Irinotecan and temozolomide in combination with dasatinib and rapamycin versus irinotecan and temozolomide for patients with relapsed or refractory neuroblastoma (RIST-rNB-2011): a multicentre, open-label, randomised, controlled, phase 2 trial.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumour in children. Relapsed or refractory neuroblastoma is associated with a poor outcome. We assessed the combination of irinotecan-temozolomide and dasatinib-rapamycin (RIST) in patients with relapsed or refractory neuroblastoma. METHODS: The multicentre, open-label, randomised, controlled, phase 2, RIST-rNB-2011 trial recruited from 40 paediatric oncology centres in Germany and Austria. Patients aged 1-25 years with high-risk relapsed (defined as recurrence of all stage IV and MYCN amplification stages, after response to treatment) or refractory (progressive disease during primary treatment) neuroblastoma, with Lansky and Karnofsky performance status at least 50%, were assigned (1:1) to RIST (RIST group) or irinotecan-temozolomide (control group) by block randomisation, stratified by MYCN status. We compared RIST (oral rapamycin [loading 3 mg/m2 on day 1, maintenance 1 mg/m2 on days 2-4] and oral dasatinib [2 mg/kg per day] for 4 days with 3 days off, followed by intravenous irinotecan [50 mg/m2 per day] and oral temozolomide [150 mg/m2 per day] for 5 days with 2 days off; one course each of rapamycin-dasatinib and irinotecan-temozolomide for four cycles over 8 weeks, then two courses of rapamycin-dasatinib followed by one course of irinotecan-temozolomide for 12 weeks) with irinotecan-temozolomide alone (with identical dosing as experimental group). The primary endpoint of progression-free survival was analysed in all eligible patients who received at least one course of therapy. The safety population consisted of all patients who received at least one course of therapy and had at least one post-baseline safety assessment. This trial is registered at ClinicalTrials.gov, NCT01467986, and is closed to accrual. FINDINGS: Between Aug 26, 2013, and Sept 21, 2020, 129 patients were randomly assigned to the RIST group (n=63) or control group (n=66). Median age was 5·4 years (IQR 3·7-8·1). 124 patients (78 [63%] male and 46 [37%] female) were included in the efficacy analysis. At a median follow-up of 72 months (IQR 31-88), the median progression-free survival was 11 months (95% CI 7-17) in the RIST group and 5 months (2-8) in the control group (hazard ratio 0·62, one-sided 90% CI 0·81; p=0·019). Median progression-free survival in patients with amplified MYCN (n=48) was 6 months (95% CI 4-24) in the RIST group versus 2 months (2-5) in the control group (HR 0·45 [95% CI 0·24-0·84], p=0·012); median progression-free survival in patients without amplified MYCN (n=76) was 14 months (95% CI 9-7) in the RIST group versus 8 months (4-15) in the control group (HR 0·84 [95% CI 0·51-1·38], p=0·49). The most common grade 3 or worse adverse events were neutropenia (54 [81%] of 67 patients given RIST vs 49 [82%] of 60 patients given control), thrombocytopenia (45 [67%] vs 41 [68%]), and anaemia (39 [58%] vs 38 [63%]). Nine serious treatment-related adverse events were reported (five patients given control and four patients given RIST). There were no treatment-related deaths in the control group and one in the RIST group (multiorgan failure). INTERPRETATION: RIST-rNB-2011 demonstrated that targeting of MYCN-amplified relapsed or refractory neuroblastoma with a pathway-directed metronomic combination of a multkinase inhibitor and an mTOR inhibitor can improve progression-free survival and overall survival. This exclusive efficacy in MYCN-amplified, relapsed neuroblastoma warrants further investigation in the first-line setting. FUNDING: Deutsche Krebshilfe.

Detection of a 7SL RNA-derived small non-coding RNA using Molecular Beacons in vitro and in cells.

Small non-coding RNAs (sncRNA) are involved in many steps of the gene expression cascade and regulate processing and expression of mRNAs by the formation of ribonucleoprotein complexes (RNP) such as the RNA-induced silencing complex (RISC). By analyzing small RNA Seq data sets, we identified a sncRNA annotated as piR-hsa-1254, which is likely derived from the 3'-end of 7SL RNA2 (RN7SL2), herein referred to as snc7SL RNA. The 7SL RNA is an abundant long non-coding RNA polymerase III transcript and serves as structural component of the cytoplasmic signal recognition particle (SRP). To evaluate a potential functional role of snc7SL RNA, we aimed to define its cellular localization by live cell imaging. Therefore, a Molecular Beacon (MB)-based method was established to compare the subcellular localization of snc7SL RNA with its precursor 7SL RNA. We designed and characterized several MBs in vitro and tested those by live cell fluorescence microscopy. Using a multiplex approach, we show that 7SL RNA localizes mainly to the endoplasmic reticulum (ER), as expected for the SRP, whereas snc7SL RNA predominately localizes to the nucleus. This finding suggests a fundamentally different function of 7SL RNA and its derivate snc7SL RNA.

Role of the RNA-binding protein La in cancer pathobiology.

RNA-binding proteins are important regulators of RNA metabolism and are of critical importance in all steps of the gene expression cascade. The role of aberrantly expressed RBPs in human disease is an exciting research field and the potential application of RBPs as a therapeutic target or a diagnostic marker represents a fast-growing area of research.Aberrant overexpression of the human RNA-binding protein La has been found in various cancer entities including lung, cervical, head and neck, and chronic myelogenous leukaemia. Cancer-associated La protein supports tumour-promoting processes such as proliferation, mobility, invasiveness and tumour growth. Moreover, the La protein maintains the survival of cancer cells by supporting an anti-apoptotic state that may cause resistance to chemotherapeutic therapy.The human La protein represents a multifunctional post-translationally modified RNA-binding protein with RNA chaperone activity that promotes processing of non-coding precursor RNAs but also stimulates the translation of selective messenger RNAs encoding tumour-promoting and anti-apoptotic factors. In our model, La facilitates the expression of those factors and helps cancer cells to cope with cellular stress. In contrast to oncogenes, able to initiate tumorigenesis, we postulate that the aberrantly elevated expression of the human La protein contributes to the non-oncogenic addiction of cancer cells. In this review, we summarize the current understanding about the implications of the RNA-binding protein La in cancer progression and therapeutic resistance. The concept of exploiting the RBP La as a cancer drug target will be discussed.

Comparing mTOR inhibitor Rapamycin with Torin-2 within the RIST molecular-targeted regimen in neuroblastoma cells.

The prognosis for patients with relapsed or refractory high-risk neuroblastoma remains dismal and novel therapeutic options are urgently needed. The RIST treatment protocol has a multimodal metronomic therapy design combining molecular-targeted drugs (Rapamycin and Dasatinib) with chemotherapy backbone (Irinotecan and Temozolomide), which is currently verified in a phase II clinical trial (NCT01467986). With the availability of novel and more potent ATP competitive mTOR inhibitors, we expect to improve the RIST combination therapy. By comparing the IC50 values of Torin-1, Torin-2, AZD3147 and PP242 we established that only Torin-2 inhibited cell viability of all three MycN-amplified neuroblastoma cell lines tested at nanomolar concentration. Single treatment of both mTOR inhibitors induced a significant G1 cell cycle arrest and combination treatment with Dasatinib reduced the expression of cell cycle regulator cyclin D1 or increased the expression of cell cycle inhibitor p21. The combinatorial index depicted for both mTOR inhibitors a synergistic effect with Dasatinib. Interestingly, compared to Rapamycin, the combination treatment with Torin-2 resulted in a broader mTOR pathway inhibition as indicated by reduced phosphorylation of AKT (Thr308, Ser473), 4E-BP (Ser65), and S6K (Thr389). Furthermore, substituting Rapamycin in the modified multimodal RIST protocol with Torin-2 reduced cell viability and induced apoptosis despite a significant lower Torin-2 drug concentration applied. The efficacy of nanomolar concentrations may significantly reduce unwanted immunosuppression associated with Rapamycin. However, at this point we cannot rule out that Torin-2 has increased toxicity due to its potency in more complex systems. Nonetheless, our results suggest that including Torin-2 as a substitute for Rapamycin in the RIST protocol may represent a valid option to be evaluated in prospective clinical trials for relapsed or treatment-refractory high-risk neuroblastoma.

Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation.

The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation.

Imatinib resistance and microcytic erythrocytosis in a KitV558Δ;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor.

Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the Kit receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequently develop imatinib resistance as a result of second-site Kit mutations. To investigate the consequences of second-site Kit mutations on GIST development and imatinib sensitivity, we engineered a mouse model carrying in the endogenous Kit locus both the Kit(V558Δ) mutation found in a familial case of GIST and the Kit(T669I) (human KIT(T670I)) "gatekeeper" mutation found in imatinib-resistant GIST patients. Similar to Kit(V558/+) mice, Kit(V558;T669I/+) mice developed gastric and colonic interstitial cell of Cajal hyperplasia as well as cecal GIST. In contrast to the single-mutant Kit(V558/+) control mice, treatment of the Kit(V558;T669I/+) mice with either imatinib or dasatinib failed to inhibit oncogenic Kit signaling and GIST growth. However, this resistance could be overcome by treatment of Kit(V558;T669I/+) mice with sunitinib or sorafenib. Although tumor lesions were smaller in Kit(V558;T669I/+) mice than in single-mutant mice, both interstitial cell of Cajal hyperplasia and mast cell hyperplasia were exacerbated in Kit(V558;T669I/+) mice. Strikingly, the Kit(V558;T669I/+) mice developed a pronounced polycythemia vera-like erythrocytosis in conjunction with microcytosis. This mouse model should be useful for preclinical studies of drug candidates designed to overcome imatinib resistance in GIST and to investigate the consequences of oncogenic KIT signaling in hematopoietic as well as other cell lineages.

Evaluating the RIST Molecular-Targeted Regimen in a Three-Dimensional Neuroblastoma Spheroid Cell Culture Model.

BACKGROUND: The outcome for patients with high-risk neuroblastoma remains poor and novel treatment strategies are urgently needed. The RIST protocol represents a novel metronomic and multimodal treatment strategy for high-risk neuroblastoma combining molecular-targeted drugs as 'pre-treatment' with a conventional chemotherapy backbone, currently evaluated in a phase II clinical trial. For preclinical drug testing, cancer cell growth as spheroid compared to mo-nolayer cultures is of advantage since it reproduces a wide range of tumor characteristics, including the three-dimensional architecture and cancer stem cell (CSC) properties. The objective of this study was to establish a neuroblastoma spheroid model for the rigorous assessment of the RIST treatment protocol. METHODS: Evaluation of CSC marker expression was performed by mRNA and protein analysis and spheroid viability by luminescence-based assays. Aberrant expression of RNA-binding protein La in neuroblastoma was assessed by tissue microarray analysis and patients' data mining. RESULTS: Spheroid cultures showed increased expression of a subgroup of CSC-like markers (CXCR4, NANOG and BMI) and higher Thr389 phosphorylation of the neuroblastoma-associated RNA-binding protein La when compared to monolayer cultures. Molecular-targeted 'pre-treatment' of spheroids decreased neoplastic signaling and CSC marker expression. CONCLUSIONS: The RIST treatment protocol efficiently reduced the viability of neuroblastoma spheroids characterized by advanced CSC properties.

Heparanase wildtype is associated with a reduced incidence of transplant-associated systemic vasculopathies.

Some of the early complications of hematopoietic stem cell transplantation (HSCT) concerning the small vessels can be summarized as transplant-associated systemic vasculopathies (TASV). One enzyme known to play a major role in inflammation, tissue remodeling, and repair processes as well as tumor metastasis is heparanase (HPSE). HPSE genetic variants have recently been associated with significant influence on the risk of developing certain TASV such as a sinusoidal obstruction syndrome. This study aimed to validate the two known HPSE single nucleotide polymorphisms (SNPs)-rs4693608 and rs4364254-as a genetic predictor of TASV in a cohort of 494 patients and were correlated retrospectively with the clinical course post-HSCT. Significant association was revealed for rs4364254, showing that the incidence of TASV (38.0% vs. 57.8%, p = .009) and in particular of acute graft-versus-host disease (aGvHD) (36.3% vs. 54.0%, p = .0138) was lower in wildtype CC carriers than in TC/TT carriers. Moreover, compared with all other genotypes, the allelic combination GG-CC had the lowest incidence of TASV (34.9% vs. 57.4%, p = .0109) and aGvHD in particular (34.9% vs. 53.5%, p = .0315). A competing risk regression analysis confirmed a significantly reduced risk for a TASV in patients with GG (subhazard ratio [SHR] = 0.670, p = .043) and CC (SHR = 0.598, p = .041) compared with the corresponding homozygote SNP as well as for allelic combinations correlated with low HPSE gene expression (SHR = 0.630, p = .016) and in correlation with clinical risk factors. In summary, our study emphasizes an association of HPSE gene SNPs with TASV, in particular with aGvHD, which could be implementable as pre-transplant risk stratification if validated prospectively.

RNA-Binding Protein La Mediates TGFß-Induced Epithelial to Mesenchymal Transition and Cancer Stem Cell Properties.

BACKGROUND: the aberrant overexpression of predominantly nuclear localizing RNA-binding protein (RBP) La contributes to proliferation, mobility, and chemoresistance of cancer cells and tumor growth in mice. METHODS: studies included cancer tissue microarrays (TMAs) analyses, cancer tissue data mining, transforming growth factor ß (TGFß)-induced cancer cell plasticity studies, three dimensional sphere growth, epithelial to mesenchymal transition (EMT) assays, analysis of cancer stem cell (CSC) marker expression, and post-translational modification of cancer-associated La protein. RESULTS: we demonstrated that significant overexpression of RBP La in lung and head and neck cancer tissue correlates with poor overall survival. Furthermore, small interfering RNA-mediated depletion of La reduced proliferation and migration of cancer cells, blocked TGFß-induced EMT, and diminished both EMT and CSC marker expression. Rescue experiments with La wildtype but not RNA chaperone domain activity-defective La mutant increased the expression of those cancer progression markers, suggesting a critical role of La's RNA chaperone activity in this process. La depletion in cancer cells also significantly decreased sphere growth in the presence of TGFß. Interestingly, TGFß treatment induced phosphorylation of La at threonine 389 (pLaT389) only in adherents but not in 3D growing cultures. CONCLUSION: our study suggests that the TGFß/AKT/pLaT389 signaling pathway regulates cancer cell plasticity.

Critical role for Kit-mediated Src kinase but not PI 3-kinase signaling in pro T and pro B cell development.

The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.

Salt-Dependent Modulation of the RNA Chaperone Activity of RNA-Binding Protein La.

It is well established that the RNA-binding protein La has RNA chaperone activity. Recent work suggests that the La protein has two distinct RNA chaperone domains (RCD-A and RCD-B) assisting structural changes in diverse groups of RNA molecules such as RNA polymerase III transcripts (e.g., pre-tRNA, U6 snRNA), cellular messenger, and viral RNAs. In this protocol we focus on the RNA chaperone domain RCD-B, which is located in the carboxy-terminal domain of La. It has been shown that this RNA chaperone domain assists structural changes in predicted RNA hairpins folded in the 5'-untranslated regions of cyclin D1 and Bcl2 mRNAs. Besides RNA helicases, which are implicated in melting RNA hairpin structures in an ATP-dependent manner, RNA chaperones fulfil a similar function in an ATP-independent manner. Aiming to study the RNA chaperon activity of La, we established a La-dependent molecular beacon-based RNA chaperone assay and systematically tested the various salt conditions. Herein we describe the assay format and design to study the salt dependency of RNA chaperones. This protocol can be easily adapted to test the RNA chaperone activity of other RNA-binding proteins and to optimize assay conditions.

Posttranscriptional control of HBV gene expression.

Acute and chronic hepatitis B virus (HBV) infection is the cause of about 1 million deaths each year worldwide. Although a vaccine for HBV is available, new HBV infections are appearing at alarming rates and maternal-fetal transmission is a major cause of viral spread. Thus, new and effective antiviral strategies are necessary for the 300 million chronically HBV-infected individuals to reduce their morbidity and mortality. Precise processing of viral RNAs is essential for the hepatitis viral life cycle, and, to our knowledge, the processing, nuclear export, and stabilization/degradation of viral RNAs are exclusively mediated by host factors. Thus, identification of host factors required for viral RNA metabolism and subsequent molecular analysis of the interactions between viral RNAs and corresponding host factors might represent novel avenues for the development of new antiviral strategies. In this review, we summarize the current knowledge about the posttranscriptional control of HBV RNA metabolism.

The hepatitis B virus PRE contains a splicing regulatory element.

The posttranscriptional regulatory element (PRE) is considered to enhance hepatitis B virus (HBV) gene expression by facilitating the nuclear export of intronless viral subgenomic RNAs. Its role in the RNA metabolism of the viral pregenomic RNA (pgRNA) is currently unknown. We identified a positively cis-acting splicing regulatory element (SRE-1) and present two lines of evidence for its functionality. Firstly, in a heterologous context SRE-1 functionally substitutes for a retroviral bidirectional exonic splicing enhancer (ESE). As expected, SRE-1 is a splicing enhancer also in its natural viral sequence context, since deletion of SRE-1 reduces splicing of pgRNA in cell culture experiments. Secondly, we show that stimulation of HBV RNA splicing by the splicing factor PSF was repressed by the PRE. Analysis of a variety of PSF mutants indicated that RNA-binding and protein-protein interaction were required to enhance splicing. In addition, we show that the PRE contributed to pgRNA stability, but has little influence on its nuclear export. Herein, we report for the first time that the PRE harbors splicing stimulating and inhibiting regulatory elements controlling processing of the viral pregenome. We discuss a model in which the regulation of pgRNA splicing depends on cellular factors interacting with the PRE.

SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability.

The cancer-associated RNA-binding protein La is posttranslationally modified by phosphorylation and sumoylation. Sumoylation of La regulates not only the trafficking of La in neuronal axons but also its association with specific mRNAs. Depletion of La in various types of cancer cell lines impairs cell proliferation; however, the molecular mechanism whereby La supports cell proliferation is not clearly understood. In this study, we address the question of whether sumoylation of La contributes to cell proliferation of HEK293 cells. We show that HEK293 cells stably expressing green fluorescent protein (GFP)-tagged wild-type La (GFP-LaWT) grow faster than cells expressing a sumoylation-deficient mutant La (GFP-LaSD), suggesting a proproliferative function of La in HEK293 cells. Further, we found that STAT3 protein levels were reduced in GFP-LaSD cells due to an increase in STAT3 ubiquitination and that overexpression of STAT3 partially restored cell proliferation. Finally, we present RNA sequencing data from RNA immunoprecipitations (RIPs) and report that mRNAs associated with the cell cycle and ubiquitination are preferentially bound by GFP-LaWT and are less enriched in GFP-LaSD RIPs. Taken together, results of our study support a novel mechanism whereby sumoylation of La promotes cell proliferation by averting ubiquitination-mediated degradation of the STAT3 protein.

Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells.

The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells.

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

The La protein counteracts cisplatin-induced cell death by stimulating protein synthesis of anti-apoptotic factor Bcl2.

Up-regulation of anti-apoptotic factors is a critical mechanism of cancer cell resistance and often counteracts the success of chemotherapeutic treatment. Herein, we identified the cancer-associated RNA-binding protein La as novel factor contributing to cisplatin resistance. Our data demonstrate that depletion of the RNA-binding protein La in head and neck squamous cell carcinoma cells (HNSCC) increases the sensitivity toward cisplatin-induced cell death paralleled by reduced expression of the anti-apoptotic factor Bcl2. Furthermore, it is shown that transient expression of Bcl2 in La-depleted cells protects against cisplatin-induced cell death. By dissecting the underlying mechanism we report herein, that the La protein is required for Bcl2 protein synthesis in cisplatin-treated cells. The RNA chaperone La binds in close proximity to the authentic translation start site and unwinds a secondary structure embedding the authentic AUG. Altogether, our data support a novel model, whereby cancer-associated La protein contributes to cisplatin resistance by stimulating the translation of anti-apoptotic factor Bcl2 in HNSCC cells.

Pleomorphic characteristics of a germ-line KIT mutation in a large kindred with gastrointestinal stromal tumors, hyperpigmentation, and dysphagia.

PURPOSE: Somatic mutations that result in the activation of the growth factor receptor KIT are commonly found in gastrointestinal stromal tumors (GISTs). Six families have been reported in which a germ-line mutation in KIT is associated with an autosomal dominant predisposition to the development of GISTs. Hyperpigmentation, urticaria pigmentosa, and dysphagia have been described in some, but not all, families. Preliminary correlations between the site of mutation and the clinical phenotype have been proposed, but the strength of these associations is not defined. DESIGN: A large kindred with multiple GISTs, hyperpigmentation, and dysphagia was identified after the index case presented with multiple GISTs. A germ-line mutation in KIT (W557R) was identified in an affected cousin, after which a large family meeting was held and testing offered. Clinical data were obtained by interview and, whenever possible, medical record documentation. RESULTS: To date, 19 individuals have been tested, and the mutation has been shown to cosegregate with the syndrome. The phenotypic expression, however, is variable. GISTs, often presenting as upper gastrointestinal bleeding, and hyperpigmentation are common, but not diagnosed in all documented or obligate carriers. Dysphagia is a less prevalent complaint. The diagnosis of GISTs appears to be made at a younger age in more recent generations. Metastatic disease is uncommon. CONCLUSIONS: A germ-line mutation in KIT resulting in an amino acid substitution in the juxtamembrane region is associated with a syndrome of GIST, hyperpigmentation, and dysphagia, although the prominence of each component varies.

Association of KIT exon 9 mutations with nongastric primary site and aggressive behavior: KIT mutation analysis and clinical correlates of 120 gastrointestinal stromal tumors.

PURPOSE: Activating mutations of the KIT juxtamembrane region are the most common genetic events in gastrointestinal stromal tumors (GISTs) and have been noted as independent prognostic factors. The impact of KIT mutation in other regions, such as the extracellular or kinase domains, is not well-defined and fewer than 30 cases have been published to date. EXPERIMENTAL DESIGN: One hundred twenty GISTs, confirmed by KIT immunoreactivity, were evaluated for the presence of KIT exon 9, 11, 13, and 17 mutations. The relation between the presence/type of KIT mutation and clinicopathological factors was analyzed using Fisher's exact test and log-rank test. RESULTS: Forty-four % of the tumors were located in the stomach, 47% in the small bowel, 6% in the rectum, and 3% in the retroperitoneum. Overall, KIT mutations were detected in 78% of patients as follows: 67% in exon 11, 11% in exon 9, and none in exon 13 or 17. The types of KIT exon 11 mutations were heterogeneous and clustered in the classic "hot spot" at the 5' end of exon 11. Seven % of cases showed internal tandem duplications (ITD) at the 3' end of exon 11, in a region that we designate as a second hot spot for KIT mutations. Interestingly, these cases were associated with: female predominance, stomach location, occurrence in older patients, and favorable outcome. There were significant associations between exon 9 mutations and large tumor size (P < 0.001) and extragastric location (P = 0.02). Ten of these 13 patients with more than 1-year follow-up have developed recurrent disease. CONCLUSIONS: Most KIT-expressing GISTs show KIT mutations that are preferentially located within the classic hot spot of exon 11. In addition, we found an association between a second hot spot at the 3'end of exon 11, characterized by ITDs, and a subgroup of clinically indolent gastric GISTs in older females. KIT exon 9 mutations seem to define a distinct subset of GISTs, located predominantly in the small bowel and associated with an unfavorable clinical course.

Loss of expression and function of SOCS3 is an early event in HNSCC: altered subcellular localization as a possible mechanism involved in proliferation, migration and invasion.

BACKGROUND: Suppressor of cytokine signaling 3 (SOCS3) is an inducible endogenous negative regulator of signal transduction and activator of transcription 3 (STAT3). Epigenetic silencing of SOCS3 has been shown in head and neck squamous cell carcinoma (HNSCC), which is associated with increased activation of STAT3. There is scarce information on the functional role of the reduction of SOCS3 expression and no information on altered subcellular localization of SOCS3 in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We assessed endogenous SOCS3 expression in different HNSCC cell lines by RT-qPCR and western blot. Immunofluorescence and western blot were used to study the subcellular localization of endogenous SOCS3 induced by IL-6. Overexpression of SOCS3 by CMV-driven plasmids and siRNA-mediated inhibition of endogenous SOCS3 were used to verify the role of SOCS3 on tumor cell proliferation, viability, invasion and migration in vitro. In vivo relevance of SOCS3 expression in HNSCC was studied by quantitative immunohistochemistry of commercially-available tissue microarrays. Endogenous expression of SOCS3 was heterogeneous in four HNSCC cell lines and surprisingly preserved in most of these cell lines. Subcellular localization of endogenous SOCS3 in the HNSCC cell lines was predominantly nuclear as opposed to cytoplasmic in non-neoplasic epithelial cells. Overexpression of SOCS3 produced a relative increase of the protein in the cytoplasmic compartment and significantly inhibited proliferation, migration and invasion, whereas inhibition of endogenous nuclear SOCS3 did not affect these events. Analysis of tissue microarrays indicated that loss of SOCS3 is an early event in HNSCC and was correlated with tumor size and histological grade of dysplasia, but a considerable proportion of cases presented detectable expression of SOCS3. CONCLUSION: Our data support a role for SOCS3 as a tumor suppressor gene in HNSCC with relevance on proliferation and invasion processes and suggests that abnormal subcellular localization impairs SOCS3 function in HNSCC cells.