In our study, 127 participants were enrolled, and the cancerous tissue along with the adjacent non-cancerous tissue of the breast was obtained and stored at −20° for further experiments and analysis. The inclusion criteria for the specimens in the study included the histopathologically confirmed breast cancer patients of age group 20–79 years having at least 6 months of life expectancy.

Following clinical parameters were included for the study such as tumor size, age at diagnosis, histological grade, the status of lymph node (LN), reproductive history, and information (age at menarche and menopausal status), clinical staging or TNM stages, Estrogen receptor (ER) (+ or -), Progesterone receptor (PR) (+ or -) and Human Epidermal Growth Factor Receptor 2 (HER2) (+ or -).

The females (n = 127) included in the study were clinically confirmed with sporadic breast cancer and were genetically unrelated. Normal adjacent breast tissue was taken as control.

The study involved females with histopathologically confirmed primary breast cancer and having at least 6 months of life expectancy, lying between the age group 20–79 years. The participants provided consent to abide by the procedures of the study. All the females included in the study were registered in the medical record book of AIIMS, New Delhi, and their medical records were evaluated for studying various clinical and pathological parameters of the patients.

For the isolation of RNA, the breast cancer tissues, and the collected normal tissues were preserved in the RNAlater (Qiagen) kit, and afterward, RNA isolation was done by using TRIzo1Reagent (Invitrogen) by following the instruction provided by the manufacturer in the protocol. Thereafter, the complementary DNA (cDNA) from total RNA was synthesized using a cDNA kit (verso Thermo Fisher Scientific) and later storage was done at −20°C for further analysis. Subsequently, the above-prepared cDNA was used in the (qPCR) where amplification was carried out using Roche Light Cycler^® 96 SYBR Green I Master mix. By applying the primers for FOXO1: sense 5′- CCA​CAT​TCA​ACA​GGC​AGC​AG-3′ antisense 5′- GAC​GGA​AAC​TGG​GAG​GAA​GG-3′ which amplified a 152-bp. product. β actin gene was taken as an internal control and amplified in the same qPCR reaction. The primers used for qPCR reaction were sense 5′-AGA​TAG​TGG​ATC​AGC​AAG​CAG-3′ and antisense 5′-GCG​AAG​TTA​GGT​TTT​GTC​A-3′, which amplified a 160 bp. product. Standardized protocol of our laboratory (Real et al., 2018; Sadaf et al., 2018; Khan et al., 2020) was used to perform PCR. Measurements were taken in triplicates. The calculation was done for the relative amount of mRNA using Light Cycler 96 (Roche) equipped with Software 1.5. The calibrated normalized ratio was estimated as per the given standard formula: RQ = 2-∆∆Cq = [(Cq targeted gene–Cq βactin) calibration sample].

Phenol-chloroform Isoamyl (PCI) method was used for isolation of gDNA from Breast cancer and adjacent normal tissue (Russell and Sambrook, 2001). The quantity and quality evaluation of isolated genomic DNA was done using a Nanodrop spectrophotometer (ND1000), and agarose gel electrophoresis was further performed for validation.

EZ DNA Methylation-Gold™ Kit was used to carry out Bisulfite conversion following the instruction given by the manufacturer. The converted product was amplified using dual sets of methylated and unmethylated FOXO1 primers. Eukaryotic promoter database was used to retrieve the FOXO1 gene promoter sequence, and MethPrimer software was used for primer designing (Figure 1). When searched by MethPrimer, the promoter region of the FOXO1 gene was found to contain two CpG islands of 600 bp. The primer pairs that were used for the detecting methylation in the promoter region of FOXO1 were as follows: sense 5′- GGA​AAA​TCG​GGT​TTT​ATT​TAG​TTC-3′ and antisense 5′- GAC​TAC​TAC​GAC​TAC​CAA​ACC​GC-3′, for the unmethylated detection: sense 5′- TGG​AAA​ATT​GGG​TTT​TAT​TTA​GTT​T-3 and antisense 5′- CAA​CTA​CTA​CAA​CTA​CCA​AAC​CAC​C-3′. The product size for unmethylation was 172 bp, and for methylation, it was 170 bp. MS-PCR was done by the following condition: initially denaturation at 95°C for 5 min, 35 cycles of amplification at 95°C for the 30 s, annealing at 53.9°C (methylation) and 52.7°C (unmethylation) for 30 s, 72°C for 30 s and final extension at 72°C for 7 min. The pictures of the amplified product were obtained on a 2% agarose gel having EtBr, visualization was done under ultraviolet (UV) illumination with the Gel Doc (Bio-Rad Molecular Imaging System). The experiments were executed in triplicate without any disparity observed among the replicates.

Breast cancer tissue and adjacent normal tissue stored in formalin were used for Block preparation. Poly-l-lysine slides were used, and the sections of the block were taken on slides. Subsequently, different grades of xylene were used for deparaffinization, and rehydration was done using ethanol. The quenching of internal peroxide activity was done by 0.3% hydrogen peroxide, and citrate buffer boiling resulted in Ag withdrawal. To non-specific interaction of protein was ceased using serum solution as a blocking agent, and incubation at 4°C with primary antibody (CST#2880 FOXO1, 1:100) was done overnight. Furthermore, anti-rabbit biotinylated secondary antibody and streptavidin HRP incubation was done for 20–30 min, respectively. 3,3́- 3,3′-Diaminobenzidine (DAB) method was used to visualize antibody binding sites. Also, Hematoxylin counterstaining was carried out. For positive control, normal breast tissue was considered, while for the negative control, the primary antibody was skipped following the same protocol resulting in no staining. Interpretation of the staining was carried out under the guidance expert histopathologists using light microscope (magnification ×400), and the grading was done as follows: [1] 0% tumor staining with no expression, [2] 1%–10% tumor staining with mild expression (+), [3] 10%–50% tumor staining denoting moderate expression (++) [4] >50% tumor staining indicating high expression (+++ or ++++).

SPSS-IBM (version 22.0) was utilized to find the relevant association with the clinicopathological parameters. The current study data are represented as mean ± standard error (SE). the p-value of less than 0.005 was considered significant. To evaluate the significance of differential FOXO1 mRNA expression levels, a non-parametric test, i.e., Wilcoxon signed-rank test was used.

The expression of FOXO1 at the mRNA level was detected in case of breast cancer and adjacent normal tissue. Its expression was normalized against beta-actin expression. FOXO1 mRNA expression was found to be downregulated in 69.29% cases (88/127) and out of which 73.80% cases (65/88) were categorized under histological grade I and II of breast cancer. The fold change of 88 downregulated cases was examined to be 5.16 the expression of FOXO1 in breast cancer tissue was 1.16 ± 0.02 (Mean ± SE) and in the normal tissue was 1.95 ± 0.07 (Mean ± SE) (p < 0.0001). Correlating the FOXO1 mRNA expression with different clinic pathological parameters of patients indicated significant association with Age, Age at first live birth, Tumor Size, Lymph Node Status (Table 1, Table 2, Figure 2).

Promoter methylation study of FOXO1 promoter region was done through Methylation Specific PCR, the hypermethylated promoter region of FOXO1 was found in 61.41% (78/127) cases. The correlation of promoter methylation with clinical parameters revealed a significant association with the menopausal status of breast cancer patients. In progressive stages III and IV of breast cancer, 58/91 cases were found to be methylated (Figure 3; Table 3.)

FOXO1 expression analysis at the protein level was done by IHC and it was found to be absent in 77.95% (99/127) cases. However, 28 Cases exhibited the moderate or high expression of FOXO1 protein. Also, the FOXO1 protein expression pattern substantiated the mRNA expression. Furthermore, the percentage of FOXO1 protein downregulation was significant with Her2 neu status, tumor size, and histological grade of breast cancer (Figure 4; Table 4.)

The results represented a strong correlation of FOXO1 protein expression with the promoter methylation and 74 out of 78 hypermethylated cases showed low or no protein expression and 04 cases showed protein expression. In 51.02% (25/49) cases that showed no methylation had no protein expression. The cases which had downregulation of FOXO1 showed 78.40% (69/88) hypermethylation while 23.07% (9/39) cases had moderate to high-level protein expression. highly significant p-value (p= 0.0001) was found between FOXO1 methylation in the promoter region and protein expression, which represented a strong correlation Tables 5,6,7