# Prenatal alcohol exposure increases the aggressiveness of estrogen‐induced pituitary tumors in male rats

## TL;DR

Prenatal alcohol exposure makes pituitary tumors more aggressive in male rats, similar to what was found in females.

## Contribution

The study shows that prenatal alcohol exposure promotes aggressive pituitary tumors in male rats through stem cell-like changes.

## Key findings

- Prenatal alcohol-exposed male rats developed more aggressive pituitary tumors with elevated stem cell and aggressiveness markers.
- Tumor cells from alcohol-exposed males showed higher proliferation, invasiveness, and colony formation compared to controls.
- The Dppa4 gene, linked to aggressive tumors, was upregulated in alcohol-exposed male rats.

## Abstract

We have recently shown that estrogen‐induced prolactin‐secreting pituitary tumors are aggressive in prenatal alcohol‐exposed female rats. In this study, we investigated whether similar tumor aggressiveness occurs in estrogen‐treated prenatal alcohol‐exposed male rats.

Pregnant Fischer 344 rats were fed from gestational days 7 and 21 with a liquid diet containing ethanol 6.7% v/v (AF), pair‐fed with an isocaloric liquid diet (PF), or fed chow ad libitum (AD). Alcohol‐fed dams exhibited a blood alcohol concentration of 120–150 mg/dL 2 h after the last feeding. Male offspring were orchiectomized at 60 days of age and implanted subcutaneously with estradiol implants. Four months after the estradiol implants, rats were sacrificed, and pituitary tumor tissues were collected. Tumor cells were isolated and cultured for analysis.

Pituitary tumor cells from AF males exhibited stem‐like cell properties and showed elevated expression of stem cell regulatory genes and proteins (SOX‐2, OCT‐4, KLF4, SNAIL‐1, and Nestin), tumor aggressiveness markers (MMP‐9, CD44, CD34, PTTG, FGFR4, Ki‐67, N‐Cadherin), and prolactin compared to those from AD and PF controls. AF cells also had a higher cell proliferation rate, increased invasiveness, and colony formation compared to those in AD and PF cells, indicating more aggressive cancer cells than control cells. Notably, AF cells had a higher expression of developmental pluripotency‐associated 4 (Dppa4), a gene we recently identified as upregulated in aggressive tumors and in fetal alcohol‐exposed animals.

These findings are consistent with our previous observations in estrogen‐treated AF female rats. These results support the hypothesis that prenatal alcohol exposure programs the pituitary epithelium toward a mesenchymal stem cell‐like phenotype, contributing to the development of aggressive pituitary prolactinomas in both sexes.

Prenatal alcohol exposure (PAE) enhances the development of aggressive pituitary tumors (PitNETs) in female rats. In this study, we found that PAE also promotes the development of PitNETs in male rats. Like in females, PAE treatment enhances the expression of stem cell and tumor aggressiveness genes and increases tumor cell proliferation rate, invasiveness, and colony formation in the pituitary. These findings suggest that PAE reprograms pituitary cells toward a mesenchymal stem cell‐like state, promoting aggressive PitNET development in both sexes.