Human MCS5A1 candidate breast cancer susceptibility gene FBXO10 is induced by cellular stress and correlated with lens epithelium-derived growth factor (LEDGF)

Abstract

Genetic variation and candidate genes associated with breast cancer susceptibility have been identified. Identifying molecular interactions between associated genetic variation and cellular proteins may help to better understand environmental risk. Human MCS5A1 breast cancer susceptibility associated SNP rs7042509 is located in F-box protein 10 (FBXO10). An orthologous Rattus norvegicus DNA-sequence that contains SNV ss262858675 is located in rat Mcs5a1, which is part of a mammary carcinoma susceptibility locus controlling tumor development in a non-mammary cell-autonomous manner via an immune cell-mediated mechanism. Higher Fbxo10 expression in T cells is associated with Mcs5a increased susceptibility alleles. A common DNA-protein complex bound human and rat sequences containing MCS5A1/Mcs5a1 rs7042509/ss262858675 in electrophoretic mobility shift assays (EMSAs). Lens epithelium-derived growth factor (LEDGF), a stress-response protein, was identified as a candidate to bind both human and rat sequences using DNA-pulldown and mass spectrometry. LEDGF binding was confirmed by LEDGF-antibody EMSA and chromatin immunoprecipitation (ChIP). Ectopic expression of LEDGF/p75 increased luciferase activities of co-transfected reporters containing both human and rat orthologs. Over-expressed LEDGF/p75 increased endogenous FBXO10 mRNA levels in Jurkat cells, a human T-cell line, implying LEDGF may be involved in increasing FBXO10 transcript levels. Oxidative and thermal stress of Jurkat cells increased FBXO10 and LEDGF expression, further supporting a hypothesis that LEDGF binds to a regulatory region of FBXO10 and increases expression during conditions favoring carcinogenesis. We conclude that FBXO10, a candidate breast cancer susceptibility associated gene, is induced by cellular stress and LEDGF may play a role in expression of this gene.

Keywords: MCS5A; comparative genetics; complex diseases; non-protein-coding genetic variants; rs7042509.

Figure 1.

Comparative genomic analysis of human breast cancer risk associated SNP rs7042509. (A) Alignment of human genomic DNA sequence containing SNP rs7042509 with orthologous sequences from different species. Human genomic DNA sequence containing rs7042509 major allele was compared to genomic reference sequences of other mammalian species to determine the percentage (%) of base pairs that were identical within the full and boxed sequences shown. Orthologous sequences were found by aligning the human major allele sequence to the human reference genome (GRCh37/hg19) using the UCSC genome browser BLAT function followed by the convert function. Rat genomic DNA containing WF and WKY variants of SNV ss262858675, which is in the same relative genomic position compared to human SNP rs7042509, was found to have similar sequence identity to human sequence containing SNP rs7042509. (B) Human genome (GRCh37/hg19) position of rs7042509 relative to a FBXO10 5′-UTR. (C) Rat genome (Baylor 3.4/rn4) position of ss262858675 relative to an Fbxo10 5′-UTR. Both human and rat variants are approximately 3.5 kb from respective 5′-UTRs and CpG islands. The rat sequence identified using BLAT-convert is located approximately 1.7 kb from an Fbxo10 5′-UTR and does not contain dimorphisms between susceptible WF and Mcs5a1-resistant WKY rat alleles.

Figure 2.

Identification of nuclear protein–DNA interactions using EMSA. (A) Protein–DNA complexes identified in EMSA using 41 bp oligonucleotides containing human rs7042509 ± 20 bp flanking sequence, and 41bp oligonucleotides containing rat ss262858675 ± 20 bp flanking sequence with rat thymus NE. Biotin-labeled oligonucleotides (20 fmol) were incubated with 2 μg NE. The shifts marked with an arrow showed commonality between human and rat alleles. Unlabeled DS-oligonucleotides (200×) competed with labeled oligonucleotides for NE binding. (B) Human rs7042509 and rat ss262858675 alleles competed for binding to NE. Excess unlabeled 41 bp DS-oligonucleotides containing human rs7042509 major allele competed with labeled 41 bp oligonucleotides containing rat ss262858675 WKY allele for binding to rat thymus NE and vice versa. Lower-case letter m indicates human major allele of rs7042509. Upper case W indicates rat WKY allele of ss262858675. The number before letters m and W is the fold (×) excess of unlabeled oligonucleotides.

Figure 3.

LEDGF interacted with DNA sequence containing human breast cancer risk associated SNP rs7042509 and rat ss262858675. (A) LEDGF was confirmed to bind human rs7042509 and rat ss262858675 containing oligonucleotides using LEDGF-antibody compared to an IgG control. Human rs7042509 major and rat ss262858675 WKY oligonucleotides were used to bind proteins in WKY rat thymus NE. Anti-LEDGF (2 μg) or IgG control antibodies were added to reaction mixtures prior to the addition of labeled oligonucleotides. (B and C) LEDGF was confirmed to bind human rs7042509 major and rat ss262858675 WKY oligonucleotides by chromatin immunoprecipitation. Chromatin immunoprecipitation was performed using both human Jurkat cell (B) and WKY rat thymus (C) genomic DNA. Human/rat FBXO10/Fbxo10 regions of interest were amplified using species respective primers. Human/rat GAPDH/Gapdh and CDKN1B/Cdkn1b loci were included as respective negative and positive LEDGF binding controls.

Figure 4.

Genomic sequences containing rat Mcs5a1 SNV ss262858675 susceptible WF and resistant WKY alleles bound similarly by LEDGF. (A) ChIP-QPCR results showing LEDGF enrichment at WF and WKY rat genomic regions containing SNV ss262858675. Rat thymus chromatin was used in chromatin immunoprecipitation (ChIP) of LEDGF, RNAPII, or IgG bound DNA. Locus-specific primers were used in QPCR to compare relative protein binding occupancy between strains. Mcs5a1 SNV ss262858675 was contained within the Fbxo10 amplicon. Rat Cdkn1b and Gapdh loci were included as respective positive and negative LEDGF binding controls. There were no statistically significant differences between Fbxo10 ChIP-QPCR groups (P = 0.0752, Kruskal–Wallis test). A Kruskal–Wallis test was significant for Cdkn1b ChIP-QPCR groups (P = 0.0434). LEDGF was enriched more at WKY Cdkn1b compared to WF (P < 0.05, Mann–Whitney’s U-test). (B) LEDGF interacted with RNA polymerase II on both WF and WKY rat chromatin in ChIP-CoIP experiments. Chromatin complexes enriched for LEDGF, RNAPII, or IgG were collected after an immunoprecipitation wash step and before the DNA purification step of ChIP. Amounts of 5% input from initiating ChIP samples were also included. The band-labeled p52 is likely LEDGF/p52.

Figure 5.

Ectopic expression of LEDGF/p75 in Jurkat cells increased luciferase activities of reporters containing human SNP rs7042509 and rat SNV ss262858675 variants. Two copies each of human major, human minor, rat WKY and rat WF, respectively, were cloned into the pGL3-Promoter vector. Control reporter vector pRL-TK Renilla was used as a transfection control. Data are presented as mean ± SD of relative luciferase activity for each co-transfection group. (A) Each variant resulted in luciferase activities that were lower than the pGL3-Promoter parent vector, but higher than the promoterless pGL3-Basic vector. (B) Variant rs7042509 and ss262858675 respective luciferase reporters were co-transfected with pEGFP-LEDGF/p75 expression or pEGFP-C1 control vectors. Kruskal–Wallis test comparing all experimental groups was statistically significant (P = 0.0061). Asterisks indicate P-values < 0.05 of independent Mann–Whitney’s U-tests comparing each LEDGF co-transfection to the respective control. Neither co-transfection with pEGFP-LEDGF/p75 nor pEGFP-C1 vector affected pGL3-Promoter luciferase activity.

Figure 6.

Ectopic over-expression of LEDGF/p75 in Jurkat cells increased FBXO10 levels, but knockdown of LEDGF/p75 had no effect on FBXO10. (A and B) Jurkat cells were transfected with pEGFP-C1 empty or pEGFP-LEDGF/p75 expression plasmids. Following transfection (48 h), cells were sorted and acquired based on green fluorescence using fluorescence activated cell sorting (FACS). Total RNA was used to synthesize cDNA for QPCR to determine FBXO10 (A) and LEDGF/p75 (B) transcript levels. Graphed are mean Target/GAPDH mRNA ± SD for each transfection and green fluorescence sorted group. (A) Transcript levels of FBXO10 were significantly higher in green fluorescence positive cells transfected with pEGFP-LEDGF compared to pEGFP-C1 (Kruskal–Wallis test P = 0.0249 followed by a Mann–Whitney’s U-test P < 0.05). (B) Increased LEDGF transcript levels were confirmed in Jurkat cells transfected with pEGFP-LEDGF that were green fluorescence positive compared to green fluorescence negative (Kruskal–Wallis test P = 0.0249 followed by a Mann–Whitney’s U-test P < 0.05). (C and D) Knockdown of LEDGF/p75 in Jurkat cells using siRNA had no effect on FBXO10 levels. Total RNA was collected for cDNA synthesis 48 h after transfection with LEDGF/p75 specific or scramble control siRNAs. Quantitative PCR was performed to determine FBXO10 (C) and LEDGF (D) mRNA levels. The data presented are expressed as the mean Target/GAPDH mRNA ± SD for each experimental group. Jurkat cell FBXO10 transcript levels were not changed by LEDGf knockdown (P = 0.2482, Mann–Whitney’s U-test). Transcript levels of LEDGF in LEDGF siRNA transfected cells were 30% of levels in control siRNA transfected cells (P = 0.0209, Mann–Whitney’s U-test).

Figure 7.

Oxidative stress and recovery from heat stress resulted in transient increases in FBXO10 mRNA and protein levels. (A) Oxidative stress (0.2 mM H₂O₂) resulted in increased FBXO10 levels in Jurkat cells at 3, 6, and 9 h after initial exposure. Oxidative stress (0.2 mM H₂O₂) resulted in decreased FRMPD1 levels in Jurkat cells at 3 and 6 h, and increased FRMPD1 at 12 h after initial H₂O₂ exposure. Cells were collected at times (hours) shown on the x-axis after initial H₂O₂ exposure. GAPDH was used to standardize FBXO10 mRNA expression. Results are presented as the mean ± SD of Target/GAPDH mRNA levels. Asterisks indicate P-values < 0.05 of Mann–Whitney’s U-tests comparing FBXO10 or FRMPD1 between the respective time-point and 0 h following a significant Kruskal–Wallis test (P = 0.0155, FBXO10; and P = 0.0220, FRMPD1). B. Jurkat cells recovering at 37°C from 42°C heat stress had increased FBXO10 mRNA levels. Jurkat cells were heat stressed for the times (hours) indicated on the x-axis. There was no change in FBXO10 transcript levels during 6 h of heat stress; however, FBXO10 increased in Jurkat cells recovering from 6 h of heat stress. Asterisks indicate P-values < 0.05 of Mann–Whitney’s U-tests comparing FBXO10 between the respective time-point and 0 h following a significant Kruskal–Wallis test (P < 0.001). (C and D) FBXO10 and LEDGF protein levels increased in H₂O₂ exposed and heat stress recovering Jurkat cells, respectively. Relative protein abundance standardized to GAPDH was measured by densitometry and these are shown adjacent to each respective Western blot.