Glioblastomas are the most aggressive of all brain tumors. The malignant cells are able maneuver in the tightly packed brain tissue along blood vessels to form secondary and tertiary tumors at distant locales. Once metastasis has set in, the prognosis is poor, as surgery and radio- and chemotherapy are unable to destroy the scattered glioblastoma cells. Identifying molecular players that regulate this aggressive invasiveness of glioblastoma cells is therefore of critical importance, so that they can be leveraged for drug development.
Now, researchers at the Luxemburg Institute of Health Genes have identified the genes responsible for the extreme invasiveness of glioblastomas and the molecular regulator that switches them on.
“In this context, we sought to elucidate the genes responsible for glioblastoma invasiveness and the specific molecules that ‘switch’ them on,” said Simone Niclou, PhD, director of the Luxemburg Institute of Health, department of oncology, and corresponding author of the publication.
In an article titled, “AN1-type zinc finger protein 3 (ZFAND3) is a transcriptional regulator that drives Glioblastoma invasion,” published in Nature Communications, the team wrote that they progressively blocked the entire repertoire of genes activated in invasive glioblastoma cells derived from a patient, using a loss-of-function RNA interference approach.
The authors noted that they used RNA interference that target mRNAs instead of CRISPR knockout screens that target DNA because glioblastoma cells have an abnormal number of chromosomes (aneuploidy), making blocking gene expression at the transcript level a more reliable approach. Zeroing in on cells with compromised or lost invasiveness, the researchers then sequenced and analyzed genes responsible for invasiveness in glioblastoma cells using four independent bioinformatic algorithms. All four identified an overlapping set of 17 genes deduced to be essential for invasiveness. This included the colony stimulation factor, CSF1, established as a promoter of invasiveness and metastasis in other studies.
To reduce the number of gene candidates the investigators then compared the expression of the 17 genes in patient-derived glioblastoma cells with high, low, and no invasiveness. Significantly higher expression of the gene coding for the gene ZFAND3 (AN1/A20 zinc finger domain containing protein 3) was noted in highly invasive glioblastoma cells, particularly at the outer edges of the tumors, both in vitro and upon implantation of glioblastoma cells into mouse brains.
“When we deactivated the ZFAND3 gene in highly invasive GBM cells, we observed that colonization of healthy tissue was significantly impaired, indicating that ZFAND3 plays a key role in promoting glioblastoma invasiveness,” said Anne Schuster, first author and research associate at the Luxembourg Institute of Health.
“Similarly, when we overexpressed the ZFAND3 gene in noninvasive glioblastoma cells in mice, we noticed that the tumor lost the circumscribed growth pattern typical of control noninvasive cells and that the number of cells escaping the primary tumor increased considerably, further confirming that ZFAND3 expression confers invasion potential to glioblastoma cells even if they were initially noninvasive,” said Eliane Klein, second author and research engineer at the Luxembourg Institute of Health.
Probing into the location of ZFAND3 in the cell revealed mechanistic insights into how ZFAND3 regulates invasiveness of glioblastoma cells. The authors noted that ZFAND3 needs to find its way into the nucleus of the cell and have intact zinc finger motifs, a hallmark of gene regulatory proteins, to increase motility in glioblastoma cells.
RNA sequencing upon knockdown of ZFAND3 mRNA, revealed the concomitant downregulation of several genes involved in cell adhesion and motility, including COL6A2, EGFR, FN1, NRCAM, and NRP1. This, together with evidence showing ZFAND3 binds to the regulatory regions of these genes and is part of nuclear protein complexes, indicates ZFAND3 activates transcription of genes essential for invasiveness in glioblastoma cells.
“In essence, our work has brought forward ZFAND3 as a novel key regulator involved in the malignancy of glioblastoma, thereby providing a new molecular mechanism against which future drugs may be directed,” concluded Niclou.