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Lysosomal acid ceramidase ASAH1 controls the transition between invasive and proliferative phenotype in melanoma cells

Abstract

Phenotypic plasticity and subsequent generation of intratumoral heterogeneity underly key traits in malignant melanoma such as drug resistance and metastasis. Melanoma plasticity promotes a switch between proliferative and invasive phenotypes characterized by different transcriptional programs of which MITF is a critical regulator. Here, we show that the acid ceramidase ASAH1, which controls sphingolipid metabolism, acted as a rheostat of the phenotypic switch in melanoma cells. Low ASAH1 expression was associated with an invasive behavior mediated by activation of the integrin alphavbeta5-FAK signaling cascade. In line with that, human melanoma biopsies revealed heterogeneous staining of ASAH1 and low ASAH1 expression at the melanoma invasive front. We also identified ASAH1 as a new target of MITF, thereby involving MITF in the regulation of sphingolipid metabolism. Together, our findings provide new cues to the mechanisms underlying the phenotypic plasticity of melanoma cells and identify new anti-metastatic targets.

Introduction

Cancer cells including melanoma cells display a high degree of intertumoral heterogeneity as well as intratumoral heterogeneity (ITH) [1, 2]. ITH is dynamically controlled by not only the tumor microenvironment but also the intrinsic plastic capacity of the cells and is recognized as the main therapy resistance mechanism. Therefore, the molecular mechanisms behind the plastic capacity of cancer cells and the underlying heterogeneity must be fully elucidated in order to develop effective and lasting therapies.

Melanoma cells can be classified into at least two major states, i.e., proliferative and invasive. A model derived from these findings, the “phenotype-switching” model, predicts that melanoma cells may switch between these two states [3]. ITH can be, at least in part, understood by this phenotypic switch. Most importantly, melanoma phenotype switching is associated with intrinsic and acquired resistance to targeted therapies, which is the major cause for the resultant mortality of patients with cancer [4,5,6,7]. It also negatively impacts on immune checkpoint blockade and impairs immunotherapy efficiency [8, 9]. The lineage-specific transcription factor, microphthalmia-associated transcription factor (MITF), which acts as a bona fide melanoma oncogene [10,11,12,13], is a key driver of the transition between the proliferative and the invasive state of melanoma cells [14,15,16,17]. However, how MITF regulates the phenotypic switch of melanoma cells is not fully elucidated.

Cancer cells rewire their metabolic pathways to satisfy their abnormal demands for proliferation and survival [18]. In cancer cells, the most common metabolic change is the use of glycolysis even in normoxic condition, a phenomenon called “Warburg effect” [19]. There are a number of targets involved in glycolysis, which are currently exploited for potential therapeutic drug strategies [20]. However, as functionally plastic cells, malignant melanoma cells can rewire their metabolism towards oxidative phosphorylation, or glutaminolysis, two processes limiting drug efficacy [8, 21, 22]. Several lines of evidence indicate that cancer cells also show alterations in other metabolic pathways such as lipid metabolism [23, 24]. In melanoma, the role of lipid metabolism is poorly elucidated.

Here, by comparing the transcriptomic profile of invasive and proliferative melanoma cells, we found that the expression of acid ceramidase ASAH1, a key enzyme of sphingolipid metabolism [25], was among the top differentially expressed genes, suggesting that ASAH1 might be critical to the phenotypic switch. We performed a comprehensive characterization of ASAH1, function and regulation in melanoma. We found that ASAH1 loss drove the transcriptional reprogramming of proliferative melanoma cells into an invasive cell subpopulation characterized by a reduced expression of E-cadherin and activation of the integrin alphavbeta5 (αVβ5)-focal adhesion kinase (FAK) signaling cascade. Conversely, forced expression of ASAH1 in invasive melanoma cells reduced their motile features and rendered them more proliferative. We also identified ASAH1 as a target of MITF, which is a key driver in the phenotypic transition. These data also causally involve for the first time MITF in the regulation of sphingolipid metabolism.

Together, our findings reveal that lysosomal ASAH1 and the sphingolipid metabolism drive the transition between the proliferative and the invasive phenotype in melanoma cells, which is at the origin of intratumoral heterogeneity and a source of therapy resistance.

Results

ASAH1 controls the phenotype switching of melanoma cells

To identify genes differentially expressed between proliferative and invasive melanoma cells, we performed transcriptomic analysis. Using both melanoma cell lines and melanoma cells freshly isolated from human biopsies we found that the acid ceramidase ASAH1, which is located at the lysosome and which controls the sphingolipid metabolism, was among the genes most differentially expressed between the two phenotypes (Fig. 1a). We confirmed this observation using data from another source (Supplementary Fig. 1a) [26]. Further, analysis of publicly available datasets revealed significantly higher expression of ASAH1 in melanoma cells compared to other human cancer cell lines, strengthening the key role of ASAH1 in melanoma cells (Fig. 1b).

Fig. 1
figure1

Acid ceramidase ASAH1 is expressed in melanoma cells. a Heatmap showing the expression of genes in the invasive (red group) and proliferative (blue group) cells. The data were analyzed using the Morpheus software (https://software.broadinstitute.org/morpheus/). A relative color scheme based on the minimum and maximum values in each row was used to convert values to color. The relative levels are indicated by varying color intensity (low, blue; high, red). b Box plots showing the relative ASAH1 mRNA across the different tumor types, extracted from in the NCI60 line datasets. c Co-expression of MITF and ASAH1 in melanoma from the TCGA database. Gene expression data were log2 transformed. Pearson’s correlation coefficient, used to measure similarity between gene profiles, is shown. d Immunoblot to ASAH1 of total cell extracts isolated from the indicated melanoma cell lines with a non-commercial antibody showing the proform (55 kDa), the β (40 kDa) and α (14 kDa) active subunits. Detection of ERK2 serves as a loading control. e Immunoblot to MITF of total cell extracts isolated from the indicated melanoma cell lines. MITF is detected as a doublet of 55–72 kDa. Detection of ERK2 serves as a loading control. f ASAH1 activity in a panel of melanoma cells. Values represent mean + SD of two independent experiments. g Cellular lipids were extracted, and sphingolipid concentration was determined by LC/MS. Data represent the quantity of sphingosine (SPH) in ng/mg proteins and are the means + SD of two independent determinations

Analysis of publicly available datasets showed a positive correlation between ASAH1 and the transcription factor MITF (Fig. 1c and Supplementary Fig. 1B).

ASAH1 expression was next studied by quantitative polymerase chain reaction (Q-PCR) in another set of melanoma cell lines expressing MITF at different levels. Two groups of melanoma cells were distinguished, one expressing low to very low messenger RNA (mRNA) level of ASAH1 (ASAH1low) and the other expressing high mRNA level of ASAH1 (ASAH1high) (Supplementary Fig. 1C). We next assessed ASAH1 at the protein level. ASAH1 is a glycoprotein processed from a 55 kDa precursor into an active heterodimeric enzyme with α (14 kDa) and β (37 kDa) subunits [27]. Immunoblots with a homemade antibody [28] easily detected the proform and the α and β active subunits (lanes 4–6 and lanes 10–12) in melanoma cells with high ASAH1 mRNA expression, whereas the different forms were hardly detectable in cells with low ASAH1 mRNA (lanes 7–9) (Fig. 1d). Because of homemade antibody restriction, we also tested two commercially available antibodies. Only one antibody gave positive results leading to the detection of the proform and of the β-subunit (Supplementary Fig. 1D). The different melanoma cell lines analyzed for MITF expression resulted in the detection of two groups with low/absent MITF expression (MITFlow) and high MITF expression (MITFhigh) (Fig. 1e and Supplementary Fig. 1E).

Using the fluorogenic substrate Rbm14–12 [29], we measured the activity of ASAH1, which varied accordingly with the level of expression (Fig. 1f). We next performed a comprehensive lipidomic analysis, using ultra-performance liquid chromatography/mass spectrometry. Lipidomic analysis revealed higher sphingosine level in ASAH1high cells, consistent with an ASAH1 activity, compared to ASAH1low cells (Fig. 1g). Of note, we found no correlation between expression of ASAH1 and the BRAF mutational status.

Together, our results indicate that ASAH1 is highly expressed and active in about 60% of melanoma cells and its expression correlates with that of MITF.

ASAH1 expression in human melanoma samples

Immunohistochemical staining of MITF and ASAH1 proteins was performed in a series of human melanoma samples, including primary malignant melanoma and metastatic samples. Areas of high and low MITF and ASAH1 expression were observed in cutaneous metastasis of melanoma (Fig. 2a, b). Thus, the correlation between MITF and ASAH1 expression was also observed in vivo.

Fig. 2
figure2

Expression of MITF and ASAH1 in human melanoma samples. a, b IHC analyses of MITF and ASAH1 expression at superficial and deep sites of primary melanoma sample. The images exemplify correlation between MITF and ASAH1 expression within the lesions. Magnification ×4, scale bar: 500 μm and magnification ×20, scale bar: 100 μm. c IHC analyses of MITF and ASAH1 expression in a cutaneous metastatic melanoma sample. Magnification ×4, scale bar: 500 μm and magnification ×20, scale bar: 100 μm

Interestingly, within thick primary melanomas, a distinct gradient was observed, where nuclear MITF and cytoplasmic ASAH1 were strongly expressed in superficial sites with weaker or absent staining at the invasive sites of the lesion (Fig. 2c), strengthening the notion that ASAH1 loss correlates with a loss of MITF and a gain of motile ability and aggressiveness. In conclusion, our results confirm in vivo the correlation between MITF and ASAH1.

MITF drives expression of ASAH1

Our previous observations suggested that ASAH1 was a downstream target of MITF. To assess this hypothesis, we silenced MITF with two different small interfering RNAs (siRNAs). As expected, the siRNA reduced the expression of mRNA for MITF and its target genes MLANA and TYR. MITF inhibition also strongly decreased ASAH1 mRNA (Fig. 3a and Supplementary Fig. 2A-B). Similar observations were obtained at the protein level showing that MITF inhibition caused a reduction in the proform and the β-active subunit of ASAH1 (Fig. 3b). Accordingly, MITF inhibition reduced ASAH1 activity (Fig. 3c) and consequently the quantity of sphingosine (SPH) (Supplementary Fig. 2C).

Fig. 3
figure3

ASAH1 is a direct target of the MITF transcription factor. a mRNA expression of MITF and two of its target genes (Tyrosinase and MLANA) and ASAH1 in 501mel melanoma cell lines determined by qRT-PCR. Values represent mean + SD of three independent experiments performed in triplicate; ***p < 0.001. b Immunoblot to MITF and ASAH1 of 501mel melanoma cells transfected with control or Mitf-specific siRNA. Detection of ERK2 serves as a loading control. c ASAH1 activity of 501mel melanoma cells transfected with control or Mitf-specific siRNA. Values represent mean + SD of three independent experiments; **p < 0.01. d Immunoblot to MITF of Mel-ST melanocyte cells transduced with an empty adenoviral vector or a vector encoding MITF. e mRNA expression of ASAH1 in Mel-ST melanocyte cells transduced with an empty adenoviral vector or a vector encoding MITF determined by qRT-PCR. Values represent mean + SD of three independent experiments performed in triplicate; **p < 0.01. f ASAH1 activity in Mel-ST melanocyte cells transduced with an empty adenoviral vector or a vector encoding MITF. Values represent mean + SD of three independent experiments performed in triplicate; *p < 0.05. g ChIP-seq profile showing significant MITF-binding peaks in the ASAH1 genomic region (black arrowheads). The sequence under peak shows the presence of MITF-binding sites. The scale bar indicates the size of the genomic region in kilobases (kB). The H3K27ac and H3K4me3 tracks associated with active transcription, and the H3K27me3 track associated with the repression of transcription were obtained in control 501mel cells [31] and were aligned with the ChiP-seq profile of MITF [30]. h,a Cell count of 501mel melanoma cells infected with an empty vector or a vector encoding ASAH1 and transfected with control or MITF siRNA. Representative micrographs are shown. Values represent mean + SD of three independent experiments; ***p < 0.001. h,b Immunoblot of 501mel melanoma cells infected with an empty vector or a vector encoding ASAH1 and transfected with control or MITF siRNA

Conversely, we assessed whether MITF was sufficient to drive the expression of ASAH1. Forced expression of MITF in two different melanocyte cells, as shown by immunoblot (Fig. 3d and Supplementary Fig. 2D), enhanced the expression and activity of ASAH1 (Fig. 3e and Supplementary Fig. 2E) compared with cells transduced with an empty vector (Fig. 3f). MITF is a transcription factor that binds to DNA to control the expression of its targets. Using recently described MITF chromatin immunoprecipitation-sequencing (ChIP-seq) dataset [30], we identified significantly enriched MITF-binding sites in the genomic region of ASAH1 (Fig. 3g). MITF binding at the promoter region overlapped with the binding of H3K27ac and H3K4me3, two histone marks associated with active transcription [31], thereby indicating that MITF controlled the expression of ASAH1 at the transcriptional level. In line with that, MITF stimulated the activity of ASAH1 promoter (Supplementary Fig. 2F).

We next addressed the functional importance of ASAH1 in MITF effects. Forced expression of ASAH1 rescued the reduction of cell proliferation mediated by MITF inhibition. Forced expression of ASAH1 also slightly enhanced the number of cells compared to the control condition (Fig. 3ha). Further, an increase in expression in the cell cycle inhibitor p27 was observed upon MITF inhibition by siRNA, but did not occur in these cells when they were transduced with ASAH1. Together, these results demonstrate the causal role of ASAH1 in MITF proliferative activity (Fig. 3hb). Almost no cell death was found in these conditions (Supplementary Fig. 2G). Of note, immunoblot also showed that forced expression of ASAH1 was associated with an increase in MITF expression, thereby suggesting a bidirectional regulation between MITF and ASAH1.

Collectively, our findings identify ASAH1 as a new MITF target gene and demonstrate for the first time the involvement of MITF in the control of sphingolipid metabolism.

ASAH1 controls the switch between the proliferative and invasive phenotype in melanoma cells

We next assessed the impact of ASAH1 knockdown in ASAH1high cells on short-term cell growth, colony formation assay and apoptosis detection in various melanoma lines transfected with siRNA against ASAH1 versus a control siRNA. To rule out non-specific effects, different siRNAs were used. As expected, the siRNA triggered a strong decrease in expression of mRNA and protein for ASAH1 (Fig. 4a and Supplementary Fig. 3A) and translated into a decrease in ASAH1 activity (Supplementary Fig. 3B). Of note, siASAH1#2 displayed stronger effect than siASAH1#1 and was used in the following experiments as siASAH1. Electron microscopy revealed that ASAH1-inhibited melanoma cells compared to control cells were characterized by an accumulation of intracellular vacuoles rich in sphingolipid (Supplementary Fig. 3C) typically resembling the phenotype of cells from patients with lysosomal storage disorders [32]. ASAH1 inhibition dramatically reduced the number of cells in short-term cell growth (Supplementary Fig. 3D) or in colony formation assays (Supplementary Fig. 3E). Remarkably, the growth arrest caused by ASAH1 reduction was comparable to that of MITF inhibition, in agreement with ASAH1 being a target gene of MITF (Supplementary Fig. 3D-E). Fluorescence-activated cell sorting analysis of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells did not reveal a significant difference in cell death between control cells or cells in which ASAH1 or MITF were inhibited (Supplementary Fig. 3F).

Fig. 4
figure4

ASAH1 controls melanoma cell proliferation and motile features. a ASAH1 mRNA expression in melanoma cells transfected with control or two different ASAH1 (ASAH1#1 and ASAH1#2) or MITF siRNAs. Values represent mean + SD of three independent experiments performed in triplicate; **p < 0.01; ***p < 0.001. b Boyden chamber experiments of melanoma cells transfected with a control or two different ASAH1 siRNAs (ASAH1#1 and ASAH1#2) or with a MITF siRNA. Representative images are shown. Values represent mean + SD of three independent experiments; *p < 0.05, **p < 0.01 and ***p < 0.001. c Boyden chamber experiments. The 501mel melanoma cells were transfected for 48 h with ASAH1 or MITF siRNA before being exposed to 3 μM D-sphingosine (D-SPH) for 5 h. Values represent mean + SD of three independent experiments; **p < 0.01; ***p < 0.001. d Boyden chamber experiments. Invasive melanoma cells were exposed to 3 μM D-SPH for 5 h. Values represent mean + SD of three independent experiments; ***p < 0.001

Conversely, we investigated the effect of ASAH1 forced expression in ASAH1low cells. ASAH1 upregulation in WM3918 cells was confirmed by Q-PCR and immunoblot (Supplementary Fig. 4A-B). Further, forced expression of ASAH1 did not change its cellular localization (Supplementary Fig. 4C) and it translated into more ASAH1 activity (Supplementary Fig. 4D). Melanoma cells with forced expression of ASAH1 proliferated faster than native parental control cells (Supplementary Fig. 4E) and displayed a reduced motility (Supplementary Fig. 4F). Analysis of The Cancer Genome Atlas–Skin Cutaneous Melanoma (TCGA-SKCM) dataset revealed accordingly an association of ASAH1 with the proliferative signature described in Verfaillie et al. [33] (Supplementary Fig. 4G).

In addition, ASAH1-inhibited cells underwent morphological changes. They exhibited an increased dendritic morphology (Supplementary Fig. 5a). Moreover, adherence monitored either by microscopic observation after seeding (Supplementary Fig. 5B-C) or monitored in real time using the xCELLigence technology showed that cells with reduced ASAH1 expression adhered faster than that with high ASAH1 expression (Supplementary Fig. 5D). Similar observations were obtained in cells transfected with MITF siRNA (Supplementary Fig. 5D). These changes suggested that ASAH1 controlled the motile ability of melanoma cells.

Boyden chamber motility studies and scratch wound assays in different melanoma cell lines demonstrated that ASAH1 reduction enhanced cell migration compared to control cells (Fig. 4b and Supplementary Fig. 5E-F). ASAH1 reduction stimulated cell migration to the same extent as MITF inhibition. Conversely, melanoma cells with forced expression of ASAH1 displayed reduced motile ability (Supplementary Fig. 4F). In line with that, MITF and ASAH1 low melanoma cells have higher invasive capacities than MITF and ASAH1 high cells (Supplementary Fig. 5G). To demonstrate that the observed effects were dependent on ASAH1 activity, exogenous sphingosine was used to restore functional ASAH1 metabolic cascade. Short-term exposure to sphingosine at concentrations from 1 to 5 μM had no effect on cell number (Supplementary figure 6A-B). However, it dramatically reduced melanoma cell migration induced by the loss of ASAH1 or MITF (Fig. 4c and Supplementary figures 6C-D) and impaired migration of ASAH1low melanoma cells (Fig. 4d).

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors enhance mesenchymal (loss of epithelial characteristics such as E-cadherin expression) and motile features. TWIST1 is an EMT-controlling factor particularly important in melanoma [34]. Interestingly, we observed that ASAH1 reduction in ASAH1high cells enhanced TWIST1 expression (Supplementary figure 7A) and caused a loss of E-cadherin expression (Supplementary figure 7B), whereas ASAH1 forced expression in ASAH1low cells had the opposite effect, reducing TWIST1 (Supplementary figure 7C) and stimulating E-cadherin expression (Supplementary figure 7D). These features translated into an enhanced sphere formation ability of ASAH1-inhibited melanoma cells (Supplementary figure 7E). Thus, ASAH1 impacts on the mechanical and mesenchymal features of melanoma cells. Collectively, our findings demonstrate that ASAH1 acts as a rheostat to control the switch between proliferative and invasive states in melanoma cells.

ASAH1, through activation of the ITGαVβ5/FAK signaling cascade, controls motility of melanoma cells

To broadly dissect the molecular mechanisms by which ASAH1 exerts its effect in melanoma cells, we performed a gene expression profiling of ASAH1 knocked down cells compared to control cells. Top canonical pathways of the differentially expressed genes captured in the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool were related to cell migration and trafficking such as “pathways in cancer”, “focal adhesion” and “ECM-receptor interaction” (Fig. 5a), thereby strengthening the observation that ASAH1 loss is associated with an enhanced motile capacity of melanoma cells. Of note, our transcriptomic analysis also revealed a reduction in expression of several MITF target genes. The expression in MITF mRNA itself was slightly downregulated in the three cell lines but did not reach the statistical threshold. Nevertheless, at the protein level, forced expression of ASAH1 enhanced MITF (Fig. 3hb) and ASAH1 siRNA reduced MITF expression (Supplementary figure 7F).

Fig. 5
figure5

ASAH1 controls cell migration via the integrin/FAK cascade. a Top significantly deregulated pathways upon ASAH1 downregulation in three different melanoma cell lines (501mel, WM3912, WM8) transfected with control or ASAH1 siRNA. b Immunoblot to total and phosphorylated FAK (Y397) after ASAH1 downregulation (48 h) compared to control siRNA. c Confocal microscopy images of phosphorylated FAK (Y397) localization after ASAH1 downregulation (48 h) compared to control siRNA. d Immunoblot to total and phosphorylated FAK (Y397), ASAH1 and HSP90 (loading control) in WM3918 with forced expression of ASAH1 compared to the parental cells. e Immunoblot to ITGβ5, ITGαV and HSP90 (loading control) in 501mel, WM3912 and WM8 melanoma cells transfected with a control or ASAH1 siRNA. f Immunofluorescence of ITGβ5 in WM3912. g Boyden chamber experiments with control IgG Ctl) or ITGαVβ5 neutralizing antibody (ITGα β5). Values represent mean + SD of three independent experiments; ***p < 0.001. Representative images are shown

We next focused our attention on FAK which is linked to integrin signals to promote cell migration. Immunoblot showed that ASAH1 silencing in different cell types resulted in a significant increase in FAK phosphorylation (Fig. 5b). Such treatment had no effect on the levels of FAK protein. Immunofluorescence staining of cells following ASAH1 siRNA treatment revealed that FAK localized to focal adhesions compared to control cells in which FAK was more cytoplasmic (Fig. 5c and Supplementary figure 8A). Conversely, forced expression of ASAH1 inhibited FAK phosphorylation (Fig. 5d). Gene expression profiling revealed the increased expression of integrin β5 that was confirmed at the protein level (ITGβ5) (Fig. 5e). ITGβ5 can associate with integrin αV (ITGαV). Transcriptomic profiling showed a small increase in expression of mRNA for ITGAV in ASAH1-inhibited cells, even though the difference did not reach statistical significance. However, a statistically significant difference in ITGAV mRNA between control and ASAH1-inhibited cells was observed by Q-PCR and was strengthened at the protein level by western blot (Fig. 5e and Supplementary figure 8B). Further, both integrins play critical role in cell migration, invasion and tumorigenesis. Q-PCR and immunoblot revealed the recurrent increased expression in ITGαV upon ASAH1 inhibition in different melanoma cells (Fig. 5e). As shown by immunofluorescence, ITGβ5 staining was confined predominantly to the cellular periphery in ASAH1 knocked down cells compared to its cytoplasmic localization in control cells (Fig. 5f). In cells treated with MITF siRNA, ITGβ5 generated a pattern of staining similar to that obtained with ASAH1 inhibition (Fig. 5f). We next thought to determine whether ITGαVβ5 was causally involved in melanoma cell migration mediated by ASAH1 inhibition. Whereas treatment with an ITGαVβ5 blocking antibody had no effect on cell survival (Supplementary figure 8C), it dramatically impaired melanoma cell migration (Fig. 5g and Supplementary figure 8D-E). ITGαVβ5 blocking antibody exhibited similar causal effect in melanoma cell migration mediated by MITF inhibition. Altogether, our data demonstrate that ASAH1 controls melanoma cell plasticity and migration through integrin-mediated FAK activation.

Discussion

Here, we demonstrate that the lysosomal acid ceramidase ASAH1, which controls sphingolipid metabolism, acts as a driver of the phenotypic switch, thereby contributing to an intratumoral heterogeneity. We showed that whereas melanoma cells with high expression of ASAH1 displayed a proliferative activity, low ASAH1 cells had enhanced motile ability. ASAH1 was previously shown to play an important role in melanoma proliferation [35]. We also identified ASAH1 as a target of MITF in melanoma cells, thereby implying for the first time MITF in the regulation of sphingolipid metabolism. Until now, an enhanced transcription of ASAH1 has been reported by cAMP-responsive element binding protein (CREB) in human adrenal corticocarcinoma cells [36] and by estrogen receptor-α (ERα) and specificity protein-1 (Sp1) in breast cancer cells [37]. Thus, our study adds new clues to the regulation of ASAH1.

Elevated ASAH1 expression has been reported in various types of human cancers such as prostate, breast and head and neck [38, 39] and has been associated to increased malignancy and worse clinical outcome [40,41,42]. However, ASAH1 expression correlates with a good prognosis in ER-positive breast cancer [43, 44], thereby indicating that high ASAH1 expression is not necessarily an indicator of unfavorable tumor features and lower survival.

Further, defects in the ASAH1 gene result in the lysosomal storage disease Farber (FD) lipogranulomatosis, with an invariably fatal outcome [45]. FD patients are not prone to develop cancer. However, individuals with the most common “classical” form of FD die at approximately 2 years of age, before melanoma can develop. The data gathered here on the role of ASAH1 in melanoma cells highlighted genes and signaling cascades that might serve as new potential therapeutic targets in the FD patients.

The pro- or anti-tumoral role of ASAH1 can be explained by tissue-specific functions or by its involvement during specific stages of cancer. Supporting this idea, MITF expression must transiently decrease to enable melanoma cell migration and invasion but high MITF expression must be recovered to allow the growth of the metastases [14, 16, 46]. In response to microenvironmental signals, MITF exhibits an oscillatory pattern of expression and melanoma cells switch back-and-forth between proliferative and invasive to drive the metastatic disease [3, 8, 16]. ASAH1 might follow the same oscillatory model as MITF.

MITF-silenced melanoma cells also display features of senescence [30, 47]. ASAH1-depleted cells were positive for the senescence-associated β-galactosidase staining (Supplementary figure 7G), in agreement with a previous report [48], and also displayed traces of the pro-inflammatory secretome, illustrated by an increased expression of CCL2 and CYR61, as we previously reported in MITF low cells [49, 50]. Melanoma cells with reduced expression of ASAH1 exhibited a molecular program linked to invasion that partially overlapped that of MITF loss. Transcriptomic analysis of melanoma cells transfected with ASAH1 siRNA revealed no change compared to control cells in ZEB1 or SNAIL and an increase in TWIST1, BRN2 or SLUG expression, which are critical factors for melanoma progression and metastasis [34, 51, 52]. Additionally, only insulin-like growth factor receptor 1 (IGF1R), out of other critical tyrosine kinase receptors (PDGFRB, AXL, EGFR, ERBB3, EPHA2) that were reported to be deregulated in the MITF low invasive phenotype [6, 8, 53], was enhanced in ASAH1-silenced melanoma cells. Thus, the gene expression signature of ASAH1-inhibited cells appeared intermediate between the proliferative and invasive phenotypes reported for MITF. This is consistent with previous reports showing that a subgroup of profiled melanoma cell lines was intermediate between the two states [26, 54]. Further, melanoma cells with both proliferative and invasive phenotypes have been recently described [55].

Noteworthy, melanoma cells expressing classical markers of senescence were reported to retain their ability to migrate and invade [56]. This is consistent with our findings demonstrating that ASAH1 loss induced a senescence-like phenotype and enhanced the motile ability of melanoma cells. Lai et al. [48] also showed that ASAH1-null cells lose the ability to form cancer-initiating cells. Contrastingly, we demonstrated that cells in which ASAH1 expression was inhibited via siRNA gain a motile mesenchymal phenotype, which is a feature of cancer stem populations. This discrepancy can be explained by the complete compared to transient inhibition of ASAH1 that prevents adaptation through plastic ability of melanoma cells. The partial loss-of-function phenotype generated by RNA interference (RNAi) may more accurately recapitulate the effects of microenvironment than the complete loss-of-function phenotype. Furthermore, the transcriptomic analysis revealed that ASAH1 inhibition by siRNA triggered a slight decrease in expression of mRNA for MITF, yet it did not reach the statistical threshold. Nevertheless, at the protein level, forced expression of ASAH1 enhanced MITF expression and ASAH1 siRNA reduced MITF expression. These observations suggested a complex post-transcriptional regulation of MITF by ASAH1 that needs further studies to be elucidated. Migration requires the coordinated regulation of both E-cadherin-mediated cell–cell adhesions and integrin-mediated adhesions that contact the surrounding extracellular matrix (ECM) [57]. In line with that, E-cadherin inhibition and increased expression of TWIST1, a known E-cadherin transcriptional repressor [34, 58], paralleled ASAH1 loss. Supporting our data, sphingosine-1-phosphate (S1P) production, which is produced by ASAH1 activity, has been reported to suppress migration of breast cancer cells through E-cadherin re-expression and enhanced cell–cell adhesion [59]. Besides, we observed that ASAH1 loss favored anchorage-independent growth, and correlates with acquisition of mesenchymal properties and with integrin activation. Accordingly, cells with reduced ASAH1 expression displayed heightened integrin and FAK signaling. Among integrin, we identified integrin αVβ5 as the main dimer responsible for melanoma cell migration mediated by ASAH1 inhibition. In highly invasive melanoma cells, integrin αVβ5 was also shown to regulate vascular endothelial growth factor (VEGF)-A secretion and activation of the vascular endothelium, a key step in the extravasation process and formation of distant metastasis [60]. Our transcriptomic analysis revealed that VEGF-C is one of the top deregulated genes upon ASAH1 reduction. Additional candidates that may reflect aspects of migration and heterogeneity of melanoma cells include several transcription factors, such as BRN2 and NFIB, that were found increased in our transcriptomic analysis of ASAH1-inhibited cells. Recently, NFIB has been shown to mediate BRN2-driven melanoma cell migration and invasion through upregulation of EZH2 and downregulation of MITF [61, 62]. However, in our trancriptomic analysis EZH2 mRNA expression was inhibited after ASAH1 inhibition by siRNA, instead of being increased as reported in response to BRN2 forced expression [61]. Thus, these observations suggest that the increase in motile and invasive properties evoked by ASAH1 inhibition and by BRN2 forced expression are different.

Altogether, we show that ASAH1 influences the phenotypic switch in melanoma cells and that melanoma cells acquire motile properties after the loss of ASAH1. Further, our findings point out to a link between metabolism of sphingolipid and melanoma cell plasticity and open new therapeutic perspectives in melanoma.

Materials and methods

Cell cultures and reagent

All human melanoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7% fetal calf serum at 37 °C in a humidified CO2 incubator. The human melanoma cell lines WM3912, WM8 and WM3918 are a gift of Dr. M Herlyn (The Wistar Institute, Philadelphia, PA). Other melanoma cell lines were purchased from the American Tissue Culture Collection. All cell lines were regularly tested for mycoplasma contamination.

Human tumor samples and immunohistochemical analyses

Twenty BRAFV600-mutated melanoma tumor samples were obtained through the Biological Resource Center of the Hôpital Lyon Sud, Hospices Civils de Lyon, and were used with the patient’s written informed consent. This study was approved by the scientific board of the Hospices Civils de Lyon. Formalin-fixed, paraffin-embedded and 3 µm-thick tissue sections underwent immunohistochemical (IHC) staining using commercially available antibodies against MITF (C5/D5, mouse monoclonal, 1/200, Roche) and ASAH1 (ab74469, 1/100, Abcam, Cambridge, MA, USA) and Ultraview red detection or DAB revelation. For MITF, the staining was nuclear while for ASAH1 the staining was cytoplasmic. A blinded evaluation of the staining was carried out by experienced pathologists.

Gain- and loss-of-function experiments

Briefly, cells were transfected with 20 nM of siRNA plus 5 μl lipofectamineTM RNAiMAX in opti-MEM medium (Invitrogen, San Diego, CA, USA). Control (siC) and siRNAs for MITF have been previously described [63]. siRNAs to ASAH1 were from Invitrogen.

Cells were infected with lentiviral particles for ASAH1 and when indicated 24 h later they were transfected with control or MITF siRNA for 72 h.

Cell count and viability

Cell count and viability were determined in flow cytometry by adding DAPI at 1 μg/ml and for a short time to the population of cells.

Colony formation assay

The 501mel melanoma cells were transfected with control or ASAH1 siRNA for 48 h. Cells were trypsinized, seeded in 6-well plates (3 × 103) and were then placed in a 37 °C, 5% CO2 incubator. Colonies of cells were stained with 0.04% crystal violet/2% ethanol in phosphate-buffered saline for 30 min after for 14 days. Picture of crystal violet-stained wells were taken. The colony formation assay was performed in triplicate.

Anchorage-independent growth

The 501mel melanoma cells were transfected with control or siRNA to ASAH1 and next subjected to a two-layer soft agar assay in 6-well plates. Cells (1.5 × 104) were mixed with 2 ml of 1× culture medium with 10% fetal bovine serum (FBS) and 0.3% agar, cooled to 42 °C, and plated on top of a solidified bottom layer of 2 ml of 1% noble agar in cell respective 1× culture medium. Every 2 days, 1 ml of growth medium was added into the wells. After 1 week, bright-field images were taken using a Zeiss Axiovert S100. The number of colonies grown in soft agar was quantified, and the colony size (i.e., image area) was calculated using Zeiss ZEN software.

LC/MS analysis of sphingolipids and ASAH1 activity

Sphingolipids were extracted and prepared as previously published [64] and analyzed by liquid chromatography/mass spectrometry (LC/MS) as previously described [29]. ASAH1 activity was measured as previously reported [29].

Immunofluorescence and confocal experiments

Cells were fixed and permeabilized as previously described [49] before being exposed to anti-ASAH1 1:100 (Sigma, HPA005468) antibodies and then with 1:1000 dilution of anti-rabbit Alexa Fluor 594 labeled secondary antibody (Invitrogen, San Diego, CA, USA). Confocal optical sections of cells were obtained with a Zeiss LSM510Meta microscope (Zeiss, Göttingen Germany) using a 63× NA1.4 Plan-Apochromat oil immersion lens. The parameters of the system were adjusted to avoid saturation.

Western blot assays

Western blots were carried out as previously described [46]. Cell lysates (30 μg) were separated by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane and then probed with antibodies to MITF (Abcam, Ab12039), ASAH1 (Sigma, HPA005468), ERK2 (Santa Cruz Biotechnology, clone D-2), phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology Inc., #2370), anti-phosphoFAK (Cell Signaling Technology Inc., #3283), total FAK (Millipore, clone 4.47), p27 (Ozyme, #3686), ITGβ5 (Cell Signaling Technology Inc., D24A5), ITGαV (Santa Cruz biotechnology, p2W7), to integrin αVβ5 (Abcam, ab24694), E-cadherin (Ozyme, #5296) and HSP90 (Santa Cruz Biotechnology, sc-13119). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were from Dakopatts (Glostrup, Denmark). Proteins were visualized with the ECL system (Amersham). The western blots shown are representative of at least three independent experiments.

Migration assays

Cell migration was carried out using a Boyden chamber assay with 8 μm pore filter inserts (BD Bioscience). Cells were seeded on the upper chamber of a trans-well and DMEM/7% FBS placed into the lower chamber. Cells adherent to the underside of the filters were fixed with 4% paraformaldehyde, stained with 0.4% crystal violet and five random fields at ×20 magnification were counted. Results represent the average of triplicate samples from three independent experiments.

Cell migration was also studied in classical wound healing assays. Cells were grown to form a confluent monolayer and next were wounded with a p200 pipette tip. Pictures (10×) of the same areas were recorded using an Axiovert 200M microscope (Zeiss) equipped with a CoolSnap™ES camera (Photometric®, Roper Scientific).

mRNA preparation, real-time/quantitative PCR

mRNA isolation was carried out with Trizol (Invitrogen), according to standard procedure. QRT-PCR was carried out with SYBR® Green I and Multiscribe Reverse Transcriptase (Applied Biosystems) and monitored by an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Detection of SB34 gene was used to normalize the results. Primers were designed for each complementary DNA with Primer Express Software (Applied Biosystems) and are available upon request.

Gene expression profiling and bioinformatics

Total RNA of three different cell lines transfected with control scrambled siRNA or siRNA to ASAH1 was isolated with TRIZOL. Integrity of RNA was assessed using an Agilent BioAnalyser 2100 (Agilent Technologies) (RNA Integrity Number (RIN) above 8). RNA samples were then labeled with Cy3 dye using the low RNA input QuickAmp kit (Agilent) as recommended by the supplier. Then, 600 ng of labeled cRNA probe was hybridized on 8 × 60 K high-density SurePrint G3 gene expression human Agilent microarrays. Data analyses were performed using R (http://www.r-project.org/). The quality control was performed using the Bioconductor package arrayQualityMetrics and custom R scripts. We used a linear modeling approach to calculate log ratios, moderated t statistics, log odds ratios of differential expression (B statistic), and p values for each comparison of interest. The p values were adjusted for multiple testing using the Benjamini and Hochberg method, which controls the false discovery rate. Differentially expressed genes were selected based on AbsLogFC > 1 and adjusted p value < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using DAVID (http://david.abcc.ncifcrf.gov/). P < 0.05 was used as the threshold criterion.

The experimental data and microarray design have been deposited in the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE115385.

To assess the link between ASAH1 and the three phenotypes (proliferative, invasive, immune) described in Verfaillie et al. [33], we used the SKCM dataset from TCGA. RNA-sequencing (RNA-seq) and clinical data were available for 472 patients and downloaded from the TCGA data portal (https://portal.gdc.cancer.gov). RNA-seq data were normalized using the Bioconductor package DESeq2 and log2 transformed. Patients were next classified and assigned to one of the three phenotypes using molecular marker genes characteristic of each phenotype and by computing a z-score based on the following formula: z-score = (expression in sample−mean expression in all samples)/standard deviation of expression in all samples. We next computed the median of z-scores of each phenotype and then associated the phenotype with the higher value to each patient. We compared the ASAH1 expression in tumor samples, distinguished by the inferred phenotypes. The p values were calculated by the Wilcoxon rank sum test.

Statistical analysis

Data are presented as averages ± SD and were analyzed by two-sided student t-test. A p value ≤ 0.05 between the experimental and control groups was considered statistical significant. Degree of similarity analysis of genes with similar patterns of expression to MITF across the melanomas was performed using Pearson's correlation coefficient.

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Acknowledgements

This work was supported by Inserm, La Société Française de Dermatologie, and by a grant from INCA (INCA_10573). CP is a fellowship from la Ligue Nationale contre le Cancer. The authors thank Dr. M Sewer (San Diego, USA) for providing the ASAH1 promoter vector. WM3912, WM8, WM3928 and WM3918 human melanoma cell lines were a kind gift from H Meenhard and G Zhang (Wistar melanoma Institute, Philadelphia, USA).

Author contributions

CB, NA-A, RB and TL designed the research, analyzed the results and wrote the manuscript. GT, SD, JC and PB performed and analyzed the immunohistochemistry experiments. NN performed the bioinformatics analysis. JL, DG, CP, CG, KB, VG, PC, SP and BM performed all the other experiments.

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Correspondence to Corine Bertolotto.

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Leclerc, J., Garandeau, D., Pandiani, C. et al. Lysosomal acid ceramidase ASAH1 controls the transition between invasive and proliferative phenotype in melanoma cells. Oncogene 38, 1282–1295 (2019). https://doi.org/10.1038/s41388-018-0500-0

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