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, 17 (1), 113

Interleukin 32 Expression in Human Melanoma

Affiliations

Interleukin 32 Expression in Human Melanoma

Helicia Paz et al. J Transl Med.

Abstract

Background: Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma.

Methods: We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5' upstream region of the human IL32 gene into a luciferase reporter vector.

Results: A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, β, γ) of IL32. Cis-acting elements within this 5' upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature.

Conclusions: Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5' upstream region.

Keywords: IL32 transcriptional regulation; Immune infiltration; Interleukin 32 (IL32); Melanoma dedifferentiation; Myeloid polarization.

Conflict of interest statement

LHB declares Simpatica, Scientific Advisory Board Member, Jan. 2017-present; StemImmune Scientific and Medical Advisory Board, April 6, 2017-present; SapVax Advisory Board Nov. 15, 2017-present; NextCure, Scientific Advisory Board, 2018-present; Replimmune, Scientific Advisory Board, 2018-present; Western Oncolytics, Scientific Advisory Board, 2018-present; Torque Therapeutics, Consultant, 2018-present. AR has received honoraria from consulting with Bristol Myers-Squibb, Amgen, Chugai, Genentech, Merck, Novartis and Roche, and is on the scientific advisory board of Advaxis, Arcus, Bioncotech, Compugen, CytomX, Five Prime, FLX-Bio, ImaginAb, Isoplexis, Merus and Rgenix. During the conduct of this work AR was on the scientific advisory board and held stock in Kite-Pharma, and is co-founder of PACT Pharma and Tango Therapeutics. JSE is a scientific advisor to Allogene Therapeutics and Neogene Therapeutics.

Figures

Fig. 1
Fig. 1
IL32 is expressed in dedifferentiated melanomas. a Bar plot of log2 FPKM expression values across of panel of melanoma cell lines. b Bar plot showing the top 75 genes most correlated with IL32 expression. Genes highlighted in blue are melanocytic genes. (left) Scatterplot of log2 FPKM expression values between select melanocytic genes and IL32 (right). c Bar plot showing the top 75 genes most anti-correlated with IL32 expression. Genes highlighted in red are TNFα signaling and NF-κB associated genes. (left) Scatterplot of log2 FPKM expression values between select anti-correlated genes and IL32 (right)
Fig. 2
Fig. 2
TNFα and IFNγ treatment on melanoma cell lines results in dedifferentiation and IL32 gene expression. M397, M398, and M249 melanoma cell lines were treated with 1000U/mL TNFα or 100 U/mL IFNγ for 3 days and changes in gene expression were assessed by real time PCR. Gene expression for each sample was normalized to GAPDH and expressed as Delta Ct values, with the untreated M397, M398, M249 as the reference control. Error bars, standard deviation (**p < 0.01, ***p < 0.001, 95% CI, 1-way ANOVA). Figure is a representative experiment from 3 replicate experiments
Fig. 3
Fig. 3
Level of IL32 isoform expression after TNFα treatment. a IL32 gene expression (α, β, γ) after 3 day treatment with 1000 U/mL TNFα was compared to two IL32 expressing melanoma cell lines (M318 and M418) and Jurkat cells. Gene expression was normalized to GAPDH and expressed as Delta Ct values, with the Jurkat cells as the reference control. b Induction of IL32 gene expression with 24, 48, and 72 h treatment with 1000 U/mL TNFα. Gene expression was normalized to GAPDH and Delta Ct values are compared to the day 0 reference control
Fig. 4
Fig. 4
IL32 Promoter Activity. a Encode data set, b Promoter region of IL32. TSS determined from 5′RACE are indicated at − 464 bp and − 175 bp from the ATG. Two promoter sites identified by The Eukaryotic Promoter Database are identified as NK4_1 and IL32_1. c Promoter constructs used in luciferase assays. d Promoter constructs were co-transfected with pGL4.73 (Renilla) into melanoma cell lines (M318, M397, and M249). Post-transduction cells were treated for 24 h with 1000 U/mL TNFα and 100 U/mL IFNγ. Luciferase activity was assessed using Dual-Glo according to the manufactures directions. Data was normalized to Renilla luciferase expression and fold changes were calculated against the pGL3 empty control vector. Luciferase assays were performed in triplicate and plotted data is a representative experiment of three independent experiments

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