To identify novel mechanisms of resistance to E- deprivation, we first generated five LTED cell lines. wt- MCF7, wt-ZR75.1, wt-T47D, wt-HCC1428 and wt-SUM44 cells were cultured in the absence of E2 until their growth rate was shown to be independent of the exogenous E2 (Fig. 1a). At the point of resistance, ZR75.1 LTED and T47D LTED cells lost expression of ER whilst MCF7 LTED, SUM44 LTED and HCC1428 LTED cells retained or even upregulated ER expression (Fig. 1b). Pellets derived from the wt cells (representing primary diagnosis) 1-week post E2-deprivation (representing clinical response to AI therapy) and LTED (modelling relapse on an AI) were har- vested and global gene expression was interrogated for each cell line using a triangular pairwise comparison (FDR < 5 %, univariate p < 0.001 and absolute fold-change ≥1.5) (Fig. 1c). Varying numbers of genes were altered at each time point and differed among cell types, indicating hetero- geneity in response to E2-deprivation (Additional file 1: Table S1). As noted previously, comparison between gene changes in wt cells and those deprived of E2 for 1 week were dominated by proliferation, as was the comparison between 1-week deprivation and LTED . We therefore restricted our pathway analysis to comparison of the wt cell lines with their corresponding LTED derivative, in order to remove the confounding effect of proliferation. To identify common adaptive pathways, the IPA software was employed. Strikingly, the cholesterolbiosynthesis pathway (Additional file 2: Figure S1) [37–40] was upregulated ex- clusively in all LTED derivatives that retained expression of ER both in 2D and 3D culture (Table 1). Further interroga- tion of the gene expression data showed that genes encod- ing enzymes within the cholesterolbiosynthesis pathway were increased significantly (Additional file 3: Table S2).
cholesterolbiosynthesis. Studies have also highlighted a strong association between adipocyte cholesterol content and fat cell size and triglyceride content and bigger the fat cell, the more cholesterol it has been shown to contain [8,13,47]. The rate of de novo cholesterol synthesis in adi- pose tissue is only 4% compared to that of liver  and therefore, may not contribute significantly to the circula- tory cholesterol but current literature suggests an import- ant role for adipose tissue in regulation of circulatory HDL-C by mediating HDL lipidation [2,48]. Chung et al.  have shown that specific deletion of cholesterol trans- porter ABCA1 in adipose tissue significantly reduces HDL-C levels. The increased biosynthesis in adipose tissue therefore, may interfere with the lipidation process in vita- min B 12 -restricted conditions. The aim of this manuscript
In this study, we used microarray analysis to compare gene expression during acute and persistent infection with MV and to identify pathological mechanisms unique to per- sistent infection that could be targeted for further analyses. We then used a gene set enrichment analysis (GSEA) (29) to identify Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways (13) and Gene Ontology (GO) (2) functions that were more perturbed during persistent infection than acute infection. Since several of the most perturbed pathways involved cholesterolbiosynthesis, we chose to focus on the cholesterolbiosynthesis pathway in general. We then verified the proposed mechanisms using various experimental protocols. Our results suggest an un- expected role for cholesterolbiosynthesis in the regulation of MV infection persistence. These data represent not only the first study utilizing microarrays to examine gene expres- sion in cells that are persistently infected with MV but also the first global transcriptional comparison of persistent and acute viral infection.
Abstract: Standard treatment for primary prostate cancer includes systemic exposure to chemotherapeutic drugs that target androgen receptor or antihormone therapy (chemical castration); however, drug-resistant cancer cells generally emerge during treatment, limiting the continued use of systemic chemotherapy. Patients are then treated with more toxic standard therapies. Therefore, there is an urgent need for novel and more effective treatments for prostate cancer. The cholesterol biosynthetic pathway is an attractive therapeutic target for treating endocrine-dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as the metabolic precursor of endogenous steroid hormones. In this study, we have examined the effects of RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate; Roche Pharmaceuticals internal reference: RO0488071) (RO), which is an inhibi- tor of 2, 3-oxidosqualene cyclase (a key enzyme in the cholesterol biosynthetic pathway), on prostate cancer cells. Exposure of both hormone-dependent and castration-resistant human prostate cancer cells to RO reduced prostate cancer cell viability and induced apoptosis in vitro. RO treatment reduced androgen receptor protein expression in hormone-dependent prostate cancer cells and increased estrogen receptor β (ERβ) protein expression in both hormone- dependent and castration-resistant prostate cancer cell lines. Combining RO with an ERβ agonist increased its ability to reduce castration-resistant prostate cancer cell viability. In addition, RO effectively suppressed the growth of aggressive castration-resistant human prostate cancer cell xenografts in vivo without any signs of toxicity to experimental animals. Importantly, RO did not reduce the viability of normal prostate cells in vitro. Our study is the first to demonstrate that the cholesterolbiosynthesis inhibitor RO effectively suppresses growth of human prostate cancer cells. Our findings suggest that cholesterolbiosynthesis inhibitors such as RO, when used in combination with commonly used chemotherapeutic drugs or ERβ specific ligands, could represent a novel therapeutic approach to prevent the growth of prostate cancer tumors. Keywords: prostate cancer, cholesterolbiosynthesis inhibitor, cell viability, xenograft, castration resistant
Intracellular cholesterolbiosynthesis plays a crucial role in chondrocyte development. This process is regulated by the protein sterol regulatory element–binding protein cleavage-activating protein (SCAP). When intracellular cholesterol level is low, SCAP cleaves and activates the transcription factors known as SREBPs (ref. 10). After cleavage, SREBPs are further processed and translocate to the nucleus to activate genes responsible for cholesterolbiosynthesis (10). Genetic deletion of Scap in chondrocytes affected their differentiation and viability (11). In addition, pharmacological inhibition of cholesterol synthesis by statin drugs caused reduced endochondral bone growth and decreased height of the growth plate (12). Further- more, statin drugs rescued chondrocyte differentiation and bone length in models of achondroplasia (13).
transplantation was identified. Accelerated atherosclerosis, unrestricted intestinal sterol absorption, increased plasma and tissue plant sterol concentrations, and low cholesterol synthesis characterize this disease. Mean total microsomal HMG-CoA reductase (rate- control controlling enzyme for cholesterolbiosynthesis) activity was sevenfold higher (98.1 +/- 28.8 vs. 15.0 +/- 2.0 pmol/mg protein per min) and microsomal enzyme protein mass was eightfold larger (1.43 +/- 0.41 vs. 0.18 +/- 0.04 relative densitometric U/mg protein) in 11 controls than the average for two sitosterolemic liver specimens. HMG-CoA reductase mRNA probed with pRED 227 and pHRED 102 was decreased to barely detectable levels in the sitosterolemic livers. In addition, there was a 50% decrease in the rate [2-
Adipose tissue (AT) plays a central role in integrating energy metabolism and glucose homeostasis . Besides being the major site of fatty acid storage as triglycerides (TG), AT is also the body’s largest cholesterol pool. In AT, nearly all cholesterol (>95%) exists as free, non- esterified form and resides in the plasma membrane or at the cytosolic interface of lipid droplets [2-4]. In con- trast, other specialised cells and tissues store small pools of esterified cholesterol, such as adrenal cells or foam cells, which have the capacity to accumulate consider- able quantities of excess cholesterol esters . A delicate balance exists between uptake, synthesis, and storage which tightly controls the abundance of free cholesterol in peripheral cells  while excess free cholesterol is in fact deleterious to cells . AT from ob/ob mice exhibits an increase in cholesterolbiosynthesis , and hypertro- phied adipocytes from obesity rodent model, Zucker rats have elevated mRNA expression of SREBP-2, 3-hydroxy- 3-methylglutaryl-CoA reductase (HMGCR), and the LDL receptor (LDLR) [9,10]. Studies in Zucker rats  and 3T3F442A cells  also highlight that with an in- crease in triglyceride storage, cholesterol is redistributed from the plasma membrane to the surface of the lipid droplet and adipocyte cholesterol levels appear to in- crease in proportion to the triglyceride content . There is also accumulating evidence that adipose choles- terol imbalance is closely associated with adipocyte dys- function and obesity-mediated metabolic complications and insulin resistance [9,13,14].
o f lipid secretion and apo B secretion was observed, with fenofibrate being more effective than clofibrate. Fibrates inhibit lipid biosynthesis, especially fatty acid synthesis in liver (Adams et d , 1971). Early reports showed clofibrate (1.25 mM) produced direct inhibition in assays o f ACC purified fi*om chicken livers by 50% (Maragoudakis, 1969). Maragoudakis and Hankin, (1971) observed that clofibrate brought about direct inhibition of ACC by preventing ACC polymerisation by citrate. Sanchez et d, (1993) also reported inhibition of ACC in crude liver extracts assayed in the presence o f the fibric acid derivatives: gemfibrozil, bezafibrate and clofibrate. As well as reports that demonstrate the potential of fibrates to inhibit de novo fatty acid synthesis, Landriscina et d (1975) showed that clofibrate (<10 mM) reduced microsomal fatty acid chain elongation when added directly to rat liver cell extracts. This is the main system responsible for hepatic synthesis o f saturated and unsaturated fatty acids with chain length longer than Cjg. Cohen et d , (1974) and Castillo et d , (1990) noted chronic treatment of rats with clofibrate resulted in inhibition of hepatic cholesterol synthesis accompanied by 50% and 90% inhibition o f HMG-CoA reductase, respectively. Animal studies have demonstrated that fibrates induce fatty acid oxidation which diverts them fi*om the pathway of estérification and VLDL production and contributes to the hypolipidaemic effect o f reducing VLDL output, (Lazarow and De Duve, 1976.; Milton et d , 1990.; Reddy and Azamofif, 1980). Rats fed clofibrate (5 g/kg of chow diet) for 1 week showed a 10-fold increase in peroxisomal fatty acyl-CoA oxidation, (Lazarow and De Duve, 1976).
receiving MCF had a FSR that was intermediate between those of the HM- and CF-fed infants, which suggests that the cholesterol supplementation brought the FSR of the MCF-fed infants closer to the physiologic range seen among breastfed infants. These results demonstrate that adaptive regulatory mechanisms in early infancy enable human infants to respond to differences in cholesterol intakes. Theo- retically, these homeostatic mechanisms could pre- vent excess cholesterol accumulation during high cholesterol intakes or, conversely, provide for an in- crease in cholesterol availability during instances of low or negligible intake. With the assumption that HM is the standard for infant nutrition and values for FSR among HM-fed infants are considered to be “normal,” then these data indicated that CF-fed in- fants had a 53% increase in FSR, compared with HM-fed infants, during the first 4 months of life. This is indicative of the need for cholesterol in early in- fancy, a period of rapid growth. The results seen for MCF-fed infants are in contrast to those seen in the study by Bayley et al, 18 in which infants fed choles-
Abstract: Clinical trials and studies have shown that postmenopausal women undergoing com- bination hormone replacement therapy containing estrogen and progestin have an increased risk of breast cancer compared with women taking estrogen or placebo alone. Using animal models, we have previously shown that synthetic progestins, including medroxyprogesterone acetate (MPA), which is widely used clinically, accelerate breast cancer tumor growth and promote metastasis. Furthermore, we have found that MPA elevates CD44 protein expression and alde- hyde dehydrogenase (ALDH) activity, two markers of cancer stem cells (CSCs), and increases mammosphere formation, another hallmark of stem cells, in hormone-dependent T47-D human breast cancer cells. Herein, we show that RO 48-8071 (RO), an inhibitor of cholesterol synthesis, reduced MPA-induced CD44 protein expression in two hormone-dependent human breast cancer cell lines, T47-D and BT-474. Because we have previously shown that MPA induction of CD44 is progesterone receptor (PR) dependent, we examined RO’s effects on PR protein and mRNA expressions in T47-D cells. PR mRNA levels remained unchanged after RO treatment; however, RO significantly reduced the protein expression of both PR receptor isoforms, PR-A and PR-B. Using the proteasome inhibitor MG-132, we demonstrated that RO decreases PR protein expres- sion in T47-D cells via the proteasomal degradation pathway. Importantly, treatment of T47-D cells with RO abolished MPA-induced mammosphere formation. Based on our observations, we contend that RO may represent a novel means of preventing MPA-induced CSC expansion. RO could be used clinically to both treat and prevent hormone-dependent breast cancers, which represent the majority of human breast cancers. RO may also have clinical utility in reducing resistance to antihormone therapy.
activation of mevalonate pathway to meet the requirement of cholesterol for membrane biogenesis and assembly of lipid rafts for facilitating cellular growth signaling. Increase in HMGCR expression is directly linked to abnormal RAS activity that results in enhanced growth signaling in tumour cells . Gene expression of both LDLR and HMGCR is dependent on SREBP2 transcription factor hence it was logical for us to analyze the expression of SREBPs in tumour. Several studies in the past have shown that regulation of lipid and cholesterol pathway by SREBPs is critical for growth and survival of cancer cells. Under hypoxic conditions of the tumour microenvironment, SREBPs expression is upregulated to increase lipid and cholesterolbiosynthesis in brain tumour cells . Depletion of SREBPs in glioblastoma cell lines, induced apoptosis due to ER stress and activation of unfolded protein response (UPR) pathway. Thus, SREBPs are critical for tumour growth and survival because of the role they play in coordinating lipid and protein biosynthetic pathways. In our study, we found elevated expression of transcriptionally active mature form of both SREBP1 and SREBP2 in tumours in comparison with normal mucosa. From these results, it is evident that the elevated expression of HMGCR and PCSK9 in tumour tissues can be attributed to upregulation of SREBP2. However, the downregulation of LDLR protein despite overexpression of SREBP2 suggests the involvement of another pathway in post translationally depleting LDLR protein levels in tumour. One of the proteins that is responsible for LDLR turn over in the cell, is PCSK9. C-terminal domain of PCSK9 binds to the extracellular domain of LDLR at the cell surface to form LDLR-PCSK9 complex that enters the endosomal pathway and is ultimately degraded in the lysosome. Hence LDLR protein abundance is inversely related to PCSK9 expression. In our study, we observed PCSK9 elevated in tumours when compared to mucosa. This suggests that PCSK9 might play a role in downregulation of LDLR protein in certain tumour phenotype. The significance of PCSK9 upregulation in colonic tumour, other than affecting LDLR protein levels, is yet to be understood. In some of the earlier reports, it was reported that PCSK9 deficiency reduces melanoma metastasis in liver by reducing serum cholesterol .
To test this hypothesis, we used the ear punch model to study the rate of wound healing in 1,378 out- bred mice. We integrated phenotypic and genotypic analyses in this population (i.e., ear area closure after healing and genome-wide single-nucleotide polymorphism [SNP] genotyping) with whole-genome mRNA sequencing (RNA-seq) in primary macrophages from mice with extreme wound healing phenotypes (i.e., fast and slow healers from normal distribution of the rate of wound healing). This led to the identification of a gene network enriched for fatty acid and cholesterolbiosynthesis that (a) associates with the rate of wound healing and (b) is under strong genetic control in trans by the transcription factor Runx2 located on mouse chromosome 17. We showed that inhibiting the DNA binding activity of Runx2 affects the expres- sion of the genes in this network. We then targeted Fasn, which catalyzes the synthesis of long-chain fatty acids and also shows a regulatory role in cholesterol synthesis (22). In vivo pharmacological blockage of Fasn results in delayed wound healing and increased macrophage recruitment in the skin wound, suggest- ing the persistence of an unresolved inflammation. These results suggest the importance of fatty acid/ cholesterolbiosynthesis in the healing process.
myeloperoxidase-treated LDL (53); importantly, these forms of oxidatively modified LDL were shown to induce strong cellular responses in macrophages and endothelial cells to a level similar to oxLDL prepared by copper oxidation in vitro (54–56). OxLDL levels in the circulation are low relative to the total amount of LDL (48), due to efficient clearance by the hepatic reticuloendo- thelial system (57), blood antioxidants, and immune responses generated by autoantibodies against oxLDL (58). However, it is now widely accepted that oxidized forms of LDL do exist in vivo and at detectable levels in the circulation, even in healthy patients. While the role of oxLDL in adipocytes has not been well studied, adipocytes are known to express scavenger receptors, such as SR-B1 and CD36 (59, 60), and are capable of recognizing and degrading oxLDL in part through CD36 (61). Our discov- ery that OLR1 is upregulated in adipocytes by TZDs provides further evidence that oxLDL may play an important, though as- yet-undefined, role in fat cell metabolism. Adipocytes are poor at de novo cholesterolbiosynthesis (13) and obtain a majority of their sterols from lipoproteins in the circulation (10). Since oxLDL is a rich source of cholesterol, scavenger receptors could be essential for adipocyte uptake of the cholesterol required for normal signal transduction, lipid raft function, and vesicular
Oxysterols derivatives of cholesterol and sterol intermediates in cholesterolbiosynthesis have been found to be potent inhibitors of sterol biosynthesis in animal cell culture. The reported inhibition of cholesterolbiosynthesis in mammalian cells by oxygenated derivatives of cholesterol and lanosterol has been shown in most cases to decrease cellular levels of HMG-CoA reductase, a key regulatory enzyme in sterol biosynthesis [7-12]. These studies suggest a regulatory mechanism which, by analogy to steroid hormone receptors and bacterial induction-repression systems, requires a binding protein to recognize oxysterols and mediate subsequent cellular events.
the activation of Ampk (Wolfgang and Lane, 2006). Significantly, while Ampk inhibits cholesterol synthesis, the proteomic analysis suggested that cholesterolbiosynthesis was upregulated (Figure 6B and quantification in Figure S1G), with most of the enzymes driving cholesterol biogenesis found in higher abun- dance, although DHCR7, which catalyzes the conversion of 7-dehydrocholesterol to cholesterol, was in lower abundance (Figure 6B and quantification in Figure S1G). In agreement with upregulation of cholesterolbiosynthesis, the abundance of the Ldl receptor (Ldl-R, Log2FC 1.31) and Pcsk9 (Log2FC 1.97), involved in cholesterol uptake and receptor recycling, were elevated in infected IECs. These responses are typical of cells suffering sterol deficiency (Spann and Glass, 2013). This hypoth- esis was supported by the fact that, based on the fold change of its target proteins, the transcription factor Srebp2, which upre- gulate expression of genes involved in cholesterolbiosynthesis and uptake (Spann and Glass, 2013), was predicted to be strongly activated (Z score: 2.032, p value: 2.28 3 10 8 ) in in- fected IECs.
To search for new agents that can lower cellular chol- esterol, and do not negatively interfere with CD-20 and rituximab-mediated activity, we chose three compounds that can inhibit squalene synthase (YM-53601 and TAK- 475) or oxidosqualene cyclase (BIBB-515). These enzymes are further downstream steps in cholesterol biosynthetic pathway (Figure 2). By confocal microscopy, our results indicate that treatment of MEC-2 cells with YM-53601, BIBB-515 or TAK-475 did significantly up-regulate CD-20 surface expression and membrane-association (Figures 4 and 6). One recent study has shown that farnesyltransfer- ase inhibitors may up-regulate CD-20 at mRNA and protein levels and improve anti-CD-20 monoclonal antibody-mediated activation of complement-dependent cytotoxicity . Earlier reports demonstrated that cy- tokines (interleukin-4, granulocyte-macrophage colony- stimulating factor, or interferon-α) also increase CD-20 surface expression [46,49]. Taken together, our data and those results indicate that the agents inhibiting downstream steps of cholesterolbiosynthesis and other cholesterol- associated pathways could positively regulate CD-20 expression and its antibody-mediated signaling.