Differential DNA methylation patterns in primary and recurrent glioblastomas : Protumorigenic roles of Tumor Endothelial Marker 8 (TEM8) and ASAP1 genes

Abstract

Glioblastomas (GBMs) are clinically aggressive malignant brain tumors in adults with a median survival of 14 months on diagnosis. Therapeutic interventions such as debulking surgeries, chemotherapy, and ionizing radiation, while relieving symptomatic disease, are not useful in preventing subsequent relapse (or recurrence). Recurrences are virtually inevitable in glioblastoma, due to the challenges in total surgical resection and/or tumors being highly invasive. Recurrent tumors are chemoradioresistant, and prove to be fatal in most cases.

Several lines of evidence point to the fact that both Temozolomide (the chemotherapeutic drug of choice) and ionizing radiation, can bring about stable changes in glioblastoma cells. As glioblastoma cells exhibit inherent plasticity, we hypothesized that there exists DNA methylation-based epigenetic changes in therapy-exposed recurrent tumors compared to treatment-naïve tumors in patient-matched pairs. To answer this, we compared the DNA methylation profiles of 11 primary GBMs (first occurrences) and 13 recurrent GBMs, using the Illumina 450K BeadChip Array, and identified 1225 hypermethylated regions and 526 hypomethylated regions in recurrent tumors compared to their primary counterpart. We mapped the probes to gene promoters and were able to identify promoter hypermethylation of several tumor suppressor genes, and hypomethylation of several genes involved in cancer progression.

We validated transcriptional dysregulation for some of these genes by expression and immunohistochemical analysis; and functionally characterized two of them: Tumor Endothelial Marker 8 (TEM8) and Arf-GAP with SH3, Ankyrin and PH domain protein 1 (ASAP1).

In GBMs, the role and function of TEM8 remain unknown. Using immunohistochemistry, we found that while TEM8 expression is non-detectable in control brain tissues, it is elevated in some lower-grade gliomas and in all GBM tumors. In a seperate retrospective cohort of 30 pairs of patient-matched primary and recurrent glioblastomas, increased expression of TEM8 was found in recurrent tumors. In silico analyses revealed TEM8 overexpression in glioblastoma patients is related to a worse prognosis. In-vitro, using overexpression and knockdown approaches, we found that TEM8 expression conferred proliferation advantage, invasive and migratory properties in glioma cells apart from chemo- and radioresistance, and stemness. Mechanistically, we demonstrated that TEM8 expression stabilized β-catenin levels and its’ target gene expression in glioblastoma cells. Using small molecule inhibitors, we demonstrated that TEM8 activates β-catenin via Src/PI3K/GSK3β pathway in glioblastomas.

Likewise, we explored the role and function of the ASAP1 gene in glioblastomas. We demonstrated that ASAP1 expression in GBM confers poorer survival and is upregulated in recurrent tumors. Knockdown of ASAP1 resulted in reduced proliferation, migration, and reduced cell size in glioblastoma cells. We observed that ASAP1 knockdown leads to impaired mTOR and ERK signaling, especially in response to growth factors such as EGF. We, therefore, demonstrate that ASAP1 is a key player in integrating and transmitting receptor tyrosine kinase signaling to mediate growth in glioblastoma cells. Together these studies suggest that preferential upregulation of protumorigenic genes by promoter hypomethylation may aid the aggressiveness of recurrent GBM tumors.