Background
The disco-interacting protein 2 homolog C (
DIP2C), an uncharacterised gene expressed at high level in most human solid tissues and adult tumour types [
1], was identified by us as a putative cancer gene in exome-wide mutational analyses of hormone-receptor negative breast tumours [
2,
3]. Further studies have estimated the
DIP2C somatic mutation prevalence at ~5% of breast cancer cases [
4]. Recently,
DIP2C was also found mutated in 9-14% of small-cell lung cancers [
5], strengthening the evidence for a role in tumorigenesis.
Conserved across species, the human DIP2 family proteins DIP2A, DIP2B and DIP2C are highly similar, with DIP2C and DIP2B sharing 72.2% amino acid identity [
6]. All three proteins are predicted to contain DMAP1 binding (pfam06464) and AMP binding (pfam00501) domains, which give properties of binding to the transcriptional co-repressor DNA methyltransferase 1 associated protein 1 (DMAP1), and acting enzymatically via an ATP-dependent covalent binding of AMP to their substrate, respectively. The most studied family member, DIP2A, is a potential cell membrane receptor for Follistatin-like 1 (FSTL1), a secreted protein with possible role in e.g. regulation of embryonic tissue formation, joint inflammation and allograft tolerance [
7,
8]. Nervous-system specific expression of Dip2 protein has been shown in mouse and Drosophila during embryonic development [
9], which is interesting considering that all three isoforms are associated with neurodevelopmental disorders. The
DIP2A gene is a candidate for developmental dyslexia and autism [
10,
11], DIP2B deficiency has been associated with mental retardation [
6], and DIP2C has been implicated in developmental delay [
12]. While
DIP2A lacks known association to cancer development, an SNP associated with
DIP2B expression has been proposed to affect colorectal cancer risk [
13]. Thus far
DIP2C is the only family member that has been identified as a candidate cancer gene through somatic mutation analysis.
Mutations found in breast cancers are predicted to inactivate DIP2C function [
4]. To investigate the role of
DIP2C inactivation in human cancer and identify processes affected by the activity of this gene we engineered and characterised human
DIP2C knockout cell lines which revealed that loss of DIP2C affects cell growth, cell cycle regulation, and migratory capacity, potentially through regulation of DNA methylation.
Discussion
Genomic profiling has revealed a large subset of genes as likely drivers of breast tumorigenesis, including the hitherto non-characterized
DIP2C gene [
2‐
4] which is interesting in association to cancer development since it may belong to the growing number of epigenetic regulators implicated in cancer. Here we exploited rAAV-mediated gene targeting to knock out one or two alleles of
DIP2C in human cancer cells, enabling
DIP2C to be studied under control of its endogenous promoter. Gene editing was attempted in two cell lines well established for use with the rAAV-targeting method, with the low targeting efficiency (no viable clones for untransformed mammary MCF10a cells and <1% targeted RKO cells) potentially owing to the significant changes in growth and transcription depending on loss of
DIP2C that were observed in targeted RKO cells. This could mean that inactivating
DIP2C mutations are dependent on additional genetic perturbation for cells to be able to survive their introduction. In support of this theory,
DIP2C mutation was reported as a late event when the timing of mutations and chromosome rearrangements were investigated in a breast cancer genome [
43]. If this is a general observation in affected tumours has however not been studied.
Even though the
DIP2C mRNA level remained at ~50% of that of parental RKO, heterozygous
DIP2C
+/−
cells also to some degree adopted the stretched morphology and decreased growth rate seen in
DIP2C
−/−
cells, which suggests possible haploinsufficiency or a dominant negative effect of the damaged allele. The potentially functional DMAP1 binding domain spans amino acids 9-119 of DIP2C, encompassing sequence from exons 1-4. In the knockout cells a large part of DIP2C exon 9 was deleted, possibly generating a truncated protein with a preserved DMAP1 binding domain which could cause binding of non-functional DIP2C and blocking of normal DIP2C function in heterozygous clones. The predicted inactivating missense and frameshift mutations in breast cancer that motivated this study and the mutations later identified in lung cancer are all heterozygous and localized downstream of the DMAP1 binding domain [
4] (Fig.
1a), suggesting that such a mechanism could be active also in patient tumours. Furthermore this observation is interesting as decreased expression levels of both
DIP2C and
DIP2B are associated with mental retardation [
6,
12].
Disruption of DNA methylation patterns is a hallmark of cancer, and both promoter hypermethylation and global loss of DNA methylation is observed in cancers [
44]. In
DIP2C knockout cells hypermethylation was the dominating effect at CpG islands and in sites located closely upstream of transcription start sites, which agrees with the DNA methylation pattern associated with gene silencing [
44,
45]. Typically heavily methylated in normal tissue [
44], isolated CpGs in the genome were instead preferentially hypomethylated. Differential gene promoter methylation was shown correlated to changes in gene expression, particularly for those genes with more than fourfold differential expression, suggesting that
DIP2C KO methylation defects directly influence gene expression. In contrast, as expected [
44,
45], gene expression was not correlated to differential methylation at gene bodies. Although a physical interaction has not been demonstrated between DIP2C and DMAP1, for which DIP2C has a putative binding site, based on these results we cannot rule out that DIP2C is involved in the regulation of DNA methylation through this pathway or by another mechanism. Although important in cancer development [
46], DNMT regulation during tumorigenesis is poorly understood. Both DNMT1 and DMAP1 have multiple known interaction partners [
41,
47,
48], and DMAP1 has suggested roles not only in DNA methylation but also in histone acetylation and DNA repair [
47,
49], suggesting areas of investigation for future studies of DIP2C function.
In
DIP2C knockout cells, 10-15% of the functionally assigned differentially expressed genes were involved with regulation of cell death processes (Additional file
2: Table S5). The
DIP2C knockout cells did not show signs of apoptosis but displayed multiple markers for cellular senescence, a mechanism activated in ageing cells or by different forms of cellular stress, such as oncogenic signalling, as protection against inappropriate growth signals [
50]. Seemingly contradictory, senescent cells can secrete factors that e.g. promote EMT and inflammation, which could stimulate tumorigenic processes [
51]. Functional consequences of cellular senescence induction in
DIP2C
−/−
cells cannot be determined from this cell system, but interestingly EMT and inflammation were among processes enriched for in the differentially expressed and/or methylated genes. Notably,
DIP2C is among seven genes on chromosome 10p14-15 whose loss has been associated with the ability to escape from senescence in cervical cancer [
52]. Such ability is suggested to be an important mechanism in the progression from pre-malignant to malignant cells [
50]. Overexpression of
DIP2C in CRC cells did not induce senescence markers in the present study, which is consistent with literature on overexpression in primary human fibroblasts and keratinocytes [
52].
Epithelial-mesenchymal transition is a reversible spectrum of transitory cell states where cells express different levels of epithelial and mesenchymal markers [
37,
53]. The RKO cell line has increased mesenchymal characteristics compared to several other colorectal cancer cell lines, with low expression of cytoskeletal structure and cell adhesion proteins and high migration and invasive capability [
54]. The EMT inducer ZEB1 is previously reported expressed in RKO cells [
39], meaning that loss of
DIP2C may augment the RKO EMT phenotype by further ZEB1 upregulation as suggested by the data presented herein. Furthermore, high
CD44 and low
CD24 expression, characteristics associated with the breast cancer stem cell phenotype and the EMT state, was revealed in
DIP2C KO cells, with potential implications for treatment and metastasis ability [
37,
38]. The epithelial and mesenchymal states impact the stages of tumorigenesis differently [
37,
53], suggesting that timing may influence the effect of
DIP2C mutations on tumour development. Here, DIP2C KO caused increased migration in the scratch assay, suggesting possible impact on e.g. the ability to metastasize.