Christopher Kingsley

Tumor necrosis factor-like weak inducer of apoptosis stimulation of glioma cell survival is dependent on Akt2 function

Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and significant technical advances in surgical and radiation treatment, the impact on clinical outcome for patients with malignant gliomas is disappointing. We have previously shown that tumor necrosis factor–like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell survival via binding to the Fn14 receptor, activation of the NF-κB pathway, and upregulation of BCL-XL gene expression. Here, we show that TWEAK treatment of glioma cells leads to phosphorylation of Akt and BAD. TWEAK stimulation results in the phosphorylation of both Akt1 and Akt2. However, small interfering RNA (siRNA)–mediated depletion of either Akt1 or Akt2 showed that BAD serine 136 phosphorylation is dependent specifically on Akt2 function. Depletion of Akt2 expression by siRNA also abrogates TWEAK-stimulated glioma cell survival, whereas no effect on glioma cell survival was observed after siRNA-mediated depletion of Akt1 expression. Surprisingly, although siRNA-mediated depletion of BAD in glioma cells abrogates cytotoxic- and chemotherapy-induced apoptosis, TWEAK still displays a strong protective effect, suggesting that BAD serine 136 phosphorylation plays a minor role in TWEAK-Akt2–induced glioma cell survival. We also report here that AKT2 gene expression levels increased with glioma grade and inversely correlate with patient survival. Additionally, immunohistochemical analysis showed that Akt2 expression positively correlates with Fn14 expression in glioblastoma multiforme specimens. We hypothesize that the TWEAK-Fn14 signaling axis functions, in part, to enhance glioblastoma cell survival by activation of the Akt2 serine/threonine protein kinase. (Mol Cancer Res 2009;7(11):1871–81)

Elevated Expression of Fn14 in Non-Small Cell Lung Cancer Correlates with Activated EGFR and Promotes Tumor Cell Migration and Invasion

Lung cancer is the leading cause of cancer deaths worldwide; approximately 85% of these cancers are non-small cell lung cancer (NSCLC). Patients with NSCLC frequently have tumors harboring somatic mutations in the epidermal growth factor receptor (EGFR) gene that cause constitutive receptor activation. These patients have the best clinical response to EGFR tyrosine kinase inhibitors (TKIs). Herein, we show that fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is frequently overexpressed in NSCLC tumors, and Fn14 levels correlate with p-EGFR expression. We also report that NSCLC cell lines that contain EGFR-activating mutations show high levels of Fn14 protein expression. EGFR TKI treatment of EGFR-mutant HCC827 cells decreased Fn14 protein levels, whereas EGF stimulation of EGFR wild-type A549 cells transiently increased Fn14 expression. Furthermore, Fn14 is highly expressed in EGFR-mutant H1975 cells that also contain an EGFR TKI-resistance mutation, and high TKI doses are necessary to reduce Fn14 levels. Constructs encoding EGFRs with activating mutations induced Fn14 expression when expressed in rat lung epithelial cells. We also report that short hairpin RNA-mediated Fn14 knockdown reduced NSCLC cell migration and invasion in vitro. Finally, Fn14 overexpression enhanced NSCLC cell migration and invasion in vitro and increased experimental lung metastases in vivo. Thus, Fn14 may be a novel therapeutic target for patients with NSCLC, in particular for those with EGFR-driven tumors who have either primary or acquired resistance to EGFR TKIs.

Prospective molecular profiling of canine cancers provides a clinically relevant comparative model for evaluating personalized medicine (PMed) trials

Background Molecularly-guided trials (i.e. PMed) now seek to aid clinical decision-making by matching cancer targets with therapeutic options. Progress has been hampered by the lack of cancer models that account for individual-to-individual heterogeneity within and across cancer types. Naturally occurring cancers in pet animals are heterogeneous and thus provide an opportunity to answer questions about these PMed strategies and optimize translation to human patients. In order to realize this opportunity, it is now necessary to demonstrate the feasibility of conducting molecularly-guided analysis of tumors from dogs with naturally occurring cancer in a clinically relevant setting. Methodology A proof-of-concept study was conducted by the Comparative Oncology Trials Consortium (COTC) to determine if tumor collection, prospective molecular profiling, and PMed report generation within 1 week was feasible in dogs. Thirty-one dogs with cancers of varying histologies were enrolled. Twenty-four of 31 samples (77%) successfully met all predefined QA/QC criteria and were analyzed via Affymetrix gene expression profiling. A subsequent bioinformatics workflow transformed genomic data into a personalized drug report. Average turnaround from biopsy to report generation was 116 hours (4.8 days). Unsupervised clustering of canine tumor expression data clustered by cancer type, but supervised clustering of tumors based on the personalized drug report clustered by drug class rather than cancer type. Conclusions Collection and turnaround of high quality canine tumor samples, centralized pathology, analyte generation, array hybridization, and bioinformatic analyses matching gene expression to therapeutic options is achievable in a practical clinical window (<1 week). Clustering data show robust signatures by cancer type but also showed patient-to-patient heterogeneity in drug predictions. This lends further support to the inclusion of a heterogeneous population of dogs with cancer into the preclinical modeling of personalized medicine. Future comparative oncology studies optimizing the delivery of PMed strategies may aid cancer drug development.

Identification of Causal Sequence Variants of Disease in the Next Generation Sequencing Era

Over the last decade, genetic studies have identified numerous associations between single nucleotide polymorphism (SNP) alleles in the human genome and important human diseases. Unfortunately, extending these initial associative findings to identification of the true causal variants that underlie disease susceptibility is usually not a straightforward task. Causal variant identification typically involves searching through sizable regions of genomic DNA in the vicinity of disease-associated SNPs for sequence variants in functional elements including protein coding, regulatory, and structural sequences. Prioritization of these searches is greatly aided by knowledge of the location of functional sequences in the human genome. This chapter briefly reviews several of the common approaches used to functionally annotate the human genome and discusses how this information can be used in concert with the emerging technology of next generation high-throughput sequencing to identify causal variants of human disease.

FN14 expression correlates with MET in NSCLC and promotes MET-driven cell invasion.

The five-year survival rate in advanced non-small cell lung cancer (NSCLC) remains below ten percent. The invasive and metastatic nature of NSCLC tumor cells contributes to the high mortality rate, and as such the mechanisms that govern NSCLC metastasis is an active area of investigation. Two surface receptors that influence NSCLC invasion and metastasis are the hepatocyte growth factor receptor (HGFR/MET) and fibroblast growth factor-inducible 14 (FN14). MET protein is over-expressed in NSCLC tumors and associated with poor clinical outcome and metastasis. FN14 protein is also elevated in NSCLC tumors and positively correlates with tumor cell migration and invasion. In this report, we show that MET and FN14 protein expressions are significantly correlated in human primary NSCLC tumors, and the protein levels of MET and FN14 are elevated in metastatic lesions relative to patient-matched primary tumors. In vitro, HGF/MET activation significantly enhances FN14 mRNA and protein expression. Importantly, depletion of FN14 is sufficient to inhibit MET-driven NSCLC tumor cell migration and invasion in vitro. This work suggests that MET and FN14 protein expressions are associated with the invasive and metastatic potential of NSCLC. Receptor-targeted therapeutics for both MET and FN14 are in clinical development, the use of which may mitigate the metastatic potential of NSCLC.

Identification of novel clinical factors associated with hepatic fat accumulation in extreme obesity.

Objectives. The accumulation of lipids stored as excess triglycerides in the liver (steatosis) is highly prevalent in obesity and has been associated with several clinical characteristics, but most studies have been based on relatively small sample sizes using a limited set of variables. We sought to identify clinical factors associated with liver fat accumulation in a large cohort of patients with extreme obesity. Methods. We analyzed 2929 patients undergoing intraoperative liver biopsy during a primary bariatric surgery. Univariate and multivariate regression modeling was used to identify associations with over 200 clinical variables with the presence of any fat in the liver and with moderate to severe versus mild fat accumulation. Results. A total of 19 data elements were associated with the presence of liver fat and 11 with severity of liver fat including ALT and AST, plasma lipid, glucose, and iron metabolism variables, several medications and laboratory measures, and sleep apnea. The accuracy of a multiple logistic regression model for presence of liver fat was 81% and for severity of liver fat accumulation was 77%. Conclusions. A limited set of clinical factors can be used to model hepatic fat accumulation with moderate accuracy and may provide potential mechanistic insights in the setting of extreme obesity.

The Human Genome Project: Where Are We Now and Where Are We Going?

In the 1970s an explosion in our understanding of genetics and biochemistry led to the development of novel laboratory techniques such as restriction enzymes, cloning vectors, nucleic acid hybridization, and DNA sequencing, which revolutionized molecular biology research. In this fertile atmosphere, the seeds of genome sequencing were sown. The progressive spirit pervading research in the life sciences at this time helped to fuel the conception of the Human Genome Project (HGP), whose primary aims were to determine the identity of the three billion nucleotides comprising the human genome and characterize the full repertoire of genes encoded therein. Delineation of the human genome sequence has consequently enhanced our understanding of human genetics, and ignited the development of such diverse disciplines as comparative genomics, pharmacogenetics, and nutrigenomics. The fruits of the HGP directly contributed to the creation of the HapMap and 1,000 Genomes projects, which has since provided the basis for whole genome association (WGA) studies, thereby facilitating elucidation of the genetic variants that underlie the development of complex diseases including cancer, diabetes, autoimmune syndromes, and neurological disorders. Thus, the HGP continues to exert a significant influence upon our comprehension of human genetics, our understanding of complex disease susceptibility, and ultimately, the manner in which scientific research is currently conducted.

Abstract 3785: Elevated expression of Fn14 in non-small cell lung cancer correlates with c-MET and promotes tumor cell invasion and metastasis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor invasiveness and therapeutic resistance strongly contribute to the low (<10%) five-year survival rate for advanced stage lung cancer; thus, identification of novel interventions targeting metastasis and therapeutic resistance is a necessity. Hepatocyte growth factor receptor (c-Met) and fibroblast growth factor-inducible 14 (Fn14) are two cell surface receptors known to be associated with lung cancer invasiveness and cell survival. Our lab recently demonstrated that Fn14 was over-expressed in non-small cell lung cancer (NSCLC), correlated with activated epidermal growth factor receptor, and that expression of Fn14 modulated NSCLC cell invasiveness and metastasis. We hypothesize that elevated Fn14 expression can be maintained by c-Met activation, and that blockade of Fn14 can suppress NSCLC invasiveness driven by c-Met. Here we demonstrate that Fn14 protein expression significantly correlated with c-Met expression in primary NSCLC patient tumors. Fn14 and c-Met were more highly expressed in metastatic tumors compared to patient-matched primary NSCLC tumors, suggesting a role in the metastatic phenotype. To further elucidate the relationship between Fn14 and c-Met in NSCLC invasiveness, we employed two cell lines derived from a primary tumor (H2073) and metastatic lymph node (H1993) of the same patient. H1993 cells (c-Met receptor amplification) expressed significantly elevated protein levels of Fn14 compared to H2073 (low c-Met), which were significantly diminished in H1993 cells upon c-Met inhibition through SU11274 treatment. Moreover, exposure of H2073 cells to HGF induced the protein expression of Fn14. The elevated expression of Fn14 through HGF stimulation (H2073) or c-Met receptor amplification (H1993) was attenuated by pretreatment of the cells with U0126, a potent MEK inhibitor, suggesting that c-Met-induced Fn14 expression may depend on MAPK signaling. To further elucidate the effect of Fn14 on global c-Met-induced expression, genome-wide expression analysis was employed to determine genes both over-expressed in H1993 compared to H2073 and subsequently suppressed in Fn14 depleted H1993 cells. RALGPS2, a gene over-expressed in lung tumors that induces of c-fos and OPRL1, a gene elevated in metastatic melanoma, were elevated in the metastatic cell line and subsequently suppressed with Fn14 depletion. In addition, shRNA-mediated depletion of Fn14 in H1993 cells attenuated cell migration and invasion by 45% and 40%, respectively. This work demonstrates that c-Met and Fn14 may play a critical role in NSCLC metastasis, and suggests that targeting Fn14 may impact c-Met-driven NSCLC invasiveness. Successful targeting of NSCLC invasiveness would have significant clinical impact on a disease that is deadly in its advanced stages. Citation Format: Timothy G. Whitsett, Shannon P. Fortin, Paul A. Kurywchak, Julianna Ross, Janine LoBello, Christopher B. Kingsley, Jeffrey W. Allen, Glen J. Weiss, Nhan L. Tran. Elevated expression of Fn14 in non-small cell lung cancer correlates with c-MET and promotes tumor cell invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2013-3785

Abstract 1338: Chemokine (CXC motif) Ligand 5 protein expression in pancreatic tumors: Exploring chemostatic gradients in the tumor microenvironment

Due to lack of early presenting symptoms, pancreatic cancer is often diagnosed late when the tumor is highly invasive with aggressive clinical course and conventional therapies are limited in their effectiveness. Identifying molecular targets which contribute to tumor aggressiveness and metastasis could lead to development of better therapies to treat pancreas cancer patients. To identify potential molecular targets, we applied a specialized bioinformatics Breakpoint/Gene Aberration algorithm to analyze aCGH data and microarray expression data from 16 low-passage pancreas xenograft samples. Genes which were located near a putative breakpoint and whose expression was altered in a majority of the xenografts samples were considered putative targets. We report here preliminary data concerning one target, Chemokine (CXC motif) Ligand 5 (CXCL5) and its receptor CXCR2/IL8Rb. CXCL5 is a member of the ELR+ Chemokine family, a family of small, secreted ligands which are generally involved in cellular responses to injury and inflammation, but are also implicated in cancer progression and angiogenesis. CXCL5 is up regulated in 12 out of 16 xenograft samples analyzed (75%), with an average fold increase in expression of 41 (range 18 to 59). CXCL5 over-expression was further confirmed by IHC analysis of a custom made primary pancreas tumor microarray. Median IHC score (range 0-3) for CXCL5 in adenocarcinoma epithelial cells is 1.6, versus a median of 0.8 in the matched adjacent normal samples (N=34; p This work was supported by NIH P01 Grant CA109552. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1338.

Abstract A30: Integrated analysis of genomic data and annotations in cancer research

Many cancer researchers are currently generating heterogeneous genomic data sets containing mRNA expression, array based comparative genomic hybridization (aCGH), and other information from multiple patients. In the near future, data sets will likely include proteomics, methylomics, and high throughput sequencing data. Analytical tools that integrate these different data types to prioritize genes for further study have lagged, however. With this in mind, we have implemented a standalone software application that integrates CGH and mRNA expression profiling data. This application implements several different methods to generate ranked lists of genes and genomic regions based on breakpoint frequency, aberration frequency, and the impact of copy number changes on mRNA expression. The software is Java GUI based, with abstracted object models that can be extended as new genomic data types and annotations become available. We used this application to analyze aCGH datasets generated from 22 primary pancreatic tumor xenografts and mRNA expression datasets from 16 of the 22 samples. Genes which were located near a putative breakpoint and whose expression levels were high in a majority of the xenografts samples were considered putative targets. Preliminary IHC analysis of pancreas primary tumor tissue microarrays confirms one of these putative targets, Chemokine (CXC motif) Ligand 5 (CXCL5). By allowing cancer researchers to correlate genomic aberrations with expression data, we believe that this application will generate more meaningful targets for further biomarker identification and/or drug target discovery. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A30.