The discovery and simulation analysis of a novel mutation c.40 G < T (V14F) in the NRAS gene in patients with colorectal cancer in Saudi Arabia

1 Introduction

Colorectal cancer(CRC) is the third most common cancer in humans worldwide and ranks second for the most leading lethal malignancy (Siegel et al., 2019, Arnold et al., 2017). Unfortunately, approximately 20 % of CRC are initially diagnosed when they have already metastasized to other organs (Arnold et al., 2013). According to the World Health Organization (WHO) statistics the CRC is the third leading cause of death in Saudi Arabia aged less than 70 years of age group and is responsible for one in eight deaths due to the CRC related malignancies (Bray et al., 2018, WHO 2014).

The study conducted by Peeters et al., on five-year survival of CRC patients demonstrated that the possibility of survival with stage I is 90 % as compared to stage IV patients who showed only 10 % survival, and thus overall five-year survival appears to depend on the CRC stage (Peeters et al., 2015). Notably, the Saudi CRC patient’s overall survival (OS) rate was lower than the internationally reported survival rates based on tumor stage (WHO, 2010).

Despite the availability of various treatment modalities to attenuate CRC development and progression, early diagnosis and correct identification of cancer stages appear to be the most effective strategies to reduce mortality rates, alteration in bowel movements, sudden weight loss, abdominal and pelvic pain, and anemic conditions (Ahnen et al., 2014). These symptoms are considered key indicators for clinicians to start an early and thorough investigation to rule out any possibility of CRC; moreover, patients must also be educated to take such symptoms seriously and report clinicians to be diagnosed perfectly in earlier stages of the disease (Lucente and Polansky, 2018).

From this perspective, there is an indispensable need to implement effective and accurate early-stage screening and monitoring programs to detect malignancies. The adaptation of reliable diagnostic tools of prognostic significance will not only enable the diagnosis of the disease as early as possible but can also be beneficial in predicting chemotherapeutic response outcomes effectively in CRC patients. The in depth understanding and advent of modern molecular tools and techniques have led us to further understand the etiology of cancers at the molecular level. The deep advancement in the knowledge of cancer development and progression has further enhanced our understanding of cancer biology at the molecular level, especially the genetic and epigenetic events that are involved in tumorigenesis; thus, it enables researchers to focus on key signaling pathways involved in the development of cancer, which may be beneficial to reveal novel targets with therapeutic significance (Clarke and Kopetz, 2015).

Notably, the role of RAS family genes in carcinogenesis has been well investigated in CRC, and its oncogenic potential often arises due to the alteration of genes that play a significant role as key mediators as well as in inducing early tumorigenesis in these models. Numerous studies have reported that KRAS proto-oncogene, GTPase (KRAS) and NRAS mutations seem to arise early in the process of colorectal carcinogenesis and thus exert their effect on regulating constitutive signaling and activation of downstream effectors such as mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinases (PI3K) dependent pathways (Yoon et al., 2014, Ebi et al., 2011), and hence, act as a significant contributor to cancer progression along with other altered genes (Barbie et al., 2009). Multiple studies have demonstrated that, KRAS and NRAS mutations account for 45 % and 5–8 % of CRC cases respectively (Rodriguez-Salas et al., 2017). The majority of mutations in KRAS and NRAS were mainly reported in codons 12 and 13 (90 %) as compared to other codons such as 59, 61, 117, and 146, where they occur less frequently in both KRAS and NRAS genes. However, among the RAS gene family NRAS is the foremost oncogene identified to play a role in melanoma and these NRAS mutations account for almost 20 % of all melanomas (Cancer Genome Atlas Network, 2015). Frequent NRAS mutations were primarily reported at position 61 where amino acid substitution occurs from glutamine (Q) to arginine (R), lysine (K) or leucine (L) (Fedorenko et al., 2013). These mutations have been found to be involved in blocking NRAS into a GTP-bound state, leading to impaired GTPase activity (Simanshu et al., 2017). Furthermore, a deleterious mutation, glycine (G) to aspartic acid (D), has been reported at codons 12 or 13 and comprises 20 % of all NRAS mutations in the amino acid change form (Fedorenko et al., 2013). These functional mutations have been demonstrated to be involved in preventing the association of GAPase-activating proteins (GAP) with NRAS and are more frequently observed in mucosal melanoma than in cutaneous melanoma (Simanshu et al., 2017). Once the NRAS gene is mutated, cascade events occur in the cell signaling pathway, and a series of activations occurs on MAPK signaling as compared to mutations in PI3K–AKT pathways, where they hardly exert such effects on MAPK, indicating that NRAS could be involved in modulating this pathway effectively (Hodis et al., 2012). Alterations in these pathways lead to subsequent dysregulation of the cell cycle and cell proliferation signals (Mendoza et al., 2011).

Hence, in this context, the aim of this research was to study the NRAS gene at the molecular level and define the genetic alterations in the Saudi population to determine the genetic heterogeneity associated with CRC malignancies and better understand the disease manifestation. In this study, we analyzed mutations in the NRAS gene in a Saudi population using automated DNA sequencing and found a novel mutation associated with CRC. At codon 14 valine is substituted with Phenylalanine (V14F) in the NRAS protein. We identified a previously reported V14I mutation in another patient. To understand the functional consequences of the newly identified novel mutation, we carried out in silico modeling studies to establish its impact on cell signaling, especially NRAS downstream effector molecules involved in tumorigenesis.

2 Materials and Methods

3 Results

To investigate and validate the NRAS gene mutation spectrum and its functional correlation with disease susceptibility and treatment outcome in patients with CRC originating from Saudi Arabia, we first amplified exons 2 and 3 of the NRAS gene with a specific pair of primers. After purification of the amplicon cycle, sequencing was performed, and all 89 tumor samples were subjected to DNA sequencing. We identified a novel NRAS mutation (V14F) in a patient with CRC. The identified mutations are depicted in the representative chromatograms in Fig. 1. At codon 14, valine is replaced by phenylalanine (V14F). Notably, we also identified a previously reported mutation in the same codon, where valine is converted into isoleucine (V14I).

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Fig. 1

Chromatograms of the two patients with heterozygous mutations c.40 G > T (V14F), c.40G > A (V14I) and control sample.

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Homology models for the mutant NRAS proteins (V14F and V14I) were generated using the SWISS-MODEL web server. A total of 11,249 and 11,290 templates were found for NRAS (V14F) and NRAS (V14I), respectively. The crystal structure of the NRAS protein (PDB ID: 6ZIZ) was used as a template to create NRAS (V14F) and NRAS (V14I) homology models (Fig. 2). Docking studies of GTP binding with wild-type NRAS protein (6ZIZ), NRAS V14F and NRAS V14I using the SWISSDOCK web server showed differences in free energy change (kcal/mol) of −14.03, − 5.16 and − 5.80, respectively. The three-dimensional molecular structures of NRAS protein (6ZIZ), NRAS V14F and NRAS V14I bound with GTP, and the two-dimensional structures representing the interaction of amino acids to GTP are shown in Figs. 3, 5 and 7. In the MD simulation experiments (each repeated three times) for the complexes of NRAS (wild type), NRAS V14F, and NRAS V14I with GTP, the contacts (protein–ligand) were plotted against simulation time to understand how the binding forms evolved over the course of the simulation. Hydrogen bonds, hydrophobic contacts and ionic interactions are indicated by red, green and blue dots, respectively. The 'Solute RMSD from the starting structures', namely NRAS WT, NRAS V14F and NRAS V14I with GTP, plotted against the simulation times. The RMSDs for Calpha [RMSDCa], backbone [RMSDBb], and all heavy atoms [RMSDAll] plotted against simulation time for NRAS WT, NRAS V14F, and NRAS V14I with GTP are shown in Figs. 4, 6, and 8, respectively. In addition, analysis of the effects of the mutations on biological function using the PROVEAN web tool revealed that the V14F mutation was deleterious (PROVEAN score: − 4.267), whereas the V14I mutation was classified as neutral (PROVEAN score: − 0.830). However, mutation analysis using Missense 3D software did not reveal any structural changes or damage caused by amino acid substitutions in the NRAS.

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Fig. 2

NRAS Protein Structure. (A) Wild type NRAS (B) NRAS (V14F), and (C) NRAS (V14I). The amino acid at the 14th position in NRAS (WT), NRAS (V14F), NRAS (V14I) are shown in as space-filling [(Calotte or Corey, Pauling, and Koltun (CPK)] models and visualized using CLC Drug Discovery Workbench 4.0.

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Fig. 3

Structure of NRAS-GTP Complex (A) The three-dimensional structure of NRAS (WT) with GTP (B) Two dimensional (2D)-diagram of binding interactions for the complex between GTP and NRAS (WT).

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Fig. 4

Solute RMSD from the starting structure (vertical axis) as a function of simulation time. The Calpha [RMSDCa], backbone [RMSDBb], and all-heavy atom [RMSDAll] RMSDs were plotted against the simulation time for NRAS WT interactions with GTP.

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Fig. 5

Structure of NRAS (V14F)-GTP Complex (A) The three-dimensional structure of NRAS (V14F) with GTP (B) Two dimensional (2D)-diagram of binding interactions for the complex between GTP and NRAS (V14F).

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Fig. 6

Solute RMSD from the starting structure (vertical axis) as a function of simulation time. The Calpha [RMSDCa], backbone [RMSDBb], and all-heavy atom [RMSDAll] RMSDs were plotted against the simulation time for NRAS mutant protein (V14F) interactions with GTP.

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Fig. 7

Structure of NRAS (V14I)-GTP Complex (A) The three-dimensional structure of NRAS (V14I) with GTP (B)Two dimensional (2D)-diagram of binding interactions for the complex between GTP and NRAS (V14I).

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Fig. 8

Solute RMSD from the starting structure (vertical axis) as a function of simulation time. The Calpha [RMSDCa], backbone [RMSDBb], and all-heavy atom [RMSDAll] RMSDs were plotted against the simulation time for NRAS mutant protein (V14I) interactions with GTP.

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4 Discussion

Currently, one of the research priorities is to identify the key signalling pathways associated with tumorigenesis, as well as to analyse potential drug resistantmutant phenotype variants in cancer cells. It would be promising for personalized medicine in terms of evaluating and correlating the drug response in individual phenotype variants with distinct sets of genetic alterations might be beneficial in adopting suitable chemotherapeutic strategies effective for the management of such diverse phenotype-related CRC patients (Rasool et al., 2021; Rasool et al., 2024). The recently proposed model by Fearon and Vogelstein for CRC is a multistep genetic process and is considered a benchmark for elucidating the involvement of multiple genes in the development and progression of carcinogenesis, particularly in solid tumours (Fearon and Vogelstein, 1990). According to this model, the initiation of carcinogenesis occurs with the rise of adenomatous polyposis (APC), a tumor suppressor gene that is inactivated and consequently leads to alterations in the KRAS gene. Furthermore, many consequential mutations in the TP53, PIK3CA, and TGF-β pathways that lead to changes in the downstream signalling network are more often correlated with malignant transformation and, as a result, the development and progression of cancer (Samuels and Velculescu, 2004). Although the above-mentioned model described seven distinct mutations that play a vital role in the development and progression of colorectal carcinoma, modern advancements in molecular techniques, especially single-cell sequencing, have revealed more than 80 genes in a single CRC cell (Samuels and Velculescu, 2004). Despite this, recent findings have confirmed the involvement of only 15 genetic mutations as leading drivers associated with carcinogenesis (Wood et al., 2007, Leary et al., 2008).

SwissDock is an integrated in silico tool that can be effectively used for docking simulation studies via a web interface (Grosdidier et al., 2011, Naseer et al., 2016). In this study, we performed docking simulations with the NRAS protein structure (6ZIZ) and mutant structures (V14F and V14I) using the SwissDock web tool. The replacement of valine at amino acid position 14 with phenylalanine or isoleucine in the NRAS protein decreased the binding affinity of these proteins to GTP. Molecular docking studies also confirmed that although both mutations caused structural instability, the V14F interaction with GTP was more unstable than that with V14I. Based on PROEAN and missense 3D web tools, we found that the V14F mutation was deleterious compared to V14I, but neither mutation caused structural damage in the NRAS protein; however, they both functionally damage the protein and resulted in impaired efficiency.

5 Conclusion

Currently, there are no significant efforts to understand the treatment approach for various genetic mutation backgrounds affecting individual CRC patients with altered NRAS genes. Near-term treatment strategies will include combining MEK inhibitors with inhibitors of key intracellular signaling pathways (either involved in RAS signaling or not) or other drugs that disrupt cell cycle checkpoints or epigenetics. To develop personalized therapies, it is important to understand in detail the molecular etiology of each patient. In this study, we investigated the NRAS gene at the molecular level and identified a novel mutation, V14F, in a Saudi patient. This mutation disrupts structural affinity as evaluated using in silico modeling. Further studies with more patient samples and animal models are needed to reach a consensus for therapeutic interventions based on this novel mutatoin in CRC patients.

Disclosure of Funding

This research work was funded by Institutional Fund Projects under grant no (IFPNC-013-141-2020).

CRediT authorship contribution statement

Mahmood Rasool: Writing – original draft, Supervision, Resources, Project administration, Investigation, Data curation. Absarul Haque: Writing – review & editing, Writing – original draft, Conceptualization. Mohammed Alharthi: Investigation, Data curation. Abdulrahman Sibiany: Investigation, Data curation. Mohammed Saad Alamri: Investigation, Data curation. Samer Mohammed Hassan Alqarni: Investigation, Formal analysis. Irfan A. Rather: Validation, Supervision, Project administration. Adeel Gulzar Chaudhary: Visualization, Supervision, Software. Peter Natesan Pushparaj: Writing – review & editing, Resources, Conceptualization. Sajjad Karim: Software, Methodology, Investigation, Formal analysis.