Promoter Methylation Status Modulate the Expression of Tumor Suppressor (RbL2/p130) Gene in Breast Cancer

Background Aberrant expression of tumor suppressor genes may correspond to the abnormal cell development and tumorigenesis. Rbl2/p130, a member of retinoblastoma family of proteins, has growth suppressive properties. Numerous studies reported de-regulation of Rbl2/p130 in various types of cancer as a consequence of a number of genetic alterations. However, role of epigenetic mechanisms like DNA methylation in Rbl2/p130 expression remains elusive. Methods In the current study, 76 breast cancer tumors along with normal tissues (n = 76), blood (n = 76) of respective individuals and control blood (n = 50) were analyzed. Rbl2/p130 expression was analyzed by quantitative real time PCR (syber green method). Promoter methylation status was studied through methylation specific PCR of bisulfite converted genomic DNA. Data was analyzed using various statistical tests. Results We report significantly reduced Rbl2/p130 expression (P = 0.001) in tumors tissues as compared to control samples. Similarly, Rbl2/p130 expression varies with age and disease stages (P = 0.022), which suggest its involvement in tumor progression. Aberrant promoter methylation (Δmeth) was found in almost all the diseased samples and that was significantly different (P<0.001) with control samples. Similarly, methylation status varies significantly with tumor progression stages (P = 0.022). Hyper-methylation was observed at -1, +3, +15 and +75 of Rbl2/p130 promoter flanking around the TSS. Statistical analysis revealed that Rbl2/p130 expression negatively correlates to its promoter methylation (r = -0.412) in tumor tissues. Our results reflect an epigenetic regulation of Rbl2/p130 expression in breast cancer. This highlights the importance of Rbl2/p130 promoter methylation in breast cancer pathogenesis.


Identification of Patients and Tissue Samples Collection
Fresh tissue (n = 76) and their adjacent normal control tissues (ANCT) (n = 76) along with blood sample (n = 76) from the same patients were collected from local hospitals at the time of surgery. ANCT control samples were selected microscopically, with uninvolved areas more than 2 cm away from the tumor areas based on clinical observations made by oncologist. Tissue samples were collected and preserved in RNA Later solution to avoid RNA degradation, whereas blood samples were collected in EDTA tubes. Moreover blood samples from healthy individuals (n = 50) were also collected. The inclusion criterion for the control samples was the absence of a prior history of cancer or a pre-cancerous lesion. Patients and controls suffering from familial diseases (i.e., diabetes, blood pressure and cardiovascular impairment) were excluded from this study. Samples were collected from the patients after signing the informed consent during their clinical visits immediately after surgery, from Holy Family Hospital Rawalpindi, Pakistan Institute of Medical Sciences Islamabad, Leady Reading Hospital Peshawar and Khyber Teaching Hospital Peshawar. Tumor tissues and adjacent normal tissue were confirmed by an oncologist. All the samples were stored at -20°C until further analysis. This study was conducted with prior approval from ethical committees of both CIIT and collaborating hospitals. Patient were categorized into two age groups, first cohort comprised of patients below the age of 45(<45) years, and the 2 nd cohort included patients with age of 45 or greater than 45 years ( 45).
The current study was conducted on 76 breast cancer patients including tumors their normal control, and blood of respective individuals. Majority of the patients belonged to Punjab and Khyber-Pakhtunkhwa province. Patient's data was evaluated on different parameters e.g. age, tumor staging and grade.~43% of the patients belonged to 1 st study cohort that was below the age of 45(<45) years, whereas~57% of the patients belong to 2 nd cohort (of or greater than the age of 45 years; 45). Among them~64% cases were premenopausal and the remaining were postmenopausal. Other demographic and clinical characteristics of the cancer patients included in this cohort are mentioned in the Table 1.

RNA Extraction and Real Time PCR Quantification
RNA extraction from tissues (tumor + ANCT) was carried out using standard Trizol method [29] with slight modifications as per requirement. RNA was stored and at -80°C until further analysis. Reverse transcription polymerase chain reaction (RT-PCR) was carried out using super script first-strand cDNA synthesis kit (Invitrogen, USA). Gene specific primers for Rbl2/ p130 (Left primer; AGAGGATGCTGAGGAGGAAA, Right primer; CAATAGCCTGGGTTG GATCT) and β-actin (internal control) (Left primer; CACTCTTCCAGCCTTCCTTC, Right primer; TGATCTCCTTCTGCATCGTG) were used for quantitative real time PCR analysis using Syber dye base approach. qPCR was performed on Step One Plus Real-Time PCR system (Applied Biosystems). Thermo-cycler conditions were 95°C for 10 min (initial denaturation) followed by 40 cycles of 95°C for 15 seconds, 54°C for 60 seconds and 72°C for 20 seconds with a final extension of 72°C for 1 min and the results were interpreted using their delta delta ct values.

DNA Extraction and Bisulphite Modification
DNA extraction from tissue (tumor and ANCT) as well as blood samples were performed separately using standard phenol-chloroform method [30] and stored at -20°C for further use. 200-500 ng of genomic DNA was used for bisulphite conversion of un-methylated cytosine`C`into thymine`T`using commercially available EpiJET bisulfite conversion kit (Cat 6 ¼ K 1461) as per manufacturer's instructions.

Primer design for Methylation Analysis
A set of DNA methylation independent primers (Left Primer; GGTGGGGAGGTACGTGTTT A, Right Primer; GCCACAGTCCTGATCCTAGT) were designed for~1.2 kb fragment (NCBI,53434268-53435426) flanking around Rbl2/p130 TSS (transcription start site) using primer blast online tool [31]. These primers were designed from a region where CpG's contents were less. Moreover, for detection of DNA methylation status a pair of methylation/un-methylation specific primers (Left Meth; TTTGAATCGTCGGTTTGGATTAC, Right meth; CT CGAAACGACTAAAAACCTCGAA, and Left Un-Meth; TTTTTTTGAATTGTTGGTTT GGATTATG, Right Un-Meth; TTAACCCTCAAAACAACTAAAAACCTCA) around TSS of Rbl2/p130 was designed using MethPrimer online tool [32].

Methylation Specific PCR (MSP) and Sequencing
The targeted promoter region around Rbl2/p130 TSS was first amplified from bisulphite converted genomic DNA using methylation independent primers. Bisulphite converted genomic DNA (~100 ng) was amplified using following cycle conditions; 98°C for 30 sec (initial denaturation) followed by 35 cycles at 98°C for 10 sec, 62°C for 30 sec and 72°C for 30 sec; and a final extension at 72°C for 7 minutes. The amplified product (1 μl,~100 ng) was used as a template in methylation-specific PCR (MSP) reaction. MSP was performed to detect promoter methylation status of Rbl2/p130 using a set of each methylation and un-methylation specific primers. Maxima Hot Start Taq DNA polymerase (Thermo Scientific Cat 6 ¼ EP0601) was used to amplify MSP primers through given cycle conditions; 98°C for 3 min (initial denaturation) followed by 35 cycles at 98°C for 45 sec, 64°C for 40 sec and 72°C for 45 sec; and a final extension at 72°C for 7 minutes. CpG methylated human genomic DNA (Cat 6 ¼ SD1131) from Thermo scientific was used as positive control for methylation reaction, whereas PCR water was used as negative controls for methyl specific reaction. For un-methylated-specific PCR unconverted DNA (without bisulphite treatment) was used as positive control. Moreover, amplified MSP products were sequenced by Macrogen (Korea) and the results were analyzed using BioEdit and ClastalW alignment tools.

Gel Electrophoresis and Quantification
The PCR products from the above reactions were visualized on 2% agarose gel under UV illuminator (BioDoc Analyze, Biometra) and the relative band intensities were quantitated using densitometer to compare the degree of methylation and un-methylation quantitatively. Moreover, for the sake of convenience change in methylation status (Δmeth) was calculated by subtracting unmethylation values of a particular sample from its methylation values considering unmethylation as default condition.

Data Analysis
Statistical analysis were performed with OriginPro 2015 (OriginLab, Northampton, MA). For analysis transcript profiles, expression data of the target gene (Rbl2/p130) was normalized against internal control gene (β-actin). The correlation among different factors was assessed by Pearson Correlation Coefficient test. Depending on experiment, the statistical significance was determined on 95% confidence intervals (CIs) using the Mann-Whitney test, an analysis of variance (ANOVA) and specific comparisons were made by Tukey's honest significant difference (HSD) test. The values of P<0.05 were considered as significant. Statistical analysis enabled to investigate the correlation of methylation frequency and expression level of RbL2/p130 gene.

Rbl2/p130 Expression is Significantly Lower in Tumor Tissues
Using quantitative real-time RT-PCR, gene expression levels of Rbl2/p130 were determined in breast tumors and adjacent control tissues. The relative expression of Rbl2/p130 in breast tumors was found to be significantly lower (P = 0.001) than control samples in all age groups ( Table 2 and Fig 1). This highlights the significance of Rbl2/p130 expression as an early prognostic tool for cancer pathogenesis. Moreover, levels of Rbl2/p130 were also found lower (P = 0.022) in stage or grade specific manner (Table B in S1 File). Patients with advance tumor stages (metastatic) had statistically significant low expression than patients with relatively early stages of tumor (Table 2 and Table A in S1 File). This observation suggests its involvement in tumor progression. Our results also suggest a decrease in Rbl2/p130 expression with growing age, but these differences are more pronounced in cancer patients ( Fig 1A) where Rbl2/p130 expression values were more dispersed at older age. Similarly, the two-study cohort constituted on the basis of age (i.e. <45 and 45 years) also showed significant differences in Rbl2/p130 expression. In both age groups reduced Rbl2/p130 expression was recorded compared to their controls ( Fig 1D; P = 0.001), although the decrease in expression was drastically low in patients below the age of 45 compared to their controls.

Aberrant Promoter Methylation Around Rbl2/p130 TSS in Tumor Patients
Promoter methylation status in breast tumors, adjacent normal tissues and blood from the same patients along with control blood samples were analyzed to correlate the effect of methylation with down regulation of RbL2/p130 gene in breast cancer patients (Fig 2). Total 80 CpG sites in upstream regulatory sequences (promoter) of RbL2/p130 (NCBI "53434362-53434967"), were targeted by methylation specific PCR. Methylation levels were found higher than un-methylation in cancer patients (Fig 3), as is also supported by gel quantitation results to measure relative band intensities (Table 3 and Fig 4A).
Hyper methylation was observed in region (-10) to (+ 80) around transcription start site (TSS) of Rbl2/p130 gene. In general the CpG positions at (-1), (+3), (+15) and (+75) were found hyper-methylated in all samples. However the CpG positions (-8), (+9), (+21), (+28), (+47) and (+52) were found methylated in > 50% patients. In contrast, the CpG positions at (+12), (+35), (+39), (+43), (+55) and (+69) were observed un-methylated in all of the recruited patients. However in control (CpG methylated commercial Human DNA) reference sample all CpG site were found methylated running through targeted promoter region (Figs 4B and 5). Promoter methylation pattern was studied among different age and disease status individuals. Patients above the age of 45 years showed increased methylation level and decreased un-methylation status when compared with second study cohort i.e. participants <45 years of age ( Fig 3A). The patients having <45 years ages showed higher un-methylation in~76% of cases, while~24% of the cases was methylated. In contrast the patients having 45 years ages were carrying higher levels of methylation (~74%) and lower level (~26%) of un-methylated promoter status. Moreover, Δmeth values were found to be significantly different (P = 0.019) in study cohort 1 i.e. <45 years (Tables 2 and 3). Similar observation was made for promoter methylation status for pre-and post-menopausal stages. In pre-menopausal stage~35% cases showed higher methylation as compared to un-methylation (~65% cases), while patients with post-menopausal stage have higher methylation (in~85% cases) compared to un-methylation (~15%) ( Table 3). Interestingly, levels of methylation were found to be increasing with advancing tumor stages, while their un-methylation status was decreasing (Fig 3B). Patients with disease stage I/ II had lower methylation (~42% cases) compared to patients at advanced disease stage III, (~86%), while 100% methylation were observed patients at stage IV. Similarly, the high-grade tumors (poor differentiated) had higher methylation (~86%) status compared to moderate (~79%) and well-differentiated (~18%) tumor (Table 3). Among different histological types, patients with Ductal Carcinoma In Situ (DCI) show higher methylation frequency (75%) followed by Invasive Ductal Carcinoma (IDC~55.36%) and Invasive Lobular Carcinoma (ILC 37.5%) compared to un-methylation status as shown in Table 3. Statistical analysis revealed a significant difference among means (Δ meth) of disease and their adjacent normal control tissues in all histological tumor types except for DCI (Table 2 and Fig 6).

Expression of Rbl2/p130 Negatively Correlates to Its Promoter Methylation
Methylation of cytosine at CpG sites around upstream regulatory (promoter) region of a gene may correspond to gene expression. It has been observed that the degree of methylation have significantly negative effect on expression level of Rbl2/p130 gene. Statistical analysis reveals a strong negative correlation between meth and un-meth levels with Rbl2 expression (r = -0.412, and r = -0.403 respectively) in breast cancer patients, whereas no significant correlation was found among Rbl2 expression and promoter methylation in control samples (Table 4). This reflects involvement of promoter methylation in regulation of Rbl2/p130 expression. In addition to that effect of aging on Rbl2 methylation status was also observed. Breast cancer patients showed strong negative correlation with meth and un-meth levels (~r = -0.579), while control patients showed a weak negative correlation with age (Table 4 and Fig 3).

Discussion
Cytogenetically, p130 maps to human chromosomal area 16q12.2, an area repeatedly altered in human cancers [3,5,33,34]. The Rbl2/p130 promoter shows a characteristic structural organization of "housekeeping" and growth control-related genes; a typical TATA or CAAT box is missing, but several GC rich zones and potential binding sites for numerous transcription factors are present [35].
Rbl2/p130 has been shown to block the cell cycle, whereas pRb overexpression sometimes fails to do so. Numerous evidences favor the role of Rbl2/p130 as a tumor suppressor protein.
Rbl2/p130 down-regulates the cell cycle and it has also been shown to block the cell cycle [9]. Rbl2/p130 have been shown to inversely correlate with cancer malignancies by immune-histochemical analysis in endometrial carcinoma, oral squamous carcinoma and in uveal melanoma [5,9,36].
Our findings corroborate with these reports and tells another aspect of the same story; significant decrease (P = 0.001) in Rbl2/p130 expression in breast tumors tissues irrespective of  Promoter Methylation and Expression of Rbl2/P130 Gene patient's age. This suggests that loss of Rbl2 expression is a frequent phenomenon in breast cancer pathogenesis. Furthermore Rbl2/p130 expression was drastically reduced to~4 times in patients below the age of 45, although the down regulation in Rbl2/p130 expression was also Promoter Methylation and Expression of Rbl2/P130 Gene present in patients above the age of 45 years (Fig 1D). Though our findings are in contradiction with an earlier study that reported decrease in Rbl2 expression in older patients than younger ones, the differences in Rbl2 expression among different ages groups in their study were statistically non-significant [37]. Moreover, their definition of younger age doesn't correspond to ours, as they considered patients below 68 years as younger. Nevertheless, decrease in Rbl2/ p130 expression seems to be an important event in tumor initiation among patients below the age of 45 years. Similarly our results also support previous assumption regarding loss of RbL2/ p130 expression in age specific manner [15,38]. Furthermore the frequency of Rbl2/p130 expression was significantly decreased with increasing tumor stage and grade (Tables 2 and 4 and Table A in S1 File). This highlights not only the significance of Rbl2/p130 expression as an early prognostic tool for breast cancer pathogenesis but also suggests its involvement in tumor progression. Loss of Rb2/p130 expression have been reported to be an important parameter for risk assessment of various cancers including non-small cell lung cancer progression [39,40], endometrial and mammary carcinomas [37], ovarian cancers [15,16] in their initial stages. Several reports, not only discuss the loss of Rbl2/p130 in various types of cancer; but also give an insight on genetic alterations leading to silencing of Rbl2/p130 but epigenetic mechanisms involved in Rbl2/p130 silencing remains elusive. Although Rbl2/p130 has been shown to play role in ER-α promoter methylation by recruiting DNMTI at ER-α promoter [41], which might be an important event in deregulated expression of ER-α in various types of cancer, no comprehensive data is available on Rbl2/p130 promoter methylation itself. Here we report that aberrant promoter methylation at CpG sites around TSS of Rbl2/p130 might play a vital role in regulating its expression. DNA methylation is an important epigenetic phenomenon, responsible for enhanced expression of proto-oncogenes as well as silencing of tumor suppressor genes leading to abnormal proliferation and de-differentiation of cancer cells [28,42].
Hyper-methylation in the region -10 to + 80 around TSS of Rbl2/p130 in tumor sample was observed. Interestingly these hyper methylated samples showed reduced Rbl2/p130 expression which further may lead the disease to metastasize. Previously it was investigated that the region of the retinoblastoma gene flanking over +13 to +18 near TSS is crucial for DNA methylation and thereby gene silencing [28]. It has been reported that aberrant promoter methylation may Promoter Methylation and Expression of Rbl2/P130 Gene correspond to gene silencing and thus loss of function [43,44]. Usually promoter methylation occur more frequently at CpG site located 5'UTR of genes [44]. It has been reported that DNA methylation of tumor suppressor gene increases twofold in stage III and IV thereby causing gene silencing in human esophageal squamous cell carcinoma as well as in breast cancer [21]. Our results reflect a strong negative correlation (Pearson coefficient r = -0.412; Table 4) among methylation status and Rbl2/p130 expression in diseased tissues. Circumstantial evidence supports the idea that aberrant promoter methylation might be the causative agent for reduced Rbl2/p130 expression and tumor initiation, and is worth exploring phenomenon as a new prognostic tool for breast cancer pathogenesis. This hypothesis is also supported by the fact that CpG cytocines at -1 and +3 positions around TSS were found hyper-methylated in almost all the samples. Intact TSS is required for efficient binding of the transcription machinery. Methylation in or around TSS might perturb this binding leading to reduced expression of Rbl2/p130. Similarly, 5'UTR (+15-+50) of the target candidate gene were found to be heavily methylated, has been reported previously for retinoblastoma protein (pRb) [28]. UTR play important role in transcript stability and it has been reported that 5'UTR play pivotal role to develop pathogenesis [45]. Similarly post transcriptional regulation of mRNA may also correspond to aberrant gene expression. Methylation in the promoter regions might affect the expression and stability of Rbl2/p130. Numerous single nucleotide polymorphisms (SNPs) have already been reported at 16q 12.2 region >gi|568815582: 53434362-53434967 flanking over TSS of Rbl2/p130. Interestingly, a CpG C >T (SNP "rs144092904" NCBI) conversion at -1 position of TSS has been reported. This signifies the importance of methylation at this position. This conversion protects fraction of the population to undergo methylation at this very crucial position. However no data is available on disease susceptibility for population with this SNP. Based on these findings, screening of C >T conversion in normal individuals is an interesting domain to explore. Therefore it has been concluded from this genetic and epigenetic characterization, that promoter methylation status may prove to be one of the important prognostic factor for the etiology of breast cancer.

Ethical Standard
The study was conducted with a prior approval from the institutional ethical review board of COMSTAS Institute of Information