Identification of Disease Susceptibility Alleles in the Next Generation Sequencing Era.

The development of next generation sequencing (NGS) technologies has transformed the study of human genetic variation. In less than a decade, NGS has facilitated the discovery of causal mutations in both rare, monogenic diseases and common, heterogeneous disorders, leading to unprecedented improvements in disease diagnosis and treatment strategies. Given the rapid evolution of NGS platforms, it is now possible to analyze whole genomes and exomes quickly and affordably. Further, emerging NGS applications, such as single-cell sequencing, have the power to address specific issues like somatic variation, which is yielding new insights into the role of somatic mutations in cancer and late-onset diseases. Despite limitations associated with current iterations of NGS technologies, the impact of this approach on identifying disease-causing variants has been significant. This chapter provides an overview of several NGS platforms and applications and discusses how these technologies can be used in concert with experimental and computational strategies to identify variants with a causative effect on disease development and progression.

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.

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.