Interferon (IFN)-stimulated gene 15 (ISG15) is a 15 kDa protein composed of two ubiquitin-like (UBL) domains. A hinge sequence connects the N-terminal UBL domain to the C-terminal UBL domain, which has a motif containing lysine, arginine and glycine residues (LRLRGG) [1–4]. Through this sequence, ISG15 is covalently associated with its target proteins on lysine (Lys) residues by the sequential actions of the E1-activating enzyme (UBE1L), the E2-conjugating enzyme [ubiquitin-conjugating enzyme E2 L6 (UBCH8)], and the E3 ligases [HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), ariadne RBR E3 ubiquitin protein ligase 1 (HHARI), and tripartite motif containing 25 (TRIM25)] [5–8]. This process is known as ISGylation and occurs in three steps similar to the protein ubiquitination process: (A) UBE1L mediates the formation of an adenosine triphosphate (ATP)-dependent thioester bond with ISG15; (B) ISG15 is transferred from UBE1L to UBCH8 through a transesterification reaction, forming a thioester bond between ISG15 and UBCH8; (C) from the ISG15-E2 enzyme complex, the E3 ligases promote the transfer and covalent attachment of ISG15 to the Lys residue of the target proteins. Hence, the E3 ligases HERC5, HHARI, and TRIM25 mediate the substrate specificity for ISGylation [5–8]. Protein ISGylation is regulated by a de-ISGylase enzyme named ubiquitin-specific peptidase 18 (USP18) that removes ISG15 from target proteins, reducing ISGylation and increasing free ISG15 levels (Figure 1) [9–11].

Interestingly, many proteins may be ISGylated by HERC5 in a co-translational manner since HERC5 is localized in the polysomes and associates with the 60S ribosomal subunit [12]. It has been proposed that ISGylation occurs in proteins that suffer premature translation termination and in misfolded proteins to remove them from functional proteins [13, 14]. Moreover, compared with other posttranslational modifications, only a few ISGylated proteins have been reported. Consequently, the E3 ligase and the Lys residue or residues where ISGylation occurs are known only for some ISGylated proteins. This modification has been shown to occur mainly as monoISGylation on one, two, or multiple Lys residues [15, 16]. Some ISGylated proteins within the cancer context are displayed in Table 1.

ISGylation is a posttranslational modification related to changes in protein stability, increasing or decreasing protein levels, by competing with or promoting degradation via the ubiquitin-proteasome system (UPS) or lysosome-associated pathway. ISGylation can also modify the protein interaction pattern. The molecular actions associated with protein ISGylation are described in Figure 2.

Whereas the ISGylation system promotes the covalent binding of ISG15 to its target proteins, USP18 is a deISGylase protein that removes ISG15 from its modified proteins, maintaining ISG15 in its free form (non-conjugated). Some studies have demonstrated that USP18^–/– models display enhanced ISGylation levels [9, 51–53]. By contrast, increased USP18 activity can enhance free ISG15 levels [10, 11]. Interestingly, ISG15 is a protein modifier and a cytokine-like protein, since non-conjugated/free ISG15 is secreted from some immunologic cells, such as lymphocytes and monocytes, and recognized by natural killer (NK) cells and CD3+ T cells [54–58]. These cells express an integrin receptor containing αL and β2 integrin subunits (LFA-1) integrin-type receptor. LFA-1 receptor recognition of ISG15 induces the secretion of IFN-α and interleukin-10 (IL-10) [59]. In the context of cancer, free ISG15 may also be secreted and may act as a potential factor in the microenvironment of malignant tumors [60, 61]. Furthermore, free ISG15 seems to have intracellular actions via protein-protein interactions, which are mentioned briefly in Table 2 and Table 3.

ISG15 expression is increased in most cancer types. High levels of ISG15 mRNA have been detected by RNA-sequencing (RNA-seq) in several malignant neoplasias from patients’ samples (Figure 3) [71]. High levels of ISG15 protein have also been determined by immunochemistry, including breast, nasopharyngeal, and oral carcinomas [72–76]. These results indicate an upregulation of ISG15 in cancer, suggesting a pro-tumor role of ISG15.

Some studies have shown that the depletion of ISG15 expression reduces the proliferation and migration of breast carcinoma-derived cells [42, 77]. ISG15 expression is also deregulated in other cancer types developed in the central nervous system. For instance, glioblastoma displays high levels of ISG15, which are associated with poor survival [78]. ISG15 upregulation also seems important for the ISGylation of essential proteins, such as OCT4, and the cell stemness of glioblastoma cells [34]. Nevertheless, ISG15-dependent antitumor activities, including the reduction of proliferation, tumor growth, and the induction of apoptosis, have been reported in some cancers, such as ovarian cancer and leukemia [79, 80]. For instance, reduced ISG15 expression in ovarian cancer seems related to a poor prognosis for patients with this disease [81]. Therefore, the deregulation of ISG15 expression may display pro- and anti-tumor activities depending on the cancer type.

The dual activities of ISG15 in cancer may be related to the crosstalk of ISG15 with other molecular pathways. For example, ISG15 is increased in the cervical cancer context, and ISG15 depletion inhibits proliferation and migration, suggesting its pro-tumor role [82]; however, ISG15 also displays antitumor activities in cervical cancer cells but in a p53-dependent manner [83]. Another example is ISG15 upregulation in endometrial carcinoma to promote MYC proto-oncogene (MYC) signaling and changes in the DNA methylation profile, leading to pro-tumor actions and a poor prognosis [84].

Notably, the expression of the ISGylation enzymes and the USP18 deISGylase are critical in determining the relationship between ISGylation and free ISG15 levels and, consequently, their functions in cancer. Although type I IFNs α and β are the classical inductors of ISG15 expression [2, 54–56, 85], IFN-γ also induces ISG15 expression, depending on the cell type [86]. The ISGylation system (UBA7, UBE2L6, HERC5, and TRIM25) can also be regulated in response to type I IFNs [22, 83, 87–89]. Moreover, several molecular pathways can modulate ISG15 expression in a cancer context, some of which are summarized in Table 4. Thus, protein ISGylation can be increased by several deregulated signaling pathways in cancer.

Further, some components of the ISGylation system, such as UBCH8 and TRIM25, can be shared with the ubiquitination system. UBE2L6 has been demonstrated to be the primary target gene for IFN-α and IFN-β in A549 lung epithelial cells, HepG2 hepatoma cells, and NK-92 cells [102]. UBE2L6 encodes UBCH8, which is also implicated in ubiquitination reactions for protein degradation via the UPS [103]. Histone deacetylation inhibitors (e.g., LBH589) increase UBCH8 levels, favoring the degradation of a mutated form of fms related receptor tyrosine kinase 3 (FLT3) associated with acute myeloid leukemia [104]. These data suggest that alterations in other posttranslational modifications may also affect protein ISGylation in the cancer context. TRIM25 is also an E3-ubiquitin ligase associated with the ubiquitination and degradation of some tumor suppressors. However, TRIM25 is one of more than 600 E3 ligases in the ubiquitination system [105, 106]. It is unclear whether there is an interplay between ubiquitination and ISGylation mediated by TRIM25.

The high levels of ISG15 in several cancer types suggest the potential of ISG15 as a biomarker. Moreover, some cancer therapies, such as chemotherapy, radiotherapy, and targeted therapy, seem to be affected when ISG15 levels are deregulated, suggesting that ISG15 may be implicated in the response to cancer therapies (Table 5). In addition, the ISG15 protein has been considered a novel tumor-associated antigen to generate a Listeria-based vaccine targeting ISG15 (Lm-LLO-ISG15) [107, 108]. Nevertheless, more studies are required to understand the role of ISG15 in cancer therapies. The association between ISG15 and cancer therapies is summarized in Table 5.

In summary, protein ISGylation is a posttranslational modification implicated in malignant neoplasm progression. ISGylation can modulate protein stability, either positively or negatively, by promoting or inhibiting ISGylated protein degradation via the UPS or lysosomes. Furthermore, ISGylation can modulate molecular interactions by generating or disassembling protein complexes. The mechanisms, targets, and functional consequences of ISGylation seem to be defined by cancer type. Several proteins have been identified as ISGylation targets, and the Lys residues where this modification occurs have only been reported in some of them. Thus, identifying and characterizing new ISGylation target proteins and exploring the molecular bases of ISGylation and its functional repercussions are still necessary. Protein ISGylation levels and the ISGylated protein types can affect the response to chemotherapeutic treatments; studies in more depth are required to understand the role of ISG15/ISGylation in cancer therapies. Moreover, it is important to consider that ISGylation levels are related to the deregulation of ISG15, ISGylation system enzymes, and USP18 de-ISGylase expression in cancer. These elements implicated in the modulation of ISGylation can also be helpful for the novel design of therapeutic strategies, but more investigation is required.

ISGylation is a key posttranslational modification in malignant neoplasms, with implications for their progression, affecting adaptation in the tumor microenvironment and the ability to respond to cancer therapies. In most cancer types, protein ISGylation seems to be related to a pro-tumor effect, but its interplay with other molecular pathways depending on the cell type may determine the actions of this modification in promoting or reducing malignant neoplasm progression. Thus, ISG15/ISGylation can be over- or down-regulated depending on the type of cancer, and the identification of ISGylated proteins will help in understanding the novel molecular pathways associated with ISG15. Thus, protein ISGylation emerges as a central factor in cancer, which demands further investigation. Novel findings about the molecular mechanisms of ISG15 in tumorigenesis may be useful in preventing, treating, and controlling cancer.