before any tangible link can be made between broad level PTC development and progression (56). Finally, in a few cases, H-Ras mutations were found in conjunction with GNAQ and TERT mutations, both of which have been characterized as potentially oncogenic (57, 58). These findings are not so much paradigm shifting as they are a reflection of the degree to which ongoing work continues to shed light on new areas of H-Ras biology.

H-Ras and RNA interference

One such area of particular interest is that of a theorized link between mechanisms of RNA interference (RNAi) and Ras oncology. Specifically, it is now believed that Ras mutants share a degree of coordination with Argonaute 2 (AGO2), a key regulator of RNA-based genetic silencing. Original work conducted by Yang and colleagues found higher levels of tyrosine 393-phosphorylated AGO2 in H-RasG12V–expressing cells, resulting in premature, oncogene-induced senescence (59). Whether this is a cancer-driving event is unclear; however, parallel studies in K-RasG12V mutant cancer cells found a definitive, tumorigenic function arising from Ras-Ago2 binding (60). In brief, AGO2 phosphorylation impacts the overall efficacy of the RNA-induced silencing complex, translating to a rise in expression of both oncogenic K-Ras as well as oncogenic miRNA. It remains to be seen if these findings might be recapitulated in H-Ras.

Conversely, there have also emerged reports of certain miRNA transcripts serving a regulatory function against H-Ras gene expression. Specifically, miR-203 was found to be downregulated in H-Ras mutant skin cancer samples. Induced restoration of miR-203 in tumor-forming cells subsequently inhibited proliferation of the cell population. Identification of miR-203 targets, which included critical Ras pathway regulators among them, pointed to a link between H-Ras driven tumor formation and concurrent loss of miR-203 regulation (61).

The data discussed herein, among others, have already fostered a host of new research efforts aimed at dissecting the relationship between Ras, miRNA, and RNAi (62, 63).

Cancer stem cells

Another emerging field of research is that of cancer stem cells (CSC), which are thought to possess stem cell-like characteristics, including the ability to generate all cell types of a given cancer. As such, these cells could persist in tumors and initiate relapse or metastasis (64, 65). Research into the relationship between CSC initiation and Ras is still far from establishing a potential model of interaction, but there have been reports that suggest a correlation between oncogenic H-Ras and cancer stem cell-like phenotypes. Oncogenic, mutant H-Ras in urothelial cells, when coupled with p53 depletion in vivo, proved capable of enriching genes associated with stem cell genesis (66). In a different study, H-Ras G12V-transformed cells began to exhibit a CSC phenotype following serum depletion, which was meant to mimic the normally hypoxic and nutrient-deprived conditions of the tumor microenvironment. These cells began to exhibit chemoresistance, a known characteristic of CSCs, towards common drugs such as etoposide and irinotecan. Increased hexokinase and AMP kinase expression was also detected in serum-deprived mutant cells, although the common H-Ras signal transducers (MAPK, PI3K/AKT) did not display an increase (67). It remains to be seen how future research may further elucidate the relationship between CSCs and Ras, but CSCs may someday emerge as a key target for cancer treatments, especially when attempting to combat metastasis or invasion.

In the nearly four decades since it was first linked to cancer, the database of information about Ras has grown exponentially; yet, development of Ras-targeted therapies has not progressed in a concomitant manner. This is primarily due to the inherent challenges posed by the nature of Ras itself. Briefly, Ras proteins are notoriously “smooth,” presenting no obvious binding pockets for a potential drug. In addition, a molecule small enough to penetrate the cell membrane would not be large enough to affect any sort of functional interactions, especially given the high GTP-binding affinity of Ras (2, 15). The success of signal transduction therapies in conjunction with vastly improved scientific techniques and technologies has, perhaps finally, brought the ultimate goal of Ras inhibition within reach.

Presently, there exist a handful of drugs being tested for their efficacy against specific Ras isoforms (Table 1). Here, we will focus specifically on developments in the targeting of H-Ras.

Table 1.

H-Ras inhibitory compounds under development.

Compound nameDescriptionStatus
Tipifarnib Farnesyltransferase inhibitor Phase II clinical trial (69, 70) 
Lonafarnib Farnesyltransferase inhibitor Previously phase II; no trials ongoing (NCT00050336) 
NSC1011 Rce1 protease inhibitors Preclinical research (71) 
Cysmethnil Carboxyl methyl transferase inhibitors Preclinical research (72, 73) 
ISIS2503 Synthetic oligodeoxynucleotide Previously phase I; no trials ongoing (74) 
Salirasib Ras farnesylcysteine mimetic Previously phase II; no trials ongoing (75–77) 
NS1 Monobody against H-Ras allosteric region Preclinical research (33) 
KBFM123 H-Ras-GTP target Preclinical research (34) 
Compound nameDescriptionStatus
Tipifarnib Farnesyltransferase inhibitor Phase II clinical trial (69, 70) 
Lonafarnib Farnesyltransferase inhibitor Previously phase II; no trials ongoing (NCT00050336) 
NSC1011 Rce1 protease inhibitors Preclinical research (71) 
Cysmethnil Carboxyl methyl transferase inhibitors Preclinical research (72, 73) 
ISIS2503 Synthetic oligodeoxynucleotide Previously phase I; no trials ongoing (74) 
Salirasib Ras farnesylcysteine mimetic Previously phase II; no trials ongoing (75–77) 
NS1 Monobody against H-Ras allosteric region Preclinical research (33) 
KBFM123 H-Ras-GTP target Preclinical research (34) 

Farnesyltransferase inhibition

Currently, FTIs represent the most promising attempt to affect H-Ras activity. Rather than directly target the Ras protein, however, FTIs disrupt the crucial farnesylation step of Ras posttranslational modification, preventing successful membrane trafficking. Although the initial discovery of this drug class was heralded with great excitement, subsequent experiments and trials were unable to produce consistently good outcomes (20). K-Ras and N-Ras, it was ultimately revealed, could bypass FTase activity through an alternative geranylgeranyltransferase (GGTase)-directed pathway, rendering FTI treatment ineffective in this context (68).

Although this setback dampened the original optimism around FTIs, this class of drug has now been given new life as an H-Ras specific inhibitor, because H-Ras cannot be acted upon by GGTases. At present, two major compounds of this type have emerged: tipifarnib and lonafarnib. Although these compounds were initially marketed as pan-Ras inhibitors, the elucidation of the alternative geranyl pathway has reclassified them toward H-Ras–driven cancers. Although both compounds have shown promise in vitro and in vivo, the majority of studies focus on tipifarnib. In a recent preclinical study, tipifarnib exhibited a robust ability to impair progression of anaplastic thyroid cancers (69). In addition, it is presently involved in a number of ongoing clinical trials across a number of cancer types, including NSCLC, HNSCC, and peripheral T-cell lymphoma (NCT02383927, NCT03496766, NCT03719690, NCT02464228, NCT02779777, and NCT02807272). The preliminary results of one trial (NCT02383927) were recently revealed to be quite promising. As the trial intended to study the efficacy of tipifarnib in H-Ras mutated SCC, the majority of the patients enrolled had tumors of the head and neck. Among these, 35% of patients exhibited partial response; furthermore, almost all of the responsive tumors expressed mutations of either G12, G13, or Q61, suggesting an associated between mutation site and clinical benefit. Although work is still ongoing, the presented results provide proof of tipifarnib efficacy in H-Ras–mutated HNSCC (70). Whether future trials continue the momentum around FTIs remains to be seen, but as it stands, FT inhibitors remain the most well-studied and promising avenue of direct H-Ras inhibition.

Other targeting methods

Although farnesyltransferase inhibition represents an H-Ras–specific therapeutic approach, it is not the only potential route of inhibition against H-Ras–dependent cancers (Table 1). The other enzymes involved in the posttranslational modification of Ras, Rce1, and ICMT, have shown promise as targets for therapeutic agents (71–73). As it stands, though, the compounds reported to act against these enzymes exist in very early development stages, lagging far behind those of FTIs. Yet, because the targeted enzymes operate on all Ras isoforms, these novel inhibitors have the potential to operate beyond the context of H-Ras dependency, broadly impairing Ras membrane localization and signaling activity.

Other angles of direct inhibition have also been explored. In one trial, an antisense inhibitor of H-Ras proved modestly active in patients with advanced solid tumors (74). Another development comes in the form of salirasib, or farnesylthiosalicylic acid (FTS), which serves as a Ras farnesylcysteine mimetic. Mechanistically, salirasib competes with Ras for binding to escort proteins, thus disrupting membrane localization once more. Given its nature as a mimetic, FTS is postulated to hold potential as a broad-Ras inhibitor relative to FTIs, as farnesylated K-, H-, and N-Ras would all be affected by its competition. Early trials, however, have produced moderate responses, although the results do warrant further investigation (75, 76). An evaluation of salirasib in H-Ras positive bladder cancers showed mixed results in both WT and mutants: cell proliferation and migration slowed but many downstream Ras pathways remained unaffected (77). As with the introduction of any novel treatment agent, much more study is required before the true value of FTS as a new anti-Ras drug might be measured.

Efforts towards direct inhibition have also begun to evaluate whether the Ras G-domain might include areas of therapeutic value. An identified monobody named NS1 has been found to bind the allosteric region of H-Ras. More specifically, NS1 displays extensive interactions with the Arg135 residue of the Ras α4-α5 interface, an area previously purported to play a facilitating role in Ras structural stabilization, protein interactions, and nucleotide binding (16, 78). An in vitro analysis showed impaired oncogenic H-Ras dimerization following NS1 introduction. This was followed by downregulated ERK/MAPK as well as AKT activation. In addition, NS1 expression in bladder carcinoma cell lines disrupted H-RasG12V–mediated ERK phosphorylation (33). With a similar goal, researchers in Japan have reported the discovery of small molecule, KBFM123, which targets H-Ras-GTP in a pocket situated between the effector domain's switch I and II regions (34). Preliminary data relays an ability to bind and inhibit H-RasG12V–c-Raf interaction. Together, these outcomes demonstrate the therapeutic potential of Ras G-domain targeting, opening the door to a yet-untouched horizon of Ras inhibition.

Apoptosis

In the context of anticancer therapeutics, the interplay between H-Ras and apoptosis is a novel area of interest. It is well-known that, as part of its oncogenic hallmarks, Ras dysregulates mechanisms of apoptosis and broadly promotes cell survival. The specific mechanisms through which this occurs has been poorly understood, though the overarching idea is that Ras induces pathways that may be both antiapoptotic and proapoptotic, with the overall sum circumstances determining cell outcomes (79). That said, a defect or disruption of Ras-antiapoptotic signaling capacity might disrupt this precarious balance, allowing proapoptotic mechanisms to take over. In one 2016 study, cyclopentenone prostaglandin A1 (PGA1) triggered apoptosis in NIH3T3 cells via a mechanism of H-Ras–induced caspase activation. In cells that were either H-Ras deficient or possessed, a mutant H-Ras-C118S incapable of PGA1 binding, apoptosis did not occur. Interestingly, in H-Ras and N-Ras knockout cell lines, apoptosis again did not occur, suggesting the mechanism to be independent of K-Ras activation (80). In another study done in urinary bladder cancer J82 cells, the presence of oncogenic H-Ras rendered cells more susceptible to histone deacetylase inhibitor (HDACI) cytotoxicity and subsequent apoptosis (81). Both studies suggest that, in the presence of these compounds, there is altered H-Ras regulation of Raf-Mek-Erk and caspase activation pathways, which likely contributes to the apoptotic effect. Already, other compounds are being discovered which demonstrate an ability to induce caspase-dependent apoptosis in H-Ras mutant cancers (82).

Although much work has been done on H-Ras, there remain many questions and uncertainties. Key to a better understanding of this protein is an understanding of its signaling mechanism, specifically to what degree membrane localization or compartmentalization into lipid domains affects its ability to interact with and activate its signal transduction pathways. There are also certain roles of H-Ras that are as of yet poorly understood, including its relationship with miRNA, its potential function in cancer stem cells, and its co-occurrence with PI3K/AKT mutations. All told, this information might further an ability to target and inhibit its function in the context of cancer. Given the recent encouraging clinical data of H-Ras inhibitors in various cancers, we believe the future may be promising as far as clinical applications of single agents H-Ras inhibitors or their use within the context of future combination approaches.

The innate complexity of Ras-family–mediated signaling, tumorigenesis, and drug resistance remains an area of significant challenge and active research. In H-Ras–related studies, the simple infrequency with which mutant H-Ras–driven cancers are naturally encountered only serves to compound existing obstacles. Yet, that has not resulted in stagnation. Rather, robust and multifaceted research efforts have resulted in a wave of emerging insights and areas of study, many of which are still inadequately understood. That said, the propagation of new data surrounding H-Ras effector interactions, membrane localization, and tumorigenic settings has opened the door towards novel strategies of Ras disruption. Taken together, efforts to study H-Ras have contributed to delivering renewed hope that the ultimate goal of Ras inhibition might be one day realized.

No potential conflicts of interest were disclosed.

The authors thank Dr. Anthea Hammond for her editing of the manuscript.

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