Evidence of synergism among three genetic variants in a patient with LMNA-related lipodystrophy and amyotrophic lateral sclerosis leading to a remarkable nuclear phenotype.

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), can be clinically heterogeneous which may be explained by the co-inheritance of multiple genetic variants that modify the clinical course. In this study we examine variants in three genes in a family with one individual presenting with ALS and lipodystrophy. Sequencing revealed a p.Gly602Ser variant in LMNA, and two additional variants, one each in SETX (g.intron10-13delCTT) and FUS (p.Gly167_Gly168del). These latter genes have been linked to ALS. All family members were genotyped and each variant, and each combination of variants detected, were functionally evaluated in vitro regarding effects on cell survival, expression patterns and cellular phenotype. Muscle biopsy retrieved from the individual with ALS showed leakage of chromatin from the nucleus, a phenotype that was recapitulated in vitro with expression of all three variants simultaneously. Individually expressed variants gave cellular phenotypes there were unremarkable. Interestingly the FUS variant appears to be protective against the effects of the SETX and the LMNA variants on cell viability and may indicate loss of interaction of FUS with SETX and/or R-loops. We conclude that these findings support genetic modifications as an explanation of the clinical heterogeneity observed in human disease.

Characterization and correlates of exercise among adolescents with anorexia nervosa and bulimia nervosa.

OBJECTIVE: To characterize exercise behaviors among adolescents with anorexia nervosa (AN), atypical AN, or bulimia nervosa (BN), and determine associations between exercise and medical risk. STUDY DESIGN: Cross-sectional electronic medical records of all patients evaluated by the Eating Disorder Program at Stanford between January 1997 and February 2011 were retrospectively reviewed. RESULTS: 1,083 subjects (961 females, 122 males; mean age 15.6) met eligibility criteria. Most patients (89.7%) reported exercise (mean 7.0 h per week over mean 5.4 days per week) prior to presentation. Running (49.9%), calisthenics (40.7%), walking (23.4%), soccer (20.9%), and swimming (18.2%) were the most common exercises; a majority (60.6%) reported team sport participation. Males were less likely to report team exercise (p = .005). Bradycardia (heart rate <50) at presentation was associated with team sport participation (adjusted odds ratio [AOR] 1.66, 95% confidence interval [CI] 1.02-2.72) and hours of exercise per week (AOR 1.05, 95% CI 1.02-1.09), controlling for diagnosis, sex, age, duration of illness, rate of weight loss, and percent median body mass index (%mBMI). DISCUSSION: Adolescents with AN, atypical AN, and BN reported high levels of exercise. Females reported more team sport participation. Greater exercise frequency and team sport participation were associated with bradycardia. Further studies assessing the relationship between exercise and bradycardia may help inform the medical management of adolescents with these eating disorders who are more physically active.

Genome-wide maps of circulating miRNA biomarkers for ulcerative colitis.

Inflammatory Bowel Disease--comprised of Crohn's Disease and Ulcerative Colitis (UC)--is a complex, multi-factorial inflammatory disorder of the gastrointestinal tract. In this study we have explored the utility of naturally occurring circulating miRNAs as potential blood-based biomarkers for non-invasive prediction of UC incidences. Whole genome maps of circulating miRNAs in micro-vesicles, Peripheral Blood Mononuclear Cells and platelets have been constructed from a cohort of 20 UC patients and 20 normal individuals. Through Significance Analysis of Microarrays, a signature of 31 differentially expressed platelet-derived miRNAs has been identified and biomarker performance estimated through a non-probabilistic binary linear classification using Support Vector Machines. Through this approach, classifier measurements reveal a predictive score of 92.8% accuracy, 96.2% specificity and 89.5% sensitivity in distinguishing UC patients from normal individuals. Additionally, the platelet-derived biomarker signature can be validated at 88% accuracy through qPCR assays, and a majority of the miRNAs in this panel can be demonstrated to sub-stratify into 4 highly correlated intensity based clusters. Analysis of predicted targets of these biomarkers reveal an enrichment of pathways associated with cytoskeleton assembly, transport, membrane permeability and regulation of transcription factors engaged in a variety of regulatory cascades that are consistent with a cell-mediated immune response model of intestinal inflammation. Interestingly, comparison of the miRNA biomarker panel and genetic loci implicated in IBD through genome-wide association studies identifies a physical linkage between hsa-miR-941 and a UC susceptibility loci located on Chr 20. Taken together, analysis of these expression maps outlines a promising catalog of novel platelet-derived miRNA biomarkers of clinical utility and provides insight into the potential biological function of these candidates in disease pathogenesis.

LYN is a mediator of epithelial-mesenchymal transition and a target of dasatinib in breast cancer.

Epithelial-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is considered a key process driving tumor cell invasiveness and metastasis. Using breast cancer cell lines as a model system, we sought to discover gene expression signatures of EMT with clinical and mechanistic relevance. A supervised comparison of epithelial and mesenchymal breast cancer lines defined a 200-gene EMT signature that was prognostic across multiple breast cancer cohorts. The immunostaining of LYN, a top-ranked EMT signature gene and Src-family tyrosine kinase, was associated with significantly shorter overall survival (P = 0.02) and correlated with the basal-like ("triple-negative") phenotype. In mesenchymal breast cancer lines, RNAi-mediated knockdown of LYN inhibited cell migration and invasion, but not proliferation. Dasatinib, a dual-specificity tyrosine kinase inhibitor, also blocked invasion (but not proliferation) at nanomolar concentrations that inhibit LYN kinase activity, suggesting that LYN is a likely target and that invasion is a relevant end point for dasatinib therapy. Our findings define a prognostically relevant EMT signature in breast cancer and identify LYN as a mediator of invasion and a possible new therapeutic target (and theranostic marker for dasatinib response), with particular relevance to clinically aggressive basal-like breast cancer.

Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues.

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.

Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery.

BACKGROUND: Breast cancer cell lines have been used widely to investigate breast cancer pathobiology and new therapies. Breast cancer is a molecularly heterogeneous disease, and it is important to understand how well and which cell lines best model that diversity. In particular, microarray studies have identified molecular subtypes-luminal A, luminal B, ERBB2-associated, basal-like and normal-like-with characteristic gene-expression patterns and underlying DNA copy number alterations (CNAs). Here, we studied a collection of breast cancer cell lines to catalog molecular profiles and to assess their relation to breast cancer subtypes. METHODS: Whole-genome DNA microarrays were used to profile gene expression and CNAs in a collection of 52 widely-used breast cancer cell lines, and comparisons were made to existing profiles of primary breast tumors. Hierarchical clustering was used to identify gene-expression subtypes, and Gene Set Enrichment Analysis (GSEA) to discover biological features of those subtypes. Genomic and transcriptional profiles were integrated to discover within high-amplitude CNAs candidate cancer genes with coordinately altered gene copy number and expression. FINDINGS: Transcriptional profiling of breast cancer cell lines identified one luminal and two basal-like (A and B) subtypes. Luminal lines displayed an estrogen receptor (ER) signature and resembled luminal-A/B tumors, basal-A lines were associated with ETS-pathway and BRCA1 signatures and resembled basal-like tumors, and basal-B lines displayed mesenchymal and stem/progenitor-cell characteristics. Compared to tumors, cell lines exhibited similar patterns of CNA, but an overall higher complexity of CNA (genetically simple luminal-A tumors were not represented), and only partial conservation of subtype-specific CNAs. We identified 80 high-level DNA amplifications and 13 multi-copy deletions, and the resident genes with concomitantly altered gene-expression, highlighting known and novel candidate breast cancer genes. CONCLUSIONS: Overall, breast cancer cell lines were genetically more complex than tumors, but retained expression patterns with relevance to the luminal-basal subtype distinction. The compendium of molecular profiles defines cell lines suitable for investigations of subtype-specific pathobiology, cancer stem cell biology, biomarkers and therapies, and provides a resource for discovery of new breast cancer genes.

Genomic profiling identifies GATA6 as a candidate oncogene amplified in pancreatobiliary cancer.

Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.

Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth.

Hormone antagonists can be effective tools to delineate receptor signaling pathways and their resulting downstream physiological actions. Mutation of the receptor binding domain (RBD) of human H2 relaxin (deltaH2) impaired its biological function as measured by cAMP signaling. In a competition assay, deltaH2 exhibited antagonistic activity by blocking recombinant H2 relaxin from binding to receptors on THP-1 cells. In a flow cytometry-based binding assay, deltaH2 demonstrated weak binding to 293T cells expressing the LGR7 receptor in the presence of biotinylated H2 relaxin. When human prostate cancer cell lines (PC-3 and LNCaP) were engineered to overexpress eGFP, wild-type (WT) H2, or deltaH2, and subsequently implanted into NOD/SCID mice, tumor xenografts overexpressing deltaH2 displayed smaller volumes compared to H2 and eGFP controls. Plasma osmolality readings and microvessel density and area assessment suggest that deltaH2 modulates physiological parameters in vivo. In a second murine model, intratumoral injections of lentivectors engineered to express deltaH2/eGFP led to suppressed tumor growth compared to controls. This study provides further evidence supporting a role for H2 relaxin in prostate tumor growth. More importantly, we report how mutation of the H2 relaxin RBD confers the hormone derivative with antagonistic properties, offering a novel reagent for relaxin research.

RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes.

DNA amplification is a frequent occurrence in cancer genomes. While tumor amplicons may harbor known oncogenes "driving" amplification, amplicons rarely comprise only single genes. The potential functional contribution of coamplified genes remains largely unexplored. In breast cancer, 20-30% of tumors exhibit amplification within chromosome band 17q12, containing the ERBB2 oncogene. Analysis of array-based comparative genomic hybridization and expression profiling data indicate that the minimum region of recurrent amplification (i.e., the amplicon "core") at 17q12 includes two other genes, GRB7 and STARD3, which exhibit elevated expression when amplified. Western blot analysis confirms overexpression of each at the protein level in breast cancer cell lines SKBR3 and BT474 harboring amplification. In these cell lines (but not in control MCF7 breast cancer cells lacking 17q12 amplification), targeted knockdown of ERBB2 expression using RNA interference (RNAi) methods results in decreased cell proliferation, decreased cell-cycle progression, and increased apoptosis. Notably, targeted knockdown of either GRB7 or STARD3 also leads to decreased cell proliferation and cell-cycle progression, albeit to a lesser extent compared with ERBB2 knockdown. We conclude that the amplification and resultant overexpression of genes coamplified with ERBB2 at 17q12 can contribute to proliferation levels of breast cancer cells. Our findings validate the utility of RNAi in the functional interrogation of tumor amplicons, and provide evidence for a contribution of coamplified genes to tumor phenotypes.