Investigating the effect of MAP2K1 gene (MEK1) in MAPK pathway in the induction of adult T-cell leukemia/lymphoma (ATLL)

Abstract

Background and Objectives:

HTLV-1 is responsible for two important diseases, HAM/TSP and ATLL. Approximately 10 to 20 million people are infected with HTLV-1 worldwide. Identifying altered genes in different cancers is crucial for finding potential treatment strategies. One of the proteins of the RAS/MAPK signaling pathway is MEK1, which is made from the MAP2K1 gene. The effects of the MAP2K1 gene on the MAPK signaling pathway are not yet fully elucidated. The current study aims to determine the MAP2K1 gene mutations and the level of MAP2K1 gene expression in ATLL patients compared to healthy individuals.

Materials and Methods:

Ten ATLL and 10 healthy control individuals were investigated in this study. We used ELISA test to screen anti-HTLV-I antibodies and PCR for confirmation of infection. Then, we extracted total RNA from fresh whole blood, and cDNA was synthesized. The expression levels of the MAP2K1 gene were examined by qRT-PCR, and to check possible mutations in the MAP2K1 gene; all samples were sequenced and analyzed by BioEdite Software.

Results:

MAP2K1 gene expression in the ATLL group was significantly higher than in the healthy control (P=0.001). The mutational sequencing analysis showed nucleotide 212 (S→R) change and identification mutations at different nucleotides that were entirely different from the nucleotide mutations defined in the UniProt database.

Conclusion:

These results could be a perspective in the prevention, prognosis, and targeted treatment of diseases in which the MAP2K1 gene plays a vital role.

INTRODUCTION

The discovery of human T-cell lymphotropic virus type 1 (HTLV-1) in 1980 identified the first human oncoretroviral (1, 2). HTLV-1 is known as a member of the Retroviridae family and belongs to the Orthoretrovirinae subfamily and the Deltaretrovirus genus. The HTLV-1 infection rate is estimated at 10 to 20 million individuals globally. The two serious diseases are HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATLL) caused by HTLV-1. Adult T-cell leukemia (ATLL) is a T-cell malignancy and an aggressive lymphoproliferative disorder of mostly CD4 T-cells caused by HTLV-1 (1, 3, 4). Approximately 4 to 5% of HTLV-1 carriers experience ATLL disorder (3). In HAM/TSP, inflammatory reactions play an important role in affecting the corticospinal tracts. Moreover, irreversible paraparesis and chronic neuromyopathy are the result of these inflammatory reactions and can affect the lower organs (5). In addition, HTLV-1 is involved in the development of a variety of autoimmune diseases and several other inflammatory diseases, such as bronchiectasis, arthritis, infective dermatitis, myopathies, and multiple different uveitides (6, 7). HTLV-1 is transmitted in various routes such as blood transfusion, intravenous injection, sexual contact, vertical transmission from mother to baby, breastfeeding, and organ transplant (8, 9).

In cancer, genetic changes occur in specific genes that lead to the ability of cells to grow and divide uncontrollably. In the last decade, the systematic study of these genetic alterations has been facilitated by the possibility of DNA sequencing. Now a better understanding of the processes of the signaling pathways has been developed (10). Given that specific medication targets genetic changes, DNA sequencing plays an important role in routine clinical surveillance (11–14). Furthermore, in different tumors, the altered genes and pathways are very diverse, and accurate identification of the altered genes and pathways in different cancers is critical to explore therapeutic options. In various cancers, different significant signaling pathways such as phosphatidylinositol 3 kinase (PI3K)/protein kinase B(Akt) signaling, Receptor Tyrosine Kinase (RTK)/Ras GTPase/MAP kinase (MAP-Kinase), etc. have been recognized in the process of genetic change. The frequency of gene alteration in key pathways is not equal, some specific genes frequently changed that can be identified in cancer, while some other genes are rarely or never changed (10).

Mitogen-activated protein kinase (MAPK) pathways play an essential and critical role in cancer initiation and development. Specifically, mutations in RAF and RAS proteins cause improper activation of MAPK pathways, and as a result, constitutive activation occurs in the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway (15). In various cancers, including thyroid, neuroblastoma, breast, and non-small cell lung cancer, the MAPK pathway has been identified as a primary cell signaling pathway (16). The MEK1 protein kinase is made by providing instructions from the MAP2K1 gene. This protein is one of the components of the RAS/MAPK signaling pathway, which is involved in the transmission of extracellular chemical signals into the nucleus (17). Nevertheless, a perfect understanding of the functions of MAP2K1 gene regulation of oncogenesis and host cellular proteins is yet to be determined. Our knowledge of these signaling pathways faces several challenges. The exact functions of the MAP2K1 gene and its effects on the MAPK signaling pathway are still unknown. On the other hand, the insufficient knowledge of possible mutations that may have occurred in the MAP2K1 gene in the MAPK signaling pathway is important and controversial. In addition, no study has been conducted to measure the levels of MAP2K1 gene expression in individuals with ATLL. To the best of our knowledge, no comprehensive study has yet investigated the possible mutations of the MAP2K1 gene in the MAPK signaling pathway. This is the first study to examine the challenges in the functions of the MAP2K1 gene in the oncogenic signaling pathways in ATLL. The present study aimed to understand the possible mutations in the MAP2K1 gene in the MAPK signaling pathway of ATLL carcinogenesis. Also, this research investigates the expression levels of the MAP2K1 gene in healthy control compared to patients with ATLL.

MATERIALS AND METHODS

Samples

A cross-sectional study was done from 2021 to 2022 on 20 samples. In this study, there were two investigated groups. The first group included 10 ATLL patients collected from Shariati Hospital, Tehran, Iran., The inclusion criteria for this group were no autoimmune infectious or immune deficiency diseases, and the second group comprising 10 healthy control was collected from the Alborz Blood Transfusion Organization, Alborz, Iran. The inclusion criteria for this group were HTLV-1 negative, no autoimmune, infectious, or immune deficiency diseases. We utilize serological test enzyme-linked immunosorbent assay (ELISA, Diapro, Italy) (18, 19) to conduct the seropositive test for HTLV-1. The positive specimens were confirmed by the Polymerase chain reaction (PCR) technique (20, 21). This study was approved by the Ethical Committee of Tehran University of Medical Sciences, Tehran, Iran (IR.TUMS.SPH. REC.1400.090).

Quantitative real-time PCR

Total RNA was extracted from fresh whole blood utilizing TriPure isolation reagent following the manufacturer’s instructions (Roche, Germany). DNase treatment was used to remove genomic DNA contamination for all purified RNA samples before constructing complementary DNA (cDNA). Subsequently, cDNA was synthesized utilizing RT-ROSET Kit (ROJE Technologies, Iran). According to the manufacturer's protocol, H₂O 6 µl, Master Mix 10 µl, Primer Forward 1 µl, Primer Revers 1 µl, cDNA Template 2 µl. SYBR Green-based (TaKaRa, Otsu, Japan), and then RT-qPCR was used according to the manufacturer’s instructions.

The following primers were designed and utilized to determine the expression levels of MEK1: (forward primer: 5ʹ-GGGACCAGCTCTGCGGAGA-3ʹ, reverse primer: 5ʹ-GCCCCCAGCTCACTGATCTTCT -3ʹ) which amplified 185 bp. The optimum annealing temperature was 30 seconds at 61°C and the extension time was 25 seconds at 72°C, repeated in 42 cycles. The 5 standard curves real-time PCR was conducted on the cDNA samples using the Q-6000 machine (Qiagen, Germany). We utilized the RPLP0 as a housekeeping gene (22) to normalize the mRNA expression levels and to control the sampling error (23). The normalized value of the expression for each gene was calculated as the ratio of relative copies number of the mRNA of interest/relative mRNA copies number of reference gene, indicated as the expression index.

Statistical analysis

Analysis was done with GraphPad Prism Software Version 8.0.2 (GraphPad Software, Inc). The output was analyzed using the statistical Mann-Whitney U test for statistical differences between gene expressions. We considered p-Value <0.05 as significant.

Sequencing and mutational

The following primers were designed and used to PCR test for Full MEK1: (forward primer: 5ʹ-GATCAGTGAGCTG-GGGGCT-3ʹ, reverse primer: 5ʹ-CTGCTCTCTCTGCGGGGTTT-3ʹ) which amplified 840 bp. The extension time was 40 seconds at 94°C, optimum annealing temperature was 35 seconds at 61°C and the extension time was 30 seconds at 72°C, repeated in 45 cycles.

All PCR products were sequenced bidirectionally using the specific MEK1 primer pairs. The raw sequence data were visualized and edited by the Clustal W program in BioEdit (24) software, we manually edited all acquired sequencing chromatograms to achieve contigs.

RESULTS

Demographic data

In this study, no significant differences were observed in the ages of the groups (25).

MAP2K1 mRNA expression

The mean MAP2K1 mRNA expression level in the healthy control and ATLL was found as 0.006 ± 0.005 and 0.056 ± 0.042, respectively. The expression of the MAP2K1 gene in ATLL was higher than in the healthy control (P=0.001).

Mutational analysis

All the samples were compared with the original sequence of the MAP2K1 gene received from the NCBI database. We analyzed 280 amino acids. We used a uniport database of the MAP2K1 gene to check mutation (Table 1), and all the mutations registered in this database were compared with the sequencing results of this study. In nucleotide 212, the S→R change was observed, while the mutation recorded in Uniport for this position was S→A, so the class of amino acids had changed (Fig. 1).

Table 1.

The registered mutations of the MAP2K1 gene

Mutagenesis	Position	Description
K → A	97	Loss of catalytic function. Strongly reduce phosphorylation upon ultraviolet irradiation
K → R	97	Loss of catalytic activity. Exerts no effect on BRAF-KSR1 or BRAF-KSR2 dimerization
S → A	150	No loss of function.
S → A	212	No loss of function.
S → A	218	Loss of catalytic function. Exerts no effect on BRAF-KSR1 dimerization; when associated with A-222.
S → D	218	Exerts no effect on BRAF-KSR1 dimerization; when associated with D-222.
M → V	219	Can Increase interaction with BRAF and KSR1.
M → W	219	Can increase interaction with BRAF and KSR1; when associated with L-220.
A → L	220	Can increase interaction with KSR1 and BRAF; when associated with w-219.
N → Y	221	Can increase interaction with KSR1 and BRAF.
S → A	222	Loss of catalytic function. Exerts no effect on BRAF-KSR1 dimerization; when associated with A-218.
S → D	222	Exerts no effect on BRAF-KSR1 dimerization; when associated with D-218.
F → S	311	Loss of interaction with KSR1 and BRAF. Loss of BRAF-KSR1 dimerization.

Fig. 1.

Multiple sequence alignments (MSAs) of MAP2K1 gene in the healthy and ATLL groups to show possible mutations. In nucleotide 212, the S→R change was observed, while the mutation recorded in Uniport for this position is S→A. Therefore, the class change of amino acid was observed.

DISCUSSION

In this study, the MAP2K1 gene expression level was investigated. In addition, by sequencing samples (both ATLL patients and healthy controls), possible mutations in the MAP2K1 gene were evaluated in both groups. The most important result obtained from this research was the increase in MAP2K1 gene expression level in ATLL patients compared to healthy control subjects. In addition, an amino acid change at nucleotide position 212 (S→R) was detected.

According to the results of the present study and previous studies, although infrequently, MAP2K1 gene mutations are involved in various forms of cancers (26). Identification of one mutation in MEK1, mainly, a heterozygous missense D67N substitution, was reported in 1 / 15 ovarian cancer cell lines (27). The Q56P alteration was detected in 1 / 85 NSCLC cell lines. In addition, between 207 primary tumor samples, one somatic K57N mutation of MAP2K1 in two cases of NSCLC were identified (28). One somatic amino acid alteration (R201H or E203K) of the MAP2K1 gene in two cases of 55 colorectal cancer cases was detected. Moreover, screening of 38 breast cancer cases revealed no MAP2K1 mutations (29).

Interestingly, sequencing of our samples revealed the identification of mutations at different nucleotides that were completely different from the nucleotide mutations defined in the UniProt database. For instance, in nucleotides, position 85 (V→A), 94 (M→V), 107 (I→V), 130 (Y→C), and 136 (D→E) were detected. Thus, according to the findings of this study, the existence of novel MAP2K1 mutations is also possible.

The findings of the present study are in line with the results of former studies that showed the key role of MAP2K1 gene mutations in the progression of different types of cancers. For instance, between 172 samples or cell lines, mutations in the MAP2K1 gene were observed in 3.5% of human epithelial cancers. One mutation (D67N) and four new mutations (P306H, R47Q, R49L, and I204T) were recognized in the MAP2K1 gene (26). Among 7119 melanoma cases, small in-frame deletions were identified in 37 cases (0.5%), most likely caused by known or putative activation of the MAP2K1 gene. E102_I103del, P105_A106del, Q58_E62del, I103_K104del, I99_K104del, L98_I103del, and E41_F53del were identified in 11, 8, 6, 5, 3, 3 and 1 cases respectively (30). Fifteen patients with melorheostosis were investigated. The obtained results showed MAP2K1 gene mutation in 5/15 cases using whole exome sequencing (31). Eight cases of Langerhans cell histiocytosis were investigated by next-generation sequencing. It reported the identification of an E102_I103del mutation in the MAP2K1 gene in a BRAF wild-type subject, which was subsequently confirmed definitively by the Sanger sequencing method (32).

Despite the obvious strengths of this study, it is suggested that in the following studies, protein quantitative measurement was used for confirmation of the results.

CONCLUSION

The present study found a higher level of MAP2K1 gene expression in patients with ATLL compared to healthy individuals. In addition, mutations that differed from MAP2K1 gene data were discovered in the UniProt database. Therefore, it seems that more studies are needed to confirm these mutations. Considering the importance of ATLL in society, the obtained results can be a turning point in the prevention, prognosis, and efficient treatment of this disease and disorders in which the MAP2K1 gene plays a key role.