Cancer stem cells (CSC) are thought to play a major role in the development and metastatic progression of pancreatic ductal adenocarcinoma (PDAC), one of the deadliest solid tumors. Likewise, the tumor microenvironment contributes critical support in this setting, including from tumor stromal cells and tumor-associated macrophages (TAM) that contribute structural and paracrine-mediated supports, respectively. Here, we show that TAMs secrete the IFN-stimulated factor ISG15, which enhances CSC phenotypes in PDAC in vitro and in vivo. ISG15 was preferentially and highly expressed by TAM present in primary PDAC tumors resected from patients. ISG15 was secreted by macrophages in response to secretion of IFNβ by CSC, thereby reinforcing CSC self-renewal, invasive capacity, and tumorigenic potential. Overall, our work demonstrates that ISG15 is a previously unrecognized support factor for CSC in the PDAC microenvironment with a key role in pathogenesis and progression. Cancer Res; 74(24); 7309–20. ©2014 AACR.

The interferon (IFN)-stimulated gene 15 (ISG15) encodes one of the most abundant proteins induced by interferon, and its expression is associated with antiviral immunity. To identify protein components implicated in IFN and ISG15 signaling, we compared the proteomes of ISG15^-/- and ISG15^+/+ bone marrow derived macrophages (BMDM) after vaccinia virus (VACV) infection. The results of this analysis revealed that mitochondrial dysfunction and oxidative phosphorylation (OXPHOS) were pathways altered in ISG15^-/- BMDM treated with IFN. Mitochondrial respiration, Adenosine triphosphate (ATP) and reactive oxygen species (ROS) production was higher in ISG15^+/+ BMDM than in ISG15^-/- BMDM following IFN treatment, indicating the involvement of ISG15-dependent mechanisms. An additional consequence of ISG15 depletion was a significant change in macrophage polarization. Although infected ISG15^-/- macrophages showed a robust proinflammatory cytokine expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO) production and arginase-1 activation was detected. Accordingly, following IFN treatment, NO release was higher in ISG15^+/+ macrophages than in ISG15^-/- macrophages concomitant with a decrease in viral titer. Thus, ISG15^-/- macrophages were permissive for VACV replication following IFN treatment. In conclusion, our results demonstrate that ISG15 governs the dynamic functionality of mitochondria, specifically, OXPHOS and mitophagy, broadening its physiological role as an antiviral agent. Protein modification by ubiquitin and ubiquitin-like proteins is a key regulatory process of the innate and adaptive immune response. Interferon-stimulated gene 15 product (ISG15) is an ubiquitin-like protein modifier that can reversibly attach to different viral and cellular proteins, mediating potent antiviral responses. In turn, many viruses, including poxviruses, have evolved strategies to antagonize the antiviral and inflammatory effects of the innate immune response in order to keep infected cells alive until virus replication is complete. Here, we describe a novel role for ISG15 in the control of mitochondrial function. Post-translational modifications such as ISGylation regulate essential mitochondrial processes including respiration and mitophagy, and influence macrophage innate immunity signaling. These findings are clinically relevant since mitochondrial dysfunction is seen in many pathologies, such as infectious disease, cancer, and cardiovascular or neurological disorders, among others, underscoring the importance of the relationship between cellular metabolism and immune response.

Viruses are responsible for the majority of infectious diseases, from the common cold to HIV/AIDS or hemorrhagic fevers, the latter with devastating effects on the human population. Accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. Interferon-stimulated gene 15 (ISG15) plays an important antiviral role during viral infection. ISG15 catalyzes a ubiquitin-like post-translational modification termed ISGylation, involving the conjugation of ISG15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. Numerous biomedically relevant viruses are targets of ISG15, as well as proteins involved in antiviral immunity. Beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. In this review, we give an overview of the biological consequences of ISGylation for virus infection and host defense. We also compare several published proteomic studies to identify and classify potential mitochondrial ISGylation targets. Finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of ISG15 in the regulation of mitochondrial processes, specifically OXPHOS and mitophagy.

The Interferon Stimulated Gene 15 (ISG15), a unique Ubiquitin-like (Ubl) modifier exclusive to vertebrates, plays a crucial role in the immune system. Primarily induced by interferon (IFN) type I, ISG15 functions through diverse mechanisms: (i) covalent protein modification (ISGylation); (ii) non-covalent intracellular action; and (iii) exerting extracellular cytokine activity. These various roles highlight its versatility in influencing numerous cellular pathways, encompassing DNA damage response, autophagy, antiviral response, and cancer-related processes, among others. The well-established antiviral effects of ISGylation contrast with its intriguing dual role in cancer, exhibiting both suppressive and promoting effects depending on the tumour type. The multifaceted functions of ISG15 extend beyond intracellular processes to extracellular cytokine signalling, influencing immune response, chemotaxis, and anti-tumour effects. Moreover, ISG15 emerges as a promising adjuvant in vaccine development, enhancing immune responses against viral antigens and demonstrating efficacy in cancer models. As a therapeutic target in cancer treatment, ISG15 exhibits a double-edged nature, promoting or suppressing oncogenesis depending on the tumour context. This review aims to contribute to future studies exploring the role of ISG15 in immune modulation and cancer therapy, potentially paving the way for the development of novel therapeutic interventions, vaccine development, and precision medicine.

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15−/− macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens. Modification of proteins by ubiquitin (UB) and ubiquitin-like proteins (UBLs) are key regulatory processes of the innate and adaptive immune response. Interferon (IFN) stimulated gene product 15 (ISG15) is an ubiquitin-like protein modifier, which is reversibly conjugated to different viral and cellular proteins mediating considerable antiviral responses. In turn, many viruses, including poxviruses, have evolved strategies to block the antiviral and inflammatory effects of the innate immune responses to keep cells alive until virus replication is completed. Here, we describe a novel function of ISG15 in the control of macrophages activation, phagocytosis and apoptosis in response to viral infection. These processes are essential for the self-defense mechanism to protect animals from infectious disease and could be crucial to understand the ISG15 antiviral activity described in animal models.

Conjugation of ISG15 inhibits replication of several viruses. Here, using an expression system for assaying human and mouse ISG15 conjugations (ISGylations), we have demonstrated that vaccinia virus E3 protein binds and antagonizes human and mouse ISG15 modification. To study ISGylation importance in poxvirus infection, we used a mouse model that expresses deconjugating proteases. Our results indicate that ISGylation restricts in vitro replication of the vaccinia virus VVΔE3L mutant but unconjugated ISG15 is crucial to counteract the inflammatory response produced after VVΔE3L infection.