Loss of CITED1, an MITF regulator, drives a phenotype switch in vitro and can predict clinical outcome in primary melanoma tumours

Cell lines

Cell lines were obtained from ATCC. HT144 and SKMEL3 cells were cultured in McCoy’s5A supplemented with 10% and 15% foetal bovine serum (FBS), respectively. A2058, WM852 and WM239 were cultured in RPMI 1640 supplemented with 10% FBS; A375 and HMBC cells were cultured in DMEM supplemented with 10% FBS and SKMEL5 cells were cultured in MEM media supplemented with 10% FBS. Cells were grown in the presence of penicillin and streptomycin (50 I.U./mL) purchased from Invitrogen. Cell media and FBS were purchased from ThermosScientific, HyClone range. As of March 2014, these are part of the Life Technologies (Thermo Fisher Scientific) product portfolio.

Gene expression analysis

RNA was isolated (4 replicates for each treatment) using a Qiagen RNeasy Plus mini-kit and the quality determined using a Bioanalyser (Agilent). Replicates were cell samples from separate wells, but plated on the same day and derived from the same passage number. Gene expression experiments were performed using the Illumina HT-12 array covering more than 47,000 transcripts and known splice variants across the human transcriptome. The raw data was quantile normalized and Illumina control probes were removed from subsequent analysis using BASE (Vallon-Christersson et al., 2009). The data were exported to MeV, log2 transformed and gene and sample centred (Saeed et al., 2003). SAM (significance of microarray analysis) was performed using a two-group comparison; for the siRNA experiment the groups corresponded to siNEG vs. #1 & #3 siCITED1 and for the TGFβ1 experiment the groups corresponded to cells with or without TGFβ1 treatment. In both cases there was a median false discovery risk of 10 false-positive transcripts. Hierarchical clustering was performed to visualize the data. 1009 probes were significantly altered by the TGFβ1 treatment while 312 probes were found to be significantly altered in the siRNA experiment (208 upregulated and 104 downregulated). These data can be found in Files S1 and S2, respectively. DAVID was used to assist in functional annotation of the final gene lists (Huang et al., 2007).

For the publically available data cited, 120 melanoma cell lines from three cohorts (Johansson, Pavey & Hayward, 2007; Hoek et al., 2006; Greshock et al., 2010) analysed by Affymetrix gene expression microarrays were collected, individually MAS5 normalized, and merged into a single cohort. Probe sets were collapsed into single genes and mean-centred across the entire cohort. These 120 cell lines and their associated normalized expression data can be found in Data S2. Data from Harbst et al. were classified using nearest centroid and Pearson correlation. Survival analysis and multivariate cox regression methods were performed in R.

Transient transfections, promoter-reporter assay and TGFβ1-treatment

Transient transfections were performed using Lipofectamine2000 and Opti-MEM reduced serum media (Life Technologies, Waltham, Massachusetts, USA) according to the manufactures recommendations. The siRNA was purchased from Applied Biosystems and the notations in the text: siNEG, #1 siCITED1 and #3 siCITED1 correspond to the catalogue ID numbers #4390843, #s8965 and #s224062 respectively. For the MITF targeting siRNA; N, siM1 and siM3 correspond to the catalogue ID numbers #4390843, #s8790 and #ss8792, respectively. For the luciferase reporter assay, a Dual-Luciferase Reporter assay system #E1910 (Promega, Southampton, UK) was used to measure relative reporter activity on a FLUOstar Omeaga microplate reader (BMG Labtech, Offenburg, Germany). A375 cells were transfected with a luciferase reporter construct harbouring 2.3kb of the MITF-M specific promoter in a PGL2 vector (Wellbrock et al., 2008). A pRL-Renilla Luciferase reporter vector was used as a control for each transfection. CITED1 was overexpressed using a pRc/CMV containing a N-terminal HA-tagged human CITED1 (transcript isoform 1) referred to as pCITED1 in the text (Shioda, Fenner & Isselbacher, 1996). An empty CMV-promoter expression plasmid, pcDNA3.1 (+) was used a negative control. Recombinant human transforming growth factor-β1 (TGFβ1), #PHG9203 was purchased from Invitrogen. For the A2058 gene expression experiment, cells were exposed to either 5 or 10 ng/ml TGFβ1 in serum-free media for 24 h. In the case of the Luciferase reporter assay, cells were serum starved the day after transfection for 3 h and exposed to 5 ng/ml TGFβ1 in serum free media for 24 h prior to harvesting.

Antibodies and immunoblotting

The following antibodies were used: anti-CITED1, #AB15096 from Abcam; anti-MITF (C5 clone), # MA5-14146 from ThermoScientific; anti-MITF (D5 clone) from Dako, #M3621, (used in Fig. 4C); anti-CDKN1A/P21, #2947 and anti-CDKN1C/P57, #2557 were purchased from CellSignaling Technology and anti- β-Actin (AC-15), #A5441 from Sigma-Aldrich. Cell lysates were resolved by SDS-PAGE (pre-cast gels purchased from Life Technologies) and transferred to 0.45 µm PVDF membranes purchased from Millipore by electroblotting. The membranes were blocked in 5% non-fat milk in TBST prior to incubation with primary antibodies diluted 2.5% non-fat milk. The blots were probed with the appropriate secondary antibodies (Pierce Biotechnology, Waltham, Massachusetts, USA) in 5% non-fat milk. The membranes were developed using ECL (GE Healthcare, Buckinghamshire, UK).

A note on reproducibility: Western blots were performed for a variety of purposes; in cases where it was simply a control to establish if siRNA downregulation had been successful, or confirm that an observation seen on mRNA level was also reflective of protein level, then there may have been only one Western blot performed on those particular samples (although often technical repeats were performed). In contrast, observations that were key to the hypothesis presented were performed a number of times to ensure reproducibility e.g., The effect of siCITED1 on upregulation of MITF and CITED1 overexpression on MITF downregulation was seen on samples for multiple experiments performed (i.e., >3): protein samples were used to confirm the initial observations from the GEX (using two siRNAs at two different timepoints) and two transfection of different amounts of pCITED1 plasmid; subsequently, two sets of timecourse experiments were performed to establish the effect independently, at both mRNA and protein level. The effect on P57 was initially observed using transfection of two different amounts pCITED1 plasmid and subsequently confirmed at multiple timepoints in both of two separate sets of timecourse experiments. The P21 effect was observed at single timepoint following siCITED1 treatment in both of two separate timecourse experiments. For the reciprocal effect of siMITF on CITED1 it should be noted that the effect was observed in 3 different cells lines, using at least two separate transfections.

Cell cycle analysis

Flow cytometry was performed on a FACSCalibur (BD Biosciences, San Jose, California, USA) and subsequently analysed using ModFit (Verity House Software, Topsham, Maine, USA). Briefly, following transfection, confluent cells were detached, washed in 1XPBS and fixed in 70% ethanol. Prior to analysis they were stained with a propidium iodide solution and a 20G syringe was used to obtain a homogenous single cell solution. All events were saved (up to 20,000 events per replicate) ungated, using BD Cell Quest and the data exported to ModFit where following selection of the appropriate ploidy status, a standard auto-analysis fit using autolinerarity was performed. We found that a 2-cycle aneuploid-dip/tetraploid was appropriate for HT144 and A2058 while 1-cycle diploid was suitable for A375.

Alamar blue assay

The Alamar blue assay reagent was purchased from Invitrogen (Life Technologies, Waltham, Massachusetts, USA) and used according to the manufactures’ instructions. Briefly, following transfection cells were seeded into 96-well plates at 5,000 cells/well. In each experiment, for each of the treatments i.e.: siNEG, #1 siCITED1 and #3 siCITED1, 8 wells spread over 3 rows were used. At the indicated time points (4, 72, 96 and 120 h post-transfection), Alamar blue was added and the cells incubated at 37 °C for 2 h. Fluorescence was measured (544 nm) on a FLUOstar Omeaga microplate reader (BMG Labtech, Offenburg, Germany). The values obtained at the 4-h time point were used to normalize the fluorescence readings to account for any initial cell counting error. Cells were also seeded in parallel for Western blot analysis (72, 96, 120 h) to ensure successful CITED1 down regulation.

Droplet digital PCR

RNA was isolated from cells using a Qiagen RNeasy Plus mini-kit and quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). cDNA was generated from 50–100 ng total RNAs using ‘iScript Advanced cDNA synthesis for RT-qPCR’ (Bio-Rad). Bio-RAD’s ‘ddPCR Supermix for Probes’ was then used with predesigned TaqMan gene expression assays (Applied Biosystems, Carlsbad, California, USA) consisting of specific primers and FAM labelled probes for MITF (#Hs01117294_m1), MITF-M isoform specific transcript (Hs00165165_m1)*, CITED1 (#Hs00918445_g1) and IPO8 (#Hs00183533_m1). (*There appeared to be no advantage in using the MITF-M isoform specific transcript over the MITF probe that could measure multiple isoforms). A manual cut-off for positive/negative droplets was selected using the Bio-Rad QuantaSoft™ data analysis suite to calculate the relative copies/µl of each transcript.

TGFβ induces expression of the invasive signature genes while suppressing a cohort of proliferative signature genes including CITED1

Hoek et al. (2006) noted that many of the genes that defined the invasive phenotype were commonly TGFβ-driven while at the same time only the proliferative signature phenotype cells were sensitive to TGFβ growth inhibition in vitro. That MITF levels decrease and invasiveness is enhanced in response to TGFβ stimulation was also confirmed subsequently (Pierrat et al., 2012; Pinner et al., 2009). In agreement, we showed that the melanoma cell line A2058 upregulates WNT5A in response to TGFβ exposure and that exogenous Wnt-5a in turn, increased their invasive potential (Jenei et al., 2009). For the present study, in an effort to examine what other phenotype specifying genes were directly regulated by TGFβ, we performed gene expression analysis and found TGFβ treatment resulted in both upregulation of invasive signature genes and suppression of genes characterizing the proliferative phenotype using a SAM two-group comparison (Fig. 1A and Material & Methods section). The effect is most pronounced if only those signature genes that were deemed significantly altered by TGFβ treatment are examined. The original signature set defined by Hoek et al., was redefined as more public datasets became available and has a slightly different but overlapping gene profile based on the top ranked differentially expressed genes (Fig. 1B). Both MITF and CITED1 are in the proliferative cohort and their response to TGFβ treatment was confirmed at protein level in A2058 cells (Fig. 1C).

CITED1 expression positively correlates with the expression of MITF

Examination of publically available gene expression data on 120 melanoma cell lines demonstrated a consistent positive correlation between CITED1 and MITF expression (r = 0.6543). Each cell line was assigned as either ‘proliferative’ or ‘invasive’ based on a score derived from the averaged expression values of the approximately 50 genes in each defining signature set that had matching gene symbols in our data (Fig. 2A). We also confirmed the correlation in cell lines derived from our own lab (Fig. S1). This was important as inconsistency in interlaboratory phenotype signatures has previously been reported (Widmer et al., 2012). We could additionally confirm expression at the protein level (Fig. 2B).

Gene expression analysis reveals CITED1 silencing can induce a phenotype-switch

To investigate the function of CITED1 in melanoma, we transiently downregulated its expression using CITED1 targeting siRNA. We choose the HT144 cell line as it had a relatively high level of detectable CITED1 mRNA and protein expression. A scatterplot of the 120 cell lines assigned as either ‘proliferative’ or ‘invasive’ based on the maximum matching gene signature score, demonstrates the shift in phenotype that occurs following CITED1 downregulation (Figs. 3A and 3B). A heatmap of the expression profiles clearly illustrates that the shift is due to a general induction of the ‘proliferative’ and suppression of the ‘invasive’ cohort (Fig. 3C). It was apparent that the #3 siCITED1 siRNA was not as effective at switching the cells as the #1 siCITED1, this was observed consistently throughout our experiments and may be due to the fact that #3 siCITED1 was not as successful at silencing CITED1 (Fig. 3B, inset).

CITED1 is a reciprocal regulator of MITF and impacts MITF target gene expression

A heatmap highlights the identity of only the significantly differentially induced transcripts between siNEG and both #1 & #3 siCITED1 (Fig. 4A). Of most relevance, we found MITF, a known driver of the proliferative phenotype switch and many of its previously known downstream targets, these also included genes categorized by Gene Ontology annotation (GO) as related to pigmentation and UV/DNA damage response (Fig. 4A) (Hoek et al., 2008b; McGill et al., 2006; Sánchez-Martín et al., 2002; Strub et al., 2011). We could confirm that indeed MITF protein levels were affected by siCITED1 in HT144 cells and that conversely, overexpression of CITED1 in A2058 cells, resulted in downregulation of MITF (Fig. 4B). Strub et al. (2011) identified a large number of genomic targets of MITF by ChIP-seq analysis. A comparison of the genes differentially expressed by siCITED1 compared to siNEG, revealed that there was significant enrichment of these potential targets (Fig. S2A). Notably, genes both up and down regulated by siCITED1 are represented among genes defined as having MITF-occupied promoters (Fig. S2B). We also found that downregulation of MITF using siRNA in HT144 cells (Fig. 4C) and in WM293A, and SKMEL5 cells (Figs. S3A and S3B) resulted in decreased protein expression of CITED1 suggesting reciprocity between these factors.

Induction of MITF by CITED1 silencing transiently restrains cell cycle progression and impacts cell viability

To investigate the effect of CITED1 silencing on melanoma cells behaviour we analysed the cell cycle distribution following siRNA treatment by flow cytometry. In siCITED1 treated HT144 cells we saw G1 accumulation as indicated by an increase in the diploid G1 fraction and a concomitant reduction in the total S-phase fraction peaking at 33 h but also observed at 48 and 72 h post-transfection in comparison to siRNA control HT144 cells. Again, the effect was apparent but not as pronounced using the #3 siCITED1 (Fig. S4A). Similar effects were seen in #1 and #3 siCITED1 treated A2058 and A375 cells (Figs. S4B and S4C).

Owing to the previously reported dependency of MITF-induced cell cycle arrest on CDKN1A/P21 we investigated the levels of several cyclin-dependant kinase inhibitors following CITED1 silencing (Carreira et al., 2005). We found that CDKN1A/P21 was transiently increased in siCITED1 treated HT144 cells relative to the siNEG treated HT144 cells (Fig. 5B). In contrast, in A2058 cells, which do not have detectable levels of CDKN1A/P21 (Fig. S5), the levels of CDKN1C/P57 were supressed in response to CITED1 overexpression (Fig. 5B). We hypothesised therefore that melanoma cells can utilise either CDKN1A/P21 or CDKN1C/P57 to mediate cell cycle arrest induced by MITF and this is reflected in the expression levels of the alternate CDK inhibitors in different melanoma cell lines (Fig. S5).

In agreement with the cell cycle data, an Alamar Blue assay revealed a significant reduction in cell viability as measured by metabolic activity over 5 days in HT144 cells treated with siCITED1 (Fig. 5C). The effect was apparent but not as pronounced in the #3 siCITED1 sample.

The effect of CITED1 silencing on MITF is transient and mediated via promoter activation

We observed that the peak upregulation of MITF and CDKN1A/P21 protein following siCITED1 treatment varied from transfection to transfection, being seen between 24–48 h post-transfection but appearing as unchanged or even downregulated after this time (Fig. 6A). In agreement, later timepoints of the cell cycle analysis (=/>72 h) exhibited little or no change in G1/S-phase distribution or even a reverse pattern (Fig. 5A HT144, and data not shown: A2058, A375). We therefore sought to examine the transcriptional dynamics more closely, map the changes in MITF following CITED1 silencing and see if they corresponded to cell behaviour and changes at the protein level. We used a quantitative droplet digital PCR based assay (Bio-Rad) to measure mRNA in HT144 cells transfected with siCITED1#1 and siNEG as well as A2058 cells transiently overexpressing CITED1 compared to an empty vector control. MITF, CITED1 and IPO8 specific primers and probes were used to measure exact copies/µl of each mRNA from aliquots of the same cDNA solution. Plots of siCITED1(copies/µl)/siNEG(copies/µl) and EV(copies/µl)/pCITED1(copies/µl) show the directional change in MITF and CITED1 relative to the housekeeper IPO8. CITED1 expression is rapidly supressed following siCITED1 treatment of HT144 cells, concomitant with an upregulation of MITF that diminishes over time and in fact is supressed by 100 h in accordance with observations at the protein level (Figs. 6A and 6B). In contrast, overexpression of CITED1 in A2058 cells results in transient suppression of MITF at both protein and transcript level (Figs. 6C and 6D).

The rapid MITF transcriptional response to CITED1 manipulation suggested to us that the effect could be directly mediated at the promoter level. To test this hypothesis, we over expressed an MITF-M promoter-reporter construct and CITED1 in A375 cells. We chose A375 cells, as while they had less endogenous CITED1 and MITF than HT144 or A2058 so as not to cause interference with the assay, we also knew that they could respond adequately as they had an identical G1 accumulation/S-phase decrease to both HT144 and A2058 cells following CITED1 silencing (Fig. S4C). TGFβ treatment was used as a positive control for repression of the MITF-M promoter. CITED1 transfection led to significant suppression of the MITF-M promoter luciferase activity relative to the empty vector control, as did TGFβ treatment alone or combination with CITED1 overexpression (Fig. 6E). There did not appear to be an additive or synergistic effect using both TGFβ treatment and CITED1 overexpression suggesting TGFβ may be dependent on CITED1 for MITF suppression.

The CITED1-silenced gene signature predicts outcome in primary melanoma

The ‘proliferative’ and ‘invasive’ signature phenotypes have served to define the gene expression classification of melanoma cell lines. However, primary tumours and metastatic lesions have also been molecularly classified into several distinct groups by gene expression profiling (Harbst et al., 2012; Jonsson et al., 2010). The four-class structure found in tumours consists of the ‘pigmentation,’ ‘proliferative,’ ‘high-immune’ and ‘normal-like’ subgroups with a subset falling into an unclassifiable cohort (Jonsson et al., 2010). We used the same tumour classification to subtype the 120 cell lines that had publically available expression data and could show that the tumour ‘pigmentation’ subgroup that highly expresses MITF, corresponds to the cell line ‘proliferative’ phenotype described by Hoek et al. Accordingly, the tumour ‘proliferative’ and ‘high-immune’ subgroups comprise the cell line ‘invasive’ phenotype (Fig. 7A). It is worth noting that the names of the tumour subgroups were derived from a description of the differentially expressed genes that comprised each molecular classification while the ‘invasive-proliferative’ switching phenotypes were named to reflect the behaviour exhibited by lines classified by one or other signature. This distinction helps to explain the confusing occurrence that both classifications have a group referred to as ‘proliferative’ although they are not equivalent.

The overlap between the primary tumour classifying and cell line classifying systems allows us to infer that CITED1 expression is most likely restricted to a subset of MITF high ‘pigmentation’ subtype tumours. As the tumour subtype classification was shown to be prognostically significant in primary melanomas we were interested to know if CITED1 expression itself was independently predictive of outcome. Previously we reported on the analysis of 223 primary lesions using the Illumina WG-DASL protocol (Harbst et al., 2012). As the CITED1 probe in this assay did not produce reliable data we instead derived a CITED1-silenced gene signature score based on the differentially expressed genes from the HT144 siCITED1 experiment (Fig. 3). We therefore effectively created a multi-gene surrogate expression signature rather than using CITED1 gene expression itself. We subsequently interrogated the gene expression data on the primary melanoma lesions using a nearest centroid approach derived from the CITED1-silenced gene signature. This revealed that primary melanomas with a gene expression signature most similar to the CITED1-silenced signature (CITED1low-class) had a significantly better outcome than those with a signature most disparate from the CITED1-silenced signature (CITED1high-class) (Fig. 7B). Importantly, the CITED1 signature classing had independent prognostic information (HR 1.85, CI [0.30–0.98], p = 0.044) from the AJCC staging system (HR 5.05, CI [2.42–10.55], p = 1.64 × 10 − 5). Accepting the caveat that we depend here on a proxy gene-signature, these data indirectly imply that CITED1 expression is a potential prognostic indicator in primary melanomas and the transcriptional program influenced by CITED1 expression determines tumour behaviour in vivo.