Abstract
The diffuse-type histologic variant of gastric cancer is characterized by highly invasive growth patterns and lack of cellular cohesion. Two recent studies have identified highly recurrent mutations of the gene encoding the small GTPase RhoA and suggest that RhoA activity may have a tumor suppressive role in this disease.
Gastric cancer is the third leading cause of cancer mortality worldwide and, when diagnosed, carries a dismal prognosis. Although gastric carcinoma has marked heterogeneity, the two most salient subtypes are intestinal gastric cancer (IGC) and diffuse gastric cancer (DGC). The dichotomization of IGC and DGC emerged following recognition of their distinct histopathologic growth patterns. IGC is the more common variant and typically emerges following Helicobater pylori infection, which leads to atrophic gastritis, intestinal metaplasia, dysplasia, and finally carcinoma. Similar to most adenocarcinomas, IGC typically shows cohesive groups of tumor cells with a glandular architecture. DGC, by contrast, owes its name to its characteristic lack of cellular cohesion, invasion throughout the stroma, and poor cellular differentiation (often with a signet-ring cell morphology). Clinically, DGC’s propensity for invasion translates into early metastasis and poor survival. Moreover, unlike in IGC where tumors with ERBB2-amplification are treated with trastuzumab, we lack effective targeted therapies for DGC.
In addition to their histopathologic features, there are underlying biologic and genomic distinctions between DGC and IGC. A key finding shaping our understanding of DGC was the discovery that families with a hereditary form of DGC carried a mutation in CDH1, which encodes E-cadherin (Guilford et al., 1998). Beyond hereditary DGC, the vastly more common sporadic form of DGC has also been associated with E-cadherin loss, either through somatic mutation or promoter hypermethylation. Recently, a pair of studies published in Nature Genetics expands our understanding of DGC by describing novel recurrent mutations of RHOA, encoding the small GTPase RhoA, in 14.3%–25.3% of DGC patients (Kakiuchi et al., 2014; Wang et al., 2014).
Wang et al. (2014) performed whole-genome sequencing, DNA copy number, gene expression, and DNA methylation analyses of 100 tumor and nontumor paired samples, spanning both IGC and DGC. Their analysis revealed frequent mutations in TP53 in both subtypes, ARID1A in EBV-related or microsatellite instability-related cancers, and CDH1 in DGC. RHOA mutation was identified recurrently within DGC and, following sequencing in a larger DGC cohort, was found in 14 of 98 DGC patients (14.3%). Kakiuchi et al. (2014) initially performed whole-exome sequencing within 30 DGCs and focused sequencing in another 57 cases, finding RHOA mutations in 22 of 87 (25.3%) cases.
These findings and similar results emerging from The Cancer Genome Atlas study of gastric adenocarcinoma (unpublished data) implicate RHOA as a novel candidate driver of DGC. RhoA is a member of the Rho family of small GTPases-Ras-like proteins that act as an intermediary between cell surface receptors and different intracellular signaling proteins. Similar to other GTPases, RhoA cycles between an inactive, GDP-bound configuration and an active GTP-bound configuration that interacts with downstream effectors, such as ROCK, that impact the structure and dynamics of the actin cytoskeleton, cell migration, cytokinesis, and the cell cycle. RhoA overexpression has been observed in various cancers, and RhoA activity has been implicated in tumorigenesis and tumor cell invasion (Karlsson et al., 2009).
Given the characteristic invasive growth patterns that are a hallmark of DGC, mutations in RHOA could be predicted to lead to constitutive activation of RhoA, enhancing activity of downstream mediators and increasing cellular invasion. Among the mutations in RHOA identified by these two studies, one led to a truncated protein, while the others were missense events with dramatic clustering in the amino terminal portion of the protein. However, the specific RHOA mutations identified in DGC were not at sites analogous to oncogenic mutations in RAS-family GTPases that cause RAS to become locked into its active-GTP bound state. These RHOA mutations were noted in hotspot sites, including Y42C, G17E, R5Q/W, and L57V. The most common alteration, Y42C, seen in 14 cases, lies in the effector-binding region of RhoA. Although not previously identified in cancer, the Y42C substitution in RhoA had been evaluated in earlier biochemical studies, which revealed attenuated activation of protein kinase N (Sahai et al., 1998). RhoA-Y42 notably corresponds to Y40 on HRas, where mutations selectively reduce HRas activation of RAF, but not other RAS effectors (Joneson et al., 1996), suggesting that the Y42 RHOA mutation may similarly modulate RhoA activity. Intriguingly, G17E mutations of RHOA were identified in five patients. Recent genomic sequencing studies in T cell neoplasms identified highly recurrent RHOA G17V mutations and demonstrated functionally that these mutants fail to bind GTP and act in a dominant-negative fashion to inhibit RhoA GTP loading (Palomero et al., 2014; Sakata-Yanagimoto et al., 2014; Yoo et al., 2014).
To functionally interrogate these novel RHOA mutations found in DGC, Kakiuchi et al. (2014) studied several cancer cell lines harboring RHOA mutations: the OE19 cell line (adenocarcinoma of the gastric cardia), the breast cancer cell line BT474, and the colorectal cancer line SW948. They showed that small interfering RNA (siRNA)-mediated silencing of RHOA significantly impairs in vitro proliferation in these mutant cell lines but does not similarly impact gastric cancer cell lines with wild-type RHOA. Furthermore, they demonstrated that reintroduction of the codon 17 or 42 RHOA mutants, but not reintroduction of wild-type RHOA rescued cell proliferation effects of RHOA siRNA, suggesting tumor-promoting activity for these RHOA mutants.
The results from Wang et al. (2014) provide additional insights into the potential role of mutant RHOA. Using a Rho binding domain assay to immunoprecipitate RhoA-GTP, the authors showed that both the Y42C and L57V mutants significantly attenuate the GTP-associated form compared to wild-type protein, indicating a potential defect in RhoA activation with these mutants. They further utilized primary mouse intestinal organoids to study the impact of RHOA mutants Y42C and L57V upon anoikis (cell death induced when anchorage-dependent cells detach from the surrounding extracellular matrix). Inhibition of anoikis may represent a key requirement for DGC, because loss of E-cadherin leading to reduction in cellular adhesion has been shown to result in acute cell death via anoikis (Kantak and Kramer, 1998). With dissociation of the mouse intestinal organoids, the introduction of Y42C or L57V RHOA mutants enhanced organoid reformation. While treatment with ROCK inhibitor Y-27632 also enhanced colony growth, wild-type RHOA induction reduced the colony forming efficiency.
Through comprehensive genomic characterization, these studies demonstrate that, along with CDH1 mutations, RHOA mutations are quite common in DGC but not in other variants of gastric cancer. Intriguingly, these results suggest a model whereby wild-type RhoA activity has a tumor suppressive role in the pathophysiology of DGC and that RHOA mutations inhibit this tumor suppressive function, suggesting these mutants are not merely loss of function, but may repress RhoA activity. It remains to be clarified, however, whether RHOA mutations merely attenuate physiologic RhoA activity or, alternatively, if these mutations result in a gain of function. Given the pressing need for new therapeutic targets for DGC, further research will be required to determine if the activity of these novel RHOA mutants and the deleterious role of RhoA activity in this disease can be exploited as a therapeutic vulnerability.
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