Mutant p53s and chromosome 19 microRNA cluster overexpression regulate cancer testis antigen expression and cellular transformation in hepatocellular carcinoma

A subset of hepatocellular carcinoma (HCC) overexpresses the chromosome 19 miRNA cluster (C19MC) and is associated with an undifferentiated phenotype marked by overexpression of cancer testis antigens (CTAs) including anti-apoptotic melanoma-A antigens (MAGEAs). However, the regulation of C19MC miRNA and MAGEA expression in HCCs are not understood. Here we show that, C19MC overexpression is tightly linked to a sub-set of HCCs with transcription-incompetent p53. Using next-generation and Sanger sequencing we found that, p53 in Hep3B cells is impaired by TP53-FXR2 fusion, and that overexpression of the C19MC miRNA-520G in Hep3B cells promotes the expression of MAGEA-3, 6 and 12 mRNAs. Furthermore, overexpression of p53-R175H and p53-R273H mutants promote miR-520G and MAGEA RNA expression and cellular transformation. Moreover, IFN-γ co-operates with miR-520G to promote MAGEA expression. On the other hand, metals such as nickel and zinc promote miR-526B but not miR-520G, to result in the suppression of MAGEA mRNA expression, and evoke cell death through mitochondrial membrane depolarization. Therefore our study demonstrates that a MAGEA-promoting network involving miR-520G, p53-defects and IFN-γ that govern cellular transformation and cell survival pathways, but MAGEA expression and survival are counteracted by nickel and zinc combination.


Transcription competent (TC) and Transcription incompetent (TI) p53 sample clustering of iCluster sub-set and heatmaps for SLC family and cell death
RmiDataset (described above) is subjected to heatmap analysis using 30 signature genes that represent p53 transcription competence (p53-induced and p53-repressed genes) 1 were subjected to cluster analysis (Filtering: SD=30, log transformed, genes and arrays centered; Hierarchical: Genes clustered-correlation centered, arrays clustered-correlation centered, average linkage) to generate heatmap and the two largest clusters that shown clear differences between p53-induced genes and p53-repressed genes were designated as p53 transcription competent (p53-TC) and p53 transcription incompetent (p53-TI) clusters. For the list of patient TCGA IDs of p53-TC and p53-TI clusters (p53-TCTI dataset) please refer Supplementary table-1.

Cancer Cell Line Encyclopedia (CCLE) and cell line p53TCTI dataset details
RNA-seq and miRNA-seq data of cancer cell lines were from Broad Institute CCLE database (https://portals.broadinstitute.org/ccle). The miRNA-seq data was integrated to RNAseq data and cell lines that express similar levels of cumulative C19MC miRNAs were chosen and subjected to cluster analysis using 30 signature genes that represent p53 transcription competence (p53-induced and p53-repressed genes) 1 . Fourteen p53TC and 14 p53TI cell lines were chosen to examine the expression level of MAGEA-3, MAGEA-6 and MAGEA-12 using Graphpad Prism software (v7.04; La Jolla, CA, USA), and represented as 10-90% boxplots.

Immunofluorescence analysis and microscopy
Stable miR-520G GFP expressing cells were plated in 24 well plates at high density (200,000 cells/ml) and cultured for 48hrs with a media change at 24hrs. The cells were methanol fixed in -20°C for a period of 24-48hrs, washed with PBS, blocked for 30 minutes at room temperature with blocking buffer (1% BSA and 0.3% Triton-X100 in PBS). Then the cells were incubated overnight with REST/NRSF Antibody

Mitochondria polarization status analyses
Cells in triplicates after indicated treatment/culture duration were added JC-1 dye (Cayman Chemicals #15003) and Hoechst-33342 (Cayman Chemicals #15547) as per manufacturer's instructions. Depolarized mitochondria, polarized mitochondria and DNA were imaged using green, red and blue channels respectively and pseudo-colored as indicated in figures/legends. The cells with depolarized mitochondria+ DNA condensation and total number of cells per frame were counted and the percentages of cells with depolarized mitochondria were calculated. Image processing was done as described under immunofluorescence section.

Chromatin condensation and DNA fragmentation analyses
Chromatin condensation assay: Cells in triplicates after indicated treatment/culture duration were added 10 nM Hoechst-33342. DNA was imaged using blue channel. The cells with DNA condensation and total number of cells per frame were counted and the percentages of cells with DNA condensation were calculated. Image processing was done as described under immunofluorescence section.

DNA Fragmentation assay:
Cells were treated with 250 M of NiCl 2 , ZnCl 2 or their combination for 24 hrs. The cells were scrapped in existing media, pelleted, washed with PBS and subjected to DNA isolation using QIAamp DNA mini kit (Qiagen # 51304) as per manufacturer's protocol. The DNA was then resolved in 2% agarose gel in TAE buffer alongside of marker (GeneRuler 100 bp DNA Ladder: ThermoFisher Scientific # SM0243).