There are 8 different human syndromes caused by mutations in the cholesterol synthesis pathway. A subset of these disorders such as Smith-Lemli-Opitz disorder, are associated with facial dysmorphia. However, the molecular and cellular mechanisms underlying such facial deficits are not fully understood, primarily because of the diverse functions associated with the cholesterol synthesis pathway. Recent evidence has demonstrated that mutation of the zebrafish ortholog of HMGCR results in orofacial clefts. Here we sought to expand upon these data, by deciphering the cholesterol dependent functions of the cholesterol synthesis pathway from the cholesterol independent functions. Moreover, we utilized loss of function analysis and pharmacological inhibition to determine the extent of sonic hedgehog (Shh) signaling in animals with aberrant cholesterol and/or isoprenoid synthesis. Our analysis confirmed that mutation of hmgcs1, which encodes the first enzyme in the cholesterol synthesis pathway, results in craniofacial abnormalities via defects in cranial neural crest cell differentiation. Furthermore targeted pharmacological inhibition of the cholesterol synthesis pathway revealed a novel function for isoprenoid synthesis during vertebrate craniofacial development. Mutation of hmgcs1 had no effect on Shh signaling at 2 and 3 days post fertilization (dpf), but did result in a decrease in the expression of gli1, a known Shh target gene, at 4 dpf, after morphological deficits in craniofacial development and chondrocyte differentiation were observed in hmgcs1 mutants. These data raise the possibility that deficiencies in cholesterol modulate chondrocyte differentiation by a combination of Shh independent and Shh dependent mechanisms. Moreover, our results describe a novel function for isoprenoids in facial development and collectively suggest that cholesterol regulates craniofacial development through versatile mechanisms.

Inborn errors of cholesterol metabolism occur as a result of mutations in the cholesterol synthesis pathway (CSP). Although mutations in the CSP cause a multiple congenital anomaly syndrome, craniofacial abnormalities are a hallmark phenotype associated with these disorders. Previous studies have established that mutation of the zebrafish hmgcs1 gene (Vu57 allele), which encodes the first enzyme in the CSP, causes defects in craniofacial development and abnormal neural crest cell (NCC) differentiation. However, the molecular mechanisms by which the products of the CSP disrupt NCC differentiation are not completely known. Cholesterol is known to regulate the activity of WNT signaling, an established regulator of NCC differentiation. We hypothesized that defects in cholesterol synthesis are associated with reduced WNT signaling, consequently resulting in abnormal craniofacial development. To test our hypothesis we performed a combination of pharmaceutical inhibition, gene expression assays, and targeted rescue experiments to understand the function of the CSP and WNT signaling during craniofacial development. We demonstrate reduced expression of four canonical WNT downstream target genes in homozygous carriers of the Vu57 allele and reduced axin2 expression, a known WNT target gene, in larvae treated with Ro-48-8071, an inhibitor of cholesterol synthesis. Moreover, activation of WNT signaling via treatment with WNT agonist I completely restored the craniofacial defects present in a subset of animals carrying the Vu57 allele. Collectively, these data suggest interplay between the CSP and WNT signaling during craniofacial development.

One in 8 women will develop breast cancer in their lifetime. Yet, the burden of disease is greater in Black women. Black women have a 40% higher mortality rate compared to White women, and a higher incidence of breast cancer at age 40 and younger. While the underlying cause of this disparity is multifactorial, exposure to endocrine disrupting chemicals (EDCs) in hair and other personal care products has been associated with an increased risk of breast cancer. Parabens are known EDCs that are commonly used as preservatives in hair and other personal care products, and Black women are disproportionately exposed to products containing parabens. Studies have shown that parabens impact breast cancer cell proliferation, death, migration/invasion, and metabolism, as well as gene expression in vitro. However, these studies were conducted using cell lines of European ancestry; to date, no studies have utilized breast cancer cell lines of West African ancestry to examine the effects of parabens on breast cancer progression. Like breast cancer cell lines with European ancestry, we hypothesize that parabens promote pro-tumorigenic effects in breast cancer cell lines of West African ancestry. Luminal breast cancer cell lines with West African ancestry (HCC1500) and European ancestry (MCF-7) were treated with biologically relevant doses of methylparaben, propylparaben, and butylparaben. Following treatment, estrogen receptor target gene expression and cell viability were examined. We observed altered estrogen receptor target gene expression and cell viability that was paraben- and cell-line specific. This study provides greater insight into the tumorigenic role of parabens in the progression of breast cancer in Black women.

BackgroundInborn errors of cholesterol metabolism occur as a result of mutations in the cholesterol synthesis pathway (CSP). Although mutations in the CSP cause a multiple congenital anomaly syndrome, craniofacial abnormalities are a hallmark phenotype associated with these disorders. Previous studies have established that mutation of the zebrafish hmgcs1 gene (Vu57 allele), which encodes the first enzyme in the CSP, causes defects in craniofacial development and abnormal neural crest cell (NCC) differentiation. However, the molecular mechanisms by which the products of the CSP disrupt NCC differentiation are not completely known. Cholesterol is known to regulate the activity of WNT signaling, an established regulator of NCC differentiation. We hypothesized that defects in cholesterol synthesis reduce WNT signaling, consequently resulting in abnormal craniofacial development.MethodsTo test our hypothesis we performed a combination of pharmaceutical inhibition, gene expression assays, and targeted rescue experiments to understand the function of CSP and WNT signaling during craniofacial development.ResultsWe demonstrate reduced expression of axin2, a WNT downstream target gene in homozygous carriers of the Vu57 allele and in larvae treated with Ro-48-8071, which inhibits the synthesis of cholesterol. Moreover, activation of WNT signaling via treatment with a WNT agonist completely restored the craniofacial defects present in the Vu57 allele.ConclusionsCollectively, these data suggest interplay between the CSP and WNT signaling during craniofacial development.

One in 8 women will develop breast cancer in their lifetime. Yet, the burden of disease is greater in Black women. Black women have a 40% higher mortality rate compared to White women, and a higher incidence of breast cancer at age 40 and younger. While the underlying cause of this disparity is multifactorial, exposure to endocrine disrupting chemicals (EDCs) in hair and other personal care products has been associated with an increased risk of breast cancer. Parabens are known EDCs that are commonly used as preservatives in hair and other personal care products, and Black women are disproportionately exposed to products containing EDCs. Studies have shown that parabens impact breast cancer cell proliferation, death, migration/invasion, and metabolism, as well as gene expression in vitro. However, these studies were conducted using cell lines of European ancestry; to date, no studies have utilized breast cancer cell lines of West African ancestry to examine the effects of parabens on breast cancer progression. Like breast cancer cell lines with European ancestry, we hypothesize that parabens promote pro-tumorigenic effects in breast cancer cell lines of West African ancestry. Luminal breast cancer cell lines with West African ancestry (HCC1500) and European ancestry (MCF-7) were treated with biologically relevant doses of methylparaben, propylparaben, and butylparaben. Following treatment, estrogen receptor target gene expression and cell viability were examined. We observed altered estrogen receptor target gene expression and cell viability that was paraben- and cell-line specific. This study provides greater insight into the tumorigenic role of parabens in the progression of breast cancer in Black women.