Presentation Authors: Jan Rudzinski*, Natasha Govindasamy, Konstantin Stoletov, Adrian Fairey, John Lewis, Edmonton , Canada
Introduction: Metastatic RCC is incurable, and the current therapeutic paradigm is centered around controlling the disease burden to prolong overall survival. We conducted a whole human genome short hairpin RNA (shRNA) screen on human squamous cell carcinoma (Hep3) cells to identify a panel of novel functional genes that are required for productive cell motility and successful metastatic dissemination. One such novel protein target encodes for chromosome 14 open reading frame 142 (C14orf42), which has been demonstrated to be up regulated in metastatic clear cell RCC. The objective of our study was to characterize the impact of C14orf142 on clear cell RCC in vitro motility, invasion, and in vivo vascular extravasation.
Methods: Benign proximal convoluted tubule cells (PCT) and clear cell RCC cell lines 786-0 (derived from renal tissue) were obtained from American Type Culture Collection (ATCC). Targeted genomic editing to knockout (KO) C14orf142 was achieved with CRISPR-Cas9 system. Successful protein knockdown was validated using western blot analysis. To measure impact of gene KO on in vitro invasion we conducted the FITC-gelatin degradation assay. To measure the combined effect of invasion and productive cell migration in vitro we utilized the modified Boyden chamber assay coated with 0.1% gelatin. To study cancer cell vascular extravasation in vivo, 786-0 cells were injected IV into fertilized avian embryos. For statistical analysis, t-test was used to evaluate differences between groups with p value of â‰¤0.05 accepted as statistically significant.
Results: The baseline expression of C14orf142 was significantly higher in 786-0 (1.078AUDÂ±0.11) compared to PCT(0.12AUDÂ±0.04) (pâ‰¤0.05). The CRISPR-Cas9 targeted genomic editing resulted in generation of 786-0 clones with complete KO of C14orf142. The FITC-gelatin degradation assay demonstrated significant difference in gelatin degradation between 786-0 scramble and 786-0 CRISPR KO clones (100% vs 27%Â±5.56%, respectively, pâ‰¤0.05). The modified Boyden chamber assay also showed significant difference in productive cell migration between 786-0 scramble and 786-0 CRISPR KO clones (100% vs 42Â±3.46%, respectively, pâ‰¤0.05). Compared to 786-0 scramble clones, 786-0 CRISPR KO clones demonstrated significant reduction in extravasation on the avian embryo model (100% vs 46.33Â±8.37%, respectively, pâ‰¤0.05).
Conclusions: Up regulation of novel target protein, C14orf142, in 786-0 clear cell RCC cells may play an important role in cancer cell invasion, productive cell migration, and vascular extravasation.
Source of Funding: Kidney Cancer Research Network (KCRN)Kidney Cancer Canada (KCC)Canadian Institute of Health Research (CIHR)