MX 2 is a novel regulator of cell cycle in melanoma cells

MX2 protein is a dynamin‐like GTPase2 that has recently been identified as an interferon‐induced restriction factor of HIV‐1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome‐wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context‐dependent.


| INTRODUC TI ON
Cutaneous melanoma makes up approximately 4% of skin cancers, yet it is responsible for more than 70% of skin cancer-related deaths (Sample & He, 2018). Somatic melanoma genetics are complex with tumors exhibiting high mutational load mostly attributed to UVinduced DNA damage (Hodis et al., 2012). New germline genetic variants and genes contributing to melanoma susceptibility and progression are continually being discovered (Amos et al., 2011;Barrett et al., 2011;Bishop et al., 2009). Recently, genome-wide association studies (GWAS) have linked rs45430 SNP, a major T to minor C allele change, intronic to MX2 (myxovirus resistance 2) gene with reduced risk to cutaneous melanoma, and multiple primary tumors (Barrett et al., 2011;Gibbs et al., 2015). However, the functional role of this SNP or MX2 gene itself in the tumorigenesis has so far not been elucidated. MX2 protein is a dynamin-like GTPase2 identified as an interferon (IFN)-induced restriction factor for several primate lentiviruses including HIV-1 (Buffone, Schulte, Opp, & Diaz-Griffero, 2015; Goujon et al., 2013). Humans possess two MX genes, MX1 and MX2, with a high level of homology (Haller, Staeheli, Schwemmle, & Kochs, 2015). While MX1 protein is mainly induced after type I IFN (IFNα/β) stimulation during the antiviral response (Haller & Kochs, 2010;Kim, Shenoy, Kumar, Bradfield, & MacMicking, 2012), MX2 can be expressed at significant levels even in the absence of IFN (King, Raposo, & Lemmon, 2004). Unlike MX1, MX2 has an extended N-terminal domain and exists as two isoforms. While the longer 78 kDa isoform displays antiviral activity and is associated with the nuclear envelope, the shorter 76 kDa isoform is cytoplasmic without clearly defined cellular activity to date (Haller et al., 2015). MX2 found in association with nuclear pores contributes to the regulation of viral DNA nuclear import and/or integration into the host cell genome (Kane et al., 2013). One previous study suggested that MX2 could have additional, viral-independent cellular functions including regulation of cell cycle progression (King et al., 2004).
Here, we show for the first time, to best of our knowledge, that MX2 is functionally involved in cancer-related processes in melanoma. It is differentially expressed in melanoma tumors and cell lines, and it is a predictor of better patient survival. Interestingly, our data further show that MX2 function is complex, with both tumor-suppressive and oncogenic features depending on the cellular context.

| Cell lines and culture conditions
Primary human melanocytes (NHM9, NHM134, and NHM160) were isolated and cultured as previously described (Magnussen et al., 2012).

| siRNA knockdown
Described in the Supporting Information Data S1.

| Double thymidine block
Cells were synchronized at G1/S using a double thymidine block.
At approximately 30% confluency, MM382 cells were subjected to culturing media supplemented with 2 mM thymidine for 16 hr (first block). Afterward, thymidine was washed off twice with PBS and cells were allowed to grow for 8 hr in normal conditions. Thymidine at final concentration of 2 mM was added for additional 15 hr before final release. Cells were collected at 0-, 2-, 4-, 6-, 8-, 10-, and 12-hr time points after release.

| Cell viability
Two x 10 5 cells per well were seeded into 6-well plates 24 hr before treatment with siRNA. Cells were trypsinized and collected, and the total number was counted after 72 hr of treatment with siRNA. Viability values are presented as a mean percentage ± SE of three independent experiments normalized to the negative control siRNA.

| RNA sequencing and analysis
The RNA-seq files (fastq) prior to analysis were treated with Trimmomatic-0.38 (Bolger, Lohse, & Usadel, 2014) to remove sequence adapters. After trimming, the reads were (quasi)-mapped directly to the transcriptome using human (GRCh38, Ensemble version 94), Salmon software (Patro, Duggal, Love, Irizarry, & Kingsford, 2017). The DESeqDataSet was constructed by importing transcript abundance estimates from Salmon using the R txtimport package (Soneson et al., 2015), differentially expressed genes detected by R DESeq2 package (Love, Huber, & Anders, 2014). For the selection of differently expressed genes, a significance threshold based on adjusted p-value <.01 was applied. To further strengthen the selection, significantly (p < .01) expressed genes from three groups, combined (knockout 1 and knockout 2) and individually, were compared. From these, a core of 520 genes was selected based on overlapping expression between the groups.

| Data
Sequence data are stored at Services for Sensitive Data (TSD)-University of Oslo. Access can be arranged by contacting the corresponding author (Ana S.) upon request. Graphical presenstations: Heatmaps were constructed using aheapmap function in R package

Significance
The study provides the first evidence that antiviral MX2 gene is associated with the tumorigenesis process in melanoma. It has an IFN independent role in the regulation of cell cycle and the PI3K/AKT pathway. However, MX2 function is clearly cell type-and context-dependent. Our findings are adding a functional explanation to previous genome-wide association studies that reported an association between MX2 gene and reduced risk for melanoma.

| Incucyte growth rate assessment
Cells overexpressing MX2 and GFP as a control were seeded into 24-well plate at a density of 25,000 cells per well. Cell proliferation was measured by a confluence assay using IncuCyte TM FLR (Essen Instruments) live-cell imaging system. Phase-contrast images were generated every 3 hr over a period of 3 days (for melanoma cells) or 4 days (for melanocytes). Cell proliferation was determined by analyzing cell confluence over time. The experiment was repeated three times in triplicate. Confluence values were normalized to an initial time point; data are presented as a mean value at a given time point ± SE.

| Flow cytometric analysis
For cell cycle analysis, 2 × 10 5 cells per well were seeded into 6-well plates 24 hr before treatment with siRNA. Forty-eight hours after transfection, cells were harvested by trypsinization, washed twice in ice-cold PBS, and fixed resuspending cell pellets in 1 ml 70% icecold methanol. Fixated cells were stained with a ready-to-use DNA Labelling Solution (Cytognos, cat. no. CYT-PIR-25). Flow cytometric experiments were performed on BD FACSCalibur TM Flow cytometer (BD Biosciences). Data were analyzed with FlowJo v.7.6.1 software (Treestar Inc. Ashland).

| Quantitative real-time PCR
Described in the Supporting Information Data S1. cycles of PCR at 95ºC for 15 s, and 60ºC for 1 min. Genotypes of the samples were determined from the allelic discrimination and amplification plots.

| Generation of MX2 and GFP expression constructs
MX2 cDNA was purchased from OriGene, catalog no. SC127459.
Entry vector encoding GFP-pENTRY-GFP-was a gift from William Hahn (Addgene plasmid #15301). A destination vector pLenti-CMV-Puro-DEST (w118-1) was a gift from Eric Campeau and Paul Kaufman; Addgene plasmid #17452. pCW57.1 construct was a gift from David Root; Addgene plasmid #41393. Detailed procedures for plasmid construction are described in the Supporting Information Data S1.

| Lentivirus production and generation of stable cell lines
Described in the Supporting Information Data S1.

| In vivo animal studies
WM983b cells (2 × 10 6 ) stably expressing MX2 or GFP diluted in 200 µl serum-free RPMI-1640 media were subcutaneously injected in the right flank of nude mice (athymic nude foxn1 nu). Tumor sizes were measured once a week using a caliper, and the volume V was calculated as follows: V = W 2 × L × 0.5 (where W and L are tumor width and length, respectively). The experimental protocol was evaluated and approved by the National Animal Research Authority and conducted in accordance with regulations of the European Laboratory Animals Science Association.

| Clinical melanoma specimens for IHC
Formalin-fixed, paraffin-embedded tissue from 42 benign nevi, 154 primary melanomas, and 60 metastases was examined for expression of MX2 protein. Clinical follow-up was available for all patients, 72 male and 82 female, with the mean age of 55.6 (range 19-97). The follow-up period ranged from 1 to 361 months (mean = 104.8 months, median = 126.5 months). The Regional Committee for Medical Research Ethics South of Norway (S-06151) and The Social and Health Directorate (06/2733) approved the current study protocol.
By multiplying intensity score with percentage positive cell score, a total immunoreactivity score was calculated ranging from 0 to 12. Immunoscore >3 was considered as high in the statistical analyses.

| Mitotic rate classification
Mitotic rate was histologically assessed by count of mitoses per mm 2 , also described in Poźniak et al., (2019).

| Statistical analysis
Statistical analysis was performed applying SPSS package version 18, (SPSS Inc.) and Stata 14.2. Comparison between variables was performed using the chi-square test or Fisher exact test. Two-tailed paired Student's t test and Wilcoxon matched-pairs signed-rank test was used for the evaluation of in vitro results. A p value of less than .05 was considered statistically significant. In the Leeds Melanoma Cohort (LMC) (Nsengimana et al., 2018), the relationship between

| Transcriptomic data
Generation of gene expression data from 703 FFPE tumors of the LMC was as described elsewhere (Nsengimana et al., 2018). These data were deposited in the European Genome-phenome Archive

| MX2 is constitutively and differentially expressed in melanoma tumors and cell lines
To investigate the potential role of MX2 in melanoma, we first examined its RNA and protein expression in a panel of human melanocytes, established primary and metastatic melanoma cell lines. Immunoblot analysis revealed constitutive, yet differential MX2 protein expression that correlated with RNA levels (Figure 1a,b). Most metastatic lines expressed lower levels of MX2 compared to the primary melanoma and cultured melanocyte lines. Furthermore, an apparent reduction of MX2 protein level was seen in metastatic WM239 line compared to primary WM115 line, both derived from the same patient, suggesting that MX2 is downregulated during disease progression.
Interestingly, the highest MX2 protein expression was seen in the recently established early passage metastatic MM382 line. To rule out that this could be an in vitro culturing artifact, we also examined MX2 expression in the original tumor sample that was histologically dissected and evaluated to contain more than 80% of tumor cells, and found it to be comparable to the cell line ( Figure 1c (Table S2).
We also investigated whether rs45430 SNP is associated with MX2 expression in both cell lines and metastatic melanoma samples ( Figure 1a,e). While we observed a tendency for TT genotype to be associated with a higher MX2 expression, it was not statistically significant.
In other cell types, MX2 expression is shown to be induced by IFN signaling. To examine whether this is valid in melanoma, we in-  Antiviral functions of MX2 have been associated with its localization to the nuclear envelope; however, cytoplasmic localization has also been reported (Dicks et al., 2018;Melén et al., 1996).
Cytoplasmic and nuclear fractionation of melanoma cell lines showed that MX2 protein is mainly found in the nuclear fraction, but a weak cytoplasmic localization was also detected (Figure 1g).

| MX2 expression is associated with longer melanoma-specific survival
Using previously described whole transcriptome data derived from 703 primary melanomas from the Leeds Melanoma Cohort (LMC) (Nsengimana et al., 2018), we investigated the expression and association of MX2 mRNA level with melanoma-specific survival (MSS).
A Kaplan-Meier curve was generated after dichotomization of MX2 expression to high and low groups with respect to the median showing that higher MX2 expression was associated with longer mela-  TT CT CT CT  CT TT  CC  TT CC TT TT TT TT CT   BRAF V600E  rs45430  CT  TT  We also tested whether rs45430 SNP is associated with MX2 expression in the primary melanomas. The SNP data were generated as previously described (Law et al., 2015). The expression of MX2 was significantly lower in participants homozygous for the C allele in comparison with the CT or TT genotype (Figure 2d).
Protein expression of MX2 was also analyzed by immunohistochemistry in a second melanoma dataset consisting of 42 paraffin-embedded nevi, 154 primary melanomas, and 60 metastatic melanomas. As shown in Figure 2e, cytoplasmic and/or nuclear expression was observed. Note that a variable MX2 staining was also observed in infiltrating immune cells.

| MX2 function in melanoma is cell linedependent
Since we observed that a subset of melanoma cell lines displays high constitutive MX2 expression, it is possible that these cells have adapted to circumvent its growth inhibitory effects or that MX2 has a different functional role in these cells. To investigate these possibilities, we downregulated MX2 using two different MX2 targeting siRNA oligos. A clear reduction in MX2 mRNA levels was seen after siRNA transfection without effecting MX1 ( Figure S4a,b).
Interestingly, 72 hr post-transfection, a significant viability decrease was seen in high MX2 expressing WM115 and MM382 cells, while subtle or no effects were seen in low MX2 expressing WM1366 cells ( Figure 4a). To identify whether the observed decrease in viability was due to decreased proliferation or apoptosis, we examined the expression of mitosis marker phospho-Histone H3 (pHH3) and the suggesting cell cycle-related effect in these lines. The activation of the AKT and MAPK signaling pathways was also examined, but surprisingly no significant changes were observed ( Figure S5).

Effects of MX2 downregulation on the cell cycle distribution
were assessed by flow cytometry 48 hr post-siRNA transfections.
The analysis revealed that MX2 knockdown increased the proportion of cells in G1 phase, including weak effect in WM1366 suggesting induction of the cell cycle arrest (Figure 4c). As expected, G1 arrest was accompanied with decreased levels of cyclin D1 and kinase Cdk2 expression and increased levels of Cdk inhibitory proteins p27Kip1 and p21Cip1 (Figure 4d). Again, observed effects were more prominent in WM115 and MM382 cell lines than in WM1366.
Due to these notable effects on the cell cycle, we investigated whether MX2 expression itself could be oscillating during the cycle.
Synchronization of the cells at G1/S boundary by double thymidine block showed expected oscillation of the cyclins and mitotic phospho-Histone H3 after release. MX2 levels, however, did not change during the cell cycle progression suggesting that its expression is cell cycle phase-independent (Figure 4f).
To further investigate which cellular processes are influenced by MX2 downregulation, we also performed RNA-seq of MM382 cells 48 hr after siRNA transfection. A core of 520 differentially expressed genes (Table S3) overlapping between two siRNA oligos was selected for further GO enrichment analysis (Figure 5a), while fifty most upand downregulated genes are presented in Figure 5b. The analysis showed that highly over-represented GO terms included processes

| D ISCUSS I ON
In recent years, several GWAS have identified novel melanoma susceptibility SNPs, including in the intron of MX2 gene that have no previously defined functional roles in cancer-related processes.
Thus, the overall objective of our study was to investigate whether and how MX2 function can influence melanoma tumorigenesis. So far, MX2 has been mainly defined by its antiviral functions, highlighting its induction by type I IFN and ability to interfere with the replication of different types of negative-stranded RNA viruses.
Our expression data from melanocytes, and primary and metastatic melanoma show that MX2 can be constitutively expressed independently of IFN induction, which is in agreement with two previous studies in HeLa and T98G cells (King et al., 2004;Melén et al., 1996).
While we detected MX2 expression in all melanocyte and primary melanoma cell lines, 8 out of 10 metastatic cell lines showed lower or lack of expression. There was also an apparent reduction of expression in a metastatic versus primary cell line derived from the same patient suggesting that MX2 is downregulated during disease progression. Furthermore, an increasing percentage of MX2 IHC negative samples was observed in metastatic lesions.
The exact mechanism of this downregulation needs further elucidation, but inactivation of the IFN pathway and suppression of its target genes during disease development has been reported in melanoma as well as in other cancers (Katlinskaya et al., 2016;Katlinski et al., 2017;Walter et al., 2017). Interestingly, a study of breast cancer by Han et al. (Han, Russo, Kohwi, & Kohwi-Shigematsu, 2008) found that transcription factor and chromatin organizer SATB1 reprograms gene expression profile of cancer cells to promote tumor growth and that MX2 is among the repressed genes.
Here, we also showed that reintroduction of MX2 expression in endogenously low expressing cell lines leads to downregulation of AKT activity and inhibition of tumor growth in vitro and in vivo.
These effects were profound in a metastatic WM983b suggesting that downregulation of MX2 is important during disease progression. Since it is demonstrated that type I and II IFNs used in melanoma treatment due to their antiproliferative effects can regulate AKT activity in a complex manner (Kaur, Sassano, Dolniak, et al., 2008;Kaur, Sassano, Joseph, et al., 2008), one can speculate if some of these effects can partially be mediated by MX2. Inheritance of the minor C allele rs45430 SNP in the intron of MX2 was reported to be protective for melanoma and multiple primaries in the GWAS (Barrett et al., 2011;Gibbs et al., 2015). Here, we found that the homozygous C allele is associated with lower expression of MX2 in primary melanoma tumors, and a similar trend was seen in metastatic samples. Since we also report that higher expression levels of MX2 are seen in thinner primaries with a lower mitotic rate and better survival, these data seem somewhat difficult to explain. However, it is known that expression quantitative trait loci (eQTL) can display opposite directional effects in a tissue-specific manner (Mizuno & Okada, 2019). Indeed, minor C allele is associated with lower MX2 expression in whole blood, while the opposite is seen for sun-exposed skin ( Figure S6) (TheGTExConsortium, 2015).
Currently, it is unclear what functional role MX2 plays in different immune cell types or how this relates to melanoma risk; therefore, further studies are warranted.
Interestingly, we have observed similar discrepancies previously for an inherited SNP in the PARP1 gene. The SNP was associated with higher PARP1 levels, increased risk of melanoma, and related to PARP1 induced cell proliferation mediated through MITF (Choi et al., 2017). Yet, the same SNP was found to be associated with a lower risk of death from melanoma (Davies et al., 2014).
Remarkably, despite its growth inhibitory effects and downregulation in metastatic cell lines, a subset of melanoma lines in our panel exhibited high endogenous MX2 expression. Knockdown of MX2 in these lines decreased proliferation and lead to perturbation of the cell cycle, which is inconsistent with observations from our overexpression experiments. However, MX2 belongs to dynamin-like GTPase family proteins, which are also known to be involved in the regulation of cell cycle progression and it is likely that MX2 function is complex and cell type-and context-dependent. In support of these observations, one previous study has reported that depletion of endogenous MX2 in cancer cells results in delayed progression through G1/S phase of the cell cycle (King et al., 2004). We have observed similar G1 arrest accompanied with cyclin D1 degradation and cyclin E upregulation in p21-dependent manner as reported (Sandor et al., 2000). In addition, our RNA-seq analysis revealed that MX2 is also involved in DNA replication and mitosis processes partially by regulation of Aurora A and PKL1. A study by Kane et al. (2013) investigating MX2 potency to inhibit HIV-1 showed that arresting the cell cycle in osteosarcoma and myelogenous leukemia cells increases MX2 viral inhibitory activity. We can speculate that antiviral MX2 potency in non-dividing cells increases when it does not engage in other cellular processes, including DNA replication and/or mitosis as suggested by our study. These results further support the hypothesis of a cellular type-and setting-dependent MX2 function.
In summary, we have demonstrated that widely accepted antiviral MX2 gene has tumor-suppressive features in melanoma, where it regulates the growth of tumor cells partially through negative modification of AKT activity, and it is downregulated during disease progression. However, its role seems to be complex and cell contextdependent since we found that in a subset of melanoma cell lines, it is highly expressed and necessary for cell cycle progression. Further elucidation of this dual mechanism of action is needed to understand its complex roles in tumorigenesis.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interests to disclose.