Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma

Abstract

Introduction: Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.

Results: We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.

Conclusion: We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in 'silent' metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.

Fig 1. IFNα-2b (IntronA) stimulates TAP1 expression in peripheral blood mononuclear cells (PBMC) of patients (n = 18) with malignant melanoma receiving adjuvant high-dose immunotherapy.

(A) The actual administered dose of IFNα-2b was about 20 million IU/m² dependent on the clinically observed side effects. Only days of IFNα treatment are shown. (B) mRNA expression of TAP1 in PBMCs of patients treated with adjuvant high-dose IFNα-2b. Statistical analysis of TAP expression was performed using the Statistical Analysis System of the SAS Institute Inc. (Cary, NC, USA).

Fig 2. IFNα stimulates TAP1 expression in mouse blood and tumor tissue and suppresses subcutaneous melanoma metastasis in a murine model for malignant melanoma.

C57BL/6 mice were inoculated subcutaneously with B16F1 malignant melanoma cells (day 0). The mice received either no treatment (control group, n = 3) or recombinant murine IFNα (n = 3). IFNα treatment (10.000 IU) started on day +3 after application of B16F1 cells for 5 consecutive days. (A) Measurement of tumor growth in control mice and IFNα treated mice. (B) On day +7.5, three IFNα treated and three control mice were sacrificed and blood was collected for qRT-PCR analysis of TAP1 expression. (C) Three days after the last injection of IFNα (day +11), three mice of the IFNα treated and three mice of the control group were sacrificed and tumors were excised for qRT-PCR analysis of TAP1 expression. Statistical analysis was performed using unpaired Student’s t-test (* p<0.05; not significant (n.s.)).

Fig 3. Stimulatory effects of IFNα on TAP expression, peptide binding and transport.

THP1 cells and A375 cells were treated with the indicated concentrations of IFNα for 3 hours (A, B). After stimulation, TAP1 mRNA and 18SrRNA mRNA (as internal reference) were measured by qRT-PCR analysis. Depicted are -fold changes relative to untreated cells = standard error of mean (SEM) of three independent experiments. (C) TAP1 protein expression is induced by IFNα in THP1 cells measured by western blot analysis. (D) TAP dependent peptide-binding sites are increased significantly by IFNα in THP1 cells (p = 0.0072). (E) ATP-dependent peptide transport is stimulated significantly by IFNα in THP1 cells (p = 0.0006). Statistical analysis was performed using unpaired Student’s t-test (** p<0.01, *** p<0.001).

Fig 4. IFNα-activated signalling pathways in THP1 and A375 cells.

THP1 (A) and A375 (B) cells were stimulated with the indicated concentrations of IFNα. As controls, THP-1 cells were additionally stimulated with IL-6/sIL-6R and LPS; A375 cells were stimulated with OSM or IL-1β. The phosphorylation levels of STAT1, STAT3, ERK1/2 and Akt were detected via Western blot analysis. Displayed are mean values of at least three independent experiments.

Fig 5. The inhibition of Janus kinases abrogates the IFNα-upregulated TAP1 expression in THP1 and A375 cells.

(A) THP1 (left) or A375 (right) cells were pre-incubated with the indicated amounts of JAK inhibitor I (JI-1), followed by stimulation with 100 U/ml or 1000 U/ml IFNα, respectively. Western blot analysis was performed using a specific antibody against phosphorylated STAT1 (STAT1-pY701), a STAT1 antibody recognizing the protein irrespective of its phosphorylation status and with an α-tubulin antibody. (B) Phosphorylation intensities were quantified by chemiluminescence analysis and normalization to loading controls. Shown are the means (n = 3) with standard error of mean (SEM, two-tailed, paired Student’s t-test, * p<0.05, ** p<0.01, *** p<0.001). (C) THP1 (left) or A375 (right) cells were pretreated with JI-1, and subsequently exposed to 100 U/ml or 1000 U/ml IFNα. Relative expression levels of TAP1 were analyzed by qRT-PCR and normalized to 18SrRNA as internal reference. Shown are -fold changes relative to unstimulated control = SEM (n = 3). Statistical significance was evaluated by performing a two-tailed, paired Student’s t-test (** p<0.01, *** p<0.001).