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THE IMPACT OF GLOBAL DNA METHYLATION AND HYPOXIA-INDUCIBLE FACTOR 1 ALPHA LEVELS IN THE PROGRESSION OF BREAST CANCER
B.R. Sahar1*, R.M.Kh. Al-Jumaily2*
1 Department of Biotechnology, College of Science, University of Baghdad, Baghdad, Iraq;
2 Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq.
* Corresponding authors: basimrsahar@gmail.com, rakad.aljumaily@sc.uobaghdad.edu.iq
Abstract. Breast cancer (BC) is one of the most common causes of death among women in the world. This study investigated the role of global DNA methylation (5mC) and hypoxia-inducible factor 1 alpha (HIF-1a) levels in BC disease progression. Blood samples were collected from 40 patients with benign breast tumors, 40 patients with malignant breast tumors, and 40 healthy subjects. Patients with malignant breast tumors were divided into two groups: women in stage II (low-level), and patients in stages III and IV (high-level). Genomic DNA was isolated from whole blood samples from the subjects and used for global DNA methylation. Furthermore, the levels of HIF-1a expression were measured. The results showed that the levels of 5mC in patients with BC and benign breast tumors were considerably lower (0.538 ± 0.03 and 0.432 ± 0.04%, respectively), compared to the control (0.619 ± 0.05%). Furthermore, there was a significant (p < 0.05) decrease in levels of 5mC in BC patients at stages III and IV compared to control. However, there were no significant differences between low-level and high-level stages. The HIF-1a levels of patients in both the benign breast tumors and BC were insignificant (923.35 ± 72.42 and 1386.03 ± 102.01pg/ml, respectively), compared to the control (825.5 ± 62.36 pg/ml). Although, BC patients at low levels showed no significant difference in HIF-1a levels compared with patients at high levels. The findings indicated that variations in 5mC levels across different stages and types of breast tumors may serve as a prognostic indicator for the development of BC and also implicated HIF-1a in the development of BC.
Keywords: DNA methylation, HIF-1a, breast cancer, benign breast tumor.
List of Abbreviations
BC - Breast cancer
DNMTs - DNA methyltransferases
MBD - Methyl-binding domain
TF - Transcription factor
HIF-1a - Hypoxia-inducible factor 1 alpha
5mC - 5-methylcytosine
HRP - Horseradish peroxidase
BMI - Body mass index
TSGs - Tumor suppressor genes
Introduction
Breast tumors (malignant and benign) are the most common tumors among women in the world. The percentage of benign breast tumors is ten times higher than malignant breast tumors and is considered the main cause of breast diseases in women (Alamri et al, 2020; Sahan, 2022). The term «benign breast disease» is used to refer to several non-cancerous breast disorders. Invasive breast cancer is associated with proliferative breast lesions as a risk factor
(Waldman et al., 2019). Early and accurate diagnosis of breast tumors is essential for effective treatment because breast cancer (BC) is the leading cause of cancer-related deaths (Prihan-tono et al, 2021; Salman et al, 2022).
Epigenetic modifications, such as histone modifications, DNA methylation, and mi-croRNAs, are master regulators of gene expression. It is also considered an important predictive biomarker for detecting cancer (de Almeida et al., 2019). DNA methylation occurs when a group of DNA methyltransferases (DNMTs) adds a methyl group to the ring of cytosine py-rimidine in CpG dinucleotides (Rodríguez-Paredes & Esteller, 2011; Lafta et al., 2023). Gene expression is supposed to be reduced by the recruitment of methyl-binding domain (MBD) proteins, which prevent transcription factor (TF) binding to DNA by changing chromatin conformation (Kouzarides, 2007; Mik-kelsen et al., 2007). One of the main risk factors for BC is abnormal DNA methylation. The re-
suits of several genome-wide DNA methylation studies show that higher levels of DNA methylation are associated with a higher risk of BC in women overall and in women from high-risk families (van Veldhoven et al., 2015). On the other hand, even though there is a correlation between rising global methylation and a decreased risk of breast cancer, there is also a correlation between increased methylation levels inside functional promoters and an increased risk of BC (Joo et al., 2018).
Hypoxia-inducible factor 1 alpha (HIF-1a) is a protein composed of basic helix-loop-helix PAS domains. It is widely recognized as the preeminent transcriptional regulator that governs cellular and developmental reactions to hypoxia (Gunton, 2020). Hypoxia-induced dysregulation and overexpression of HIF1A have been extensively linked to cancer biology and numerous other pathophysiologies, including energy metabolism, cellular survival, and tumor invasion, in addition to vascularization and angiogenesis (Semenza, 2003). Concerning BC, hypoxia, which is characterized by low in-tratumoral oxygen levels is linked to aggressive tumor activity, metastasis, and resistance during treatment. It has been almost thirty years since the initial in vivo measurements of oxygen content and subsequent observations of hypoxia in BC patients were documented (Vaupel et al., 1991). Later studies found that HIF-1a controls how cells adapt to low oxygen levels (Wang et al., 1995; Giatromanolaki et al., 2022). However, using different diagnostic techniques to identify the underlying cause of various diseases is necessary to better understand the mechanism of the development of different diseases (AL-Jumaily et al., 2023).
Therefore, this research aimed to ascertain the relationship between DNA methylation and HIF-1a levels in women with breast malignancies and demographic factors.
Materials and Methods
Subjects
The blood samples were collected from 40 women with benign breast tumors and 40 with malignant breast tumors during their attendance at Al-Karama Hospital, Oncology Center,
Wasit, and Oncology Center, Al-Hussein Teaching Hospital, Karbala, Iraq. The age of BC women ranged from 33-75 (52.17 ± 1.48) years, and the age of benign breast tumor women ranged from 16-65 (30.00 ± 1.99) years. Forty healthy women, ages ranging from 24 to 54 (39.50 ± 2.77) years, also participated in this study. Malignant breast tumors were divided into two groups: patients in stage II (low level) and those in stages III and IV (high level), respectively.
Ethical clearance
The Ethical Committee of the Department of Biotechnology, College of Science, University of Baghdad, approved the research (CSEC/0623/0044 on June 8, 2023) after receiving written informed consent from all participants. The studies conformed to the standards set by the latest revision of the Declaration of Helsinki.
Genomic DNA extraction
Genomic DNA was extracted from whole blood samples by using the Quick-DNA™ Blood Miniprep kit (Zymo, USA). The purity, integral, and concentration of genomic DNA were estimated by Nanodrop at A260/A280 ab-sorbance and the ratio between 1.8 to 2.0 indicates that genomic DNA is high quality.
Global DNA methylation
The global DNA methylation (5mC) was estimated after several steps of incubation with primary and secondary antibodies according to the manufacturer's instructions of the MethylFlashTM methylated DNA quantification kit (Epigentek) and by using the Elisa technique. The amount of total 5mC content was measured at 450 nm.
Measurement of HIF-1a levels
The level of HIF-1a was evaluated using the Sandwich-ELISA (Elabscience, USA) method. Serum samples were added to the wells of the ELISA microplate and combined with the specific antibody. Human HIF-1a specific bioti-nylated detection antibody and avidin-horse-radish peroxidase (HRP) conjugated antibody were added to each microplate well and incu-
bated. The constituents had been washed. A substrate solution was added to each well. Only those wells containing human HIF-1a, bio-tinylated detection antibodies, and avidin-HRP conjugate appeared blue. The reaction of the enzyme with the substrate was terminated by adding a stop solution until the color turned yellow. The optical density (OD) was calculated spectro-photometrically at 450 nm wavelength.
Statistical analysis
The statistical analysis was conducted utilizing the Statistical Package for the Social Sciences (SPSS; version 23). The information was presented as (Mean ± SE). Significant statistical comparisons between groups were identified using the t-test, with a P-value of 0.05 or less indicating significance. In contrast, analyses of variance (ANOVA) were employed to examine the differences between more than two groups.
Results
Characteristics of the study groups
The analysis of the different demographic and clinicopathological parameters in patients
Baseline characterist
with breast tumors is illustrated in Table 1. In the age group of patients who were 50 years or above, the peak age frequency of BC patients was 62.50%; only 37.50% of patients were under 50 years old, whereas 90% of patients with benign breast tumors were under 50 years old, and only 10% were over 50 years old. The average body mass index (BMI) was 29.79 ± 0.93 in BC while 25.14 ± 0.84 kg/m2 in benign breast tumors. The results of the current study showed that most patients with a lesion of the tumor in the left breast (52.5%) and 42.5% in the right breast, with only 5% in bilateral. Breast cancer was prevalent among married women (95%). The results of BC patients revealed that 60% of women were in premenopausal age and about 40% of women in postmenopausal age with significant differences found at p < 0.05 (Table 1). Smokers made up 7.5% of patients, nonsmokers made up 92.5%, a statistically significant difference at the p < 0.05 level. Tumor grades were low level (I+II) and high level (III+ IV), 45 and 55%, respectively, as shown in Table 1.
Table 1
of breast tumor patients
Variables Type of Breast Tumor Percentage % P-value
Malignant No = 40 Benign No = 40
Age (yrs) Average age 32-72 16-52
Age Mean ± SD 5.675 ** 0.0001
52.17 ± 1.48 30.00 ± 1.99
Age G. <50 15 (37.50%) 36 (90.00%) 0.050
>50 25 (62.50%) 4 (10.00%) 0.0001
BMI (Kg/m2 ) 29.79 ± 0.93 25.14 ± 0.84 2.678 ** 0.0004
Tumor site Right 21 (52.5%) 24 (60%)
Left 17 (42.5%) 16 (40%)
Bilateral 2 (5%) 0
Marital status Married 38 (95%) 23(57.5%)
Single 2 (5%) 17 (42.5%)
Menstruation Premenopausal 16 (40%) 4 (10%)
Postmenopausal 24 (60%) 36 (90%)
Family History YES 7 (17.50%) 0.0001
NO 33 (82.50%)
Smoking YES 3 (7.5%)
NO 37 (92.5%)
Tumor stage Low L. (I+II) 18 (45.00%) 0.527
High L. (III+ IV) 22 (55.00%)
Table 2
Variations in the global concentrations of 5-methylcytosine (5mC) among patients with breast malignancies
Group Mean ± SE
DNA methylation (% 5mC)
Control 0.619 ± 0.05 a
Benin tumor 0.538 ± 0.03 ab
Malignant tumor 0.432 ± 0.04 b
LSD value 0.123**
P-value 0.0090
Note: Different alphabets in the same column indicated significant differences; **: p < 0.01
5-methylcytosine level in breast tumors As shown in Table 2, a statistically significant difference (p < 0.009) was observed in the present study among BC patients whose 5mC levels were lower (0.432 ± 0.04%) than those in the benign breast and control groups (0.538 ± 0.03 and 0.619 ± 0.05%, resp ec-tively).
Determination of HIF-1a in breast tumors The level of HIF-1a increased significantly (p < 0.01) in BC patients (1386.03 ± ± 102.01 pg/ml) compared to benign breast tumor patients and control (923.35 ± 72.42 and 825.5 ± 62.36 pg/ml, respectively), as illustrated in Table 3. However, no significant differences (p>0.05) between benign breast tumor patients and control groups were observed (Table 3).
DNA methylation andHIF-1a levels according to breast cancer stages
As shown in Fig. 1, there was a statistically significant difference (p > 0.05) in the 5mC level for BC patients with low levels (0.446 ± ± 0.06) compared to the control group (0.619 ± ± 0.05). Also, the level of 5mC dropped (0.419 ± 0.04) compared to the control group, which was also statistically significant (p > > 0.01). This was observed only in BC patients in the high-level stage. Furthermore, no significant difference was observed between low and high levels.
Also, the results of HIF-1a of BC patients at low levels showed that there were significant differences in HIF-1a level (825.5 ± 62.36 pg/ml) compared with the low and high-level stages (1231.4 ± 120.7 and 1643.8 ± 167.6 pg/ml, respectively) as shown in Fig. 2.
Table 3
Changes in HIF-1a levels in breast tumor patients
Groups Mean ± SE
HIF-1a (pg/ml)
Control 825.5 ± 62.36b
Benign tumor 923.35 ± 72.42 b
Malignant tumor 1386.03 ± 102.01 a
LSD value 280.45 **
P-value 0.0001
Note: Different alphabets in the same column indicated significant differences; **: p < 0.01
Fig. 1. Comparison of 5-methylcytosine (5mC) levels in low- and high-level breast cancer patients compared to controls
*: Significant difference (p < 0.5)
Fig. 2. Comparison of HIF-1a levels in different stages of breast cancer (low level and high level) *: Significant difference (p < 0.5)
Table 4
The correlation coefficient between DNA methylation and HIF-1a levels in different groups of breast tumors
Parameter Correlation coefficient ® with D )NA methylation
Control Benign tumor Malignant tumor
HIF-1a -0.24 -0.35 -0.21
The patient correlation coefficient between DNA methylation and HIF-1a
As shown in Table 4, the correlation coefficient between DNA methylation and HIF-1a levels did not exhibit any significant relationship according to the groups.
Discussion
The findings of this research agree with other studies that showed that most Iraqi pa-
tients have tumors on the left side (Mahmoud & Mahmoud, 2014; Hussein & Mohseen, 2019; AL-Bedairy et al., 2020). However, other studies indicated that BC patients carried tumors on the right side (Mustafa et al., 2016; Mutar et al., 2019; Sabri et al., 2020). The findings of family history agree with Mahmoud and Mahmoud (2014) who found that about 17.50% of patients with BC had a positive family history of BC while 82.5% of BC patients had no history of
BC with a significant difference observed at p < 0.05. Also, these findings agree with Ma-lekzadeh Moghani et al. (2022). The risk of BC is approximately 1.5 times higher in women with one affected first-degree female relative and 2-4 times higher in women with multiple affected first-degree female relatives (Kha-razmi et al., 2014; Liu et al., 2021). In addition, smokers made up 7.5% of patients, nonsmokers made up 92.5%, a statistically significant difference at the p < 0.05 level. Breast cancer risk was 18% greater among women who started smoking more than 10 years before having their first child (Jones et al., 2017).
Breast cancer exhibits substantial molecular and histological heterogeneity. At the outset, cancer was conceptualized as an intricate, multistage phenomenon predicated on genetic modifications, cellular proto-oncogenes, and the in-activation of tumor suppressor genes (TSGs). The interplay between environmental and genetic risk factors is governed by particular epi-genetic programs, such as modifications in DNA methylation. As a consequence, essential molecular pathways implicated in the development of BC become dysregulated (Pasculli et al., 2018; AL-Rubaye & AL-Jumaily, 2023). The findings of this research are consistent with those of Soares et al. (1999) and Euhus et al. (2008), who discovered that the global DNA methylation of BC and benign breast tumors was distinct from one another. According to the findings of the research, BC had a lower 5mC than benign tumors, and there was a very significant correlation between the levels of global DNA methylation and the stages of cancer. An experiment by Hon et al. (2012) found that DNA hypomethylation in BC patients, along with higher levels of DNA methylation in certain genes linked to the promoter site, may play a major role in the illness getting worse. Furthermore, research conducted by Marino and colleagues (2022) discovered many regions of aberrant DNA methylation in BC, as well as how these sites are related to the regulation of gene expression.
Gene expression abnormalities associated with cancer are orchestrated by aberrant DNA methylation, which can suppress tumor sup-
pressor genes through hypermethylation or activate prometastatic genes through hypomethyl-ation. Since adding or removing the methyl group from CpG islands is a dynamic and reversible process, targeting the methylate may be an effective anti-cancer therapy (Mahmood et al. , 2020). Some malignancies, including those of the stomach, kidney, pancreas, lungs, and intestines, have been related to a reduction in global DNA methylation. Hypomethylation is associated with both tumorigenesis and tumor progression. In addition, it has been demonstrated that proto-oncogenes are hypo-methylated, particularly in liver tumors and leukemia (Skvortsova et al., 2019).
Two types of abnormal DNA methylation may be discovered in cancer cells: the first is site-specific CpG island promoter hypermeth-ylation and the second is global DNA hypomethylation. After examining the tumor cells, these two abnormalities were discovered. Many studies have also shown that tumor suppressor genes suffer from CpG island promoter hyper-methylation in cancer cells. On the other hand, it has been discovered that in repetitive DNA sequences, there is global DNA hypomethyla-tion (Kerachian & Kerachian, 2019). However, hypermethylation and hypomethylation of CpG have been demonstrated to have significant associations, and both are crucial stages in the progression of BC. The loss of methylation, especially in repetitive regions like LINE-1, has been linked to more genomic instability and may play a part in mutations and chromosomal recombination (Dumitrescu, 2012; Tsai et al, 2018).
Global DNA methylation-based signatures for determining the methylation pattern in benign and malignant breast tumors revealed that epigenetic cell modification is responsible for the development of breast tumors and cancers over an individual's lifetime, Epigenetic aberrations or abnormalities (DNA methylation) found in around 75% of the participants with benign tumors, while 96% of the subjects with malignant tumors exhibited epigenetic aberrations. This is consistent with the finding that about 5-10% of cancers are caused by gene abnormalities that are inherited from parents and
that around 90-95% of instances are linked to epigenetic factors, which may be thought of as environmental factors (Ehrlich, 2002; Jackson et al., 2004).
The findings of this study agree with those of Khadem et al. (2022), who found that patients with invasive ductal carcinoma had significantly higher levels of HIF-1a in their serum compared to those with benign breast tumors and the control group. Also, the current study is in line with the findings of Cai et al. (2016), who reported that BC patients had significantly higher levels of HIF-1a than those with benign breast tumors. However, the data of these studies suggest that HIF-1a may be a promising bi-omarker with a high potential for early identifying subsets of patients with poor prognoses. Another study concluded that the level of HIF-1 rose with the progression of the pathological stage. Furthermore, the level of HIF-1 was found to be greater in poorly differentiated lesions compared to the same variety of well-differentiated lesions, confirming the results of the present study (Bos et al., 2001).
One of the most important processes that enable cancer cells to survive, continue, and develop in a hypoxic environment is an adaptive mechanism called tumor hypoxia. In this process, HIF-1 is often involved, especially in solid tumors, and leads to increased stress-induced cell division and reduces apoptosis (Collin et al., 2021). A complex form of "inflammatory hypoxia" that distinguishes itself by inhibiting HIF-1 hydroxylation and ubiquitination is one notable characteristic that the majority of solid tumors share (Corrado & Fontana, 2020; Gong et al., 2020). Enhanced production of inflammatory mediators, enzymes, and cytokines, along with elevated hypoxia at sites of inflammation, have been associated with chronic inflammation (Eltzschig & Carmeliet, 2011). In-tracellular oxygen concentration influences the range of HIF-1a expressed in cells (Huang et al., 1996; Jiang et al., 1996). Also, intracellular oxygen concentration influences the range of HIF-1a expressed in cells (Huang et al., 1996; Jiang et al., 1996). Under conditions of normal cellular oxygen concentrations, HIF-1 a undergoes continuous degradation via the ubiquitin
pathway (Semenza, 2001). On the other hand, when oxygen levels are low (hypoxia), HIF-1a cannot diffuse. This prevents the protein from stabilizing and increases the amount of protein within the cells. Meanwhile, the exact mechanism by which a cell detects oxygen is still unknown; it is hypothesized that reactive oxygen species are responsible. As a result, oxidation-induced stabilization of the HIF-1 a protein occurs (Srinivas et al., 1998).
It has been reported by Jackson et al. (2004) that about 500,000 copies of LINE-1 are spread out across the human genome, and most of them are generally heavily methylated. On the other hand, hypomethylation of LINE-1 is associated with low levels of DNA methylation in BC tumor tissue. Hypomethylation in BC patients is associated with decreased survival and an increased risk of disease recurrence. Moreover, it is related to its histological grade, disease stage, and tumor size. According to the results, hypo-methylation was observed globally, even in low-grade tumors, suggesting that demethyla-tion is an early stage in BC development (Deroo et al., 2014). The location of the tumor, the size of the tumor, and the amount of blood flow that affects oxygen accessibility are a few important factors that contribute to the presence of hypoxemia. In many cases, the fast development of solid tumors results in the establishment of regions that are deficient in oxygen. This is because ongoing growth and vascular abnormalities cause the tumor bulk to get inadequate perfusion (Yehia et al., 2015; Emami Nejad et al., 2021). This effect, along with the possibility that it is linked to more aggressive tumors and higher microvascular density, helps explain why HIF-1a levels are higher when the tumor has gone beyond grade III and stage T3. Hypoxia is an essential factor in the progression of tumors, encompassing invasion and metastasis. The presence of the positive LNM subset is linked to a significant increase in HIF-1 a, which is a key regulator of lymphangiogenesis and lymph node growth (Moon et al., 2021).
The correlation coefficient between DNA methylation and HIF-1 a (Table 4) does not exhibit any significant relationship depending on the group. Hypoxic conditions can change
DNA methylation patterns, which suggests that there is a connection between the extracellular environment, epigenetic modifications, and the advancement of cancer (Liu et al., 2011). Thus, the progression of breast tumors may be influenced by local epigenetic alterations under hy-poxic microenvironmental conditions, resulting in aberrant silencing and activation of cancer-causing genes. Hypoxia has an impact on the expression of DNMT enzymes in malignancies like breast, prostate, and liver. When there is not enough oxygen in the blood, more HIF1 binds to the promoters of DNMT1 and DNMT3B. This increases methylation and decreases the expression of tumor suppressor genes, such as SPRY2 (Watson et al., 2014; Gao et al., 2017).
Furthermore, DNMTs are also capable of inhibiting HIF expression through a negative feedback mechanism. In fetal lung fibroblasts, the hypoxic response was stopped by methylat-ing the HIF2A promoter after HIF2a increased DNMT1 expression (Xu et al., 2018). In renal cell carcinomas and glioblastoma cell lines,
lower DNMT3A expression was linked to higher HIF2A expression and lower methylation of the HIF2A promoter. Previous research has shown that under hypoxic conditions (1% O2), ectopic expression of DNMT3A inhibits viability and cell proliferation by reducing the mRNA expression and protein activity of HIF2A (Humphries et al., 2023). To make the most of epigenetic treatments in the treatment of BC, it will be necessary to have a deeper knowledge of the interactions that occur between these factors.
Conclusion
Based on the findings of this research, it can be inferred that the variations in the levels of 5mC at different stages and types of breast tumors have the potential to serve as a risk factor for the development of BC. Furthermore, these findings also imply that HIF-1a has a role in the advancement of BC. A deeper understanding of these interactions, on the other hand, will be necessary to make the most effective use of epigenetic medicines in the treatment of BC.
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