DNA methylation changes in response to neoadjuvant chemotherapy are associated with breast cancer survival.

BACKGROUND: Locally advanced breast cancer is a heterogeneous disease with respect to response to neoadjuvant chemotherapy (NACT) and survival. It is currently not possible to accurately predict who will benefit from the specific types of NACT. DNA methylation is an epigenetic mechanism known to play an important role in regulating gene expression and may serve as a biomarker for treatment response and survival. We investigated the potential role of DNA methylation as a prognostic marker for long-term survival (> 5 years) after NACT in breast cancer. METHODS: DNA methylation profiles of pre-treatment (n = 55) and post-treatment (n = 75) biopsies from 83 women with locally advanced breast cancer were investigated using the Illumina HumanMethylation450 BeadChip. The patients received neoadjuvant treatment with epirubicin and/or paclitaxel. Linear mixed models were used to associate DNA methylation to treatment response and survival based on clinical response to NACT (partial response or stable disease) and 5-year survival, respectively. LASSO regression was performed to identify a risk score based on the statistically significant methylation sites and Kaplan-Meier curve analysis was used to estimate survival probabilities using ten years of survival follow-up data. The risk score developed in our discovery cohort was validated in an independent validation cohort consisting of paired pre-treatment and post-treatment biopsies from 85 women with locally advanced breast cancer. Patients included in the validation cohort were treated with either doxorubicin or 5-FU and mitomycin NACT. RESULTS: DNA methylation patterns changed from before to after NACT in 5-year survivors, while no significant changes were observed in non-survivors or related to treatment response. DNA methylation changes included an overall loss of methylation at CpG islands and gain of methylation in non-CpG islands, and these changes affected genes linked to transcription factor activity, cell adhesion and immune functions. A risk score was developed based on four methylation sites which successfully predicted long-term survival in our cohort (p = 0.0034) and in an independent validation cohort (p = 0.049). CONCLUSION: Our results demonstrate that DNA methylation patterns in breast tumors change in response to NACT. These changes in DNA methylation show potential as prognostic biomarkers for breast cancer survival.

Targeting choline phospholipid metabolism: GDPD5 and GDPD6 silencing decrease breast cancer cell proliferation, migration, and invasion.

Abnormal choline phospholipid metabolism is associated with oncogenesis and tumor progression. We have investigated the effects of targeting choline phospholipid metabolism by silencing two glycerophosphodiesterase genes, GDPD5 and GDPD6, using small interfering RNA (siRNA) in two breast cancer cell lines, MCF-7 and MDA-MB-231. Treatment with GDPD5 and GDPD6 siRNA resulted in significant increases in glycerophosphocholine (GPC) levels, and no change in the levels of phosphocholine or free choline, which further supports their role as GPC-specific regulators in breast cancer. The GPC levels were increased more than twofold during GDPD6 silencing, and marginally increased during GDPD5 silencing. DNA laddering was negative in both cell lines treated with GDPD5 and GDPD6 siRNA, indicating absence of apoptosis. Treatment with GDPD5 siRNA caused a decrease in cell viability in MCF-7 cells, while GDPD6 siRNA treatment had no effect on cell viability in either cell line. Decreased cell migration and invasion were observed in MDA-MB-231 cells treated with GDPD5 or GDPD6 siRNA, where a more pronounced reduction in cell migration and invasion was observed under GDPD5 siRNA treatment as compared with GDPD6 siRNA treatment. In conclusion, GDPD6 silencing increased the GPC levels in breast cancer cells more profoundly than GDPD5 silencing, while the effects of GDPD5 silencing on cell viability/proliferation, migration, and invasion were more severe than those of GDPD6 silencing. Our results suggest that silencing GDPD5 and GDPD6 alone or in combination may have potential as a new molecular targeting strategy for breast cancer treatment. Copyright © 2016 John Wiley & Sons, Ltd.