LADON, a Natural Antisense Transcript of NODAL, Promotes Tumour Progression and Metastasis in Melanoma

The TGFβ family member NODAL, repeatedly required during embryonic development, has also been associated with tumour progression. Our aim was to clarify the controversy surrounding its involvement in melanoma tumour progression. We found that the deletion of the NODAL exon 2 in a metastatic melanoma cell line impairs its ability to form tumours and colonize distant tissues. However, we show that this phenotype does not result from the absence of NODAL, but from a defect in the expression of a natural antisense transcript of NODAL, here called LADON. We show that LADON expression is specifically activated in metastatic melanoma cell lines, that its transcript is packaged in exosomes secreted by melanoma cells, and that, via its differential impact on the expression of oncogenes and tumour suppressors, it promotes the mesenchymal to amoeboid transition that is critical for melanoma cell invasiveness. LADON is, therefore, a new player in the regulatory network governing tumour progression in melanoma and possibly in other types of cancer.


Introduction
Metastasis is responsible for up to 90% of cancer deaths [1], making the determination of the molecules involved in this process crucial to the improvement of diagnosis and treatment.The NODAL gene, which encodes a TGFβ family member, was identified as a possible player in the acquisition of metastatic behaviour [2,3].The NODAL ligand binds a receptor complex containing type I (ALK4 or 7) and type II (ActRIIA or B) serine/threonine kinase receptors, the activation of which leads to the phosphorylation of the signal transducers SMAD2 or SMAD3.NODAL is best known for its role during development, where it is required both to maintain the undifferentiated state of embryonic precursors and to specify the identity of specific cell types, including several motile cell types [4].It is also expressed in the adult, notably in tissues that undergo periodic renewal or remodelling under the control of hormonal stimuli, such as the endometrium and the mammary gland [5,6].The NODAL expression in tumour cells has been correlated with their plasticity and invasive behaviour [2,7], in line with its functions in embryonic and adult tissues.
The involvement of NODAL in tumour progression and metastasis was first described in melanoma cell lines [3], but although evidence supporting such a role for this factor has been obtained in other cancer types, its actual contribution to metastasis in melanoma has been disputed [8,9], as its expression was not always detected there.A review of available NODAL expression data in cancer cells suggested the existence of distinct splice variants [10], all including the second exon of the gene but sometimes, notably in melanoma cell lines, lacking its third (and last) exon and therefore lacking the capacity to produce a functional ligand.A previous study had already shown that the expression of CRIPTO, an obligatory co-receptor of NODAL, was weak in metastatic melanoma cell lines, and restricted to a small cell subpopulation [11].These observations raised the possibility in this context of a different mode of action of NODAL in conferring metastatic properties to cells, independent of its known signalling function.
We therefore used genetic and pharmacological approaches, both in vivo and in vitro, to reassess the importance of NODAL in mediating the invasive properties of melanoma cell lines.Our results argue that the NODAL protein plays no role, but that a natural antisense transcript, which we have named LADON, and which overlaps with the NODAL exon 2, promotes tumour progression and invasion.Our study identifies LADON as a novel regulator of melanoma progression.

NODAL Exon 2 Promotes Tumour Growth and Metastasis in A375 Cells
We used RT-PCR analysis to characterize the NODAL expression in a panel of human cell lines relevant to cancer and melanoma: melanocytes, non-metastatic melanoma (MNT1), metastatic melanoma (A375M-noted A375 in the rest of the study, FO1, 888 Mel, SLM8), breast cancer (MCF7), and embryonic kidney (HEK293).A primer pair specific for the NODAL exon 2 amplified the expected band in all of them (Figures 1A and S1A), whereas primers spanning the junction of exons 2 and 3 detected a transcript only in the embryonic kidney cell line (Figures 1A and S1B).The primers spanning the junction of exons 1 and 2 also failed to detect a band in all cell lines, except in the HEK293 line (Figure S1B).These results are consistent with studies that have detected the expression of the NODAL exon 2, but not the NODAL exon 3, in melanoma cell lines [8,10], and further suggest that the NODAL exon 1 is not expressed either.
To test for the possible contribution of the NODAL exon 2 to the properties of melanoma cells, we used genome editing to delete it in all copies of the gene present in A375 cells (Figure 1A).This exon encodes more than half of the mature signalling protein and its deletion prevents the production of a functional ligand [12].Five of the independent mutant clones thus obtained, designated A375∆E2a to e, were characterized (Figure S1C,D).The absence of the NODAL exon 2 was found to impair their ability to close the gap in a 2D wound-healing (scratch) assay (Figures 1B and S1E).This effect was detected in less than 12 h of culture, which suggests that it results from a decrease in cell motility, not a decrease in mitosis.Measurements allowed us to plot the respective gap closure rates of mutant and unmodified A375 cells (Figure 1C).Exposure to the exogenously supplied recombinant NODAL or ACTIVIN, another TGFβ-related ligand that signals via the same receptors as NODAL, did not rescue the motility of the A375∆E2 cells in the same assay, and did not affect that of unmodified A375 cells either (Figures 1C and S1F).Treatment with the SB431542, a pharmacological inhibitor of the ACTIVIN/NODAL type I receptors ALK4, 5 and 7, also had no effect (Figure 1C), confirming that ACTIVIN/NODAL signalling is not involved in whatever is determining gap closure rates.
To assess the impact of the mutation on tumour progression and metastasis in vivo, chorioallantoic membrane (CAM) assays [13] were performed in fertilized chicken eggs using unmodified A375 cells and A375∆E2d mutant cells (Figure S1G).1.10 6 cells were deposited on the upper CAM of E9 chick embryos.Tumour progression was assessed 9 days later (E18) by two criteria: (1) by measuring the weight of the tumour formed on the upper CAM; and (2) by measuring the presence of human cells in the lower CAM and the chick embryo liver.The A375∆E2d mutant cells gave rise to tumours that were only about 1/9 the size of those derived from unmodified A375 cells (Figure 1D,E), and their metastatic presence in the lower CAM and the liver was reduced to 1/10 and 1/2 of the levels seen with the unmodified cells, respectively (Figure 1F).Histological analyses showed that primary tumours derived from mutant A375∆E2 cells had a higher mitotic index but were also far more necrotic than those derived from unmodified cells (Figures 1D and S1H).These results indicate that A375 cells require the NODAL exon 2 to promote normal tumour growth and invasion.However, the lack of response of the cells to either the exogenous NODAL and ACTIVIN or to an inhibitor of their signalling pathway, as well as the absence of full-length NODAL transcripts, strongly argue that the mode of action of the gene in this context does not rely on the production of the mature ligand.

A Natural Antisense Transcript of NODAL Exon 2 Is Expressed in Melanoma Cells
An update of the Ensembl database (GRCh38.p13primary assembly) revealed the presence of a third transcript at the human NODAL locus, in addition to the two NODAL splice variants already known.This 1728nt long RNA (AC022532; ENSG00000280401) is transcribed from the plus strand (opposite to NODAL) and is annotated as non-coding (Figures 2A and S2A).The corresponding transcription unit is hereafter designated as LADON.A full-length cDNA was first obtained from a human teratocarcinoma cDNA library [14].It starts in NODAL intron 2 and ends in NODAL intron 1 and therefore includes a sequence complementary to that of the entire NODAL exon 2. Its 5 and 3 regions contain short interspersed nuclear elements (SINEs) Alu and MIR.Homologous transcripts of similar size were found in a chimpanzee and an orangutan (CK820-G0007942 and CR201-G0049649, respectively).A longer homolog has been found in pigs (ENSSSCG00000051352), but none has been reported in mice and rats, nor in any of the other classical vertebrate animal models (zebrafish, xenopus, and chick), which may suggest that it arose within the mammalian lineage.
To find out whether changes in the LADON expression correlate with tumour progression and prognosis in humans we used the GEPIA web server to analyze transcriptomic data collected by The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) projects [15].Dramatic differences in the LADON expression levels between tumours and their corresponding healthy tissues were detected in a majority (21/31) of the cancer types analyzed, with the largest differential-a 96% reduction in normal expression level-found in skin cutaneous melanoma (SKCM; Figure 2B).However, if we look at how this expression varies with the stage of the cancer, the data show that it peaks at stage II when the tumour is growing locally and preparing to metastasize (Figure 2C).Finally, SKCM patients with higher levels of LADON expression survived longer than patients with lower expression levels (panel Figure 2D).No copy number alterations were found at the NODAL locus in these patients.
sequence complementary to that of the entire NODAL exon 2. Its 5′ and 3′ regions contain short interspersed nuclear elements (SINEs) Alu and MIR.Homologous transcripts of similar size were found in a chimpanzee and an orangutan (CK820-G0007942 and CR201-G0049649, respectively).A longer homolog has been found in pigs (ENSSSCG00000051352), but none has been reported in mice and rats, nor in any of the other classical vertebrate animal models (zebrafish, xenopus, and chick), which may suggest that it arose within the mammalian lineage.performed in keratinocytes (HaCat), melanocytes, non-metastatic (MNT1, DAJU, SK28) or metastatic melanoma cells (A375, FO1, 888 Mel, and SLM8), after 24 h and 96 h of culture.The LADON expression was normalized to that of endogenous RPL13.For each cell line, the value at 24 h was then set to 1. (G) The RT-qPCR measurements (dots) of LADON expression after 24 h culture were performed on the same panel as in (F) but normalized to the endogenous RPL13 expression.The histograms display mean values ± SD from a minimum of two independent replicates, p-values were calculated using the Student's t-test, ** < 0.01.These data are consistent with the possibility that LADON plays a role in melanoma progression and metastasis, but the observation that higher expression levels delay cancer progression was at first difficult to reconcile with our in vivo results.

Metastatic Melanoma Cell Lines Share the Ability to Upregulate LADON
To confirm the presence of LADON transcripts in A375 cells, a total of A375 RNA was reverse transcribed using either a reverse (N2R) or a forward (N2F) primer concerning the orientation of its transcription (Figure 1A).The resulting cDNAs were used to PCR-amplify sequences spanning the exon 2 and the adjoining 5 or 3 regions (Figure 2A).The fact that a band of the expected size was only obtained with cDNAs transcribed using the reverse primer N2R confirmed the expression of LADON and the absence of NODAL transcripts (Figure S2B).
We then used a specific pair of primers (L1F-L1R, Figure 2A) to detect LADON transcripts, and amplified a band of the expected size in all the cell lines of our panel (Figure 2E).The RT-qPCR analysis of LADON expression in melanocyte and melanoma cell lines revealed big variations in the expression of LADON within individual metastatic melanoma cell lines but not in the melanocyte and non-metastatic cell lines.The extent of these variations was correlated with the length of time the cells had been cultured, as a 2-to 4-fold increase in the LADON expression was observed between 24 h and 96 h after seeding the culture (Figure 2F).However, the basal level of LADON expression was not predictive of the metastatic or non-metastatic nature of the cell lines in our panel (Figure 2G).This basal level was 5-to 20-fold higher in keratinocytes than in melanoma cell lines, which at least partly explains the high level of the LADON expression detected in skin tissue (Figure 2B).Interestingly, A375∆E2 clones, which expressed a truncated LADON transcript (LADON-∆E2) reduced to the 5 and 3 parts that flank exon 2 (Figures 2A and S2C), showed no sign of increased expression after 96 h in culture (Figure S2D), implying that the deletion deprived LADON of a critical regulatory input.
These results confirm that the absence of NODAL did not cause the changes in behaviour we have characterized in cells deleted for the NODAL exon 2. They identify LADON as a candidate and suggest a correlation between the propensity of metastatic cell lines to upregulate its expression and their ability to adopt more aggressive behaviour.Such a correlation would be in line with the expression data obtained in SKCM patients.

Melanoma Cells Secrete Factors That Promote LADON Expression
To investigate what is triggering the increase in LADON expression in the A375 cell line, we first tested its dependence on cell density [16].The LADON transcript levels measured after 24 h culture in cells seeded at high (80%) density were actually no different from those measured in cells seeded at low (20%) density (Figure 3A).The increase in LADON expression found after 72 h culture was therefore more likely to depend on the duration of the culture.To determine whether the LADON expression is dependent on secreted factors accumulating in the culture medium, A375 cells were grown in standard medium (SM), with or without 10% fetal calf serum (FCS), and the media were collected 3 days later when the cells had reached high density.These A375-conditioned media (CM) were then used to culture cells seeded at low density for 24 h.For A375 cells, this resulted in a 2.4-fold increase in the expression of LADON (Figure 3B), compared to its expression level when cultured in SM.This increase occurred regardless of the presence of FCS, thus eliminating nutrient depletion as a possible cause (Figure S3A).Other metastatic melanoma cell lines, FO1 and 888 MEL were also found to increase the LADON expression when cultured in their own CM (Figure S3B).In contrast, cells from non-tumorigenic nonmetastatic cell lines exposed to their own CM did not affect the LADON expression (MNT1) or even reduce it (DAJU, SK28).Interestingly, the MNT1 cells did not respond to A375-CM, but the MNT1-CM did trigger an increase in LADON expression in A375 cells (Figure 3B).Thus, while both the A375 and MNT1 cells secrete and accumulate LADON-inducing factors in their culture medium, only the A375 cells are able to respond to these factors, suggesting that this ability is specific to metastatic cell lines.Analysis of the published content of the A375 cell-derived exosomes (GSE35388) [17] revealed a relative enrichment in the LADON transcripts (Figure S3C).This raised the possibility that secreted exosomes contribute to the inductive capacity of the CM.We purified exosomes from the A375-CM and confirmed that, compared with cells, they show a 7-fold enrichment in the quantity of the LADON transcripts (Figure S3C).However, neither these purified exosomes nor the supernatant left after their separation could on their own induce an increase of the LADON expression in A375 cells similar to that obtained with A375-CM (Figure 3C).It is only after these two separate fractions were combined that such a response was obtained, suggesting that this induction requires factors from both fractions.We then found that the exon 2-depleted A375∆E2-CM had the same capacity to induce LADON expression as that obtained from the A375 cells (Figure 3D), implying that a full-length LADON is not required in the CM to increase its own expression and that the production of LADON-inducing factors is LADON-independent.Cells from the A375∆E2 clones, however, failed to increase the expression of the LADON-∆E2 transcript when exposed to the same CM (Figure 3D), confirming that the capacity of the locus to respond is dependent on its integrity.

LADON Expression Promotes Invasion
Migrating melanoma cells rely on two inter-convertible modes of migration, designated mesenchymal and amoeboid [18,19] with the faster amoeboid mode being the most efficient to drive invasion [20][21][22].We therefore investigated the effect of the A375-CM on cell morphology and invasiveness.The mesenchymal to amoeboid transition (MAT), which involves a change from an elongated cell to a smaller and rounded cell morphology, was monitored via F-actin staining (Figure 3E) [23].In the SM, no clear difference was seen between cultures of the mutant and unmodified A375 cell populations, both showing similar cell size ranges (Figure S3D).However, when grown in the CM, the average size of the A375 cells decreased, with no cells covering an area greater than 50 µm 2 , and their roundness index increased (Figures 3F and S3E).When the capacity of these cells to transmigrate through a collagen layer was quantified over 24 hours, this change in the composition of the population was associated with a 7-fold increase in the rate of trans-migration (Figures 3G and S3G).The FO1 cells grown in their own CM showed a similar decrease in mean cell size and an increase in their transmigration rate (Figures S3F and 3H).In contrast, the A375∆E2 cells, whose transmigration rate in SM was one-tenth that of the A375 cells, showed no improvement in transmigration capacity when cultured in the CM (Figures 3G and S3G).LADON is therefore endowing the A375 cells with the capacity to undergo CM-induced changes in cell behaviour.Again, to assess the contribution of the exon 2 sequences to this particular effect of the CM we analyzed the effect of the A375∆E2-CM on the A375 cells.This treatment had no significant impact on the cell size of the A375 cells (Figure 3F) and although their transmigration rate showed a slight increase, it was about a quarter of that of cells treated with the A375-CM (Figures 3G and S3G).This suggested that, unlike the increase in LADON expression, changes in cell shape and behaviour are induced by an exon 2-dependent component of the CM.The A375 cells treated with the A375∆E2-CM would therefore have to increase their expression of LADON before they could secrete this component themselves and respond to it.It follows that after just 24 h (Figure 3C,F,G) their response could be delayed, but that given sufficient time they should undergo the same changes as those treated with the A375-CM, and that the differences observed between them should disappear.This is indeed what we observed when we repeated the experiment but cultured the cells for 48 h instead of 24 h (Figure S3I).
To confirm that it is the mutation of the LADON transcript and not another unrelated consequence of the deletion that is causing the drop in invasiveness, we examined the behaviour of the A375 and FO1 cells following siRNA-mediated depletion (knockdown) of the LADON expression.Three different siRNAs located at the 5 end of the gene were tested.The RT-PCR results showed a significant decrease in LADON expression with two of them, used alone or together (Figure S3J).The knockdown (KD) of LADON severely impaired the ability of the A375 and FO1 cells to transmigrate through a collagen monolayer over 24 h, behaviour reminiscent of the A375∆E2 clones (Figure 3I).We also noticed that in both cell lines LADON KD cells systematically reached confluence earlier than controls.They indeed had higher cell counts at the end of the culture (Figure S3K), suggesting that their proliferation rate was positively affected.We confirmed that the A375∆E2 clones have higher proliferation rates than their parental cell line (Figure S3L), an observation consistent with the histological analysis of the primary tumours they gave rise to in the CAM assay (Figure S1H).Finally, to confirm that LADON promotes invasion independently of NODAL we assessed whether its forced expression in the A375∆E2 cells could rescue their transmigration rate.Transient transfection experiments of cells from two mutant clones indeed showed a marked improvement in the transmigration rates of those that were transfected with a plasmid expressing LADON (Figure 3J).
Taken together, these results demonstrate that the expression of LADON in melanoma cells conditions both their capacity to produce factors that promote changes conducive to metastasis (i.e., MAT, increase in invasiveness, decrease in proliferation), and their capacity to respond to such factors.

LADON Affects the Expression of Oncogenes and Suppressors of Tumour Progression
To identify factors acting downstream of LADON, we used mass spectrometry to compare the proteomes of the A375 and A375∆E2 cells (volcano plot, Figure 4A).We focused on proteins that were identified by at least 2 independent peptides and with a fold change superior to 1.5.A Gene Ontology (GO) term analysis of the 28 proteins thus obtained showed significant enrichment in components of filamentous actin (PDLIM4, FERMT2, and NCKAP1), stress fibers (PDLIM4, Dynactin, and FERMT2), as well as lamellipodia (PDLIM4, PLCG1, FERMT2, and NCKAP1) (Figure 4A; Table 1).All of these were upregulated in the A375∆E2 cells, suggesting a repressive role of LADON in their expression.Among the six proteins that had a fold change superior to 2 (Table 2), 5 were overexpressed in the A375∆E2 cells and turned out to be known tumour (FAM107B, ACPP) or metastasis (NDRG1) suppressors [24][25][26][27], or transcription factors known to activate these proteins (p65, also known as NF-κB regulatory subunit RELA, is an activator of ACPP) [28].Conversely, Figure 4. Model for the role of LADON in melanoma tumour progression and metastasis.In metastatic melanoma cells, the LADON transcript (in blue) promotes the transition from a proliferative cell identity to a less proliferative and more invasive cell identity.In the course of a 4-day culture, an increase of LADON expression promotes the expression of known oncogenes and represses that of tumour or metastasis suppressor, which in turn affects the expression of downstream targets, such as cytoskeleton components.The LADON expression also affects the potency of the conditioned medium produced by the cells, presumably via its impact on the exosomes (quantity, content) and the secreted factors they release.The LADON-dependent exosomes and secreted factors (in blue) promote the progression of exposed melanoma cells toward their more invasive identity.The LADON transcript is itself enriched in exosomes.

Discussion
Our investigations found no evidence that NODAL could influence the behaviour of metastatic melanoma cells but revealed instead their reliance on the expression of LA-DON, a natural antisense transcript of the NODAL locus.Our findings support a critical role for its transcript in the network of interactions that governs tumour progression and metastasis in melanoma, notably in the mechanisms underlying how cells produce and respond to pro-metastatic signals.
The absence of the NODAL transcripts in melanoma cell lines reported here is consistent with the results of other studies [8,10], as well as with a recent characterization of transcript diversity at the human NODAL locus [31].This latter report and our own study show that most exon 2-containing RNA species present in melanoma cells are in fact transcribed from the strand opposite to NODAL and correspond to LADON.This finding explains some of the inconsistencies found in previous studies of the role of NODAL in melanoma, as an exon 2-based assay was used to track the expression of the gene in several of them [10,11,31,32].The error of confusing LADON expression with NODAL expression was compounded by the use of commercial antibodies cross-reacting with non-specific proteins that were mistakenly identified as NODAL, as was later demonstrated [8].Donovan et al. showed that while there is evidence that TGFβ and ACTIVIN-A, both signalling via SMAD2,3, do promote tumour progression in melanoma in various ways, no such case can be made for NODAL.Model for the role of LADON in melanoma tumour progression and metastasis.In metastatic melanoma cells, the LADON transcript (in blue) promotes the transition from a proliferative cell identity to a less proliferative and more invasive cell identity.In the course of a 4-day culture, an increase of LADON expression promotes the expression of known oncogenes and represses that of tumour or metastasis suppressor, which in turn affects the expression of downstream targets, such as cytoskeleton components.The LADON expression also affects the potency of the conditioned medium produced by the cells, presumably via its impact on the exosomes (quantity, content) and the secreted factors they release.The LADON-dependent exosomes and secreted factors (in blue) promote the progression of exposed melanoma cells toward their more invasive identity.The LADON transcript is itself enriched in exosomes.Together, these results suggest that LADON promotes the MAT and cell motility in the metastatic melanoma cell line A375 at least partially via its impact on the expression of known promoters and suppressors of tumour progression.

Discussion
Our investigations found no evidence that NODAL could influence the behaviour of metastatic melanoma cells but revealed instead their reliance on the expression of LADON, a natural antisense transcript of the NODAL locus.Our findings support a critical role for its transcript in the network of interactions that governs tumour progression and metastasis in melanoma, notably in the mechanisms underlying how cells produce and respond to pro-metastatic signals.
The absence of the NODAL transcripts in melanoma cell lines reported here is consistent with the results of other studies [8,10], as well as with a recent characterization of transcript diversity at the human NODAL locus [31].This latter report and our own study show that most exon 2-containing RNA species present in melanoma cells are in fact transcribed from the strand opposite to NODAL and correspond to LADON.This finding explains some of the inconsistencies found in previous studies of the role of NODAL in melanoma, as an exon 2-based assay was used to track the expression of the gene in several of them [10,11,31,32].The error of confusing LADON expression with NODAL expression was compounded by the use of commercial antibodies cross-reacting with non-specific proteins that were mistakenly identified as NODAL, as was later demonstrated [8].Donovan et al. showed that while there is evidence that TGFβ and ACTIVIN-A, both signalling via SMAD2,3, do promote tumour progression in melanoma in various ways, no such case can be made for NODAL.
The absence of NODAL expression in melanoma cells means that the changes in behaviour we characterized in the A375 cells deleted for NODAL exon 2, both in vivo and in vitro, resulting from the impact of the deletion on the expression of genes other than NODAL.The only disturbance we could identify in the expression of the different transcription units in the vicinity of exon 2 is that of LADON, which expresses a truncated transcript and can no longer respond to inductive treatment.The knockdown of LADON in the A375 and FO1 cells results in a loss of invasiveness and an increase in cell proliferation similar to those obtained by the deletion of exon 2. Finally, the forced expression of LADON in exon 2-deleted cells reversed the impact of the deletion on their transmigration rate, demonstrating that the transition to a more invasive behaviour is dependent on the LADON transcript itself, acting independently of NODAL.These results, in line with its impact on the proteome of melanoma cells, show that LADON is involved in the transition from a proliferative cell identity to a less proliferative but more invasive cell identity, a change of phenotype inherent in the metastatic process in melanoma [33][34][35].
An interesting aspect of LADON's involvement in melanoma is that it seems to require tight regulation of its expression.Our own results show that melanoma cells are at first expressing much lower levels of the transcript than healthy skin cells, but that this expression reaches a peak at stage II before going back to low levels.This observation finds some echo in the fact that the capacity to increase LADON expression correlates with a metastatic identity in our panel of melanoma cell lines.
There are now multiple examples of lncRNAs being involved in tissue physiology and in the regulation and misregulation of cellular processes, which often result in cancer [36][37][38].This is a major reason for the interest in lncRNAs as biomarkers for diagnosis and prognosis, but also as potential targets for therapy or as tools to correct defective gene expression underlying cancer and other pathologies [39,40].Even at its peak, the expression of LADON never reaches a level that would allow it to be an effective miRNA sponge [41,42].The low level of expression of LADON is actually a common feature of lncRNAs and recent evidence suggests that it may be a key determinant of their specificity [43,44].An interesting feature of the transcript is the presence in its 3 region of the short interspersed elements (SINEs) Alu and MIR.The presence of Alu elements in lncRNAs has been associated with the capacity of such transcripts to regulate RNA transcription, decay, and splicing [45][46][47], while the presence of other SINEs has been associated with the regulation of other processes such as translation [48,49].In that respect, it will be interesting to investigate whether or not the changes in protein expression highlighted by our mass spectrometry analysis are preceded by changes in the expression of the corresponding genes.
LADON expression has so far only been reported in melanoma and breast cancer cell lines [31], and this study, but it is likely to be present in other cancer cell lines.There is evidence that it is present in healthy human cell types and tissues, where we currently have no indication of its function.Most of these cells, whether cancerous or not, do not express NODAL.For those that do express NODAL, future studies should investigate whether LADON has the ability to modulate NODAL signalling.

Plasmids
pcDNA3.1 and GFP pcDNA 3.1 are from Thermo Fisher Scientific (Waltham, MA, USA).LADON corresponding to AK001176.1,1725bp was synthesized and inserted in pCDN3.1 (Genscript, Leiden, The Netherlands).This vector was used for the transfection of the A375∆E2 a and d for rescue experiments.

Cell Lines and Cell Culture
The melanoma cell line A375M was purchased from the ATCC.The normal melanocyte cell line was kindly provided by Nathalie Andrieu (Centre de Recherches en Cancérologie de Toulouse).The SLM8 cell line, kindly provided by M Viguier, is derived from lymph node metastasis.The MNT1 and FO1 melanoma cell lines were a gift from Lionel Larue (Institute Curie CNRS UMR3347, INSERM U1021, Institute Curie).The 888 Mel cell line, a gift from A. Mauviel (Institut Curie/CNRS UMR 3347/INSERM U1021), is derived from the lung metastatic WM793 melanoma cell line.Cells were grown in DMEM/F12 Glutamax (Table 3, Invitrogen, Cergy-Pontoise, France) supplemented with antibiotics and 10% fetal calf serum (FCS) in a 5% CO 2 atmosphere.
To prepare the conditioned medium, melanoma cell lines were seeded at 70% confluence and then incubated for 72 h in DMEM medium with or without FCS, as stated.This melanoma-conditioned medium was collected, centrifuged at 11,000× g for 15 min to remove cell debris, and stored at −80 • C.
The source and identifier of all the plasmids and cell lines are listed in the reagent and resource Table 3. ) were seeded on the upper compartments of 2 mg/mL type I collagen-coated culture inserts (8 µm pores -Greiner Bio-One SAS, Courtaboeuf, France).The DMEM supplemented with 10% FCS was used as a chemoattractant.The A375 and A375∆E2 cells were allowed to migrate at 37 • C and 5% CO 2 for 24 h.The non-migrating cells on the upper face of the filter were removed by gently scraping them off using a cotton swab.The cells on the lower face were washed in PBS, fixed with 4% formaldehyde for 10 min, and washed in PBS.The nuclei were then labeled with Hoechst for 5 min and washed again.The migrating cells were observed under an epifluorescence microscope using a 10× magnification.Five to fifteen pictures of adjacent fields of the central zone of each Transwell were taken.Fluorescence was quantified with the IMAGEJ software [50] (US National Institutes of Health, Bethesda, MD, USA).Histograms display the data obtained from three independent experiments, and all the experiments were performed in duplicate.p-values were calculated using an ANOVA.Data are displayed as normalized results, where the nuclei of melanoma cells are set to 1 (or 100%) when grown in control conditions.

A375 Staining
For immunofluorescence experiments, the A375 cells were seeded onto glass coverslips in twelve-well plates and incubated overnight.Next, the cells were fixed in a 4% formaldehyde solution and incubated with Alexa Fluor 647-coupled phalloidin to visualize F-actin, and Hoechst 33342 to label nuclei.Coverslips were then further washed in PBS, treated with Fluoromount-G anti-fade (Southern Biotech), and analyzed by confocal microscopy.The shape of the A375 cells (n > 20), elongated or round was analyzed by the IMAGEJ software, and the proportion of each corresponding cell type was quantified.

Wound Healing/Scratch Assay
Cell migration was examined using a wound-healing assay.In brief, 0.2 × 10 6 cells were seeded in a well of twelve-well plates, and at the confluence, a scratch wound was made with a 10 µL pipette tip and then washed twice with PBS to remove cell debris.Wells were photographed under phase-contrast microscopy (time = 0) while cells were allowed to migrate into the scratch wound area for up to 18 h at 37 • C 5% CO 2 atmosphere using an Essenbio IncuCyte apparatus.The speed of the closure area was calculated over time by the IMAGEJ software.The data are represented as a ratio of migratory cells normalized to A375 cells migration in the control condition.All the experiments were performed in duplicate.

CRISPR/CAS9 Genome Editing
The CRISPR/Cas9 genome editing was performed with the GeneArt CRISPR Nuclease Vector Kit according to the manufacturer's instructions (Life Technologies).To select the target sequence for genome editing, the genomic sequences surrounding the NODAL exon 2 were submitted to an online CRISPR Design Tool (https://portals.broadinstitute.org/gppx/crispick/public (accessed on 9 October 2023)) Two target sites were selected upstream and downstream of this sequence.The oligonucleotides used to construct the gRNAs for the human NODAL gene exon 2 (deletion of 698 pb) are listed in Table S1.
The ds oligonucleotides generated were cloned into the GeneArt CRISPR Nuclease Vector.Competent E. coli cells were transfected with 3 µL of the ligation reaction, and then 50 µL from the transformation reaction was spread on a pre-warmed LB agar plate containing 100 µg/mL ampicillin.The plates were incubated overnight at 37 • C. The identity of the ds oligonucleotide insert in positive transformants was confirmed by sequencing.For the A375 cell line transfection, the cationic lipid-based Lipofectamine 2000 Reagent was used.The A375 cells positive for the transfection were sorted by FACS using OFP, a fluorescent protein present in the GeneArt CRISPR Nuclease Vector.Genotyping was performed on 96 clones to detect the deletion.Primers used to assess the efficacy of the CRISPR/Cas9 deletion are listed in Table S1.

RNA Extraction, Reverse Transcription (RT) and Quantitative PCR (Q-PCR)
For PCR analysis, 10 5 transfected GFP-positive cells were sorted by FACS analysis and collected into RNAse-free tubes.RNA from different cell lines was extracted with a Macherey Nagel kit for RNA purification.The first strand cDNA was synthesized with the SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific).RT-PCR amplification mixture (10 µL) contained 1/20 of cDNA product, 10X SYBR Green I Master Mix buffer, and 10 nM forward and reverse primers.Reactions were run on a Light Cycler PCR apparatus (Roche, Basel, Switzerland).Cycle conditions were 10 min at 95 • C and 45 cycles at 95 • C for 10 s, 61 • C for 10 s and 72 • C for 10 s.Each assay included a standard curve of three serial dilution points of A375 cDNA (ranging from 100 ng to 1 ng).All PCR efficiencies were above 95%.For each gene and a given RT-PCR, values were normalized to the level of expression of the reference genes RLP13 and GAPDH.No significant differences in the final ratios were found between the two reference genes; therefore, only RPL13 was used for normalization.Primers and annealing temperatures for all genes are indicated in Table S1.For each gene, the values were averaged over at least three independent measurements.
Sequences for all the siRNAs and oligos used in this study can be found in Table S1.

CAM Assay
All the elements of this assay were carried out at INOVOTION (Société: 811310127), La Tronche-France.According to decree n • 2013-118 of French legislation, 2010/63/UE European directive and Animal Welfare Act of United States, all assays used or provided by INOVOTION are not considered animal testing.Fertilized White Leghorn eggs were incubated at 37.5 • C with 50% relative humidity for 9 days.More than 15 eggs were processed for each experimental condition.At stage E9, the chorioallantoic membrane (CAM) was lowered by drilling a small hole through the eggshell into the air sac, and a 1 cm 2 window was cut in the eggshell above the CAM.A375 and A375∆E2d cells were cultivated in DMEM/F12 glutamax medium supplemented with 10% FBS and 1% penicillin/streptomycin.On day E9, cells were detached with trypsin, washed with a complete medium, and suspended in graft medium.An inoculum of 1.10 6 cells was added onto the CAM of each egg (E9) and then eggs were randomized into groups.On day E18, to quantitatively evaluate tumour growth, the upper portion of the CAM was removed, washed with PBS buffer, and fixed in PFA for 48 h.After that, tumours were carefully cut away from normal CAM tissue and weighed.On day E18 to evaluate the number of metastatic cells, a 1 cm 2 portion of the lower CAM and liver were collected, 8 samples per group (n = 8).Genomic DNA was extracted from the CAM and liver samples (commercial kit) and analyzed by qPCR with specific primers for human Alu sequences.Calculation of Cq for each sample, mean Cq, and relative amounts of metastases for each group were directly managed by the Bio-Rad ® CFX Maestro software.Non-injected eggs were also evaluated in parallel, as a negative control for specificity.A one-way ANOVA analysis with post-tests was performed on the data.

Histology
Also carried out at INOVOTION, a total of 10 tumours (5 per genotype) were harvested, fixed in 4% formaldehyde for 48 h, trimmed, and embedded in paraffin.Paraffin blocks were sectioned at approximately 4 microns thickness.The sections were put on glass slides and stained with Hematoxylin & Eosin (H&E).Stained tumour sections were examined by an expert pathologist.Ten non-overlapping fields (HPF) from each tumour section were assessed and graded using a semi-quantitative scoring system for several parameters.Pictures were taken using an Olympus microscope (BX60, serial NO. 7D04032) and its camera (Olympus DP73, serial NO.OH05504).Protein extracts (60 µg) were precipitated with acetone at −20 • C; the protein extracts were then incubated overnight at 37 • C with 20 µL of 25 mM NH 4 HCO 3 containing sequencing-grade trypsin (12.5 µg/mL, Promega, Madison, WI, USA).The resulting peptides were desalted using ZipTip µ-C18 Pipette Tips (Millipore, Burlington, MA, USA) and analyzed either in technical triplicates or individually by a Q-Exactive Plus coupled to a Nano-LC Proxeon 1000 equipped with an easy spray ion source (all from Thermo Scientific).Peptides were separated by chromatography with the following parameters: Acclaim PepMap100 C18 pre-column (2 cm, 75 µm i.d., 3 µm, 100Å), Pepmap-RSLC Proxeon C18 column (50 cm, 75µm i.d., 2µm, 100 Å), 300 nl/min flow rate, gradient from 95% solvent A (water, 0.1% formic acid) to 35% solvent B (100% acetonitrile, 0.1% formic acid) over a period of 98 min, followed by a column regeneration for 23 min, giving a total run time of 2 h.Peptides were analyzed in the Orbitrap cell, in full ion scan mode, at a resolution of 70,000 (at m/z 200), with a mass range of m/z 375-1500 and an AGC target of 3 × 10 6 .Fragments were obtained by high collision-induced dissociation (HCD) activation with a collisional energy of 30%, and a quadrupole isolation window of 1.4 Da.MS/MS data were acquired in the Orbitrap cell in a Top20 mode, at a resolution of 17,500 with an AGC target of 2 × 10 5 , with a dynamic exclusion of 30 s. MS/MS of the most intense precursor were first acquired.Monocharged peptides and peptides with unassigned charge states were excluded from the MS/MS acquisition.The maximum ion accumulation times were set to 50 ms for MS acquisition and 45 ms for MS/MS acquisition.

LC-MS/MS Data Processing
The LC-MS/MS.rawfiles were processed using the Mascot search engine (version 2.5.1)coupled to Proteome Discoverer 2.2 (Thermo Fisher Scientific) for peptide identification with both a custom database and the database Swissprot (from 2017) with the Homo sapiens taxonomy.The following post-translational modifications were searched on proteome Discoverer 2.2: Oxidation of methionine, acetylation of protein N-term, phosphorylation of serine/threonine, and phosphorylation of tyrosine.Peptide Identifications were validated using a 1% FDR (False Discovery Rate) threshold calculated with the Percolator algorithm.

Statistical Analysis
Data was collected and graphed using GraphPad Prism software.A 1-way ANOVA or two-sided student's t-test was used to determine statistical significance where appropriate.p-values of less than 0.05 were considered statistically significant.Information about the statistics used for each experiment, including sample size, experimental method, and specific statistic test employed, can be found in the relevant figures or figure legends.

Data Collection and Analysis
The HG-U133 plus 2 arrays (Affymetrix) data presented are accessible through GEO Series accession GSE35388 analyzed by GEO2R at the National Center for Biotechnology Information Gene.

Figure 1 .
Figure 1.The NODAL exon2 promotes tumour progression and metastasis in A375 cells (A) Schematic of the human NODAL locus with its 3 exons (E1 to E3), showing the full-length NODAL mRNA above.The primers used to detect its expression are represented by arrows.(B) Representative images, acquired

Figure 2 .
Figure 2. LADON, a natural antisense transcript of NODAL is a candidate oncogene in human melanoma.(A) Schematic of the human NODAL locus with its 3 exons (E1 to E3), showing the LADON transcript transcribed from the opposite strand below.The truncated LADON-∆E2 transcript is expressed in A375 cells deleted for the NODAL exon2.The arrows represent the primers used to track these transcripts.The black boxes show the location of short interspersed nuclear elements (SINE), which are mostly Alu and MIR sequences.(B) The bar plot of the LADON expression profile across all tumour samples and paired normal tissue was obtained from TCGA and GTEx data.The height of the bars represents the median expression of tumour types and normal tissues.The tumour abbreviation chart is provided in supplementary Table S1.The arrow indicates the high differential of LADON expression found in skin cutaneous melanoma (SKCM).(C) Pathological stage plot of LADON expression in SKCM.(D) The overall survival plot of SKCM patients with tumours expressing high or low levels of LADON.The hazard ratio (HR) calculation is based on the Cox PH model.(E) The RT-PCR with primers (L1F-L1R) amplifying a 130 nt band from the 5 regions of LADON detects the transcript in all cell lines of the panel: melanocytes, non-metastatic melanoma (MNT1), metastatic melanoma (A375, 888 mel, SLM8, FO1), breast cancer (MCF7), and an embryonic kidney.(HEK293).(F) The RT-qPCR measurements of the LADON expression were

21 Figure 3 .
Figure 3.The melanoma cells secrete factors that promote LADON expression, and invasion.(A) The RT-qPCR analysis of the LADON expression in the A375 cells seeded at low (LD) or high density (HD) and cultured for 24 or 72 h.Cell density does not affect LADON's expression level whereas cell culture duration does.The values were normalized to that of the LD condition after 24 h, which was set to 1. (B) The RT-qPCR analysis of the LADON expression in the A375 and MNT1 cells cultured for 24 h in the standard culture medium (SM), A375-conditioned medium (A375-CM) or MNT1-conditioned medium (MNT1-CM).The values were normalized to that of the SM condition, which was set at 1. (C) The RT-qPCR analysis of the LADON expression in the A375 cells cultured for 24 h in the SM, A375-CM, supernatant fraction of A375-CM, exosome fraction of A375-CM or a combination of these two fractions.The values were normalized to that of the SM condition, which was set at 1. (D) The RT-qPCR analysis of the LADON and LADON-ΔE2 expression in the A375 or A375ΔE2a cells cultured for 24 h in the SM, A375-CM or A375ΔE2-CM.The values were normalized

Figure 3 .
Figure 3.The melanoma cells secrete factors that promote LADON expression, and invasion.(A) The RT-qPCR analysis of the LADON expression in the A375 cells seeded at low (LD) or high density (HD) and cultured for 24 or 72 h.Cell density does not affect LADON's expression level whereas cell culture duration does.The values were normalized to that of the LD condition after 24 h, which was set to 1. (B) The RT-qPCR analysis of the LADON expression in the A375 and MNT1 cells cultured for

Figure 4 .
Figure 4. Model for the role of LADON in melanoma tumour progression and metastasis.In metastatic melanoma cells, the LADON transcript (in blue) promotes the transition from a proliferative cell identity to a less proliferative and more invasive cell identity.In the course of a 4-day culture, an increase of LADON expression promotes the expression of known oncogenes and represses that of tumour or metastasis suppressor, which in turn affects the expression of downstream targets, such as cytoskeleton components.The LADON expression also affects the potency of the conditioned medium produced by the cells, presumably via its impact on the exosomes (quantity, content) and the secreted factors they release.The LADON-dependent exosomes and secreted factors (in blue) promote the progression of exposed melanoma cells toward their more invasive identity.The LADON transcript is itself enriched in exosomes.

Table 1 .
Proteins were significantly enriched using the Gene Ontology approach.

Table 2 .
Proteins with a fold change superior to 2.