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XLS file - 34K, ER stress relation of genes up-regulated in mouse embryonic fibroblasts upon deletion of the AT/RT tumor suppressor gene Snf5 and also up-regulated in human AT/RT (SNF5KO_GS_UP genes, Isakoff et al., 2005)

XLS file - 147K, Six groups of genes (A-F), which show the highest correlation to one of the three tumor types

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XLS file - 46K, ER stress relation of Group A genes (more highly expressed in AT/RT-like cells as compared to glioma and PNET cells).

Podocalyxin (PODXL) is a highly glycosylated and sialylated transmembrane protein that is up-regulated in various types of tumors and whose expression levels positively correlate with tumor grade. We previously found Podxl to be highly expressed in murine tumorigenic neural stem/progenitor cells (NSPs). Here we investigated the effects of elevated Podxl levels in these cells. NSPs overexpressing Podxl did not form brain tumors upon intracranial transplantations, indicating that high levels of this gene alone are not sufficient for tumor initiation. However, Podxl overexpression had a positive effect on cell number, sphere formation and cell viability, indicating that it might in this way contribute to the development and/or maintenance of tumors. Proteome analyses of Podxl-overexpressing and control NSPs revealed increased levels of Annexin A2 (ANXA2). We also found increased transcript levels, indicating that PODXL stimulates expression of the Anxa2 gene. Lack of Anxa2 in Podxl-overexpressing NSPs resulted in reduced viability of these cells, suggesting that PODXL-mediated pro-survival effects can at least in part be explained by increased ANXA2 levels. Finally, our data indicate that Podxl overexpression activates the MAP kinase (MAPK) pathway which in turn up-regulates Anxa2 expression. Our data indicate a novel molecular connection between PODXL and ANXA2: both exert pro-survival effects in NSPs, and PODXL positively regulates ANXA2 expression through the MAPK pathway.

Bmi1 was originally identified as a gene that contributes to the development of mouse lymphoma by inhibiting MYC-induced apoptosis through repression of Ink4a and Arf. It codes for the Polycomb group protein BMI-1 and acts primarily as a transcriptional repressor via chromatin modifications. Although it binds to a large number of genomic regions, the direct BMI-1 target genes described so far do not explain the full spectrum of BMI-1-mediated effects. Here we identify the putative tumor suppressor gene EphA7 as a novel direct BMI-1 target in neural cells and lymphocytes. EphA7 silencing has been reported in several different human tumor types including lymphomas, and our data suggest BMI1 overexpression as a novel mechanism leading to EphA7 inactivation via H3K27 trimethylation and DNA methylation.

SUMMARY Monoamine and acetylcholine neurotransmitters from the autonomic nervous system (ANS) regulate insulin secretion in pancreatic islets. The molecular mechanisms controlling neurotransmitter signaling in islet β cells and their impact on diabetes development are only partially understood. Using a glucose-intolerant, MafA-deficient mouse model, we demonstrate that MAFA controls ANS-mediated insulin secretion by activating the transcription of nicotinic (ChrnB2 and ChrnB4) and adrenergic (Adra2A) receptor genes, which are integral parts of acetylcholine-and monoamine-signaling pathways. We show that acetylcholine-mediated insulin secretion requires nicotinic signaling and that nicotinic receptor expression is positively correlated with insulin secretion and glycemic control in human donor islets. Moreover, polymorphisms spanning MAFA-binding regions within the human CHRNB4 gene are associated with type 2 diabetes. Our data show that MAFA transcriptional activity is required for establishing β cell sensitivity to neurotransmitter signaling and identify nicotinic signaling as a modulator of insulin secretion impaired in type 2 diabetes., Graphical abstract

Connexins (Cx) form gap junctions and allow direct cell-to-cell communication. Cx through gap junctions or by themselves play regulatory roles on cell growth and differentiation. Using genetically modified mice, we previously found that Cx32 acts as a down-regulator of growth in normal thyroid gland. In this study, we examined the impact of Cx32 ablation on oncogene-driven thyroid growth and neoplastic transformation. Cx32 knockout (Cx32-KO) mice were crossed with transgenic mice expressing, selectively in the thyroid gland, either the E7 or RET/PTC3 (RP3) oncogene. As already described, Cx32-KO mice had no detectable thyroid alteration in physiological conditions and mice expressing E7 or RP3 exhibited time-dependent thyroid hypertrophy and variable changes in expression of differentiation. The thyroid of E7 mice evolved towards a large colloid goitre whereas RP3 mice developed a hyperplastic thyroid of variable size, and the largest glands (about 40% of total) represented a profound tissue remodeling with proliferative papillary formations. E7-induced thyroid hypertrophy was reduced by about 40% in Cx32-KO mice as compared with wild-type (WT) littermates. On the contrary, thyroid hypertrophy induced by thyrotropin stimulation (in response to goitrogen treatment) was enhanced by about 40% in Cx32-KO mice as compared with WT mice. Thyroid hypertrophy of RP3 mice and the proportion of glands showing extensive tissue remodeling were drastically reduced in mice devoid of Cx32. Our data show that Cx32, which negatively controls thyroid growth activated by thyrotropin via the cAMP pathway, would act as a positive effector of thyroid growth triggered by oncogenes acting through other signaling cascades.

Although brain tumors are classified and treated based upon their histology, the molecular factors involved in the development of various tumor types remain unknown. In this study, we show that the type and order of genetic events directs the development of gliomas, central nervous system primitive neuroectodermal tumors, and atypical teratoid/rhabdoid-like tumors from postnatal mouse neural stem/progenitor cells (NSC/NPC). We found that the overexpression of specific genes led to the development of these three different brain tumors from NSC/NPCs, and manipulation of the order of genetic events was able to convert one established tumor type into another. In addition, loss of the nuclear chromatin-remodeling factor SMARCB1 in rhabdoid tumors led to increased phosphorylation of eIF2α, a central cytoplasmic unfolded protein response (UPR) component, suggesting a role for the UPR in these tumors. Consistent with this, application of the proteasome inhibitor bortezomib led to an increase in apoptosis of human cells with reduced SMARCB1 levels. Taken together, our findings indicate that the order of genetic events determines the phenotypes of brain tumors derived from a common precursor cell pool, and suggest that the UPR may represent a therapeutic target in atypical teratoid/rhabdoid tumors. Cancer Res; 72(13); 3381–92. ©2012 AACR.

Thyroid epithelial cells communicate through gap junctions formed from connexin (Cx)32, Cx43, and Cx26. We previously reported that reexpression of Cx32 in “gap junction-deficient” FRTL-5 and FRT thyroid cell lines induces a reduction of cell proliferation rate and an activation of expression of cell differentiation. The present study aimed at determining whether Cx32 could exert similar regulatory functions in vivo. We investigated morphological and functional characteristics of thyroid gland of Cx32-deficient mice (Cx32-KO), mice overexpressing Cx32 selectively in the thyroid (Cx32-T+), and Cx32-KO mice with a thyroid-selective Cx32 complementation obtained by crossing Cx32-KO and Cx32-T+ mice. In basal conditions, Cx32-KO mice did not present any detectable thyroid alteration, whereas Cx32-T+ mice showed a thyroid hypoplasia (20% reduction) associated with a slight increase in thyroid functional activity. Under thyrotropin stimulation (following sodium perchlorate treatment), Cx32-KO mice developed a larger goiter (≤65% increase) than wild-type littermates, whereas Cx32-T+ mice exhibited the same thyroid hyperplasia as wild-type mice. Restoration of Cx32 expression in the thyroid of Cx32-KO mice abrogated the thyroid growth increase related to Cx32 deficiency. All together, these data show that Cx32 acts as a downregulator of growth of thyroid gland; an excess of Cx32 limits growth of thyroid cells in the basal state, whereas a lack of Cx32 confers an additional growth potential to TSH-stimulated thyroid cells.

Podocalyxin (PODXL) is a highly glycosylated and sialylated protein found mainly on podocytes and up-regulated in various types of tumors. Known to be a marker of hematopoietic progenitors and pluripotent stem cells, PODXL is also increasingly considered a marker of malignancy in several aggressive tumor types. Though the exact role of PODXL in tumors is not yet established, it has increasingly been shown in vitro to be involved in cell migration and invasion. We found Podxl to be highly expressed in murine tumorigenic neural stem/progenitor cells. To investigate effects of elevated Podxl levels in neural stem/progenitor cells (NSPs), Podxl or an empty control construct were over-expressed in these cells. Podxl-overexpressing NSPs did not form brain tumors upon intracranial transplantations, indicating that high levels of this gene alone are not sufficient for tumorigenesis. In vitro studies however revealed how Podxl might contribute to the development and /or maintenance of tumors. Podxl overexpression had a positive effect on cell number, sphere formation and cell viability. To find out how Podxl exerts its pro-survival effects, the proteome of Podxl-overexpressing and empty control NSPs was analysed by mass spectrometry. These experiments revealed an up-regulation of Annexin A2, a calcium-regulated phospholipid binding protein interacting with the cytoskeleton and extracellular matrix. In addition to elevated Annexin A2 protein levels, we also found increased transcript levels indicating that PODXL stimulates expression of the Annexin A2 gene. NSPs lacking Annexin A2 and overexpressing Podxl are less viable than wild type Podxl-overexpressing NSPs, suggesting that PODXL-mediated pro-survival effects can at least in part be explained by increased Annexin A2 levels. We are now investigating how Podxl and Annexin A2 mechanistically interact to enhance cell viability Citation Format: Isabelle V. Leefa Chong San, Gaelle Prost, Ulrike Nuber. Effects of Podocalyxin on neural stem/progenitor cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A09.