Pamela Tan

Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention.

siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells.

Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinADeltaE). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinADeltaE when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinADeltaE mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinADeltaE message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinADeltaE acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinADeltaE to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.

Abstract 3126: Tumor heterogeneity in glioblastoma is an active process driven by a mutant EGFR-induced paracrine circuit

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Amplification and mutation of the epidermal growth factor receptor gene (EGFR), resulting in the common and oncogenic EGFRvIII (ΔEGFR) variant, is a signature pathogenetic event in GBM. Strikingly, despite its greater biological activity than wildtype EGFR (wtEGFR), only a minority of cancer cells in the primary tumor possess the hallmark ΔEGFR lesion, while the remainder of cancer cells express wtEGFR. We hypothesized that the ΔEGFR-expressing subpopulation provides enhanced tumorigenicity to the entire tumor cell population through a paracrine mechanism. Using a combination of mixed tumor engraftments and biochemical analysis of paracrine factors and signaling pathways activation, we determined that human glioma tissues, glioma cell lines, glioma stem cells and primary mouse astrocytes, that express ΔEGFR each secrete IL-6 and/or LIF cytokines. This then prompts a novel interaction between the receptor that is common to these cytokines, gp130, and wtEGFR in neighboring cells that express amplified levels of EGFR, resulting in co-receptor activation and tumor growth enhancement. Ablating IL-6, LIF or gp130 uncouples this cellular cross-talk and potently attenuates tumor growth enhancement. These findings demonstrate that the heterogeneity which characterizes GBM, and perhaps other tumors with this feature, does not occur stochastically, but instead can be an actively maintained feature and illuminates a heterotypic cancer cell interaction of potential therapeutic significance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3126.

Abstract A180: Tumor heterogeneity in glioblastoma is actively maintained by a paracrine circuit driven by mutant EGFR

Glioblastoma is typified by a heterogeneous composition of diverse cytological subtypes carrying a variety of gene alterations. An example of heterogeneity that is present in >50% of cases is amplification of the epidermal growth factor receptor (EGFR) gene. This amplification is often accompanied by structural alterations leading to expression of a variant form of the gene, EGFR (also referred to as EGFRvIII, deltaEGFR, EGFR‐de2‐7 and EGFR*), which conveys enhanced tumor aggressiveness. This potent tumor‐promoting function of EGFR would suggest that it should be the predominant amplified receptor in clinical samples, however, paradoxically, EGFR is usually present as minor and focal populations within tumors having more homogeneous wtEGFR amplification. This disconnect between tumorigenic potential and the frequencies and proportions of the amplified mutant EGFR and wtEGFR in GBM might arise if mutant EGFR occurs later in tumor progression where it not only enhances the tumorigenicity of cells which express it, but also potentiates, the proliferation of neighboring cells expressing amplified wtEGFR. If this were so, the potentiation loop might provide an attractive and novel therapeutic target. Here, we tested and validated the hypothesis that engraftment of a minor population of EGFR‐expressing glioma cells can dramatically enhance the tumorigenicity of tumors cells expressing wtEGFR through paracrine stimulation. In support of this general notion, EGFR‐driven pro‐tumorigenic enhancement was lost upon siRNA‐mediated knockdown of IL‐6, and conversely, glioma cells engineered to over‐express IL‐6 enhanced the tumorigenicity of wtEGFR‐expressing cells. These findings demonstrate that the molecular heterogeneity of GBM can be actively maintained, suggesting a coordinated strategy for therapeutic intervention. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A180.