Silencing of heparanase in human glioblastoma cells utilizing CRISPR/Cas9 methodology uncovers heparanase gene signature

Introduction. Heparanase is an endo-β-D-glucuronidase that cleaves heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPG). This activity is responsible for remodeling of the extracellular matrix (ECM) underlying epithelial and endothelial cells, thereby promoting cell dissemination associated with tumor metastasis, angiogenesis, and inflammation. Heparanase expression is low in normal epithelial and mesenchymal cells but its expression is up-regulated markedly in many carcinomas as well as sarcomas, hematological malignancies, and gliomas. Notably, cancer patients exhibiting high levels of heparanase had a significantly shorter postoperative survival time than patients whose tumors exhibit low levels of heparanase, thus supporting its pro-metastatic function. More recent results implicate heparanase in all steps of tumor development, including tumor initiation, growth, metastasis and chemo/radio-resistance, making it an attractive target for the development of anti-cancer drugs, some of which are currently entering advanced (Phase II) clinical trials. The mechanism by which heparanase promotes tumorigenesis is not completely understood and is thought to combine enzymatic and signaling aspects that likely involve a unique gene signature. Here, we have utilized a CRISPER/Cas9 methodology to reveal the significance of heparanase in tumor growth and identify the gene signature involved.

Methods. U87MG human glioblastoma cells were transfected with a CRISPR/Cas9 plasmid aimed to target and mutate the 2^nd exon of heparanase. GFP-positive cells were sorted and individual cell clones were sequenced to confirm homozygous frame-shift genomic mutations. RNA-SEQ analysis was applied to determine differential gene expression in null vs wild-type (wt) cell clones.

Results. Five heparanase null clones were validated for homozygous frame-shift mutations. The null cells exhibited no heparanase enzymatic activity or gene transcription (qPCR), and exhibited reduced cellular invasion. These traits were re-gained following transfection of the null clones with heparanase cDNA (rescue). Total RNA was extracted from parental U87 cells and wt clones vs heparanase null and rescued U87 cell clones, and subjected to RNAseq methodology. We found 53 genes that are affected by heparanase expression in glioma cells. Some of the affected genes are known to play a significant role in cancer development and are currently being validated for their regulation by heparanase and relevance to cancer.

Conclusions. We have identified a unique gene signature under the regulation of heparanase in U87 glioma cells. These genes and pathways will hopefully pave a way for novel treatment modalities in glioma patients.