The antitumor effects of IFN-α are abrogated in a STAT1-deficient mouse
Gregory B. Lesinski, Mirela Anghelina, Jason Zimmerer, Timothy Bakalakos, Brian Badgwell, Robin Parihar, Yan Hu, Brian Becknell, Gerard Abood, Abhik Ray Chaudhury, Cynthia Magro, Joan Durbin, William E. Carson III
Gregory B. Lesinski, Mirela Anghelina, Jason Zimmerer, Timothy Bakalakos, Brian Badgwell, Robin Parihar, Yan Hu, Brian Becknell, Gerard Abood, Abhik Ray Chaudhury, Cynthia Magro, Joan Durbin, William E. Carson III
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Article Oncology

The antitumor effects of IFN-α are abrogated in a STAT1-deficient mouse

Abstract

IFN-α activates the signal transducer and activator of transcription (STAT) family of proteins; however, it is unknown whether IFN-α exerts its antitumor actions primarily through a direct effect on malignant cells or by stimulating the immune system. To investigate the contribution of STAT1 signaling within the tumor, we generated a STAT1-deficient melanoma cell line, AGS-1. We reconstituted STAT1 into AGS-1 cells by retroviral gene transfer. The resulting cell line (AGS-1STAT1) showed normal regulation of IFN-α–stimulated genes (e.g., H2k, ISG-54) as compared with AGS-1 cells infected with the empty vector (AGS-1MSCV). However, mice challenged with the AGS-1, AGS-1STAT1, and AGS-1MSCV cell lines exhibited nearly identical survival in response to IFN-α treatment, indicating that restored STAT1 signaling within the tumor did not augment the antitumor activity of IFN-α. In contrast, STAT1–/– mice could not utilize exogenous IFN-α to inhibit the growth of STAT1+/+ melanoma cells in either an intraperitoneal tumor model or in the adjuvant setting. The survival of tumor-bearing STAT1–/– mice was identical regardless of treatment (IFN-α or PBS). Additional cell depletion studies demonstrated that NK cells mediated the antitumor effects of IFN-α. Thus, STAT1-mediated gene regulation within immune effectors was necessary for mediating the antitumor effects of IFN-α in this experimental system.

Authors

Gregory B. Lesinski, Mirela Anghelina, Jason Zimmerer, Timothy Bakalakos, Brian Badgwell, Robin Parihar, Yan Hu, Brian Becknell, Gerard Abood, Abhik Ray Chaudhury, Cynthia Magro, Joan Durbin, William E. Carson III

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Characterization of the AGS-1 cell line. AGS-1 has the appearance of a m...
Characterization of the AGS-1 cell line. AGS-1 has the appearance of a malignant melanoma by (a) hematoxylin and eosin staining and immunohistochemistry for (b) S-100 and (c) HMB-45. (d) Immunoblot analysis confirmed that the AGS-1 cell line was STAT1 deficient (lane 3). Cell lysates from the A431 cell line (lane 1) and B16F1 cell line (lane 2) were used as positive controls. The protein content of the lysates was quantitated, and equal amounts were loaded onto the gel. (e) EMSA analysis revealed no activation of STAT1 in IFN-α–treated AGS-1 cells. Lysates from IFN-α–stimulated B16F1 cells were used as positive controls in this assay. SIS, SIS-inducible element. (f) Flow cytometric analysis of H2k expression. Treatment of B16F1 cells for 48 hours with 104 U/ml muIFN-α resulted in increased H2k expression. Loss of STAT1 significantly reduced the induction of H2k in the AGS-1 cell line after IFN-α treatment. The minor induction of H2k expression by IFN-α in the AGS-1 cell line can be attributed to STAT1-independent signaling through NF-κB (28, 29). (g) Northern blot analysis indicated a loss of ISG-54 gene regulation in AGS-1 cells after stimulation with 104 U/ml muIFN-α for 16 hours. Probes specific for GAPDH (housekeeping gene) were used as controls and did not vary across samples. (h) Northern blot findings were confirmed by PCR using primers specific for muISG-54 and showed increased ISG-54 expression only in B16F1 cells (positive control) after IFN-α treatment. Primers specific for 28s rRNA (housekeeping gene) were used as a controls in this assay. Neg, negative control.