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

Burks J, Reed RE, Desai SD - Oncotarget (2015)

Bottom Line: 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.We conclude that free ISG15 may have antitumor and immunoregulatory function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, LSU Health Sciences Center-School of Medicine, New Orleans, LA, USA.

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.

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IFNβ increases MHC class I surface expression in T47D breast cancer cellsA, 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).
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Figure 3: IFNβ increases MHC class I surface expression in T47D breast cancer cellsA, 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).

Mentions: ISG15 is a potential tumor antigen [22]. The effective antigen presentation by MHC class I molecules is essential to activate the adaptive arm (T cell activation) of the immune system [23]. To test the potential role of ISG15 in activating the adaptive arm of the immune system, we assessed MHC class I surface expression, a marker for efficient antigen presentation, on T47D breast cancer cells devoid of free ISG15 expression and IFNβ-treated T47D cells expressing high levels of ISG15. Figure 3A shows that the ISG15 pathway is induced in the IFNβ-treated T47D cells. The same cells were used for assessing MHC class I surface expression. The MHC class I surface expression was assessed by flow cytometry analysis using an anti-human HLA-ABC PE antibody. As shown in Figure 3B, IFNβ-treated T47D cells overexpressing the ISG15 pathway displayed a 2-fold increase in levels of surface MHC class I expression (lower panels) compared to untreated T47D cells (upper panels). The experiment was independently repeated three times and the mean values of the median MHC class I fluorescence intensity are plotted in the accompanying bar graph (Figure 3B, right panel). Increased MHC class I surface expression suggested that IFNβ, a major inducer of the ISG15 pathway, promotes antigen presentation. This study corroborates the literature that elevated IFNβ signaling stimulates MHC class I expression in breast cancer cells [24] and other studies that IFNs induces MHC class I surface expression on cancer cells [25,26].


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

Burks J, Reed RE, Desai SD - Oncotarget (2015)

IFNβ increases MHC class I surface expression in T47D breast cancer cellsA, 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466680&req=5

Figure 3: IFNβ increases MHC class I surface expression in T47D breast cancer cellsA, 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).
Mentions: ISG15 is a potential tumor antigen [22]. The effective antigen presentation by MHC class I molecules is essential to activate the adaptive arm (T cell activation) of the immune system [23]. To test the potential role of ISG15 in activating the adaptive arm of the immune system, we assessed MHC class I surface expression, a marker for efficient antigen presentation, on T47D breast cancer cells devoid of free ISG15 expression and IFNβ-treated T47D cells expressing high levels of ISG15. Figure 3A shows that the ISG15 pathway is induced in the IFNβ-treated T47D cells. The same cells were used for assessing MHC class I surface expression. The MHC class I surface expression was assessed by flow cytometry analysis using an anti-human HLA-ABC PE antibody. As shown in Figure 3B, IFNβ-treated T47D cells overexpressing the ISG15 pathway displayed a 2-fold increase in levels of surface MHC class I expression (lower panels) compared to untreated T47D cells (upper panels). The experiment was independently repeated three times and the mean values of the median MHC class I fluorescence intensity are plotted in the accompanying bar graph (Figure 3B, right panel). Increased MHC class I surface expression suggested that IFNβ, a major inducer of the ISG15 pathway, promotes antigen presentation. This study corroborates the literature that elevated IFNβ signaling stimulates MHC class I expression in breast cancer cells [24] and other studies that IFNs induces MHC class I surface expression on cancer cells [25,26].

Bottom Line: 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.We conclude that free ISG15 may have antitumor and immunoregulatory function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, LSU Health Sciences Center-School of Medicine, New Orleans, LA, USA.

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.

Show MeSH
Related in: MedlinePlus