Investigating the functional significance of an FGFR2 intronic SNP in Breast Cancer

PhDSingle nucleotide polymorphisms present in the second intron of the fibroblast growth factor receptor 2 (FGFR2) gene have been linked with increased risk of breast cancer in several genome wide association studies. The potential effect of those SNPs appeared to be mediated through the differential binding of cis-regulatory elements, such as transcription factors, since all the SNPs in linkage disequilibrium were located in a regulatory DNA region. Preliminary studies have shown that a Runx2 binding site is functional only in the minor, disease associated allele of rs2981578, resulting in increased expression of FGFR2 in cancers from patients homozygous for that allele. Moreover, the increased risk conferred by the minor FGFR2 allele is associated most strongly in oestrogen receptor alpha positive (ERα) breast tumours, suggesting a potential interaction between ERα and FGFR signalling. Here, we have developed a human cell line model system to study the effect of those SNPs on cell behaviour. In an ERα positive breast cancer cell line, rs2981578 was edited using Zinc Finger Nucleases. Unexpectedly, the acquisition of the single risk allele in MCF7 cells failed to affect proliferation or cell cycle progression. Binding of Runx2 to the risk allele was not observed. However FOXA1 binding, an important ERα partner, appeared decreased at the rs2981578 locus in the risk allele cells. Additionally, differences in allele specific expression (ASE) of FGFR2 were not observed in a panel of 72 ERα positive breast cancer samples. Thus, the apparent increased risk of developing ERα positive breast cancer is not caused by rs2981578 alone. Rather, the observed increased risk of developing breast cancer might be the result of a coordinated effect of multiple SNPs forming a risk haplotype in the second intron of FGFR2

KAP1 regulates endogenous retroviruses in adult human cells and contributes to innate immune control

Endogenous retroviruses (ERVs) have accumulated in vertebrate genomes and contribute to the complexity of gene regulation. KAP1 represses ERVs during development by its recruitment to their repetitive sequences through KRAB zinc-finger proteins (KZNFs), but little is known about the regulation of ERVs in adult tissues. We observed that KAP1 repression of HERVK14C was conserved in differentiated human cells and performed KAP1 knockout to obtain an overview of KAP1 function. Our results show that KAP1 represses ERVs (including HERV-T and HERV-S) and ZNF genes, both of which overlap with KAP1 binding sites and H3K9me3 in multiple cell types. Furthermore, this pathway is functionally conserved in adult human peripheral blood mononuclear cells. Cytosine methylation that acts on KAP1 regulated loci is necessary to prevent an interferon response, and KAP1-depletion leads to activation of some interferon-stimulated genes. Finally, loss of KAP1 leads to a decrease in H3K9me3 enrichment at ERVs and ZNF genes and an RNA-sensing response mediated through MAVS signaling. These data indicate that the KAP1-KZNF pathway contributes to genome stability and innate immune control in adult human cells

Functional Analysis of a Breast Cancer-Associated FGFR2 Single Nucleotide Polymorphism Using Zinc Finger Mediated Genome Editing

The authors thank Barts Charity for funding and Breast Cancer Campaign for funding the Barts Breast Tissue Bank

DMBA-induced tumorigenesis study.

<p>Cohorts of female mice (n = 10) were subjected to classical two-step skin carcinogenesis treatment. A) At 58 weeks post initiation, when the experiment was terminated, 100% of DMBA treated wild type mice had developed papillomas while only 50% of knockout treated mice had papillomas. Control TPA treated mice of both genotypes never developed any neoplastic skin lesions. B) Average number of papillomas per mouse at the indicated times after the start of DMBA/TPA treatment. With the exception of one outlier knockout female that developed five papillomas, the remaining knockout mice never developed more than one papilloma per mouse. The maximum number of papillomas on a DMBA treated wild type female was two. In addition to regression of some papillomas over time, when mice died, the average papilloma count was re-calculated for the remaining live mice, thus the average count could go up and down over the course of the experiment. Circles represent mice killed due to papillomas reaching the maximum size specified by Home Office licence. Asterisks represent deaths due to unrelated reasons. C) H&E staining of 4 µm sections from papillomas of wild type and <i>fgf22</i> knockout mice showed identical histology, with exophytic growth and no sign of epithelial invasion (CS cornified squames; Ep epithelium; S stroma). D) Cartoon illustrating difference in response of wild type, <i>fgf22</i> knockout and skin-specific <i>fgfr2b</i> knockout mice to chemically induced carcinogenesis and a proposed model for the phenotypes seen. We hypothesise that lack of downstream cytoprotective signalling mediated via Fgfr2b could potentially promote a more pro-tumorigenic phenotype.</p

Normal wound repair in <i>fgf22</i> knockout mice.

<p>A) H&E stained tissue sections of 3 mm punch biopsy wounds 5 days after wounding. B1–B2) Tissue sections immunostained for 5-bromo-2-deoxyuridine. High power fields in B2) focus on BrdU-positive cells (brown) at the wound site. C and D) Sirius red staining of sections from wild type and <i>fgf22</i> knockout male mice skin 14 days post-wounding. C) represents the same section in bright field as D) in dark field. The spectrum of colours from green, yellow, orange and red progressively exhibits the packing of collagen molecules. (D dermis, HE hyperproliferative epidermis, HF hair follicle, G granulation tissue, BC blood clot. Wound margins are indicated by arrows). Scale bar (200 µm) in A, B1, C, D; (50 µm) in B2. E). The percentage positive BrdU score was calculated from immunostained sections from B1 by counting the total number of positive and negative cells within the wound margins and then deriving the percentage of positive cells. Plots represent mean of three independent wounds per genotype per time point where two slides per wound were counted. Error bars represent SEM. F) and G) Morphometric analysis of wound repair. Morphometric measurements of wounds were performed on three independent wounds for each genotype and each time point. F) Wound gap was calculated as the distance between two margins of the inward growing epithelium and was measured three times for each wound section. G) The area of migrating epithelial tongue was also measured. Error bars represent SEM.</p

FGF signaling in HaCaT cells.

<p>A) HaCaT keratinocytes that had been serum-starved overnight were stimulated with FGF7, FGF10 or FGF22 (100 ng/ml) for 0, 15, 30 or 60 minutes and lysed in sample buffer prior to Western blotting. FGF7 and FGF10 elicited rapid ERK phosphorylation, whereas FGF22 yielded no significant response (phospho-ERK staining appears stronger due to increased length of exposure – to allow visualization of bands). B and C) Serum-starved HaCaT cells were stimulated as above with 100 ng/ml FGF7 (B) or FGF10 (C) either following a 4 h pre-treatment with FGF22 (100 ng/ml) or concomitant with FGF22 treatment. In each case, experiments were repeated (n = 5) and bands quantified using ImageJ (bars represent SEM). No reduction in ERK signalling was observed in either case. D) Forty-eight hours post-transfection with scrambled control non-targeting siRNA or FGF22 siRNA (both at 10 nM), serum-starved HaCaT cells were stimulated with either FGF7 or FGF10 (both 100 ng/ml) for 15 or 60 minutes and levels of phospho-ERK and total ERK analysed by Western blot as described above. No difference in ERK phosphorylation was detected in either case, with experiments repeated (n = 3) and bands quantified using Image (bars represent SEM).</p

Normal skin and pelage hair in <i>fgf22</i> knockout mice.

<p>A) Histological analysis of H&E stained back (A1 and A2) and tail (A3 and A4) skin sections from four-month-old <i>fgf22</i> wild type and knockout mice. No differences were observed in skin thickness or morphology, when back skin or tail skin sections from control and <i>fgf22</i> null mice were compared. (EP epidermis, DE dermis, AD adipose tissue, PC <i>panniculus carnosus</i>, HF hair follicle, * sebaceous gland). Scale bar (200 µm). B) Comparison of pelage hair structure. All major hair shaft types (zigzag, guard and awl/auchene) were present in both wild type and knockout animals. The morphology of different hair types showed no difference between wild type and <i>fgf22</i> knockout mice as shown by light microscopic analysis. Hairs were plucked from eight-week-old wild type mice (n = 100 hairs) and compared with hairs of <i>fgf22</i> knockout mice (n = 100 hairs). Scale bar  = 200 µm. C) Tail epidermis whole-mount preparations from 10 weeks old mice, stained with Mayer’s haemalum, revealed no difference between wild type and <i>fgf22</i> knockout mice in terms of sebaceous gland number and morphology. Hair follicle is indicated by arrow and sebaceous gland by asterisk.</p

<i>In vitro</i> wound healing.

<p>Scratch wound assay was performed on primary keratinocytes isolated from wild type and <i>fgf22</i> knockout male back skin. A) Photographs show the migration of wild type and knockout primary keratinocytes to wounded regions of the monolayer culture at 0, 12, 18 and 32 h post wounding. Scale bar (200 µm). B) Wound closure was monitored, photographed and measured at all timepoints after wounding. Error bars represent SEM.</p

Generation of <i>fgf22</i> knockout mice.

<p>A) Targeting strategy for the <i>fgf22</i> knockout mouse. Schematic representations of the endogenous mouse <i>fgf22</i> locus (middle), targeting construct (top) and disrupted allele (bottom). Black boxes in the wild type allele represent exons 1 to 3. E indicates <i>EcoRI</i> restriction enzyme recognition sites. PCR primers used for genotyping are represented by arrows. B) Southern blots showing successful recombination in ES cells used for generation of <i>fgf22</i> null mice. Of 192 ES clones analysed, two scored positive for homologous recombination (1C12 and 1E1) and displayed identical hybridisation patterns. C) PCR analysis of genomic DNA isolated from ear snips of an heterozygous breeding pair of both identified mutant clones. <i>Fgf22</i> wt (+/+) samples show a single band at 286 bp (mice 1, 3 and 4), <i>fgf22</i> ko (−/−) display a single ko allele at 130 bp (mice 2 and 7) and heterozygous (+/−) samples amplify both wt and ko alleles (mice 5, 6 and 8). D) Confirmation of gene deletion. RT-PCR analysis of cDNA generated from mouse brain. Samples from wild type mice (+/+) display an intense <i>fgf22</i> band, <i>fgf22</i> heterozygous samples (+/−) display same size band of decreased intensity and <i>fgf22</i> knockout samples (−/−) lack the presence of a correct size band. GAPDH primers were used as a control for RNA quality and concentration. Blank represents PCR reaction mix without cDNA.</p