Eukaryotic initiation factor 4E-binding protein as an oncogene in breast cancer.

BACKGROUND: Eukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy. METHODS: Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1. RESULTS: Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes. CONCLUSIONS: These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.

Deletion of the murine ortholog of the 8q24 gene desert has anti-cancer effects in transgenic mammary cancer models.

BACKGROUND: The gene desert on human chromosomal band 8q24 harbors multiple genetic variants associated with common cancers, including breast cancer. The locus, including the gene desert and its flanking genes, MYC, PVT1 and FAM84B, is also frequently amplified in human breast cancer. We generated a megadeletion (MD) mouse model lacking 430-Kb of sequence orthologous to the breast cancer-associated region in the gene desert. The goals were to examine the effect of the deletion on mammary cancer development and on transcript level regulation of the candidate genes within the locus. METHODS: The MD allele was engineered using the MICER system in embryonic stem cells and bred onto 3 well-characterized transgenic models for breast cancer, namely MMTV-PyVT, MMTV-neu and C3(1)-TAg. Mammary tumor growth, latency, multiplicity and metastasis were compared between homozygous MD and wild type mice carrying the transgenes. A reciprocal mammary gland transplantation assay was conducted to distinguish mammary cell-autonomous from non-mammary cell-autonomous anti-cancer effects. Gene expression analysis was done using quantitative real-time PCR. Chromatin interactions were evaluated by 3C. Gene-specific patient outcome data were analysed using the METABRIC and TCGA data sets through the cBioPortal website. RESULTS: Mice homozygous for the MD allele are viable, fertile, lactate sufficiently to nourish their pups, but maintain a 10% lower body weight mainly due to decreased adiposity. The deletion interferes with mammary tumorigenesis in mouse models for luminal and basal breast cancer. In the MMTV-PyVT model the mammary cancer-reducing effects of the allele are mammary cell-autonomous. We found organ-specific effects on transcript level regulation, with Myc and Fam84b being downregulated in mammary gland, prostate and mammary tumor samples. Through analysis using the METABRIC and TCGA datasets, we provide evidence that MYC and FAM84B are frequently co-amplified in breast cancer, but in contrast with MYC, FAM84B is frequently overexpressed in the luminal subtype, whereas MYC activity affect basal breast cancer outcomes. CONCLUSION: Deletion of a breast cancer-associated non-protein coding region affects mammary cancer development in 3 transgenic mouse models. We propose Myc as a candidate susceptibility gene, regulated by the gene desert locus, and a potential role for Fam84b in modifying breast cancer development.

Two members of the TRiC chaperonin complex, CCT2 and TCP1 are essential for survival of breast cancer cells and are linked to driving oncogenes.

Gene amplification is a common mechanism of oncogene activation in cancer. Several large-scale efforts aimed at identifying the comprehensive set of genomic regions that are recurrently amplified in cancer have been completed. In breast cancer, these studies have identified recurrently amplified regions containing known drivers such as HER2 and CCND1 as well as regions where the driver oncogene is unknown. In this study, we integrated RNAi-based functional genetic data with copy number and expression data to identify genes that are recurrently amplified, overexpressed and also necessary for the growth/survival of breast cancer cells. Further analysis using clinical data from The Cancer Genome Atlas specifically identified candidate genes that play a role in determining patient outcomes. Using this approach, we identified two genes, TCP1 and CCT2, as being recurrently altered in breast cancer, necessary for growth/survival of breast cancer cells in vitro, and determinants of overall survival in breast cancer patients. We also show that expression of TCP1 is regulated by driver oncogene activation of PI3K signaling in breast cancer. Interestingly, the TCP1 and CCT2 genes both encode for components of a multi-protein chaperone complex in the cell known as the TCP1 Containing Ring Complex (TRiC). Our results demonstrate a role for the TRiC subunits TCP1 and CCT2, and potentially the entire TRiC complex, in breast cancer and provide rationale for TRiC as a novel therapeutic target in breast cancer.

Acute posterior multifocal placoid pigment epitheliopathy: clinical presentation and risk of stroke and transient ischaemic attack.

BACKGROUND/AIMS: Acute posterior multifocal placoid pigment epitheliopathy is a rare but important disease that can be associated with life-threatening complications due to cerebral vasculitis. The primary objective was to determine the incidence of neurological complications and risk factors for stroke and transient ischaemic attack (TIA) associated with acute posterior multifocal placoid pigment epitheliopathy. Secondary objectives included the clinical presentation, visual outcomes and recurrence rates. METHODS: This was a multicentre retrospective case series including 111 eyes from 60 subjects presenting from January 2009 to June 2020. RESULTS: Median age at presentation was 29 years (IQR 24.7-35.1) and 36 subjects (60.0%) were male. 20 subjects (33.3%) reported a viral prodrome. Stroke and TIA were observed in seven subjects (11.7%). Older age was the only significant risk factor for stroke/TIA (p=0.042). Vision loss occurred in seven eyes, with four eyes (3.6%) having final visual acuity 6/15-6/60 and three eyes (2.7%) having visual acuity of 6/60 or worse. Recurrence occurred in 10 subjects (16.7%). CONCLUSIONS: The presence of headache cannot reliably predict those at risk of stroke/TIA. Individuals presenting with acute posterior multifocal pigment epitheliopathy should therefore undergo a clinical neurological review and work-up for cerebral vasculitis as deemed appropriate by the treating ophthalmologist and collaborating neurologist.

DroID 2011: a comprehensive, integrated resource for protein, transcription factor, RNA and gene interactions for Drosophila.

DroID (http://droidb.org/), the Drosophila Interactions Database, is a comprehensive public resource for Drosophila gene and protein interactions. DroID contains genetic interactions and experimentally detected protein-protein interactions curated from the literature and from external databases, and predicted protein interactions based on experiments in other species. Protein interactions are annotated with experimental details and periodically updated confidence scores. Data in DroID is accessible through user-friendly, intuitive interfaces that allow simple or advanced searches and graphical visualization of interaction networks. DroID has been expanded to include interaction types that enable more complete analyses of the genetic networks that underlie biological processes. In addition to protein-protein and genetic interactions, the database now includes transcription factor-gene and regulatory RNA-gene interactions. In addition, DroID now has more gene expression data that can be used to search and filter interaction networks. Orthologous gene mappings of Drosophila genes to other organisms are also available to facilitate finding interactions based on gene names and identifiers for a number of common model organisms and humans. Improvements have been made to the web and graphical interfaces to help biologists gain a comprehensive view of the interaction networks relevant to the genes and systems that they study.

Ophthalmic surgery in New Zealand: analysis of 410,099 surgical procedures and nationwide surgical intervention rates from 2009 to 2018.

BACKGROUND: Surgical intervention rates (SIR) provide a proxy measure of disease burden, surgical capacity, and the relative risk-benefit ratio of surgery. The current study assessed decade trends in ophthalmic surgery and calculated SIRs for all major classes of commonly performed ophthalmic procedures in New Zealand. METHODS: Retrospective population-based analysis of all ophthalmic surgical procedures performed in New Zealand from 2009 to 2018. National and regional datasets from public and private health sectors and industry were analysed. SIRs were calculated for all major ophthalmic procedures, and subgrouped by patient demographics. RESULTS: There were 410,099 ophthalmic surgical procedures completed with a 25.3% overall increase over 10 years. Procedures were mostly government-funded (51%, n = 210,830) with 71% of patients aged over 64 years. Cataract surgery (78%, n = 318,564) had the highest mean SIR (703/100,000/year) and increased by 25% during the study period, consistent with population growth in the over 64 years old age group. Vitrectomy surgery had the second highest mean SIR (67/100,000/year) and increased by 50%, well above national population growth during the study period. Other SIRs included conjunctival lesion-biopsy (38/100,000/year), glaucoma (33/100,000/year), strabismus (20/100,000/year), dacryocystorhinostomy (10/100,000/year), and keratoplasty surgery (4/100,000/year). CONCLUSIONS: This comprehensive review of New Zealand ophthalmic surgery reports increasing SIRs that cannot be explained by population growth alone. Cataract surgery numbers increased year on year consistent with the increase in the over 64 years old population. Vitrectomy surgery growth exceeded that of the national population, including those over 64 years.

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Pars Plana Vitrectomy With or Without Circumferential Scleral Buckling for Primary Repair of Retinal Detachments Secondary to Retinal Dialysis.

PURPOSE: The aim of this study was to evaluate the anatomical and functional outcomes after pars plana vitrectomy with or without circumferential scleral buckling, in patients with retinal detachment secondary to retinal dialysis. DESIGN: Case series. METHODS: A retrospective consecutive case series involving 30 eyes of 30 patients with retinal detachments secondary to retinal dialysis was completed. All 30 patients underwent primary pars plana vitrectomy, with the addition of a circumferential scleral buckle in 24 patients. The main outcome measure was the primary reattachment rate at 6 months after a single surgery. RESULTS: Primary anatomical success was achieved in 90% of patients. The final anatomical success rate was 93%, with 4 of 30 eyes still having silicone oil in situ. The main indication for vitrectomy surgery was an inadequate fundal view for scleral buckle surgery alone. The macula was detached in 50% of patients and proliferative vitreoretinopathy grade C was found in 23% of cases. The mean preoperative visual acuity was 20/814 (range 20/20-light perception) and the mean postoperative visual acuity was 20/258 (range 20/20-hand movements). CONCLUSIONS: Good final anatomical success rates can be achieved with pars plans vitrectomy, plus or minus circumferential buckling, in patients with retinal detachments secondary to retinal dialysis.

Full color-tunable vertically stacked quantum dot light emitting diodes for next-generation displays and lighting.

We report solution-processed color tunable vertically stacked electroluminescent red, green, and blue quantum dot light emitting diodes (QLEDs). These QLEDs can be independently driven to produce all primary, secondary, and white lights. We have fabricated the device by chemical and electrical isolation of each QLED with transparent polymers and by the use of transparent electrodes. These stacked QLEDs can be used for next-generation display and lighting applications that need high pixel density along with quantum dots' intrinsic benefits such as low turn-on voltage, color purity, and solution processability.

Development and implementation of the SUM breast cancer cell line functional genomics knowledge base.

Several years ago, the SUM panel of human breast cancer cell lines was developed, and these cell lines have been distributed to hundreds of labs worldwide. Our lab and others have developed extensive omics data sets from these cells. More recently, we performed genome-scale shRNA essentiality screens on the entire SUM line panel, as well as on MCF10A cells, MCF-7 cells, and MCF-7LTED cells. These gene essentiality data sets allowed us to perform orthogonal analyses that functionalize the otherwise descriptive genomic data obtained from traditional genomics platforms. To make these omics data sets available to users of the SUM lines, and to allow users to mine these data sets, we developed the SUM Breast Cancer Cell Line Knowledge Base. This knowledge base provides information on the derivation of each cell line, provides protocols for the proper maintenance of the cells, and provides a series of data mining tools that allow rapid identification of the oncogene signatures for each line, the enrichment of KEGG pathways with screen hit and gene expression data, an analysis of protein and phospho-protein expression for the cell lines, as well as a gene search tool and a functional-druggable signature tool. Recently, we expanded our database to include genomic data for an additional 27 commonly used breast cancer cell lines. Thus, the SLKBase provides users with deep insights into the biology of human breast cancer cell lines that can be used to develop strategies for the reverse engineering of individual breast cancer cell lines.

Primary Ocular Toxoplasmosis Presenting to Uveitis Services in a Non-endemic Setting.

PURPOSE: This study sought to describe the different clinical features and presentations of primary ocular toxoplasmosis in a setting not demonstrating an outbreak of disease. METHODS: This was a retrospective review of patients presenting to uveitis management services in Auckland and Hamilton, New Zealand between 2003 to 2018 with uveitis and positive toxoplasmosis immunoglobulin M serology. RESULTS: We identified 16 patients with primary acquired toxoplasmosis infection and ocular involvement. The mean age was 53 years. Systemic symptoms were reported in 56% (9 / 16). Visual acuity was reduced to 20 / 30 or less in 50% of patients (8 / 16). A single focus of retinitis without a pigmented scar was the salient clinical feature in 69% (11 / 16). Optic nerve inflammation was the sole clinical finding in 19% (3 / 16). Bilateral arterial vasculitis was the sole clinical finding in 13% (2 / 16). A delay in the diagnosis of toxoplasmosis of more than two weeks occurred in 38% (6 / 16) due to an initial alternative diagnosis. Antibiotic therapy was prescribed in all cases. Vision was maintained or improved in 69% (11 / 16) at the most recent follow-up visit (15 months to 10 years). Relapse occurred in 69% (11 / 16), typically within four years from the initial presentation. CONCLUSIONS: Primary ocular toxoplasmosis presenting in adulthood is a relatively uncommon cause of posterior uveitis in New Zealand. This condition should be considered in any patient presenting with retinitis or optic nerve inflammation without a retinochoroidal scar. This disease tends to relapse; thus, close follow-up is required.

Long-term survival following diabetic vitrectomy.

AIM: To update long-term survival data on patients with proliferative diabetic retinopathy undergoing vitrectomy and to identify associated risk factors. METHODS: Retrospective clinical record review at a single New Zealand tertiary referral centre. A total of 182 eyes that underwent a vitrectomy for a diabetic vitreous haemorrhage and/or tractional retinal detachment between March 2000 and December 2010 were included. Kaplan-Meier survival curves and Cox-regression analyses were performed for survival rates and associated risk factors. RESULTS: The mean age of patients was 55 years (range 22 to 85) at time of surgery. The three-year survival rate following diabetic vitrectomy was 83.5%, and the five-year survival rate (N=154) was 70.1%. Increasing age, dialysis and high serum creatinine were associated with poorer survival on multivariate Cox regression analyses (hazard ratio of 1.035, 4.216 and 1.930 respectively with p-values of 0.018, <0.001 and 0.046). CONCLUSION: Survival rates after diabetic vitrectomy remain relatively poor but comparable to earlier New Zealand and international reports. However, there remain significant differences between ethnic groups within New Zealand that need to be addressed in addition to renal disease, which appears to be a major risk factor for poor survival. Overall, the contemporary survival outcomes observed in this study may influence decision making by patients and clinicians as well as encourage a review of current healthcare resource allocation in diabetes care.

A database and tool, IM Browser, for exploring and integrating emerging gene and protein interaction data for Drosophila.

BACKGROUND: Biological processes are mediated by networks of interacting genes and proteins. Efforts to map and understand these networks are resulting in the proliferation of interaction data derived from both experimental and computational techniques for a number of organisms. The volume of this data combined with the variety of specific forms it can take has created a need for comprehensive databases that include all of the available data sets, and for exploration tools to facilitate data integration and analysis. One powerful paradigm for the navigation and analysis of interaction data is an interaction graph or map that represents proteins or genes as nodes linked by interactions. Several programs have been developed for graphical representation and analysis of interaction data, yet there remains a need for alternative programs that can provide casual users with rapid easy access to many existing and emerging data sets. DESCRIPTION: Here we describe a comprehensive database of Drosophila gene and protein interactions collected from a variety of sources, including low and high throughput screens, genetic interactions, and computational predictions. We also present a program for exploring multiple interaction data sets and for combining data from different sources. The program, referred to as the Interaction Map (IM) Browser, is a web-based application for searching and visualizing interaction data stored in a relational database system. Use of the application requires no downloads and minimal user configuration or training, thereby enabling rapid initial access to interaction data. IM Browser was designed to readily accommodate and integrate new types of interaction data as it becomes available. Moreover, all information associated with interaction measurements or predictions and the genes or proteins involved are accessible to the user. This allows combined searches and analyses based on either common or technique-specific attributes. The data can be visualized as an editable graph and all or part of the data can be downloaded for further analysis with other tools for specific applications. The database is available at http://proteome.wayne.edu/PIMdb.html CONCLUSION: The Drosophila Interactions Database described here places a variety of disparate data into one easily accessible location. The database has a simple structure that maintains all relevant information about how each interaction was determined. The IM Browser provides easy, complete access to this database and could readily be used to publish other sets of interaction data. By providing access to all of the available information from a variety of data types, the program will also facilitate advanced computational analyses.

Functional oncogene signatures guide rationally designed combination therapies to synergistically induce breast cancer cell death.

A critical first step in the personalized approach to cancer treatment is the identification of activated oncogenes that drive each tumor. The Identification of driver oncogenes on a patient-by-patient basis is complicated by the complexity of the cancer genome and the fact that a particular genetic alteration may serve as a driver event only in a subset of tumors that harbor it. In this study, we set out to identify the complete set of functional oncogenes in a small panel of breast cancer cell lines. The cell lines in this panel were chosen because they each contain a known receptor tyrosine kinase (RTK) oncogene. To identify additional drivers, we integrated functional genetic screens with copy number and mutation analysis, and cancer genome knowledge databases. The resulting functional oncogene signatures were able to predict responsiveness of cell lines to targeted inhibitors. However, as single agents, these drugs had little effect on clonogenic potential. By contrast, treatment with drug combinations that targeted multiple oncogenes in the signatures, even at very low doses, resulted in the induction of apoptosis and striking synergistic effects on clonogenicity. In particular, targeting a driver oncogene that mediates AKT phosphorylation in combination with targeting the anti-apoptotic BCL2L1 protein had profound effects on cell viability. Importantly, because the synergistic induction of cell death was achieved using low levels of each individual drug, it suggests that a therapeutic strategy based on this approach could avoid the toxicities that have been associated with the combined use of multiple-targeted agents.

Amplification of WHSC1L1 regulates expression and estrogen-independent activation of ERα in SUM-44 breast cancer cells and is associated with ERα over-expression in breast cancer.

The 8p11-p12 amplicon occurs in approximately 15% of breast cancers in aggressive luminal B-type tumors. Previously, we identified WHSC1L1 as a driving oncogene from this region. Here, we demonstrate that over-expression of WHSC1L1 is linked to over-expression of ERα in SUM-44 breast cancer cells and in primary human breast cancers. Knock-down of WHSC1L1, particularly WHSC1L1-short, had a dramatic effect on ESR1 mRNA and ERα protein levels. SUM-44 cells do not require exogenous estrogen for growth in vitro; however, they are dependent on ERα expression, as ESR1 knock-down or exposure to the selective estrogen receptor degrader fulvestrant resulted in growth inhibition. ChIP-Seq experiments utilizing ERα antibodies demonstrated extensive ERα binding to chromatin in SUM-44 cells under estrogen-free conditions. ERα bound to ERE and FOXA1 motifs under estrogen-free conditions and regulated expression of estrogen-responsive genes. Short-term treatment with estradiol enhanced binding of ERα to chromatin and influenced expression of many of the same genes to which ERα was bound under estrogen-free conditions. Finally, knock-down of WHSC1L1 in SUM-44 cells resulted in loss of ERα binding to chromatin under estrogen-free conditions, which was restored upon exposure to estradiol. These results indicate the SUM-44 cells are a good model of a subset of luminal B breast cancers that have the 8p11-p12 amplicon, over-express WHSC1L1, and over-express ERα, but are independent of estrogen for binding to chromatin and regulation of gene expression. Breast cancers such as these, that are dependent on ERα activity but independent of estradiol, are a major cause of breast cancer mortality.

Oncogenic signaling in amphiregulin and EGFR-expressing PTEN-null human breast cancer.

A subset of triple negative breast cancer (TNBC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) and loss of PTEN, and patients with these determinants have a poor prognosis. We used cell line models of EGFR-positive/PTEN null TNBC to elucidate the signaling networks that drive the malignant features of these cells and cause resistance to EGFR inhibitors. In these cells, amphiregulin (AREG)-mediated activation of EGFR results in up-regulation of fibronectin (FN1), which is known to be a mediator of invasive capacity via interaction with integrin ß1. EGFR activity in this PTEN null background also results in Wnt/beta-catenin signaling and activation of NF-κB. In addition, AKT is constitutively phosphorylated in these cells and is resistant to gefitinib. Expression profiling demonstrated that AREG-activated EGFR regulates gene expression differently than EGF-activated EGFR, and functional analysis via genome-scale shRNA screening identified a set of genes, including PLK1 and BIRC5, that are essential for survival of SUM-149 cells, but are uncoupled from EGFR signaling. Thus, our results demonstrate that in cells with constitutive EGFR activation and PTEN loss, critical survival genes are uncoupled from regulation by EGFR, which likely mediates resistance to EGFR inhibitors.