Oxidized LDL Receptor 1 (OLR1) as a Possible Link between Obesity, Dyslipidemia and Cancer

Recent studies have linked expression of lectin-like ox-LDL receptor 1 (OLR1) to tumorigenesis. We analyzed microarray data from Olr1 knockout (KO) and wild type (WT) mice for genes involved in cellular transformation and evaluated effects of OLR1 over-expression in normal mammary epithelial cells (MCF10A) and breast cancer cells (HCC1143) in terms of gene expression, migration, adhesion and transendothelial migration. Twenty-six out of 238 genes were inhibited in tissues of OLR1 KO mice; the vast majority of OLR1 sensitive genes contained NF-κB binding sites in their promoters. Further studies revealed broad inhibition of NF-kB target genes outside of the transformation-associated gene pool, with enrichment themes of defense response, immune response, apoptosis, proliferation, and wound healing. Transcriptome of Olr1 KO mice also revealed inhibition of de novo lipogenesis, rate-limiting enzymes fatty acid synthase (Fasn), stearoyl-CoA desaturase (Scd1) and ELOVL family member 6 (Elovl6), as well as lipolytic phospholipase A2 group IVB (Pla2g4b). In studies comparing MCF10A and HCC1143, the latter displayed 60% higher OLR1 expression. Forced over-expression of OLR1 resulted in upregulation of NF-κB (p65) and its target pro-oncogenes involved in inhibition of apoptosis (BCL2, BCL2A1, TNFAIP3) and regulation of cell cycle (CCND2) in both cell lines. Basal expression of FASN, SCD1 and PLA2G4B, as well as lipogenesis transcription factors PPARA, SREBF2 and CREM, was higher in HCC1143 cells. Over-expression of OLR1 in HCC1143 cells also enhanced cell migration, without affecting their adherence to TNFα-activated endothelium or transendothelial migration. On the other hand, OLR1 neutralizing antibody inhibited both adhesion and transmigration of untreated HCC1143 cells. We conclude that OLR1 may act as an oncogene by activation of NF-kB target genes responsible for proliferation, migration and inhibition of apoptosis and de novo lipogenesis genes

A list of transformation related genes influenced by <i>Olr1</i> deletion in mice.

<p>A list of transformation related genes influenced by <i>Olr1</i> deletion in mice.</p

Phenotypic consequences of <i>OLR1 overexpression or inhibition</i>.

<p><b>A.</b> Wound healing assay. Upper panel - representative images of wound healing assay performed using HCC1143 cells transfected with either empty plasmid or <i>OLR1</i> cDNA vector; Lower panel – graph depicting the distance between edges of the wound after 36 hours of incubation. (*) – p<0.01; <b>B.</b> Adhesion assay. Upper panel- representative images of adherent non-transfected HCC1143 cells loaded with CellTracker Red CMTX (Invitrogen, Carlbad, CA) and applied to non-activated or activated (50 µg/ml oxLDL, 4 hrs) confluent HUVECs transfected with OLR1 Silencer or scrambled siRNA. Lower panel - graph depicting the number of adherent cells averaged from multiple fields of view in triplicate cultures. (*) – p<0.05 compared to non-activated control (“scrRNA”); (†) - p<0.05 compared to scrambled RNA; <b>C.</b> Colorimetric transendothelial migration assay. Upper panel – verification of the confluence of HUVECs on the membranes by staining cells with CellTracker Red CMTX. Lower panel – absorbance values of stain extracted from the cells migrated through TNFα-activated endothelial monolayer in presence of <i>OLR1</i> neutralizing antibody or human IgG (Control).</p

<i>Olr1</i> deletion results in broad inhibition of NF-κB target genes.

<p>A diagram depicting a set of overlapping genes between transformation and <i>Olr1</i> KO transcriptomes. From the set of 238 genes upregulated during transformation, 26 genes were found to be inhibited in <i>Olr1</i> KO mice. Vast majority of these genes carried NF-κB sites in their proximal promoter sequences. In total, 86 NF-κB target genes were found to be inhibited in <i>Olr1</i> KO mice with enrichment for regulation of apoptosis (p = 0.0002), proliferation (p = 0.00003), wound healing (p = 0.0002), defense response (p = 0.0011), immune response (p = 0.0003) and cell migration (p = 0.0009).</p

NF-κB target genes outside of transformation pool significantly inhibited in Olr1 knockout mice (more than 1.2-fold).

<p>Legend: (^a)- a list of NFkB target genes was compiled from the following web-based databases: 1 - <a href="http://bioinfo.lifl.fr/NF-KB/" target="_blank">http://bioinfo.lifl.fr/NF-KB/</a>; 2 -<a href="http://people.bu.edu/gilmore/nf-kb/target/index.html#cyto" target="_blank">http://people.bu.edu/gilmore/nf-kb/target/index.html#cyto</a>; and 3 - <a href="http://www.broadinstitute.org/mpr/publications/projects/Lymphoma/FF_NFKB_suppl_revised.pdf" target="_blank">http://www.broadinstitute.org/mpr/publications/projects/Lymphoma/FF_NFKB_suppl_revised.pdf</a>. The genes whose expression was significantly altered by OLR1 deletion was analyzed for enrichment themes using DAVID bioinformatic database. The enriched themes included regulation of apoptosis, “A”; Wound healing,“W”; Cell proliferation, “P”; Defense response,“D”; Cell migration, “M”.</p

SKA2 Methylation is associated with Decreased Prefrontal Cortical Thickness and Greater PTSD Severity among Trauma-Exposed Veterans

Methylation of the SKA2 gene has recently been identified as a promising biomarker of suicide risk. Based on this finding, we examined associations between SKA2 methylation, cortical thickness, and psychiatric phenotypes linked to suicide in trauma-exposed veterans. 200 trauma-exposed white non-Hispanic veterans of the recent conflicts in Iraq and Afghanistan (91% male) underwent clinical assessment and had blood drawn for genotyping and methylation analysis. 145 participants also had neuroimaging data available. Based on previous research, we examined DNA methylation at the CpG locus cg13989295 as well as DNA methylation adjusted for genotype at the methylation-associated SNP (rs7208505) in relationship to whole-brain cortical thickness, posttraumatic stress disorder symptoms (PTSD), and depression symptoms. Whole-brain vertex-wise analyses identified three clusters in prefrontal cortex that were associated with genotype-adjusted SKA2 DNA methylation (methylationadj). Specifically, DNA methylationadj was associated with bilateral reductions of cortical thickness in frontal pole and superior frontal gyrus, and similar effects were found in the right orbitofrontal cortex and right inferior frontal gyrus. PTSD symptom severity was positively correlated with SKA2 DNA methylationadj and negatively correlated with cortical thickness in these regions. Mediation analyses showed a significant indirect effect of PTSD on cortical thickness via SKA2 methylation status. Results suggest that DNA methylationadj of SKA2 in blood indexes stress-related psychiatric phenotypes and neurobiology, pointing to its potential value as a biomarker of stress exposure and susceptibility