Epigenetic Alteration and its Association With Downregulated FOXP3 Gene in Indian Breast Cancer Patients

Background: FOXP3 gene, known to be a potential tumor suppressor, has been identified to interact with HER2 in mammary cancer. Moreover, the high expression of FOXP3 serves as a good predictor of the survival of patients in breast cancer, prostate cancer, and gastric cancer. The expression and epigenetic alterations were evaluated in female breast cancer patients. Material and Methods: Expression studies at the mRNA level and protein level were conducted in 140 breast cancer cases by real-time PCR and immunohistochemistry, respectively. Epigenetic studies were also conducted by analyzing the methylation status at the promoter region of the gene using MS-PCR. Results: FOXP3 mRNA expression and protein expression were downregulated in breast cancer patients. The absence of FOXP3 protein expression is significantly associated with promoter methylation, where 70 methylated cases exhibited protein loss (70/95, 73.6%). Statistically, we also found a significant correlation between FOXP3 protein expression and TNM stage, promoter methylation, and histological grade. The methylated FOXP3 cases that did not express protein were also significantly associated with positive lymph node metastasis and HER-2 status. Conclusion: The expression profile of FOXP3 may serve as a prognostic factor. In short, FOXP3 may stand in the most crucial list of biomarkers for breast cancer, bringing compelling results in terms of treatment and management of the disease.


INTRODUCTION
It is a well-known fact that females around the world are mostly affected by breast cancer (1.7 million cases, 11.9%); however, it positions fifth as the cause of death (6.4%) because of the comparatively conducive prognosis. Whereas breast cancer accounted for the most prominent cause of mortality in females in undeveloped and developing regions of the world (Ferlay et al., 2015), the development of biomarkers and their clinical use in therapy for prediction and expected response hold a remarkable potential.
FOXP3(Forkhead box P3), located on Xp11.23, is a member of the Forkhead/winged-helix family of transcription factors which is responsible for X-linked autoimmune diseases in mice as well as humans (Wildin et al., 2001;Brunkow et al., 2001;Schubert et al., 2001). Transcription factor FOXP3 regulates the development and function of Treg cells, and Treg cells are known to regulate homeostasis  and immunosuppression (Müller et al., 2010) and also recognized as the most peculiar marker for Treg (Hori et al., 2003). Before the advanced research on FOXP3 expression, it was thought to be expressed only in hematopoietic cells but now seems to be present in human tumors, particularly tumors of the breast (Merlo et al., 2009). In mammary cancers, FOXP3 is found to regulate HER-2 and SKP2 by repressing their expression, and importantly these genes are linked to a poor prognosis in the cases with breast carcinoma (Martin et al., 2010). The downregulation and many functional somatic mutations in the FOXP3 gene were usually found in human breast cancer samples. These mutations may also account for the overall down-regulation at the protein level (Karanikas et al., 2008).
FOXP3 halts the transcription of HER-2 by attaching to the promoter region of the ERBB2 gene (Redpath et al., 2011;Zuo et al., 2007), and it is a well-known fact that HER-2 is a potent marker in terms of prediction and effective therapy (Presson et al., 2011;Weigelt and Reis-Filho, 2010). The study has also pointed out the high expression of FOXP3 as a good predictor of the survival of a patient in prostate cancer, breast cancer, gastric cancer, and bladder cancer Wang et al., 2009;Winerdal et al., 2011;He et al., 2013;Fiori Lopes et al., 2014;Hao et al., 2014;Ma et al., 2014). Previous studies also reported that many SNPs in the FOXP3 gene had been associated with breast cancer (Jiang and Ruan, 2014).
FOXP3 is also linked to p21 and LATS2, where it is involved in transcriptional control (Li et al., 2011). Due to these captivating characteristics of FOXP3, the present work examines the correlation of FOXP3 protein expression with the clinicopathological variables to strengthen its role as a putative biomarker for breast carcinoma in the Indian population.

Ethical Statement
The University Ethical Committee of Jamia Millia Islamia (JMI), New Delhi, and the Ethical Committee for Human Study of AIIMS (All India Institute of Medical Sciences), New Delhi, have officially approved the study. The experimental work had been undertaken with written consent obtained from each subject, and the study complies with the rules and standards set by the Ethics Code of the Medical Association of the world, which have been noted as per the Declaration of Helsinki as published in British Medical Journal (1964).

Sample Collection
A total of 140 participants were included in the present case-control study. Cancer tissue from the breast and noncancerous adjacent tissue were both obtained from the surgical oncology department of the collaborating institute (AIIMS). The samples were collected in three vials containing RNALater, phosphate-buffered saline (PBS), and formalin, respectively, for further processing.
The classification of breast cancer stages was done under the TNM staging system, and the histological grading of tumors was classified based on the Nottingham grade system. The exclusion criteria in the current study included familial cancer, any previous type of cancer, other metastasized cancer that has spread from different organs, and chemotherapy and radiotherapy exposure. In addition, included were various clinicopathological variables such as tumor distinctiveness [age, tumor size, metastasis at the lymph node level, TNM staging, grade of tumor, molecular subtype of tumor, hormonal receptor status (ER, PR, and Her2neu), and reproductive history (menopausal status parity)]. Quantitative-PCR RNALater (Qiagen) was used to store excised tissues from normal and breast cancer patients, and then RNA was extracted by using the TRIzol method as per the manual. cDNA was synthesized using a Thermo Fisher verso kit from the extracted RNA. The qPCR is processed using Roche LightCycler ® 96 machine with SYBR Green I Master mix reagent (Roche) with the help of FOXP3 primers (Fwd-5′-TCCCAGAGTTCCTCCACAAC-3′ and Rev-5′ATTGAGTGTCCGCTGCTTCT-3′) that give an amplified 122-bp product. The internal control used was GAPDH gene which was also amplified in the same PCR reactions. The program used for the amplification cycles was as follows: preheating at 95°C for 1 min, 30 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 15 s, extension at 72°C for 15 s, and further elongation at 72°C for 7 min. The experiments were repeated thrice. The relative quantification of expression was calculated as the calibrator normalized ratio using LightCycler 96 (Roche) Software 1.5. The formula used, RQ 2 -ΔΔC t, was according to MIQE.

Genomic DNA Extraction
The phenol-chloroform extraction method was used to isolate highmolecular-weight total gDNA from both tumor and normal tissues stored in PBS. The genomic DNA isolated was quantified on a Nanodrop spectrophotometer, and its quality was also assessed using an A260/280 ratio. It was further visualized on the 1% agarose gel stained with ethidium bromide under a UV transilluminator.

MS-PCR for Epigenetic Analysis
Isolated gDNA from the tissues were given bisulfite treatment using Zymo research EZ DNA Methylation-Gold ™ Kit per the instructions.
The treated gDNA was amplified using two sets of methylated and unmethylated primers for the FOXP3 promoter. MethPrimer tool was used to design the set of primers for methylation and unmethylation. (Li and Dahiya, 2002). The methylated primer pairs for the promoter region of the FOXP3 gene were: forward 5′-TGTAGGGGGTGT AGAATTTTTTTC-3′ and reverse 5′-AAACTAAATTCCCAA AAACCTCG-3′ and for the unmethylated were forward 5′-GTA GGGGGTGTAGAATTTTTTTTGT-3 and reverse 5′-TAAAAC TAAATTCCCAAAAACCTCA-3′. The positive controls used in the experiment were commercially available Completely methylated and unmethylated human genomic DNA.
MS-PCR was performed under the following cycles: First denaturation at 95°C for 7 min, denaturation at 95°C for 30s 52.5°C annealing for both types of primers for 30 s, 72°C for 30 s and final elongation at 72°C for 7 min which was amplified for 35 cycles. Then, 2% agarose gel stained with EtBr was used to visualize and analyze the PCR product, which was finally photographed using the Bio-Rad Gel Documentation system. All experiments were conducted in triplicates.

Immunohistochemistry
The tissue biopsies of the breast carcinoma and adjoining noncancerous tissue were conserved in formalin-fixed blocks. The blocks were then sectioned and engraved on slides coated with poly-L-lys and exposed to deparaffinization by dipping in different concentrations of xylene and further rehydrated with grades of ethanol. By using 0.3% H 2 O 2 for 30 min, the endogenous peroxidase activity was blocked, and sodium citrate buffer (pH 9.0) was used for Ag retrieval.
The sections were treated for 30 min with TENG-T (10 mM Tris; 5 mM ethylenediaminetetraacetic acid, 0.15 mol/L NaCl, 0.25% gelatin, and 0.05% (v/v) Tween20 (pH 8.0) to block the samples. Bovine serum albumin was used to limit unspecific binding to the protein. The slides were treated with the primary antibody (mAbCam#ab22510 FOXP3 1:50) and incubated overnight at 4°C. The slides were then treated for 30 min with a biotinylated anti-mouse secondary antibody along with streptavidin-horseradish peroxidase conjugate. The DAB chromogen was finally used as a substrate to give a brown-colored precipitate. The slides were also treated with hematoxylin as a counterstain for better contrast.
Histopathologists interpreted the slides after immunohistochemistry; the slides were photographed under a light microscope at ×400 magnification. The pathologist further graded the expression on the number scale 0-4, with 0 as no expression and 4 as highest expression; the slides with >50% protein staining were considered in the highest scale.  Figure 1A), out of which nearly 86.5% (77/89) of the cases registered in the study were linked to histological grade type 1 and type 2. As per the fold change analysis, 89 cases out of 140 samples seemed to be downregulated (5.09-fold), while the expression pattern of FOXP3 at the level of mRNA, when normalized accordingly with the internal control GAPDH in tumor and non-tumor tissues, was 2.07 ± 0.7 (mean ± standard error) and 2.51 ± 0.4 (mean ± standard error) ( Figure 1B), (p-value of <0.0001; Table 1) respectively. However, there was no significant association observed between FOXP3 mRNA level and various clinical variables.

Expression of FOXP3 Protein is Either Lost or Low in Breast Cancer
The expression of FOXP3 at the protein level was found to be either low or absent in 95 cases of the total 140 samples involved in the study (67.85%) (Figure 2), while in the other 45 cases, the expression pattern was either in the high or moderate range as interpreted by a histopathologist on the basis expression scale (45/140, 32.14%). The protein expression pattern was in relation to the mRNA expression. The FOXP3 protein, as visualized by immunohistochemistry, was mainly located in the nuclear region.

FOXP3 Protein Expression and Its Significant Correlation With Clinicopathological Parameters
As revealed by immunohistochemistry staining, the majority of FOXP3 proteins in the samples were found to be significantly downregulated. Moreover, when we tried to statistically associate the protein expression with the clinical parameters of the patients, we observed a significant correlation between TNM staging and FOXP3 protein expression (p < 0.035) ( Table 2). However, with other parameters, no significant association was obtained statistically ( Table 2), though most cases with tumor grades 1 and 2 seem to have protein loss (82/95, 86.3%).

FOXP3 Promoter Methylation and Its Association With Clinical Variables
Methylation at various CpG present in the upstream promoter area of FOXP3 gene was observed in 73 cases (73/140, 52.14%) ( Figure 3) and, once linked with clinical parameters, revealed a significant association with the Nottingham histological grades 1 and 2 type tumors of breast cancer patients  Frontiers in Genetics | www.frontiersin.org November 2021 | Volume 12 | Article 781400 (0.031). Though no significant associations were seen with other parameters included in the study (Table 3), we did note a significantly higher number of methylated cases in metastatic lymph node (59/73, 80.8%), estrogen receptorpositive (52/73, 71.2%), and menopausal female patients (51/73, 69.8%) ( Table 3).

Convincing Association of FOXP3 Protein Expression With FOXP3 Promoter Methylation in the Cases of Breast Carcinoma
The correlation study of methylated FOXP3 gene and its respective protein expression displayed the significant link, in which out of 95 cases with protein loss, 70 cases possessed methylation at the promoter region (70/95, 73.68%) (

Association of Methylated FOXP3 Gene Exhibiting Loss of Protein With Numerous Clinicopathological Variables
The methylated promoter region of FOXP3 cases demonstrating either the absence or presence of protein exhibited a statistically significant relation with Her 2 neu receptor (p 0.004) and metastatic lymph node tumors (p 0.01) ( Table 5). Moreover, 95.7% of methylated cases (67/70) with lymph node metastasis have protein loss. (Table 5). Additionally, the cases having protein loss exhibiting either a methylated or unmethylated FOXP3 promoter region shows a convincing association with positive Her-2 receptor (p 0.03) and tumors of grades 1 and 2 (p 0.01) ( Table 5). Furthermore, it is seen that there is a strong statistical relation between FOXP3 protein loss and the promoter methylation with the various clinicopathological parameters ( Table 6), where most of the features were associated in a highly significant manner (p-value < 0.05).

DISCUSSION
FOXP3 expression is identified in tumors of the breast, prostate, lung, gastric, and thyroid Yang et al., 2017;Ma et al., 2014;the Chu et al., 2015) suggesting its crucial role in the biology of cancer. The previous study demonstrated the inverse correlation between breast cancer angiogenesis and nuclear FOXP3 expression. Adding to the same observation, the significant downregulation of FOXP3 also resulted in the reduced survival in breast cancer (Li et al., 2018) FOXP3 has been reported to modulate the expression of various genes involved in the process of carcinogenesis to exert its suppressing role in tumor development (Szylberg et al., 2016). At the same time, we cannot forget that significant studies have suggested the positive association between FOXP3 expression and better survival in patients and the tumor-suppressive role of the FOXP3 gene in breast cancer. Therefore, the present work investigated the FOXP3 expression pattern and its correlation with various clinicopathological variables to strengthen its prognostic value and tumor-suppressive property. An earlier study demonstrated a quantitative method to assess the methylation status of FOXP3 to understand the role of Treg cells in immunomodulation (Wieczorek et al., 2009). In our study transcription factor, FOXP3 promoter methylation and expression were studied and analyzed in breast cancer patients of the northern region of India using methylation-specific PCR, real-time PCR, and immunohistochemistry to assess its role as a potential biomarker. The study correlated the findings with the clinicopathological variables (age, histological grade, ER status, HER2 status, etc.) of the procured cases.
In our study, at the mRNA level, nearly 63.5% (89/140) of cases were found to be downregulated (5.09 fold), and interestingly 86.5% (77/89) were linked with the histological grades I and II, suggesting the possible role of FOXP3 in the early development of the disease. The study is supported by the previous studies on FOXP3 expression at the transcript level (Zhang and Sun, 2010;Hinz et al., 2007). Furthermore, apart from breast cancer, one of the studies pointed out that the upregulation of FOXP3 in gastric cancer cells put a brake on GC cell growth in both in vivo and in vitro studies (Hao et al., 2014), unraveling the crucial role of FOXP3 expression in different carcinomas. The protein expression profile exhibited low or no expression in nearly 69% (95/140) of breast cancer cases, followed by either moderate or high expression in 32% (45/ 140) of the cases. The expression was either cytoplasmic or nuclear, which was demonstrated in different types of cancer (Merlo et al., 2009;Karanikas et al., 2008;Zhang and Sun, 2010;Hinz et al., 2007;Ladoire et al., 2011;Tao et al., 2012).
We did find a significant association between FOXP3 protein expression and the TNM stage (p-value, 0.035). Interestingly almost 74% (95/140) of the cases of stage (III and IV) harbored protein loss. Because of the above mentioned condition, it has been observed earlier that, in the most aggressive cancer of epithelial tissues, FOXP3 may help in the suppression of cancer as these aggressive cancer tissues harbored very low or no expression of FOXP3 at the transcript and protein levels (Wang et al., 2009;Jiang and Ruan, 2014;Li et al., 2011).
The promoter methylation of FOXP3 was observed in 52% of the cases (73/140) and significantly associated  with Nottingham histological grades 1 and 2 (p-value 0.031). The finding depicts a strong association between promoter hyper-methylation and mRNA expression in deactivating or down-regulating the possible role of FOXP3 in the suppression of breast cancer (Esteller, 2007;Li et al., 2007).
While analyzing an association between protein loss and hypermethylated promoter cases, we found a compelling association as out of 95 protein loss cases, 70 cases possessed methylation at the promoter region (70/95, 73.68%), whereas in a total of 67 unmethylated cases, 62.6% (42/67) exhibited the presence of protein. More intriguing results came out while analyzing methylation and protein loss with each other, as 95.7% methylated cases (67/70) with lymph node metastasis displayed protein loss. The findings are strongly supported by previous studies that have shown the association to be significantly lower in tumor grade and lymph node involvement in the breast tumor cells with positive FOXP3 expression (Ladoire et al., 2012). Thus, the loss in FOXP3 due to epigenetic change like methylation may serve as a potential biomarker in cases with lymph node metastasis.
In summary, our data provide some intriguing findings of FOXP3 expression and its association with different clinicopathological parameters. However, the present study on a smaller sample size may weaken the statistical power. Therefore, further investigation on different sets of the population with a larger sample size is required to establish FOXP3 as a potential cancer biomarker for diagnostic and prognostics purposes.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ supplementary material.

ETHICS STATEMENT
This study was in agreement with the ethical standards of the host institute Jamia Millia Islamia University and AIIMS, New Delhi, India, and also as per the guidelines of the Helsinki Declaration in 1964 and its amendments. The patients/ participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
Sa and SH took charge of the experimental design and execution. The experiments were executed by Sa. The reagents, materials, and analysis tools were contributed by Sa, MN, MK, and SD. Manuscript preparation was carried out by Sa and co-authors.