Protein ISGylation: a posttranslational modification with implications for malignant neoplasms

Interferon (IFN)-stimulated gene 15 (ISG15) is a member of the ubiquitin-like (UBL) protein family that can modify specific proteins via a catalytic process called ISGylation. This posttranslational modification can modulate the stability of the ISGylated proteins and protein-protein interactions. Some proteins modified by ISG15 have been identified in malignant neoplasms, suggesting the functional relevance of ISGylation in cancer. This review discusses the ISGylated proteins reported in malignant neoplasms that suggest the potential of ISG15 as a biomarker and therapeutic target in cancer.

Figure 1.Enzymatic cascade reactions of ISGylation.UBE1L is associated with ISG15 by forming a thioester bond, activating ISG15 in an ATP-dependent manner.Next, E2 ligase (UBCH8) has a cysteine active site where ISG15 is transferred.Finally, E3 ligase (HERC5, HHARI, or TRIM25) catalyzes the covalent binding of ISG15 to its target protein.USP18 removes ISG15 from the ISGylated protein, mediating a de-ISGylation process.PPi: inorganic pyrophosphate 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.

Molecular actions of ISGylation
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.

Effect of ISGylation on protein stability
The effect of ISGylation is related to increased protein stability by competing with or blocking ubiquitination and degradation via the UPS.For instance, IFN-stimulated cells treated with proteasome inhibitors enhance ISGylation levels [35].In addition, when ISGylation levels are decreased by reducing the expression of ISG15, ubiquitination detection is enhanced in breast cancer cells.Similarly, when the expression of the E2 ISG15 conjugating enzyme (UBE2L6) is reduced, the ISGylation levels decrease, while ubiquitination marks increase [36].Another study showed that Lys 29 and Lys 48 from ubiquitin are substrates for ISG15, forming ubiquitin-ISG15 chains that are not degradable and affecting the binding of ubiquitin to its target proteins [37].ISGylation maintains the stability of some proteins by inhibiting their polyubiquitination and degradation; these include pSTAT1, EMD, and YAP.Hence, pSTAT1 is ISGylated to stabilize its activity as a transcription factor [38]; the stability of EMD conferred by ISGylation promotes glucose metabolism in lung adenocarcinoma (LUAD) [31]; and the ISGylation of YAP confers stability to promote pro-tumor actions [32].By contrast, an increase in ubiquitination levels was observed with ISG15 overexpression in HepG2 cells [39].Some studies have shown that ISGylation can also promote the degradation of proteins, but the molecular details are not completely clear.Some examples are Promotes glioma cell stemness [34] ND: not determined; MAPK: mitogen-activated protein kinase; JNK: c-Jun N-terminal kinase; PARK: parkin; ΔNp63α: alternative splice variant of phosphoprotein 63; BECN1: beclin 1; 4EHP: eukaryotic translation initiation factor 4E homologous protein; 14-3-3σ: stratifin; PCNA: proliferating cell nuclear antigen; TLS: translesion DNA synthesis; p53: phosphoprotein 53; HIF-1α: hypoxia-inducible factor 1 subunit α; FOXO3A: forkhead box O3A; PTEN: phosphatase and tensin homolog; EMD: skeletal protein emerin; YAP: Yes-associated protein; EGFR: epidermal growth factor receptor; Akt: Akt kinase; Ki-ras: KRAS protooncogene, GTPase; GDI2: guanosine diphosphate (GDP) dissociation inhibitor 2; OCT4: POU class 5 homeobox 1 (also known as POU5F1) β-catenin [27,28], FOXO3A, and PTEN [29,30], which seem to present ISGylation-associated ubiquitination for their degradation in cancer contexts (Table 1).

ISGylation is associated with lysosomal pathways in cancer
Autophagy is a catabolic process that delivers cellular components to lysosomes and organelles for macromolecule destruction.Not only is protein ISGylation related to the UPS but this modification has also been associated with lysosomal pathways.ISG15 overexpression in U251 glioma cells increases the ISGylation and autophagic degradation pathways [40].ISGylation has been shown to inhibit exosome secretion, leading to lysosomal degradation of multivesicular bodies (MVBs) proteins [41].Nevertheless, ISG15 enhances the stability of Ki-Ras, inhibiting its lysosomal degradation in breast cancer cells [42].Furthermore, ISG15 or UBE2L6 depletion leads to increased autophagy in esophageal cancer cells, suggesting that ISGylation can inhibit autophagy [43].Interestingly, type I IFN-induced ISGylation at residues Lys 117, 263, 265, and 266 of BECN1 was observed in 293T and HepG2 cell lines.The ISGylation of BECN1 inhibits autophagy, but the de-ISGylase USP18 facilitates autophagy and the degradation of EGFR by promoting the de-ISGylation of BECN1 [15].

ISGylation modulates molecular interactions
Protein-protein interactions that form multiprotein complexes also seem to be affected by ISGylation modifications.Filamin B has been demonstrated to be a scaffold for Rac family small GTPase 1 (RAC1), MAPK/extracellular signal-regulated kinase (ERK) kinase kinase 1 (MEKK1), and MAPK kinase 4 (MKK4) proteins, which are part of IFN-α/β-induced JNK signaling to induce apoptosis.When filamin B is ISGylated, its scaffold functions are interrupted, decreasing JNK signaling and its actions [18].Further, the scaffold protein IQGAP1 and the cytoskeletal protein, non-muscle myosin IIA (NMIIA), are targets of ISGylation in the breast cancer context.However, the ISGylation of these proteins has not been related to changes in protein stability, suggesting that the effects of this modification may affect protein-protein interactions [44][45][46].

ISGylation modulates the activity of some proteins
Other molecular interactions affected by ISGylation may affect cellular processes.For example, TLS via DNA polymerase N is induced when the PCNA is monoubiquitinated and then ISGylated in response to DNA damage by ultraviolet (UV) light.As a result of PCNA ISGylation, the ubiquitination mark is removed, leading to the release of polymerase N from PCNA for TLS termination [24].Furthermore, the 4EHP [messenger RNA (mRNA) 5' cap structure-binding protein] is modified by HHARI-dependent ISGylation, increasing its binding to the cap and competing with the eukaryotic translation initiation factor 4E (eIF4E) translation initiation factor [21].Further, when the E2-ubiquitin conjugation enzyme UBC13 is ISGylated, its activity decreases, affecting the ubiquitination process [47].However, ISGylation enhances the activity of the CHIP and PARK, two E3 ubiquitin ligases that mark and induce the degradation of their substrates [19,48].
Furthermore, proteins can display several changes due to their ISGylation.For example, the ISGylation of EMD confers stability but is also required for the interaction between EMD and pyruvate dehydrogenase E1 α subunit (PDHA) protein to inhibit aerobic oxidation [31].Similarly, YAP ISGylation results in its stability, reducing its interaction with E3-ubiquitin ligase β-transducin repeat containing E3-ubiquitin protein ligase (βTrCP); however, this modification of YAP favors its activity promoting the transcription of genes, such as PGLS that encodes 6-phosphogluconolactonase (6PGL) of the pentose phosphate pathway (PPP).This glucose metabolism pathway promotes tumor growth in LUAD [32].
It has been proposed that genomic stability may be conferred through ISGylated proteins by mitigating DNA replication stress [49].Moreover, the metabolic plasticity and mitophagy of pancreatic cancer (PC) stem cells also seem modulated by protein ISGylation.ISG15 depletion reduces ISGylation in mitochondria, impairing mitophagy and reducing oxidative phosphorylation [50].

Free ISG15: another face of ISG15
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 (nonconjugated).Some studies have demonstrated that USP18 -/-models display enhanced ISGylation levels [9,[51][52][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][55][56][57][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.

Deregulation of ISG15 expression and its implications in cancer
ISG15 expression is increased in most cancer types.High levels of ISG15 mRNA have been detected by RNAsequencing (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][73][74][75][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 OSCC Free ISG15 interacts with Rac1-GDP, promoting cell migration in an ISGylation-independent manner.This event has been related to lymphatic metastasis of OSCC [66] PDA: pancreatic ductal adenocarcinoma; TAMs: tumor-associated macrophages; ESCC: esophageal squamous cell carcinoma; MHCI: major histocompatibility complex I; OSCC: oral squamous cell carcinoma; CSCs: cancer stem cells Table 3. Effects of free ISG15 that may be relevant in a cancer context

Effect Description of free ISG15-associated effects Reference
Regulation of IFN signaling In humans, JAK1-IFNAR2 interaction is disrupted by USP18, affecting IFN-α/β signaling Free ISG15 interacts with USP18, inhibiting its degradation by SKP2 [67]

Protein complex disassembly
Intracellular free ISG15 interrupts the USP18-SKP2 interaction promoting the stability of USP18 [68]

Regulation of E3-ubiquitin ligase activity
Free ISG15 binds to the E3-ubiquitin ligase NEDD4 to interrupt its interaction with the E2 biquitin-conjugating enzyme, decreasing ubiquitination [69] Participation in selective autophagy LRRC25 inhibits the IFN-signaling by promoting lysosomal degradation of ISG15associated RIG-1 [70] JAK1-IFNAR2: janus kinase 1-IFN α and β receptor subunit 2; SKP2: S-phase kinase-associated protein 2; NEDD4: NEDD4 E3 ubiquitin protein ligase (also known as NEDD4-1 or RPF1, neural precursor cell expressed, developmentally down-regulated 4); LRRC25: leucine-rich repeat containing 25; RIG-1: RNA sensor RIG-1 essential proteins, such as OCT4, and the cell stemness of glioblastoma cells [34].Nevertheless, ISG15dependent 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][55][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][88][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.Graph analyzed using gene expression profiling interactive analysis (GEPIA, http://gepia.cancer-pku.cn/index.html).TPM: transcripts per kilobase of exon model per million mapped reads; ACC: adrenocortical carcinoma; BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: lymphoid neoplasm diffuse large B cell lymphoma; ESCA: esophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LIHC: liver hepatocellular carcinoma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: testicular germ cell tumor; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; UCS: uterine carcinosarcoma 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 IFNβ 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.

ISG15 as a potential therapeutic target for cancer
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.

Conclusions
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.

Figure 2 .
Figure 2. Target proteins for ISGylation.Several proteins associated with different molecular activities are modified by ISGylation (circles of different colors).The numbers 1-7 indicate the effect of ISGylation (the arrows indicate increase or decrease) on its target proteins.pSTAT1: phosphorylated signal transducer and activator of transcription 1 (STAT1); IQGAP1: IQ motif containing GTPase activating protein 1; UBC13: E2 ubiquitin-conjugating protein UBC13; CHIP: C-terminus of the Hsc70-interacting protein

Table 1 .
Examples of ISGylation target proteins

Table 2 .
Actions of free ISG15 in some cancer types

Table 4 .
Molecular pathways that modulate ISG15/ISGylation levels in a cancer context