Nuclear respiratory factor 1 transcriptomic signatures as prognostic indicators of recurring aggressive mesenchymal glioblastoma and resistance to therapy in White American females.

PURPOSE: The mechanisms contributing to recurrence of glioblastoma (GBM), an aggressive neuroepithelial brain tumor, remain unknown. We have recently shown that nuclear respiratory factor 1 (NRF1) is an oncogenic transcription factor and its transcriptional activity is associated with the progression and prognosis of GBM. Herein, we extend our efforts to (1) identify influential NRF1-driven gene and microRNA (miRNA) expression for the aggressiveness of mesenchymal GBM; and (2) understand the molecular basis for its poor response to therapy. METHODS: Clinical data and RNA-Seq from four independent GBM cohorts were analyzed by Bayesian Network Inference with Java Objects (BANJO) and Markov chain Monte Carlo (MCMC)-based gene order to identify molecular drivers of mesenchymal GBM as well as prognostic indicators of poor response to radiation and chemotherapy. RESULTS: We are the first to report sex-specific NRF1 motif enriched gene signatures showing increased susceptibility to GBM. Risk estimates for GBM were increased by greater than 100-fold with the joint effect of NRF1-driven gene signatures-CDK4, DUSP6, MSH2, NRF1, and PARK7 in female GBM patients and CDK4, CASP2, H6PD, and NRF1 in male GBM patients. NRF1-driven causal Bayesian network genes were predictive of poor survival and resistance to chemoradiation in IDH1 wild-type mesenchymal GBM patients. NRF1-regulatable miRNAs were also associated with poor response to chemoradiation therapy in female IDH1 wild-type mesenchymal GBM. Stable overexpression of NRF1 reprogramed human astrocytes into neural stem cell-like cells expressing SOX2 and nestin. These cells differentiated into neurons and form tumorospheroids. CONCLUSIONS: In summary, our novel discovery shows that NRF1-driven causal genes and miRNAs involved in cancer cell stemness and mesenchymal features contribute to cancer aggressiveness and recurrence of aggressive therapy-resistant glioblastoma.

Nuclear Respiratory Factor 1 (NRF1) Transcriptional Activity-Driven Gene Signature Association with Severity of Astrocytoma and Poor Prognosis of Glioblastoma.

Despite tremendous progress in understanding the pathobiology of astrocytoma, major gaps remain in our knowledge of the molecular basis underlying the aggressiveness of high-grade astrocytoma (glioblastoma - GBM). Recently, we and others have shown nuclear respiratory factor 1 (NRF1) transcription factor being highly active in human cancers, but its role in astrocytoma remains unknown. Therefore, the purpose of this study was to uncover the role of NRF1 in the progression of GBM. NRF1 has higher mRNA expression and transcription factor activity in astrocytoma compared to non-tumor brain tissue. NRF1 activity also correlated with the aggressiveness of cancer. Increased NRF1 TF activity coupled with overexpression of RHOG was associated with poor survival of GBM patients. NRF1 activity was associated with transcriptomic signatures of neurogenesis, cell stemness, epithelial-mesenchymal transition and cell cycle progression. Overexpression of CDK4, AKT1, APAF1, HDAC1, NBN, TGFB1, & TNFRSF1A and downregulation of CASP3, IL7, STXBP1 and OPA1 predicted GBM malignancy in high expressors of NRF1 activity. Increased expression of the NRF1 motif containing genes, H6PD, NAT10, NBEAL2, and RNF19B predicted poor survival of IDH1 wild-type GBM patients. Poor survival outcomes and resistance to Temozolomide therapy were associated with higher NRF1 expression including its targets - LDHA, ZMAT3, NSUN2, ARMC5, NDEL1, CLPTM1L, ALKBH5, YIPF5, PPP2CA, and TFG. These findings suggest that aberrant NRF1 activity may contribute to the pathogenesis of GBM and severity of astrocytoma. Further analyses of NRF1 gene signatures will pave the way for next generation targeted therapies and drug combination strategies for GBM patients.

Interplay between NRF1, E2F4 and MYC transcription factors regulating common target genes contributes to cancer development and progression.

BACKGROUND: Nuclear respiratory factor 1 (NRF1), historically perceived as a protein regulating genes controlling mitochondrial biogenesis, is now widely recognized as a multifunctional protein and as a key player in the transcriptional modulation of genes implicated in various cellular functions. Here, we present emerging data supporting novel roles of NRF1 in cancer development and progression through its interplay with the transcription factors E2F4 and MYC. To identify common human NRF1, E2F4 and MYC target genes, we analyzed the Encyclopedia of DNA Elements (ENCODE) NRF1 ChIP-Seq data. By doing so, we identified 9253 common target genes with NRF1, E2F4 and MYC binding motifs. NRF1 binding motifs were found to be present in genes operating in signaling pathways governing all hallmarks of malignant transformation and progression, including proliferation, invasion, self-renewal and apoptosis. CONCLUSIONS: In addition to controlling mitochondrial biogenesis NRF1, in conjunction with E2F4 and MYC, may play a critical role in the acquisition of human cancer characteristics. Additionally, NRF1 may orchestrate both MYC and E2F4 to regulate common target genes linked to multiple networks in the development and progression of cancer. A comprehensive understanding of this dynamic interplay will set the stage, not only for the design of novel treatment strategies, but also for the discovery of pan-cellular transcription factor regulatory strategies to predict cancer risk, therapy response and patient prognosis.

Microarray Analysis in Glioblastomas.

Microarray analysis in glioblastomas is done using either cell lines or patient samples as starting material. A survey of the current literature points to transcript-based microarrays and immunohistochemistry (IHC)-based tissue microarrays as being the preferred methods of choice in cancers of neurological origin. Microarray analysis may be carried out for various purposes including the following: i. To correlate gene expression signatures of glioblastoma cell lines or tumors with response to chemotherapy (DeLay et al., Clin Cancer Res 18(10):2930-2942, 2012). ii. To correlate gene expression patterns with biological features like proliferation or invasiveness of the glioblastoma cells (Jiang et al., PLoS One 8(6):e66008, 2013). iii. To discover new tumor classificatory systems based on gene expression signature, and to correlate therapeutic response and prognosis with these signatures (Huse et al., Annu Rev Med 64(1):59-70, 2013; Verhaak et al., Cancer Cell 17(1):98-110, 2010). While investigators can sometimes use archived tumor gene expression data available from repositories such as the NCBI Gene Expression Omnibus to answer their questions, new arrays must often be run to adequately answer specific questions. Here, we provide a detailed description of microarray methodologies, how to select the appropriate methodology for a given question, and analytical strategies that can be used. Experimental methodology for protein microarrays is outside the scope of this chapter, but basic sample preparation techniques for transcript-based microarrays are included here.

Somatic mutations lead to an oncogenic deletion of met in lung cancer.

Activating mutations in receptor tyrosine kinases play a critical role in oncogenesis. Despite evidence that Met kinase is deregulated in human cancer, the role of activating mutations in cancers other than renal papillary carcinoma has not been well defined. Here we report the identification of somatic intronic mutations of Met kinase that lead to an alternatively spliced transcript in lung cancer, which encodes a deletion of the juxtamembrane domain resulting in the loss of Cbl E3-ligase binding. The mutant receptor exhibits decreased ubiquitination and delayed down-regulation correlating with elevated, distinct Met expression in primary tumors harboring the deleted receptor. As a consequence, phospho-Met and downstream mitogen-activated protein kinase activation is sustained on ligand stimulation. Cells expressing the Met deletion reveal enhanced ligand-mediated proliferation and significant in vivo tumor growth. A hepatocyte growth factor competitive Met antagonist inhibits receptor activation and proliferation in tumor cells harboring the Met deletion, suggesting the important role played by ligand-dependent Met activation and the potential for anticancer therapy. These results support a critical role for Met in lung cancer and somatic mutation-driven splicing of an oncogene that leads to a different mechanism for tyrosine kinase activation through altered receptor down-regulation in human cancer.

An automated image capture and quantitation approach to identify proteins affecting tumor cell proliferation.

To facilitate the characterization of proteins that negatively regulate tumor cell proliferation in vitro, the authors have implemented a high-throughput functional assay that measures S-phase progression of tumor cell lines. For 2 tumor cell lines-human melanoma A375 and human lung carcinoma A549-conditions were established using the cyclin-dependent kinase inhibitor, p27kip; the tumor suppressor p53, a kinase-inactive allele of the cell cycle-regulated serine/threonine kinase Aurora2; and the G1/S drug block, aphidicolin. For screening purposes, gene libraries were delivered by adenoviral infection. Cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on a Cellomics ArrayScanII was used to quantify the effects of these treatments on cell proliferation. The assay can be used to identify novel proteins involved in proliferation and serves as a more robust, reproducible, and sensitive alternative to enzyme-linked immunosorbent assay (ELISA)-based technologies.

Real-time impedance assay to follow the invasive activities of metastatic cells in culture.

Here we present research detecting the invasive activities of metastatic cells in vitro using electric cell-substrate impedance sensing (ECIS). The assay is based on previous microscopic observations, where metastatic cells added over established endothelial cell layers were observed to attach to and invade the cell layer. Human umbilical vein endothelial cells (HUVECs) werefirst grown to confluence on small gold electrodes. The impedance of these electrodes was followed after the addition of suspensions of different sublines of the Dunning murine prostatic adenocarcinoma series (G, AT1, AT2, AT3, ML, and MLL). For highly metastatic sublines, within an hour after being challenged, the impedance of the confluent HUVEC layer was substantially reduced. The effect of the weakly metastatic sublines was less pronounced, and the extent and the rate of this drop in impedance could be correlated with the metastatic potential of each of six sublines tested. The real-time assay is effective in both normal and low (1%) serum concentrations, and the detected activity requires the presence of viable transformed cells. In addition to the murine cell lines, similar behavior was observed using four established human prostatic cancer lines (DU145, PC3, TSU, and PPC1). These results suggest that this ECIS-based assay might be used with primary human cultures to establish the metastatic abilities of cells isolated from biopsies.