Nutritional ketosis modulates the methylation of cancer-related genes in patients with obesity and in breast cancer cells

Abstract

Scientific evidence demonstrates that a very low-calorie ketogenic diet (VLCKD) is effective and beneficial in the treatment of obesity, capable of reversing the methylome associated with obesity and has immunomodulatory capacity. This effect is in part promoted by nutritional ketosis and could be involved in counteracting obesity-related cancer. The aim of this study was to evaluate the effect of nutritional ketosis on the methylation of genes related to tumor processes in patients with obesity and in breast cancer cells. Based on methylome data (Infinium MethylationEPIC BeadChip, Illumina) from patients with obesity treated with a VLCKD for weight loss (n = 10; n = 5 women, age = 48.8 ± 9.20 years, BMI = 32.9 ± 1.4 kg/m2), genes belonging to cancer-related pathways were specifically evaluated and further validated in vitro in MDA-MB-231 (triple negative) and MCF7 (RE positive) breast tumor cells pretreated for 72 h with βOHB, the main ketone body, secretome from visceral (VATs) or subcutaneous (SATs) adipose tissue of patients with obesity. The cell tumoral phenotype was evaluated by proliferation assay and expression of cancer-related genes. VLCKD-induced nutritional ketosis promoted changes in the methylation of 18 genes (20 CpGs; 17 hypomethylated, 3 hypermethylated) belonged to cancer-related pathways with MAPK10, CCN1, CTNNA2, LAMC3 and GLI2 being the most representative genes. A similar pattern was observed in the MDA-MB-231 cells treated with β-OHB, without changes in MCF7. These epigenetic changes paralleled the tumoral phenotype modulated by the treatments. Taking together these results highlight the potential role of VLCKD as an adjuvant to anticancer treatment in groups more susceptible to the development of cancer such as patients with obesity, exerting epigenetic regulation through nutritional ketosis and weight loss.

Keywords: Adipose tissue; Breast cancer; DNMTs; Epigenetics; Ketogenic diet; Ketone bodies; Oncogenes; Sirtuins; Tumor suppressors.

Fig. 1

Analysis of cancer-related genes in methylome associated with a very low-calorie ketogenic diet (VLCKD). (A) Methylation changes in cancer-related genes induced by nutritional ketosis on a VLCKD. P-value was calculated by means of paired Student’s t-test respect to baseline. (B) Network enriched in protein interactions of cancer-related genes regulated by methylation. (C) Supervised clustering of the CpGs that were found to be differentially methylated between baseline and maximum ketosis with changes > 4%. MMP9, gene encoding matrix metalloproteinase 9; HRAS, gene encoding the H-Ras protein; CHUK, conserved helix-loop-helix ubiquitous kinase gene; ERBB2, gene encoding the receptor tyrosine kinase; LAMA2, gene encoding the laminin alpha 2 subunit; IGF1R, gene encoding the factor receptor insulin-like growth 1; CCND1, gene encoding the cyclin D1 protein; TGFB3, gene that encodes transforming growth factor beta 3; WNT2, WNT family member 2 gene; MAPK10, gene encoding mitogen-activated protein kinase 10; CRK, proto-oncogene CRK, adapter protein; LAMC1, gene encoding the gamma 1 subunit of laminin; CTNNA2, gene encoding alpha 2 catenin; LAMC3, gene encoding the gamma 3 subunit of laminin; WNT3, gene encoding WNT family member 3; HDAC1, gene encoding histone deacetylase 1; GLI2, glioma-associated oncogene 2; LAMB1, gene encoding laminin beta 1 subunit

Fig. 2

Gene expression levels of cell lines exposed to β-hydroxybutyrate (β-OHB). (A) MDA-MB-231 and (B) MCF7. Asterisk (*) denotes statistically significant differences (p < 0.05) compared to the control evaluated by Student’s t-test. BRCA1, breast cancer 1; PTEN, homolog of phosphatase and tensin; TP53, tumor protein 53; SIRT, sirtuin; GSTM2, glutathione S-transferase Mu 2; DNMT, DNA methyltransferase; BIRC5, baculoviral inhibitor of apoptosis repeat-containing 5; MYC, myelocytomatosis; ALDH3A1, aldehyde dehydrogenase 3A1

Fig. 3

Gene expression levels of cell lines exposed to visceral adipose tissue (VAT) secretome. (A) MDA-MB-231 and (B) MCF7. Asterisk (*) denotes statistically significant differences (p < 0.05) compared to the control evaluated by Student’s t-test. BRCA1, breast cancer 1; PTEN, homolog of phosphatase and tensin; TP53, tumor protein 53; SIRT, sirtuin; GSTM2, glutathione S-transferase Mu 2; DNMT, DNA methyltransferase; BIRC5, baculoviral inhibitor of apoptosis repeat-containing 5; MYC, myelocytomatosis; ALDH3A1, aldehyde dehydrogenase 3A1

Fig. 4

Gene expression levels of cell lines exposed to subcutaneous adipose tissue (SAT) secretome. (A) MDA-MB-231 and (B) MCF7 Asterisk (*) denotes statistically significant differences (p < 0.05) compared to the control evaluated by Student’s t-test. BRCA1, breast cancer 1; PTEN, homolog of phosphatase and tensin; TP53, tumor protein 53; SIRT, sirtuin; GSTM2, glutathione S-transferase Mu 2; DNMT, DNA methyltransferase; BIRC5, baculoviral inhibitor of apoptosis repeat-containing 5; MYC, myelocytomatosis; ALDH3A1, aldehyde dehydrogenase 3A1

Fig. 5

Methylation pattern in breast cancer cell lines of the cancer-related genes previously identified in patients with obesity treated with a VLCKD. (A) Methylation pattern induced by in vitro treatment with β-hydroxybutyrate (β-OHB) or SAT or VAT secretome in breast cancer cell lines. The data represent the changes in methylation β values in the treated cells compared to the untreated control cells. (B) Methylation levels of GLI2 gene in treated MDA-MB-231 and MCF7 tumor cells. The data are presented as the mean; error bars represent standard error. (*) Denotes differences statistically significant (p < 0.05) in relation to the control evaluated using the U Mann-Whitney test. CCND1, gene encoding the cyclin D1 protein; MAPK10, gene that encodes the protein mitogen-activated kinase 10; GLI2, glioma-associated oncogene 2; LAMC3, gene encoding the subunit laminin gamma 3; CTNNA2, gene that encodes alpha 2 catenin