Over the past decades, the DOHaD concept has provided causal relationships between early-life exposure to environmental risk factors and the developmental origins of non-communicable chronic diseases, including PCa [7, 21, 22]. Although the molecular mechanisms involved in this process remain elusive, the imbalance of sex hormones in both dams and offspring submitted to maternal malnutrition has been pointed to as a key factor related to prostate carcinogenesis in the progeny [6, 9, 10, 21, 22].
To further elucidate how maternal malnutrition interferes with the balance of sex hormones in male offspring, we evaluated the serum cholesterol profile and their intermediate metabolites involved in the steroidogenesis pathways. An increase in total cholesterol was observed in GLLP animais at PND 21. Sohi et al. (2011) have demonstrated an increase in cholesterol levels in male rat offspring submitted to MPR (8% protein) at the same age and associated this result with permanent epigenetic silencing of the hepatic Cyp7a1 promoter, which metabolizes cholesterol to bile acids . The offspring from the GLLP group also showed an imbalaced in plasma concentrations of steroid hormones; while testosterone and estradial were elevated, pregnenolone, progesterone, and DHEA were reduced, suggesting that these intermediate metabolites could be consumed to sustain the higher levels of testosterone and estradiol.
Although largely driven by androgens, the developing prostate is sensitive to estrogens. Epidemiological and experimental data have demonstrated that abnormal exposure to an excessive estrogenization early in life may disrupt molecular mechanisms governing prostate developmental biology, with long-lasting consequences for prostate diseases with aging [9, 21, 22, 50]. As such, African-American men are at higher risk of prostatic carcinoma than their Caucasian counterparts, and it has been postulated that this higher risk is related, in part, to the higher estrogens levels observed during pregnancy in the former population [9, 10]. These conditions, termed "developmental estrogenization" or "estrogen imprinting", states that inappropriate exposure to estrogen (due to ethnicity, exposure to the endocrine disruptor, environmental chemicals, maternal malnutrition, among other individual and environmental factors) is memorized ("imprinted") by cells and tissues [12, 13, 51–56]. Understanding the relationship between exposure to an estrogenic environment with long-lasting consequences for prostate diseases is essential for the DOHaD concept. It has been demonstrated in rodent models that exposure to high levels of estrogens early in life blocks epithelial cell differentiation and increases the number of basal epithelial cells [15, 57]. Using proteomic approaches, Santos et al., 2020 identified estrogenic signaling pathways, endoplasmic reticulum functions, oxidative stress, and deregulation of insulin/IGF signaling pathways as the main deregulated mechanisms involved in the VP response to maternal malnutrition in both young and older male offspring . We further demonstrated that the impairment of the glandular compartment in young rats was associated with an increase in collagen fiber deposition in the stroma. The increase in collagen content can be explained, at least in part, by the reduction of the gelatinolytic activity of the active form of the MMP-2, a class of enzyme responsible for extracellular matrix remodeling in the prostate gland [27, 58, 59].
To give further translational insights into the relationship between deregulated miRNAs networks early in life and the developmental origins of PCa, we identified 5 miRNAs with similar nucleotide sequences commonly deregulated in the VP of maternally malnourished offspring and patients with PCa. Of these, miR-206 was selected for further functional validation. RT-qPCR confirmed the upregulation of this miRNA in the VP of young offspring. Accordingly, experimental upregulation of miR-206 led to retardation of mammary gland development through modulation of Wnt and transcription factors Tbx3 and Lef1 . The upregulation of miR-206 in the GLLP group can be associated with stromal collagen accumulation in the impaired VP of young rats since the miR-206/Plg network, which modulates the extracellular matrix remodeling and angiogenesis processes [61, 62], was downregulated in these animals.
It is noteworthy that the prostate is particularly more sensitive to estrogens during critical windows of development . Estrogen actions on the prostate are mainly mediated through major canonical estrogen receptors (ER), including ERα and ERβ. Studies involving knockout mice and developmental exposure to estrogenic compounds indicate that paracrine signaling involving stromal ERα is the dominant form of estrogen-mediated imprinting in the developing uterus , ovary , mammary gland , lung , and prostate [51, 66, 67]. Consistently, the deletion of the ERα gene in the stromal fibroblast leads to a reduction in prostate branching morphogenesis . Additionally, Lee et al., (2013) demonstrated that miR-206 epigenetically regulates downregulated ESR1 expression during normal mammary gland development . Although there is no data regarding the Esr1 modulation by miR-206 in the developing prostate, our data demonstrated the potential relationship of miR-206 in modulating ER signaling since miR-206-ERs networks have opposite expression profiles in the VP of young offspring in the GLLP group.
In addition to the miR-206 potential role as an important modulator of gene expression during the developmental process, a body of evidence highlighted its key role as a tumor suppressor miRNA in PCa [35, 36, 64]. However, there is no data regarding the epigenetic modulation of ERs by miR-206 in PCa. Studies with knockout mice for estrogen receptors indicate that Esr1 and Esr2 can have opposite actions on prostate carcinogenesis since Esr1 is recognized to promote cell proliferation, while Esr2 has been described as anti-proliferative and proapoptotic. Altogether, these results suggest that the overall proliferative response to estrogen results from a balance between ERα and ERβ signaling [68–70]. Our functional validation experiments demonstrated downregulation of ESR1 expression in PNT2 cells transfected with miR-206. These results are consistent with deregulation of the miR-206-ESR1 network observed in the VP of young and older offspring rats from the GLLP group. Moreover, it has been demonstrated that in vitro inhibition of miR-206 in PC-3 cells increases cell invasion through upregulated ANXA2 and E-cadherin, and downregulation of N-cadherin and vimentin . Wang et al., (2018) also described the tumor suppressor effect of miR-206 in PCa by negatively regulating cell proliferation and migration by targeting CXCL11 . These authors also demonstrated experimentally that up-regulation of miR-206 inhibits proliferation, migration, invasion, and induced G1/G0 arrest of PCa cells. The expression profile of miRNA extracted from PRAD-TCGA demonstrated the downregulation of miR-206 in patients with PCa. Interestingly, we observed a reduction in the expression profile of miR-206 and an increase in Plg gene expression in the VP of older offspring rats from the GLLP groups, which developed prostate carcinoma in situ [22–24]. Functional validation confirmed the modulation negative correlation of the miR-206/PLG expression, as well as their participation in modulating cell migration and invasion in PNT2 cells. Furthermore, we provide new evidence on the participation of miR206-ESR1 network mediating prostate carcinogenesis in maternally malnourished offspring rats. Overall, our data provide new insights into the participation of an estrogenized environment associated with deregulation of miR-206-PLG-ESR1 in the developmental origin of prostate diseases in maternally malnourished offspring rats. Understanding these mechanisms can encourage the adoption of a governmental policy for the early life prevention of non-communicable chronic diseases, as proposed by the DOHaD concept.