Abstract 4440: Genetic knockdown of the E3 ubiquitin ligase Itch increases pancreatic cell line sensitivity to cancer therapeutics: A comparison between different gene editing techniques
Authors: Read Oliver J. , Harrison David J. , Reynolds Paul A. 
Published: August 2019

Introduction: E3 ligases are responsible for tagging ubiquitin onto substrates to either activate, relocate or target the substrate for degradation via the proteasome. Previous studies have shown that genetic knockdown of the E3 ubiquitin ligase Itch caused an increase in sensitivity to gemcitabine in pancreatic cancer models both in-vitro (cell lines) and in-vivo (mouse xenografts). Here we test the hypothesis that Itch knockdown sensitises pancreatic cell lines to existing cancer therapeutics, using both transient and stable knockdown methods.

Experimental Procedures: Knockdown of Itch in MiaPaCa2 (p53 mutant) and Capan 2 (p53 WT) cells was performed using both siRNA and CRISPR-Cas9 techniques; validated by western blot and RT-PCR. Alongside parental and scrambled controls, cells were treated with either doxorubicin, gemcitabine or γ-radiation. For doxorubicin and gemcitabine treatment, cells were exposed to the drug for 24 hours before it was removed. In the case of irradiation; cells were subject to 0-4Gy of radiation before being passaged onto plates. Cell viability was assessed in 96-well plates using a combination of a Sulforhodamine B assay (SRB) to quantify live cells 4 days post-treatment, and automated cytometry analysis (Celigo) to assess well confluence over each of the 4 days. Clonogenicity was also assessed by aliquoting cells at low initial density and treating the cells with distinct doses dependent on mode of treatment and cell line. After 7 days number of colonies using a colony formation algorithm on Celigo.

Data Summary: Transient knockdown of Itch in p53-/- cells was sufficient to decrease cell viability. This decrease in cell viability was further potentiated by the application of gemcitabine, doxorubicin or radiation. Stable knockdown using CRISPR-Cas9 increased MiaPaCa2 cell sensitivity to low-dose doxorubicin and radiation (5-10nM and 0.5-1Gy respectively). Whilst statistically significant, the change in response was not as large as that observed with the transient knockdown. In p53 WT cells there was no significant difference in cell sensitivity upon Itch knockdown.

Conclusions: The data from this investigation support the hypothesis of Itch being a possible target for sensitising pancreatic cell lines to cancer therapeutics. However the discrepancies in response to treatment between the transient and stable Itch knockdown scenarios raises the possibility that, in the case of the stable knockdown, the cells have adapted to the absence of Itch. This presents a potential weakness in using CRISPR-Cas9 in long-term experiments where the downstream consequences of gene silencing are not immediate.

Citation Format: Oliver J. Read, David J. Harrison, Paul A. Reynolds. Genetic knockdown of the E3 ubiquitin ligase Itch increases pancreatic cell line sensitivity to cancer therapeutics: A comparison between different gene editing techniques [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4440.