Colorectal cancer (CRC) ranks third in incidence and fourth in cancer mortality globally (1). Most of the studies on CRC have focused on genetics and the genetic alterations of genes, such as KRAS proto-oncogene, GTPase (KRAS), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), adenomatous polyposis coli (APC), tumor protein 53 (p53) or phosphatase and tensin homolog (PTEN), among others, as well as their effects on molecular pathways, such as phosphatidylinositol-4,5-biphosphate 3-kinase (PI3K)/PTEN/ serine/threonine kinase 1 (AKT) and Rat sarcoma (RAS) gene family/rapidly accelerated fibrosarcoma (RAF) gene family/mitogen-activated protein kinase (MEK) gene family/ extracellular-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), that lead to the development of CRC (2-4). In particular, the KRAS gene has been identified as a proto-oncogene activated by point mutations in more than 40% of CRC cases (5); its mutational status has been associated with a worse disease prognosis and lower response to treatments, such as immunotherapy (6). Nevertheless, patients wild-type KRAS gene also have a low response to treatments such as cetuximab (26-41%) or panitumumab (11-17%) (5). Knowing the methylation status as well as other epigenetic modifications associated with proto-oncogenes might offer new information contributing to the development of new therapeutic strategies in CRC, given that these modifications are reversible and that there are currently drugs that re-establish the function of these genes and thus improve the response to conventional treatments such as chemotherapy or radiotherapy (7,8). However, little is known about epigenetic modifications in wild-type proto-oncogenes.

The KRAS protein is a GTPase whose function is the transduction of signals that activate signaling cascades, such as PI3K, MAPK and T-cell lymphoma invasion and metastasis-inducing factor 1 (TIAM1), which cause cell proliferation and survival due to evasion of apoptosis and activate metabolism and cell growth, migration, and invasion (9). The role of wild-type KRAS protein in CRC is unknown; it is also unknown whether protein overexpression may have a similar effect to gene mutation, by promoting tumor progression. Regarding the regulation of KRAS gene expression, its distal promoter is involved in the transcription of the gene (10), and presents a large number of CpG islands and has several regions for anchoring specificity protein 1 (Sp1) and neurofibromin 1 (NF1) protein, associated with transcription initiation complexes (10,11). There are also microRNAs (miRNAs), such as miR-18a-3p and miR-143, in which a direct interaction with KRAS mRNA has been demonstrated by it binding to the 3’-untranslated region end of the mRNA, leading to its degradation or prevention of the translation process (12,13). The relationship of these miRNAs to the KRAS gene has been extensively studied (12-18).

This study aimed to determine whether there is a relationship between the expression of the wild-type KRAS gene (mRNA and protein), methylation of the distal promoter of this gene, and miR-143 and miR-18a-3p levels in formalin-fixed and paraffin-embedded tissues (FFPE) from patients with sporadic CRC. Information that allows determining whether epigenetic factors such as DNA methylation or miRNAs regulate the expression of wild-type KRAS may have important implications in understanding its role in the mechanism of CRC carcinogenesis, as well as possible applications in the prevention, diagnosis, or treatment of this type of cancer.

Definition of study subjects. This study included FFPE tissue blocks from 51 patients with sporadic CRC and wild-type KRAS gene who were treated at the gastroenterology service of the National Cancer Institute, Bogotá, Colombia between 2008 and 2015.

The amplification and detection of mutations in the KRAS gene was initially carried out using a Therascreen^® KRAS RGQ PCR Kit (Qiagen, Hilden, Germany) and subsequently using Cobas^® KRAS mutation test (Roche, Basel, Switzerland). For both kits, a Cobas z 480 analyzer (Roche) was used, following manufacturer instructions. FFPE tissue samples from three patients with CRC with KRAS mutations in codons 12 and 13 were used as reference.

DNA/RNA extraction and quantification. Extraction of genomic DNA and total RNA was performed by manual microdissection technique and 2-mm diameter punch of the tumor regions of the FFPE tissues selected by two pathologists, without including non-tumor tissue, using AllPrep DNA/RNA FFPE Kit (Qiagen), following the instructions given by the manufacturer. The extracted nucleic acids were quantified using Qubit RNA HS Assay, Qubit RNA BR Assay, Qubit DNA BR Assay, and Qubit DNA HS Assay Kits (Thermo Fisher Scientific-Invitrogen, Waltham, MA, USA), following manufacturer protocols.

KRAS mRNA expression levels. The expression levels of wild-type KRAS mRNA were determined by reverse transcriptase-quantification polymerase chain reaction (qPCR) with commercial KAPA SYBR^® FAST One-Step qRT-PCR Master Mix (2X) Kit (Kapa Biosystems, Wilmington, MA, USA) on a LightCycler 480 II platform (Roche), following manufacturer indications and using previously described primers for KRAS (19) and ubiquitin C (UBC) (20) as a reference gene. Absolute copy number quantification was performed.

Expression of miR-18a-3p and miR-143. For this analysis, miRNAs were retrotranscribed using miScript II RT Kit (Qiagen). Quantification was performed by real-time PCR using miScript SYBR Green PCR Kit (Qiagen) and specific primers obtained from the miScript Primer Assays Kit (Qiagen) for miR-18a-3p, miR-143, and small nucleolar RNA, C/D box 61 (SNORD61) as reference gene, on the LightCycler 480 II platform (Roche), following the manufacturer indications. Absolute copy number quantification was performed.

Wild-type KRAS protein expression. KRAS protein expression was evaluated by immunohistochemistry (IHC). The tissues were blocked by 0.3% hydrogen peroxide and incubated for 20 min at 37˚C with rabbit polyclonal antibody to KRAS (HPA049830; Atlas Antibodies, Voltavägen, Bromma, Sweden) dilution 1:20 in phosphate-buffered saline. The primary antibody was identified using UltraView Universal DAB Detection Kit (Ventana Medical Systems, Inc.). Immunodetection was carried out with diaminobenzidine, while counterstaining was performed with hematoxylin, followed the Ventana automated procedure (Roche).

IHC evaluation. The presence/absence of cells staining for KRAS in the cell membrane and in the cytoplasm; the intensity of staining (weak, moderate, or intense); and the percentage of cells staining in the observed tissue were assigned values between 0% and 100%. For the analysis of protein expression, an IHC score was generated, both for membranous and the cytoplasmic expression, resulting from multiplying the staining percentage score by the score for staining intensity (weak=1, moderate=2, or intense=3). Normal colon mucosa was used as positive staining control.

Methylation analysis of the distal promoter of the KRAS gene. Bisulfite conversion of the DNA, PCR amplifications of the distal promoter of the KRAS gene and amplimer sequencing were performed as previously described (21,22). The sequences obtained were aligned against the reference sequence (GenBank: X07536.1), using the free CLC Sequence Viewer software (version 8.0) (CLC Bio-Qiagen, Aarhus, Denmark).

Statistical analysis. The results obtained were treated as categorical variables. Frequency tables were constructed and based on data distribution, analysis groups were established and subsequently used to find possible associations between the variables analyzed in this study using Fisher’s exact test. The following categorical variables were considered: Normalized KRAS mRNA expression (absent/low: <0.01 and high: ≥0.01); normalized miR-18a-3p expression (absent or present); normalized miR-143 expression level (absent/low: <0.01 and high: ≥0.01); KRAS protein level evaluated as IHC score: absent or low: ≤10 in membrane and ≤100 in cytoplasm; and high: >10 in membrane and >100 in cytoplasm); methylation level in one or more of the four CpG islands evaluated in the distal promoter of the KRAS gene. Additionally, multivariate analyses were performed to determine the strength of association between the selected variables. For these, missing data were discarded; consequently, analyses were performed for death from 41 patients. The variable ‘stage’ was re-categorized into early (stage I and II: T1N0M0 to T4bN0M0) and advanced (stage III and IV: T1N1M0 - any T, any N, M1c) (23). A significance level of α<0.05 and a marginal significance of α<0.1 were established for all variables. Data were analyzed using R-Project software (version 4.0.3).

Baseline status. The mutational status for the KRAS gene, codons 12 and 13, was evaluated in 51 patients and none were revealed to have mutations in these codons. The demographic and clinicopathological characteristics of the patients included in this study are presented in Table I.

Expression of wild-type KRAS mRNA. In four cases (7.8%), it was not possible to determine KRAS mRNA expression since there was no expression of UBC. These cases were excluded from subsequent analyses (Table I). Based on the observed data, three categories were constructed for KRAS mRNA expression levels to facilitate subsequent analyses: Absent: 23 cases (48.94%); low level (values <0.01): 8 cases (17.02%); and high level (values ≥0.01): 16 cases (34.04%) (Table I).

Expression of miR-143 and miR-18a-3p. Considering the expression level distributions for miR-18a-3p, data were categorized as expression absent and present, while for miR-143, data were categorized as low or absent (Table I). Expression levels were different for each analyzed miRNA; 64.7% of cases did not express miR-18a-3p, whereas only 19.6% of cases did not express miR-143.

Expression of wild-type KRAS protein. The immunoreactivity to the KRAS antibody observed in one of the cases used in this study in the membrane and in the cytoplasm is illustrated in Figure 1. Wild-type KRAS protein was most frequently expressed in cytoplasm (98.4%) in comparison to the membrane (31.4%) (Table I). Regarding the IHC score, in most cases, scores represented absent or low KRAS expression (Table I).

Correlation between KRAS mRNA and protein expression levels. No statistically significant associations were found between KRAS mRNA expression category (absent, low, and high) and the IHC score for KRAS protein expression in the membrane (p=0.08) and the cytoplasm (p=0.79).

Correlation between wild-type KRAS expression and miR-18a-3p and miR-143 expression. No correlation was found between KRAS mRNA expression and miR-18a-3p or miR-143 expression, miR-18a-3p and protein expression in the membrane and the cytoplasm, nor miR-143 expression and protein levels in the cytoplasm measured as IHC score in the univariate or multivariate analyses, including other variables (age, sex, degree of differentiation, and stage). When performing logistic regression, categorizing by clinical variables, IHC score and miRNA expression, no statistically significant association was found between miR-18a-3p and KRAS protein expression in the cytoplasm or the membrane. There was no statistically significant relationship between miR-143 and cytoplasmic or membranous KRAS expression (data not shown).

Methylation of the distal promoter of the KRAS gene. Methylation status was analyzed in 30 cases. In 28 of these (93.3%), no methylation was detected in any of the CpG islands in the analyzed region (Figure 2A), whereas in two cases (6.7%), partial methylation was detected in some of the CpG islands evaluated (Figure 2B and C). In the two cases with partial methylation in the promoter region of the wild-type KRAS gene, no membranous expression of KRAS protein was detected, while high cytoplasmic KRAS protein expression was found (IHC scores were 180 and 200, respectively). In these two cases, KRAS mRNA expression was low, miR-18a-3p expression was absent, while miR-143 expression was high.

This study highlights novel information on the methylation status of the distal promoter of the wild-type KRAS gene, the expression of the protein at the cellular level and how it relates to the expression of two miRNAs whose direct targets are the wild-type KRAS gene.

By qPCR analysis, 51 patients with a diagnosis of CRC who did not have mutations in the KRAS gene at codons 12 and 13 were selected. However, mutations at codons 61 and 146 were not taken into account due to their low frequency (24). It is important to consider that despite the sensitivity of qPCR in the diagnosis of KRAS gene mutations, detection is limited by the cellular heterogeneity present in the tumor, the region from which the sample is extracted, and the integrity of the DNA used to perform the analysis, given that in most cases samples from FFPE tissues are used (25). Therefore, it is possible that there were cell subpopulations that may have had mutations in the KRAS gene and were not detected (26).

In this study, expression of KRAS protein in the cytoplasm was higher than that in the membrane. In other studies of CRC, KRAS expression has been reported with highly variable values, between 16% and 100% in the membrane (27-29) and between 42% and 100% in the cytoplasm (29-33), which is probably due to the lack of unified criteria that would allow comparisons between them and the different types of antibodies used in each study compared to ours. Contrary to what was observed in the present study, some studies have reported higher KRAS protein expression in the membrane than in the cytoplasm (28,32), which may be caused by KRAS ‘turnover’ mediated by endosomes from the endoplasmic reticulum, which might contain a higher proportion of KRAS not yet available at the membrane level (34). In turn, these proportions may vary between the analyzed samples. It is important to consider that these studies do not mention whether the patients analyzed had mutations in the KRAS gene, a fact that might have an impact on the cellular location of the protein, given that in mutational states, the protein is constitutively active in membrane signal transduction. Another possible cause of these discrepancies may be the lack of specificity of our antibody for the KRAS protein (34,35), given that the antibody used in this study also recognizes HRAS and NRAS, which might lead to overestimation in the IHC results obtained.

Interestingly, The Human Protein Atlas shows that KRAS protein expression in normal colon tissue using the antibody used in the present study is 100%, with an intense staining pattern, while in CRC samples, a 75% positivity and different staining intensities are reported, which indicates that KRAS protein expression decreases during carcinogenesis (36). It has also been reported that KRAS mutation does not alter the expression level or subcellular localization of KRAS protein (28). Although the overexpression of the wild-type KRAS gene can be considered a mechanism comparable to a gain-of-function due to mutations, the high expression of wild-type KRAS protein in normal tissue rules out the possibility that an increase in its expression has a similar effect to that of activating mutations and suggests opposite effects given its decrease during tumor progression. Some studies conducted in murine and in vitro models demonstrated the role of wild-type KRAS as a tumor-suppressor gene in the lung (37) and in the colon and rectum (38,39). These data suggest that wild-type KRAS protein has tumor-suppressive activity, which is frequently lost during tumor progression (37,38,40,41).

No statistically significant association was found between KRAS mRNA and protein expression levels in the membrane and the cytoplasm, which is in agreement with what was reported by Chen et al. (12) and the information given by the producer of the antibody used in this study (https://www.proteinatlas.org/ENSG00000133703-KRAS/pathology). Lack of correlation may be explained by considering that mRNA is very labile, and it is possible that part of the nucleic acid is degraded during its extraction. These results may also be affected by the stability of both mRNA and proteins, which is regulated by mechanisms not evaluated in this study, such as post-transcriptional mRNA methylation, post-translational modifications of proteins, among others (42-46).

Regarding miR-18a-3p expression, the frequency of positive patients (35.3%) in our study is lower than previously reported for this miRNA, with expression levels between 77% and 92% in FFPE tumor tissues and in fresh CRC tissue samples, respectively (18,46,47). It is important to consider that this study used FFPE material collected between 2008 and 2015 for RNA extraction, a fact that may have some impact on the results obtained due to the instability of the RNA extracted from these tissues, even though miRNAs are considered more stable than mRNA due to their size. In this study, no significant association was found between the expression levels of miR-18a-3p and KRAS mRNA.

Regarding miR-143, the expression found in this study was similar to other studies in which low values between 57% and 88% or absence have been reported (14-17,48-51). Evidence suggests that a decrease in miR-143 in CRC is independent of the transcription process, and it was hypothesized that this might be due to reduced Dicer-processing activity or to reduced miRNA stability, possibly as a result of diminished retention in a ribonucleoprotein complex (49).

Recently, a study was published evaluating the expression levels of this miRNA in samples from patients diagnosed with CRC, but without mutations in the KRAS gene, in which no significant differences were observed between the expression levels of this miRNA and normal tissue adjacent to the tumor (52), a fact that is contradictory to several studies showing the role of this miRNA with miR-145 in tumor suppression (53).

No significant association was found between the expression of miR-143 and KRAS mRNA or protein. On the contrary, other studies of patients diagnosed with CRC and of colon adenocarcinoma cell lines have determined an inverse correlation between KRAS protein and miR-143 expression levels (12), but to our knowledge, there are no studies on patient samples with which the results of our study can be compared.

The distal promoter region in which the methylation analysis was carried out is an anchoring site for the transcription factor Sp1, which has a preponderant role in the transcription of the RAS genes (11,54,55). In this study, a low frequency of partial methylation was observed, which would be consistent with the high expression of KRAS protein. The low methylation frequencies observed in this study may be related to the type of sample analyzed, since the integrity of DNA may be affected by long exposure to formaldehyde and the low pH to which the sample is subjected for preservation. In future studies, it would be interesting to apply sample restoration methods that have been shown to be effective in improving DNA quality for subsequent molecular applications (56). Another explanation may be the detection limit reported in the literature for direct sequencing, which is approximately 10 to 20%, and if heterogeneous methylation exists in the tissue, methylated variants below this level would not be detected with the described methodology (57). The low number of cases with methylation did not allow evaluation of whether partial methylation in the analyzed CpG islands affects the expression of the KRAS gene at the mRNA and protein levels. However, the partial methylation in the CpG regions evaluated in the distal promoter of KRAS is of interest since it might be assumed that methylation, being a reversible event, occurs during some phases in the progression of the disease and is then reversed in most cases. However, in a few cases, partial methylations in the gene promoter would be retained. If so, it would be interesting to determine what the function of these partial methylations is and what impact they have on disease progression and treatment.

No association was found between the expression levels of KRAS mRNA, miR-18a-3p, miR-143 and methylation status. Methylation status was detected with low frequency, thus being the first report of methylation in wild-type KRAS.

The Authors declare that they have no competing interests.

All Authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Jehison Alirio Herrera-Pulido, Orlando Ricaurte Guerrero, Jinneth Acosta Forero, Pablo Moreno-Acosta, Alfredo Romero, Carolina Sanabria, Gustavo Hernández, and Martha Lucía Serrano. The first draft of the article was written by Jehison Alirio Herrera-Pulido and Martha Lucía Serrano and all Authors commented on previous versions of the article. All Authors read and approved the final article.

The Authors express their deepest gratitude to Dr. Miguel López and Dr. Humberto Arboleda for all their contributions during the development of this study; to Dr. Hernán Hernández for his valuable contributions to the construction of primers used in this study; to Marcela Nuñez for carrying out the statistical analyses; and to the National Cancer Institute of Colombia for providing physical spaces and financial resources to carry out this research.