Differentiating radiation necrosis from tumor recurrence: a systematic review and diagnostic meta-analysis comparing imaging modalities.

PURPSOSE: Cerebral radiation necrosis (RN) is often a delayed phenomenon occurring several months to years after the completion of radiation treatment. Differentiating RN from tumor recurrence presents a diagnostic challenge on standard MRI. To date, no evidence-based guidelines exist regarding imaging modalities best suited for this purpose. We aim to review the current literature and perform a diagnostic meta-analysis comparing various imaging modalities that have been studied to differentiate tumor recurrence and RN. METHODS: A systematic search adherent to PRISMA guidelines was performed using Scopus, PubMed/MEDLINE, and Embase. Pooled sensitivities and specificities were determined using a random-effects or fixed-effects proportional meta-analysis based on heterogeneity. Using diagnostic odds ratios, a diagnostic frequentist random-effects network meta-analysis was performed, and studies were ranked using P-score hierarchical ranking. RESULTS: The analysis included 127 studies with a total of 220 imaging datasets, including the following imaging modalities: MRI (n = 10), MR Spectroscopy (MRS) (n = 28), dynamic contrast-enhanced MRI (n = 7), dynamic susceptibility contrast MRI (n = 36), MR arterial spin labeling (n = 5), diffusion-weighted imaging (n = 13), diffusion tensor imaging (DTI) (n = 2), PET (n = 89), and single photon emission computed tomography (SPECT) (n = 30). MRS had the highest pooled sensitivity (90.7%). DTI had the highest pooled specificity (90.5%). Our hierarchical ranking ranked SPECT and MRS as most preferable, and MRI was ranked as least preferable. CONCLUSION: These findings suggest SPECT and MRS carry greater utility than standard MRI in distinguishing RN from tumor recurrence.

Whole-genome cartography of estrogen receptor alpha binding sites.

Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation.

Differential estradiol and selective estrogen receptor modulator (SERM) regulation of Keratin 13 gene expression and its underlying mechanism in breast cancer cells.

Expression of the Keratin 13 (KRT13) gene, which encodes a cytoskeletal protein thought to play important roles in breast cancer growth and metastasis, is differentially regulated by estradiol (E2) and the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene. While stimulation of KRT13 by tamoxifen is robust and prolonged, stimulation by E2 is more transient and raloxifene has virtually no effect. To investigate the mechanistic basis for the differential ligand regulation of KRT13, we have defined the regulatory regions of KRT13, compared gene expression by E2 and SERMs, and explored the magnitudes and time courses of estrogen receptor (ER) and cofactor recruitment patterns on these regions. Using a ChIP scanning approach and reporter transactivation assays, we identified a 2.5 kb upstream ER-binding regulatory region for KRT13. Directed composite mutations in this region revealed that three estrogen response elements and three Sp1 sites were involved in its ligand-dependent regulation. Differential recruitment of ERalpha and cofactors to the KRT13 regulatory sites paralleled the different time course and magnitude of regulation by these ligands: there was almost no ERalpha or cofactor recruitment with raloxifene, whereas there was strong, prolonged ER recruitment and histone acetylation with tamoxifen, and an early and more transient recruitment with E2. Taken together, our results suggest that the different ligand regulations of KRT13 are due to ligand-differential recruitment of ER and coactivators, and they provide insight into the mechanisms responsible for the different agonistic activities and differential gene regulation by estradiol and the SERMs tamoxifen and raloxifene.

Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus.

Estrogen is of great importance in the regulation of uterine function. The aim of this study was to examine the individual physiological roles of each of the two receptors for estradiol, estrogen receptor (ER) alpha and ERbeta, and their potential comodulatory effects on gene expression and uterine growth using recently developed ER subtype-selective agonist ligands. The use of ER subtype-selective ligands provides an alternative, complementary approach to the use of receptor knockout mice. Administration of the ERalpha-selective ligand propyl pyrazole triol (PPT) to immature mice resulted in a significant increase in uterine weight, as well as bromodeoxyuridine incorporation and proliferating cell nuclear antigen expression in luminal epithelial cells. PPT also increased complement component 3, lactoferrin, and glucose-6-phosphate dehydrogenase (G6PDH), and decreased androgen receptor (AR) and progesterone receptor (PR) mRNA levels in uterine tissue, as did estradiol (E(2)). However, when compared with E(2), PPT was less effective in stimulating uterine weight, complement component 3, and G6PDH expression but was as effective as E(2) in regulating lactoferrin, AR, and PR expression. In contrast to the action of the ERalpha agonist PPT, the ERbeta agonist diarylpropionitrile (DPN) did not increase uterine weight or luminal epithelial cell proliferation at a dose that reduced G6PDH and elicited a decrease in PR and AR mRNA and protein expression. Interestingly, DPN reduced the uterine weight stimulation by PPT, and enhanced the effect of PPT in decreasing uterine PR and AR mRNA. These findings with ER subtype-selective ligands indicate that ERalpha is the major regulator of estrogen function in the uterus, but that ERbeta does exert effects on some uterine markers of estrogen action. In addition, ERbeta can modulate ERalpha activity in a response-specific and dose-dependent manner.

Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) alpha or ERbeta in human osteosarcoma cells: distinct and common target genes for these receptors.

Estrogens exert many important effects in bone, a tissue that contains both estrogen receptors alpha and beta (ERalpha and ERbeta). To compare the actions of these receptors, we generated U2OS human osteosarcoma cells stably expressing ERalpha or ERbeta, at levels comparable with those in osteoblasts, and we characterized their response to 17beta-estradiol (E2) over time using Affymetrix GeneChip microarrays to determine the expression of approximately 12,000 genes, followed by quantitative PCR verification of the regulation of selected genes. Of the approximately 100 regulated genes we identified, some were stimulated by E2 equally through ERalpha and ERbeta, whereas others were selectively stimulated via ERalpha or ERbeta. The E2-regulated genes showed three distinct temporal patterns of expression over the 48-h time course studied. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ERbeta, in keeping with the selective E2 enhancement of the motility of ERbeta-containing cells. On genes regulated equally by E2 via ERalpha or ERbeta, the phytoestrogen genistein preferentially stimulated gene expression via ERbeta. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation.

Activities of estrogen receptor alpha- and beta-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression.

Estrogens exert their regulatory transcriptional effects, which can be stimulatory or repressive, at diverse gene sites via two estrogen receptors, ERalpha and ERbeta. Since these two ERs have different tissue distributions, ligands that have the capacity to selectively activate or inhibit these two ERs would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity. We have developed several ligands that showed ERalpha or ERbeta selectivity at promoter-gene sites containing consensus estrogen response elements (EREs): ERalpha-selective agonist (propyl-pyrazole-triol (PPT)), ERalpha-selective antagonist (methyl-piperidino-pyrazole (MPP)), ERbeta-potency selective agonist (diarylpropionitrile (DPN)) and ERbeta-selective antagonist/ERalpha-agonist (R,R-tetrahydrochrysene (R,R-THC)). In this study, we have examined the activity of these compounds at a range of gene sites where ER stimulates gene expression through non-consensus EREs (complement C3), or multiple half-EREs (NHE-RF/EBP50), or by tethering to DNA via other proteins (TGF beta3 and progesterone receptor A/AP-1), and at gene sites where ER represses gene transcription (interleukin-6). At all of these genes, PPT showed full stimulation through ERalpha while displaying no agonism through ERbeta. MPP antagonized estradiol actions on gene transactivation and transrepression through ERalpha, with little or no effect on transcription mediated through ERbeta. DPN displayed subtype-selective agonism, being ca. 30-fold more potent through ERbeta. R,R-THC was a complete antagonist through ERbeta and displayed agonism through ERalpha, the level of which was promoter dependent. Because these ligands maintain their agonist or antagonist character and ER subtype-selectivity at gene sites of diverse nature, where estradiol is either stimulatory or inhibitory, these compounds should prove useful in elucidating the biological functions of ERalpha and ERbeta.

The estrogen-regulated transcription factor PITX1 coordinates gene-specific regulation by estrogen receptor-alpha in breast cancer cells.

The estrogen receptor α (ERα) is a master regulator of gene expression and works along with cooperating transcription factors in mediating the actions of the hormone estradiol (E2) in ER-positive tissues and breast tumors. Here, we report that expression of paired-like homeodomain transcription factor (PITX1), a tumor suppressor and member of the homeobox family of transcription factors, is robustly up-regulated by E2 in several ERα-positive breast cancer cell lines via ERα-dependent interaction between the proximal promoter and an enhancer region 5' upstream of the PITX1 gene. Overexpression of PITX1 selectively inhibited the transcriptional activity of ERα and ERß, while enhancing the activities of the glucocorticoid receptor and progesterone receptor. Reduction of PITX1 by small interfering RNA enhanced ERα-dependent transcriptional regulation of a subset of ERα target genes. The consensus PITX1 binding motif was found to be present in 28% of genome-wide ERα binding sites and was in close proximity to estrogen response elements in a subset of ERα binding sites, and E2 treatment enhanced PITX1 as well as ERα recruitment to these binding sites. These studies identify PITX1 as a new ERα transcriptional target that acts as a repressor to coordinate and fine tune target-specific, ERα-mediated transcriptional activity in human breast cancer cells.

Estrogen receptor regulation of carbonic anhydrase XII through a distal enhancer in breast cancer.

The expression of carbonic anhydrase XII (CA12), a gene that encodes a zinc metalloenzyme responsible for acidification of the microenvironment of cancer cells, is highly correlated with estrogen receptor alpha (ER alpha) in human breast tumors. Here, we show that CA12 is robustly regulated by estrogen via ER alpha in breast cancer cells, and that this regulation involves a distal estrogen-responsive enhancer region. Upon the addition of estradiol, ER alpha binds directly to this distal enhancer in vivo, resulting in the recruitment of RNA polymerase II and steroid receptor coactivators SRC-2 and SRC-3, and changes in histone acetylation. Mutagenesis of an imperfect estrogen-responsive element within this enhancer region abolishes estrogen-dependent activity, and chromosome conformation capture and chromatin immunoprecipitation assays show that this distal enhancer communicates with the transcriptional start site of the CA12 gene via intrachromosomal looping upon hormone treatment. This distal enhancer element is observed in the homologous mouse genomic sequence, and the expression of the mouse homologue, Car12, is rapidly and robustly stimulated by estradiol in the mouse uterus in vivo, suggesting that the ER regulation of CA12 is mechanistically and evolutionarily conserved. Our findings highlight the crucial role of ER in the regulation of the CA12 gene, and provide insight into the transcriptional regulatory mechanism that accounts for the strong association of CA12 and ER in human breast cancers.

Sonic hedgehog signaling regulates the expression of insulin-like growth factor binding protein-6 during fetal prostate development.

At the onset of ductal morphogenesis in the developing prostate, Shh expression condenses at evaginations of urogenital sinus epithelium and activates Gli transcription factors in the adjacent mesenchyme. Abrogation of Hedgehog signaling disrupts proper prostatic budding, ductal growth, and branching. We now show that Hedgehog signaling regulates the expression of insulin-like growth factor binding protein-6 (Igfbp-6) in the developing mouse prostate. Igfbp-6 is a secreted factor that specifically binds insulin-like growth factor-II (IGF-II), prevents its binding to the IGF-I receptor, and is thought to regulate the activity of IGF-II in growth and differentiation. Igfbp-6 is expressed in both the developing and adult prostate. In the urogenital sinus, Igfbp-6 mRNA colocalized with Ptc1 and Gli1 mRNA in the mesenchyme, while Igfbp-6 protein was found in both the mesenchymal and epithelial layers. Exogenous Shh peptide induced expression of Igfbp-6 in the developing prostate while the chemical inhibitor of Hedgehog signaling, cyclopamine, reduced its expression. These studies show that Igfbp-6 is an actual target of Shh signaling in the urogenital sinus and provide the first evidence for a linkage between the Hedgehog and IGF signaling pathways in prostate development.

The human prostate expresses sonic hedgehog during fetal development.

PURPOSE: The keynote event of prostate ductal development is the formation of epithelial buds that invade the urogenital sinus mesenchyma. Studies in mice have shown that budding requires the signaling peptide, which is expressed in the epithelium of the prostatic anlagen. We report our characterization of (SHH) expression in the human fetal prostate. MATERIALS AND METHODS: Reverse transcriptase-polymerase chain reaction was performed in fetal prostate RNA isolated at 15.5 and 18 weeks of gestation, respectively. Immunostaining was performed on sections from 7 male fetuses at 9.5 to 34 and in 4 female fetuses at 9 to 18 weeks of gestation. RESULTS: Weak staining for was seen in the prostatic urethra at 9.5 weeks. Intense staining was seen at 11.5 and 13 weeks in the prostatic urothelium and nascent prostatic buds. Staining was slightly diminished at 16.5, further diminished at 18 to 20 and absent at 34 weeks. expression at 15.5 and 18 weeks was confirmed by reverse transcriptase-polymerase chain reaction assay of freshly isolated prostate tissue. Comparative immunostaining in the female showed urothelial staining at 9 and 12 weeks with staining greatest above the entrance of the müllerian ducts. Staining diminished earlier in the female (14 weeks) than in the male and was almost absent at 18 weeks. CONCLUSIONS: expression in the human fetal prostate is contemporaneous with the fetal testosterone surge and with ductal budding of the prostatic urothelium. expression is also present in the female urogenital sinus but in the absence of testosterone it is not associated with ductal budding.

Sonic hedgehog activates mesenchymal Gli1 expression during prostate ductal bud formation.

Ductal budding in the developing prostate is a testosterone-dependent event that involves signaling between the urogenital sinus epithelium (UGE) and urogenital sinus mesenchyme (UGM). We show here that ductal bud formation is associated with focused expression of Sonic hedgehog (Shh) in the epithelium of nascent prostate buds and in the growing tips of elongating prostate ducts. This pattern of localized Shh expression occurs in response to testosterone stimulation. The gene for the Shh receptor, Ptc1, is expressed in the UGM, as are the members of the Gli gene family of transcriptional regulators (Gli1, Gli2, and Gli3). Expression of Ptc1, Gli1, and Gli2 is localized primarily to mesenchyme surrounding prostate buds, whereas Gli3 is expressed diffusely throughout the UGM. A strong dependence of Gli1 (and Ptc1) expression on Shh signaling is demonstrated by induction of expression in both the intact urogenital sinus and the isolated UGM by exogenous SHH peptide. A similar dependence of Gli2 and Gli3 expression on Shh is not observed. Nonetheless, the chemical inhibitor of Shh signaling, cyclopamine, produced a graded inhibition of Gli gene expression (Gli1>Gli2>Gli3) in urogenital sinus explants that was paralleled by a severe inhibition of ductal budding.