Role Of The Cholesterol Metabolite 27-Hydroxycholesterol In Breast And Prostate Cancer

The cholesterol metabolites known as oxysterols play an important role in maintaining cell homeostasis. They play vital roles in inflammation, cell growth, cell signaling, immunity, oxidative stress and aging. Following the discovery that the most abundant oxysterol, 27-hydroxycholesterol (27-OHC) is a Selective Estrogen Receptor Modulator (SERM), it has been implicated in hormonal cancers such as breast and uterine cancer. 27-OHC is a ligand of estrogen receptors (ER), a nuclear hormone receptor involved in cell growth and proliferation. The downstream events followed by 27-OHC-induced ER activation in the context of breast cancer is unknown. Also, the role of 27-OHC in prostate cancer is ill-defined given the involvement of ER in prostate cancer (PCa) and benign prostatic hyperplasia (BPH). Our studies delineate for the first time a potential cellular mechanism of action of 27-OHC in the context of ER+ breast cancer, whereby 27-OHC induced ER activation in ER+ MCF7 cells increases cell proliferation via perturbing the p53-MDM2 axis. We demonstrate that 27-OHC, through ER, exacerbated p53 inactivation via MDM2 resulting in an increase in cell proliferation in ER+ breast cancer cells. Next, we address the possibility of 27-OHC exacerbating prostate cancer cell proliferation. In the context of BPH and PCa, we show that 27-OHC not only increased cell proliferation in tumorigenic cell lines, LNCaP and PC3 but also in the non-tumorigenic cells, RWPE-1. We further demonstrate that 27-OHC- induced cell proliferation in prostate cells is specifically through ERβ. Given the tissue dependent selective ER modulation of 27-OHC, our novel findings suggest that 27-OHC activates ER signaling in the prostate. Altogether, our findings elucidate and establish the novel role and cellular mechanism of action of the oxysterol 27-OHC in the context of breast and prostate cancer. In the interest of discovering new therapeutic avenues for breast and prostate cancers, our work may aid in the development of novel therapies that could mitigate/halt/alleviate the progression of ER+ breast cancers and prostate cancers

Gadd153 and NF-κB crosstalk regulates 27-hydroxycholesterol-induced increase in BACE1 and β-amyloid production in human neuroblastoma SH-SY5Y cells.

β-amyloid (Aβ) peptide, accumulation of which is a culprit for Alzheimer's disease (AD), is derived from the initial cleavage of amyloid precursor protein by the aspartyl protease BACE1. Identification of cellular mechanisms that regulate BACE1 production is of high relevance to the search for potential disease-modifying therapies that inhibit BACE1 to reduce Aβ accumulation and AD progression. In the present study, we show that the cholesterol oxidation product 27-hydroxycholesterol (27-OHC) increases BACE1 and Aβ levels in human neuroblastoma SH-SY5Y cells. This increase in BACE1 involves a crosstalk between the two transcription factors NF-κB and the endoplasmic reticulum stress marker, the growth arrest and DNA damage induced gene-153 (gadd153, also called CHOP). We specifically show that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter and subsequent increase in BACE1 transcription and Aβ production. The NF-κB inhibitor, sc514, significantly attenuated the 27-OHC-induced increase in NF-κB-mediated BACE1 expression and Aβ genesis. We further show that the 27-OHC-induced NF-κB activation and increased NF-κB-mediated BACE1 expression is contingent on the increased activation of gadd153. Silencing gadd153 expression with siRNA alleviated the 27-OHC-induced increase in NF-κB activation, NF-κB binding to the BACE1 promoter, and subsequent increase in BACE1 transcription and Aβ production. We also show that increased levels of BACE1 in the triple transgenic mouse model for AD is preceded by gadd153 and NF-κB activation. In summary, our study demonstrates that gadd153 and NF-κB work in concert to regulate BACE1 expression. Agents that inhibit gadd153 activation and subsequent interaction with NF-κB might be promising targets to reduce BACE1 and Aβ overproduction and may ultimately serve as disease-modifying treatments for AD

27-OHC increases the activation of NF-κB via the phosphorylation of IKKα/β which is dependent on gadd153 expression.

<p>27-OHC induces a significant increase in the phosphorylation of IKKα/β, and siRNA to gadd153 reverses the 27-OHC-induced increase in phosphorylation of IKKα/β (a,b). 27-OHC induces a significant decrease in IKBα levels and a significant increase in the phosphorylation of IKBα; siRNA to gadd153 reverses the 27-OHC-induced alterations in IKBα and p- IKBα levels (c,d). Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). ***p<0.001 versus control; ^†† p<0.01 versus 27-OHC and ^††† p<0.001 versus 27-OHC.</p

Schematic representation of the molecular mechanisms involved in the 27-OHC-induced increase in BACE1 expression.

<p>27-OHC activates gadd153 (1) which evokes the phosphorylation of the IKK-complex (2). Phosphorylation IKK complex (3) results in the phosphorylation and subsequent proteasomal degradation of IκB (4), thereby releasing the p65-p50 NF-κB dimer from inhibitory sequestration in the cytosol and allowing the p65-p50 NF-κB dimer to translocate to the nucleus (5). The translocated p65-p50 NF-κB dimer binds to distinct κB sites in the BACE1 promoter region and upregulates BACE1 expression (5). Increased gadd153 expression by 27-OHC may also induce NF-κB activation, nuclear translocation, and subsequently increase BACE1 expression by other mechanism that are yet to be elucidated (6). siRNA to gadd153 reduces the 27-OHC-induced nuclear translocation of NF-κB and thereby attenuates the increase in BACE1(7). The NF-κB inhibitor sc514 also decreases the 27-OHC-induced increase in BACE1 expression by inhibiting the nuclear translocation of NF-κB and subsequent increase in NF-κB-mediated transcription of BACE1 (8).</p

Primers designed and used for RT-PCR, EMSA, and ChIP analyses.

<p>Primers designed and used for RT-PCR, EMSA, and ChIP analyses.</p

Gadd153 expression is necessary for 27-OHC-induced increase in the levels of Aβ40 and Aβ42.

<p>(a,b) ELISA immunoassay demonstrates that siRNA to gadd153 expression attenuates the 27-OHC-induced increase in levels of intracellular and secreted Aβ42 and Aβ40. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.05, **p<0.01, and ***p<0.001 versus control, ^†† p<0.01 and ^††† p<0.001 versus 27-OHC.</p

27-OHC-induced NF-κB-mediated increase in BACE1 transcription and expression is contingent on gadd153 expression.

<p>(a–c) siRNA to gadd153 mitigates the 27-OHC-induced increase in BACE1 protein levels and mRNA expression. (d) EMSA shows that 27-OHC increases the binding of NF-κB to the exogenous DNA sequence that corresponds to the NF-κB binding site in the BACE1 promoter; silencing gadd153 expression reduces the increase in the binding of NF-κB. (e) ChIP analysis shows that siRNA to gadd153 decreases the 27-OHC-induced increase in binding of NF-κB to the BACE1 promoter. (f,g) Dual luciferase assay demonstrates that siRNA to gadd153 decreases the 27-OHC-induced increase in NF-κB transcriptional activity and BACE1 promoter activity. (h) Levels of gadd153 are detectable in hippocampus of 3, 6, and 12 month-old 3xTg-AD, while NF-κB levels are detectable in hippocampus of 6 and 12month-old, and BACE1 levels increasing only in 12 month-old 3xTg-AD. Data in a-g is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.05, **p<0.01, and ***p<0.001 versus control, ^†† p<0.01 and ^††† p<0.001 versus 27-OHC.</p

27-OHC induces NF-κB activation and subsequently increases NF-κB-mediated BACE1 expression.

<p>(a,b) 27-OHC significantly increases the levels of the p65 and the p50 subunits of the NF-κB in the nuclear homogenates, and (c–e) increases the protein and mRNA levels of BACE1; treatment with the NF-κB inhibitor sc514 attenuates the 27-OHC-induced increase in protein and mRNA expression of BACE1. (f) ChIP analysis shows that 27-OHC increases the binding of NF-κB in the BACE1 promoter region. (g) Dual luciferase assay demonstrates that 27-OHC increases the NF-κB transcriptional activity as measured by a significant increase in NF-κB reporter activity. (h) Dual luciferase assay demonstrates that 27-OHC significantly increases the BACE1 promoter activity, while the NF-κB inhibitor sc514 decreases the 27-OHC-induced increase in BACE1 promoter activity. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.05, **p<0.01, and ***p<0.001 versus control, ^† p<0.05, ^†† p<0.01, and ^††† p<0.001 versus 27-OHC.</p

27-OHC-induced increase in the levels of intracellular as well as secreted forms of Aβ42 and Aβ40 is contingent on NF-κB activation.

<p>(a–d) ELISA immunoassay demonstrates that 27-OHC increases the levels of intracellular and secreted Aβ42 and Aβ40, while the NF-κB inhibitor sc514 mitigates the 27-OHC-induced increase in both intracellular and secreted Aβ42 and Aβ40. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.05, **p<0.01, and ***p<0.001 versus control, ^†† p<0.01 and ^††† p<0.001 versus 27-OHC.</p