The TWEAK receptor Fn14 is a potential cell surface portal for targeted delivery of glioblastoma therapeutics

Abstract

Fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is the cell surface receptor for the tumor necrosis factor (TNF) family member TNF-like weak inducer of apoptosis (TWEAK). The Fn14 gene is normally expressed at low levels in healthy tissues but expression is significantly increased after tissue injury and in many solid tumor types, including glioblastoma (GB; formerly referred to as 'GB multiforme'). GB is the most common and aggressive primary malignant brain tumor and the current standard-of-care therapeutic regimen has a relatively small impact on patient survival, primarily because glioma cells have an inherent propensity to invade into normal brain parenchyma, which invariably leads to tumor recurrence and patient death. Despite major, concerted efforts to find new treatments, a new GB therapeutic that improves survival has not been introduced since 2005. In this review article, we summarize studies indicating that (i) Fn14 gene expression is low in normal brain tissue but is upregulated in advanced brain cancers and, in particular, in GB tumors exhibiting the mesenchymal molecular subtype; (ii) Fn14 expression can be detected in glioma cells residing in both the tumor core and invasive rim regions, with the maximal levels found in the invading glioma cells located within normal brain tissue; and (iii)

Tweak: Fn14 engagement as well as Fn14 overexpression can stimulate glioma cell migration, invasion and resistance to chemotherapeutic agents in vitro. We also discuss two new therapeutic platforms that are currently in development that leverage Fn14 overexpression in GB tumors as a way to deliver cytotoxic agents to the glioma cells remaining after surgical resection while sparing normal healthy brain cells.

Figure 1. The TWEAK/Fn14 signaling dyad

Both membrane-anchored TWEAK and soluble TWEAK monomers assemble into trimers. TWEAK binding to the Fn14 extracellular domain promotes Fn14 trimerization and binding of TRAF adaptor proteins to the Fn14 cytoplasmic tail. This interaction triggers the activation of various downstream protein kinases, only some of which are shown here, and signaling cascades, including the classical (c) and alternative (a) NF-κB pathways, resulting in transcription factor binding to genomic DNA and the expression of numerous TWEAK-inducible genes, including Fn14.^, TWEAK:Fn14 engagement stimulates various cellular responses such as apoptosis, migration, and differentiation.

Figure 2. Analysis of Fn14 mRNA expression in GB molecular subtypes

Fn14 (queried as TNFRSF12A) mRNA expression data in 195 GB tumors was downloaded as z-scores from cbioportal.org.^, Z-Scores were computed by cbioportal.org relative to the expression distribution of each gene in tumors that are diploid for this gene. Patients used in this analysis were limited to the provisional TCGA dataset. The provisional dataset using the Affymetrix U113 microarray platform is the only GB dataset with annotation for classical, mesenchymal, neural, and proneural expression clusters on cbioportal.org. Patient/sample sets for each expression cluster were plotted as box and whisker plots. Classical n= 54, Mesenchymal n=56, Neural n=29, and Proneural n=56. Whiskers of the plot map the maximum and minimum z-score for each expression cluster. Bar in box represents the mean value for the Fn14 z-score of each group. Top and bottoms of the box represent the 25^th and 75^th percentile, respectively, of the Fn14 z-score values for each group. Each of the mean expression values are significantly different from one another (p< 0.05) as determined using unpaired Student's t test (two-tailed).

Figure 3. Analysis of Fn14 protein expression in normal brain and GB invasive rim tissue

Non-neoplastic brain tissue from a male 45-yr-old epilepsy patient (panel A) and right frontal tumor tissue from a female 59-yr-old GB patient (panel B) was immunostained using an anti-Fn14 rabbit mAb (EPR3179; Abcam). The GB image shows the invasive rim region, with the Fn14-positive glioma cells (brown stain, several positive cells are denoted with arrows) interspersed with normal brain tissue. Scale bar = 200 um.

Figure 4. Summary diagram of signaling pathways and NF-κB-inducible gene products implicated in Fn14-triggered glioma cell migration, invasion and survival

TWEAK:Fn14 binding and Fn14 overexpression stimulate similar intracellular signaling cascades and downstream cellular responses. Some of the pathways and molecules that play a role in Fn14-mediated glioma cell migration, invasion and resistance to chemotherapeutic and pro-apoptotic agents are shown here.

Figure 5. Adjuvant therapy for GB patients using engineered nanoparticles

(A) MRI images obtained from a female 59-yr-old GB patient before and after surgical resection are shown. Therapeutic nanoparticles delivered into the invasive rim region at the time of surgery using advanced CED technology could potentially reach the invasive glioma cells outside the immediate resection margin and deliver their cytotoxic payload. (B) Conventional, drug-loaded NPs without a specialized surface coating penetrate poorly through brain extracellular space after CED (normal brain cells indicated with white circles containing black lines; catheter is indicated using dark black V) and Fn14 targeting is restricted to those glioma cells close to the catheter entry point (white circles; Fn14 molecules indicated with rectangles). In contrast, NPs engineered for enhanced brain-penetrating capacity move deeper into the adjacent brain parenchyma and surface decoration with an Fn14 targeting moiety may increase NP binding to the invasive glioma cells.