Mutations in the RAS gene family are common in many cancer types. The point mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene typically affect the hotspots at codons 12 and 13 [1,2] However, at lower frequencies, KRAS mutations can also occur in codons 18, 61, 117, and 146. RAS GTPase-activating protein 1 (is a small guanosine triphosphatase (GTPase) that acts as a molecular switch and interacts with more than 20 effector proteins through localization to the inner surface of the cell membrane [1,2]. The point mutation in KRAS can impair the intrinsic GTPase activity of KRAS protein, preventing its conversion from an active form “guanosine triphosphate” (GTP) to its inactive form “guanosine diphosphate” (GDP). Consequently, KRAS remains permanently bound to GTP resulting in activation of downstream signaling pathways [1,2].

KRAS mutations are predominant in most cancers, such as pancreatic ductal adenocarcinoma (PDAC) (86%), colorectal cancer (CRC) (85%), and lung cancer (30%) [3]. This is followed by NRAS (12%) mutations, which are predominant in cutaneous melanoma and acute myelogenous leukemia. However, HRAS mutations that are found in bladder and head and neck squamous cell carcinomas are infrequently seen in other types of cancers [4]. According to the COSMIC v94 database, 99% of KRAS mutations are missense mutations, mainly with a gain of function.

In this review, we will first discuss the pathobiology of PDAC. Then, the significance of KRAS mutations in PDAC will be discussed. In addition, we will show how modulation of the immune response and promotion of angiogenesis by oncogenic KRAS can alter the tumor microenvironment (TME). We will finally highlight the link between diabetes and PDAC, as well as the importance of vitamin D for effective targeted therapies.

Tumors of the exocrine pancreas are, by far, the most common type of pancreatic cancers, of which PDAC is the most common type (90%). PDAC is an epithelial tumor, and its formation requires a stepwise progression over many years. In other words, it requires the transition of a normal pancreatic duct to a pre-invasive precursor lesion, a frank malignant, invasive cancer, then a metastatic tumor. Histologically, there are three morphological noninvasive precursor lesions of PDAC, including pancreatic intraepithelial neoplasms (PanIN), intraductal papillary mucinous neoplasms (IPMN), and mucinous cystic neoplasms (MCN), of which PanIN is the most studied one. PanIN may advance cancer that exhibit invasion, metastasis, and therapeutic resistance through a dense stromal microenvironment (desmoplastic) establishment in addition to the development of genetic variability [5]. The PDAC TME comprises a myriad of cells in addition to hyaluronic acid, cytokines, chemokines, and a variety of collagens. The cellular component includes macrophages, dendritic cells, T cells, and B cells [6]. Local immunity is always suppressed, resulting in an ideal milieu for tumor initiation, progression, as well as distant metastasis. The cold tumor with dominant CD4+ regulatory T-cells usually evades the immune system and dense desmoplastic TME hinders the access of therapeutic agents [7].

Several gene alterations have been identified during tumor progression and interaction with the TME. The whole-exome sequencing analysis of PDAC revealed around 60 genetic alterations, most of which are point mutations [8]. According to several studies, KRAS is the most frequently mutated oncogene in PDAC (from 70% to 95%). In addition to KRAS, mutations were identified in other well-known genes, e.g., CDKN2A (encoding p16), TP53, ARID1A, SMAD4, as well as in novel genes, e.g., ATM (one of the key genes of DNA repair), EPC1 and ARID2 (involved in chromatin modification), and KDM6A and PREX2 (involved in carcinogenesis) [9].

KRAS mutations in exon 3 have a remarkably favorable prognosis. Coexistent KRAS mutations were detected in the same pancreatic neoplastic mass more frequently than in other tumors. KRAS mutations coexistent with TP53 alterations and/or loss of SMAD4 protein herald a worse PDAC prognosis [10]. The sensitivity of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in the diagnosis of pancreatic malignant lesions can be improved by implementing the evaluation of the TP53 gene [11].

TP53 alterations have been detected in 50–75% of PDACs. The disease outcome is worsened with loss of normal p53 protein, mainly if combined with KRAS mutations and loss of expression of SMAD4. The coexisting mutations lead to one of the aberrant signaling nodes in PDAD that shows an enhanced activity of hepatocyte growth factor receptor (HGFR) and its respective tyrosine kinase, epidermal growth factor receptor (EGFR), and an increased expression of neuropilin 1, CD44, and β1 integrin. Such activity is augmented by heterodimerization of HGFR and EGFR [12]. Approximately 50% of pancreatic cancers harbor inactivated SMAD4 due to intragenic mutations or homozygous deletion, which occur late in PDAC. The loss of SMAD4 protein is associated with an increased risk of metastases and a worse prognosis [10,13]. In PDAC, SMAD4 mutations result in suppression of TGF-β signal transduction and may lead to altered sensitivity to gemcitabine [11,14]. Similarly, approximately 95% of sporadic pancreatic carcinomas have inactivated CDKN2A as a result of intragenic mutation [15,16]. CDKN2A is linked to familial pancreatic cancer. Suppressed p16 expression is associated with larger tumors and with a poorer prognosis [11,17]. It is noteworthy that CDK4 inhibitors have shown promising results for the treatment of CDKN2A-deficient tumors in preclinical PDAC models [18]. BRCA1/2 mutations have been identified in, 5 to 10% of PDAC. Such mutations may lead to either sporadic or familial disease [8,19].

Infrequent genetic alterations and events in PDAC include microsatellite instability (MSI), also known as defective DNA mismatch repair (dMMR), BRAFV600E mutations, and MGMT promoter hypermethylation [11]. In addition to these genetic alterations, other factors serve as fuel for aggressive pancreatic cancer development. This includes dysregulated stromal-associated factors, signaling pathways, and microRNAs (Figure 1), [20].

Subgroups of PDAC were defined according to the presence of mutations/genomic alterations/events. Intriguingly, the locally rearranged subgroup is characterized by >50 events limited to one to three chromosomes. These events are typically oncogene amplifications that target existing therapeutics or genomic catastrophes such as chromothripsis [21].

Approximately 86% of somatic alterations in PDAC target KRAS. G12D and G12V variants account for approximately 80% of KRAS mutations and hence the initiation of most PDAC cases [22]. G12 mutation is followed by that of G13 (9%) and Q61 (1%) in PDAC [23]. Mutations of the KRAS exon 2 codons G12 and G13 exist in almost all PDAC cases (in more than 95% of PDAC cases). Other mutations, such as Q61 (<1%) in KRAS exon 3 and K117 and A146 (<1%) in exon 4, seem to be additional hotspots associated with constitutively activated KRAS in pancreatic cancer [24].

In normal cells, the active state KRAS is bound to GTP, while it is bound to GDP in the inactive state. RAS proteins keep switching “on” and “off” through conformational changes through binding of GTP and GDP. GEF (guanine nucleotide exchange factor) promotes dissociation of GDP and acts as a positive regulator; GAP (GTPase-activating protein) promotes hydrolysis of GTP and acts as a negative regulator helping to keep most of KRAS in an inactive GDP-bound state (Figure 2) [25]. Most RAS mutations involve GAP-mediated inactivation of RAS. For example, substitutions in residues G12 prevent van der Waals bond formation between RAS and the GAP, leading to perturbation of Q61 (or the catalytic glutamine) orientation in RAS. This results in the pronounced attenuation of GTP hydrolysis, with enduring activation of RAS-driven downstream pathways [26]. Activated KRAS induces a myriad of downstream signaling pathways and effector proteins, such as mitogen-activated protein kinase (MAPK)–MAPK kinase (MEK), phosphoinositide 3-kinase (PI3K)–AKT–the mechanistic target of rapamycin (mTOR), rapidly accelerated fibrosarcoma (RAF)–MEK–extracellular signal-regulated kinase (ERK), and Nuclear factor-κB (NF-κB) pathway (among other nuclear transcription factors). These factors can enhance the survival, proliferation, transformation, and invasion of cancer cells [27]. Additionally, mutant KRAS results in the autonomous release of type I cytokine complexes by cancer cells. Subsequently, a cascade of events follows that leads to metabolic reprogramming (see Section 5) [28]. The signaling pathways of KRAS are discussed comprehensively in previous review articles [27,29,30,31]. The aforementioned studies point to the potential role of KRAS mutations in modulating the immune status of the TME.

The modulation of the immune response through several cytokines, as well as the promotion of angiogenesis by oncogenic KRAS, can alter the TME [27]. KRAS mutations are likely to coexist with mutations of other genes in PDAC, as previously described. The summative effect on the TME shapes the immune status of the tumor surrounding, a crucial factor that determines the capacity of the tumor to metastasize and to respond to therapeutic agents [21]. As an example, a worse PDAC prognosis is expected when KRAS mutations coexist with TP53 alterations and/or loss of SMAD4 protein. In addition, the combination of KRAS mutations and loss of SMAD4 enhances the activity of HGFR and EGFR, together with an increased neuropilin 1, CD44, and β1 integrin expression [21,32,33].

Yu and coworkers (2015) showed that RAS signaling regulates pathological inflammation in severe acute pancreatitis. Their study indicated that RAS signaling controls CXC chemokine formation, indirectly affecting neutrophil recruitment and tissue injury in the inflamed pancreatic tissue. Inhibition of RAS signaling resulted in the decreased taurocholate-induced pancreatic activity of myeloperoxidase, which indicates the suppression of neutrophil recruitment [34]. KRAS was involved in CXC chemokine formation and the induction of VEGF, which plays a critical role in pancreatic angiogenesis. Furthermore, RAS was shown to upregulate COX2, which, in turn, promotes tumor formation via MEK/c-Jun pathway and human umbilical vein endothelial cells (HUVEC) invasion [34,35].