DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in
cancer.
Malignant gliomas are the most common
primary brain tumors in adults and the second most common
tumor in children. Morbidity and mortality are high in
glioma patients because
tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted
proteases (eg,
cathepsins) and their endogenous inhibitors (
cystatins). In our previous studies we identified
cystatin E/M (CST6) as a frequent target of epigenetic silencing in
glioma.
Cystatin E/M is a potent inhibitor of
cathepsin B, which is frequently overexpressed in
glioma. Here, we study the expression of
cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR,
bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28
primary brain tumors demonstrated reduced/absent
cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a
glioma tumor initiating cell (
TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric
brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of
cystatin E/M in
glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric
brain tumors and occurs in
TICs, which are thought to give rise to the
tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in
TICs.