The transglutaminase 2 gene is aberrantly hypermethylated in glioma

Abstract

Transglutaminase 2 (TG2) is a ubiquitously expressed protein that catalyzes protein/protein crosslinking. Because extracellular TG2 crosslinks components of the extracellular matrix, TG2 is thought to function as a suppressor of cellular invasion. We have recently uncovered that the TG2 gene (TGM2) is a target for epigenetic silencing in breast cancer, highlighting a molecular mechanism that drives reduced TG2 expression, and this aberrant molecular event may contribute to invasiveness in this tumor type. Because tumor invasiveness is a primary determinant of brain tumor aggressiveness, we sought to determine if TGM2 is targeted for epigenetic silencing in glioma. Analysis of TGM2 gene methylation in a panel of cultured human glioma cells indicated that the 5' flanking region of the TGM2 gene is hypermethylated and that this feature is associated with reduced TG2 expression as judged by immunoblotting. Further, culturing glioma cells in the presence of the global DNA demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor Trichostatin A resulted in re-expression of TG2 in these lines. In primary brain tumors we observed that the TGM2 promoter is commonly hypermethylated and that this feature is a cancer-associated phenomenon. Using publically available databases, TG2 expression in gliomas was found to vary widely, with many tumors showing overexpression or underexpression of this gene. Since overexpression of TG2 leads to resistance to doxorubicin through the ectopic activation of NFκB, we sought to examine the effects of recombinant TG2 expression in glioma cells treated with commonly used brain tumor therapeutics. We observed that in addition to doxorubicin, TG2 expression drove resistance to CCNU; however, TG2 expression did not alter sensitivity to other drugs tested. Finally, a catalytically null mutant of TG2 was also able to support doxorubicin resistance in glioma cells indicating that transglutaminase activity is not necessary for the resistance phenotype.

Fig. 1

TGM2 is aberrantly hypermethylated in many cultured brain tumor lines. a Protein extracts were formed from cultures of human glioma lines U87MG, U118, T98G and A172. Extracts were immunoblotted with anti-TG2 (top panel) or anti-tubulin (bottom). b The structure of the 5′ flank of the human TGM2 gene is shown. Depicted is the first transcribed exon (exon 1), the boundaries of the CpG island located within this locus, the translational (ATG) and transcriptional start sites (TSS), and the location of oligonucleotide primers used for amplification (Forw, Rev) and sequencing (Seq). c Genomic DNA was isolated from the glioblastoma cell lines U87MG, U118, T98G and A172. After bisulfite conversion, the methylation status of the TGM2 gene was analyzed by pyrosequencing. Shown are resultant pyrograms and mean CpG methylation for the region of the TGM2 gene analyzed. d TGM2 methylation was measured by pyrosequencing for each of the indicated glioma cell lines, graphed is the mean of at least 5 independent pyrosequencing assays

Fig. 2

Global DNA demethylation results in TG2 re-expression in cultured glioma cell lines. a The glioma lines U87MG, U118, T98G and A172 were cultured in the presence or absence of the global DNA demethylating agent 5-azadC as indicated. Total RNA was isolated, cDNA synthesized, and PCR conducted using TG2 specific primers (top) or GAPDH specific primers (bottom). b. Protein extracts were obtained from the glioblastoma cell lines U118, T98G and A172 cultured in the presence or absence of 5-azadC. Cell extracts were immunoblotted with anti-TG2 (top) or anti-tubulin (bottom)

Fig. 3

TGM2 is aberrantly hypermethylated in primary human brain tumors. a Analysis of TGM2 methylation was conducted by pyrosequencing on genomic DNA isolated from various normal human brain specimens including two samples of adult cortex, one from fetal cortex, and two independent samples of dissected white and gray matter. Additionally, genomic DNA harvested from two neural progenitor cell lines (SC-23 and SC-27) was analyzed for methylation of the TGM2 gene. b DNA was isolated from archival specimens of glioblastoma multiformae (GBM) and anaplastic oligodendroglioma (AO) tumors and TGM2 methylation assayed by pyrosequencing. The %CpG methylation graphed for each sample is at least the mean of two independent experiments

Fig. 4

TG2 expression is heterogeneous in human brain tumors. a Relative TG2 expression on normal brain tissue, glioblastoma multiforme (stage IV), oligodendroglioma (stage II, III) or astrocytoma (stage II, III) specimens was obtained by query of the Rembrandt database. b Brain tumor samples were stratified into tumor groups that show ≥ 2-fold upregulation (dashed line), ≤ 2-fold downregulation (gray circles), and intermediate levels (gray squares) of TG2 expression. Kaplan–Meier analysis was subsequently conducted with these groupings, as well as mean TG2 expression among all glioma samples (black diamonds), to compare the association of TG2 expression with patient survival

Fig. 5

TG2 expression in the glioblastoma cell line U118MG induces chemoresistance to doxorubicin and CCNU but not to other glioma therapeutics. a The glioma cell line U118MG was stably transfected with an expression vector (pcDNA3.1) containing a full-length human TG2 cDNA or empty vector. Immunoblot analysis of extracts U118MG clone expressing TG2 (clone B5) or vector only (Vector) with anti-TG2 (top) or anti-tubulin (bottom) is shown. b U118MG Clone B5 (closed squares) or vector only (open diamonds) were treated with the indicated dose (0–0.5 mg/ml) of doxorubicin and 72 h after drug assayed for viability by MTT analysis. c. Clone B5 and vector only cells were also subject to viability analysis with a range of CCNU (0–0.64 mg/ml). Chemosensitivity of clone B5 and vector cells were also assayed following treatment with Vincristine (0–500 ng/ml) (d); Temozolomide (0.5–500 μM) (e); Cisplatin (0–0.23 mg/ml) (f); Cyclophosphamide (0–4 mg/ml) (g). Error bars = ±1.0 SD

Fig. 6

TGase activity is not necessary for TG2-induced chemoresistance to doxorubicin in glioma cells. a U118MG cells were stably transfected with an expression vector containing human TG2 cDNA encoding the TGase null mutation W241Q. Shown is an immunoblot of extracts from U118MG cells expressing wild type TG2 (Clone B5), empty vector (Vector), or U118MG cells expressing TG2 W241Q mutants (clone 4B1 and clone 4C4). Extracts were probed with anti-TG2 (top) or anti-tubulin (bottom). b Indicated cell lines were treated with 7.8 μg/ml doxorubicin and 72 h after drug addition cell viability was assayed by MTT. Graphed is the mean of 8 independent data points/cell line, error bars = ±1.0 SD