Factor interaction analysis for chromosome 8 and DNA methylation alterations highlights innate immune response suppression and cytoskeletal changes in prostate cancer

Abstract

Background: Alterations of chromosome 8 and hypomethylation of LINE-1 retrotransposons are common alterations in advanced prostate carcinoma. In a former study including many metastatic cases, they strongly correlated with each other. To elucidate a possible interaction between the two alterations, we investigated their relationship in less advanced prostate cancers.

Results: In 50 primary tumor tissues, no correlation was observed between chromosome 8 alterations determined by comparative genomic hybridization and LINE-1 hypomethylation measured by Southern blot hybridization. The discrepancy towards the former study, which had been dominated by advanced stage cases, suggests that both alterations converge and interact during prostate cancer progression. Therefore, interaction analysis was performed on microarray-based expression profiles of cancers harboring both alterations, only one, or none. Application of a novel bioinformatic method identified Gene Ontology (GO) groups related to innate immunity, cytoskeletal organization and cell adhesion as common targets of both alterations. Many genes targeted by their interaction were involved in type I and II interferon signaling and several were functionally related to hereditary prostate cancer genes. In addition, the interaction appeared to influence a switch in the expression pattern of EPB41L genes encoding 4.1 cytoskeleton proteins. Real-time RT-PCR revealed GADD45A, MX1, EPB41L3/DAL1, and FBLN1 as generally downregulated in prostate cancer, whereas HOXB13 and EPB41L4B were upregulated. TLR3 was downregulated in a subset of the cases and associated with recurrence. Downregulation of EPB41L3, but not of GADD45A, was associated with promoter hypermethylation, which was detected in 79% of carcinoma samples.

Conclusion: Alterations of chromosome 8 and DNA hypomethylation in prostate cancer probably do not cause each other, but converge during progression. The present analysis implicates their interaction in innate immune response suppression and cytoskeletal changes during prostate cancer progression. The study thus highlights novel mechanisms in prostate cancer progression and identifies novel candidate genes for diagnostic and therapeutic purposes. In particular, TLR3 expression might be useful for prostate cancer prognosis and EPB41L3 hypermethylation for its detection.

Figure 1

Bioinformatic analysis of microarray expression data. A: The distributions of the raw p-values of all genes for the main effects hypomethylation of LINE-1 retrotransposons (hypo), alteration of chromosome 8 (chrom8) and for the interaction effect (hypo AND chrom8). In each graph the numbers of genes with p-values in the indicated ranges (in increments of 0.02) are shown. The uniform distributions of the p-values for the two main single effects indicate that not more genes are declared significant than expected at random, whereas for the interaction effect a skewed distribution is observed, i.e. significantly small p-values are assigned to a large number of genes. B: The subgraph induced by the top 15 GO terms identified by the classic algorithm for scoring GO terms for enrichment. In this graph, nodes represent GO terms and edges represent parent-child relationships, i.e. an arrow from node A to node B indicates that the genes in B are a subset of the genes in A. Black arrows indicate is-a relationships and red arrows part-of relationships, as defined in the Gene Ontology nomenclature. In general, GO terms are represented by ellipses with the corresponding GO IDs plotted inside. GO IDs surrounded by boxes instead of ellipses indicate the 10 most significant GO terms as identified by enrichment analysis. Color represents the relative significance of enrichment, ranging from dark red (most significant) to light yellow (least significant). Interesting areas in the GO graph defined by significant related GO terms are highlighted by different underlying colors (e.g. red for immune response). C: The subgraph induced by the top 15 GO terms identified by the weight algorithm for scoring GO terms for enrichment. For a detailed description see Fig. 1B. Circles instead of ellipses indicate GO terms that are found significant by the classic algorithm but not by the weight algorithm. see Additional file 3 for a listing of all GO groups in Fig. 1B and 1C

Figure 2

Box plot representation of microarray analysis results for selected genes. A: HOXB13; B: BCCIPA; C: HMG20B; D: GADD45A; E: MX1; F: MX2; G: CCL5; H: IFNB1; I: IRF3; J: STAT3; K: TLR2; L: TLR3; M: EPB41L3; N: EPB41L4B; O: LAMA2; P: SPON2; Q: FBLN1. In each graph, the expression values (log-scale) for the respective genes are depicted for the cancers with neither hypomethylation of LINE-1 retrotransposons nor alteration of chromosome 8 (null), hypomethylation only (hypo), alteration of chromosome 8 only (chrom8) and both alterations (chrom8 AND hypo). The p-values refer to the result of the interaction analysis (see Methods for details).

Figure 3

Box plot comparison of expression of selected genes in prostate cancer vs. benign tissues by real-time quantiative RT-PCR. A: HPN; B: HOXB13; C: BCCIP; D: GADD45A; E: MX1; F;TLR3; G: EPB41L3; H: EPB41L4B; I: FBLN1. Expression values were determined for each sample in duplicate with < 10% variation. They are indicated relative to the reference gene TBP determined in the same fashion. T: cancer samples (n = 47), N: benign tissue samples (n = 13); p-values according to Mann-Whitney tests.

Figure 4

DNA methylation analysis of EPB41L3 and GADD45A. A: Bisulfite sequencing of the EPB41L3 promoter in selected cell lines and prostate tissue samples. Du145, PC3, 22Rv1, and LNCaP: prostate cancer cell lines. T: tumor tissues; N: benign tissues, UP: normal urothelial cells. B: Examples of Methylation-specific PCR for EPB41L3. M: primers specific for methylated promoter sequence, U: primers specific for unmethylated promoter sequence. T: prostate carcinoma tissue, N: benign prostate tissue, Bl: blood leukocyte DNA as unmethylated control, Du: Du145 DNA as methylated control. C: Effect of 5-aza-dC and SAHA on GADD45A expression. For each cell line from left to right: untreated (white bars), 5-aza-dC (horizontal stripes), SAHA (vertical stripes), 5-aza-dC + SAHA (black bars). D: Bisulfite sequencing of GADD45A in selected cell lines and prostate tissue samples. Du145, PC3, 22Rv1, and LNCaP: prostate cancer cell lines. T: tumor tissues; N: benign tissues