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. 2015 Mar 30;6(9):7221-31.
doi: 10.18632/oncotarget.3372.

Free ISG15 triggers an antitumor immune response against breast cancer: a new perspective

Julian Burks  1   2 , Ryan E Reed  1 , Shyamal D Desai  1
Affiliations

Free ISG15 triggers an antitumor immune response against breast cancer: a new perspective

Julian Burks et al. Oncotarget. .

Abstract

Interferon-Stimulated Gene 15 (ISG15), an antagonist of the canonical ubiquitin pathway, is frequently overexpressed in various cancers. In cancer cells, ISG15 is detected as free (intracellular) and conjugated to cellular proteins (ISGylation). Free ISG15 is also secreted into the extracellular milieu. ISGylation has protumor functions and extracellular free ISG15 has immunomodulatory properties in vitro. Therefore, whether ISG15 is a tumor suppressor or tumor promoter in vivo remains controversial. The current study aimed to clarify the role of free ISG15 in tumorigenesis. Breast cancer cells stably expressing control, ISG15, and UbcH8 (ISG15-specific E2 ligase) shRNAs were used to assess the immunoregulatory and antitumor function of free ISG15 in cell culture (in vitro) and in nude mice (in vivo). We show that extracellular free ISG15 suppresses breast tumor growth and increases NK cell infiltration into xenografted breast tumors in nude mice, and intracellular free ISG15 enhances major histocompatibility complex (MHC) class I surface expression in breast cancer cells. We conclude that free ISG15 may have antitumor and immunoregulatory function in vivo. These findings provides the basis for developing strategies to increase systemic levels of free ISG15 to treat cancer patients overexpressing the ISG15 pathway.

Keywords: ISG15; antitumor; breast cancer; immune system; ubiquitin/proteasome.

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Conflict of interest statement

Conflicts of interest

We declare no conflict of interest.

Figures

Figure 1
Figure 1. ISG15 inhibits ZR-75-1 breast tumor growth in nude mice and induces NK cell filtration into xenograft tumors
A, ZR/control and ISG15 shRNA xenografts were established in female nude mice, and tumor volume was measured two weeks after implantation as indicated in Methods. Each bar represents average volume calculated from eight mice (four mice/group in two different experiments) (Bars: +/− SEM). B, Frozen tumor sections (from A) were immunostained using anti-CD49b-DX5 antibodies (upper panels) or anti-ISG15 antibodies (lower panels) as described in Methods.
Figure 2
Figure 2. ISG15 inhibits MDA-MB-231 breast tumor growth in nude mice and induces NK cell filtration into xenograft tumors
A, ISG15 expression in MDA/LV-control and ISG15 shRNA was analyzed using anti-ISG15 antibodies in Western analysis as described in Methods. B, MDA/LV-control (Group I) and ISG15 (Group II) shRNA xenografts were established in female nude mice. On the same day, recombinant free ISG15 (188 ug) (Boston Biochemical) was injected near the site of xenograft implantation in Group III. Tumor volume was measured from 0-3 weeks after implantation as indicated in Methods. Each point represents average volume (+/− SEM) calculated from eight different tumors for Group I and II, and four different tumors for Group III. Representative photographs of tumor-bearing mice after three weeks of all test groups are shown in the right panel. C, Frozen tumor sections (from Group I, II, and III shown in A) were immunostained using anti-CD49b-HHMα2 antibodies as described in Methods.
Figure 3
Figure 3. IFNβ increases MHC class I surface expression in T47D breast cancer cells
A, T47D breast cancer cells were left untreated or treated with human IFNβ (1000 units/ml) for 24 hrs. Cell lysis and immunoblotting analysis using anti-ISG15 antibodies was carried out as described in Methods. B. Flow cytometric analysis of MHC class I (HLA class I ABC-PE) surface expression on T47D and IFNβ-treated T47D breast cancer cells (from A) is shown. Mean values of the median fluorescence intensity from three independent experiments are plotted in the bar graph (Bars: +/− SEM).
Figure 4
Figure 4. MHC class I surface expression is increased in ISG15 overexpressing breast cancer cells
A, A representative flow cytometric analysis of MHC class I surface expression on MDA/LV-control and ISG15 shRNA cells is shown. Bar graph shows the quantification of the flow cytometric data shown in A. B, Experiment shown in panel A was repeated three times and the mean values of the median fluorescence intensity are plotted in the bar graph (Bars: +/− SEM).
Figure 5
Figure 5. MHC class I surface expression is dependent on the function of 26S proteasome in ISG15 overexpressing breast cancer cells
A, Cell surface MHC class I molecules on MDA/LV-control shRNA cells were removed by acid stripping for 90s. Reappearance of MHC class I surface expression was then measured in the presence/absence of MG132 (5 μM) after 6 hr by flow cytometric analysis as described in Figure 4. A representative flow cytometric graph for MHC class I surface reappearance on MDA/LV-control cells in the presence/absence of MG132 is shown. B, Experiment shown in panel A was repeated three times and the mean values of the median fluorescence intensity are plotted in the bar graph (Bars: +/− SEM).
Figure 6
Figure 6. Free ISG15 and not ISGylation contributes to increased MHC class I surface expression in breast cancer cells
A, A representative graph of flow cytometric analysis of MHC class I surface expression on MDA/LV-control and UbcH8 shRNA cells is shown. Bar graph shows the quantification of the flow cytometric data shown in A. B, Experiment shown in panel A was repeated three times and the mean values of the median fluorescence intensity are plotted in the bar graph. B) Reappearance of MHC class I surface expression was measured (three experiments) in the presence/absence of MG132. C, Expression of ISG15 and conjugates using anti-ISG15 antibodies is shown. All bars: +/− SEM.
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