Unravelling the proteomic landscape in Interferon Stimulated Gene 15 deficient cancer cellular models

Abstract

The Interferon Stimulated Gene 15 (ISG15) is a ubiquitin-like protein that can be found both conjugated to target proteins, in a process known as ISGylation, and in a non-conjugated “free” form where it can act as a cytokine. Even though ISG15 has been widely studied for its antiviral properties, recent research has implicated ISG15 in many more biological processes, including the development and progression of cancer. In this area, re search is divided between studies that suggest ISG15 is a tumour suppressor and immune system enhancer, and those that claim it relates to tumour aggressiveness and treatment resistance. The extensive number of modification targets and variety of roles of ISG15 makes it challenging to determine its molecular mechanisms in cancer.

The aim of this study is to shed light on the role of this ubiquitin-like protein in cancer cell models and to provide insights into the mechanisms of action that leads to ISG15 function in interferon (IFN) resistance. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 gene-editing technology was used to ablate ISG15 expression in a cervical cancer cellular model (SiHa cells). Once a stable knock-out was obtained, a variety of analysis were performed with and without interferon treatment to try to identify any biological difference between the wild type cells and the ISG15 deficient cells, including cell growth and cell cycle analysis and colony formation assays. After identifying ISG15 deficient cells as sensitive to IFNα treatment, which was confirmed in a variety of cellular models, three different proteomic approaches were used to try to address the proteomic landscape in two ISG15 deficient cell backgrounds in the presence and absence of IFNα treatment. a) First, a stable isotope labelling by amino acids in cell culture (SILAC) technique was used in wild-type (WT) and ISG15 deficient cervical cancer (SiHa) cells, either untreated or treated for 24 or 48 hours, to look at recently synthesised protein changes.

Results showed significant increased expression of other ISGs like IFIT1 and MX1, but also of other less expected proteins such as NQO1, MT1G and MT1F, all of which are involved in oxidative stress response and detoxification. This was reflected in the analysis of the dysregulated biological processes performed with the identified targets, which revealed upregulated response to stress. This analysis also revealed upregulated processes related to antigen processing and presentation, downregulated processes related to response to wounding and wound healing and various dysregulated metabolic processes. b) Second, in order to find out what proteins and processes were commonly altered upon depletion of ISG15 in different cancer models, a steady state whole proteomics approach was used in ISG15 deficient patient-derived glioblastoma stem cells (GSCs). Again, the upregulation of a IFN stimulated signature was detected in the absence of ISG15, as well as processes related to the positive regulation of IFN production. Several metabolic processes were also found to be dysregulated, and processes related to antigen processing and presentation were also detected to be upregulated upon IFN treatment. c) Finally, as the previous results suggested the involvement of ISG15 in MHC class I antigen presentation, a state-of-the-art immunopeptidomic method was used to elute and analyse the peptides presented by MHC-I in ISG15 deficient versus wild-type GSCs in the presence or absence of IFNα treatment. The presentation of peptides derived from several proteins were found significantly altered in the absence of ISG15 (e.g. GAPDH), and peptide length distribution analysis suggested an increased and sustained expression of MHC-I presented peptides upon IFN treatment.

Overall, results suggest that the loss of ISG15 results in an amplified IFN response promoting sensitivity, probably due to the loss of the role of ISG15 as a negative regulator of this pathway. At the same time, the absence of ISG15 seems to be involved with mitochondrial and metabolic dysregulation, as well as altered antigen peptide expression. Further researching these findings could be useful in the field of immune based and peptide targeted therapies.