Epigenetic silencing of beta-spectrin, a TGF-beta signaling/scaffolding protein in a human cancer stem cell disorder: Beckwith-Wiedemann syndrome

Abstract

Hereditary cancer syndromes provide powerful insights into dysfunctional signaling pathways that lead to sporadic cancers. Beckwith-Wiedemann syndrome (BWS) is a hereditary human cancer stem cell syndrome currently linked to deregulated imprinting at chromosome 11p15 and uniparental disomy. However, causal molecular defects and genetic models have remained elusive to date in the majority of cases. The non-pleckstrin homology domain β-spectrin (β2SP) (the official name for human is Spectrin, beta, nonerythrocytic 1 (SPTBN1), isoform 2; the official name for mouse is Spectrin beta 2 (Spnb2), isoform 2), a scaffolding protein, functions as a potent TGF-β signaling member adaptor in tumor suppression and development. Yet, the role of the β2SP in human tumor syndromes remains unclear. Here, we report that β2SP(+/-) mice are born with many phenotypic characteristics observed in BWS patients, suggesting that β2SP mutant mice phenocopy BWS, and β2SP loss could be one of the mechanisms associated with BWS. Our results also suggest that epigenetic silencing of β2SP is a new potential causal factor in human BWS patients. Furthermore, β2SP(+/-) mice provide an important animal model for BWS, as well as sporadic cancers associated with it, including lethal gastrointestinal and pancreatic cancer. Thus, these studies could lead to further insight into defects generated by dysfunctional stem cells and identification of new treatment strategies and functional markers for the early detection of these lethal cancers that otherwise cannot be detected at an early stage.

FIGURE 1.

Wild type versus β2SP^+/− BWS-like phenotype. A–D, gross comparison of wild type (A, left) versus mutant β2SP^+/− (A, right) mouse, wild type (B, left) versus β2SP^+/− (B, right) tongue, wild type (C, left) versus β2SP^+/− (C, right) kidney, wild type (D, left) versus β2SP^+/− (D, right) ear. E, wild type (left) versus β2SP^+/− (right) liver. F, wild type (left) versus β2SP^+/− (right) spleen. G, wild type (left) versus β2SP^+/− (right) stomach. H, wild type (left) versus β2SP^+/− (right) lung. I, wild type (left) versus β2SP^+/− (right) testis. J, wild type (left) versus β2SP^+/− (right) brain. K, wild type (left) versus β2SP^+/− (right) heart.

FIGURE 2.

Loss of β2SP protein expression in BWS. A, expression of β2SP RNA is decreased greater than ∼50% in all tested human BWS cells compared with HepG2 cells assessed by quantitative PCR analyses. B, immunohistochemical labeling of β2SP in normal and BWS kidney tumor revealed loss of β2SP expression in BWS kidney tumor compared with normal kidney. C, Western blot analysis human of BWSC demonstrates loss of β2SP expression. D, Western blot analysis of β2SP in human BWST is shown. Results shown in A reflect a mean ± S.E. from three independent experiments, performed in triplicate. ***, p < 0.001 compared with control values, determined by t test.

FIGURE 3.

DNA methylation pattern of β2SP gene promoter in BWS. A, schematic representation of β2SP promoter and CpG islands. B, methylation status of the β2SP promoter in BWS cell lines and tissues detected by MS-PCR. C, DNA methylation pattern of the β2SP gene promoter in BWS cell lines and tissues identified by bisulfite sequencing. D, methylation status of the β2SP promoter in BWS tumor tissues (BWSTM) detected by MS-PCR (I) and bisulfite sequencing (II). Genomic DNA isolation from seven formaldehyde-fixed paraffin-embedded BWS tumor tissues is shown.

FIGURE 4.

Reactivation of β2SP gene expression by DNA methylation inhibition. A, effect of 5-aza-dC on β2SP gene expression in BWS cell line by immunoblotting assay. B, DNA methylation pattern of the β2SP gene promoter in BWSC-3 treated with 5 μm 5′-aza-dC for 6 days identified by bisulfite sequencing. Results shown in A reflects a mean ± S.E. (error bars) from three independent experiments. **, p < 0.01 and ***, p < 0.001 compared with untreated (control) values determined by t test.

FIGURE 5.

Increased IGF2 expression in β2SP^+/− mice is similar to that observed in human BWS. A, immunohistochemical analysis examining IGF2 (I) and IGF2R (II) in wild type and mutant β2SP liver and pancreas. Increased IGF2 expression in β2SP^+/− tissues is shown (arrows). B, p57^Kip2 expression in wild type (I) and β2SP^+/− (II) mouse liver tissues assessed by immunohistochemistry. p57^Kip2 expression increased in β2SP^+/− liver tissues compared with wild type. Potassium voltage-gated channel (KCNQ1) expression in wild type (III) and β2SP^+/− (IV) mouse heart tissues was assessed by immunohistochemistry. KCNQ1 expression increased in β2SP^+/− liver tissues compared with wild type. C, BWSC-3 showing a high level of IGF2 RNA by quantitative PCR. IGF2 RNA levels decrease in cells transfected with full-length β2SP plasmid. D, GH protein level in β2SP^+/− mice serum measured by ELISA. E, IGF1 mRNA level in β2SP^+/− liver tissues (I) and BWS liver (II). Results shown in C reflect a mean ± S.E. (error bar) from three independent experiments. ***, p < 0.001 compared with untreated (control) values determined by t test.