Controlled Access. Restriction lift date: 2030-09-30
Investigation into the role of Ubiquitin Specific Peptidase 18 in oesophageal cancer
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Date
2025-09-30
Authors
Carey, Maria
Journal Title
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Publisher
University College Cork
Published Version
Abstract
Oesophageal cancer is the seventh most common cancer and the sixth leading cause of cancer-related deaths worldwide, with a five-year survival rate of 20%. There are two main histological subtypes: squamous cell carcinoma and adenocarcinoma. Treatment of both involves chemotherapy, chemo-radiotherapy, and/or surgery, depending on tumour stage and patient status. Unfortunately, resistance to chemotherapy is common and often contributes to this poor prognosis. Novel therapeutic targets are therefore urgently required to improve treatment regimens for people with oesophageal cancer.
Earlier research by this group found that a network of interferon-stimulated genes (ISGs), known as the ISGylation network, is differentially expressed in drug-sensitive and resistant oesophageal cancer cells. The ISGylation network is similar to the ubiquitin network, involving the conjugation of Interferon-Stimulated Gene 15 (ISG15), a ubiquitin-like protein (Ubl), to target proteins in a sequential manner, mediated by E1, E2, and E3 enzymes. Ubiquitin Specific Peptidase 18 (USP18) selectively removes ISG15 in a process referred to as deISGylation. USP18 also negatively regulates the type I interferon (IFN) response, independent of its enzymatic activity. USP18 is expressed at high levels in a panel of chemo-sensitive and apoptosis-competent oesophageal cancer cells, and expressed at low levels in chemo- and apoptosis-resistant oesophageal cancer cells.
We investigated whether the expression levels of USP18 can affect the response of two chemo-resistant oesophageal cancer cell lines, KYSE450 and KYSE140, to chemotherapy. Treatment with IFN-α alone enhanced the sensitivity of KYSE450 cells to 5-fluorouracil (5-FU), but had no effect in KYSE140 cells and did not alter oxaliplatin sensitivity in either cell line. In contrast, silencing USP18 in the presence of IFN-α significantly increased the sensitivity of both cell lines to 5-FU and oxaliplatin and induced extensive apoptosis in these cell lines, which were previously considered to be apoptosis-resistant.
USP18 and its substrate ISG15 were induced by IFN-α treatment alone. However, ISG15 was predominantly present in the unconjugated form due to the co-upregulation of USP18. Knockdown of USP18 led to significant accumulation of ISG15-conjugated proteins, demonstrating the efficiency of USP18 in removing this modification. This accumulation of conjugates, together with the occurrence of cell death, raises the possibility that ISG15-modified proteins play an important role in the activation of cell death signalling. In addition, USP18 depletion in the presence of IFN-α increased LC3 II expression and autophagosome formation, both alone and in combination with chemotherapeutic agents, demonstrating negative regulation of autophagy. Together, these findings indicate that targeting USP18 could reactivate cell death signalling and restore sensitivity to chemo-resistant oesophageal cancer.
We then investigated whether Poly(I:C), a synthetic double-stranded RNA analogue that mimics viral infection, could provide an alternative to IFN-α. We demonstrated that Poly(I:C) induced ISGs, including USP18 and its substrate ISG15, in KYSE450 and KYSE140 cells. After depleting USP18 with siRNA, Poly(I:C) induced apoptosis to a level similar to IFN-α in KYSE450 cells. In contrast, KYSE140 cells were unresponsive, showing no significant decrease in viability. Notably, there was no induction of ISG15 conjugates in KYSE140 cells, suggesting that this modification may be important for cell death competency.
These findings demonstrate that combining USP18 depletion with an IFN-like stimulus to promote ISG15 conjugation represents a promising strategy to overcome resistance to cell death, and indeed also induces significant cell death without chemotherapy. While IFN-α remains the most reliable partner in this therapeutic strategy, Poly(I:C) or similar agents could expand therapeutic options if predictive biomarkers are established.
As USP18 depletion had such a profound effect on chemosensitivity, we wanted to create USP18 gene-knockout cell lines to validate the siRNA data and further explore the downstream signalling linked to ISGylation. We employed a CRISPR/Cas9 knock-out protocol that had been successful in other cell lines. Despite optimisation of CRISPR/Cas9 protocols, no knockout was generated in either the KYSE450 or KYSE140 cell lines. It is unclear why the CRISPR protocols did not achieve a knockout in these cell lines. Possible explanations include limited chromatin accessibility to the Cas9 nuclease or deficiencies in DNA repair pathways. By contrast, a knockout of USP18 was successfully generated in the OE21 cell line, providing a drug-sensitive USP18 knockout model to further expand this body of work.
The increased chemosensitivity and apoptosis in the oesophageal cancer cell lines was associated with elevated levels of ISGylation. However, the identity of the ISGylated proteins, which may mediate chemosensitivity, remains unknown. We sought to identify these ISG15-modified proteins. Following USP18 depletion with siRNA and treatment with IFN-α, ISG15 conjugates were purified by immunoprecipitation from the KYSE450 cell line. Three independent test samples were then analysed by mass spectrometry and filtered against a control IP using a non-specific antibody and the CRAPome database. We identified 117 ISG15-conjugated proteins in three independent test samples. Of these, 36 were common to databases or published IP studies, underscoring the novelty of our dataset. Gene ontology and network analyses of the ISG15 interactome revealed functional clusters enriched in protein synthesis and targeting, a role not previously associated with ISGylation. In addition, we identified components of the proteasome, consistent with prior reports and further supporting ISGylation's role in protein degradation. Importantly, three candidate targets were independently validated by co-immunoprecipitation, supporting the mass spectrometry findings.
Collectively, this thesis provides new insights into the role of USP18 in signalling pathways that influence cell viability. We have demonstrated a novel strategy to re-engage apoptosis in resistant oesophageal cancer cells by impeding USP18 in the presence of type I IFN signalling. Additionally, we have identified specific ISG15 targets that may mediate these effects. This research highlights a new approach to significantly improve therapeutic regimens by targeting USP18 or a downstream mediator of its effects. Expanding this research could lead to new treatment options for oesophageal cancer, especially for patients with limited response to current therapies. Further research of this pathway may enable ‘precision oncology’ to be available in oesophageal cancer, bringing more effective, tailored therapies within reach.
Description
Controlled Access
Keywords
USP18 , ISG15 , IFN-α , Oesophageal cancer , Apoptosis , Autophagy , Drug resistance
Citation
Carey, M. M. 2025. Investigation into the role of Ubiquitin Specific Peptidase 18 in oesophageal cancer. PhD Thesis, University College Cork.