Epithelial and Neutrophil Interactions and Coordinated Response to Shigella in a Human Intestinal Enteroid-Neutrophil Coculture Model.

Polymorphonuclear neutrophils (PMN) are recruited to the gastrointestinal mucosa in response to inflammation, injury, and infection. Here, we report the development and the characterization of an ex vivo tissue coculture model consisting of human primary intestinal enteroid monolayers and PMN, and a mechanistic interrogation of PMN-epithelial cell interaction and response to Shigella, a primary cause of childhood dysentery. Cellular adaptation and tissue integration, barrier function, PMN phenotypic and functional attributes, and innate immune responses were examined. PMN within the enteroid monolayers acquired a distinct activated/migratory phenotype that was influenced by direct epithelial cell contact as well as by molecular signals. Seeded on the basal side of the intestinal monolayer, PMN were intercalated within the epithelial cells and moved paracellularly toward the apical side. Cocultured PMN also increased basal secretion of interleukin 8 (IL-8). Shigella added to the apical surface of the monolayers evoked additional PMN phenotypic adaptations, including increased expression of cell surface markers associated with chemotaxis and cell degranulation (CD47, CD66b, and CD88). Apical Shigella infection triggered rapid transmigration of PMN to the luminal side, neutrophil extracellular trap (NET) formation, and bacterial phagocytosis and killing. Shigella infection modulated cytokine production in the coculture; apical monocyte chemoattractant protein (MCP-1), tumor necrosis factor alpha (TNF-α), and basolateral IL-8 production were downregulated, while basolateral IL-6 secretion was increased. We demonstrated, for the first time, PMN phenotypic adaptation and mobilization and coordinated epithelial cell-PMN innate response upon Shigella infection in the human intestinal environment. The enteroid monolayer-PMN coculture represents a technical innovation for mechanistic interrogation of gastrointestinal physiology, host-microbe interaction, innate immunity, and evaluation of preventive/therapeutic tools. IMPORTANCE Studies of mucosal immunity and microbial host cell interaction have traditionally relied on animal models and in vitro tissue culture using immortalized cancer cell lines, which yield nonphysiological and often unreliable results. Herein, we report the development and characterization of an ex vivo enteroid-PMN coculture consisting of normal human intestinal epithelium and a mechanistic interrogation of PMN and epithelial cell interaction and function in the context of Shigella infection. We demonstrated tissue-driven phenotypic and functional adaptation of PMN and a coordinated epithelial cell and PMN response to Shigella, a primary cause of dysentery in young children in the developing world.

Depletion of the apical endosome in response to viruses and bacterial toxins provides cell-autonomous host defense at mucosal surfaces.

Polarized epithelial cells form an essential barrier against infection at mucosal surfaces. Many pathogens breach this barrier to cause disease, often by co-opting cellular endocytosis mechanisms to enter the cell through the lumenal (apical) cell surface. We recently discovered that the loss of the cell polarity gene PARD6B selectively diminishes apical endosome function. Here, we find that in response to the entry of certain viruses and bacterial toxins into the epithelial cells via the apical membrane, PARD6B and aPKC, two components of the PARD6B-aPKC-Cdc42 apical polarity complex, undergo rapid proteasome-dependent degradation. The perturbation of apical membrane glycosphingolipids by toxin- or virus-binding initiates degradation of PARD6B. The loss of PARD6B causes the depletion of apical endosome function and renders the cell resistant to further infection from the lumenal cell surface, thus enabling a form of cell-autonomous host defense.

Occurrence of Ulcerative Lesions in Sea Stars (Asteroidea) of theNorthern Gulf of California, USA.

To assess the variation of sea star (Asteroidea) populations in the Northern Gulf of California and look for evidence of sea star ulcerative lesions, counts of sea star species were recorded in 2019 using a standard belt-transect technique of 30 m2. During surveys, scuba divers also measured sea stars' diameter and examined them for ulcerative lesions. Ulcerative lesions were noted on Phataria unifascialis only. To verify changes in abundance and size of the two most abundant species, P. unifascialis and Pharia pyramidata, historical data from 2009, 2010, and 2016 were used as comparison and using the same methodology. To evaluate differences in abundance or diameter in sea star species over time, analysis of variance tests were used. We found a significant reduction in diameter for the species P. unifascialis and P. pyramidata over time (<0.0001), but only P. unifascialis also showed a significant decrease in abundance (P=0.018). The decrease in diameter recorded for these two species, along with the signs of ulcerative lesions found on one of them in 2019, suggest that a potential mortality event occurred and, as a result, new younger (i.e., smaller) recruits could be recovering the population. These results highlight the importance of population monitoring to understand complex reef community dynamics.

Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

Improved Radium-223 Therapy with Combination Epithelial Sodium Channel Blockade.

Background: Radium-223 dichloride ([223Ra]RaCl2) is the first approved alpha particle-emitting therapy and is indicated for treatment of bone metastatic castrate resistant prostate cancer. Approximately half of the dose is absorbed into the gastrointestinal (GI) tract within minutes of administration, limiting disease-site uptake and contributing to toxicity. Here, we investigate the role of enteric ion channels and their modulation for improved therapeutic efficacy and reduced side effects. Methods: Utilizing primary human duodenal organoids (enteroids) as in vitro models of the functional GI epithelium, we found that Amiloride (ENaC blocker) and NS-1619 (K+ channel activator) presented significant effects in 223Ra membranal transport. The radioactive drug distribution was evaluated for lead combinations in vivo, and in osteosarcoma and prostate cancer models. Results: Amiloride shifted 223Ra uptake in vivo from the gut, to nearly double the uptake at sites of bone remodeling. Bone tumor growth inhibition with the combination as measured by bioluminescent and X-ray imaging was significantly greater than single agents alone, and the combination resulted in no weight loss. Conclusion: This combination of approved agents may be readily implemented as a clinical approach to improve outcomes of bone metastatic cancer patients with the benefit of ameliorated tolerability.

Epithelial WNT2B and Desert Hedgehog Are Necessary for Human Colonoid Regeneration after Bacterial Cytotoxin Injury.

Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli-secreted cytotoxin EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

Evaluating Shigella flexneri Pathogenesis in the Human Enteroid Model.

The enteric pathogen Shigella is one of the leading causes of moderate-to-severe diarrhea and death in young children in developing countries. Transformed cell lines and animal models have been widely used to study Shigella pathogenesis. In addition to altered physiology, transformed cell lines are composed of a single cell type that does not sufficiently represent the complex multicellular environment of the human colon. Most available animal models do not accurately mimic human disease. The human intestinal enteroid model, derived from LGR5+ stem cell-containing intestinal crypts from healthy subjects, represents a technological leap in human gastrointestinal system modeling and provides a more physiologically relevant system that includes multiple cell types and features of the human intestine. We established the utility of this model for studying basic aspects of Shigella pathogenesis and host responses. In this study, we show that Shigellaflexneri is capable of infecting and replicating intracellularly in human enteroids derived from different segments of the intestine. Apical invasion by S. flexneri is very limited but increases ∼10-fold when enteroids are differentiated to include M cells. Invasion via the basolateral surface was at least 2-log10 units more efficient than apical infection. Increased secretion of interleukin-8 and higher expression levels of the mucin glycoprotein Muc2 were observed in the enteroids following S. flexneri infection. The human enteroid model promises to bridge some of the gaps between traditional cell culture, animal models, and human infection.

Enterohemorrhagic E. coli (EHEC)-Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers.

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

Label-free Raman spectroscopy provides early determination and precise localization of breast cancer-colonized bone alterations.

Breast neoplasms frequently colonize bone and induce development of osteolytic bone lesions by disrupting the homeostasis of the bone microenvironment. This degenerative process can lead to bone pain and pathological bone fracture, a major cause of cancer morbidity and diminished quality of life, which is exacerbated by our limited ability to monitor early metastatic disease in bone and assess fracture risk. Spurred by its label-free, real-time nature and its exquisite molecular specificity, we employed spontaneous Raman spectroscopy to assess and quantify early metastasis driven biochemical alterations to bone composition. As early as two weeks after intracardiac inoculations of MDA-MB-435 breast cancer cells in NOD-SCID mice, Raman spectroscopic measurements in the femur and spine revealed consistent changes in carbonate substitution, overall mineralization as well as crystallinity increase in tumor-bearing bones when compared with their normal counterparts. Our observations reveal the possibility of early stage detection of biochemical changes in the tumor-bearing bones - significantly before morphological variations are captured through radiographic diagnosis. This study paves the way for a better molecular understanding of altered bone remodeling in such metastatic niches, and for further clinical studies with the goal of establishing a non-invasive tool for early metastasis detection and prediction of pathological fracture risk in breast cancer.

CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells.

The transcriptional co-regulator Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2 (CITED2) may promote breast tumor growth; however, the mechanisms by which its effects are mediated remain to be fully elucidated. Tumor-associated macrophages serve an important function in tumor development and progression and are recruited by chemotactic factors produced by cells within the tumor microenvironment. The present study assessed the effects of CITED2 silencing on macrophage recruitment in two xenograft mouse models of human breast cancer, one in which tumor growth was sensitive to CITED2 silencing (MDA-MB-231) and one in which it was insensitive (MDA-MB-468). The present study identified that silencing CITED2 significantly attenuated macrophage infiltration in MDA-MB-231 but not MDA-MB-468 orthotopic tumors, concordant with its effect on tumor growth. Correspondingly, conditioned media obtained from CITED2-silenced MDA-MB-231 cells exhibited a significantly decreased ability to induce macrophage recruitment by Transwell migration assay, whereas the chemotactic effect of MDA-MB-468 conditioned media was unaffected. Examining the expression of macrophage chemoattractants within orthotopic tumors and tumor cell-conditioned media revealed a significant decrease in C-C motif chemokine ligand (CCL)20 mRNA and protein expression following CITED2-silencing in MDA-MB-231 cells, compared with that in cells transfected with scramble shRNA. However, mRNA and protein expression was unaffected by CITED2-silencing in MDA-MB-468 cells. Furthermore, chromatin immunoprecipitation analysis revealed that CITED2 was localized to the CCL20 promoter in MDA-MB-231 cells, suggesting that it serves a direct function in its regulation, which is consistent with the effect of CITED2 silencing on CCL20 expression. Lastly, neutralizing CCL20 in the conditioned media of MDA-MB-231 cells significantly inhibited macrophage recruitment. Collectively, these results suggest that CITED2 is involved in modulating macrophage recruitment, representing a novel mechanism through which it may influence tumor growth. This may be partly mediated by regulating tumor cell production of the chemokine CCL20.

Enterotoxigenic Escherichia coli is phagocytosed by macrophages underlying villus-like intestinal epithelial cells: modeling ex vivo innate immune defenses of the human gut.

There is a paucity of information on diarrheagenic enterotoxigenic Escherichia coli (ETEC)'s interaction with innate immune cells, in part due to the lack of reliable models that recapitulate infection in human gut. In a recent publication, we described the development of an ex vivo enteroid-macrophage co-culture model using human primary cells. We reported that macrophages residing underneath the epithelial monolayer acquired "resident macrophage" phenotype characterized by lower production of inflammatory cytokines and strong phagocytic activity. These macrophages extended projections across the epithelium, which captured ETEC applied to the apical side of the epithelium and reduced luminal bacterial load. Additional evidence presented in this addendum confirms these findings and further demonstrates that macrophage adaptation occurs regardless of the stage of differentiation of epithelial cells, and that ETEC uptake arises rapidly after infection. The enteroid-macrophage co-culture represents a novel and relevant tool to study host-cell interactions and pathogenesis of enteric infections in humans.

Down-regulation of CITED2 attenuates breast tumor growth, vessel formation and TGF-ß-induced expression of VEGFA.

While we previously demonstrated that CITED2 expression in primary breast tumor tissues is elevated relative to normal mammary epithelium and inversely correlated with patient survival, its functional impact on primary tumor development and progression remained unknown. To address this issue, we examined the effect of CITED2 silencing on the growth of human breast cancer cell lines MDA-MB-231 and MDA-MB-468 following orthotopic administration in vivo. Here, we show that CITED2 silencing significantly attenuated MDA-MB-231 primary tumor growth concordant with reduced tumor vascularization, while MDA-MB-468 primary tumor growth and tumor vascularization remained unaffected. Correspondingly, expression of VEGFA was significantly reduced in shCITED2-expressing MDA-MB-231, but not MDA-MB-468 tumors. Consistent with the observed pattern of vascularization and VEGFA expression, we found that TGF-ß stimulation induced expression of VEGFA and enhanced CITED2 recruitment to the VEGFA promoter in MDA-MA-231 cells, while failing to induce VEGFA expression in MDA-MB-468 cells. Further supporting its involvement in TGF-ß-induced expression of VEGFA, CITED2 silencing prevented TGF-ß induction of VEGFA expression in MDA-MB-231 cells. Collectively, these data indicate that CITED2 regulates primary breast tumor growth, likely by influencing tumor vasculature via TGF-ß-dependent regulation of VEGFA.

The hypoxia-inducible factor-1α activates ectopic production of fibroblast growth factor 23 in tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome in which ectopic production of fibroblast growth factor 23 (FGF23) by non-malignant mesenchymal tumors causes phosphate wasting and bone fractures. Recent studies have implicated the hypoxia-inducible factor-1α (HIF-1α) in other phosphate wasting disorders caused by elevated FGF23, including X-linked hypophosphatemic rickets and autosomal dominant hypophosphatemia. Here we provide evidence that HIF-1α mediates aberrant FGF23 in TIO by transcriptionally activating its promoter. Immunohistochemical studies in phosphaturic mesenchymal tumors resected from patients with documented TIO showed that HIF-1α and FGF23 were co-localized in spindle-shaped cells adjacent to blood vessels. Cultured tumor tissue produced high levels of intact FGF23 and demonstrated increased expression of HIF-1α protein. Transfection of MC3T3-E1 and Saos-2 cells with a HIF-1α expression construct induced the activity of a FGF23 reporter construct. Prior treatment of tumor organ cultures with HIF-1α inhibitors decreased HIF-1α and FGF23 protein accumulation and inhibited HIF-1α-induced luciferase reporter activity in transfected cells. Chromatin immunoprecipitation assays confirmed binding to a HIF-1α consensus sequence within the proximal FGF23 promoter, which was eliminated by treatment with a HIF-1α inhibitor. These results show for the first time that HIF-1α is a direct transcriptional activator of FGF23 and suggest that upregulation of HIF-1α activity in TIO contributes to the aberrant FGF23 production in these patients.

CITED2 Modulates Breast Cancer Metastatic Ability through Effects on IKKα.

UNLABELLED: Previously, we identified the transcriptional coactivator CITED2 as a potential facilitator of bone metastasis using a murine mammary cancer model. Extending these studies to human breast cancer, it was observed that CITED2 mRNA expression was significantly elevated in patient specimens of metastatic breast cancer relative to primary tumors, with highest levels in metastasis to bone relative to non-bone sites. To further evaluate CITED2 functions in breast cancer metastasis, CITED2 expression was stably reduced in the human breast cancer cell lines MDA-MB-231 and MDA-MB-468, which are metastatic in animal models. While CITED2 knockdown had no effect on cell proliferation, cell migration and invasion were significantly reduced, as was the establishment of metastasis following intracardiac administration in athymic nude mice. To explore the mechanism behind these effects, gene expression following CITED2 knockdown in MDA-MB-231 cells by cDNA microarray was performed. As confirmed at the mRNA and protein levels in both MDA-MB-231 and MDA-MB-468 cells, expression of the NF-κB regulator IKKα was significantly reduced, along with several NF-κB targets with known roles in metastasis (OPN, MMP9, uPA, SPARC, IL11, and IL1ß). Furthermore, ChIP assay revealed recruitment of CITED2 to the promoter of IKKα, indicating a direct role in regulating its expression. Consistent with reduced IKKα expression, CITED2 knockdown inhibited both canonical and noncanonical NF-κB signaling. Finally, restoration of IKKα expression following CITED2 knockdown in MDA-MB-231 and MDA-MB-468 cells rescued their invasive ability. Collectively, these data demonstrate that CITED2 modulates metastatic ability in human breast cancer cells, at least in part, through the regulation of IKKα. IMPLICATIONS: The current study highlights the role of CITED2 in facilitating breast cancer metastasis, partly via regulation of IKKα. Mol Cancer Res; 14(8); 730-9. ©2016 AACR.

CCL20/CCR6 Signaling Regulates Bone Mass Accrual in Mice.

CCL20 is a member of the macrophage inflammatory protein family and is reported to signal monogamously through the receptor CCR6. Although studies have identified the genomic locations of both Ccl20 and Ccr6 as regions important for bone quality, the role of CCL20/CCR6 signaling in regulating bone mass is unknown. By micro-computed tomography (µCT) and histomorphometric analysis, we show that global loss of Ccr6 in mice significantly decreases trabecular bone mass coincident with reduced osteoblast numbers. Notably, CCL20 and CCR6 were co-expressed in osteoblast progenitors and levels increased during osteoblast differentiation, indicating the potential of CCL20/CCR6 signaling to influence osteoblasts through both autocrine and paracrine actions. With respect to autocrine effects, CCR6 was found to act as a functional G protein-coupled receptor in osteoblasts and although its loss did not appear to affect the number or proliferation rate of osteoblast progenitors, differentiation was significantly inhibited as evidenced by delays in osteoblast marker gene expression, alkaline phosphatase activity, and mineralization. In addition, CCL20 promoted osteoblast survival concordant with activation of the PI3K-AKT pathway. Beyond these potential autocrine effects, osteoblast-derived CCL20 stimulated the recruitment of macrophages and T cells, known facilitators of osteoblast differentiation and survival. Finally, we generated mice harboring a global deletion of Ccl20 and found that Ccl20(-/-) mice exhibit a reduction in bone mass similar to that observed in Ccr6(-/-) mice, confirming that this phenomenon is regulated by CCL20 rather than alternate CCR6 ligands. Collectively, these data indicate that CCL20/CCR6 signaling may play an important role in regulating bone mass accrual, potentially by modulating osteoblast maturation, survival, and the recruitment of osteoblast-supporting cells. © 2016 American Society for Bone and Mineral Research.

Whole-Body and Microenvironmental Localization of Radium-223 in Naïve and Mouse Models of Prostate Cancer Metastasis.

BACKGROUND: Bone-metastatic, castration-resistant prostate cancer (bmCRPC) represents a lethal stage of the most common noncutaneous cancer in men. The recent introduction of Radium-223 dichloride, a bone-seeking alpha particle (α)-emitting radiopharmaceutical, demonstrates statistically significant survival benefit and palliative effect for bmCRPC patients. Clinical results have established safety and efficacy, yet questions remain regarding pharmacodynamics and dosing for optimized patient benefit. METHODS: We elucidated the biodistribution of (223)Ra as well as interaction with the bone and tumor compartments in skeletally mature mice (C57Bl/6 and CD-1, n = 3-6) and metastasis models (LNCaP and PC3, n = 4). Differences in uptake were evaluated by µCT and histological investigation. Novel techniques were leveraged on whole-mount undecalcified cryosections to determine microdistribution of Radium-223. All statistical tests were two-sided. RESULTS: (223)Ra uptake in the bones (>30% injected activity per gram) at 24 hours was also accompanied by non-negligible remnant activity in the kidney (2.33% ± 0.36%), intestines (5.73% ± 2.04%), and spleen (10.5% ± 5.9%) Skeletal accumulation across strains did not correspond with bone volume or surface area but instead to local blood vessel density (P = .04). Microdistribution analysis by autoradiography and α camera revealed targeting of the ossifying surfaces adjacent to the epiphyseal growth plate. In models of PCa metastasis, radioactivity does not localize directly within tumors but instead at the apposite bone surface. Osteoblastic and lytic lesions display similar intensity, which is comparable with uptake at sites of normal bone remodeling. CONCLUSIONS: Profiling the macro- and microdistribution of (223)Ra in healthy and diseased models has important implications to guide precision application of this emerging α-therapy approach for bmCRPC and other bone metastastic diseases.

Diagnostic imaging learning resources evaluated by students and recent graduates.

Many learning resources can help students develop the problem-solving abilities and clinical skills required for diagnostic imaging. This study explored veterinary students' perceptions of the usefulness of a variety of learning resources. Perceived resource usefulness was measured for different levels of students and for academic versus clinical preparation. Third-year (n=139) and final (fifth) year (n=105) students and recent graduates (n=56) completed questionnaires on perceived usefulness of each resource. Resources were grouped for comparison: abstract/low complexity (e.g., notes, multimedia presentations), abstract/high complexity (e.g., Web-based and film case repositories), concrete/low complexity (e.g., large-group "clicker" workshops), and concrete/high complexity (e.g., small-group interpretation workshops). Lower-level students considered abstract/low-complexity resources more useful for academic preparation and concrete resources more useful for clinical preparation. Higher-level students/recent graduates also considered abstract/low-complexity resources more useful for academic preparation. For all levels, lecture notes were considered highly useful. Multimedia slideshows were an interactive complement to notes. The usefulness of a Web-based case repository was limited by accessibility problems and difficulty. Traditional abstract/low-complexity resources were considered useful for more levels and contexts than expected. Concrete/high-complexity resources need to better represent clinical practice to be considered more useful for clinical preparation.

CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells.

Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator of ER in breast cancer cells and that its increased expression in tumors may result in estrogen-independent ER activation, thereby reducing estrogen dependence and response to anti-estrogen therapy.

Enpp1: a potential facilitator of breast cancer bone metastasis.

Bone is the most common site of breast cancer metastasis and once established, it is frequently incurable. Critical to our ability to prevent and treat bone metastasis is the identification of the key factors mediating its establishment and understanding their biological function. To address this issue we previously carried out an in vivo selection process to isolate murine mammary tumor sublines possessing an enhanced ability to colonize the bone. A comparison of gene expression between parental cells and sublines by genome-wide cDNA microarray analysis revealed several potential mediators of bone metastasis, including the pyrophosphate-generating ectoenzyme Enpp1. By qRT-PCR and Western analysis we found that expression of Enpp1 was elevated in human breast cancer cell lines known to produce bone metastasis in animal models compared to non-metastatic and normal mammary epithelial cell lines. Further, in clinical specimens, levels of Enpp1 were significantly elevated in human primary breast tumors relative to normal mammary epithelium, with highest levels observed in breast-bone metastasis as determined by qRT-PCR and immunohistochemical analysis. To examine the potential role of Enpp1 in the development of bone metastasis, Enpp1 expression was stably increased in the breast cancer cell line MDA-MB-231 and the ability to colonize the bone following intracardiac and direct intratibial injection of athymic nude mice was determined. By both routes of administration, increased expression of Enpp1 enhanced the ability of MDA-MB-231 cells to form tumors in the bone relative to cells expressing vector alone, as determined by digital radiography and histological analysis. Taken together, these data suggest a potential role for Enpp1 in the development of breast cancer bone metastasis.

MIP-1δ activates NFATc1 and enhances osteoclastogenesis: involvement of both PLCγ2 and NFκB signaling.

Pathological bone resorption is a source of significant morbidity in diseases affecting the skeleton such as rheumatoid arthritis, periodontitis, and cancer metastasis to bone. Evidence indicates that elevated levels of inflammatory mediators such as IL-1, IL-6, and TNF-α play a role in this process by promoting the formation of bone-resorbing osteoclasts. Additionally, current studies have identified inflammatory chemokines of the macrophage inflammatory protein (MIP) family as potential mediators of pathological bone resorption, where both MIP-1α and -3α have been shown to enhance osteoclast (OCL) development. In this study we provide evidence that MIP-1δ, whose expression is associated with renal cell carcinoma bone metastasis and rheumatoid arthritis, enhances OCL formation in vitro via a direct effect on OCL precursors. Consistent with this ability, exposure of OCL precursors to MIP-1δ resulted in the activation of PLCγ2 and NF-κB, two signaling pathways known to regulate OCL differentiation. Moreover, MIP-1δ induced expression and nuclear translocation of NFATc1, a master regulator of osteoclastogenesis, which was dependent on activation of both the PLCγ2 and NFκB signaling pathways. Lastly, consistent with in vitro studies, in vivo administration of MIP-1δ significantly increased OCL number and resorption area as determined using a murine calvarial bone resorption model. Taken together, these data highlight the potential of MIP-1δ as a mediator of pathological bone resorption and provide insight into the molecular mechanism through which MIP-1δ enhances osteoclastogenesis.