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Howlin J, Cirenajwis H, Lettiero B, Staaf J, Lauss M, Saal L, Borg Å, Gruvberger-Saal SK, Jönsson GB.2014. Loss of CITED1, an MITF regulator, drives a phenotype switch in vitro and can predict clinical outcome in primary melanoma tumours. PeerJ PrePrints2:e686v1https://doi.org/10.7287/peerj.preprints.686v1
CITED1 is a non-DNA binding transcriptional co-regulator whose expression can distinguish the ‘proliferative’ from ‘invasive’ signature in the phenotype-switching model of melanoma. We have found that, in addition to other 'proliferative' signature genes, CITED1 expression is repressed by TGFβ while the ‘invasive’ signature genes are upregulated. In agreement, CITED1 positively correlates with MITF expression and can discriminate the MITF-high/pigmentation tumor molecular subtype in a large cohort (120) of melanoma cell lines. Interestingly, CITED1 overexpression significantly suppressed MITF promoter activation, mRNA and protein expression levels while MITF was transiently upregulated following siRNA mediated CITED1 silencing. Conversely, MITF siRNA silencing resulted in CITED1 downregulation indicating a reciprocal relationship. Whole genome expression analysis identified a phenotype shift induced by CITED1 silencing and driven mainly by expression of MITF and a cohort of MITF target genes that were significantly altered. Concomitantly,we found changes in the cell-cycle profile that manifest as transient G1 accumulation, increased expression of CDKN1A and a reduction in cell viability. Additionally, we could predict survival outcome by classifying primary melanoma tumors using our in vitro derived ‘CITED1-silenced’ gene expression signature. We hypothesize that CITED1 acts a regulator of MITF, functioning to maintain MITF levels in a range compatible with tumourigenesis.
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A scatter plot of CITED1/MITF expression
(a) A scatter plot of CITED1 expression versus MITF expression in our in-house cell lines subjected to gene expression analysis in agreement with the observation in the public data set, ( see Fig 2a. (b) Figure 2a reproduced ) with additional labelling to indicate the common cell lines.
(a) Of the 5578 unique occupied genes (those associated with genomic regions bound by MITF) identified by Strub et al., 5169 could be found in the Illumina HT12 gene set, mapping to 8272 probes by searching on gene symbol (Strub et al. 2011) . Of the 312 probes significantly changed by siCITED1, 41% or 128 are found in this list. (b) A Venn diagram indicating the number of genes significantly up or down regulated by siCITED1 represented in the MITF occupied gene list.
The effect of silencing MITF on CITED1 in WM239A and SKMEL5 cells
(a) Western blot showing the effect of silencing MITF using two siRNAs (siM1, siM3) on both MITF and CITED1 levels in WM239A cells relative to a negative control siRNA (N) at 48 hours post-transfection. β-Actin is used as a loading control. (b) Western blot showing the effect of silencing MITF using the siRNA siM3 on both MITF and CITED1 levels in SKMEL5 cells relative to a negative control siRNA (N) at 48 hours post-transfection. β-Actin is used as a loading control.
Additional data for % changes in cell cycle distribution
(a) A bar chart showing the % change in cell cycle distribution in both #1 and #3 siCITED1 treated HT144 cells relative to siNEG treated HT144 cells. The reduction in total S-phase is shown at 33 hours, 48 hours and 72 hours post-transfection in addition to the corresponding increase in the diploid G1 fraction. (b) A bar chart showing the % change in cell cycle distribution in both #1 and #3 siCITED1 treated relative to siNEG treated A2058 cells. The reduction in total S-phase and corresponding increase in G1 is shown at 48 hours post-transfection. (c) A bar chart showing the % change in cell cycle distribution in both #1 and #3 siCITED1 treated relative to siNEG treated A375 cells. The reduction in total S-phase and corresponding increase in G1 is shown at 48 hours post-transfection.
The rheostat model of MITF/CITED1 action in melanoma
The rheostat model of MITF action in melanoma indicating the hypothesised role of CITED1 in the regulation of MITF expression and melanoma cell behaviour. The model is an original adaptation of those previously proposed by others, now incorporating our data (Hoek & Goding 2010; Carreira et al. 2006; Cheli et al. 2011).