Oncogenic and tumor suppressor genes expression in myeloproliferative neoplasms: The hidden side of a complex pathology

Abstract Background The present study aimed to explore the changes in the expressions of six tumor‐related genes in myeloproliferative neoplasms (MPNs). The study population included 130 patients with MPNs (52 with chronic myeloid leukemia (CML), 49 with essential thrombocythemia (ET), 20 with polycythemia vera (PV), and 9 with primary myelofibrosis (PMF)) and 51 healthy individuals. Methods The expression profiling of six genes (ADAMTS18, CMTM5, CDKN2B, DCC, FHIT, and WNT5B) in the peripheral blood granulocyte cells was explored by real‐time quantitative reverse transcription polymerase chain reaction. Results The patients with MPNs showed significant downregulation of CMTM5 (EFC = 0.66) and DCC (EFC = 0.65) genes in contrast to a non‐significant upregulation of ADAMTS18, CDKN2B, FHIT, and WNT5B genes. Downregulation of DCC was consistent in all subtypes of MPN (EFC range: 0.591–0.860). However, CMTM5 had a 1.22‐fold upregulation in PMF in contrast to downregulation in other MPN subtypes (EFC range: 0.599–0.775). The results revealed a significant downregulation in CMTM5 and DCC at below 60‐years of age. Furthermore, female patients showed a clear‐cut downregulation in both CMTM5 and DCC (EFC DCC: 0.436 and CMTM5: 0.570), while male patients presented a less prominent downregulation with a borderline p‐value only in DCC (EFC: 0.69; p = 0.05). Conclusions Chronic myeloid leukemia cases showed a significant upregulation of WNT5B, as a known oncogenesis gene. Two tumor suppressor genes, namely DCC and CMTM5, were downregulated in the patients with MPNs, especially in females and patients below 60 years of age.


| INTRODUC TI ON
Since the discovery of Jak2V617 followed by CALR and other mutations in patients with MPNs, a great number of genomic studies have revealed more somatic alterations in the majority of these patients. Although the mutational events involved in MPNs pathogenesis have been comprehensively determined, the impact of different somatic alterations on gene expression and transcriptional output has not been evaluated yet. In the MPNs family, especially in CML, some downstream signaling pathways account for the progression of the disease. This factor alongside molecular events alters the expression profiles of several important genes that may play a crucial role in the evolution and pathogenesis of MPNs. Therefore, the present study aimed to assess the expression levels of a number of genes associated with DNA methylation, as the common epigenetic change in hematological cancers. For this purpose, the raw data from GeoDataSet NCBI (GSE87806) and the previous studies were reviewed to identify the most likely genes involved in this process.

| MATERIAL S AND ME THODS
In this cross-sectional study, patients with MPNs referred to the hematology-oncology department of Namazee Hospital, Shiraz, Iran from May 2018 to May 2019 were selected as the patient group. A group of age-and sex-matched volunteers was also selected as the control group. The patients were diagnosed based on the WHO's criteria and clinical, laboratory, and molecular analyses.
Written informed consent was obtained from all the participants. for WNT5B, ADAMTS18, and CMTM5 genes as the annealing step, and 20 s at 72°C. Each PCR reaction was done in triplicates. After completing the polymerase cycle, a melting curve analysis was performed to identify the non-specific PCR products and primer dimer formation. The relative expression data of the genes relative to the internal control gene was obtained using the 2 (−ΔΔCT) method (Livak method). The fold changes were further converted to the log 10 scale. Then, the mean values of the relative fold changes in the patient group and healthy controls were calculated, analyzed, and compared.

| Statistical analysis
The value of ΔCts was analyzed for normality of distribution by the Shapiro-Wilk test. For normally distributed data, mean and SD were calculated. Additionally, analysis of variance was used to compare different groups under investigation. Non-normally distributed data were analyzed by non-parametric tests, and medians and ranges were compared using Mann-Whitney test. All data analyses were carried out using the SPSS 18 software and p < 0.05 was considered statistically significant.  *p = 0.09 between the patient and control groups (no significant difference). However, a significant difference was found between the patients with PV and other patient groups in terms of mean age (p = 0.004).

| RE SULTS
63.4 ± 13.9 years, which was significantly higher compared to other MPN categories and the control group (p = 0.021). The male to female ratio was equal to one in the patient group (Table 2).

| Gene expression
The results showed the normal ΔCts distribution of CMTM5, DCC,  (Table 4 and Figure 1).
In the next step, we tried to explore the relationship between gene expression and age and sex Tables 5 and 6. In this study, the participants were divided into lower and higher than 60 years age groups and were compared concerning gene expression. The results revealed no significant difference in this regard in the patient and control groups. However, a statistically significant difference was found in the means of delta CMTM5 and delta DCC between the individuals younger than 60 years in the MPN and control groups ( A comparison of the sex-related gene expression in the study population has been depicted in Table 6. The male and female par-  Table 6).
The BCR-ABL1 mutation effect has been shown in Table 7 (normally distributed data) and Figure 2 (non-normal distributed data).
The positive BCR-ABL1 mutation led to the overexpression of the one oncogene) were found to be more likely to be affected by the methylation process. In the present study, therefore, the ECF of the selected genes was explored as the first step to explore the possible association between these genes and MPN pathogenesis and clinical outcome. 7,16,17,21,22 To the best of our knowledge, the present study is one of the few studies exploring the expression levels of some tumor suppressors and oncogenes in a population of patients with MPNs.
Among the five genes mainly known as tumor suppressor genes, two (CMTM5 and DCC) showed a significant downregulation. These genes are involved in many biological processes in the human body. For instance, CMTM5 has a tumor inhibitory function, an immune system modulation capability, and an active role in the male reproductive system. 16,22 Many cancers such as myeloid leukemia, prostate cancer, and cervical cancer are associated with the downregulation of this gene. 16,22,23 Another tumor suppressor gene, which is primarily known as "deleted in colorectal carcinoma" or less than 30% DCC gene absence or downregulation was reported in CML cases. 28,29 In colorectal cancer, the absence or downregulation of this gene was associated with poor prognosis and an increased rate of metastasis and progression from the benign to a malignant form of colorectal tumors. It also played an important role in the loss of homogeneity, which contributed to poor prognosis in these tumors. [30][31][32][33] Up to now, little attention has been paid to the role of this gene and its expression. Considering the 0.652 DCC downregulation among the cases with MPNs ( Table 3) (Table 5). Some other studies in different contexts and malignancies did not report any correlation between age and the gene expression of CMTM5. 43 As expected, the present study findings also showed the ineffectiveness of age in the gene expressions of ADAMTS18, CDKN2B, FHIT, and WNT5B.
Furthermore, the role of sex in the expression of these genes was evaluated in this study. Based on the results, females showed a more prominent downregulation of DCC and CMTM5 in the setting of MPNs (Table 6). However, none of these genes had a significantly different expression between the males and females in the control group. Thus, the sex difference in the expression rate was ruled out. In the current investigation, the CML patients presented the overexpression of the WNT5B gene (p = 0.048). Considering the non-normal distribution of the data represented in Figure 2, Mann-Whitney test was employed. The results revealed that EFC was close to one among the patients with negative BCR-ABL1 mutation, which implied no change in gene expression.
Nevertheless, the positive cases had the highest value of overexpression (EFC = 1.6). It seemed that the BCR-ABL1 mutation had a synergistic effect on the expression of the WNT5B gene. The overexpression of this gene, as an oncogene, has been reported in many studies, some of which indicated BCR-ABL+as an aggravating factor for its overexpression. 44,45 This gene could promote cell migration and invasion, eventually leading to poor outcomes 17,46 thus, it may be reasonable to investigate the role of WNT5B overexpression in the prognosis of BCR-ABL1+ cases.

| CON CLUS ION
The present study was one of the few studies investigating the role

ACK N OWLED G EM ENTS
The authors would like to thank Ms. A. Keivanshekouh at the

Research Consultation Center (RCC) of Shiraz University of Medical
Sciences for improving the use of English in the manuscript.

CO N FLI C T O F I NTE R E S T
None declared.

I N FO R M E D CO N S E NT
The study was explained to the patients and their informed consent forms were obtained.

D ECL A R ATI O N
To the best of our knowledge, the present study is one of the few studies exploring tumor suppressor genes or oncogenes in a popu-