Characterizing Genetic Susceptibility to Breast Cancer in Women of African Ancestry.

Background: Genome-wide association studies have identified approximately 100 common genetic variants associated with breast cancer risk, the majority of which were discovered in women of European ancestry. Because of different patterns of linkage disequilibrium, many of these genetic markers may not represent signals in populations of African ancestry.Methods: We tested 74 breast cancer risk variants and conducted fine-mapping of these susceptibility regions in 6,522 breast cancer cases and 7,643 controls of African ancestry from three genetic consortia (AABC, AMBER, and ROOT).Results: Fifty-four of the 74 variants (73%) were found to have ORs that were directionally consistent with those previously reported, of which 12 were nominally statistically significant (P < 0.05). Through fine-mapping, in six regions (3p24, 12p11, 14q13, 16q12/FTO, 16q23, 19p13), we observed seven markers that better represent the underlying risk variant for overall breast cancer or breast cancer subtypes, whereas in another two regions (11q13, 16q12/TOX3), we identified suggestive evidence of signals that are independent of the reported index variant. Overlapping chromatin features and regulatory elements suggest that many of the risk alleles lie in regions with biological functionality.Conclusions: Through fine-mapping of known susceptibility regions, we have revealed alleles that better characterize breast cancer risk in women of African ancestry.Impact: The risk alleles identified represent genetic markers for modeling and stratifying breast cancer risk in women of African ancestry. Cancer Epidemiol Biomarkers Prev; 26(7); 1016-26. ©2017 AACR.

Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer.

Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2'-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner.

Genome-wide association studies in women of African ancestry identified 3q26.21 as a novel susceptibility locus for oestrogen receptor negative breast cancer.

Multiple breast cancer loci have been identified in previous genome-wide association studies, but they were mainly conducted in populations of European ancestry. Women of African ancestry are more likely to have young-onset and oestrogen receptor (ER) negative breast cancer for reasons that are unknown and understudied. To identify genetic risk factors for breast cancer in women of African descent, we conducted a meta-analysis of two genome-wide association studies of breast cancer; one study consists of 1,657 cases and 2,029 controls genotyped with Illumina's HumanOmni2.5 BeadChip and the other study included 3,016 cases and 2,745 controls genotyped using Illumina Human1M-Duo BeadChip. The top 18,376 single nucleotide polymorphisms (SNP) from the meta-analysis were replicated in the third study that consists of 1,984 African Americans cases and 2,939 controls. We found that SNP rs13074711, 26.5 Kb upstream of TNFSF10 at 3q26.21, was significantly associated with risk of oestrogen receptor (ER)-negative breast cancer (odds ratio [OR]=1.29, 95% CI: 1.18-1.40; P = 1.8 × 10 − 8). Functional annotations suggest that the TNFSF10 gene may be involved in breast cancer aetiology, but further functional experiments are needed. In addition, we confirmed SNP rs10069690 was the best indicator for ER-negative breast cancer at 5p15.33 (OR = 1.30; P = 2.4 × 10 − 10) and identified rs12998806 as the best indicator for ER-positive breast cancer at 2q35 (OR = 1.34; P = 2.2 × 10 − 8) for women of African ancestry. These findings demonstrated additional susceptibility alleles for breast cancer can be revealed in diverse populations and have important public health implications in building race/ethnicity-specific risk prediction model for breast cancer.

Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter.

Basal-like breast cancer is a molecularly distinct subtype of breast cancer that is highly aggressive and has a poor prognosis. MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in basal-like breast cancer. We here report that epigenetic modifications at the miR-29c promoter, rather than copy number variation of the gene, may drive the lower expression of miR-29c in basal-like breast cancer. Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c. Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes. Furthermore, inhibition of CpG methylation with 5-aza-CdR increases miR-29c expression in basal-like breast cancer cells. Flourescent In Situ Hybridization (FISH) revealed chromosomal abnormalities at miR-29c loci in breast cancer cell lines, but with no correlation between copy number variation and expression of miR-29c. Our data demonstrated that dysregulation of miR-29c in basal-like breast cancer cells may be in part driven by methylation at CpG sites. Epigenetic control of the miR-29c promoter by epigenetic modifiers may provide a potential therapeutic target to overcome the aggressive behavior of these cancers.

Functional promoter variants in sphingosine 1-phosphate receptor 3 associate with susceptibility to sepsis-associated acute respiratory distress syndrome.

The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor S1PR3 are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African- and ED samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Because of limited sample size, conclusive findings could not be drawn in African-descent ARDS subjects; however, significant associations were found for two promoter SNPs (rs7022797 -1899T/G; rs11137480 -1785G/C), across two ED samples supporting the association of alleles -1899G and -1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter; reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge; and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.

An intronic MYLK variant associated with inflammatory lung disease regulates promoter activity of the smooth muscle myosin light chain kinase isoform.

Intronic single-nucleotide polymorphisms (SNPs) are commonly associated with complex diseases but exhibit unknown biologic functionality. Myosin light-chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK, plays a key pathophysiological role in complex diseases including acute lung injury (ALI) and asthma. We studied the potential regulatory roles of two intronic MYLK SNPs (rs936170 and rs820336) previously associated with ALI and asthma. Due to their genomic location at the junction encoding the non-muscle and smooth muscle MLCK (smMLCK) isoforms, we first identified the transcription start site (TSS) of the smMLCK isoform, and isolated a 2,954-bp DNA fragment upstream of the smMLCK TSS. Serial 5' deletion of the fragment revealed a proximal promoter region exhibiting strong promoter activity with potential inhibitory elements in the distal region. Site-directed mutageneses and luciferase reporter assays showed no effect of the distal promoter SNP rs936170 on smMLCK promoter activity. In contrast, SNP rs820336, located in an enhancer/repressor region downstream of TSS, was identified to regulate smMLCK promoter activity in an allelic-dependent manner. The A allele interrupted the binding site for Forkhead box protein N1 (FOXN1), a transcription factor governing expression of immune response genes. Silencing of FOXN1 expression (siRNA) reduced FOXN1 interaction with cis-regulatory elements in proximity to rs820336 and significantly decreased smMLCK expression. These functional insights into the involvement of intronic MYLK SNPs further strengthen the concept that MYLK contributes to inflammatory disease susceptibility and represents an attractive molecular target in complex inflammatory disorders.

A transcribed pseudogene of MYLK promotes cell proliferation.

Pseudogenes are considered nonfunctional genomic artifacts of catastrophic pathways. Recent evidence, however, indicates novel roles for pseudogenes as regulators of gene expression. We tested the functionality of myosin light chain kinase pseudogene (MYLKP1) in human cells and tissues by RT-PCR, promoter activity, and cell proliferation assays. MYLKP1 is partially duplicated from the original MYLK gene that encodes nonmuscle and smooth muscle myosin light chain kinase (smMLCK) isoforms and regulates cell contractility and cytokinesis. Despite strong homology with the smMLCK promoter (∼ 89.9%), the MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. Moreover, MYLKP1 and smMLCK exhibit negatively correlated transcriptional patterns in normal and cancer cells with MYLKP1 strongly expressed in cancer cells and smMLCK highly expressed in non-neoplastic cells. For instance, expression of smMLCK decreased (19.5 ± 4.7 fold) in colon carcinoma tissues compared to normal colon tissues. Mechanistically, MYLKP1 overexpression inhibits smMLCK expression in cancer cells by decreasing RNA stability, leading to increased cell proliferation. These studies provide strong evidence for the functional involvement of pseudogenes in carcinogenesis and suggest MYLKP1 as a potential novel diagnostic or therapeutic target in human cancers.

Regulation of myosin light chain kinase expression by angiotensin II in hypertension.

BACKGROUND: Increased growth and contraction of vascular smooth muscle cells (VSMCs) are major abnormalities in many vascular disorders. To investigate the signaling pathways that mediate these processes, we studied the expression of smooth muscle myosin light chain kinase (smMLCK) in VSMCs. METHODS: Primary cultured VSMCs isolated from normotensive Wistar-Kyoto (WKY) rats were treated with angiotensin II (Ang II). smMLCK expression was examined in the cells using western blot analysis. In vivo, a specific inhibitor of smMLCK or MAP kinase kinase (MEK) was delivered to spontaneously hypertensive rats (SHRs) using an osmotic pump, and their blood pressures were measured using tail-cuff sphygmomanometry. RESULTS: Expression of smMLCK protein is rapidly increased by Ang II, an important agonist responsible for increased vasoconstriction and vascular remodeling, in concert with increased myosin light chain phosphorylation. Inhibiting Ang II type 1 (AT1) receptor, Ras, or MEK blocked the Ang II-induced increase in smMLCK expression. In vivo, inhibiting MEK decreased smMLCK expression, blood pressure, and vascular thickening in SHRs. Moreover, inhibiting smMLCK activity decreased blood pressure and smooth muscle mass in arteries in SHRs. CONCLUSIONS: The regulation of smMLCK expression by Ang II via Ras signaling is important in the regulation of vascular remodeling and blood pressure. Targeting this pathway could be an effective strategy for developing novel therapeutics to treat hypertension.

Naturally extended CT . AG repeats increase H-DNA structures and promoter activity in the smooth muscle myosin light chain kinase gene.

Naturally occurring repeat sequences capable of adopting H-DNA structures are abundant in promoters of disease-related genes. In support of this, we found (CT)(22) . (AG)(22) repeats in the promoter of smooth muscle myosin light chain kinase (smMLCK), a key regulator of vascular smooth muscle function. We also found an insertion mutation that adds another six pairs of CT . AG repeats and increases smMLCK promoter activity in spontaneously hypertensive rats (SHR). Therefore, we used the smMLCK promoters from normotensive and hypertensive rats as a model system to determine how CT . AG repeats form H-DNA, an intramolecular triplex, and regulate promoter activity. High-resolution mapping with a chemical probe selective for H-DNA showed that the CT . AG repeats adopt H-DNA structures at a neutral pH. Importantly, the SHR promoter forms longer H-DNA structures than the promoter from normotensive rats. Reconstituting nucleosomes on the promoters, in vitro, showed no difference in nucleosome positioning between the two promoters. However, chromatin immunoprecipitation analyses revealed that histone acetylations are greater in the hypertensive promoter. Thus, our findings suggest that the extended CT . AG repeats in the SHR promoter increase H-DNA structures, histone modifications, and promoter activity of the smMLCK, perhaps contributing to vascular disorders in hypertension.

Involvement of Ras-regulated myosin light chain phosphorylation in the captopril effects in spontaneously hypertensive rats.

BACKGROUND: Early treatment with captopril prevents the development of hypertension by inhibiting the generation of angiotensin II and smooth muscle contraction. Although smooth muscle contraction is regulated by myosin light chain phosphorylation (MLC-P), the role of MLC-P in captopril effects in hypertension has not been described. Therefore, we treated spontaneously hypertensive rats (SHR) with captopril and investigated the effects of this agent on downstream signaling. METHODS: Male SHR (n = 12) were treated with captopril (3.7 mmol/L in drinking water) beginning in utero and continuing up to 12 weeks of age. Age- and sex-matched untreated SHR and Wistar-Kyoto (WKY) rats were used as controls. Rats were split into three subgroups and were sacrificed at 12, 18, or 24 weeks of age. Systolic blood pressure, left ventricular weight, and body weight were measured. Mesenteric arteries were removed for histologic and biochemical studies. RESULTS: At 12 weeks, captopril significantly decreased systolic blood pressure (from 198 +/- 10 to 125+/-16 mm Hg), reduced left ventricular weight-to-body weight ratios (from 2.94 +/- 0.06 to 2.17 +/- 0.08 mg/g), and prevented vascular remodeling in mesenteric arteries in SHR. Ras expression, extracellular receptor kinase phosphorylation (ERK-P), myosin light chain kinase (MLCK) expression, and MLC-P were all significantly increased in mesenteric arteries in untreated SHR compared with WKY rats. Early captopril treatment in SHR significantly inhibited Ras and MLCK expression at all ages and decreased ERK-P and MLC-P at 12 and 18 weeks in mesenteric arteries. CONCLUSIONS: These data demonstrate that the antihypertensive effects of captopril are correlated with inhibition of Ras-regulated ERK activation, MLCK expression, and MLC-P.

Increased myosin light chain kinase expression in hypertension: Regulation by serum response factor via an insertion mutation in the promoter.

Regulation of gene transcription in vascular smooth muscle cells (VSMCs) by serum response factor (SRF) plays a crucial role in vascular development and in the pathophysiology of vascular diseases. Nevertheless, the regulation of specific genes by SRF in vascular diseases is poorly understood. Therefore, we investigated the regulation of smooth muscle myosin light chain kinase (smMLCK) by using spontaneously hypertensive rats (SHR) as an experimental model. We found that smMLCK expression in blood vessels increases during the development of hypertension and is always greater in blood vessels from SHR compared with normotensive rats. Analysis of the DNA sequences of the promoters isolated from SHR and normotensive rats revealed that SHR contain a 12-base pair insertion adjacent to the CArG box. This insertion increases SRF binding to the CArG box and positively regulates SRF-dependent promoter activity. The increase in smMLCK expression was blocked by dominant-negative SRF, dominant-negative Ras, or antisense oligonucleotides to ERK. In vivo, inhibiting MEK decreased smMLCK expression and blood pressure in SHR partly by decreasing SRF binding to the smMLCK promoter. These data provide novel insight into the regulation of smMLCK expression at the molecular level and demonstrate the importance of SRF in regulating smMLCK promoter activity in SHR.