Identification of driver mutations in human diseases is often limited by cohort size and availability of appropriate statistical models. We propose a framework for the systematic discovery of genetic alterations that are causal determinants of disease, by prioritizing genes upstream of functional disease drivers, within regulatory networks inferred de novo from experimental data. We tested this framework by identifying the genetic determinants of the mesenchymal subtype of glioblastoma. Our analysis uncovered KLHL9 deletions as upstream activators of two previously established master regulators of the subtype, C/EBP? and C/EBP?. Rescue of KLHL9 expression induced proteasomal degradation of C/EBP proteins, abrogated the mesenchymal signature, and reduced tumor viability in vitro and in vivo. Deletions of KLHL9 were confirmed in > 50% of mesenchymal cases in an independent cohort, thus representing the most frequent genetic determinant of the subtype. The method generalized to study other human diseases, including breast cancer and Alzheimer's disease.
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.
Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr(176), but not Trp(231), resulted in the loss of TWEAK binding to Fn14 substantiating Tyr(176) as the anchoring residue. Importantly, mutation of TWEAK at Tyr(176) did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor ?-light chain enhancer of activated B cell (NF-?B) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.
The long-term survival of patients with glioblastoma is compromised by the proclivity for local invasion into the surrounding normal brain, escaping surgical resection and contributing to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the Rho guanosine triphosphatase family member Rac1. Here, we demonstrate that TWEAK acts as a chemotactic factor for glioma cells, a potential process for driving cell invasion into the surrounding brain tissue. TWEAK exposure induced the activation of Src family kinases (SFKs), and pharmacologic suppression of SFK activity inhibited TWEAK-induced chemotactic migration. We employed a multiplexed Luminex assay and identified Lyn as a candidate SFK activated by TWEAK. Depletion of Lyn suppressed TWEAK-induced chemotaxis and Rac1 activity. Furthermore, Lyn gene expression levels increase with primary glioma tumor grade and inversely correlate with patient survival. These results show that TWEAK-induced glioma cell chemotaxis is dependent upon Lyn kinase function and, thus, provides opportunities for therapeutic targeting of this deadly disease.
Glioma cell migration correlates with Pyk2 activity, but the intrinsic mechanism that regulates the activity of Pyk2 is not fully understood. Previous studies have supported a role for the N-terminal FERM domain in the regulation of Pyk2 activity as mutations in the FERM domain inhibit Pyk2 phosphorylation. To search for novel protein-protein interactions mediated by the Pyk2 FERM domain, we utilized a yeast two-hybrid genetic selection to identify the mammalian Ste20 homolog MAP4K4 as a binding partner for the Pyk2 FERM domain. MAP4K4 coimmunoprecipitated with Pyk2 and was a substrate for Pyk2 but did not coimmunoprecipitate with the closely related focal adhesion kinase FAK. Knockdown of MAP4K4 expression inhibited glioma cell migration and effectively blocked Pyk2 stimulation of glioma cell. Increased expression of MAP4K4 stimulated glioma cell migration; however, this stimulation was blocked by knockdown of Pyk2 expression. These data support that the interaction of MAP4K4 and Pyk2 is integrated with glioma cell migration and suggest that inhibition of this interaction may represent a potential therapeutic strategy to limit glioblastoma tumor dispersion.
Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the "Go or Grow" hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the "Go or Grow" hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens in situ (n?=?44) revealed higher proliferation (Ki67 labeling index) in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-?B activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-?B- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-?B activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-?B staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation in vitro, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-?B by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration in vitro and invasion ex vivo. Notably, inhibition of NF-?B was found to have no effect on the proliferation rate of glioma cells. These findings suggest that the reciprocal and coordinated suppression/activation of transcription factors, such as c-Myc and NF-?B may underlie the shift of glioma cells from a "growing-to-going" phenotype.
Brain metastasis (BM) can affect ? 25% of nonsmall cell lung cancer (NSCLC) patients during their lifetime. Efforts to characterize patients that will develop BM have been disappointing. microRNAs (miRNAs) regulate the expression of target mRNAs. miRNAs play a role in regulating a variety of targets and, consequently, multiple pathways, which make them a powerful tool for early detection of disease, risk assessment, and prognosis. We investigated miRNAs that may serve as biomarkers to differentiate between NSCLC patients with and without BM. miRNA microarray profiling was performed on samples from clinically matched NSCLC from seven patients with BM (BM+) and six without BM (BM-). Using t-test and further qRT-PCR validation, eight miRNAs were confirmed to be significantly differentially expressed. Of these, expression of miR-328 and miR-330-3p were able to correctly classify BM+ vs. BM- patients. This classifier was used on a validation cohort (n = 15), and it correctly classified 12/15 patients. Gene expression analysis comparing A549 parental and A549 cells stably transfected to over-express miR-328 (A549-328) identified several significantly differentially expressed genes. PRKCA was one of the genes over-expressed in A549-328 cells. Additionally, A549-328 cells had significantly increased cell migration compared to A549 cells, which was significantly reduced upon PRKCA knockdown. In summary, miR-328 has a role in conferring migratory potential to NSCLC cells working in part through PRKCA and with further corroboration in additional independent cohorts, these miRNAs may be incorporated into clinical treatment decision making to stratify NSCLC patients at higher risk for developing BM.
Novel thermoreversible copolymers of N-isopropylacrylamide (NIPAAm) with collagenase-sensitive solubility behavior were synthesized by radical polymerization of poly(NIPAAm-co-NASI) and nucleophilic substitution of custom peptides GAPGL-NH(2) and GAPGLF-NH(2). The materials were characterized by nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography in conjunction with static light scattering, differential scanning calorimetry (DSC), and cloud point determination. Successful synthesis and specific degradation by collagenase above and below the material LCST was confirmed by NMR. The LCST behavior of the polymers was affected by collagenase. The LCST of the copolymers, as measured by cloud point determination, increased by 1 and 9 degrees C, respectively, after enzymatic degradation. DSC thermographs indicated increased polymer solubility after enzymatic degradation because of a reduced energy of gelation. These results demonstrate the significant impact of a single amino acid on the LCST behavior of thermosensitive copolymers. Furthermore, the results suggest that comonomers in similar systems could be designed to elicit phase transitions or conformation changes in response to a variety of enzymes for which the substrate structure is known.
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