Deregulation of ABCG1 early in life contributes to prostate carcinogenesis in maternally malnourished offspring rats.

AIMS: The developmental Origins of Health and Disease (DOHaD) concept has provided the framework to assess how early life experiences can shape health and disease throughout the life course. Using a model of maternal exposure to a low protein diet (LPD; 6% protein) during the gestational and lactational periods, we demonstrated changes in the ventral prostate (VP) transcriptomic landscape in young rats exposed to maternal malnutrition. Male offspring Sprague Dawley rats were submitted to maternal malnutrition during gestation and lactation, and they were weighed, and distance anogenital was measured, followed were euthanized by an overdose of anesthesia at 21 postnatal days. Next, the blood and the ventral prostate (VP) were collected and processed by morphological analysis, biochemical and molecular analyses. RNA-seq analysis identified 411 differentially expressed genes (DEGs) in the VP of maternally malnourished offspring compared to the control group. The molecular pathways enriched by these DEGs are related to cellular development, differentiation, and tissue morphogenesis, all of them involved in both normal prostate development and carcinogenesis. Abcg1 was commonly deregulated in young and old maternally malnourished offspring rats, as well in rodent models of prostate cancer (PCa) and in PCa patients. Our results described ABCG1 as a potential DOHaD gene associated with perturbation of prostate developmental biology with long-lasting effects on carcinogenesis in old offspring rats. A better understanding of these mechanisms may help with the discussion of preventive strategies against early life origins of non-communicable chronic diseases.

Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats.

The Developmental Origins of Health and Disease (DOHaD) concept has provided the framework to assess how early life experiences can shape health and disease throughout the life course. While maternal malnutrition has been proposed as a risk factor for the developmental programming of prostate cancer (PCa), the molecular mechanisms remain poorly understood. Using RNA-seq data, we demonstrated deregulation of miR-206-Plasminogen (PLG) network in the ventral prostate (VP) of young maternally malnourished offspring. RT-qPCR confirmed the deregulation of the miR-206-PLG network in the VP of young and old offspring rats. Considering the key role of estrogenic signaling pathways in prostate carcinogenesis, in vitro miRNA mimic studies also revealed a negative correlation between miR-206 and estrogen receptor α (ESR1) expression in PNT2 cells. Together, we demonstrate that early life estrogenization associated with the deregulation of miR-206 networks can contribute to the developmental origins of PCa in maternally malnourished offspring. Understanding the molecular mechanisms by which early life malnutrition affects offspring health can encourage the adoption of a governmental policy for the prevention of non-communicable chronic diseases related to the DOHaD concept.

Increased oxidative stress and cancer biomarkers in the ventral prostate of older rats submitted to maternal malnutrition.

The concept of Developmental Origins of Health and Disease (DOHaD) states that exposure to malnutrition early in life increase the incidence of non-communicable chronic diseases throughout the lifespan. In this study, a reduction in serum testosterone and an increase in estrogen levels were shown in older rats born to protein malnourished dams (6% protein in the diet) during gestation and lactation. Intraprostatic levels of reduced glutathione were decreased, while tissue expression of glutathione S-transferase pi and sulfiredoxin-1 were increased in these animals. Strong immunostaining for alfametilacil CoA racemase (AMACR), vascular endothelial growth factor-A (VEGF-A), and aquaporin-1 (AQP1) was also observed. In silico analysis confirmed commonly deregulated proteins in the ventral prostate of old rats and patients with prostate cancer. In conclusion, the increase in oxidative stress associated with an imbalance of sex hormones may contribute to prostate carcinogenesis in offspring, highlighting early-life malnutrition as a key risk factor for this malignance.

Maternal Low-Protein Diet Deregulates DNA Repair and DNA Replication Pathways in Female Offspring Mammary Gland Leading to Increased Chemically Induced Rat Carcinogenesis in Adulthood.

Studies have shown that maternal malnutrition, especially a low-protein diet (LPD), plays a key role in the developmental mechanisms underlying mammary cancer programming in female offspring. However, the molecular pathways associated with this higher susceptibility are still poorly understood. Thus, this study investigated the adverse effects of gestational and lactational low protein intake on gene expression of key pathways involved in mammary tumor initiation after a single dose of N-methyl-N-nitrosourea (MNU) in female offspring rats. Pregnant Sprague-Dawley rats were fed a normal-protein diet (NPD) (17% protein) or LPD (6% protein) from gestational day 1 to postnatal day (PND) 21. After weaning (PND 21), female offspring (n = 5, each diet) were euthanized for histological analysis or received NPD (n = 56 each diet). At PND 28 or 35, female offspring received a single dose of MNU (25 mg/kg body weight) (n = 28 each diet/timepoint). After 24 h, some females (n = 10 each diet/timepoint) were euthanized for histological, immunohistochemical, and molecular analyses at PDN 29 or 36. The remaining animals (n = 18 each diet/timepoint) were euthanized when tumors reached ≥2 cm or at PND 250. Besides the mammary gland development delay observed in LPD 21 and 28 groups, the gene expression profile demonstrated that maternal LPD deregulated 21 genes related to DNA repair and DNA replication pathways in the mammary gland of LPD 35 group after MNU. We further confirmed an increased γ-H2AX (DNA damage biomarker) and in ER-α immunoreactivity in mammary epithelial cells in the LPD group at PND 36. Furthermore, these early postnatal events were followed by significantly higher mammary carcinogenesis susceptibility in offspring at adulthood. Thus, the results indicate that maternal LPD influenced the programming of chemically induced mammary carcinogenesis in female offspring through increase in DNA damage and deregulation of DNA repair and DNA replication pathways. Also, Cidea upregulation gene in the LPD 35 group may suggest that maternal LPD could deregulate genes possibly leading to increased risk of mammary cancer development and/or poor prognosis. These findings increase the body of evidence of early-transcriptional mammary gland changes influenced by maternal LPD, resulting in differential response to breast tumor initiation and susceptibility and may raise discussions about lifelong prevention of breast cancer risk.

Impact of gestational low protein diet and postnatal bisphenol A exposure on chemically induced mammary carcinogenesis in female offspring rats.

This study evaluated the effect of gestational low protein diet (LPD) and/or postnatal bisphenol A (BPA) exposure on mammary gland development and carcinogenesis in female offspring. Pregnant Sprague-Dawley rats were fed a normal protein diet (NPD, 17% protein) or LPD (6% protein). At weaning, female offspring were distributed in four groups (NPD, LPD, NPD + BPA, and LPD + BPA) and received vehicle or BPA in drinking water (0.1%), during postnatal day (PND) 21 to 51. On PND 51, some female offspring were euthanized or received a single dose of 7,12-dimethylbenzoanthracene (DMBA, 30 mg/kg, i.g.) and were euthanized on PND 250. On PND 51, neither gestational LPD nor postnatal BPA exposure, individually or in combination, significantly altered the development of mammary gland tree, mean number of terminal structures or estrogen receptor beta (ER-ß), proliferating cell nuclear antigen (PCNA) or caspase-3 protein expression in the mammary tissue. A significant reduction in mammary epithelial area (%) was observed in both LPD groups and a significant increase in ER-α protein expression was detected only in LPD group. In LPD + BPA group was observed a significant increase in both fat pad area (%) and in mean number of mammary epithelial cells positive for progesterone receptor (PR). On PND 250, the groups that received BPA presented lower latency and higher tumor incidence and tumor multiplicity and LPD + BPA group more aggressive tumors. These findings suggest that postnatal BPA exposure associated with gestational LPD is able to induce morphological changes in the mammary gland and increase susceptibility to mammary carcinogenesis.

Maternal Low-Protein Diet Impairs Prostate Growth in Young Rat Offspring and Induces Prostate Carcinogenesis With Aging.

Carcinogenesis is frequently linked to genetic background, however, exposure to environmental risk factors has gained attention as the etiologic agent for several types of cancer, including prostate. The intrauterine microenvironment has been described as a preponderant factor for offspring health; and maternal exposure to insult has been linked to chronic disease in older offspring. Using a model of maternal exposure to low-protein diet (LPD; 6% protein), we demonstrated that impairment of offspring rat prostatic growth on postnatal day (PND) 21 was associated with prostate carcinogenesis in older offspring (PND 540). One explanation is that maternal LPD consumption exposed offspring to an estrogenic intrauterine microenvironment, which potentially sensitized prostate cells early during glandular morphogenesis, increasing cellular response to estrogen in older rats. The onset of accelerated prostatic growth, observed on PND 21, associated with an unbalanced estrogen/testosterone ratio and increased circulating IGF-1 in older offspring appears to contribute to the development of prostate carcinoma in groups on gestational low protein and gestational and lactational low protein diets (33 and 50%, respectively). Our study strongly indicated maternal exposure to LPD as a potential risk factor for induction of slow-growing prostate carcinogenesis in rat offspring later in life.

Influence of postnatal prolactin modulation on the development and maturation of ventral prostate in young rats.

Besides androgenic dependence, other hormones also influence the prostate biology. Prolactin has been described as an important hormone associated with maintenance of prostatic morphophysiology; however, there is a lack of information on the involvement of prolactin during prostate development and growth. This study aimed to evaluate whether perinatal prolactin modulation interferes with rat ventral prostate (VP) development and maturation. Therefore, prolactin or bromocriptine (an inhibitor of prolactin release from the pituitary) were administered to Sprague Dawley rats from postnatal Day (PND) 12 to PND 21 or 35. Animals were then killed and serum hormonal quantification, VP morphological-stereological and immunohistochemical analyses and western blotting reactions were employed. Our results demonstrate that prolactin blockage increased serum testosterone on PND 21, which reflected an increase in anogenital distance. Although prolactin modulation did not interfere with VP weight, it modified VP morphology by dilating the acinar lumen and reducing epithelial cell height. Prolactin activated the signal transducer and activator of transcription (STAT) downstream pathway, increased androgen receptor expression and epithelial proliferation. In addition, prolactin and bromocriptine also increased expression of cytokeratin 18, a marker of luminal-differentiated cells. In conclusion, the VP responds to prolactin modulation through a mechanism of increasing the epithelial proliferative response and dynamics of cell differentiation, especially in animals treated for a more prolonged period.

The prostate response to prolactin modulation in adult castrated rats subjected to testosterone replacement.

Despite the androgenic dependence, other hormones, growth factors, and cytokines are necessary to support prostatic growth and maintain the glandular structure; among them, prolactin is a non-steroidal hormone secreted mainly by the pituitary gland. However, extra-pituitary expression of prolactin, such as in the prostate, has also been demonstrated, highlighting the paracrine and autocrine actions of prolactin within the prostate. Here, we investigated whether prolactin modulation alters ventral prostate (VP) morphophysiology in adult castrated rats. Sprague Dawley rats were castrated and after 21 days, divided into ten experimental groups (n = 6/group): castrated control: castrated animals that did not receive treatment; castrated+testosterone: castrated animals that received T (4 mg/kg/day); castrated+PRL (PRL): castrated animals receiving prolactin (0.3 mg/kg/day); castrated+T+PRL: castrated animals that received a combination of testosterone and prolactin; and castrated+bromocriptine (BR): castrated animals that received bromocriptine (0.4 mg/kg/day). The control group included intact animals. The animals were treated for 3 or 10 consecutive days. At the end of experimental period, the animals were euthanized, and the blood and VP lobes were collected and analyzed by different methods. The main findings were that the administration of prolactin to castrated rats did not exert anabolic effects on the VP. Although we observed activation of downstream prolactin signaling after prolactin administration, this was not enough to overcome the prostatic androgen deficiency. Likewise, there was no additional glandular involution in the castrated group treated with bromocriptine. We concluded that despite stimulating the downstream signaling pathway, exogenous prolactin does not act on VP in the absence or presence of high levels of testosterone.

Impairment of microvascular angiogenesis is associated with delay in prostatic development in rat offspring of maternal protein malnutrition.

Experimental data demonstrated the negative impact of maternal protein malnutrition (MPM) on rat prostate development, but the mechanism behind the impairment of prostate growth has not been well understood. Male Sprague Dawley rats, borned to dams fed a normal protein diet (CTR group, 17% protein diet), were compared with those borned from dams fed a low protein diet (6% protein diet) during gestation (GLP group) or gestation and lactation (GLLP). The ventral prostate lobes (VP) were removed at post-natal day (PND) 10 and 21, and analyzed via different methods. The main findings were low birth weight, a reduction in ano-genital distance (AGD, a testosterone-dependent parameter), and an impairment of prostate development. A delay in prostate morphogenesis was associated with a reduced testosterone levels and angiogenic process through downregulation of aquaporin-1 (AQP-1), insulin/IGF-1 axis and VEGF signaling pathway. Depletion of the microvascular network, which occurs in parallel to the impairment of proliferation and differentiation of the epithelial cells, affects the bidirectional flux between blood vessels impacting prostatic development. In conclusion, our data support the hypothesis that a reduction in microvascular angiogenesis, especially in the subepithelial compartment, is associated to the impairment of prostate morphogenesis in the offspring of MPM dams.

Streptozotocin-Induced Maternal Hyperglycemia Increases the Expression of Antioxidant Enzymes and Mast Cell Number in Offspring Rat Ventral Prostate.

Gestational diabetes mellitus (GDM) has increased in recent years. Although the cellular and molecular mechanisms involved in GDM-increased risk factors to offspring remained poorly understood, some studies suggested an association between an increase in oxidative stress induced by maternal hyperglycemia and complications for both mothers and newborns. Here, we investigated the impact of maternal hyperglycemia followed by maternal insulin replacement during lactation on the expression of antioxidant enzymes and mast cell number in offspring ventral prostate (VP) at puberty. Pregnant rats were divided into three groups: control (CT); streptozotocin-induced maternal hyperglycemia (MH); and MH plus maternal insulin replacement during lactation (MHI). Male offspring were euthanized at postnatal day (PND) 60 and the VP was removed and processed for histology and Western blotting analyses. Maternal hyperglycemia delayed prostate maturation, and increased mast cell number catalase (CAT), superoxide dismutase (SOD), glutatione-s-transferase (GST-pi), and cyclooxygenase-2 (Cox-2) expression in the offspring of hyperglycemic dams. Maternal insulin replacement restored VP structure, mast cell number and antioxidant protein expression, except for Cox-2, which remained higher in the MHI group. Thus, an increase in oxidative stress induced by intrauterine hyperglycemia impacts prostate development and maturation, which persists until puberty. The overall improvement of maternal metabolism after insulin administration contributes to the restoration of prostate antioxidant enzymes and secretory function. Taken together, our results highlighted that imbalanced physiological maternal-fetal interaction contributes to the impairment of reproductive performance of the offspring from diabetic mothers. Anat Rec, 300:291-299, 2017. © 2016 Wiley Periodicals, Inc.