CRISPR-Mediated VHL Knockout Generates an Improved Model for Metastatic Renal Cell Carcinoma

Abstract

Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1α in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC.

Deletion of Von Hippel–Lindau Interferes with Hyper Osmolality Induced Gene Expression and Induces an Unfavorable Gene Expression Pattern

“…Loss of Vhl function induced stabilization of Hif1a. The deletion of Vhl was associated with loss of epithelial structure that is similar to the phenotype observed in RENCA cells [25]. Similar to RENCA cells, loss of Vhl induces a more metastatic phenotype in mpkCCD cells as they migrate faster.…”

“…The CRISPR/Cas9 technology has been used in a study before to delete VHL in the RENCA renal cancer cell line [25] where the authors described an EMT like phenotype due to a Vhl knock out. To our knowledge, this study is the first one that used a healthy renal epithelial cell line to introduce CRISPR/Cas9-mediated Vhl deletion and characterizes the phenotype of the cells.…”

“…More than 4700 genes were differentially expressed compared to control cells under isosmotic conditions. Loss of Vhl function was associated with more than 8500 differentially expressed genes in the RENCA RCC cell line after CRIPSR/Cas9 deletion of Vhl [25]. Unfortunately, the total list of genes and the used significance level is not published to compare the list of genes.…”

Endoglin Is Essential for the Maintenance of Self-Renewal and Chemoresistance in Renal Cancer Stem Cells

“…Interestingly, HIF1A is one of the critical factors involved in kidney cancer development and is known to suppress tumor cell growth ( Shen et al., 2011 ). The upregulation of HIF1A in CD105 + cells could in part contribute to the slow proliferation phenotype of this population ( Schokrpur et al., 2016 ).…”

Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment

“…Up to the present, studies using CRISPR/Cas9 in relation to tumor development and research into treatment involving tumor suppressor genes have yielded some positive results. The main anti‐tumor research results of tumor suppressor genes are listed in Table (Part A‐D) …”