Kzar et al (2021): Factor-1 alpha mRNA expression in women with © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24464

Hypoxia inducible factor-1 alpha mRNA and estrogen index as prognostic indicator in women with breast cancer

Hamzah H. Kzar1*, Moaed E. Al-Gazally2, Moshtak A. wtwt3

1Veterinary Medicine collage, Al-Qasim Green University, Iraq 2 College of Medicine, Babylon University (works now at university of Al-Ameed, Iraq 3College of Medicine, University of Babylon, Iraq

*Author for Correspondence: [email protected] (Kzar)

Abstract Breast cancer (BC) is a one of highly prevalent and mortality type of cancers among women worldwide. The overexpression of certain such as HIF-1A in blood of breast cancer tumors may be useful to investigate the prognosis and selecting therapies. Many are now known to be regulation by HIF-1A through activation. HIF-1A plays important roles in breast cancer metastasis by mediating hypoxia-induced expression of mRNA-encoding genes. The cellular response to estrogen is mediated by two different receptors subtype, alpha (ERA) and beta (ERB). One hundred and twenty women with BC involved in this study as a patients group as well as 120 apparently healthy women as control group were investigated. Relative quantitative qRT-PCR was used to assessment the HIF-1A mRNA gene expression in patients and control groups. ERA and ERB levels were investigated by ELISA. The results of this study suggested significant statistical difference in mean± SD ofERA (p-value <0.05) between patients and control groups but non-significant when compare both groups for ERB. When comparing of HIF-1A mRNA gene expression fold between subgroups, we found that IDC have a higher gene expression value compare to ILC and this was also applied on Grade1+2 and Grade 3 subgroups. This study showed that (target) HIF-1A mRNA gene expression to be 21.1folds relative togene of interest (GAPDH) gene expression (p-value<0,005) and the results also showing the ratio of ERA/ERB or ERI was in the range of (8-12.9) in patients group, but the index was (0.5-7.9) in control group.HIF-1A gene expression and ERI is useful prognostic biomarkers for diagnosis and following-up women with BC treated with .

Keywords: Breast cancer, Estrogen receptors, HIF-1A, Estrogen receptor index

DOI: http://doi.org/10.36295/ASRO.2021.24464

Page(s): 552-559 Volume/ Issue: Volume: 24/ Issue: 04

Ethical issue This study was performed depending on ethical instructions of the department of community medicine / college of medicine / Babylon University. The approval was taken from all women participate in this study.

Introduction Recent global estimates mention that 2 million women were diagnosed with breast cancer (BC) in 2018, and the incidence rate is expected to continue to increase to over 3 million patients per year by 2040(1). In Iraq, breast cancer is an important public health problem in women and the number of new cases diagnosed is increasing every year(2). BC is a heterogeneous disease associated with diverse clinic-pathological and molecular features. In its pathological features, some instances of BC show slow growth with good prognosis, whereas others are aggressive and invasive. Although there are many medical treatments for breast cancer patients, such as operation, chemotherapy, endocrine therapy and targeted therapy, the greatest challenges remain in effective diagnosis and prognosis (3). Hence, hypoxia inducible factor-1 alpha (HIF-1A) and estrogen receptors (ER) are playing important role in the diagnosis, prognosis, and treatment and follow up of women with new BC cases. The mitotic arrest is caused by severe hypoxia and halting replication at the G1/S phase (4). The previous experimental settings showing that this arrest was mediated by HIF-1A(5). Many genes are now known to be regulated by HIF-1A through transcription activation(6). Hypoxia regulates HIF- 1A through post-translational modification. In the presence of oxygen, prolyl hydroxylase domain (PHD) proteins hydroxylate residues on HIF-1A. After , pVHL, the product of the von Hippel Lindau , binds and ubiquitinates of HIF-1A. Ubiquitinated HIF-1A is then targeted for proteasomal destruction(7,8). HIF-1A can be stabilized in the hypoxic core of rapidly expanding, poorly vascularized solid tumors where the partial pressure of oxygen may be <10 mmHg (9). As little as three hours of hypoxia in vitro stabilize HIF-1A in cancer cells(10).HIF-1A overexpression is suggested to be

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Kzar et al (2021): Factor-1 alpha mRNA gene expression in women with breast cancer © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24464

associated with increased women with BC mortality (11). Increased HIF-1A levels have been demonstrated by immunohistochemistry in biopsies analyzed from both lymph node-negative (12) and lymph node-positive (13) of women with BC. Estrogens exert crucial tissue-selective effects on cellular homeostasis and play a pivotal role in the pathogenesis of breast cancer (14). The cellular response is mediated by two different ERsubtypes alpha and beta (ERA and ERB)(15). The affinity of estrogens to the ER can be modulated by tissue-specific shifts in the expression of splice variants of ERA and/or ERB (16). ERA and ERB differs in their ligands affinities, are expressed in a tissue-specific fashion and may act antagonistically (17). The aim of this study is investigate the role of HIF-1A gene expression and the ERA/ERB index for using as prognostic indicator inwomen with BC have receivingchemotherapy regimen.

Materials and methods Specimen’s collection: The samples were collected during the period from 1st December, 2018 to 30th April, 2019 from oncology center/ Merjan teaching hospital. The clinic-pathological of one hundred and twenty women with BC enrolled in this study was obtained from baseline information sheets of patients and self-questionnaire. All patients are diagnosis with BC by Histopathology laboratory examination.

Determination of ERA and ERB by ELISA: ERA and ERB levels was estimated by sandwich ELISA depending on the instructions of the manufacture that provided with kits(Sunlong/China).

Total RNA extraction: AccuZol™ Total RNA Extraction Solution from Bioneer/ Korea (Cat#3066) was used to extraction of RNA from fresh whole blood of 240 samples of patients and control group. After extraction, RNA purityand quality were assessed by scandrop(Analytikjena/Germany) device to estimate the purity of RNA by the ratio of 260/280 and total yielding to be 1.89±0.01, 60±12 µg/ml respectively. cDNA synthesis: HiSenScript™ RH(-) cDNA Synthesis Kit from iNtRON/ Korea(Cat#26013) was used to cDNA Synthesis from extract RNA. The total volume reaction was 20µl Consist of oligo(dT) primer(poly A tail), RNA template, RNase inhibitor , reverse transcriptase (Revoscript™ RH(-) RTase), dNTP Mix, MgCl2, and RNase free water. The protocol used to first strand cDNA synthesis was 42C° for 30 min and 95C° for 5 min to inactivate RTase. The cDNA obtained was stored into -20C for quantitative real time –polymerase chain reaction (qRT-PCR) Analysis. Real Time Quantitative qRT-PCR Analysis RealMOD™ Green qRT-PCR mix (2X SYBR green dye) kit iNtRON/ Korea(Cat#26103) was used to qPCR analysis. The primers were designed depending on the following website: https://www.ncbi.nlm.nih.gov/gene and optimized to qPCR analysis as showing in table-1. The 25µl total volume consist of 5 µl cDNA , 2µl of both reverse and forward primers, 16µl DEPC water. The protocol of thermocycler (Exicycler™ 96 – Bioneer/ Korea) was 95C starting denaturation to 5 min following by 40 cycles, 95Cº denaturation to 30 sec, 57Cº for annealing to 30 sec, and 72Cº for extension to 20 sec and the melting curve scanning was from performed at 55Cº-95Cº for 1 sec in each temperature following by final incubation to 25Cº for 1 min. This protocol optimized to use for tow genes, gene of interest GOI (HIF-1A) and housekeeping gene HKG (GAPDH).

Statistical analysis The gene expression analysis of target and reference genes based on determination of threshold value (Ct) for real amplification of HIF-1A and GAPDH in patients and control groups. The Ct value was calculated as average of triplicate. The relative expression ratio (RGR) calculated depending on the following equation: ΔCt HIF-1A (treated-control) ΔCtGAPDH (treated-control) RGR= (EHIF-1A) / (EGAPDH) E= Efficiency is calculated from standard curve of HIF-1A and GAPDH depending on following equations: E of RT-PCR = (10 (-1/slope) -1)*100 Gene Expression Fold (GEF) =2-ΔΔCt Statistical analysis for assessment of ERA and ERB in different groups was performed by ANOVA test with using SPSS software (version 17). Significant differences were established at p < 0.05.

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Kzar et al (2021): Factor-1 alpha mRNA gene expression in women with breast cancer © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24464

Table (1): Primers sequence of HIF-1A and GAPDH

PCR Gene Primer Sequence (5'---->3') product size F: CCTATGACCTGCTTGGTGCT HIF-1A R:TATCCAGGCTGTGTCGACTG

103bp F: AGATTTGGTCGTATTGGGCG GAPDH R: TCACCATGTAGCACTCACCAT

120bp

Results The clinic-pathological of women with BC participate in this study is shows in table-2:

Table (2): Clinic-pathological characteristics of patients group

Clinic-pathological Number (%) Total patients N=120 Age 56(47) <50 64(53) ≥50 BMI ≥25 67(56) <25 53(44)

Family history 40(33) YES 80(67) NO Histological type 80(67) IDC 40(33) ILC Histological grade 72(60) Grade 1+2 48(40) Grade 3 ER status 55(46) P 65(54) N PR status 66(55) P 54(45) N Her-2 status 36(30) P 84(70) N Menopausal status 69(57.5) Pre 51(42.5) Post Site of cancer 57(47.5) Right 63(52.5) Left Metastasis status 42(35) Yes 78(65) No

IDL: Invasive ductal carcinoma ILC: Invasive lobular carcinoma, P: Positive, N: Negative The results of this study suggest significant statistical difference in mean± SD of ERA (p-value<0.05) between patients and control groupsbut non-significant when compare both groups for ERB, as shows in table-3:

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Kzar et al (2021): Factor-1 alpha mRNA gene expression in women with breast cancer © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24464

Table (3): Levels of ERA and ERB in patients and control groups

ERA(pg/ml) ERB(pg/ml) Groups P-value P-value mean± SD mean± SD Patients 124.9±14 12.3±4 N=120 Control 0.000 0.126 111.7±10.2 11.6±3 N=120

The results also showing the ratio of ERA/ERB or ERI was in the range of (8-12.2) in patients group, but the index was (3-7.9) in control group. For PCR efficiency we evaluated standard curve to optimization of RT-PCR analysis by blotting Ct values vs. differences in fluorescent (ΔF/Δt). This was carried by amplification of serial dilutions(1/10,1/100,1/1000,1/10000, and 1/100000) of cDNA that synthesized from RNA for both target(HIF-1A) and reference(GAPGH) genes, as shown in figures-1A ,B,C, and D.

Figure (1): A: Standard curve to optimization of GAPDH gene, B: standard curve to optimization of HIF- 1A gene, C: Amplification curve of GAPDH gene, D: Amplification curve of HIF-1A gene

The relative quantification results of HIF-1A mRNA gene expression as gene of interest(GOI) was compare with GAPDH gene expression as housekeeping gene(HKG) in both women with BC group was to be 21.1by applying the equation (2-ΔΔCt ) when-ΔΔCt was (-4.4)( the data not shown here) and the efficiency of PCR for HIF-1A and GAPDH were 80% and 94% respectively ,asshown in table-4 and figure-2:

Table (4): Gene expression fold (GEF)of HIF-1A mRNA and GAPDH in women with BC

Type of Efficiency RGR Gene p-value gene % GEF HIF-1A GOI 80 0.7221.1 0.000

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GAPDH HKG 94 -1

Figure (2): HIF-1A mRNA gene expression fold in 120 cases of women with BC compared to HKG

When comparing of HIF-1A mRNA gene expression fold between subgroups, we found IDC havea higher gene expression value compare to ILC. This also applied on Grade1+2 and Grade 3 subgroups, figure-3 showing the differences in gene expression fold of HIF-1A mRNA gene in these subgroups(p-value <0.05).

Figure (3): HIF-1A mRNA gene expression fold in different subgroups Receiver Operator Characteristic (ROC) analysis of GEF of HIF-1A mRNA as molecular marker in BC

The ROC curve analysis of GEF of HIF-1A mRNA as molecular marker in women with BC group showed an area under the curve (AUC) of (0.852)(95% CI, 0.803-0.901) with a sensitivity of (87%) and specificity of (62%) at a cut off value 20.7, as shown in figure 4.

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Kzar et al (2021): Factor-1 alpha mRNA gene expression in women with breast cancer © Annals of Tropical Medicine & Public Health DOI: http://doi.org/10.36295/ASRO.2021.24464

Figure (4): ROC curve of GEF HIF-1A mRNA in patients against control group

Table 5, showing the AUC for ROC curve corresponding to the diagnostic value of GEF of HIF-1A mRNA depending on clinic-pathological variables.

Table (5): AUC for ROC curve corresponding to the diagnostic value of GEF of HIF-1A mRNA Variables AUC ST. ER 95% CI Family history 0.7572 0.04 0.66-0.85 Histological type 0.605 0.05 0.49-0.71 Histological grade 0.634 0.05 0.52-0.74 Menopausal status 0.684 0.04 0.59-0.77 Site of cancer 0.586 0.05 0.48-0.68 Metastasis status 0.833 0.03 0.76-0.90

Discussion The second most common cancer in women in worldwide is breast cancer and accounted about 23% of all types of cancers (18). There were over 2 million new cases in 2018. The disease is the most frequently diagnosed cancer in the vast majority of the countries and is also the leading cause of cancer death in over 100 countries (19). According to Iraqi cancer registry, BC remain the most prevalence cancer in female. For this reason we suggest increase needing to find new biomarkers to diagnosis and follow up of new cases of BC. In this study we extracted total RNA from whole blood of patients and control groups and converting to cDNA, this products were amplification with unique primers designed for HIF-1A and GAPDH genes to get optimal amplification at specific Ct(threshold). In this study, we observed the gene expression fold of HIF- 1A mRNA gene is expressed by 21.1 folds more than GAPDH, this indicate of that the target genes controlling by HIF-1A were highly expression and increase the incidence of BC.High levels of HIF-1A in VHL syndrome leads to over-expression of growth factors such as vascular endothelial growth factor VEGF (20). HIF-1A overexpression is associated with increased patient mortality in many different cancers. The previous studies suggested that VEGF induced tumorigenesis and increase breast cancer risk (21,22).Normal depends on the coordination of several independent and temporally ordered processes (23,24). In BC, increased HIF-1A levels have been demonstrated by immunohistochemistry in biopsies analyzed from both lymph node-negative (25) and lymph node-positive (26)BC patients. In this study we found IDC subgroup have a higher gene expression value compare to ILC and the results was also applied on Grade1+2 and Grade 3 subgroups, this may be because of overexpression of HIF-1A in women with breast cancer. Sex steroids, especially estrogens, are pivotal for the development of the mammary gland, the sexual organs, the skeleton, the blood vessels and other tissues. This influence is mediated by the two estrogen receptors (ER) ERA and ERB which control the transcription of a greater number of genes. The deregulation of these signaling cascades can contribute to carcinogenesis (27-28). ERA can be used as a target of anti-hormone therapy because of over two third of all newly diagnosed breast cancers are primarily ERA positive (29,30). ERA/ERB index (ERI) was assessing in this study to use with over expression of HIF1A as a biomarker to diagnosis and follow up women with BC. ERI was 8-12.9(≥ 8) in women with BC group and ERI was 0.5-7.9(< 8) in control group, this suggests a new classification of BC cases depending on ERI (less than 8 or more than 8), this may be helps to first diagnosis of women by these cut-off values. Inhibition of HIF-1A may be useful when combined with other chemotherapeutic agents or to treating of patients with BC. Another strategy is to inhibit the proteins regulated by HIF-1A target genes. VEGF is now targeted for many types of cancer therapies and this protein level is controlled by the transcription factor, HIF-1A (31). The results of present study show that the sensitivity and specificity for diagnostic of BC by evaluation of GEF of HIF-1A mRNA of patient compare to control group were 87% and 62%, respectively. Warwick et al (2014) were using ROC analysis and showed the AUC was 0.62 by using body mass index and age to evaluation the risk factors of BC, lower than the AUC of present results (32). Chen et al (2008) reported that by using ROC analysis that miR-199a is involved in tumor progression and chemo-resistance in by regulating IKKβ expression (33). To the best of the present study knowledge, this is the first study reporting the association of HIF-1A mRNA expression (GEF) with the clinic-pathological profile in Iraqi women with BC, The expression of HIF-1A mRNA of metastasis status yielded a significant AUC of 0.833 (95 % CI 0.76–0.90) with a sensitivity of 82 % and specificity of 63 % from an optimal cutoff value of 20.5. The results of this study showed that the GEF of HIF-1A mRNA may be had higher sensitivity, specificity, and diagnostic value of metastasis status subgroup than other types of Iraqi women with BC and this results may be useful in diagnosis and following up of chemotherapy treatment. In conclusion, HIF-1A gene expression and ERI are useful prognostic biomarkers for diagnosis and following-up women with breast cancer those treated with chemotherapy.

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Data Availability The data used to support the findings of this study are available from the corresponding author upon request. Funding This work did not receive funding from any organization or institution. Authors’ contributions HHK designed the experiments, data analysis, and the manuscript writing. MEA supervising on the experiments. MAW participated in conducting some experiments. All authors read and approved the final manuscript. Conflict of interest No potential conflict of interest relevant to this article was reported. Acknowledgement We thanks all women participated in this study and the staff of oncology center in Merjan teaching Hospital.

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