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

<div><p>Background</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p></div

Relative expression profiling of Rbl2/p130 gene in different age groups and tumor stages.

<p>(<b>A)</b> Changes in relative Rbl2/p130 expression (ΔCT values) with age, both in normal and tumor tissues; (<b>B)</b> Observed changes in Rbl2/p130 in tumor and control tissues; (<b>C)</b> Differences in relative expression of Rbl2/p130 transcript as observed among control and different disease stages (<b>D)</b> Showing Rbl2 expression in control and disease samples, in both the study cohort (<45 and ≥45 years).</p

Map of Rbl2/p130 promoter showing methylated and un-methylated CpG’s cytosine ranging from −10 to +80 with reference to TSS (transcription start site).

<p>Map of Rbl2/p130 promoter showing methylated and un-methylated CpG’s cytosine ranging from −10 to +80 with reference to TSS (transcription start site).</p

Rbl2/p130 promoter methylation and transcript expression analysis among various histological types of breast cancer.

<p><b>(A)</b> Relative expression of Rbl2/p130 in control and tumor tissues among various histological types of breast cancer. <b>(B)</b> Change in promoter methylation (ΔMeth) status in control and diseased tissues among different histological different types of breast cancer. <b>DCI;</b> ductal carcinoma in situ. <b>IDC;</b> invasive ductal carcinoma. <b>ILC;</b> invasive lobular carcinoma.</p

Mean comparison of Rbl2/p130 promoter methylation status using tukey test.

<p>Mean comparison of Rbl2/p130 promoter methylation status using tukey test.</p

Statistical relation of Rbl2/p130 expression and its promoter methylation.

<p>(<b>A)</b> Differences in Rbl2/p130 promoter methylation status of tumor and normal tissues in study cohort I (<45 years) and study cohort II (≥45 years); (<b>B)</b> Observed differences in Rbl2/p130 promoter methylation status of various tumor tissues at different disease stages; (<b>C & D)</b> Correlation of relative expression (ΔCT values) and promoter methylation status as observed. Unmeth (C panel) and Meth (D panel) values are plotted separately. Adjusted R-values are shown in respective panels.</p

Promoter methylation analysis of Rbl2/p130 gene in normal individuals and breast cancer patients.

<p><b>(A)</b> 2% agarose gel showing amplified products of methyl specific PCR (MSP). “M” represents methylation, “UM” represents un-methylation and L represents DNA size ladder. (<b>B)</b> Upper panel shows methylation of CpG “C” in tumors, whereas lower panel highlights un-methylation in control individuals. <u><b>T</b></u> shows position of non CpG “C” converted into <b>T</b> as a results of bisulfite conversion while arrows indicates the position of methylated cytosine at CpGs sites.</p