Estrogen Induces c-myc Transcription by Binding to Upstream ERE Element in Promoter

: Estrogen Receptor α (ER α ) is reported to regulate the expression of many target genes by binding to speciﬁc estrogen response elements (EREs) in their promoters. c-myc is known to be over-expressed in most of the human carcinomas due to dysregulated transcription, translation, or protein stability. Estrogen (E) can induce the c-myc expression by binding to an upstream enhancer element in its promoter. This suggests that elevated estradiol (E2), a potent form of estrogen, levels could induce the expression of c-myc in breast cancer (BC). The expression of c-myc and estradiol were induced at Stage III and Stage IV of breast cancer. c-myc and estradiol expression was also associated with the established risk factors of breast cancer, such as BMI. Age at the time of the disease was alsocorrelated with the relative expression of c-myc and estradiol ( p < 0.0007 and p < 0.000001). The correlation coefﬁcient (R = 0.462) shows a positive relationship between estradiol bound ER, ER, and c-myc. Docking energy − 229 kJ/mol suggests the binding afﬁnity of estradiol bound ER binding to 500 bp upstream of proximal promotor of c-myc at three distinct positions. The data presented in this study proposed that the expression of c-myc and estradiol are directly correlated in breast cancer. The prognostic utility of an induced level of c-myc associated with the normal status of the c-myc gene and estradiol for patients with metastatic carcinoma should be explored further.


Introduction
Estrogen is a steroid hormone that has critical roles in reproductive development, bone homeostasis, cardiovascular remodeling, and brain functions. However, estrogen also promotes mammary, ovarian, and endometrial tumorigenesis. Estrogen antagonists and drugs that reduce estrogen biosynthesis have become highly successful therapeutic agents for breast cancer. The three major forms of estrogen that have been reported are estrone (E1), the primary form of estrogen that the body makes after menopause, estradiol, (E2) In this article, we discuss the co relation of c-myc, ER, and estradiol in BC and their correlation/inter-dependence in response to epidemiological factors.

Study Design
Blood samples of total 142 female BC patients who alluded to the N.O.R.I. Cancer Hospital and P.I.M.S. Hospital Islamabad were contacted for this non-randomized research design (Table 1). Besides, 77 healthy age-matched female volunteers with no history of BC filled in as the control group. A total of 5 mL of the blood was collected for isolation of RNA in EDTA tubes and stored at −4°C till research analysis. Informed consent was signed and approved by all individuals who participated in this study. All individuals under study were given questionnaires to fill out their clinic-pathological and lifestyle parameters (Table S2).

. Inclusion Criteria
Breast cancer patients were histologically confirmed for different stages of cancer (Stages I, II, III, IV). All epidemiological parameters of inclusion are enlisted in Table S1. In this article, we discuss the co relation of c-myc, ER, and estradiol in BC and their correlation/inter-dependence in response to epidemiological factors.  (Table 1). Besides, 77 healthy age-matched female volunteers with no history of BC filled in as the control group. A total of 5 mL of the blood was collected for isolation of RNA in EDTA tubes and stored at −4 • C till research analysis. Informed consent was signed and approved by all individuals who participated in this study. All individuals under study were given questionnaires to fill out their clinic-pathological and lifestyle parameters (Table S2).

Exclusion Criteria
Patients who were positive for HIV and HCV or any other infection were excluded in this study. Patients diagnosed with multiple diseases and terminally ill patients were also excluded.

Expression Analysis
RNA Extraction/Quantitative RT-PCR Total RNA was extracted using TRIzol ® reagent (Invitrogen) according to manufacturer's protocol. Gene-specific primers were designed using primer3 online tool and optimized before use ( Table 2). Nucleotide blast was done using NCBI blast tool. In silico PCR was carried out using serial cloner and UCSC web browser. Product size was confirmed by confirmatory gel electrophoresis. mRNA quality and quantity were measured by the IMPLEN ® P300 Nanophotometer P3 Implen GmbH, München, Germany, and cDNA synthesis was carried out using 500 ng of total RNA using Oligo (dT) primers and reverse transcriptase.
Gene expression analysis of c-myc and estradiol was carried out on Applied Biosys-tems™ 7500 real-time PCR, Thermo Fisher Scientific, Waltham, MA, USA using SYBR green master mix. RT-PCR conditions for c-myc were set as holding stage at 95 • C for 10 min, single cycling stage at 95 • C for 30 s and 57 • C for 1 min followed by melt curve cycle at 95 • C for 10 s with 10 • C difference for each step. To obtain Ct value for β-globin, same as mentioned above was used, except for Tm change, which is 59 • C in case of β-globin. Melt curve for c-myc generated by RT-PCR is shown in (Figure 2).
Relative gene expression is calculated using comparative Ct by Livak method. Ct is normalized to the housekeeping gene β-globin (Tm = 59 • C). Data was expressed in 2-fold standard means and RE means with ±SEM.

Enzyme-Linked Immunosorbent Assay
Estradiol in the blood samples of BC patients was extracted by using dichloromethane (Wako, Osaka, Japan). Estradiol is measured according to the manufacturer protocols (High Sensitivity Salivary 17β-estradiol Enzyme Immunoassay Kit, Salimetrics LLC, Carlsbad, CA, USA). MTP-300 microplate reader is used to measure absorbance in each well at 450 nm. R values were above 0.99 for the control dilution range of estradiol.

DNA Molecular Docking&Protein-Ligand Docking
There are few studies where DNA intercalator are being used for theraputic potential. Molecular docking was carried out to explain the structural affinity between 17β-estradiol and c-myc.

Preparation of Ligand and Receptor Molecule
Molecular properties of the ligand are extracted by using Molinspiration tool ® . SDF file is converted to pdbqt format before use.
Receptor molecule for DNA docking was 500 bp genetic fragment upstream of cmyc proximal promotor. Firstly, we searched for the promotor binding sites of c-myc (NG_007161.2 Homo sapiens MYC proto-oncogene, on chromosome 8, 14,518 nucleotides) using primer 2.0 tool, which is freely available on http://www.cbs.dtu.dk/services/ Promoter/ (accessed on 25 June 2021) [16]. It gave us 11 marginal predicted sites docking score < 0.6 and three highly likely predicted sites at positions 700, 5900, and 7300 with predicted scores of 1.186, 1.246, and 1.019, respectively (jobid = 60CE40B1000036637FE816BA). Results were confirmed by another tool fprom.pl on Linux-based server; it delivered an enhancer at position 5262 with +9.0 docking score [17]. As reported earlier by [15], half ERE binding sites were available ≈67 kb away from promotor regions, so we utilized 500 bp sequence around 2nd promotor region and 5262 bp enhancer position for DNA and ligand molecular docking to align these with the structure of ER bound estradiol (2j7x pdb format) on HDOCK server. Relative gene expression is calculated using comparative Ct by Livak method. Ct is normalized to the housekeeping gene β-globin (Tm = 59 °C). Data was expressed in 2-fold standard means and RE means with ±SEM.

Enzyme-Linked Immunosorbent Assay
Estradiol in the blood samples of BC patients was extracted by using dichloromethane (Wako, Osaka, Japan). Estradiol is measured according to the manufacturer protocols (High Sensitivity Salivary 17β-estradiol Enzyme Immunoassay Kit, Salimetrics LLC, Carlsbad, CA, USA). MTP-300 microplate reader is used to measure absorbance in each well at 450 nm. R values were above 0.99 for the control dilution range of estradiol.

DNA Molecular Docking&Protein-Ligand Docking
There are few studies where DNA intercalator are being used for theraputic potential. Molecular docking was carried out to explain the structural affinity between Receptor molecule for protein docking is prepared by removing water and adding hydrogen to the pdb structure. Further we did add kollman charges to our protein and grid it and saved in pdbqt format for docking studies.

Statistical Analysis
Microsoft Excel is used to arrange data in spreadsheets, and csv format is used for R import and GraphPad prism import. Excel add-in "DATA ANALYSIS" was used to create bar graphs, poly-regression analysis, one-way ANOVA, and Welch's t test. Livak method of normalizing Ct values was used to calculate expression analysis [18]. Box and whisker graphs were made using software GraphPad prism 7 ® version 7.04 by GraphPad software Inc., San Diego, CA, USA. Correlation data analyses were done using psych and ggplot2 packages in RStudio software version 1.3.1093 Boston, MA, USA [19].

BMI Association with c-myc and Estradiol
BMI and menopausal status (data not mentioned here) show a strong affiliation with the elevated expression of estradiol and c-myc in breast cancer patients. BMI is calculated using the standard BMI Reference Range as given by the US Department of Health and Human Services ( Figure 3). Out of 142 patients, only 126 could answer about their weight and height. The patients who knew only about their height and not weight and vice versa were excluded. BMI index was calculated on the basis of the World Health Organization's (WHO) recommendations. We observed that patients had significantly higher BMI, with 30% obese and 27% overweight patients. Collectively, this amounts to 57% of total patients. Correlation studies suggested that c-myc and estradiol are affected by BMI by the correlation coefficients of 0.46 and 0.50, respectively. These results are significant at p < 0.05, df = 95%, n = 126 (chi square = 1.6 × 10 −10 ).

Statistical Analysis
Microsoft Excel is used to arrange data in spreadsheets, and csv format is used for R import and GraphPad prism import. Excel add-in "DATA ANALYSIS" was used to create bar graphs, poly-regression analysis, one-way ANOVA, and Welch's t test. Livak method of normalizing Ct values was used to calculate expression analysis [18]. Box and whisker graphs were made using software GraphPad prism 7 ® version 7.04 by GraphPad software Inc., San Diego, CA, USA. Correlation data analyses were done using psych and ggplot2 packages in RStudio software version 1.3.1093 Boston, MA, USA [19].

BMI Association with c-myc and Estradiol
BMI and menopausal status (data not mentioned here) show a strong affiliation with the elevated expression of estradiol and c-myc in breast cancer patients. BMI is calculated using the standard BMI Reference Range as given by the US Department of Health and Human Services (Figure 3). Out of 142 patients, only 126 could answer about their weight and height. The patients who knew only about their height and not weight and vice versa were excluded. BMI index was calculated on the basis of the World Health Organization's (WHO) recommendations. We observed that patients had significantly higher BMI, with 30% obese and 27% overweight patients. Collectively, this amounts to 57% of total patients. Correlation studies suggested that c-myc and estradiol are affected by BMI by the correlation coefficients of 0.46 and 0.50, respectively. These results are significant at p < 0.05, df = 95%, n = 126 (chi square = 1.6 × 10 −10 ).

Age Related c-myc Regulation in Breast Cancer Lymphocytes
The relative expression of c-myc in patients was normalized against healthy control. Patients were divided in four distinct age groups (average age at onset of the disease = ±46.6 years): age group 21-35 ( Figure 4A), age group 36-45 ( Figure 4B), age group 46-50 ( Figure 4C), and age group > 60 ( Figure 4D). We observed that overall, the mean RE of c-myc is relatively high in patients as compared to healthy control, as shown in Figure 4E (p-value < 0.0001). Research analysis provided a clearer view of elevated c-myc RE in an age-related fashion. We observed that after certain age, i.e., age > 60, the c-myc-related expression is less elevated as compared to age groups < 60 years, which could be due to the decreased level of estrogen. We calculated the relative risk from binomial pairwise comparison after performing the Pearson chi-square goodness of fit test, and results showed that the relative risk for age group 36-45 and age group 46-60 was >1, while the relative risk for age groups 21-35 and >60 was <0.143. This is due to the age-specific expression pattern of estrogen. Estradiol level were also significantly elevated in patients with p value < 0.001 as compared to age matched healthy control ( Figure 5). expression is less elevated as compared to age groups < 60 years, which could be due to the decreased level of estrogen. We calculated the relative risk from binomial pairwise comparison after performing the Pearson chi-square goodness of fit test, and results showed that the relative risk for age group 36-45 and age group 46-60 was >1, while the relative risk for age groups 21-35 and >60 was <0.143. This is due to the age-specific expression pattern of estrogen. Estradiol level were also significantly elevated in patients with p value < 0.001 as compared to age matched healthy control ( Figure 5).   1, p-value = 0.0027. (B) 2 of age group 36 to 45 years. patients (Average 2 = 5.81) and healthy control (Average 2 ∆∆Ct = 1.95). n = 39, SD = 3.6, Sqt of sample size = 6.2, SEM = 1.5, df = 1, p-value = 0.01. (C) 2 ∆∆Ct of age group 46 to 60 years patients (Average RE = 7.2) and healthy control (Average RE = 0.36). n = 41, SD = 0.48, Sqt of sample size = 3.46, SEM = 0.14, df = 1, p-value < 0.0001. (D) RE of age group > 60 years. patients (Average RE = 2.49) and healthy control (Average RE = 0.8). n = 25, SD = 1.76, Sqt of sample size = 5, SEM = 0.07, df = 1, p-value = 0.0134. (E) Overall RE of patients (Average RE = 8.4 n = 116, SD = 14.9, SEM = 0.25) and healthy control (Average RE = 0.99, n = 76, SD = 2.17) df = 1, p-value < 0.0001 (** = p-value 0.001, *** = p-value 0.0001).

Expressional Level of ESR1 in Leucocytes of Breast Cancer Patients
We study the expression of ESR1 in leucocytes of breast cancer patients (142) and 77 healthy control and normalized it with housekeeping gene by Livak 2 ∆∆Ct method. Primer sequence used and annealing temperatures are mentioned in Table 2. The mean RE (2 ∆∆Ct ) in patient is higher than in control group. Welch's t-test shows significance of results at (p-value < 0.00623) for 142 patients and 77 control samples. We did divide the ESR1 data into different stages of the disease, and on analysis of variance (anova) bases, we find F value (2.8) with significance of Pr(>F) = 0.0237 ( Figure 6). Lastly, we compare the RE of both cmyc and ESR1 to find correlation between them and find strong spearman correlation coefficient R = 0.88 ( Figure 6).

Crosstalk between c-myc, ER and Estrogen in Breast Cancer Leucocytes
The protein expression of estradiol from the peripheral blood of the patients gave insight into the correlation of genetic parameters under study with disease progression. It was found that c-myc and estradiol were induced in ER+ve blood samples as compared to ER−ve leucocytes (Figure 7).

Correlation of c-myc and Estradiol in Breast Cancer Lymphocytes
A correlational study was conducted on twofold Ct values of c-myc and estradiol. The results presented in (Figure 8b) showed a positive correlation between the two with a correlation coefficient of 0.469.
in patient is higher than in control group. Welch's t-test shows significance of results at (p-value < 0.00623) for 142 patients and 77 control samples. We did divide the ESR1 data into different stages of the disease, and on analysis of variance (anova) bases, we find F value (2.8) with significance of Pr(>F) = 0.0237 ( Figure 6). Lastly, we compare the RE of both cmyc and ESR1 to find correlation between them and find strong spearman correlation coefficient R = 0.88 ( Figure 6).

Crosstalk between c-myc, ER and Estrogen in Breast Cancer Leucocytes
The protein expression of estradiol from the peripheral blood of the patients gave insight into the correlation of genetic parameters under study with disease progression. It was found that c-myc and estradiol were induced in ER+ve blood samples as compared to ER−ve leucocytes (Figure 7).

Correlation of c-myc and Estradiol in Breast Cancer Lymphocytes
A correlational study was conducted on twofold Ct values of c-myc and estradiol. The results presented in (Figure 8b) showed a positive correlation between the two with a correlation coefficient of 0.469. We confirmed our results on RStudio software and found similar results (R = 0.50) with the Psych package ( Figure 8B). There was a positive correlation and interdependence between the high expression of estradiol and c-my in-breast cancer patients, which validated our studies. n = 14, SD = 4.83, SEM = 1.29) and estradiol level in ER−ve patients (ME = 9.1, n = 15, SD = 6.07, SE = 1.57) showed a significant difference at df = 1, ** = p-value < 0.001. *** = p-value < 0.0001.

Correlation of c-myc and Estradiol in Breast Cancer Lymphocytes
A correlational study was conducted on twofold Ct values of c-myc and estradiol. The results presented in (Figure 8b) showed a positive correlation between the two with a correlation coefficient of 0.469.

Up-Regulation of c-myc & Estradiol at Metastatic Stages
For the next, step to check our predicted results in relation to stages of the disease, we concluded that the levels of c-myc and estradiol are induced in the metastatic phases of the disease. Stage III involves the nodal status, and Stage IV, where metastases occur in other parts of the body, showed elevated expression of biomarkers (Figure 9). We confirmed our results on RStudio software and found similar results (R = 0.50) with the Psych package ( Figure 8B). There was a positive correlation and interdependence between the high expression of estradiol and c-my in-breast cancer patients, which validated our studies.

Up-Regulation of c-myc & Estradiol at Metastatic Stages
For the next, step to check our predicted results in relation to stages of the disease, we concluded that the levels of c-myc and estradiol are induced in the metastatic phases of the disease. Stage III involves the nodal status, and Stage IV, where metastases occur in other parts of the body, showed elevated expression of biomarkers ( Figure 9).

Multilinear Regression and Correlation Analysis between Estradiol, c-myc, ER, and Stages of Breast Cancer Patients
Multilinear regression and correlation analysis were performed between estradiol, c-myc, ER, and stages of breast cancer patients. Results in Figure 10 showed that there is a negative correlation between stage and ER status, (R= −0.54) whereas a positive correla-

Multilinear Regression and Correlation Analysis between Estradiol, c-myc, ER, and Stages of Breast Cancer Patients
Multilinear regression and correlation analysis were performed between estradiol, c-myc, ER, and stages of breast cancer patients. Results in Figure 10 showed that there is a negative correlation between stage and ER status, (R= −0.54) whereas a positive correlation between c-myc and estradiol was observed (R = 0.50). Similarly, a strong positive correlation was observed between ER and estradiol (R = 0.76). Estradiol and c-myc showed a positive correlation with disease stage with (R = 0.60) and (R = 0.32), respectively ( Figure 10).

Molecular Docking
The system generated 10 models for docking affinity, with model 1 having the lowest energy. However, docking model 10 showed flexible binding and a greater number of H-bonds involved. Keeping in mind that the lowest energy bonds are best to describe the molecular docking, one of the best fit models, 2, with the lowest binding energy and lowest RMSD values (1.72A) is presented in (Figure 11). The bonding affinity between c-myc and estradiol is predicted using HDOCK and CB-DOCK tools [20,21]. HDOCK is a free available online server used to perform pro- Estradiol is also positively correlated to stage of the disease (R = 0.60), while c-myc showed a weak positive correlation with stage (R = 0.32). This figure produced R 2 value, which was then converted to R correlation coefficient. * Correlation is significant at 0.05 level (two tailed) ** Correlation is significant at 0.01 level (two tailed).

Molecular Docking
The system generated 10 models for docking affinity, with model 1 having the lowest energy. However, docking model 10 showed flexible binding and a greater number of H-bonds involved. Keeping in mind that the lowest energy bonds are best to describe the molecular docking, one of the best fit models, 2, with the lowest binding energy and lowest RMSD values (1.72A) is presented in (Figure 11).
The bonding affinity between c-myc and estradiol is predicted using HDOCK and CB-DOCK tools [20,21]. HDOCK is a free available online server used to perform protein-DNA docking. It is based on the hybrid algorithm of template-based modeling.
While CB-DOCK is used to check direct binding of estradiol to the c-myc protein crystal structure, CB-DOCK is a blind docking server available for free online. It is based on the cavity detection-guided easy approach bioinformatic tool.

Genetic c-myc and Receptor Bound Estradiol
The results confirm the reported molecular docking of both estradiol and c-myc with binding energies < −215 kcal/mol, LG scores of 5.606, and a MaxSub score < 0.8, as shown in Figure 12A-C. An aligned length of 229 of Chain A of the estradiol bonded ER with R = 0.931 was observed. The identification with sequence id was 95.8% (jobid = 60ce3ba192bf6) ( Figure 10). With this score, the strong binding could be observed between selected region of c-myc and receptor bound estradiol (Figure 12).

Molecular Docking
The system generated 10 models for docking affinity, with model 1 having the lowest energy. However, docking model 10 showed flexible binding and a greater number of H-bonds involved. Keeping in mind that the lowest energy bonds are best to describe the molecular docking, one of the best fit models, 2, with the lowest binding energy and lowest RMSD values (1.72A) is presented in (Figure 11). The bonding affinity between c-myc and estradiol is predicted using HDOCK and CB-DOCK tools [20,21]. HDOCK is a free available online server used to perform protein-DNA docking. It is based on the hybrid algorithm of template-based modeling.
While CB-DOCK is used to check direct binding of estradiol to the c-myc protein crystal structure, CB-DOCK is a blind docking server available for free online. It is based on the cavity detection-guided easy approach bioinformatic tool.

Root Mean Square Deviation (RMSD)
RMSD is commonly used quantifiable parameter of the similarity amongst two superimposed atomic aligns. We redo the docking only with catalytic domain and checked for the appropriate method to improve the DNA crystal structure. RMDS calculated by the system was <2.0 A of the ligand, aligned the same docking pose, and double-checked the coverage accuracy (Table 3). For the identification of possible target bindings of the antigen ESR1, we used online available tools (chIP-seq atlas) [22][23][24][25][26][27]. We generated targets specifically for ESR1 for specifically blood and breast cell types. In the chIP-seq data, we did not find the binding of ESR1 directly to the c-myc on any available distance of ±1 kb, ±5 kb, and ±10 kb regions, which is not inconsistent with our study. Previous studies showed the binding of a complex (ESR1, ESR2, JUND, FOS) at ERE sites of c-myc ( Figure 1) [4,28,29]. Similarly, on the other hand, we did find an experimentally determined gene neighborhood, gene fusion cooccurrence, and coregulation among c-myc and ESR1 using a string database version 11.5 https://string-db.org/ accessed on 15 April 2022 [23]. Figure 13 depicts said relationship between two experimentally studied factors. A better approach needs to be made for chIP-seq analysis to seek in-depth analysis of genetic complex binding on ERE sites.

Positive Association of c-myc and Estradiol Exists in Peripheral Bc Blood
In the present study, we investigated the role of c-myc, estradiol, and ER in breast cancer patients. We found that c-myc and estradiol are induced among all age groups as compared to the healthy control ( Figure 4). We also observed that the age groups more exposed to estrogen production showed induced expressions of both estrogen and c-myc in leukocytes (Figures 4 and 5). BMI is an indicator of the induced expression of estradiol, which in turn induces the c-myc expression in patients [24]. The luteal phase induces estradiol before menopause, and women get more exposed to the risk of breast cancer. Figure 13. Interaction of myc, ESR1, and ESR2. Red color is for fusion of genes, green for gene neighborhood, blue for gene cooccurrence, and pink is for experimentally determined interactions.

Positive Association of c-myc and Estradiol Exists in Peripheral Bc Blood
In the present study, we investigated the role of c-myc, estradiol, and ER in breast cancer patients. We found that c-myc and estradiol are induced among all age groups as compared to the healthy control ( Figure 4). We also observed that the age groups more exposed to estrogen production showed induced expressions of both estrogen and c-myc in leukocytes (Figures 4 and 5). BMI is an indicator of the induced expression of estradiol, which in turn induces the c-myc expression in patients [24]. The luteal phase induces estradiol before menopause, and women get more exposed to the risk of breast cancer. Other reasons for the induced expression of c-myc are the binding of more estradiol to estrogen receptors and increased transcription [15].

c-mycIs an Estrogen-Dependent Gene in Breast Cancer
We observed that higher estrogen levels were correlated with the expression of c-myc (Figures 4 and 5) Similarly, in ER+ve patients, both estradiol and c-myc expression are induced (Figure 7). The multilinear correlational studies in Figure 10 suggest the interdependence of c-myc and estradiol, which needs to be investigated more in further studies.
In BC cells, the c-myc oncogene is thought to be a conventional estrogen-induced gene [25]. Other significant estrogen-regulated genes in breast cancer (e.g., TFF1/pS2) have had their molecular regulation explored, but c-myc is known for its complex mode of regulation [26]. For many years, the exact method was unknown, until a putative estrogenresponsive site, ERE, was discovered in the basal promoter region of the c-myc gene in a previous project [26].
Utilizing a cellular model with inducible AP-1 predominant negative expression, they discovered that c-myc is an estrogen-induced and AP-1-dependent gene. These findings added to our knowledge of the estrogen, i.e., steroid hormone signaling pathway in breast carcinomas, allowing us to dig into the biomolecular processes of estrogen elevation of the cmyc gene expression. Many ER-binding sites are found in distal intergenic regions instead of promoter regions, according to genome-wide analyses ( Figure 12). We discovered an estradiol-bound ER cooperating factor that may modify the function of c-myc in a systematic way as distal enhancer.

Estradiol Affinity as Ligand to the DNA Structure
Previous studies suggest that estrogen induces c-myc gene expression via an upstream enhancer activated by the estrogen receptor (ER) [4] Estradiol is already established woman hormonal medicine used to treatment menopausal symptoms. Estradiol binding to DNA as a ligand molecule bring the idea of molecular docking [26] Many reports suggest that HRTs in longer run develops breast cancer due to continual deposition of estradiol in the body. We suggest an infancy stage method by expressional studies of estradiol and c-myc level in breast cancer patient via RT-PCR along with molecular docking the idea of targeting therapies at molecular level [9,22]. A future inhibitory study of direct targeting of ER bound estradiol at molecular level can help in reduction of estradiol in the body which in return reduces the cause of breast cancer in woman on HRT for long.

Anticancer Drug Screening Using Docking
Docking results in Figure 12 give support and affirmation to RT-PCR studies of c-myc and estradiol in breast cancer patients [26]. Our finding, together with those of other similar studies as performed by [26][27][28], suggest that various drugs based on estrogen used for pharmacological treatment mimic naturally existing estradiol. Investigating the DNA-based molecular docking processes of 17β estradiol and ER bound 17β estradiol would help in understanding the therapeutic efficacy of various inhibitory drugs in the case of breast cancer and enhancers in case of HRT treatments. As a result, it will offer new insight into anticancer drug screening.

ScRNA-Seq Data and Molecular Docking
Estrogen receptor ESR1 and its allies belong to the nuclear receptor family. Reactome Pathways shows the sharing of transcription regulation pathway with c-myc by binding to a half ERE site with the help of AP1, SP1, and junD. Jin Li et al. analyzed a single-cell RNA seq of a nuclear receptor family and its behavior as a ligand molecular in triple negative breast cancer [29].

Conclusions
Taken together, our data suggest that c-myc and estradiol are induced in an agerelated fashion in breast cancer patients. Strong correlations exist, which suggests that both estradiol and c-myc are cooperative and dependent on each other with the help of an estrogen receptor's presence.
Our work suggests that estradiol binds to somewhere around 5262 bp (selected region after defining a promotor for docking) away from the promotor along with complex protein structure and induces the expression of c-myc in the leukocytes of breast cancer patients. By identifying the structural and functional affinity of estradiol, the model2 in this study elucidates that estradiol behaves like an enhancer ligand for c-myc and exhibits many key structural points to be considered in future therapeutic studies for breast cancer.
A study to identify the measures to reduce the expression of estradiol and c-myc and control their interdependence could possibly reduce the chances of metastasis and disease prognosis. To establish the strong dependency between the trio, further investigation is necessary.