Mitogenic Sonic hedgehog signaling drives E2F1-dependent lipogenesis in progenitor cells and medulloblastoma

Abstract

Deregulation of the Rb/E2F tumor suppressor complex and aberrantion of Sonic hedgehog (Shh) signaling are documented across the spectrum of human malignancies. Exaggerated de novo lipid synthesis is also found in certain highly proliferative, aggressive tumors. Here, we show that in Shh-driven medulloblastomas, Rb is inactivated and E2F1 is upregulated, promoting lipogenesis. Extensive lipid accumulation and elevated levels of the lipogenic enzyme fatty acid synthase (FASN) mark those tumors. In primary cerebellar granule neuron precursors (CGNPs), proposed Shh-associated medulloblastoma cells-of-origin, Shh signaling triggers E2F1 and FASN expression, whereas suppressing fatty acid oxidation (FAO), in a smoothened-dependent manner. In the developing cerebellum, E2F1 and FASN co-localize in proliferating CGNPs. in vivo and in vitro, E2F1 is required for FASN expression and CGNP proliferation, and E2F1 knockdown impairs Shh-mediated FAO inhibition. Pharmacological blockade of Rb inactivation and/or lipogenesis inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals In vivo. These findings identify a novel mechanism through which Shh signaling links cell cycle progression to lipid synthesis, through E2F1-dependent regulation of lipogenic enzymes. These findings pertinent to the etiology of tumor metabolism also underscore the key role of the Shh→E2F1→FASN axis in regulating de novo lipid synthesis in cancers, and as such its value as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

Figure 1. Shh-induced mouse medulloblastomas have exaggerated lipogenesis, increased levels of FASN, and deregulated E2F1

(A) Triglyceride accumulation was analyzed in a NeuroD2-SmoA1 medulloblastoma using Oil Red O staining; lipids appear as red droplets. Upper row, leftmost panel shows H&E staining of the tumor and neighboring cortex and cerebellar white matter. (B) Protein lysates were prepared from NeuroD2-SmoA1 normal cerebellum adjacent to the tumor, and tumor, then analyzed by western blotting for lipogenic markers (FASN) and proliferation markers (Rb phosphorylation, E2F1). 30 µg protein/lane was loaded. (C) NeuroD2-SmoA1 medulloblastoma and adjacent non-tumor cerebellum were subjected to immunofluorescence analyses for E2F1, FASN, and the proliferation marker phospho-histone H3.

Figure 2. Shh signaling inactivates Rb and induces lipogenesis markers in primary CGNP cultures

(A) CGNP cultures were prepared from PN 4/5 mice and incubated with vehicle, Shh (3 µg/mL), and/or forskolin (10 µM) for up to 48 hours. Protein lysates were prepared and assayed for Rb phosphorylation as determined by upward mobility shift of the total protein. 50 µg protein/lane were loaded. (B) CGNP cultures were treated with vehicle or Shh (3 µg/mL) for 24 hours, then analyzed for levels of cdk 2, cdk 4, and cdk 6. (C) CGNP cultures prepared and treated as described above were analyzed for cdk2 kinase activity using immune complex kinase assays. In brief, 200 µg protein lysate were incubated with protein A sepharose beads bound with antibodies against cdk2 or p48 MAPK, whose levels of activity are known not to be altered by Shh signaling. Beads were collected by centrifugation, washed, and incubated with substrate (cdk 2, Histone H1; p48 MAPK, myelin basic protein) in the presence of γP³²-ATP. Reactions are separated by SDS page and visualized by exposure to X-ray film. Each radioactive (ie phosphorylated) substrate appears as a single band on the gel. (D) Western blot analysis of E2F1 levels in CGNPs treated with vehicle or Shh. (E) E2F1 mRNA expression after 24 or 48 hours in CGNPs treated with vehicle or Shh, measured by quantitative RT-PCR. (F) Shh pathway activation as determined by qRT-PCR for Gli1 mRNA expression in the same experiment shown in 1E. (G) CGNP cultures were treated with vehicle, Shh, or Shh and the Smoothened inhibitor cyclopamine (10 µg/mL). mRNA was prepared and analyzed by qRT-PCR for expression of ACC, FASN, and E2F1. (H) Western blot analysis of SREBP1 precursor (pr) and cleaved (cl) (active) form in CGNPs treated with vehicle or Shh.

Figure 3. Pharmacological inhibition of FASN in Shh-treated CGNPs induces FAO-promoting enzymes while Shh itself reduces FAO rates

(A) Simple schematic demonstrating enzymes and substrates regulating the balance between FAS and FAO. The compound C75 inhibits the function of FASN. (B) CGNP cultures were treated with vehicle or Shh in the presence or absence of the FASN inhibitor C75 (10 µg/mL) for 48 hours. mRNA was prepared and analyzed by qRT-PCR for markers of FAS (ACC, FASN) or FAO (ACOX1, MCAD). (C) Quantification of radiometric analysis of FAO in CGNPs treated with vehicle of Shh in the presence or absence of cyclopamine. Briefly, after 48 hours in culture the CGNPs are exposed to H³-palmitate for 2 hours. After incubation, the level of ³H₂O, the byproduct of FAO, is measured and normalized to cell number/protein content, to generate a measurement of rate.

Figure 4. E2F1 is required for FASN expression, CGNP proliferation, and Shh-mediated FAO suppression

(A) Cerebella were collected from wild type or E2F1-null PN 5 pups, then analyzed by western blotting for levels of E2F1, lipogenic enzymes SREBP1 precursor and cleaved (active) form, and FASN, and the proliferation marker cyclin D2. (B) PN 7 sagital sections of wild-type (bottom) and E2F1-null (top) cerebella were immunostained for FASN, E2F1, and phospho-histone H3. Rightmost panels show H&E staining. (C) Graph shows quantification of phospho-histone H3 staining as a measurement of proliferation. (D) Western blot analysis of E2F1, cleaved SREBP1, FASN, MCAD, and proliferation markers cyclin D2 and phospho-Rb in CGNPs treated with vehicle, Shh, or Shh in the presence of lentiviruses carrying shRNAs targeting E2F1 or a scrambled control shRNA virus. (E) Rates of FAO in CGNPs treated with vehicle, Shh+ scrambled control shRNA, or Shh+ shE2F1, determined by radiometric analysis.

Figure 5. Inhibition of E2F1 and FASN is cytotoxic to medulloblastoma cells in vitro

(A) Immunofluorescence analysis of E2F1, FASN, E2F1/Shh target Bmi1, cyclin D2, cdk 2, and cdk 4 in Pzp53med cells treated with DMSO (control) or the cdk inhibitor roscovitine (10 nM) for 18 hours. (B) Proliferation quantification in the presence of vehicle, cdk inhibitor roscovitine (10 nM), and FASN inhibitor C75 (10 µg/ml) determined by counting phospho-histone H3-positive cells. (* p value = 0.0162; ** p value = 0.0188, t-test analysis) (C) Western blot analysis of proteins regulating lipogenesis (FASN), proliferation (E2F1, cdk2, cyclin D2), and apoptosis (cleaved caspase-3) in Pzp53med cells treated with roscovitine (10 nM), C75 (10 µg/ml), or a combination of both drugs. (D) Effects of increasing doses of the FASN inhibitor C75 (0, 3, 10, 30 µg/ml) in the presence of roscovitine, 0 or 10 nM, on Pzp53med cell viability as determined by cell Titer-Glo assay, a bioluminescent analysis which reports ATP concentration.

Figure 6. Inhibition of E2F1 is toxic to Shh-mediated medulloblastomas in vivo and reduces levels of FASN

(A) Kaplan-Meyer survival curve of NeuroD2-SmoA1 medulloblastoma-bearing mice in days commencing with initiation of treatment with DMSO or the cdk inhibitor olomoucine (6 mg/kg, i.p. injection daily) (* p value < 0.0001, Mantel-Cox test analysis). (B) Immunofluorescence staining for markers of proliferation and survival in medulloblastomas collected from NeuroD2-SmoA1 mice treated with DMSO (left column) or olomoucine (right column). (C) Western blot analysis of proteins regulating proliferation and lipogenesis in adjacent non-tumor cerebellar material or medulloblastomas from NeuroD2-SmoA1 mice treated with DMSO (−) or olomoucine (+).

Figure 7. Treatment of medulloblastoma-bearing mice with FASN inhibitor C75 prolongs survival, reduces tumoral lipid accumulation, and impairs tumor cell proliferation in vivo

(A) Oil Red O staining to mark neutral lipid accumulation in adjacent cerebellum and medulloblastomas of Vehicle (DMSO)-treated or C75-treated mice (30 mg/kg, i.p. injection 3 times a week). (B) Western blot analysis for markers of lipid synthesis (FASN) and proliferation (cyclin D2, N-myc, cdk2) in samples of adjacent cerebella and medulloblastomas from DMSO treated (−) or C75-treated (+) mice. (C) Immunostaining for phospho-histone H3 in adjacent cerebellum and medulloblastomas in vehicle- or C75-treated mice. Quantification of phospho-histone H3-positive (mitotic) cells is shown in the accompanying graph. (D) Kaplan-Meyer survival curve of NeuroD2-SmoA1 medulloblastoma-bearing mice in days commencing with initiation of treatment with DMSO or the FASN inhibitor C75 (* p value < 0.0001, Mantel-Cox test analysis).