KRAS, the Kirsten Rat Sarcoma Virus, is a part of the Rat family Sarcoma Virus (RAS). It’s a signal transducer protein. That means it can regulate the various signaling pathways responsible for cellular survival, invasion, growth, and the functional regulation of various channels. KRAS is also a GTPase (Guanosine Triphosphate) activated enzyme-based RAS family oncoprotein. The structure of the KRAS is 85% homologous for the development of different types of Cancer in comparison to other isoforms of the Ras family (i.e.: Harvey Rat Sarcoma Virus, Kirsten Rat Sarcoma Virus and Neuroblastoma Rat Sarcoma Virus). That’s why it’s been regarded as the most significant among all of the RAS family onco-proteins. The bondage of the KRAS at the Receptor Tyrosine Kinase (RTK) or, more specifically, at the Effector Growth Factor Receptor (EGFR) leads to a higher intracellular concentration of the GTP than the GDP (Guanosine di-phosphate), which eventually activates the membrane-bound isopropyl based KRAS protein. Later, this activation of the KRAS stimulates the different cellular pathways such as [RAS- AKT (Adenosine Tyrosine Kinase)-mTOR] responsible for cellular proliferation, which means the growth of the cells, [RAL-Nf-kB] activates the innate immunity, necessary for the regulation of the cellular defense, and [RAF-MEK- ERK] maintains the life cycle of the cells. However, due to various genetic disorders, errors in the synthesis of the KRAS at the site of the ribosome, or the impact of external factors such as the Potential of Hydrogen (pH), temperature, and others, KRAS can go through a series of mutational challenges leading to the development of missense or point mutations. As a result, the Aspartic acid replaces Glycine, making the KRAS hyperactivated. So, whether a receptor bounds at the EGFR or not, KRAS will always remain hyperactivated, eventually impacting the transcription and the transmission to synthesize the myeloma cells or cells with infinite division and growth.
Figure 01: The occurrence of the Missense mutation (Glycine by Aspartic acid).
This figure illustrates the mechanism leading to a missense mutation or point mutation in the KRAS gene. The primary sequence of the KRAS protein is shown, with a specific focus on the codon sequence where a mutation can occur. The transition from glycine to aspartic acid, highlighted in the sequence, is a common mutation site. External factors such as pressure, temperature, and dysregulation can induce this mutation. The process is depicted as a sequence of events starting from the primary KRAS sequence, influenced by external factors, leading to a missense mutation or point mutation. This mutation results in the substitution of glycine with aspartic acid, significantly impacting the protein's function and contributing to oncogenic transformation.
These mutations can lead to the development of various types of Cancer and metabolic disorders. So, to learn about various types of KRAS structures, we must understand their thermodynamic properties first. In the KRAS, the P loop or the Switch-I is the predominant site of mutations. Alongside 7 different mutational states, the application of engineered mutations seems highly frequent (40%). According to some recent analysis, almost 53.5% contain engineered mutations. WT (Wild type), C118S, G12A, G12D, G12V, and Q61H are common. We have performed the study in two completely different ways. At first, we conducted the study, where the first part of the analysis focused on the alterations of the KRAS structure and the development of various disorders. This covers both aspects of the physiological and the functional point of analysis. In the second part of the study, we have drawn the KRAS's Primary (collected from the PDB), Secondary, 3D, and Thermodynamic states using the i-Tesser. Eventually provides us with a complete orientation of the KRAS structure and its functional characteristics.
1.2. Alterations of the KRAS structure and the functions Usually, the mutational transformation results from replacing the Glycine with Aspartic acid, leading to missense mutations or point mutations. [Note: It’s a type of mutation in which a single amino acid from the residue gets replaced by another amino acid, resulting in various undesired side effects.]. As a result, hyperactivated KRAS is developed, which can function independently to continuously synthesize cellular division and survival. Additionally, the pH of the location gets lower, and it damages various cellular organelles and generates Reactive Oxygen Species (ROS), which is responsible for the occurrence and the resurgence of Cancers and various related disorders.
Figure 02: This schematic diagram illustrates the impact of the Rat Sarcoma (RAS) gene and its variants on different cell types and the associated conditions.
1.3. The Role of KRAS in the Development of Cancer As discussed in the previous section, the KRAS mutational characteristics are responsible for tumor cell growth and the development of PDAC (Pancreatic Adenocarcinoma) and CRC (Colorectal Cancer). Metastases of these two types of solid tumors is the reason behind the mortality of thousands worldwide.
In this formula, S represents the standard deviation of the population, n is the size of the sample, xxx denotes each value from the given population, and x‾overline{x}x is the mean of the population. In the context of KRAS mutation research, calculating the standard deviation is essential for understanding the variability and dispersion of data points in experimental results, such as gene expression levels or responses to treatments. This statistical tool aids in assessing the reliability and significance of the observed data, thereby contributing to the interpretation of experimental outcomes and the development of targeted therapies.
The table gives us an idea about Lung, Pancreatic, Blood, and Colon cancers and their rate of survival in recent times.
Figure 03: Bar-chart to highlight the relationship between Cancer types and the Rate of Survival.
The bar diagram on the figure visually represents the given information from the provided table with a marginal value of standard deviation. Based upon the upwardly mentioned Table and the graphical representation of the data, the relationship between the different type of cancer and the survival rate in 2018 is resembled and points that even with the application of the various advanced treatments and therapies both Lung and Pancreatic cancer still has the highest morbidity, 95.1%, and 92%, respectively. Application of the standard deviation equation has helped us to figure out the Standard Deviation. KRAS plays a significant role in the occurrence of PDAC, having one of the highest morbidities, making it more interesting due to its sensitive location at the intersection of various blood vessels and with a success rate of 15% only the traditional therapies such as Radiotherapy and Chemotherapy are unable to function appropriately due to the rapid upregulation of alternative compensatory pathways as well as desmoplastic reaction ultimately making it more challenging.
To check the mutational and transcriptional landscape of the PDAC, the GTPase KRAS alongside tumor suppressor genes such as p53, CDKN2A (Cyclic-dependent kinase inhibitor type-2), and SMAD4(Mothers Against Decapentologenic-4) exhibited mutations. The mutated KRAS, amplification of their mutant alleles, overexpression of AKT-2, and the activity of the upstream regulator (PI3K) are often elevated in PDAC, increasing the survival of the tumor cells, which can be furtherly defined as a part of the epigenetic dysregulation resulted due to the modifications of the human DNA.
Figure 04: The developmental flowchart of Pancreatic Adenocarcinoma (PDAC)
The upward figure resembles the development of Pancreatic Adenocarcinoma (PDAC). As soon as the mutation at the KRAS results, it leads to the initiation of the Pancreatic intra-neoplasm stage 1, where the cells on the pancreas go through a series of altered modifications regarded as the initiation of tumorigenesis. The developed hyperplastic lesions are known as pancreatic intraepithelial neoplasia and the intraductal mucinous neoplasms (IPMN) and their prosperity to develop into cancer, which looks like finger (Papillae). Then it comes to stage 2 (mucinous cystic neoplasm) and stage 3 (intraductal papillary mucinous neoplasm) due to the inhibition of cyclic-dependent kinase 2A—eventually, the development of the PDAC results from inhibiting the tumor suppressor gene p53 and SMAD-4. Similarly, the KRAS has also been responsible for the development of Colorectal Cancer. It is considered the most life-threatening among solid tumors and the third most diagnosed cancer in United States of America (USA). According to the latest prediction been made by the American Cancer Society, 153,020 will be diagnosed with cancers, and 52,550 will die in 2023.Similar to Pancreatic Cancer, the initiation of the Colorectal Cancer is also started in the normal epithelial cells. The inhibition of the Adenoma Polypmatous Coli (APC) develops an abnormal growth of the cells called; Adenomatous Polyp, impacted by the missense mutation because of the KRAS. The APC initiates most of both sporadic and hereditary colorectal cancers. The molecular outcome of the APC function loss lies mainly in its inability to downregulate the oncogenic canonical Wnt signaling pathway. The abnormal cascade activation leads to the overexpression of Wnt target genes involved in proliferation. Resulting in the development of lower-grade dysplasia or disordered cellular structures, develops Higher-grade Dysplasia at the inhibition of the DCC gene [Note: both cellular changes can easily be observed in the figure entitled “The physiological development of Colorectal cancer”], a putative tumor suppressor gene. The somatic mutations of the DCC gene and the netrin-1 during the organization of the spinal cord led to the position of DCC inactivation, significantly impacting tumorigenesis. To eventually inhibit the p53 gene, resulting in Colorectal Cancer.
Figure 05: The developmental flowchart of Colorectal Cancer (CRC)
Figure 06: The KRAS regulated various cellular pathways.
1.4. The Role of KRAS in the Development of Rasopathies The hyperactivation of the KRAS can also lead to the development of Rasopathies. It’s a series of rare disorders where the alterations in the “Ras (Rat Sarcoma Virus)/MAPK (Mitogen Associated Phosphate Kinase)” signaling pathways occur. A table is given below to mention various KRAS dependent Rasopathies, their symptoms, and causes.
Table 02: KRAS-dependent Rasopathies, Symptoms, and their causes
Rasopathies
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Symptoms
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Cause
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Neurofibromatosis type 1
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Neurofibromas, problems with the eyes and the bones
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NF-1 gene mutation
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Noonan Syndrome
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Short stature, heart defects, unusual facial features.
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Altered KRAS, RAF-1 gene
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Cardio-facio-cutaneous Syndrome
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Small chin and short nose
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Altered KRAS, BRAF, MEK1/MEK2
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1.5. The Role of the KRAS in the Development of Diabetes Mellitus Type-2 In addition to Cancer and Rasopathies, the KRAS can also play a significant role in the development of Diabetes Mellitus Type-2. The bondage between the advanced glycolytic endpoints at the receptor activates the Phosphate Kinase C(PKC), eventually impacting the secretions of the NADPH oxidase. This increases the intercellular concentration of the Reactive Oxygen Species (ROS) and damages the DNA. All these results are due to the mutations in the KRAS and affect the secretion of the Cytochrome P450 and the Glycolysis process by inhibiting the Hexosamine kinase and Pentose Phosphate pathways. These alterations of the cascade develop insulin-resistant diabetes mellitus type-2, functioning in an unusual positive feedback mechanism rather than the natural positive feedback mechanism. A drop in the glucagon level in the blood leads to the rise of insulin concentrations in the peripheral tissues. This is an example of the negative feedback mechanism. However, in the positive feedback process, they work in an agonistic pattern by increasing each other’s volume, which seems to be synchronizing. That’s how the insulin-resistant diabetes develops.
Figure 07: The developmental process of the diabetes mellitus type-2, due to the mutation of the KRAS.
The figure illustrates the pathway involved in the development of type-2 diabetes mellitus as a consequence of KRAS mutation. The process begins with Advanced Glycation End-products (AGE) interacting with the Receptor for Advanced Glycation End-products (RAGE), leading to the activation of Protein Kinase C (PKC). This activation stimulates NADPH oxidase, resulting in the production of Reactive Oxygen Species (ROS). The ROS, in turn, influences various metabolic processes, including the activity of cytochrome P450 and the regulation of glucose levels. ROS also impacts the pentose phosphate pathway and hexosamine kinase activity within the inner membrane, both of which are linked to glycolysis. The interconnected pathways highlight the complex biochemical cascade triggered by KRAS mutation, contributing to the pathogenesis of type-2 diabetes mellitus.