Urinary Epidermal Growth Factor as a Marker of Disease Progression in Children With Nephrotic Syndrome.

INTRODUCTION: Childhood-onset nephrotic syndrome has a variable clinical course. Improved predictive markers of long-term outcomes in children with nephrotic syndrome are needed. This study tests the association between baseline urinary epidermal growth factor (uEGF) excretion and longitudinal kidney function in children with nephrotic syndrome. METHODS: The study evaluated 191 participants younger than 18 years enrolled in the Nephrotic Syndrome Study Network, including 118 with their first clinically indicated kidney biopsy (68 minimal change disease; 50 focal segmental glomerulosclerosis) and 73 with incident nephrotic syndrome without a biopsy. uEGF was measured at baseline for all participants and normalized by the urine creatinine (Cr) concentration. Renal epidermal growth factor (EGF) mRNA was measured in the tubular compartment microdissected from kidney biopsy cores from a subset of patients. Linear mixed models were used to test if baseline uEGF/Cr and EGF mRNA expression were associated with change in estimated glomerular filtration rate (eGFR) over time. RESULTS: Higher uEGF/Cr at baseline was associated with slower eGFR decline during follow-up (median follow-up = 30 months). Halving of uEGF/Cr was associated with a decrease in eGFR slope of 2.0 ml/min per 1.73 m2 per year (P < 0.001) adjusted for age, race, diagnosis, baseline eGFR and proteinuria, and APOL1 genotype. In the biopsied subgroup, uEGF/Cr was correlated with EGF mRNA expression (r = 0.74; P < 0.001), but uEGF/Cr was retained over mRNA expression as the stronger predictor of eGFR slope after multivariable adjustment (decrease in eGFR slope of 1.7 ml/min per 1.73 m2 per year per log2 decrease in uEGF/Cr; P < 0.001). CONCLUSION: uEGF/Cr may be a useful noninvasive biomarker that can assist in predicting the long-term course of kidney function in children with incident nephrotic syndrome.

Wnt Signaling Drives Prostate Cancer Bone Metastatic Tropism and Invasion.

Wnt signaling has been implicated as a driver of prostate cancer-related osteoblast differentiation, and previous studies have linked modifications in Wnt function with the induction of tumor metastasis. A unique aspect of prostate cancer bone metastases in mouse models is their relative predilection to the hindlimb (femur) compared to the forelimb (humerus). Comparative gene expression profiling was performed within the humerus and femur from non-tumor-bearing mice to evaluate differences in the microenvironments of these locations. This revealed the relative overexpression of the Wnt signaling inhibitors WIF1 and SOST in the humerus compared to the femur, with increased WNT5A expression in femur bone marrow, suggesting a coordinated upregulation of Wnt signals within the femur compared to the humerus. Conditioned medium (CM) from bone marrow stromal cells (HS-5 cells) was used to mimic the bone marrow microenvironment, which strongly promoted prostate cancer cell invasion (3.3-fold increase in PC3 cells, P < .05; 7-fold increase in LNCaP cells, P < .05). WNT5A shRNA knockdown within the CM-producing HS-5 cells significantly decreased PC3 (56%, P < .05) and LNCaP (60%, P < .05) cell invasion. Similarly, preincubation of CM with WIF1 significantly blocked LNCaP cell invasion (40%, P < .05). shRNA-mediated knockdown of the Wnt receptors FZD4 and FZD8 also strongly inhibited tumor cell invasion (60% inhibition shFZD4, P < .05; 63% shFZD8, P < .05). Furthermore, small molecule inhibition of JNK, which is an important component of the noncanonical Wnt signaling pathway, significantly inhibited CM-mediated tumor invasion. Overall, this study reveals a role for Wnt signaling as a driver of prostate cancer bone metastatic tropism and invasion.

Targeted NF1 cancer therapeutics with multiple modes of action: small molecule hormone-like agents resembling the natural anticancer metabolite, 2-methoxyoestradiol.

BACKGROUND: Both the number and size of tumours in NF1 patients increase in response to the rise in steroid hormones seen at puberty and during pregnancy. The size of tumours decreases after delivery, suggesting that hormone-targeting therapy might provide a viable new NF1 treatment approach. Our earlier studies demonstrated that human NF1 tumour cell lines either went through apoptosis or ceased growth in the presence of 2-methoxyoestradiol (2ME2), a naturally occurring anticancer metabolite of 17-ß estradiol. Previous reports of treatment with sulfamoylated steroidal and non-steroidal derivatives of 2ME2 showed promising reductions in tumour burden in hormone-responsive cancers other than NF1. Here we present the first studies indicating that 2ME2 derivatives could also provide an avenue for treating NF1, for which few treatment options are available. METHODS: STX3451, (2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), a non-steroidal sulphamate analogue of 2ME2, was tested in dose-dependent studies of malignant and benign NF1 human tumour cell lines and cell lines with variable controlled neurofibromin expression. The mechanisms of action of STX3451 were also analysed. RESULTS: We found that STX3451-induced apoptosis in human malignant peripheral nerve sheath tumour (MPNST) cell lines, even in the presence of elevated oestrogen and progesterone. It inhibits both PI3 kinase and mTOR signalling pathways. It disrupts actin- and microtubule-based cytoskeletal structures in cell lines derived from human MPNSTs and in cells derived from benign plexiform neurofibromas. STX3451 selectively kills MPNST-derived cells, but also halts growth of other tumour-derived NF1 cell lines. CONCLUSION: STX3451 provides a new approach for inducing cell death and lowering tumour burden in NF1 and other hormone-responsive cancers with limited treatment options.

Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear.

This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system 'inflammatory' cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development.

Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells.

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.

The role of steroid hormones in the NF1 phenotype: focus on pregnancy.

The Neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. Loss of its protein, neurofibromin, in the autosomal dominant disorder NF1 is associated with peripheral nervous system tumors, particularly neurofibromas, benign lesions in which the major cell type is the Schwann Cell (SC). Benign and malignant human tumors found in NF1 patients are heterogeneous with respect to their cellular composition. The number and size of neurofibromas in NF1 patients has been shown to increase during pregnancy, with, in some cases, post-partum regression, which suggests hormonal involvement in this increase. However, in this review, we consider evidence from the literature that both direct hormonal influence on tumor growth and on angiogenesis may contribute to these effects.

Influence of hormones and hormone metabolites on the growth of Schwann cells derived from embryonic stem cells and on tumor cell lines expressing variable levels of neurofibromin.

Loss of neurofibromin, the protein product of the tumor suppressor gene neurofibromatosis type 1 (NF1), is associated with neurofibromas, composed largely of Schwann cells. The number and size of neurofibromas in NF1 patients have been shown to increase during pregnancy. A mouse embryonic stem cell (mESC) model was used, in which mESCs with varying levels of neurofibromin were differentiated into Schwann-like cells. NF1 cell lines derived from a malignant and a benign human tumor were used to study proliferation in response to hormones. Estrogen and androgen receptors were not expressed or expressed at very low levels in the NF1+/+ cells, at low levels in NF1+/-cells, and robust levels in NF1-/-cells. A 17beta-estradiol (E2) metabolite, 2-methoxy estradiol (2ME2) is cytotoxic to the NF1-/- malignant tumor cell line, and inhibits proliferation in the other cell lines. 2ME2 or its derivatives could provide new treatment avenues for NF1 hormone-sensitive tumors at times of greatest hormonal influence.

A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation.

The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/-), or null (-/-) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant.

Efficient formation of uniform-sized embryoid bodies using a compartmentalized microchannel device.

The formation of spherical aggregates of cells called embryoid bodies (EBs) is an indispensable step in many protocols in which embryonic stem (ES) cells are differentiated to other cell types. Appropriate morphology and embryo size are critical for the sequential developmental stages of naturally conceived embryos. Likewise, regulating the size of EBs and the timing of their formation is crucial for controlling the differentiation of ES cells within the EB. Existing methods of formation of EBs, however, are tedious or provide heterogeneously-sized EBs. Here we describe a microfluidic system for straightforward synchronized formation of uniform-sized EBs, the size of which can be controlled by changing the cross-sectional size of microchannels in the microfluidic device. The device consists of two microchannels separated by a semi-porous polycarbonate membrane treated to be resistant to cell adhesion. ES cells introduced into the upper channel self-aggregate to form uniformly-sized EBs. The semi-porous membrane also allows subsequent treatment of the non-attached EBs with different reagents from the lower channel without the need for wash out because of the compartmentalization afforded by the membrane. This method provides a simple yet robust means to control the formation of EBs and the subsequent differentiation of ES cells in a format compatible for ES cell processing on a chip.

Immortalized mouse inner ear cell lines demonstrate a role for chemokines in promoting the growth of developing statoacoustic ganglion neurons.

The target-derived factors necessary for promoting initial outgrowth from the statoacoustic ganglion (SAG) to the inner ear have not been fully characterized. In the present study, conditioned medium from embryonic Immortomouse inner ear cell lines that maintain many characteristics of developing inner ear sensory epithelia were screened for neurite-promoting activity. Conditioned medium found to be positive for promoting SAG neurite outgrowth and neuronal survival was then tested for the presence of chemokines, molecules that have not previously been investigated for promoting SAG outgrowth. One candidate molecule, monocyte chemotactic protein 1 (MCP-1), was detected in the conditioned medium and subsequently localized to mouse hair cells by immunocytochemistry. In vitro studies demonstrated that function-blocking MCP-1 antibodies decreased the amount of SAG neurite outgrowth induced by the conditioned medium and that subsequent addition of MCP-1 protein was able to promote outgrowth when added to the antibody-treated conditioned medium. The use of the Immortomouse cell lines proved valuable in identifying this candidate cofactor that promotes outgrowth of early-stage SAG nerve fibers and is expressed in embryonic hair cells.

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in Schizosaccharomyces pombe.

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 degrees C and 30 degrees C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 degrees C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 degrees C compared to supplemented cells at 30 degrees C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.

The adaptor protein fish associates with members of the ADAMs family and localizes to podosomes of Src-transformed cells.

Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domain-containing proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes.