A subset of gastrointestinal stromal tumors (GISTs) lack gain-offunction mutations in c-KIT and PDGFR␣. These so-called wild-type (WT) GISTs tend to be less responsive to imatinib-based therapies and have a poor prognosis. We identified amplification of IGF1R in a SNP analysis of GIST and thus studied its potential as a therapeutic target in WT and mutant GIST. Expression of IGF1R and downstream effectors in clinical GIST samples was examined by using immunoblots and immunohistochemistry. The roles of IGF1R signaling in GIST and viability were analyzed by using NVP-AEW541, an inhibitor of IGF1R, alone and in combination with imatinib, or via siRNA silencing of IGF1R. IGF1R was strongly overexpressed, and IGF1R amplification was detected at a significantly higher frequency in WT GISTs, including a pediatric WT GIST, compared with mutant GISTs (P ‫؍‬ 0.0173 and P ‫؍‬ 0.0163, respectively). Inhibition of IGF1R activity in vitro with NVP-AEW541 or down-regulation of expression with siIGF1R led to cytotoxicity and induced apoptosis in GIST cell lines via AKT and MAPK signaling. Combination of NVP-AEW541 and imatinib in GIST cell lines induced a strong cytotoxicity response. Our results reveal that IGF1R is amplified and the protein is overexpressed in WT and pediatric GISTs. We also demonstrate that the aberrant expression of IGF1R may be associated with oncogenesis in WT GISTs and suggest an alternative and/or complementary therapeutic regimen in the clinical management of all GISTs, especially in a subset of tumors that respond less favorably to imatinib-based therapy.pediatric GIST ͉ tyrosine kinase inhibitors ͉ imatinib mesylase ͉ adult wild-type GIST ͉ NYP-AEW541

We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1␣ and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1␤ and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation.Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. Hepatitis B virus (HBV)1 infection causes acute hepatitis in humans, 10% of cases resulting in chronic hepatitis (1), which is linked to development of hepatocellular carcinoma (HCC) (1); integrated HBV DNA is present in virtually all HBV-mediated liver cancers (2, 3). However, the mechanism of HBV-mediated hepatocarcinogenesis remains elusive.HBV encodes a 16.5-kDa protein termed the X antigen (pX) (4). pX is expressed during viral infection (5-7), is required for the viral life cycle (7), and is highly conserved in all oncogenic mammalian hepadnaviruses (8, 9). In contrast, the avian hepatitis virus, which lacks oncogenic potential, is devoid of an X open reading frame (10). pX promotes liver tumor formation in transgenic mice expressing high levels of pX (11), potentiates c-Myc-induced hepatocarcinogenesis in c-myc/pX bitransgenics (12), and acts as a tumor promoter in hepatocarcinogenesis (13). While the published data clearly implicate pX in HCC, the direct oncogenic effect of pX in hepatocyte transformation has not been demonstrated. Herein, we provide evidence demonstrating the causal link of pX expression to the transformation of immortalized hepatocytes and describe a conditional pX expression system that is amenable to the study of the mechanism of pX-mediated transformation.pX is a multifunctional protein, with reported activities affecting transcription (14), cell growth (15, 16), and apoptotic cell death (17, 18). Although pX does not directly bind doublestranded DNA, pX acts as a promiscuous transactivator (reviewed in Ref. 14), via interaction with several components of the transcriptional apparatus (19 -23). In addition, specific pXresponsive cis-acting elements have been identified, e.g. NF-B (24 -28), AP-1 (29 -34), AP-2 (29), and CRE sites (35-37). Transcriptional activation of the AP-1 and NF-B sites by pX is via cytoplasmic signaling pathways, including the protein kinase C pathway (31, 32) a...

The hepatitis B virus (HBV) X protein (pX) is impli-Patients chronically infected with hepatitis B virus develop hepatocellular carcinoma (HCC) 1 in their 4th or 5th decade (1). The 16.5-kDa X protein, encoded by HBV, is implicated in HCC pathogenesis (2). Despite numerous studies describing pX activities, the molecular mechanism by which pX affects hepatocyte transformation is unknown. This study addresses aspects of the pX-mediated mechanism of hepatocyte transformation.It is well accepted that pX is a multifunctional protein affecting transcription (2, 3), cell growth (4, 5), and apoptosis (6 -8). Although not DNA binding, pX is a promiscuous transactivator of diverse cis-acting elements, including AP-1 (9 -12), NF-B (13-15), AP-2 (11), and CRE (16 -18) sites. This transcriptional promiscuity of pX is understood to be due to its dual mechanism of action (19). pX interacts directly with specific components of the basal transcriptional apparatus (20 -24) and with bZip (CREB/ATF) transcription factors (16 -18), resulting in enhanced CRE/bZip-mediated transcription (16,18). In addition to affecting direct transcriptional induction, pX activates the RAS-RAF-MAPK (19, 25) and JNK (26) pathways, resulting in enhanced transcription from AP-1 and NF-B cis-acting elements.pX-mediated activation of the RAS-RAF-MAPK pathway has been linked to accelerated entry of cells into S phase (4); however, the significance of the pX-dependent activation of this mitogenic pathway in hepatocyte transformation has not been demonstrated directly. Furthermore, the significance of the pX-dependent activation of the JNK pathway also remains unclear, since JNK pathway activation in some cases has been linked to transformation (27-29) and in other cases to apoptosis (30 -33).To understand the significance of pX-induced reprogramming of the mitogenic process in hepatocyte transformation, we employed a new cellular model, composed of immortalized hepatocytes, AML12 cells (34), expressing pX via the tetracycline-regulated expression system (35). We have characterized two hepatocyte lineages derived from AML12 cells as follows: 1) a differentiated hepatocyte (3pX-1) cell line, similar to the parental AML12 cells, and 2) a de-differentiated hepatocyte (4pX-1) cell line. We have demonstrated that conditional expression of pX leads to transformation in the differentiated 3pX-1 hepatocyte cell line; by contrast, expression of pX in the de-differentiated 4pX-1 cell line does not lead to transformation (35). Importantly, our observations agree with clinical data reporting the majority of HCC is derived from differentiated hepatocytes (36) versus the progenitor "oval cells." Thus, our cellular model system is ideal for investigating physiologically relevant, early signaling events activated by pX during hepatocyte transformation, and for comparing these to signaling events occurring in a cellular environment refractory to pXmediated cellular transformation.Herein, we characterize the mitogenic status of the 3pX-1 and 4pX-1 cell lines affected by pX, t...

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (␦Ag).During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of ␦Ag species that are nonfunctional or even inhibitory. A replication system was devised where all ␦Ag was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small ␦Ag, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express ␦Ag. Thus, replication of this RNA was under control of the TET-inducible ␦Ag. In the absence of TET, there was sufficient ␦Ag to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both ␦Ag and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G 1 /G 0 cells relative to both S and G 2 /M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.

Hepatitis B virus (HBV) X protein (pX) is implicated in Epidemiological evidence (1) links chronic hepatitis B virus (HBV)1 infection in humans to development of hepatocellular carcinoma (HCC). Evidence derived from comparative studies of mammalian and avian hepadnaviruses, transgenic animal studies, and cell culture transformation studies collectively (2, 3) support a role for the 16.5-kDa HBV X protein (pX) as a weak oncogene, implicated in HCC development. However, the mechanism(s) by which pX effects hepatocyte transformation is not yet understood. Also not entirely understood is the cell type in the liver, which is the precancerous precursor giving rise to HBV-mediated HCC. The accumulating evidence derived from rat models of hepatocarcinogenesis (4 -7), woodchuck hepatitis virus-mediated HCC (8), and human liver pathologies (9, 10) point to the undifferentiated (oval cell) or less-differentiated (transitional) hepatocyte as the precancerous precursor in HCC development.Activities ascribed to pX probably linked to HBV-mediated pathology include activation of the Ras-Raf-MAPK (11-14), JNK (15), and STAT3 pathways (16, 17); direct interactions with specific components of the basal transcriptional apparatus (18 -21) and with the CREB/ATF family of transcription factors (22-25); interaction with DNA repair proteins (26); and activation of the proteasome complex (27). Importantly, many studies have demonstrated that pX expression in different cell types (Chang cells, NIH3T3 cells, immortalized differentiated AML12 hepatocytes) results in distinct and opposing cellular responses, including cell cycle progression (28), G 1 /S phase arrest (29, 30), transformation (31, 32), and apoptosis (32-35). However, despite evidence supporting the growth-promoting (36) versus the antiproliferative or apoptotic function of pX (37), the molecular mechanisms by which pX effects these processes, for the most part, remain to be deciphered. Likewise, the significance of these pX-mediated processes in HCC development is poorly understood.In our studies, we employ a cellular model system linked to pX-mediated hepatocyte transformation (32) and suitable to molecular analyses. It is composed of two tetracycline-regulated, pX-expressing cell lines, a differentiated hepatocyte 3pX-1 cell line, and a dedifferentiated hepatocyte 4pX-1 cell line. Conditional pX expression selectively transforms the 3pX-1 cell line. We recently demonstrated (36) that an early pX-mediated event in 3pX-1 cells is sustained activation of the Ras-Raf-MAPK pathway, an activation that is causally linked to pX-mediated transformation. In the pX-nontransforming 4pX-1 cell line, pX expression results in sustained activation of the JNK pathway and only transient activation of the Ras-Raf-MAPK pathway. Since pX expression mediates distinct growth characteristics between the 3pX-1 and 4pX-1 cell lines (i.e. transformation in the differentiated 3pX-1 cells versus absence of transformation in the less differentiated 4pX-1 cells and differential activation of the Ras-Raf-M...

Background Neuroprotection for Parkinson Disease (PD) remains elusive. Biomarkers hold the promise of removing roadblocks to therapy development. The National Institute of Neurological Disorders and Stroke has therefore established the Parkinson’s Disease Biomarkers Program to promote discovery of PD biomarkers for use in phase II-III clinical trials. Methods Utilizing a novel consortium design, the Parkinson’s Disease Biomarker Program is focused on the development of clinical and laboratory-based biomarkers for PD diagnosis, progression, and prognosis. Standardized operating procedures and pooled reference samples were created to allow cross-project comparisons and assessment of batch effects. A web-based Data Management Resource facilitates rapid sharing of data and biosamples across the research community for additional biomarker projects. Results Eleven consortium projects are ongoing, seven of which recruit participants and obtain biosamples. As of October 2014, 1082 participants have enrolled (620 PD, 101 with other causes of parkinsonism, 23 essential tremor, and 338 controls), 1040 of whom have at least one biosample. There are 6898 total biosamples from baseline, 6, 12, and 18-month visits: 1006 DNA, 1661 RNA, 1419 whole blood, 1382 plasma, 1200 serum, and 230 cerebrospinal fluid (CSF). Quality control analysis of plasma, serum, and CSF samples indicates almost all samples are high quality (24 of 2812 samples exceed acceptable hemoglobin levels). Conclusions By making samples and data widely available, using stringent operating procedures based upon existing standards, hypothesis testing for biomarker discovery, and providing a resource which complements existing programs, the Parkinson’s Disease Biomarker Program will accelerate the pace of PD biomarker research.

Purpose: Gastrointestinal stromal tumors (GIST) are characterized by expressing a gainof-function mutation in KIT, and to a lesser extent, PDGFR. Imatinib mesylate, a tyrosine kinase inhibitor, has activity against GISTs that contain oncogenic mutations of KIT. In this study, KIT and PDGFRa mutation status was analyzed and protein modeling approaches were used to assess the potential effect of KIT mutations in response to imatinib therapy. Experimental Design: Genomic DNA was isolated from GIST tumors. Exons 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRa were evaluated for oncogenic mutations. Protein modeling was used to assess mutations within the juxtamembrane region and the kinase domain of KIT. Results: Mutations in KITexons 9,11, and 13 were identified in GISTs with the majority of changes involving the juxtamembrane region of KIT. Molecular modeling indicates that mutations in this region result in disruption of the KITautoinhibited conformation, and lead to gain-of-function activation of the kinase. Furthermore, a novel germ-line mutation in KIT was identified that is associated with an autosomal dominant predisposition to the development of GIST. Conclusions: We have used protein modeling and structural analyses to elucidate why patients with GIST tumors containing exon11mutations are the most responsive to imatinib mesylate treatment. Importantly, mutations detected in this exon and others displayed constitutive activation of KIT. Furthermore, we have found tumors that are both KITand PDGFRa mutation negative, suggesting that additional, yet unidentified, abnormalities may contribute to GIST tumorigenesis.

Cell signaling plays a central role in the etiology of cancer. Numerous therapeutics in use or under development target signaling proteins; however, off-target effects often limit assignment of positive clinical response to the intended target. As direct measurements of signaling protein activity are not generally feasible during treatment, there is a need for more powerful methods to determine if therapeutics inhibit their targets and when off-target effects occur. We have used the Bayesian Decomposition algorithm and data on transcriptional regulation to create a novel methodology, Differential Expression for Signaling Determination (DESIDE), for inferring signaling activity from microarray measurements. We applied DESIDE to deduce signaling activity in gastrointestinal stromal tumor cell lines treated with the targeted therapeutic imatinib mesylate (Gleevec). We detected the expected reduced activity in the KIT pathway, as well as unexpected changes in the p53 pathway. Pursuing these findings, we have determined that imatinib-induced DNA damage is responsible for the increased activity of p53, identifying a novel off-target activity for this drug. We then used DESIDE on data from resected, post-imatinib treatment tumor samples and identified a pattern in these tumors similar to that at late time points in the cell lines, and this pattern correlated with initial clinical response. The pattern showed increased activity of ETS domain-containing protein Elk-1 and signal transducers and activators of transcription 3 transcription factors, which are associated with the growth of side population cells. DESIDE infers the global reprogramming of signaling networks during treatment, permitting treatment modification that leverages ongoing drug development efforts, which is crucial for personalized medicine. [Cancer Res 2009;69(23):9125-32]