Limits...
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.

Show MeSH

Related in: MedlinePlus

MHC class I surface expression is dependent on the function of 26S proteasome in ISG15 overexpressing breast cancer cellsA, 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4466680&req=5

Figure 5: MHC class I surface expression is dependent on the function of 26S proteasome in ISG15 overexpressing breast cancer cellsA, 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).

Mentions: We first treated cells with MG132 for 6h and then assessed MHC class I surface expression in MDA/LV-control shRNA cells using flow cytometry as described above. Under these conditions, no difference in MHC class I surface expression in the absence or presence of MG132 was observed (not shown). Cells were then treated with acid to “strip-off” existing MHC class I complexes from the cell surfaces as described previously [30]. Acid-stripped cells were then cultured in the absence/presence of 5 μM MG132 for 6 hrs at 370C. The re-expression of MHC class I on MDA/LV-control/ISG15 shRNA cells was then assessed using flow cytometry as described in Figure 4. As shown in Figure 5A, MG132 treatment blocked re-expression of MHC class I by 37-40% in acid-stripped MDA/LV-control shRNA cells. Mean values of the median MHC class I fluorescence intensity from three experiments are plotted in the bar graph of Figure 5B. Involvement of the proteasome pathway in MHC-class I antigen presentation has been well documented previously [31, 32]. However, recent studies have revealed a role of the autophagy pathway in MHC class I-mediated antigen presentation [33]. Our results that MG132 blocks MHC-class I presentation thus suggest that increased MHC class I presentation in ISG15 overexpressing breast cancer cells is dependent on proteasome function but not the autophagy pathway.


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

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

MHC class I surface expression is dependent on the function of 26S proteasome in ISG15 overexpressing breast cancer cellsA, 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: MHC class I surface expression is dependent on the function of 26S proteasome in ISG15 overexpressing breast cancer cellsA, 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).
Mentions: We first treated cells with MG132 for 6h and then assessed MHC class I surface expression in MDA/LV-control shRNA cells using flow cytometry as described above. Under these conditions, no difference in MHC class I surface expression in the absence or presence of MG132 was observed (not shown). Cells were then treated with acid to “strip-off” existing MHC class I complexes from the cell surfaces as described previously [30]. Acid-stripped cells were then cultured in the absence/presence of 5 μM MG132 for 6 hrs at 370C. The re-expression of MHC class I on MDA/LV-control/ISG15 shRNA cells was then assessed using flow cytometry as described in Figure 4. As shown in Figure 5A, MG132 treatment blocked re-expression of MHC class I by 37-40% in acid-stripped MDA/LV-control shRNA cells. Mean values of the median MHC class I fluorescence intensity from three experiments are plotted in the bar graph of Figure 5B. Involvement of the proteasome pathway in MHC-class I antigen presentation has been well documented previously [31, 32]. However, recent studies have revealed a role of the autophagy pathway in MHC class I-mediated antigen presentation [33]. Our results that MG132 blocks MHC-class I presentation thus suggest that increased MHC class I presentation in ISG15 overexpressing breast cancer cells is dependent on proteasome function but not the autophagy pathway.

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