Suppression of Type I Interferon Signaling Overcomes Oncogene-Induced Senescence and Mediates Melanoma Development and Progression

Abstract

Oncogene activation induces DNA damage responses and cell senescence. We report a key role of type I interferons (IFNs) in oncogene-induced senescence. IFN signaling-deficient melanocytes expressing activated Braf do not exhibit senescence and develop aggressive melanomas. Restoration of IFN signaling in IFN-deficient melanoma cells induces senescence and suppresses melanoma progression. Additional data from human melanoma patients and mouse transplanted tumor models suggest the importance of non-cell-autonomous IFN signaling. Inactivation of the IFN pathway is mediated by the IFN receptor IFNAR1 downregulation that invariably occurs during melanoma development. Mice harboring an IFNAR1 mutant, which is partially resistant to downregulation, delay melanoma development, suppress metastatic disease, and better respond to BRAF or PD-1 inhibitors. These results suggest that IFN signaling is an important tumor-suppressive pathway that inhibits melanoma development and progression and argue for targeting IFNAR1 downregulation to prevent metastatic disease and improve the efficacy of molecularly target and immune-targeted melanoma therapies.

Keywords: BRAF; interferon receptor; melanoma; metastasis; senescence; type I interferon.

Figure 1. IFN contribute to the oncogene-induced senescence and restrict proliferation in melanocytes that harbor activated Braf (see also Figure S1)

A. Immunoblot analysis of HDF IMR90 cells stably expressing shCON or shIFNAR1 and transduced with control vector or vector expressing H-RasG^12V. Cell lysates were prepared 5 days post transduction. Levels of markers of senescence (HP1β, p21) and IFN signaling (IRF7) were analyzed; total ERK levels were used as a loading control. B. Representative SA-βgal staining of IMR90 stably expressing shCON or shIFNAR1 and transduced with control vector or vector expressing H-Ras 5 days post transduction (left panel). Quantification of the number of SA-βgal positive cells per field (right panel). Here and thereafter: data are shown as a mean ± S.D.; *p < 0.05; **p < 0.01; ***p < 0.001. Magnification bar: 100 μm. At least 20 fields randomly chosen from 3 independent experiments were quantified. C. Immunoblot analysis of IFNAR1 from the whole skin lysates of adult mice at day 30 after topical administration of 4HT is shown. Levels of phospho-ERK (to indicate activation of BRAF) are also shown. Levels of total ERK and β-actin in whole lysates of corresponding samples were used for loading control (lower panels). The expression of senescent marker p21 was also evaluated. D. Representative immunofluorescent analysis and quantification of 53BP1-positive foci, and SA-βgal and Ki67-positive cells in the skin of adult mice of indicated genotypes at day 30 after topical administration of 4HT. Magnification bar: 100 μm. At least 20 fields randomly chosen from 3 independent experiments were quantified. E. Representative images of appearance of snout/ear and the underside of the lateral skin from adult mice of indicated genotypes at day 30 after topical administration of 4HT.

Figure 2. Suppression of IFN signaling is sufficient for Braf^V600E-driven melanoma development (see also Figure S2)

A. Macroscopic appearance of tumor lesions and the sentinel lymph nodes developing in 4HT treated Braf^CA/+; Ifnar1^−/− mice. B. H&E staining of ear (left panel), lesions (middle panel) and immunohistochemistry staining of S100 in the lesions (right panel) from in Braf^V600E; Ifnar1^−/− mice. Magnification bar: 100 μm. C. Incidence of malignant melanomas (left panel) and Kaplan-Meier analysis of survival (right panel) of mice of indicated genotypes at indicated time after 4HT treatment. D. H&E staining of lymph node and lung tissues from Braf^V600E; Ifnar1^−/− mice. Magnification bar: 100 μm.

Figure 3. IFN prevent melanoma development and progression via cell-autonomous and non-autonomous mechanisms (see also Figure S3)

A. Immunoblot analysis of indicated proteins in lysates from parental (P) PVMM cells and derivatives (transduced for expressing GFP alone or together with IFNAR1^SA) 4 days after transduction. B. SA-βGal staining (blue) and percentage of SA-βGal positive PVMM cells and their derivatives described in A. Cells were grown in vitro (upper panels) or injected to form tumors (lower panels, counterstained with FastRed). Data are shown as a mean ± S.D. from 3 independent experiments (at least 30 randomly chosen fields per experiment). Here and thereafter *p < 0.05. Magnification bar: 100 μm. C. Tumor growth curves of PVMM cells (P) and its derivatives expressing GFP (GFP) or GFP with IFNAR1^SA mutant (IFNAR1^SA) after s.c. injection of 1x10⁶ cells into Ifnar1^+/+ mice. Similar results were obtained in at least two or more additional independent experiments. D. Fisher’s exact test analysis of relationship between IFNAR1 levels (analyzed by immunohistochemistry in samples harvested from melanoma patients prior to treatment with high dose IFN) and the therapy outcome. E. PVMM tumor growth in Ifnar1^+/+ (blue) or Ifnar1^SA (red) mice after s.c. injection of 1x10⁶ PVMM cells. Similar results were obtained in at least two or more additional independent experiments. F. Average tumor growth of YUMM in Ifnar1^+/+ (blue), Ifnar1^SA (red) or Ifnar1^−/− (black) mice after s.c. injection of 1x10⁶ YUMM cells. Similar results were obtained in at least two additional independent experiments.

Figure 4. Downregulation of IFN receptor promotes melanoma development, progression and metastasis (see also Figure S4)

A. Macroscopic appearance of the sentinel lymph nodes and tumor lesions on the ear/snout from Braf^CA; Pten^Δ/Δ; Ifnar1^+/+ and Braf^CA; Pten^Δ/Δ; Ifnar1^SA mice at day 20 and 60 after generalized induction of recombination by tamoxifen. B. SA-βGal/FastRed staining of skin tissues from corresponding mice. Magnification bar here and thereafter: 100 μm. C. H&E staining of the ear and skin tissues from corresponding mice. D. H&E staining of organs from corresponding mice. Metastatic lesions are indicated by arrows. E. Kaplan-Meier analysis of survival in animal cohorts described in A–C. F. Quantification of metastatic lesions in the lymph node from mice of indicated genotypes. Data are shown as a mean ± S.D. (n=5–6 per group); *p < 0.05.

Figure 5. Inactivation of IFN pathway during melanoma development decreases the efficacy of melanoma treatment (see also Figure S5)

A. Gene Set Enrichment Analysis (MOSERLE IFN RESPONSE, left panel) and heat map (right panel) of IFNα/β signaling pathway genes of the transcriptome profiles of the skin tissues from Braf^CA; Pten^Δ/Δ; Ifnar1^SA mice collected at day 20 and 60 after tamoxifen treatment. NES: normalized enrichment score. FDR: false discover rate. B. qPCR analysis of the expression of Interferon stimulated genes in the skin tissues from mice of indicated genotypes at day 20 and 60 after tamoxifen administration. Untreated mice of the same genotypes were used as a control. Data are shown as a mean ± S.D. (n=5–6 per group). Here and thereafter: *p < 0.05; **p < 0.01. C. Immunoblot analysis of indicated protein levels in the skin tissues lysates from mice of indicated genotype treated as indicated (two samples per group). Ifnar1^−/− mice skin sample serves as a negative control for IFNAR1 detection (analyzed by IP-IB). Relative levels of Ifnar1 mRNA (compared with untreated skin in Ifnar1^+/+ animals) are shown below the IFNAR1 protein panel. D. Relative local melanoma tumor volume change in mice of indicated genotype treated with control or anti-PD1 antibody. Tumors were induced by topical application of 4HT. Tumor size in each of the treatment groups was measured every other day and plotted as percentage change in tumor size compared to the starting size (50 mm²). Data are shown as a mean ± S.D. (n=5–6 per group). E. Relative local melanoma tumor volume change in mice treated with a vehicle control or Vemurafenib (Vem). Tumors were induced by topical application of 4HT. Tumor size in each of the treatment groups was measured every other day and plotted as percentage change in tumor size compared to the starting size (50 mm²). Data are shown as a mean ± S.D. (n=5–6 per group).