Aerobic exercise and DNA methylation in postmenopausal women: An ancillary analysis of the Alberta Physical Activity and Breast Cancer Prevention (ALPHA) Trial

<div><p>Physical activity is associated with a lower risk of breast, colon, and endometrial cancer. Epigenetic mechanisms such as changes in DNA methylation may help to explain these protective effects. We assessed the impact of a one year aerobic exercise intervention on DNA methylation biomarkers believed to play a role in carcinogenesis. The Alberta Physical Activity and Breast Cancer Prevention (ALPHA) Trial was a two-armed randomized controlled trial in 320 healthy, inactive, postmenopausal women with no history of cancer. In an ancillary analysis, frozen blood samples (n = 256) were reassessed for levels of DNA methylation within LINE-1 and Alu repeats as well as within the promoter regions of <i>APC</i>, <i>BRCA1</i>, <i>RASSF1</i>, and <i>hTERT</i> genes. Differences between the exercise and control arm at 12-months, after adjusting for baseline values, were estimated within an intent-to-treat and per-protocol analysis using linear regression. No significant differences in DNA methylation between the exercise and control arms were observed. In an exploratory analysis, we found that the prospective change in estimated VO₂max was negatively associated with <i>RASSF1</i> methylation in a dose-response manner (p-trend = 0.04). A year-long aerobic exercise intervention does not affect LINE-1, Alu, <i>APC</i>, <i>BRCA1</i>, <i>RASSF1</i>, or <i>hTERT</i> methylation in healthy, inactive, postmenopausal women. Changes in DNA methylation within these genomic regions may not mediate the association between physical activity and cancer in healthy postmenopausal women. Additional research is needed to validate our findings with <i>RASSF1</i> methylation.</p><p><b>Trial Registration:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT00522262" target="_blank">NCT00522262</a>.</p></div

CONSORT diagram of ALPHA trial ancillary study.

<p>This Consolidated Standards of Reporting Trials (CONSORT) diagram describes the inclusion and exclusion of participants included in the current trial.</p

Intent-to-treat analysis comparing changes in the DNA methylation outcomes between the exercise and control groups.

<p>Intent-to-treat analysis comparing changes in the DNA methylation outcomes between the exercise and control groups.</p

Per-protocol analysis comparing changes in the DNA methylation outcomes between the exercise and control groups.

<p>Per-protocol analysis comparing changes in the DNA methylation outcomes between the exercise and control groups.</p

The association between the amount of exercise, weight loss, change in VO₂ max and DNA methylation during a year-long aerobic physical activity intervention.

<p>The association between the amount of exercise, weight loss, change in VO₂ max and DNA methylation during a year-long aerobic physical activity intervention.</p

Baseline characteristics of ALPHA trial participants included in intent-to-treat analysis.

<p>Baseline characteristics of ALPHA trial participants included in intent-to-treat analysis.</p

Conventional RT-PCR and direct cDNA sequencing confirm the presence of <i>EGFRvIII</i> in glioblastoma FFPE tissue.

<p>A. Conventional RT-PCR failed for GBM 11, GBM 23 and GBM 19. GBM 9 is positive for <i>EGFRvIII</i>. EGFRvIII positive control: U87MGvIII (384 bp), negative control: U87MG (1184 bp) and no template control (NTC): water. B. Sequencing of GBM 9 cDNA confirms the presence of <i>EGFRvIII</i>. C. Real-time amplification plot showing EGFRvIII-positive OSCC patient (13A) and the positive control (U87MGvIII).</p

Specific detection of <i>EGFRvIII</i> mRNA by real-time RT-PCR.

<p>A. Real-time amplification plot of <i>EGFRvIII</i> transcript as detected in the U87MGvIII cell-line. U87MGvIII cells were used as a positive control, U87MG cells were used as negative control: water was used for no template control. B. Direct sequencing of U87MG and U87MGvIII cDNA confirms the presence of <i>EGFRvIII</i> transcript in U87MGvIII cells only. The broken arrow indicates the <i>EGFRvIII</i>-specific exon 1 and exon 8 junction. C. Linear dynamic range of the <i>EGFRvIII</i> real-time RT-PCR assay (100, 10, 1, 0.1 and 0.01 ng cDNA). D. <i>EGFRvIII</i> real-time RT-PCR assay amplification efficiency.</p

Clinicopathological characteristics of OSCC patients evaluated for EGFR protein expression and <i>EGFRvIII</i> frequency.

<p>Clinicopathological characteristics of OSCC patients evaluated for EGFR protein expression and <i>EGFRvIII</i> frequency.</p

CONSORT Diagram outlining patient selection criteria and the different stages at which specific analyses were undertaken for the OSCC study cohort.

<p>CONSORT Diagram outlining patient selection criteria and the different stages at which specific analyses were undertaken for the OSCC study cohort.</p