Paul R. Jamieson

EphA3 Maintains Tumorigenicity and Is a Therapeutic Target in Glioblastoma Multiforme

Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. We show that the receptor tyrosine kinase EphA3 is frequently overexpressed in GBM and, in particular, in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and appears critically involved in maintaining tumor cells in a less differentiated state by modulating mitogen-activated protein kinase signaling. EphA3 knockdown or depletion of EphA3-positive tumor cells reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific monoclonal antibody. These results identify EphA3 as a functional, targetable receptor in GBM.

Glioma surgical aspirate: a viable source of tumor tissue for experimental research

Brain cancer research has been hampered by a paucity of viable clinical tissue of sufficient quality and quantity for experimental research. This has driven researchers to rely heavily on long term cultured cells which no longer represent the cancers from which they were derived. Resection of brain tumors, particularly at the interface between normal and tumorigenic tissue, can be carried out using an ultrasonic surgical aspirator (CUSA) that deposits liquid (blood and irrigation fluid) and resected tissue into a sterile bottle for disposal. To determine the utility of CUSA-derived glioma tissue for experimental research, we collected 48 CUSA specimen bottles from glioma patients and analyzed both the solid tissue fragments and dissociated tumor cells suspended in the liquid waste fraction. We investigated if these fractions would be useful for analyzing tumor heterogeneity, using IHC and multi-parameter flow cytometry; we also assessed culture generation and orthotopic xenograft potential. Both cell sources proved to be an abundant, highly viable source of live tumor cells for cytometric analysis, animal studies and in-vitro studies. Our findings demonstrate that CUSA tissue represents an abundant viable source to conduct experimental research and to carry out diagnostic analyses by flow cytometry or other molecular diagnostic procedures.

ELK4 neutralization sensitizes glioblastoma to apoptosis through downregulation of the anti-apoptotic protein Mcl-1

Glioma is the most common adult primary brain tumor. Its most malignant form, glioblastoma multiforme (GBM), is almost invariably fatal, due in part to the intrinsic resistance of GBM to radiation- and chemotherapy-induced apoptosis. We analyzed B-cell leukemia-2 (Bcl-2) anti-apoptotic proteins in GBM and found myeloid cell leukemia-1 (Mcl-1) to be the highest expressed in the majority of malignant gliomas. Mcl-1 was functionally important, as neutralization of Mcl-1 induced apoptosis and increased chemotherapy-induced apoptosis. To determine how Mcl-1 was regulated in glioma, we analyzed the promoter and identified a novel functional single nucleotide polymorphism in an uncharacterized E26 transformation-specific (ETS) binding site. We identified the ETS transcription factor ELK4 as a critical regulator of Mcl-1 in glioma, since ELK4 downregulation was shown to reduce Mcl-1 and increase sensitivity to apoptosis. Importantly the presence of the single nucleotide polymorphism, which ablated ELK4 binding in gliomas, was associated with lower Mcl-1 levels and a greater dependence on Bcl-xL. Furthermore, in vivo, ELK4 downregulation reduced tumor formation in glioblastoma xenograft models. The critical role of ELK4 in Mcl-1 expression and protection from apoptosis in glioma defines ELK4 as a novel potential therapeutic target for GBM.

Nuclear factor one B ( NFIB ) encodes a subtype-specific tumour suppressor in glioblastoma

Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects.

Abstract 5024: Unmasking heterogeneity within the adult mammary stem cell compartment

Breast cancer is a highly heterogeneous disease at both the molecular and pathological levels. To understand this heterogeneity and ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial hierarchy. Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5+ versus Lgr5– basal epithelial cells, we identify a novel marker that led to the fractionation of three distinct mammary stem cell (MaSC) subsets in the adult gland. These exist in a largely quiescent state but differ in their repopulating ability, spatial localisation, and their molecular signatures. Interestingly, the dormant MaSC subset localises to the proximal region of the gland throughout life. These cells appear to originate from the embryonic mammary primordia before switching to a quiescent state post-natally but can be recruited into the cell cycle in response to hormones. Single cell gene expression analyses have also revealed unexpected complexity within the basal and luminal compartments. Moreover, analyses at different stages of development have provided insights into the earliest ‘lineage priming’ events. Citation Format: Nai Yang Fu, Anne Rios, Bhupinder Pal, Charity Law, Paul Jamieson, Francois Vaillant, Gordon K. Smyth, Matthew E. Ritchie, Geoffrey J. Lindeman, Jane E. Visvader. Unmasking heterogeneity within the adult mammary stem cell compartment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5024. doi:10.1158/1538-7445.AM2017-5024

Abstract 1197: EphA3 kinase ablation induces glioblastoma differentiation and prevents tumor progression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Eph receptors constitute the largest sub-family of receptor tyrosine kinases and interact with membrane-bound ligands termed ephrins. Eph and ephrins have many vital functions including cell adhesion, migration and axon guidance. Eph and ephrins have been found to be aberrantly expressed in many malignancies including brain tumor. The purpose of this study was to investigate EphA3 receptor function in the most common and aggressive form of brain tumor, Glioblastoma (GBM). Methods: Gene expression was investigated by Q-PCR, IHC and flow cytometry in high grade glioma (HGG) surgical specimens and primary derived serum free cell cultures. Targeted reduction of Eph expression was performed using both a constitutive and inducible shRNA system. Murine in-vivo studies were performed using both subcutaneous and orthotopic ‘intracranial’ xenografts in immuno-compromised animals. Signaling pathways were assessed by western blotting. Results: To establish whether the receptor tyrosine kinase EphA3 was over expressed in HGG we assessed 12 normal human brain specimens, 56 HGG specimens and 26 HGG primary cultures. EphA3 mRNA expression was negligible in normal brain while 30% of clinical specimens and 46% of primary cultured tumor cells expressed EphA3. EphA3 protein was also detected in HGG clinical specimens using IHC. To further investigate EphA3 function the receptor was down regulated using shRNA in an EphA3+ GBM neurosphere cell line. Constitutive and inducible down regulation of the EphA3 receptor resulted in initiation of neuronal and glial cell differentiation following activation of the ERK/MAPK pathway. A reduction in stem/progenitor cell proliferation was also observed following EphA3 knockdown by shRNA (46%) or by alternately inhibiting EphA3 function using soluble ephrin A5-Fc (33%). CFSE division tracking identified slower cell division in populations in which EphA3 signaling was attenuated. In-vivo studies were performed using a NOD/SCID mouse subcutaneous and intracranial xenograft model. Results highlighted a marked reduction in tumor formation in the EphA3 knockdown as opposed to control tumors. Subcutaneous control tumors formed with a median survival of 66 days while EphA3 knockdown animals survived beyond 100 days (p<0.05). Similar to the subcutaneous xenograft model a marked lack of intracranial tumor formation was observed when EphA3 was neutralized. Control mice formed large well vascularized invasive tumors with a median survival of 62 days. All EphA3 knockdown animals were free of tumor following autopsy at 145 days when the experiment was terminated (p<0.05). Importantly a mutant EphA3 rescue of the knockdown culture returned the tumorigenic potential of these cells. Conclusions: We propose EphA3, in part, regulates cancer stem cell self renewal and cell division rate in GBM and could prove a potential therapeutic target and marker of brain tumor initiating stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2011-1197

Abstract 242: The gliogenesis initiation factor Nuclear Factor IB induces differentiation and inhibits growth of glioblastoma

Introduction: The phylogenetically-conserved vertebrate transcription factor, nuclear factor IB (NFIB), is a key component in the differentiation of astrocytes during the process of gliogenesis in the developing mammalian central nervous system; a process that goes awry following various genetic and epigenetic alterations during the genesis of glioblastoma (GBM), the commonest and most aggressive form of primary human adult brain cancer. We found expression of NFIB to be reduced in GBM compared to normal human brain tissue so investigated what effect increased expression of NFIB had on GBM. Experimental procedures: Expression of NFIB was investigated by qPCR and western blot in normal human brain tissue, primary GBM surgical specimens and GBM cells lines. The Rembrandt database was interrogated for patient survival data relative to NFIB expression. GBM cell lines were derived from patient tumor tissue, following informed consent, and cultured under serum-free conditions to help preserve the phenotype of the original tumor. GBM cell lines were transfected with HA-tagged Nfib. Glial differentiation marker expression was investigated by qPCR and western blot. Stem/progenitor cell growth was investigated by neurosphere assay and by PKH26 staining. Cell proliferation was measured by MTS assay and Ki67 staining. Cell cycle analysis was performed by PI staining and FACS analysis. Apoptosis was investigated by cleaved caspase staining. Differential gene expression induced by Nfib expression was determined by microarray analysis. In vivo tumorigenicity was investigated using subcutaneous and intracranial xenografts in NOD/SCID mice. Results: We found NFIB expression in both GBM surgical specimens and GBM cell lines to be reduced relative to normal brain tissue. Reduced NFIB expression was associated with poorer patient survival. Increased expression of Nfib in transfected GBM cell lines induced expression of glial differentiation markers, inhibited cell proliferation, reduced stem/progenitor cell growth, altered cell cycle progression, and inhibited tumor growth in murine models of GBM. Conclusion: We have identified NFIB as a novel tumor suppressor gene in human GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 242. doi:10.1158/1538-7445.AM2011-242