Craig P. Webb

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortiums (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

Identifying disease-specific genes based on their topological significance in protein networks

BackgroundThe identification of key target nodes within complex molecular networks remains a common objective in scientific research. The results of pathway analyses are usually sets of fairly complex networks or functional processes that are deemed relevant to the condition represented by the molecular profile. To be useful in a research or clinical laboratory, the results need to be translated to the level of testable hypotheses about individual genes and proteins within the condition of interest.ResultsIn this paper we describe novel computational methodology capable of predicting key regulatory genes and proteins in disease- and condition-specific biological networks. The algorithm builds shortest path network connecting condition-specific genes (e.g. differentially expressed genes) using global database of protein interactions from MetaCore. We evaluate the number of all paths traversing each node in the shortest path network in relation to the total number of paths going via the same node in the global network. Using these numbers and the relative size of the initial data set, we determine the statistical significance of the network connectivity provided through each node. We applied this method to gene expression data from psoriasis patients and identified many confirmed biological targets of psoriasis and suggested several new targets. Using predicted regulatory nodes we were able to reconstruct disease pathways that are in excellent agreement with the current knowledge on the pathogenesis of psoriasis.ConclusionThe systematic and automated approach described in this paper is readily applicable to uncovering high-quality therapeutic targets, and holds great promise for developing network-based combinational treatment strategies for a wide range of diseases.

Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase

Mutations in the Ras family of GTP binding proteins represent one of the most frequently observed genetic alterations in human cancers. We and others have recently demonstrated that expression of Met, the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), is significantly up-regulated in Ras-transformed cells. Because HGF/SF-Met signaling is proposed to play a prominent role in tumor development and progression, we assessed the possible requirement for Met during Ras-mediated tumor growth and metastasis. To disrupt endogenous Met signaling, we constructed dominant-negative mutants of both human and murine Met and showed that these can inhibit HGF/SF-mediated Met signaling and cell invasion of ras-transformed cells in vitro. Moreover, ectopic expression of dominant-negative Met mutants reduced the s.c. tumor growth of ras-transformed cells and dramatically suppressed their ability to form lung metastases in vivo. Our data demonstrate that Met plays a prominent role during Ras-mediated tumor growth and metastasis, and further suggest that agents that inhibit HGF/SF-Met signaling may represent an important therapeutic avenue for the treatment of a variety of malignant tumors.

Normal and Malignant Prostate Epithelial Cells Differ in Their Response to Hepatocyte Growth Factor/Scatter Factor

Hepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer.

MicroRNA 92a-2*: A Biomarker Predictive for Chemoresistance and Prognostic for Survival in Patients with Small Cell Lung Cancer

Purpose: Although the majority of patients with small cell lung cancer (SCLC) respond to initial chemotherapy, those with disease progression at first response assessment (chemoresistance) have inferior outcomes. There is a need for predictive biomarkers to aid investigators in designing future clinical trials that better stratify patients beyond standard clinical and laboratory parameters and to identify new treatments for this patient subpopulation. We hypothesized that tumor microRNAs (miRNAs) could serve as predictive biomarkers for chemoresistance and prognostic biomarkers for survival of patients with SCLC treated with systemic chemotherapy. Patients and Methods: SCLC samples annotated with clinical characteristics and baseline comorbidities were available. miRNA microarray profiling was performed on diagnostic SCLC tumor samples, and analysis was performed using XenoBase, a data integration and discovery tool. Confirmation of the top 16 miRNA candidates was performed using quantitative real-time polymerase chain reaction followed by analyses to determine clinical and miRNA biomarkers associated with chemoresistance and survival. Results: miRNAs significantly associated with chemoresistance were miR-92a-2* (p = 0.010), miR-147 (p = 0.018), and miR-574-5p (p = 0.039). By stepwise multivariate analysis, only gender and miR-92a-2* contributed significantly to survival (p = 0.023) and (p = 0.015), respectively. Baseline comorbidities were not associated with chemoresistance or survival. Conclusions: Higher tumor miR-92a-2* levels are associated with chemoresistance and with decreased survival in patients with SCLC. Tumor miR-92a-2* may have application in screening patients with SCLC at risk for de novo chemoresistance in an effort to design more tailored clinical trials for this subpopulation. Further validation in independent sample sets is warranted.

Towards consensus practices to qualify safety biomarkers for use in early drug development

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Glycogene Expression Alterations Associated with Pancreatic Cancer Epithelial-Mesenchymal Transition in Complementary Model Systems

Background The ability to selectively detect and target cancer cells that have undergone an epithelial-mesenchymal transition (EMT) may lead to improved methods to treat cancers such as pancreatic cancer. The remodeling of cellular glycosylation previously has been associated with cell differentiation and may represent a valuable class of molecular targets for EMT. Methodology/Principal Findings As a first step toward investigating the nature of glycosylation alterations in EMT, we characterized the expression of glycan-related genes in three in-vitro model systems that each represented a complementary aspect of pancreatic cancer EMT. These models included: 1) TGFβ-induced EMT, which provided a look at the active transition between states; 2) a panel of 22 pancreatic cancer cell lines, which represented terminal differentiation states of either epithelial-like or mesenchymal-like; and 3) actively-migrating and stationary cells, which provided a look at the mechanism of migration. We analyzed expression data from a list of 587 genes involved in glycosylation (biosynthesis, sugar transport, glycan-binding, etc.) or EMT. Glycogenes were altered at a higher prevalence than all other genes in the first two models (p<0.05 and <0.005, respectively) but not in the migration model. Several functional themes were shared between the induced-EMT model and the cell line panel, including alterations to matrix components and proteoglycans, the sulfation of glycosaminoglycans; mannose receptor family members; initiation of O-glycosylation; and certain forms of sialylation. Protein-level changes were confirmed by Western blot for the mannose receptor MRC2 and the O-glycosylation enzyme GALNT3, and cell-surface sulfation changes were confirmed using Alcian Blue staining. Conclusions/Significance Alterations to glycogenes are a major component of cancer EMT and are characterized by changes to matrix components, the sulfation of GAGs, mannose receptors, O-glycosylation, and specific sialylated structures. These results provide leads for targeting aggressive and drug resistant forms of pancreatic cancer cells.

Evidence for a role of Met-HGF/SF during Ras-mediated tumorigenesis/metastasis

Aberrations in Met-hepatocyte growth factor/scatter factor (HGF/SF) signaling have been implicated in the acquisition of tumorigenic and metastatic phenotypes. Here we show that murine NIH3T3 and C127 cells transformed by the Ras oncogene overexpress the Met receptor, resulting in enhanced HGF/SF-mediated responses in vitro including invasion through basement membrane. Accompanying the increase in Met in ras-transformed NIH3T3 cells, there is a decrease in endogenous HGF/SF expression as previously observed in cells exogenously overexpressing Met. However, subcutaneously grown tumors and experimental lung metastases derived from these cells express significantly higher levels of endogenous HGF/SF together with high levels of Met. These results suggest Met-HGF/SF signaling enhances tumor growth and metastasis of Ras-transformed NIH3T3 cells.

Genes that regulate metastasis and angiogenesis.

Genetic instability and an accumulation of genetic and epigenetic changes during tumor progression lead to an increasingly aggressive and treatment-resistant phenotype, and ultimately metastasis. In recent years it has become well established that angiogenesis, the process by which new vasculature is formed from pre-existing vessels, is an essential component to primary tumor growth and distant metastasis. A greater understanding of the complex multitude of factors involved in tumor angiogenesis and metastasis is fundamental to the development of potential therapeutics to treat malignant disease. As highlighted throughout this review, angiogenesis and metastasis share many common cellular and molecular features. We will briefly discuss the pertinent genes involved in the regulation of angiogenesis and metastasis.