Competitive Binding Between Id1 and E2F1 to Cdc20 Regulates E2F1 Degradation and Thymidylate Synthase Expression to Promote Esophageal Cancer Chemoresistance

Purpose: Chemoresistance is a major obstacle in cancer therapy. We found that fluorouracil (5-FU)-resistant esophageal squamous cell carcinoma cell lines, established through exposure to increasing concentrations of 5-FU, showed upregulation of Id1, IGF2, and E2F1. We hypothesized that these genes may play an important role in cancer chemoresistance. Experimental Design: In vitro and in vivo functional assays were performed to study the effects of Id1–E2F1–IGF2 signaling in chemoresistance. Quantitative real-time PCR, Western blotting, immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays were used to investigate the molecular mechanisms by which Id1 regulates E2F1 and by which E2F1 regulates IGF2. Clinical specimens, tumor tissue microarray, and Gene Expression Omnibus datasets were used to analyze the correlations between gene expressions and the relationships between expression profiles and patient survival outcomes. Results: Id1 conferred 5-FU chemoresistance through E2F1-dependent induction of thymidylate synthase expression in esophageal cancer cells and tumor xenografts. Mechanistically, Id1 protects E2F1 protein from degradation and increases its expression by binding competitively to Cdc20, whereas E2F1 mediates Id1-induced upregulation of IGF2 by binding directly to the IGF2 promoter and activating its transcription. The expression level of E2F1 was positively correlated with that of Id1 and IGF2 in human cancers. More importantly, concurrent high expression of Id1 and IGF2 was associated with unfavorable patient survival in multiple cancer types. Conclusions: Our findings define an intricate E2F1-dependent mechanism by which Id1 increases thymidylate synthase and IGF2 expressions to promote cancer chemoresistance. The Id1–E2F1–IGF2 regulatory axis has important implications for cancer prognosis and treatment. ©2015 AACR.postprin

Imaging of Hereditary Hemorrhagic Telangiectasia

This pictorial review is based on our experience of the follow-up of 120 patients at our multidisciplinary center for hereditary hemorrhagic telangiectasia (HHT). Rendu-Osler-Weber disease or HHT is a multiorgan autosomal dominant disorder with high penetrance, characterized by epistaxis, mucocutaneous telangiectasis, and visceral arteriovenous malformations (AVMs). The research on gene mutations is fundamental and family screening by clinical examination, chest X-ray, research of pulmonary shunting, and abdominal color Doppler sonography is absolutely necessary. The angioarchitecture of pulmonary AVMs can be studied by unenhanced multidetector computed tomography; however, all other explorations of liver, digestive bowels, or brain require administration of contrast media. Magnetic resonance angiography is helpful for central nervous system screening, in particular for the spinal cord, but also for pulmonary, hepatic, and pelvic AVMs. Knowledge of the multiorgan involvement of HHT, mechanism of complications, and radiologic findings is fundamental for the correct management of these patients

Dysbindin Promotes the Post-Endocytic Sorting of G Protein-Coupled Receptors to Lysosomes

BackgroundDysbindin, a cytoplasmic protein long known to function in the biogenesis of specialized lysosome-related organelles (LROs), has been reported to reduce surface expression of D2 dopamine receptors in neurons. Dysbindin is broadly expressed, and dopamine receptors are members of the large family of G protein-coupled receptors (GPCRs) that function in diverse cell types. Thus we asked if dysbindin regulates receptor number in non-neural cells, and further investigated the cellular basis of this regulation.Methodology/principal findingsWe used RNA interference to deplete endogenous dysbindin in HEK293 and HeLa cells, then used immunochemical and biochemical methods to assess expression and endocytic trafficking of epitope-tagged GPCRs. Dysbindin knockdown up-regulated surface expression of D2 receptors compared to D1 receptors, as reported previously in neurons. This regulation was not mediated by a change in D2 receptor endocytosis. Instead, dysbindin knockdown specifically reduced the subsequent trafficking of internalized D2 receptors to lysosomes. This distinct post-endocytic sorting function explained the minimal effect of dysbindin depletion on D1 receptors, which recycle efficiently and traverse the lysosomal pathway to only a small degree. Moreover, dysbindin regulated the delta opioid receptor, a more distantly related GPCR that is also sorted to lysosomes after endocytosis. Dysbindin was not required for lysosomal trafficking of all signaling receptors, however, as its depletion did not detectably affect down-regulation of the EGF receptor tyrosine kinase. Dysbindin co-immunoprecipitated with GASP-1 (or GPRASP-1), a cytoplasmic protein shown previously to modulate lysosomal trafficking of D2 dopamine and delta opioid receptors by direct interaction, and with HRS that is a core component of the conserved ESCRT machinery mediating lysosome biogenesis and sorting.Conclusions/significanceThese results identify a distinct, and potentially widespread function of dysbindin in promoting the sorting of specific GPCRs to lysosomes after endocytosis

Uncoupling of the LKB1-AMPKα Energy Sensor Pathway by Growth Factors and Oncogenic BRAFV600E

BACKGROUND: Understanding the biochemical mechanisms contributing to melanoma development and progression is critical for therapeutical intervention. LKB1 is a multi-task Ser/Thr kinase that phosphorylates AMPK controlling cell growth and apoptosis under metabolic stress conditions. Additionally, LKB1(Ser428) becomes phosphorylated in a RAS-Erk1/2-p90(RSK) pathway dependent manner. However, the connection between the RAS pathway and LKB1 is mostly unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using the UV induced HGF transgenic mouse melanoma model to investigate the interplay among HGF signaling, RAS pathway and PI3K pathway in melanoma, we identified LKB1 as a protein directly modified by HGF induced signaling. A variety of molecular techniques and tissue culture revealed that LKB1(Ser428) (Ser431 in the mouse) is constitutively phosphorylated in BRAF(V600E) mutant melanoma cell lines and spontaneous mouse tumors with high RAS pathway activity. Interestingly, BRAF(V600E) mutant melanoma cells showed a very limited response to metabolic stress mediated by the LKB1-AMPK-mTOR pathway. Here we show for the first time that RAS pathway activation including BRAF(V600E) mutation promotes the uncoupling of AMPK from LKB1 by a mechanism that appears to be independent of LKB1(Ser428) phosphorylation. Notably, the inhibition of the RAS pathway in BRAF(V600E) mutant melanoma cells recovered the complex formation and rescued the LKB1-AMPKalpha metabolic stress-induced response, increasing apoptosis in cooperation with the pro-apoptotic proteins Bad and Bim, and the down-regulation of Mcl-1. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that growth factor treatment and in particular oncogenic BRAF(V600E) induces the uncoupling of LKB1-AMPKalpha complexes providing at the same time a possible mechanism in cell proliferation that engages cell growth and cell division in response to mitogenic stimuli and resistance to low energy conditions in tumor cells. Importantly, this mechanism reveals a new level for therapeutical intervention particularly relevant in tumors harboring a deregulated RAS-Erk1/2 pathway

Molecular Typing and Phenotype Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Blood in Taiwan

BACKGROUND: Staphylococcus aureus causes a variety of severe infections such as bacteremia and sepsis. At present, 60-80% of S. aureus isolates from Taiwan are methicillin resistant (MRSA). It has been shown that certain MRSA clones circulate worldwide. The goals of this study were to identify MRSA clones in Taiwan and to correlate the molecular types of isolates with their phenotypes. METHODS: A total of 157 MRSA isolates from bacteremic patients were collected from nine medical centers. They were typed based on polymorphisms in agr, SCCmec, MLST, spa, and dru. Phenotypes characterized included Panton-Valentine leucocidin (pvl), inducible macrolide-lincosamide-streptogramin B resistance (MLSBi), vancomycin (VA) and daptomycin (DAP) minimal inhibitory concentrations (MIC), and superantigenic toxin gene profiles. Difference between two consecutive samples was determined by Mann-Whitney-U test, and difference between two categorical variables was determined by Fisher's exact test. RESULTS: Four major MRSA clone complexes CC1, CC5, CC8, and CC59 were found, including 4 CC1, 9 CC5, 111 CC8, and 28 CC59 isolates. These clones had the following molecular types: CC1: SCCmecIV and ST573; CC5: SCCmecII and ST5; CC8: SCCmecIII, ST239, and ST241, and CC59: SCCmecIV, SCCmecV(T), ST59, and ST338. The toxin gene profiles of these clones were CC1: sec-seg-(sei)-sell-selm-(seln)-selo; CC5: sec-seg-sei-sell-selm-(seln)-selp-tst1; CC8: sea-selk-selq, and CC59: seb-selk-selq. Most isolates with SCCmecV(T), ST59, spat437, and dru11 types were pvl(+) (13 isolates), while multidrug resistance (≥4 antimicrobials) were associated with SCCmecIII, ST239, spa t037, agrI, and dru14 (119 isolates) (p<0.001). One hundred and twenty four isolates with the following molecular types had higher VA MIC: SCCmecII and SCCmecIII; ST5, ST239, and ST241; spa t002, t037, and t421; dru4, dru10, dru12, dru13, and dru14 (p<0.05). No particular molecular types were found to be associated with MLSBi phenotype. CONCLUSIONS: Four major MRSA clone complexes were found in Taiwan. Further studies are needed to delineate the evolution of MRSA isolates

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

The role of Id-1 in immortalization of esophageal epithelial cells

The potential to achieve limitless proliferation is one of the major hallmarks of cancer. Identifying the molecular basis responsible for regulating the replicative potential has important implications for carcinogenesis. It is well documented that the introduction of hTERT, the catalytic subunit of telomerase helps to overcome replicative senescence and acquire immortality. Recently, it has been reported that ectopic expression of Id-1 (Inhibitor of Differentiation), a helix-loop-helix protein which serves as a negative regulator in the regulation of transcription and differentiation, also leads to delayed senescence and even immortalization of human keratinocytes through activation of telomerase. On the other hand, human embryonic fibroblasts transfected with hTERT exhibit increased Id-1 expression. In this study, the role of Id-1 and its relationship with hTERT in immortalization of human esophageal epithelial cells was investigated by overexpressing Id-1 or hTERT, or both Id-1 and hTERT, in esophageal epithelial primary cell lines. Our results showed that in an hTERT-immortalized esophageal cell line, NE083, the expression of Id-1 increased with increasing passages. Knockdown of endogenous Id-1 at late passage (P35) by transfection with antisense Id-1 led to suppressed cell growth, down-regulation of Mdm2 and increased sensitivity to TGF-β-induced differentiation and growth arrest. In another hTERT-transfected primary esophageal cell line, NE2, cells transfected with Id-1 showed elevated growth rate, up-regulation of pMdm2 and down-regulation of p21, compared with cells without Id-1 transfected. These findings suggested that Id-1 may induce proliferation of esophageal epithelial cells and help hTERT promoting immortalization of esophageal epithelial cells, possibly through regulating the p21Waf/Cip/Mdm2 pathway

Immortalization of human esophageal epithelial cells by human telomerase reverse transcriptase (hTERT)

Carcinogenesis is a multistep process involving accumulation of numerous cellular abnormalities over a lengthy time-frame. One of the major steps is to achieve unlimited replicative potential (immortalization). The proliferative life-span of normal somatic cells is limited by an intrinsic mechanism associated with progressive shortening of telomeres during cell propagation, which induces the cells to undergo replicative senescence. We have established an immortal human esophageal epithelial cell line, NE2-hTERT, by ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase (a holoenzyme responsible for the maintenance of telomere length). During the immortalization process, the percentage of senescence associated β-galactosidase positive cells was monitored and was found to decrease with increasing passages as the cells escaped senescence and became immortalized. So far, the cell line has undergone over 100 population doublings. The NE2-hTERT cell line retained epithelial morphology and expressed cytokeratins typical for esophageal epithelium. It also had a near-diploid clonal karyotype. Analysis of expression profiles of cell cycle regulatory proteins in this cell line at different passages showed loss of p16, down-regulation of p53, as well as elevated p21 and p27 expressions during immortalization. Treatment with the demethylating agent, 5-aza-deoxycytidine, restored the p16 expression, which indicated that the spontaneous loss of p16 during immortalization was due to promoter methylation. Upregulation of both p53 and p21 in response to irradiation and mitomycin C treatment indicated that the cell line had intact DNA damage response. The altered p21 and p27 expressionsmight reflect a feedback mechanism to maintain certain ‘normality’ in cell-cycle regulation in these cells. Immortalized cell lines, especially ones with minimal genetic alterations, are invaluable models for studying the molecular basis of carcinogenesis. We observed that late passage NE2-hTERT cells showed increased tumorigenic potential in being able to form tightly-packed colonies in soft agar. This increased anchorage-independency suggests that the cell line may be just one step away from neoplastic transformation and is therefore particularly useful for studying genes associated with tumorigenic transformation of esophageal epithelial cells. [This study was supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China, Project No. HKU 7385/03M and HKU 7556/06M, and the HKU URC Seed Funding Programme for Basic Research, Project No. 200411159061

Role of cyclin D1 in conferring malignant phenotypes on immortalized esophageal epithelial cells

Cyclin D1 is commonly overexpressed in many types of human squamous cell carcinomas, including esophageal squamous cell carcinoma. Studies in the past mainly focused on the role of cyclin D1 in cell growth and regulation of the G1/S transition of the cell cycle. Recently, malignant transformation induced by classical oncogenes was shown to be associated with upregulation of cyclin D1. So far, there are still no reports on the direct contribution of cyclin D1 to malignant transformation of immortalized epithelial cells. In this study, the role of cyclin D1 in the early stage of esophageal carcinogenesis was investigated by stable overexpression of cyclin D1 in an hTERT-immortalized esophageal epithelial cell line, NE2-hTERT. Our results showed that ectopic expression of cyclin D1 stimulated anchorage-independent growth in soft-agar, which is a major hallmark of transformed cells. This finding was further supported by the decrease in colony number in an esophageal cancer cell line (KYSE510) with cyclin D1 knockdown by siRNA. Cyclin D1 overexpressing esophageal epithelial cells also acquired a higher ability to migrate through cellular matrix, and showed reduced adhesion to different substrates. Western blotting and pull-down assays were used to further explore the mechanism involved. The results showed that the overexpression of cyclin D1 was associated with upregulation of pERK1/2 and reduced Rac1 activity. Our findings suggest that cyclin D1 overexpression promotes invasive potential and migration ability in immortalized esophageal epithelial cells, and that these malignant phenotypes may be mediated by pERK1/2 and cytoskeletal signaling. [This study is supported by the Research Grants Council of the Hong Kong SAR, China (Central allocation Project No. HKUST 2/06C)