with its known effects on aromatase and estrogen synthesis, ..

Aromatase Cytochrome P450, The Enzyme Responsible for Estrogen Biosynthesis ...

aromatase and local estrogen synthesis ..

Aromatase is a membrane-bound hemeprotein of the endoplasmic reticulum. It is the only known enzyme to catalyze the biosynthesis of estrogens from androgens. Crystallization of aromatase (CYP19A1), arguably the most unique cytochrome P450 in vertebrates, and a major breast cancer drug target, remained elusive for decades. Our unusual approach of crystallizing the human placental enzyme, as in the case of steroid sulfatase, has finally been vindicated by the growth of the first crystal of the full-length microsomal P450. The structure provides an unprecedented glimpse into the active site of a substrate-specific estrogen-synthesizing catalytic machine, unlike those of the drug- and xenobiotic-metabolizing P450s or numerous published homology models, and has opened a floodgate of opportunities for the rational design of next generation aromatase inhibitors.

Estrogen plays a role in ovarian tumorigenesis

Currently, structure-based design, synthesis and evaluation of the next generation aromatase inhibitors constitute a major objective of the aromatase project. Several compounds synthesized by structure-guided design have shown great promise in our laboratory evaluation. We have conducted anti-proliferative assays on these promising candidates in a MCF-7 breast cancer cell line. Some show inhibitory and anti-proliferative properties better than those of the well-known breast cancer drug exemestane. For decades many laboratories have made attempts to crystallize various recombinant forms of human aromatase by deleting the N-terminal trans-membrane domain, but no crystal was reported. Using a bacterial construct of the amino terminus truncated aromatase we have now been able to address this problem. We have crystallized the recombinant aromatase and determined the crystal structure. In addition, some of the mutagenesis studies that we have already conducted raise interesting possibilities with regard to its oligomerization and functional issues. By incorporating the recombinant aromatase into our research plan, we have taken the aromatase and novel inhibitor discovery research to the next level. We have also initiated an investigation into the effects of environmental chemicals and endocrine-disrupting agents such as fungicides on aromatase and estrogen biosynthesis, and their mechanisms of action. Additionally, collaborative efforts are underway to extend our structure-function research into elucidating the possible roles of aromatase in the CNS and neuroendocrine effects of estrogen, as well as into rational design of antibodies as diagnostic tools for the detection of a super-active form of aromatase in estrogen-dependent breast tumors.

In situ estrogen synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms especially in postmenopausal women. Several recent studies demonstrated activity of aromatase, an enzyme that plays a critical role in estrogen synthesis in breast tumors. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is an estrogen receptor (ER) coregulator, and its expression is deregulated in breast tumors. In this study, we examined whether PELP1 promotes tumor growth by promoting local estrogen synthesis using breast cancer cells (MCF7) that stably overexpress PELP1. Immunohistochemistry revealed increased aromatase expression in MCF7-PELP1-induced xenograft tumors. Real-time PCR analysis showed enhanced activation of the aromatase promoter in MCF7-PELP1 clones compared with MCF7 cells. Using a tritiated-water release assay, we demonstrated that MCF7-PELP1 clones exhibit increased aromatase activity compared with control MCF-7 cells. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II, and growth factor signaling enhanced PELP1 activation of aromatase. PELP1-mediated induction of aromatase requires functional Src and phosphatidylinositol-3-kinase pathways. Mechanistic studies revealed that PELP1 interactions with ER-related receptor-α and proline-rich nuclear receptor coregulatory protein 2 lead to activation of aromatase. Immunohistochemistry analysis of breast tumor array showed increased expression of aromatase in ductal carcinoma in situ and node-positive tumors compared with no or weak expression in normal breast tissue. Fifty-four percent (n = 79) of PELP1-overexpressing tumors also overexpressed aromatase compared with 36% (n = 47) in PELP1 low-expressing tumors. Our results suggest that PELP1 regulation of aromatase represents a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.


Estrogen biosynthesis - Pathway Maps

N2 - Previous studies have shown that synapses and expression of synaptic proteins in hippocampal neurons are regulated by hippocampus-derived estradiol. Here, we compared the effects of this paracrine regulation in different hippocampal regions. In tissue sections, immunohistochemistry followed by semiquantitative image analysis revealed a three-fold higher expression of steroidogenic acute regulatory protein (StAR) and aromatase in neurons of the CA3 than that of the CA1 region and in granule cells. Next, we treated hippocampal cell cultures with letrozole, an aromatase inhibitor, which resulted in a dose-dependent decrease in the release of 17β-estradiol into the medium and in a dose-dependent downregulation of spinophilin and synaptophysin expression in dissociated hippocampal neurons. The downregulation of synaptic protein expression was restored by simultaneous application of letrozole together with estradiol. In response to a defined dose of letrozole, the downregulation of spinophilin expression was significantly stronger in CA1 neurons and in granule cells, than in cells of the CA3 region in slice cultures. With synaptophysin, downregulation was stronger in stratum lucidum of CA3 than in stratum radiaturn of CA1. Both region-specific expression of steroidogenic enzymes and region-specific downregulation of synaptic proteins in response to a defined dose of letrozole may suggest different levels of estrogen concentrations within the hippocampus. Varying concentrations of estradiol in the hippocampus in turn may contribute to region-specific differentiation of hippocampal neurons.

The activities of the aromatase, ..

AB - In situ estrogen synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms especially in postmenopausal women. Several recent studies demonstrated activity of aromatase, an enzyme that plays a critical role in estrogen synthesis in breast tumors. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is an estrogen receptor (ER) coregulator, and its expression is deregulated in breast tumors. In this study, we examined whether PELP1 promotes tumor growth by promoting local estrogen synthesis using breast cancer cells (MCF7) that stably overexpress PELP1. Immunohistochemistry revealed increased aromatase expression in MCF7-PELP1-induced xenograft tumors. Real-time PCR analysis showed enhanced activation of the aromatase promoter in MCF7-PELP1 clones compared with MCF7 cells. Using a tritiated-water release assay, we demonstrated that MCF7-PELP1 clones exhibit increased aromatase activity compared with control MCF-7 cells. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II, and growth factor signaling enhanced PELP1 activation of aromatase. PELP1-mediated induction of aromatase requires functional Src and phosphatidylinositol-3-kinase pathways. Mechanistic studies revealed that PELP1 interactions with ER-related receptor-α and proline-rich nuclear receptor coregulatory protein 2 lead to activation of aromatase. Immunohistochemistry analysis of breast tumor array showed increased expression of aromatase in ductal carcinoma in situ and node-positive tumors compared with no or weak expression in normal breast tissue. Fifty-four percent (n = 79) of PELP1-overexpressing tumors also overexpressed aromatase compared with 36% (n = 47) in PELP1 low-expressing tumors. Our results suggest that PELP1 regulation of aromatase represents a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.