Frequent Epigenetic Inactivation of DIRAS-1 and DIRAS-2 Contributes to Chemo-Resistance in Gliomas

We previously reported that DIRAS-3 is frequently inactivated in oligodendrogliomas due to promoter hypermethylation and loss of the chromosomal arm 1p. DIRAS-3 inactivation was associated with better overall survival. Consequently, we now investigated regulation and function of its family members DIRAS-1 and DIRAS-2. We found that DIRAS-1 was strongly downregulated in 65% and DIRAS-2 in 100% of analyzed glioma samples compared to non-neoplastic brain tissue (NNB). Moreover, a significant down-regulation of DIRAS-1 and -2 was detected in glioma data obtained from the TCGA database. Mutational analyses did not reveal any inactivating mutations in the DIRAS-1 and -2 coding regions. Analysis of the DIRAS-1 and -2 promoter methylation status showed significantly higher methylation in IDH-mutant astrocytic and IDH-mutant and 1p/19q-codeleted oligodendroglial tumors compared to NNB. Treatment of U251MG and Hs683 glioblastoma cells lines with 5-azacytidine led to significant re-expression of DIRAS-1 and -2. For IDH-wild-type primary gliomas, however, we did not observe significantly elevated DIRAS-1 and -2 promoter methylation levels, but still detected strong downregulation of both DIRAS family members. Additional analyses revealed that DIRAS-1 and -2 expression was also regulated by histone modifications. We observed a shift towards promoter heterochromatinization for DIRAS-1 and less promoter euchromatinization for DIRAS-2 in IDH-wild-type glioblastomas compared to controls. Treatment of the two glioblastoma cell lines with a histone deacetylase inhibitor led to significant re-expression of DIRAS-1 and -2. Functionally, overexpression of DIRAS-1 and -2 in glioblastoma cells translated into significantly higher sensitivity to lomustine treatment. Analyses of DNA damage markers revealed that DIRAS-1 and -2 may play a role in p53-dependent response to alkylating chemotherapy

SOCS3 promoter methylation is mutually exclusive to EGFR amplification in gliomas and promotes glioma cell invasion through STAT3 and FAK activation

The suppressor of cytokine signaling 3 (SOCS3) gene is one of eight structurally related genes of the SOCS family and has been suggested to function as a tumor suppressor by inhibition of the JAK/STAT signaling pathway. We investigated 60 human gliomas of different histological types for SOCS3 alterations and found frequent SOCS3 promoter hypermethylation and transcriptional downregulation. However, SOCS3 promoter hypermethylation was virtually absent in primary glioblastomas, which are characterized by frequent epidermal growth factor receptor (EGFR) amplification and overexpression. Assessment of the relationship between SOCS3 and EGFR aberrations revealed that SOCS3 promoter hypermethylation was inversely related to both the EGFR gene dosage as well as the EGFR protein expression, thus suggesting SOCS3 inactivation as a mechanism substituting for EGFR activation in a subset of gliomas. In support of this hypothesis, stable shRNA-mediated SOCS3 knock-down in U251 glioblastoma cells resulted in an activation of EGFR-related signaling pathways, i.e. an increase in the activation levels of STAT3, FAK and to a lesser extent MAPK, while the AKT phosphorylation levels remained unaffected. Functionally, SOCS3-depletion caused strongly increased tumor cell invasion with no obvious effect on tumor cell proliferation. In summary, our findings suggest that SOCS3 inactivation by promoter hypermethylation is mutually exclusive to EGFR activation in gliomas and preferentially promotes glioma cell invasion through STAT3 and FAK activation

MiR-328 promotes glioma cell invasion via SFRP1-dependent Wnt-signaling activation

Background: Diffusely infiltrative growth of human astrocytic gliomas is one of the major obstacles to successful tumor therapy. Thorough insights into the molecules and pathways signaling glioma cell invasion thus appear of major relevance for the development of targeted and individualized therapies. By miRNA expression profiling of microdissected human tumor biopsy specimens we identified miR-328 as one of the main miRNAs upregulated in invading glioma cells in vivo and further investigated its role in glioma pathogenesis. Methods: We employed miRNA mimics and inhibitors to functionally characterize miR-328, 3′ untranslated region luciferase assays, and T-cell factor/lymphoid enhancer factor reporter assays to pinpoint miR-328 targets and signaling pathways, and analyzed miR-328 expression in a large panel of gliomas. Results: First, we corroborated the invasion-promoting role of miR-328 in A172 and TP365MG glioma cells. Secreted Frizzled-related protein 1 (SFRP1), an inhibitor of Wnt signaling, was then pinpointed as a direct miR-328 target. SFRP1 expression is of prognostic relevance in gliomas with reduced expression, being associated with significantly lower overall patient survival in both the Repository of Molecular Brain Neoplasia Data (REMBRANDT) and The Cancer Genome Atlas. Of note, miR-328 regulated both SFRP1 protein expression levels and Wnt signaling pathway activity. Finally, in human glioma tissues miR-328 appeared to account for the downregulation of SFRP1 preferentially in lower-grade astrocytic gliomas and was inversely related to SFRP1 promoter hypermethylation. Conclusion: Taken together, we report on a novel molecular miR-328–dependent mechanism that via SFRP1 inhibition and Wnt activation contributes to the infiltrative glioma phenotype at already early stages of glioma progression, with unfavorable prognostic implications for the final outcome of the disease

FGF/FGFR2 signaling regulates the generation and correct positioning of Bergmann glia cells in the developing mouse cerebellum.

The normal cellular organization and layering of the vertebrate cerebellum is established during embryonic and early postnatal development by the interplay of a complex array of genetic and signaling pathways. Disruption of these processes and of the proper layering of the cerebellum usually leads to ataxic behaviors. Here, we analyzed the relative contribution of Fibroblast growth factor receptor 2 (FGFR2)-mediated signaling to cerebellar development in conditional Fgfr2 single mutant mice. We show that during embryonic mouse development, Fgfr2 expression is higher in the anterior cerebellar primordium and excluded from the proliferative ventricular neuroepithelium. Consistent with this finding, conditional Fgfr2 single mutant mice display the most prominent defects in the anterior lobules of the adult cerebellum. In this context, FGFR2-mediated signaling is required for the proper generation of Bergmann glia cells and the correct positioning of these cells within the Purkinje cell layer, and for cell survival in the developing cerebellar primordium. Using cerebellar microexplant cultures treated with an FGFR agonist (FGF9) or antagonist (SU5402), we also show that FGF9/FGFR-mediated signaling inhibits the outward migration of radial glia and Bergmann glia precursors and cells, and might thus act as a positioning cue for these cells. Altogether, our findings reveal the specific functions of the FGFR2-mediated signaling pathway in the generation and positioning of Bergmann glia cells during cerebellar development in the mouse

<i>Fgfr2</i> deficiency leads to locomotor deficits in adult mice.

<p>(<b>A–D</b>) Cresyl-violet stained brightfield (A,C) and darkfield (B,D) views of sagittal sections from adult <i>Fgfr2^lox/lox</i> (control, A,B) and <i>Nestin-Cre;Fgfr2^lox/lox</i> (<i>Fgfr2</i> cKO, C,D) cerebella, hybridized with a radioactive <i>Fgfr2 exon 5</i> riboprobe. (<b>E</b>) Western blotting detected the full-length FGFR2 protein (approx. 100 kD) in brain lysates of adult <i>Fgfr2^lox/lox</i> (c, control) and <i>Nestin-Cre;Fgfr2^+/lox</i> (het, heterozygote) but not <i>Nestin-Cre;Fgfr2^lox/lox</i> (hom, homozygote) mice. Hprt is the loading control. (<b>F–J</b>) Behavioral tests revealed an altered horizontal locomotion (maximum velocity (F) and total distance travelled (G)) and unsupported vertical locomotion (latency to first rearing (H) and number of rearings (I) on the board) of male <i>Fgfr2</i> cKO (blue squares; n = 12 males) compared with control (red circles; n = 15 males) mice in the modified hole board paradigm, but no significant differences between both genotypes in the accelerating Rotarod performance (measured by mean latency to fall, J). Values are given in Table S1. I-X, lobuli of the adult cerebellum; ChPl, choroid plexus. Scale bar (C): 500 µm.</p

FGF target gene activation is almost completely abolished in the CbA of <i>Fgfr2</i> cKO embryos.

<p>(<b>A–P</b>) Representative sagittal brightfield views of E16.5 (A–H; n = 5 embryos/genotype) and E18.5 (I-P; n = 4 embryos/genotype) control (A,C,E,G,I,K,M,O) and <i>Fgfr2</i> cKO (B,D,F,H,J,L,N,P) cerebella, hybridized with riboprobes for <i>Etv5</i> (A–D,I–L) and <i>Tnc</i> (E,F,M,N). (C,D) and (K,L) are higher magnifications of the boxed areas in (A,B) and (I,J), respectively. (G,H) and (O,P) are pseudo-colored overlays (<i>Etv5</i> in red, <i>Tnc</i> in green, overlapping expression domains appear in yellow) of the adjacent sections shown in (C–F) and (K–N), respectively. Red arrowheads in (D,F,L,N) point at ectopic <i>Etv5</i>⁺ (D,L) or <i>Tnc</i>⁺ (F,N) cells in the anterior EGL of the mutant embryos. Note that at E18.5, the ectopic <i>Etv5</i>⁺ cells are predominantly located in the outer margin of the EGL, whereas the ectopic <i>Tnc</i>⁺ cells are mostly confined to the inner EGL. EGL, external granular layer; PCL, Purkinje cell layer. Scale bars: 100 µm (B); 50 µm (H).</p

Reduced numbers and mispositioning of <i>Tnc</i>⁺ BG cells in the EGL of the <i>Fgfr2</i> cKO CbA.

<p>(<b>A–J</b>) Representative sagittal brightfield views of E16.5 (A–D, n = 4 embryos/genotype), E17.5 (E–H, n = 1 embryo/genotype) and E18.5 (I,J, n = 3 embryos/genotype) control (A,C,E,G,I) and <i>Fgfr2</i> cKO (B,D,F,H,J) cerebella hybridized with radioactive <i>Tnc</i> (A–F,I,J) and <i>Fgfr2</i> (G,H) riboprobes. (C,D) are higher magnifications of the boxed areas in (A,B). (E–H) are higher magnifications of the anterior CbA in adjacent sections from control or mutant embryos. Red arrowheads in (D,F) point at ectopically positioned <i>Tnc</i>⁺ BG cells in the mutant EGL. Note the complete absence of the <i>Fgfr2</i> ISH signal correlating with less <i>Tnc</i>⁺ and intensely Nissl-stained cells in the CbA of the <i>Fgfr2</i> cKO embryo shown in (F,H), although some <i>Fgfr2</i>⁺ cells are detected in the (non-neural) mesenchyme overlying the mutant EGL. Red dotted line in (A,I) delimits the anterior area used for quantification. (<b>K,L</b>) High magnification views of the EGL and PCL on adjacent sections from an E18.5 control (wild-type) embryo, hybridized with a radioactive riboprobe for <i>Fgfr2</i> (red in K) or <i>Tnc</i> (black in L). Black arrowheads point at intensely Nissl-stained cells showing an ISH signal for <i>Fgfr2</i> (K) or <i>Tnc</i> (L). Empty arrowheads point at larger, weakly Nissl-stained cells devoid of <i>Fgfr2</i> (K) or <i>Tnc</i> (L) ISH signals. (<b>M,N</b>) Quantification of <i>Tnc</i>⁺ cells in the anterior CbA (M) and EGL (N) of control (grey bars) and mutant (white bars) embryos at E16.5 and E18.5 (<i>Tnc</i>⁺ cells/µm² (M): E16.5: control, 6.49×10⁻⁴±2.5×10⁻⁵ (n = 4 embryos); <i>Fgfr2</i> cKO, 4.43×10⁻⁴±2.4×10⁻⁵ (n = 4 embryos); E18.5: control, 9.41×10⁻⁴±5.9×10⁻⁵ (n = 3 embryos); <i>Fgfr2</i> cKO, 6.25×10⁻⁴±5.6×10⁻⁵ (n = 3 embryos); <i>Tnc</i>⁺ cells in anterior EGL (N): E16.5: control, 3.00±0.41 (n = 4 embryos); <i>Fgfr2</i> cKO, 17.00±1.29 (n = 4 embryos); E18.5: control, 5.67±0.88 (n = 3 embryos); <i>Fgfr2</i> cKO, 13.33±2.33 (n = 3 embryos); Student's <i>t</i>-test). EGL, external granular layer; PCL, Purkinje cell layer; VZ, cerebellar ventricular zone. Scale bars: 100 µm (B); 50 µm (D,H); 30 µm (L).</p

Strong reduction and ectopic positioning of Sox2⁺/Blbp⁺ BG precursors and cells in the <i>Fgfr2</i> cKO CbA.

<p>(<b>A–J</b>) Representative confocal overviews (A,D,G,I) and close-up views (B,C,E,F,H,J) of the CbA on sagittal sections from control (A,B,C,G,H) and <i>Fgfr2</i> cKO (D,E,F,I,J) embryos at E16.5 (A–F; n = 4 embryos/genotype) and E18.5 (G–J; n = 3 embryos/genotype), immunostained for Sox2 (green) and Blbp (Brain lipid binding protein, red). A marked reduction of Sox2⁺/Blbp⁺ BG precursors/cells was observed in the <i>Fgfr2</i> cKO (D,I) compared to control (A,G) embryos at both stages. A reduction of Sox2⁺ and Blbp⁺ neural progenitors and RG/BG precursors was also apparent in the anterior (left) part of the cerebellar VZ of the mutant embryos at E16.5 (A,D), whereas at E18.5, Sox2⁺ and Blbp⁻ neural progenitor cells appeared to accumulate in the cerebellar VZ of the <i>Fgfr2</i> cKO embryos (G,I). Close-up views (B,C,E,F,H,J) of the boxed areas in (A,D,G,I) revealed an increased number of ectopically positioned Sox2⁺/Blbp⁺ BG cells (white arrowheads) within the EGL of the <i>Fgfr2</i> cKO embryos at E16.5 and E18.5. Only few Blbp⁺ BG fibers reached the pial surface (dashed line in C,F) of the mutant CbA, and these fibers were frequently arranged in a parallel (tangential) rather than perpendicular (radial) manner relative to this surface (white arrows in E,J). EGL, external granular layer; PCL, Purkinje cell layer; VZ, cerebellar ventricular zone. Scale bars: 500 µm (A), 100 µm (B,C).</p

Aberrant cellular layering and organization of the adult <i>Fgfr2</i> cKO cerebellum.

<p>(<b>A–F</b>) Representative confocal images of sagittal sections from adult control (A; n = 2 mice) and <i>Fgfr2</i> cKO (B,C; n = 5 mice) cerebella, double-immunostained for calbindin (Calb1, green) and calretinin (Calb2, red), and counterstained with DAPI (blue). (D–F) are higher magnifications of the boxed areas in (A–C). White arrowheads in (E) point at ectopically located Calb1⁺ PCs within the GL, and white arrows in (E) denote the PC “gaps” within the PCL occupied by Calb2⁺ GCs. White arrowheads in (F) point at Calb1⁺ PC clusters surrounded by Calb2⁺ GCs. (<b>G–I</b>) Representative sagittal sections from adult control (G; n = 15 mice) and <i>Fgfr2</i> cKO (H,I; n = 20 mice) cerebella hybridized with a riboprobe for <i>Gad2</i>, which is expressed in PCs and GABAergic interneurons within the ML. Black arrowheads in (H,I) point at the disorganized <i>Gad2</i>⁺ PCL in the anterior lobules (II and III) of the mutant cerebella. The cerebella shown in (B,E,H) display a milder phenotype, which was detected in 8/25 (32%) of the <i>Fgfr2</i> cKO mice. The cerebella shown in (C,F,I) display a more severe phenotype, detected in 15/25 (60%) of the mutants. (<b>J–M</b>) Representative immunostainings for S100b (J,K) and Gfap (L,M) on sagittal sections from adult control (J,L) and less severely affected <i>Fgfr2</i> cKO (K,M) cerebella. Note the reduced numbers of S100b⁺ BG cells within the PCL and ectopic positioning of S100b⁺ BG cells within the ML in the mutant cerebellum. Gfap⁺ BG fibers did not reach the pial surface of the mutant cerebellum. GL, granular layer; ML, molecular layer; PCL, Purkinje cell layer; II and III, lobuli of the adult cerebellum. Scale bars: 500 µm (A,G), 50 µm (D), 100 µm (J,L).</p

Disruption of the anterior PCL but apparently normal RG scaffold in the <i>Fgfr2</i> cKO CbA.

<p>(<b>A–N</b>) Representative confocal overviews (A,B,E,F) and close-up views (C,D,G–N) of the anterior CbA on sagittal sections from control (A,C,E,G,I,K,M) and <i>Fgfr2</i> cKO (B,D,F,H,J,L,N) embryos at E18.5 (n = 5 embryos/genotype), immunostained for Pax6 (cyan/green in A–D; a marker for GCPs) and Calb1 (red in A–D; a marker for PCs), or Ccnd1 (cyan/green in E–J; a marker for cycling GCPs and RG/BG precursors/cells) and Glast (red in E–H,K,L; a marker for RG/BG fibers), and counterstained with DAPI (blue in A–H,M,N; a nuclear marker). (C,D) and (G,H) are close-up views of the boxed areas in (A,B) and (E,F), respectively, (G,I,K,M) were taken from an adjacent section to the one shown in (E). (I–N) are single color channel views of (G,H), respectively. Yellow arrowheads in (D) delimit the lacking Calb1⁺ anterior PCL in the mutant embryos, and in (H,J) point at ectopically located Ccnd1⁺ RG/BG precursors within the mutant cerebellar VZ. White arrowheads in (H,J) delimit the distorted Ccnd1⁺ anterior outer EGL in the mutant embryos. EGL, external granular layer; PCL, Purkinje cell layer; VZ, cerebellar ventricular zone. Scale bars: 100 µm (A); 30 µm (C); 50 µm (E).</p