Sergey Mareninov

EGFR Mutation-Induced Alternative Splicing of Max Contributes to Growth of Glycolytic Tumors in Brain Cancer

Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.

The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings

Well preserved frozen biospecimens are ideal for evaluating the genome, transcriptome, and proteome. While papers reviewing individual aspects of frozen biospecimens are available, we present a current overview of experimental data regarding procurement, storage, and quality assurance that can inform the handling of frozen biospecimens. Frozen biospecimen degradation can be influenced by factors independent of the collection methodology including tissue type, premortem agonal changes, and warm ischemia time during surgery. Rapid stabilization of tissues by snap freezing immediately can mitigate artifactually altered gene expression and, less appreciated, protein phosphorylation profiles. Collection protocols may be adjusted for specific tissue types as cellular ischemia tolerance varies widely. If data is not available for a particular tissue type, a practical goal is snap freezing within 20min. Tolerance for freeze-thaw events is also tissue type dependent. Tissue storage at -80°C can preserve DNA and protein for years but RNA can show degradation at 5years. For -80°C freezers, aliquots frozen in RNAlater or similar RNA stabilizing solutions are a consideration. It remains unresolved as to whether storage at -150°C provides significant advantages relative to that at -80°C. Histologic quality assurance of tissue biospecimens is typically performed at the time of surgery but should also be conducted on the aliquot to be distributed because of tissue heterogeneity. Biobanking protocols for blood and its components are highly dependent on intended use and multiple collection tube types may be needed. Additional quality assurance testing should be dictated by the anticipated downstream applications.

Activation of autophagy in retinal ganglion cells

Autophagy has been shown to be activated in neuronal cells in response to injury and suggested to have a cell‐protective role in neurodegenerative diseases. In this study, we investigated the activation of autophagy in retinal ganglion cells (RGCs) following optic nerve transection (ONT) and evaluated its effect on RGC survival. Expression of several autophagy‐related genes, including Atg5, Atg7, and Atg12, and autophagy markers microtubule‐associated protein 1 light chain 3–II (LC3‐II) and beclin‐1 were analyzed at the transcriptional or protein level 1, 3, and 7 days after ONT. Transcription of the Atg5, Atg7, and Atg12 genes was up‐regulated 1.5‐ to 1.8‐fold in the retina 3 days after ONT compared with that in the controls. Expression of Atg12 mRNA was increased 1.6‐fold 1 day after ONT. Seven days after ONT, expression of Atg5, Atg7, and Atg12 mRNA was comparable to that in the untreated retinas. Western blot analysis of proteins isolated from RGCs showed 1.6‐, 2.7‐, and 1.7‐fold increases in LC3‐II level 1, 3, and 7 days after ONT, respectively, compared with those in the controls. Expression of beclin‐1 was 1.7‐fold higher 1 day after RGCs were axotomized, but 3 and 7 days after ONT it was comparable to that of the control. Inhibition of autophagy with bafilomycin A1, 3‐methyladenine, and Wortmannin in RGC‐5 cells under serum‐deprived conditions decreased cell viability by approximately 40%. These results suggest possible activation of autophagy in RGCs after optic nerve transection and demonstrate its protective role in RGC‐5 cells maintained under conditions of serum deprivation.

FAK activation and the role of epithelial membrane protein 2 (EMP2) in collagen gel contraction.

PURPOSE Proliferative vitreoretinopathy (PVR) occurs in approximately 10% of patients after retinal detachment. PVR results from a multiphase process that leads to an aberrant wound-healing strategy with contractile cellular forces and tractional retinal detachment (TRD). Epithelial membrane protein (EMP) 2 controls cell surface expression and function of integrin isoforms associated with cellular contraction in many cell types. Since EMP2 is highly expressed in retinal pigment epithelium, this study investigates the role of EMP2 in collagen gel contraction. METHODS EMP2 expression was recombinantly modified in the ARPE-19 cell line. Cell surface integrin expression was assessed by flow cytometry. Collagen gel contraction was assessed by using an in vitro assay and the percentage of contraction was quantified. Proliferation and migration were measured by BrdU incorporation and a wound-healing assay, respectively. Cellular invasion was investigated with polycarbonate membranes coated with collagen. RESULTS EMP2 expression levels correlated positively with the ability to contract collagen gels. Compared with wild-type ARPE-19 cells, the cells with increased EMP2 expression exhibited enhanced contraction (P = 0.02), and decreased EMP2 expression concomitantly resulted in decreased contraction (P = 0.002). EMP2 overexpression resulted in reduced proliferation, migration, and integrin alpha1 and alpha2 integrin expression. EMP2 overexpression was associated with a 70% increase in FAK activation (P = 0.0003) and relative resistance of gel contraction to inhibitors of FAK/Src activation. CONCLUSIONS ARPE-19-mediated collagen gel contraction is a multistep process that requires integrin ligation and activation of the FAK/Src complex. EMP2 positively modulates collagen gel contraction by ARPE-19 cells through increased FAK activation.

A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories.

UNLABELLED Frozen biospecimens are crucial for translational research and contain well-preserved nucleic acids and protein. However, the risks of freezer failure as well as space, cost, and environmental concerns of frozen biospecimens are substantial. OBJECTIVE The purpose of the study was to review the current status of room temperature biospecimen storage. METHODS We searched Pubmed and vendor websites to identify relevant information. RESULTS Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can now robustly preserve fresh tissue for up to 7days at room temperature. For longer term storage, commercial vendors of chemical matrices claim real time stability of nucleic acids of over 2 years and their accelerated aging studies to date suggest stability for 12years for RNA and 60years for DNA. However, anatomic pathology biorepositories store mostly frozen tissue rather than nucleic acids. Small quantities of tissue can be directly placed on some chemical matrices to stabilize DNA, however RNA and proteins are not preserved. Current lyophilization approaches can preserve histomorphology, DNA, RNA, and proteins though RNA shows moderate degradation after 1-2years. Formalin-free fixatives show improved but varying abilities to preserve nucleic acids and face validation as well as cost barriers in replacing FFPE specimens. The paraffin embedding process can degrade RNA. CONCLUSION Development of robust long-term room temperature biospecimen tissue storage technology can potentially reduce costs for the biomedical community in the face of growing targeted therapy needs and decreasing budgets.

Diabodies Targeting Epithelial Membrane Protein 2 Reduce Tumorigenicity of Human Endometrial Cancer Cell Lines

Purpose: Endometrial cancer is the most common gynecologic malignancy. One promising biomarker is epithelial membrane protein 2 (EMP2), and its expression is an independent prognostic indicator for tumors with poor clinical outcome expression. The present study assesses the suitability of EMP2 as a therapeutic target. Experimental Design: Human monovalent anti-EMP2 antibody fragments were isolated from a human phage display library and engineered as bivalent antibody fragments (diabodies) with specificity and avidity to both EMP2 peptides and native cell-surface EMP2 protein. Diabodies were assessed using cell death and apoptosis assays. In addition, the efficacy of EMP2 diabodies on endometrial cancer tumors was determined using mouse xenograft models. Results: Treatment of human endometrial adenocarcinoma cell lines with anti-EMP2 diabodies induced significant cell death and caspase-3 cleavage in vitro. These responses correlated with cellular EMP2 expression and were augmented by progesterone, which physiologically induces EMP2 expression. In vivo, treatment of subcutaneous human xenografts of HEC-1A cell lines with anti-EMP2 diabodies suppressed tumor growth and induced cell death in the xenograft. Conclusions: These findings suggest that EMP2 may be a potential pharmacologic target for human endometrial cancer.

Functional consequences of interactions between FAK and epithelial membrane protein 2 (EMP2).

PURPOSE Collagen gel contraction by ARPE-19 is controlled by epithelial membrane protein 2 (EMP2) through focal adhesion kinase (FAK) activation. The purpose of this study was to test the role of EMP2 in the cellular context of FAK activation. METHODS The ARPE-19 cell line was recombinantly modified to increase the expression of EMP2 and was used in this study. Quantification of FAK and Src phosphorylation was determined with Western blot analysis of whole cell lysates with the use of specific antibodies for different target sites of phosphorylation. Coimmunoprecipitation of whole cell lysates with an antibody against EMP2, followed by Western blot analysis and identification of FAK, was performed. Focal adhesions and their relationship to EMP2 were identified with immunofluorescence and confocal microscopy. F-actin distribution was identified using fluorescence microscopy, and alpha- smooth muscle actin (alpha-SMA) expression was quantified with Western blot analysis and specific antibodies. Adhesion to collagen type I was determined with a binding assay. RESULTS EMP2 overexpression led to increased FAK phosphorylation at all measured phosphorylation sites. Coimmunoprecipitation and confocal microscopy provided evidence for a physical association between EMP2 and FAK. Increased EMP2 was also associated with altered distribution of focal adhesions, changes in actin organization, increased alpha-SMA expression, and increased adherence to a collagen-coated surface. CONCLUSIONS The EMP2-FAK association represents a novel protein-protein interaction, not previously reported, that demonstrates significant functional cellular responses in the context of in vitro models of proliferative vitreoretinopathy (PVR).

Co-expression of heat shock transcription factors 1 and 2 in rat retinal ganglion cells.

Heat shock protein (HSP) plays an important role in the maintenance of neuronal survival during harmful conditions. Previously, we reported that metabolic stress induces HSP72 in retinal ganglion cells (RGCs) and protects against excitotoxicity, hypoxia and experimental glaucoma. To understand heat shock protein transcriptional mechanisms, we examined the cellular expression of heat shock factors 1 (HSF1) and 2 (HSF2) in the unstressed adult rat retina. Western blotting, immunohistochemistry and RT-PCR showed that mRNA and protein of HSF1 and HSF2 were present in the rat retina and predominantly expressed in RGC layer cells. Western blotting of dissociated RGC suspensions harvested with Thy-1 immuno-labeled magnetic beads confirmed that RGCs expressed HSF1, HSF2 and HSP72. Our findings suggest that both heat shock transcription factors 1 and 2 are linked to the heat shock response in retinal ganglion cells.

Ribosomal Proteins RPS11 and RPS20, Two Stress-Response Markers of Glioblastoma Stem Cells, Are Novel Predictors of Poor Prognosis in Glioblastoma Patients

Glioblastoma stem cells (GSC) co-exhibiting a tumor-initiating capacity and a radio-chemoresistant phenotype, are a compelling cell model for explaining tumor recurrence. We have previously characterized patient-derived, treatment-resistant GSC clones (TRGC) that survived radiochemotherapy. Compared to glucose-dependent, treatment-sensitive GSC clones (TSGC), TRGC exhibited reduced glucose dependence that favor the fatty acid oxidation pathway as their energy source. Using comparative genome-wide transcriptome analysis, a series of defense signatures associated with TRGC survival were identified and verified by siRNA-based gene knockdown experiments that led to loss of cell integrity. In this study, we investigate the prognostic value of defense signatures in glioblastoma (GBM) patients using gene expression analysis with Probeset Analyzer (131 GBM) and The Cancer Genome Atlas (TCGA) data, and protein expression with a tissue microarray (50 GBM), yielding the first TRGC-derived prognostic biomarkers for GBM patients. Ribosomal protein S11 (RPS11), RPS20, individually and together, consistently predicted poor survival of newly diagnosed primary GBM tumors when overexpressed at the RNA or protein level [RPS11: Hazard Ratio (HR) = 11.5, p<0.001; RPS20: HR = 4.5, p = 0.03; RPS11+RPS20: HR = 17.99, p = 0.001]. The prognostic significance of RPS11 and RPS20 was further supported by whole tissue section RPS11 immunostaining (27 GBM; HR = 4.05, p = 0.01) and TCGA gene expression data (578 primary GBM; RPS11: HR = 1.19, p = 0.06; RPS20: HR = 1.25, p = 0.02; RPS11+RPS20: HR = 1.43, p = 0.01). Moreover, tumors that exhibited unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) or wild-type isocitrate dehydrogenase 1 (IDH1) were associated with higher RPS11 expression levels [corr (IDH1, RPS11) = 0.64, p = 0.03); [corr (MGMT, RPS11) = 0.52, p = 0.04]. These data indicate that increased expression of RPS11 and RPS20 predicts shorter patient survival. The study also suggests that TRGC are clinically relevant cells that represent resistant tumorigenic clones from patient tumors and that their properties, at least in part, are reflected in poor-prognosis GBM. The screening of TRGC signatures may represent a novel alternative strategy for identifying new prognostic biomarkers.

Epithelial membrane protein-2 (EMP2) and experimental proliferative vitreoretinopathy (PVR).

Purpose: Proliferative vitreoretinopathy (PVR) is believed to result in part from de-differentiation of retinal pigment epithelium (RPE) with cellular migration in the vitreous cavity, membrane formation, and contraction in an aberrant wound-healing strategy. In an in vitro collagen-gel contraction assay, epithelial membrane protein 2 (EMP2) controls contraction through activation of focal adhesion kinase (FAK) in a RPE cell line (ARPE-19). The purpose of this study was to investigate how blocking or altering the level of EMP2 expression changed clinical PVR in an in vivo model. Methods: Using the ARPE-19 cell line, the levels of EMP2 modulated through stable transfections of an EMP2 overexpressing construct, EMP2 ribozyme, or vector alone. These transfected cell lines were used in a rabbit model of PVR. The severity of PVR was classified by two masked observers. An EMP2 blocking antibody was also used to decrease functional EMP2 in the PVR model. Immunohistochemistry was used to evaluate EMP2 expression in vivo. Results: The transfectants with lower levels of EMP2 had significantly less PVR severity than the degree of PVR induced by wild-type cells (p = 0.05). Also, the transfectants with a low-level of EMP2 expression showed a strong trend of less PVR severity than the high-levels EMP2 transfectants (p = 0.06). Blocking EMP2 with a specific polyclonal antibody significantly decreased the level of PVR severity (p = 0.02). PVR membranes were found to be positive for EMP2 expression. Conclusions: These in vivo studies support a direct correlation between EMP2 expression and severity of PVR. These results validate the potential for controlling RPE biology through a change in EMP2 expression, and provide a potential therapeutic target for this disease.