Paternal obesity and epigenetic inheritance of breast cancer: The role of systemic effects and transmission to the second generation

Abstract

21Background: While genetics explains some familial breast cancer cases, we showed that 22 environmentally-induced epigenetic inheritance of breast cancer can also occur in rodent 23 models. We previously reported that paternal consumption of a high-fat diet and ensuing obesity 24 increased breast cancer susceptibility in the offspring (F1). Nevertheless, it is still unclear 25 whether paternal-induced programming of breast cancer in daughters is associated with systemic 26 alterations or mammary epithelium-specific factors. It also remains to be determined whether 27 the ancestrally programmed breast cancer predisposition in F1 progeny can be transmitted to 28 subsequent generations. 29Methods: Male mice (F0) were fed either a control (CO) diet or an obesity-inducing diet (OID) 30 for seven weeks and then mated with female mice (F0) reared on a CO diet. The resulting 31 offspring (F1), also exclusively fed CO diet, were either used for mammary gland and tumor 32 transplantation surgeries or to generate the F2 generation. To induce the mammary tumors, 33 female mice were treated with 7,12 dimethylbenz[a]anthracene (DMBA). Total RNA extracted 34 from F0 or F1 males sperm was used for small RNA-Seq analysis. 35Results: Mammary glands from F1 CO female offspring exhibited enhanced development when 36 transplanted into OID females [OID(CO-MG)], as shown by higher mammary gland area, 37 epithelial branching and elongation, compared to CO females that received a CO mammary 38 gland [CO(CO-MG)]. Similarly, mammary tumors from F1 CO female offspring transplanted 39 into OID females [OID(CO.T)] displayed improved growth with a higher proliferation/apoptosis 40 rate. We also found that granddaughters (F2) from the OID grand-paternal germline showed 41 accelerated tumor growth compared to COxCO granddaughters (F2). Transmission of breast 42 cancer predisposition to the F2 generation through OID male germline was associated with 43 alterations in specific sperm tRNA fragments (tRF) in both F0 and F1 males. 44 Conclusions:Our findings indicate that systemic metabolic and mammary stromal alterations 45 are the most significant contributors to paternal programming of mammary gland development 46 and cancer predisposition in female offspring rather than mammary epithelium confined factors. 47Our data also show breast cancer predisposition in OID daughters can be transmitted to 48 subsequent generations and could explain some familial cancers, if confirmed in humans. 49 50 Insulin tolerance test (ITT) was performed after the mice fasted for 6 h, according to the method 113 described by Takada et al [21]. The insulin load (75 mU/100 g body weight) was injected as a 114 bolus, and the blood glucose levels were determined at 0, 3, 6, 9, 12, and 30 minutes after 115 injection in female offspring. The area under the curve (AUC) was calculated according to the 116 trapezoid rule. Differences in ITT were analyzed using two-way ANOVA (group, time), 117 followed by post-hoc analyses.