Background
Though first described in 1992 as NK4, interleukin 32 (IL32) [
1,
2] was recharacterized in 2005 as a proinflammatory cytokine differentially expressed in IL18 responsive cells. Since then, its expression has been implicated in various pathologies, including rheumatoid arthritis, pathogen responses, atherosclerosis, and several malignancies [
3‐
12]. IL32 is broadly expressed in immune cells, including natural killer cells [
13‐
15], T lymphocytes [
16‐
18], macrophages, and dendritic cells [
19,
20]. Functionally, IL32 can activate NF-κB and p38 mitogen activated protein kinase pathways and induce expression of proinflammatory cytokines including TNFα, IL8 and CCL2. In addition to its presence in immune tissues, IL32 expression can also be induced in human epithelial tissues. IL32 is also expressed in a broad range of human epithelial cancers–gastric [
3], lung [
5], breast [
4], colon [
6‐
9], pancreas [
10], and thyroid [
11,
12]—as well as hematologic malignancies such as lymphoma and leukemia [
16,
21]. IL32 expression in cancer is linked to features associated with worse prognosis, including angiogenesis, invasion, and metastasis [
4].
However, the role of IL32 in human melanoma cells is less well understood. Initial studies suggested that IL32 expression was related to a more invasive, metastatic phenotype marked by a loss of e-cadherin expression [
22]. Others reported that IL32 isoforms alpha and gamma were highly enriched in PD-L1 expressing melanoma specimens [
23].
Herein we report an unbiased analysis of IL32 expression in melanoma using RNA sequencing data from: [
1] a large in-house panel of melanoma cell lines and [
2] tumor tissues from The Cancer Genome Atlas (TCGA) dataset. IL32 expression correlated with a dedifferentiated phenotype which has been characterized by resistance to targeted therapies, escape from immune recognition, and a high AXL/low MITF genetic signature [
24‐
27]. Expression of IL32 in human melanoma can be transcriptionally regulated in the context of a proinflammatory tumor microenvironment with either induced or constitutive IL32 expression strongly, but not invariably, correlating with a dedifferentiated genetic signature.
Methods
Cell culture
The human melanoma cell lines were established from patient biopsies. These cells were grown in RPMI 1640 media supplemented with 10% FBS, 1% l-glutamine and 1% penicillin/streptomycin/actinomycin D (Life Technologies, Grand Island, NY). Melanoma cell lines were treated with 1000 IU/mL TNFα and 100 IU/mL IFNγ (PeproTech US, Rocky Hill, NJ) for the indicated time points. M397 was cultured with recombinant 100 ng/mL IL32α, -β, or -γ (R&D Systems, Minneapolis, MN) for 7 days, with fresh IL32 being added every 2–3 days. Cells were counted using Trypan blue. Jurkat T lymphocytes were cultured in RPMI 1640 supplemented with 10% FBS.
Gene expression analysis
Gene expression FPKM values for the melanoma cell line panel was obtained from the Gene Expression Omnibus (GEO) accession number GSE80829. The hallmark geneset for NF-κB and TNFα signaling (TNFA_SIGNALING_VIA_NFKB) was downloaded from the Molecular Signatures Database (MSigDB). Gene set enrichment analysis of the full ranked list of IL32 correlations was performed using the pre-ranked tool and the GO Biological Process Ontology gene sets from MSigDB v6.1. Gene expression FPKM values of skin cutaneous melanoma tumor biopsies from The Cancer Genome Atlas (TCGA SKCM) was downloaded from the Genomic Data Commons (portal.gdc.cancer.gov). For all analyses, FPKM values were log2 transformed with an offset of 1. Calculation of the enrichment overlap of the top 100 correlated genes with IL32 was performed using the MSigDB investigate gene sets tool (software.broadinstitute.org/gsea/msigdb/annotate.jsp) and the hallmark gene sets.
Retroviral vectors
IL32 isoforms were cloned into an MSCV retroviral vector with a T2A-GFP using the HiFi DNA Assembly Cloning Kit (NEB, Ipswich, MA). The retroviral vector was kindly provided by A. Ribas. Retroviruses were produced using GP2-293 cells (Clontech, Mountain View, CA) and human melanoma cells were transduced with IL32 expressing retroviral vectors with 4ug/mL Polybrene. GFP positive cells were sorted 48 h after transduction. Cells maintained approximately 95% GFP positivity over the course of the experiment. IL32 expression was confirmed by real time PCR and Western blot.
Real time PCR
RNA was extracted using PureLink RNA Mini Kit (ThermoFisher, Waltham, MA) and cDNA was synthesized using the High-Capacity RNA-to-cDNA kit (ThermoFisher, Waltham, MA). Real time PCRs were performed using the Applied Biosystems 7500 using the following primers designed using NCBI Primer-BLAST. GAPDH was used a housekeeping gene.
IL32 Primers:
IL32a Fwd: 5′-GAGGCAACAGATCCCCTGTC-3′.
IL32a Rev: 5′-GGCTCCGTAGGACTTGTCAC-3′.
IL32b Fwd: 5′-TCTCTCGGCTGAGTATTTGTGC-3′.
IL32b Rev: 5′-ATGACCCAGCTCCACTGAGA-3′.
IL32 g Fwd: 5′-TACTTCTGCTCAGGGGTTGG-3′.
IL32 g Rev: 5′-TGGGTGCTGCTCCTCATAAT-3′.
Differentiation gene set:
NGFR Fwd: 5′-TCATCCCTGTCTATTGCTCCA-3′.
NGFR Rev: 5′-TGTTCTGCTTGCAGCTGTTC-3′.
MLANA Fwd: 5′-GCTCATCGGCTGTTGGTATT-3′.
MLANA Rev: 3′-TTCTTGTGGGCATCTTCTTG-5′.
MITF Fwd: 5′-ATCAGCAACTCCTGTCCAGC-3′.
MITF Rev: 5′-GCCAGTGCTCTTGCTTCAGA-3′.
AXL Fwd: 5′-ACCTACTCTGGCTCCAGGATG-3′.
AXL Rev: 5′-CGCAGGAGAAAGAGGATGTC-3′.
GAPDH Fwd: 5′-TGCACCACCAACTGCTTAGC-3′.
GAPDH Rev: 5′-GGCATGGACTGTGGTCATGAG-3′.
5′ rapid amplification of cDNA ends (RACE)
To determine the transcriptional start site of IL32, 5′ RACE was performed according the manufacturer’s directions for the SMARTer RACE 5′/3′ kit (Takara, Mountain View, CA). Briefly, RNA was isolated from the IL32 expressing cell line, M318, using NucleoSpin RNA II kits (Takara, Mountain View, CA). First strand cDNA synthesis was done according the manufacturer’s directions. The 5′ RACE reaction using the following gene specific primer and a universal primer (Universal Primer Short, UPM, Takara) using 25 cycles of 94° for 30 s, 68° for 30 s, and 72° for 3 min. RACE products were run on an agarose gel and purified using the NucleoSpin Gel and PCR Clean-Up kit (Takara, Mountain View, CA). RACE products were cloned into the pRACE vector using the In-Fusion HD Cloning kit (Takara, Mountain View, CA) and sequenced using the M13F and M13R sequencing primers.
Luciferase constructs
The promoter region of IL32 were pcr’d from the Human BAC clone RP11 (RPC1-11 clone 473M20) using Pfusion DNA polymerase (NEB, Ipswich, MA). Promoter region greater than 3 kb were cloned into pGL3 using HiFi DNA Assembly Cloning Kit (NEB, Ipswich, MA). The promoter regions were derived using the following primers.
− 10.5 kb Fwd: 5′-ACCGAGCTCTTACGCGTGCTAGCCCGCAGGGAAGAAGGTGAGAGATG-3′.
− 7.5 kb Fwd: 5′-ACCGAGCTCTTACGCGTGCTAGCCCCTCTTTAGCGGTGAGTGGGG-3′.
− 5.5 kb Fwd: 5′-ACCGAGCTCTTACGCGTGCTAGCCCCAGTAGCTGGCTGTTTCGTG-3′.
− 2.5 kb Fwd: 5′-CTAGCCCGGGCTCGAGATCTAGAAAGTAGATGAGGCCAG-3′.
− 2 kb Fwd: 5′-CTAGCCCGGGCTCGAGATCTAAAACAGGGTACATACAGTCTG-3′.
− 1.5 kb Fwd: 5′-CTAGCCCGGGCTCGAGATCTGGAGTGCAGTGGCACCAT-3′.
− 1.0 kb Fwd: 5′-CTAGCCCGGGCTCGAGATCTGTTTGGCCCCAGGAAACC-3′.
− 0.5 kb Fwd: 5′-CTAGCCCGGGCTCGAGATCTCCCCAGCCAGCTGTCCCG-3′.
− 2.5 kb to − 0.5 kb Rev: 5′-CTCGAGCTCGAGTGGCGGCCAAAAGTTCAAGGAGC-3′.
− 10.5 kb to − 3 kb Rev: 5′-GAATGCCAAGCTTACTTAGATCGCATGGCGGCCAAAAGTTCAAG-3′.
Promoter regions were cloned into the SmaI/XhoI (less than − 3.0 kb) or HindIII (greater than − 3.0 kb) sites of pGL3 Luciferase reporter vector (Promega, Madison, WI). Promoter constructs were confirmed by Sanger sequencing. Promoter constructs were transfected into 1.8–2 × 104 cells human melanoma cells in equal molar concentrations using Lipofectamine LTX with PLUS Reagent (ThermoFisher, Waltham, MA). pGL4.74 (hRluc/TK, Promega, Madison, WI) was used for transfection normalization. Firefly and Renilla luciferase expression was assessed 48 h after transfection using the Dual-Glo Luciferase Assay system (Promega, Madison, WI). Cells were treated with 1000 IU/mL TNFα and 100 IU/mL IFNγ (PeproTech US, Rocky Hill, NJ) 24 h prior to assessing luciferase activity. All values were normalized to the pGL3 empty vector control.
Myeloid cell polarization
PBMCs were collected using a ficoll gradient (Ficoll-Paque Plus, GE Healthcare) and monocytes were isolated by a CD14 positive selection (Miltenyi, Bergisch Gladbach, Germany). Monocytes were split into five experimental groups: (1) Negative Control (no cytokines), (2) GM–CSF (Sanofi) + IL-4 (Cell Genix) at 1000 U/ml, 3) Recombinant IL32α (R&D Systems) at 100 ng/ml, 4) Recombinant IL32β (R&D Systems) at 100 ng/ml, 5) Recombinant IL32γ (R&D Systems) at 100 ng/ml, and cultured using Cell Genix Media to yield immature DCs at day 5. Immature DC were harvested and surface stained for flow cytometry analysis. Cell surface markers were observed on the double positive, HLA-DR and CD86 population of cells. Antibodies used included CD80 FITC (BD, Clone L307.4), Mouse IgG1 FITC (Beckman Coulter PN IM0639U), CD86 Pe-Cy7 (BD, Clone FUN-1), HLA-DR PerCpCy5.5 (BD, Clone G46-4), CD1B APC (BioLegend, Clone SN13), CD14 APC-Cy 7 (BD, Clone MφP9), CD68 BV 711 (BD, Clone Y1/82A), Mouse IgG2B BV 711 (BD, Clone 27-35), and Zombie Aqua Viability Dye BV 510 (BioLegend).
Statistical analyses
Gene expression bar graphs are shown as mean and standard differentiation. Gene expression was normalized to GAPDH and expressed as Delta Ct values. Comparison between the reference control (untreated M397, M398, and M249) was performed with one-way analysis of variance (ANOVA, 95% CI) with a Dunnetts’ multiple comparisons test. All calculations were done using Prism software. p-values less than 0.05 were considered statistically significant. For luciferase activity, data was normalized to Renilla luciferase expression and fold changes were calculated against the pGL3 empty control vector. Error bars represent standard deviation.
Discussion
Human melanomas produce a number of cytokines generally associated with the immune system [
38]. In an analysis of the RNA transcripts from 53 established melanoma lines, a significant proportion express IL32 isoforms. Thus, we evaluated the biology underlying IL32 expression in the context of melanoma both in cell lines and in melanoma tumor samples.
IL32 is found on human chromosome 16p13.3, is expressed in at least 6 splice variants (α, β, γ, δ, ε, ζ) and was originally isolated from activated natural killer and T cells [
16]. Which isoforms are secreted as opposed to acting intracellularly is still unresolved and hampered by an as of yet unidentified cell surface receptor [
39,
40]. The γ isoform is the only isoform that has a leader sequence, is composed of all exons, is believed to be secreted, and believed to be the most biologically active [
41]. A rodent IL32 homologue does not exist-raising the obvious question as to whether or not this gene product is dispensable or redundant-but human recombinant and transgenic IL32 appears to be biologically active in mice [
12,
42‐
44]. Despite these gaps in our knowledge, much of the published literature on the biology of IL32 comes from experiments utilizing commercially available recombinant protein, generally the α, β, and γ isoforms [
1].
Several themes emerge regarding IL32 biology: (a) IL32 can modulate the production of various cytokines including IL1β [
45], TNFα [
46], IL6 [
47], IL8 [
8], and may activate cells of the immune system [
15,
19]; (b) IL32 expression can be induced by various viruses and microbes [
2,
20,
42,
48‐
51] and may play a role in antiviral immunity [
52]; (c) IL32 may have direct effects on various cancer cells in vitro; [
8,
9,
22,
47,
53,
54]; (d) IL32 may play a role in tumor immunity [
4,
7,
12,
14,
44]; [
41]; and (e) IL32 has been associated with various inflammatory diseases [
43,
45], such as ulcerative colitis and rheumatoid arthritis [
55]. It is also curious that, largely from immunohistochemistry studies, IL32 is expressed by a broad range of epithelial and in hematopoietic malignancies and is generally correlated with aggressive biology, although no unifying mechanism has been identified.
In comparing IL32 expressing and non-expressing melanoma cell lines, we observed that IL32 expression in melanoma cell lines correlates with a high AXL/low MITF ratio, a genetic signature which has been reported to be associated with a treatment-resistant, dedifferentiation “invasive” phenotype. This dedifferentiation phenotype results in reduced expression of melanocytic-lineage antigens, which may also provide a mechanism for immune evasion [
32]. Melanomas, which progress on MAPK inhibitor therapies, acquire this genetic signature of reduced MITF and RTK upregulation [
25]. MITF controls the expression of a broad range of genes in melanocyte-lineage cells that govern differentiation, migration, and proliferation [
56‐
58]. The low MITF signature is regulated by receptor tyrosine kinases, including AXL [
59].
The plasticity of these neural crest-derived cutaneous malignancies is underscored by the ability of an inflammatory signal—TNFα or IFNγ, for example—to effect a differentiated to dedifferentiated conversion in melanoma cell lines with only a 72 h in vitro exposure, a phenomenon that reverses when the cytokine is removed from culture [
26]. We found that TNFα and IFNγ, which promote a dedifferentiated melanoma phenotype, also induce IL32 expression in non-IL32 expressing melanoma cell lines by impacting activity at the promoter level.
Palstra et al. recently reported that the DNA element encompassing rs4349147 is a strong long distance enhancer essential for the expression of IL32 in CD4 T cells located 10 kb 3′ of the IL32 promoter [
52]. Jurkat cells are heterozygous for the rs4349147-A and rs4349147-G alleles. The A allele increases the expression of IL32α, generally viewed as anti-inflammatory, whereas the G allele promoted the expression of the proinflammatory IL32γ and IL32β isoforms. These proinflammatory isoforms enhanced lymphocyte activation and susceptibility to HIV infection. This report provides considerable insight into various observations on the biology of IL32 isoforms. Two putative IL32 promoters have been identified using the Eukaryotic Promoter Database (
http://epd.vital-it.ch), which are depicted in Fig.
4. These two promoters, at least in Jurkat cells, designated EPD NK4_1 and EPD IL32_1, are thought to support the transcription of IL32γ and IL32α/β, respectively.
Our IL32 promoter constructs encompass both the identified EPD NK4_1 and EPD IL32_1 putative promoter regions and extend 10.5 kb 5′ to the ATG. The luciferase activity driven by these constructs would then necessarily reflect constitutive and induced gene expression of all three isoforms, as well as the putative
cis-acting elements residing within this 5′ upstream region. Palstra et al. performed formaldehyde-assisted isolation of regulatory elements (FAIRE) assays in Jurkat and a melanoma cell line (G361), which demonstrated significant increase in DNA accessibility in the region surrounding rs4349147 in the former but not the latter cell line [
52]. We therefore conjecture that this long-range enhancer may not play a role in melanoma IL32 transcription. In another IL32 promoter study using Akt-activated endothelial cell line constructs extending 2.5 kb 5′ to the ATG [
60]; the difference in cell lines and activation signals precludes any meaningful comparison.
Given the impact of proinflammatory cytokines on IL32 expression, we investigated the IL32 expression in immunologically rich melanoma tumors using RNA sequencing data available in the TCGA dataset. IL32 expression was highly correlated with a T cell dominant immune signature in these tumors, probably a result of immune cell IL32 production. We also observed a correlation between IL32 expression, derived from tumor cells and the surrounding microenvironment, and a high AXL/low MITF gene signature, as in the melanoma cell lines. However, we interpret this result cautiously because, unlike in the melanoma cell line dataset, IL32 expression in the melanoma tumor microenvironment did not inversely correlate with pigmentation or melanoma differentiation genes such as MLANA, TYR or PMEL. Thus, the high AXL/low MITF signature may be a result of increased IL32 expression by infiltrating immune cells, rather than IL32 expressing dedifferentiated melanoma cells in the tumor microenvironment.
There is substantial evidence that IL32 induces dedifferentiation of human monocytes towards a macrophage-like phonotype with dendritic cell-like aspects. IL32 exposed monocytes altered their morphology within 3 days (flattening with extensive pseudopodia), which was accompanied by increased expression of CD1 and CD14 [
19]. These macrophage-like cells exhibited active phagocytic properties and were also induced to express TNFα, IL-1β and IL6. Our preliminary in vitro studies indicated that IL32, in an IL32β and IL32γ isoform specific manner, can modulate the polarization of myeloid cells toward an M1-like macrophage phenotype with costimulatory molecule expression, lending additional support for its impact in the tumor microenvironment. It is plausible, then, that IL32 is expanded within the microenvironment as a byproduct of the feedback loop between the anti-tumor inflammatory response (marked by TNFα and IFNγ) and dedifferentiation of melanoma [
23].
Authors’ contributions
HP performed, analyzed, and interpreted the human cell line and IL32 promoter data. JT analyzed and interpreted the sequencing and TCGA data. AK analyzed and interpreted the TCGA data. CSG analyzed the sequencing and TCGA data. DMM performed, analyzed and interpreted the myeloid cell data. WHM, DS, LHB, AR, TGG, and JSE all contributed to the design of experiments and the interpretation of results. HP, JT, AK, DMM and JSE were significantly involved in writing of this manuscript. All authors read and approved the final manuscript.