The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma

Abstract

Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

“…Interestingly, Batf2 in cancer cells has also been reported to be involved in cancer cell growth inhibition. For example, lower BATF2 expression is associated with a poor prognosis in hepatocellular carcinoma (26), oral tongue squamous cell carcinoma (27), esophageal squamous cell carcinoma (28), and medulloblastoma (29). BATF2 is down-regulated in various types of cancers and induces apoptosis (8).…”

Epigenetic regulation in medulloblastoma

“…In addition to the repression of expression of TP53 and other tumor suppressors, zebularine also inhibits expression of SHH pathway components such as SMO and GLI1. In addition, the combinations of zebularine with the chemotherapeutic agents vincristine and cisplatin result in synergism and antagonism, respectively (Andrade et al, 2017).…”

Molecular characterization of Wnt pathway and function of β-catenin overexpression in medulloblastoma cell lines

“…The pediatric medulloblastoma cell lines UW402 and UW473 (kindly provided by Dr. Michael Bobola), classified as group 3 or 4 (Castro-Gamero et al 2013) and ONS-76 (subtypes belonging to the MB-SHH subgroup (Triscott et al 2013;Ivanov et al 2016) (purchased from ATCC and Banco de Células do Rio de Janeiro, respectively) were cultured as previously described (Andrade et al 2017). All cell lines were authenticated using STR profiling.…”

“…Cell lines were transfected and equal amounts of protein (20-60 lg) were size-fractionated by SDS-PAGE as described previously (Andrade et al 2017). Proteins were immunoblotted with b-catenin antibodies (sc-7963) and with the endogenous controls anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Histone3 (#4499) (Cell Signaling Technology, Danvers, MA, USA), all diluted according to manufacturer's instructions.…”