By Jamshed Arslan Pharm.D.
Glioblastoma is an aggressive type of cancer that forms from the star-shaped glial cells of the central nervous system, called astrocytes. Intriguingly, several genes linked to glioblastomas, including the gene for the tumor suppressor Von Hippel-Lindau protein (VHL), do not exhibit mutations. VHL prevents tumorigenesis by binding and modulating the function of hypoxia-inducible factor HIF-1 alpha and hydroxylated serine-threonine kinase Akt. Developmental processes that maintain cells in a proliferative and undifferentiated state are hypothesized to be one of the non-mutation-related mechanisms that regulate VHL and consequently take part in gliomagenesis. Working on this hypothesis, researchers from three medical institutes in Houston, Texas, focused on a major developmental regulator called Daam2, and found that Daam2-driven degradation of VHL leads to glial cell tumors.
Daam2 takes part in gliomagenesis
The researchers used a publicly available RNA database (http://cancergenome.nih.gov/) to show that Daam2 is most significantly overexpressed in two glioma subtypes: low-grade glioma and glioblastoma multiforme (GBM). These findings were corroborated by in-situ hybridization, qRT-PCR, and microarray data from human glioma samples. Similarly, Daam2 overexpression was observed in the GBM-mimicking mouse model generated by in-utero electroporation and CRISPR-mediated deletion of tumor suppressors Nf1, Pten, and Trp53.
To study whether Daam2 overexpression accelerates gliomagenesis, human GBM cell lines were infected with either Daam2- or GFP control-containing lentiviruses. Daam2 overexpression led to an increased rate of cell growth. Likewise, non-viral PiggyBac-mediated Daam2 overexpression was found to increase tumorigenesis in mice. The immunohistochemical analysis showed an increased number of BrdU-positive proliferating cells in mice when Daam2 was overexpressed. Conversely, shRNAi-mediated knockdown of Daam2 inhibited rate of growth of human GBM cell lines, and these cell lines exhibited decreased tumor growth in vivo.
All in all, the results suggested that Daam2 promotes glioma tumorigenesis in both humans and mice. The next step was to find the mechanism behind Daam2-mediated gliomagenesis.
Immunohistochemistry-Paraffin: Von Hippel Lindau Antibody [NBP2-30070] - IHC analysis in formalin fixed and paraffin embedded mouse brain tissue followed by peroxidase conjugation of the secondary antibody and DAB staining. This data demonstrates the use of the VHL Antibody (N-term) for immunohistochemistry. Clinical relevance has not been evaluated.
Daam2 promotes gliomagenesis by suppressing VHL
Upon further examination, the scientists found a cohort of proteins that was inhibited in the Daam2-overexpressing mouse glioma samples; most significantly, VHL and Daam2 had a strong negative correlation in GBM. The Daam2-knockout mice showed elevated VHL in the brain, and Daam2-overexpressing tumors had increased levels of HIF-1 alpha and pAkt, both of which are VHL’s downstream effectors. Similarly, human GBM cell lines infected with lentivirus containing only Daam2 had an increased rate of cell growth than those infected with both Daam2 and VHL-containing lentiviruses.
These data indicate that Daam2-induced gliomagenesis requires VHL suppression. The next step was to study how Daam2 inhibits VHL in gliomas.
"I believe it would be great to determine whether and how Daam2 regulates other genes including Claudin-7 or Syk…we are now equipped to study follow-up research of Daam2 function in GBM. Since we demonstrated that Daam2 promotes VHL ubiquitination during gliomagenesis, our next question is that how does Daam2 degrade VHL in this context. To this end, we plan to identify target ubiquitin E3 ligases for Daam2-VHL axis."
Daam2 ubiquitinylates VHL
The 293T cells were co-transfected with Flag-tagged VHL, hemagglutinin-tagged ubiquitin, and Myc-tagged Daam2, and the cells were treated with a proteasome inhibitor before harvesting. Immunoprecipitation and western blotting revealed that Daam2 associates with VHL and in the presence of Daam2 overexpression, VHL ubiquitination increases with a concomitant decrease in VHL.
This is the first study that defines an upstream mechanism of VHL suppression (namely, Daam2) in cancers. This investigation is a milestone in cancer therapy since it describes a new target (VHL-Daam2 interaction) for treating the deadliest brain cancer. The fact that Daam2 and VHL have also been linked to various non-cancerous diseases such as multiple sclerosis enhances the utility of current findings.
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Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans
and transgenic mice.
Zhu, Wenyi, et al. "Daam2 Driven Degradation of VHL Promotes Gliomagenesis." Elife, vol. 6, 2017, n.pag. doi: 10.7554/eLife.31926.