Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1).

Interleukin-like EMT inducer (ILEI, FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell-biological process that confers metastatic properties to a tumor cell. However, very little is known about how ILEI is regulated. Here we demonstrate that ILEI is an in vivo regulator of melanoma invasiveness and is transcriptionally up-regulated by the upstream stimulatory factor-1 (USF-1), an E-box-binding, basic-helix-loop-helix family transcription factor. shRNA-mediated knockdown of ILEI in melanoma cell lines attenuated lung colonization but not primary tumor formation. We also identified the mechanism underlying ILEI transcriptional regulation, which was through a direct interaction of USF-1 with the ILEI promoter. Of note, stimulation of endogenous USF-1 by UV-mediated activation increased ILEI expression, whereas shRNA-mediated USF-1 knockdown decreased ILEI gene transcription. Finally, we report that knocking down USF-1 decreases tumor cell migration. In summary, our work reveals that ILEI contributes to melanoma cell invasiveness in vivo without affecting primary tumor growth and is transcriptionally up-regulated by USF-1.

Genomic amplifications identified by circulating tumor DNA analysis guide prognosis in metastatic castration-resistant prostate cancer.

Purpose: Analysis of circulating tumor DNA (ctDNA) in patients with metastatic prostate cancer (mPC) provides an opportunity to identify and monitor genomic alterations during a patient's treatment course. We evaluated whether the presence of specific gene amplifications (GAs) and plasma copy number (PCN) alterations are associated with disease features. Methods: This is a single-institution retrospective study of patients with mPC who underwent ctDNA profiling using Guardant360® (Guardant Health Inc.). This test identifies single nucleotide variants (SNVs) and GAs of select genes by next-generation sequencing. A total of 155 men with mPC were studied. Patients were stratified by GA status. The Kaplan-Meier method and multivariate cox regression models were used to estimate overall survival (OS) or failure-free survival (FFS) from either the date of GA detection or the initiation of systemic therapy. The chi-square test was used to evaluate associations between clinical factors and GAs. Results: The presence of liver and/or lung metastases was associated with GAs of BRAF, CDK6, PI3KCA, and FGFR1. Survival analyses were completed on a subset of 83 patients with metastatic castration-resistant prostate cancer (mCRPC). Median OS was improved in patients with 1 GA compared to patients with ≥2 GAs, whether determined from the date of initial GA(s) detection (14.9 mo vs. 8.9 mo) or date of therapy initiation nearest to GA detection (16.7 mo vs. 9.0 mo). Patients without GAs had not reached median OS. Patients with androgen receptor (AR) GA only were also found to have better median OS compared to patients with AR GA plus at least one other additional GA (19.3 mo vs. 8.9 mo). Patients with PIK3CA GA had significantly lower median OS compared to patients with GAs that did not have a PIK3CA GA (5.9 mo vs. 16.0 mo). In patients with AR and/or MYC GA(s), median OS improved in those with reduced AR or MYC PCN during therapy compared to those without such a reduction (25.1 mo vs. 15.9 mo). Conclusions: The association of select GAs with survival provides an additional tool for assessing mCRPC prognosis and informing management. Serial monitoring of ctDNA GAs is also useful to guide prognosis and therapeutic response.

Histone deacetylase inhibition is cytotoxic to oligodendrocyte precursor cells in vitro and in vivo.

Histone deacetylase (HDAC) inhibition mediated by small molecule HDAC inhibitors (HDACi) has demonstrated divergent effects including toxicity towards transformed cell lines, neuroprotection in neurological disease models, and inhibition of oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes (OL). However, it remains unknown if transient HDAC inhibition may promote OPC survival. Using mouse cortical OPC primary cultures, we investigated the effects of the FDA approved pan-HDACi suberoylanilide hydroxamic acid (SAHA) on OPC survival. Initial studies showed differences in the HDAC expression pattern of multiple HDAC isoforms in OPCs relative to their terminally differentiated progeny cells, OLs and astrocytes. Treatment of OPCs with SAHA for up to 72h using a maximum concentration either at or lower than those necessary for cytotoxicity in most transformed cell lines resulted in over 67% reduction in viability relative to vehicle-treated OPCs. This was at least partly due to increased apoptosis as SAHA-treated cells displayed activated caspase 3 and were protected by the general caspase inhibitor Q-VD-OPH. Additionally, SAHA treatment of whole mice at postnatal day 5 induced apoptosis of cortical OPCs. These results suggest that SAHA negatively impacts OPC survival and may be detrimental to the myelinating brain and spinal cord. Such toxicity may be relevant in a clinical context as SAHA is currently involved in numerous clinical trials and is in consideration for use in the treatment of psychiatric and neurodegenerative conditions.

Isolation of cortical mouse oligodendrocyte precursor cells.

The reliable isolation of primary oligodendrocyte progenitors cells (OPCs) holds promise as both a research tool and putative therapy for the study and treatment of central nervous system (CNS) disease and trauma. Stringently characterized primary mouse OPCs is of additional importance due to the power of transgenics to address mechanism(s) involving single genes. In this study, we developed and characterized a reproducible method for the primary culture of OPCs from postnatal day 5-7 mouse cerebral cortex. We enriched an O4(+) OPC population using Magnetic Activated Cell Sorting (MACS) technology. This technique resulted in an average yield of 3.68×10(5)OPCs/brain. Following isolation, OPCs were glial fibrillary acidic protein(-) (GFAP(-)) and O4(+). Following passage and with expansion, OPCs were O4(+), A2B5(+), and NG2(+). Demonstrating their bi-potentiality, mouse OPCs differentiated into either more complex, highly arborized O4(+) or O1(+) oligodendrocytes (OLs) or GFAP(+) astrocytes. This bi-potentiality is lost, however, in co-culture with rat embryonic day 15 derived dorsal root ganglia (DRG). Following 7-14 days of OPC/DRG co-culture, OPCs aligned with DRG neurites and differentiated into mature OLs as indicated by the presence of O1 and myelin basic protein (MBP) immunostaining. Addition of ciliary neurotrophic factor (CNTF) to conditioned media from OPC/DRG co-cultures improved OPC differentiation into mature O1(+) and MBP(+) OLs. This method allows for the study of primary mouse cortical OPC survival, maturation, and function without relying on oligosphere formation or the need for extensive passaging.

Concordant association of insulin degrading enzyme gene (IDE) variants with IDE mRNA, Abeta, and Alzheimer's disease.

BACKGROUND: The insulin-degrading enzyme gene (IDE) is a strong functional and positional candidate for late onset Alzheimer's disease (LOAD). METHODOLOGY/PRINCIPAL FINDINGS: We examined conserved regions of IDE and its 10 kb flanks in 269 AD cases and 252 controls thereby identifying 17 putative functional polymorphisms. These variants formed eleven haplotypes that were tagged with ten variants. Four of these showed significant association with IDE transcript levels in samples from 194 LOAD cerebella. The strongest, rs6583817, which has not previously been reported, showed unequivocal association (p = 1.5x10(-8), fold-increase = 2.12,); the eleven haplotypes were also significantly associated with transcript levels (global p = 0.003). Using an in vitro dual luciferase reporter assay, we found that rs6583817 increases reporter gene expression in Be(2)-C (p = 0.006) and HepG2 (p = 0.02) cell lines. Furthermore, using data from a recent genome-wide association study of two Croatian isolated populations (n = 1,879), we identified a proxy for rs6583817 that associated significantly with decreased plasma Abeta40 levels (ss = -0.124, p = 0.011) and total measured plasma Abeta levels (b = -0.130, p = 0.009). Finally, rs6583817 was associated with decreased risk of LOAD in 3,891 AD cases and 3,605 controls. (OR = 0.87, p = 0.03), and the eleven IDE haplotypes (global p = 0.02) also showed significant association. CONCLUSIONS: Thus, a previously unreported variant unequivocally associated with increased IDE expression was also associated with reduced plasma Abeta40 and decreased LOAD susceptibility. Genetic association between LOAD and IDE has been difficult to replicate. Our findings suggest that targeted testing of expression SNPs (eSNPs) strongly associated with altered transcript levels in autopsy brain samples may be a powerful way to identify genetic associations with LOAD that would otherwise be difficult to detect.

Transcriptional activation of endothelial cells by TGFß coincides with acute microvascular plasticity following focal spinal cord ischaemia/reperfusion injury.

Microvascular dysfunction, loss of vascular support, ischaemia and sub-acute vascular instability in surviving blood vessels contribute to secondary injury following SCI (spinal cord injury). Neither the precise temporal profile of the cellular dynamics of spinal microvasculature nor the potential molecular effectors regulating this plasticity are well understood. TGFß (transforming growth factor ß) isoforms have been shown to be rapidly increased in response to SCI and CNS (central nervous system) ischaemia, but no data exist regarding their contribution to microvascular dysfunction following SCI. To examine these issues, in the present study we used a model of focal spinal cord ischaemia/reperfusion SCI to examine the cellular response(s) of affected microvessels from 30 min to 14 days post-ischaemia. Spinal endothelial cells were isolated from affected tissue and subjected to focused microarray analysis of TGFß-responsive/related mRNAs 6 and 24 h post-SCI. Immunohistochemical analyses of histopathology show neuronal disruption/loss and astroglial regression from spinal microvessels by 3 h post-ischaemia, with complete dissolution of functional endfeet (loss of aquaporin-4) by 12 h post-ischaemia. Coincident with this microvascular plasticity, results from microarray analyses show 9 out of 22 TGFß-responsive mRNAs significantly up-regulated by 6 h post-ischaemia. Of these, serpine 1/PAI-1 (plasminogen-activator inhibitor 1) demonstrated the greatest increase (>40-fold). Furthermore, uPA (urokinase-type plasminogen activator), another member of the PAS (plasminogen activator system), was also significantly increased (>7.5-fold). These results, along with other select up-regulated mRNAs, were confirmed biochemically or immunohistochemically. Taken together, these results implicate TGFß as a potential molecular effector of the anatomical and functional plasticity of microvessels following SCI.