Iron-responsive Element-binding Protein 2 Research Articles

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis

Background: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. Methods: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and β-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. Results: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and β-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. Discussion: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Hyperbaric Oxygen Therapy Inhibits Neuronal Ferroptosis After Spinal Cord Injury in Mice.

Basic science study investigating the potential molecular mechanisms of hyperbaric oxygen (HBO) therapy in mice with spinal cord injury (SCI). We aimed to explore the intrinsic mechanisms of HBO for SCI through the lens of ferroptosis in the subacute phase. HBO has been observed to facilitate the restoration of neurological function subsequent to SCI. Ferroptosis is a distinct cellular death mechanism that can be distinguished from apoptosis, necrosis, and autophagy. However, the precise relationship between these two phenomena remains poorly understood. We established an SCI model and employed a range of techniques, including behavioral assessments, electron microscopy, immunofluorescence, RT-qPCR, Western blotting (WB), Glutathione (GSH) measurement, and iron assay, to investigate various aspects of HBO therapy on SCI in mice. These included analyzing mitochondrial morphology, neuronal count, GSH levels, iron levels, and the expression of genes (Acyl-CoA synthetase family member-2, Iron-responsive element-binding protein-2) and proteins (Glutathione peroxidase 4; system Xc-light chain) associated with ferroptosis. The study included three groups: Sham-operated, SCI, and HBO. Group comparisons were performed using one-way analysis of variance and one-way repeated measures analysis of variance, followed by Tukey's post hoc test. Statistical significance was set at a P < 0.05. Our findings revealed that HBO therapy significantly enhanced the recovery of lower limb motor function in mice following SCI in the subacute phase. This was accompanied by upregulated expression of GPX4 and system Xc-light chain proteins, elevated GSH levels, increased number of NeuN+ cells, decreased expression of the iron-responsive element-binding protein-2 gene, and reduced iron concentration. Our research suggests that HBO therapy has the potential to be an effective treatment for SCI in the subacute phase by mitigating ferroptosis.

Abstract A028: ZEB1 confers sensitivity to ferroptosis through regulation of iron metabolism

Abstract Introduction: Epithelial to Mesenchymal Transition (EMT) is a cellular phenotype which may contribute to metastasis and therapeutic resistance in cancer. Therefore, therapeutic targeting of EMT-like phenotypes in cancer cells may afford an approach to treating metastatic disease and/or thwarting therapeutic resistance. Recently, GPX4 inhibitors (GPX4i) became of interest in this field as they were identified as potent and selective compounds for cells in a mesenchymal state through induction of lipid peroxidation induced cell death also known as ferroptosis. The sensitivity to these inhibitors was also shown to be dependent on the master EMT regulator, ZEB1. However, the link between ZEB1 expression and ferroptosis susceptibility remains unclear. Methodology: We used PC-3E and TEM 4-18 cell lines, originally isolated from the PC3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer, as our models of epithelial and EMT-like phenotypes respectively. To better understand the role of ZEB1 in ferroptosis susceptibility, we used ZEB1 KO TEM 4-18 cells as well as a panel of cancer cell lines that cover a broad spectrum of epithelial and EMT-like phenotypes. After evaluating the sensitivity to GPX4i in these cell lines, we examined different parameters affecting the lipid peroxidation pathway. We particularly focused on the regulation of iron metabolism by ZEB1. Results: The three GPX4i tested (ML210, ML162 and RSL3) displayed a greater toxicity against TEM 4-18 cells. Inhibitors of ferroptosis (ferrostatin-1, liproxstatin-1) completely reversed the effect of GPX4i indicating that ferroptosis was indeed triggered by GPX4i and responsible for their toxicity. Sensitivity to GPX4i correlated with ZEB1 expression in our panel of cancer cell lines and ZEB1 KO reversed toxicity of GPX4i in TEM 4-18 cells. Sensitivity to GPX4i correlated with a slightly higher labile iron pool in ZEB1 expressing cells. Expression of proteins with a previously established connection to iron metabolism such as Ferritin Light Chain (FtL), Ferritin Heavy Chain (FtH), Transferrin Receptor (TfR) and Prominin-2 (PROM2) were also dysregulated between resistant and sensitive cells. In particular, the ratio FtH:FtL and expression of PROM2 was positively correlated with resistance to GPX4i. In addition, using the Cancer Cell Line Encyclopedia (CCLE) database, we found that ZEB1 expression negatively correlated with PROM2 and Iron Responsive Element Binding Protein-2 (IREB2) suggesting these genes could be transcriptional targets of ZEB1. Conclusions: We validated GPX4i as potent EMT selective cytotoxic compounds and confirmed that the sensitivity to these inhibitors depends on ZEB1 expression. However, the mechanisms leading to sensitivity to GPX4i and how they connect to the EMT program have yet to be identified. As these mechanisms might be diverse, we hypothesize that ZEB1 regulates iron metabolism which is critical for ferroptosis induction. Citation Format: Marion Vanneste, Michael Darrin Henry, Akansha Jain. ZEB1 confers sensitivity to ferroptosis through regulation of iron metabolism [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A028.

Roots of Astragalus propinquus Schischkin Regulate Transmembrane Iron Transport and Ferroptosis to Improve Cerebral Ischemia-Reperfusion Injury.

Background The dried roots of the Astragalus propinquus Schischkin (RAP) plant, as a traditional Chinese medicine, has been widely used to treat stroke, cerebral ischemia, qi deficiency, and hypertension. Buyang Huanwu decoction is traditionally used to treat stroke in China for more than 200 years and has a significant effect on cerebral ischemia, and RAP is monarch medicine of Buyang Huanwu decoction. Therefore, this study was designed to observe the regulatory effect of RAP on transmembrane iron transporters and ferroptosis-related factors in cerebral ischemia-reperfusion injury (CIRI) in rats. Methods Middle cerebral artery occlusion (MCAO) was used to block blood flow in the blood supply area of the middle cerebral artery in seventy male SD rats to induce focal CIRI to establish a rat model of CIRI. RAP was administered to explore the regulatory effect of RAP on iron transmembrane transport under the condition of CIRI. The infarct size was measured using 2,3,5-triphenyl-tetrazolium chloride (TTC) staining, the pathological structure of brain tissue was observed by HE staining, and neuronal injury was evaluated by Nissl staining after treatment. Then, changes in the iron transporters ferritin (Fn), ferritin heavy chain (FHC), ferritin light chain (FLC), transferrin (Tf), transferrin receptor (TfR), divalent metal transporter 1 (DMT1), L-type calcium channel (LTCC), transient receptor potential canonical 6 (TRPC6), and ferroportin 1 (FPN1) were observed by immunohistochemistry staining (IHC) and Western blotting. The expression of key factors of ferroptosis, including the membrane sodium-dependent cystine/glutamate antiporter System Xc− (System Xc−) light chain subunit (XCT) and heavy chain subunit (SLC3A2), glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor (NRF2), heme oxygenase-1 (HO-1), and iron-responsive element-binding protein 2 (IREB2) in the brain tissues of rats was assessed by Western blotting. RAP decreased the infarct size and neuronal injury after CIRI in rats. Similarly, RAP treatment regulated the expression of iron transporters. As such, RAP was able to reduce the expression of Fn, FHC, FLC, Tf, TfR, DMT1, and TRPC6 and increase the expression of FPN1 through a Tf/TfR-independent pathway after CIRI in rats. Conclusion RAP stimulation inhibited ferroptosis by regulating the expression of the key ferroptosis factors XCT, SLC3A2, GPX4, NRF2, HO-1, and IREB2. In conclusion, RAP regulates transmembrane iron transport and ferroptosis to improve CIRI.

MiR-19a suppresses ferroptosis of colorectal cancer cells by targeting IREB2

ABSTRACT Colorectal cancer (CRC) is the most common malignant tumor occurred in digestive system. However, the prognosis of CRC patients is poor. Therefore, it is urgent to illuminate the mechanism suppressing CRC and explore novel targets or therapies for CRC treatment. MicroRNAs (miRNAs) are a class of non-coding RNAs with a length of 20–23 nucleotides encoded by endogenous genes, which are associated with the development of a variety of cancers, including CRC. Studies have shown that miR-19a is identified as oncogenic miRNA and promotes the proliferation, migration and invasion of CRC cells. However, the relationship between miR-19a and ferroptosis in CRC remains unknown. Here, we reported that iron-responsive element-binding protein 2 (IREB2), as an inducer of ferroptosis, was negatively regulated by miR-19a. IREB2 is a direct target of miR-19a. In addition, ferroptosis was suppressed by miR-19a through inhibiting IREB2. Thus, we proposed a novel mechanism of ferroptosis mediated by miR-19a in CRC cells, which could give rise to a new strategy for the therapy of CRC.

Curcumin induces ferroptosis in non-small-cell lung cancer via activating autophagy.

BackgroundEmerging studies showed curcumin can inhibit glioblastoma and breast cancer cells via regulating ferroptosis. However, the role of ferroptosis in the inhibitory effect of curcumin on non‐small‐cell lung cancer (NSCLC) remains unclear.MethodsCell counting kit‐8 (CCK‐8) assay was used to measure the viability of A549 and H1299 cells under different conditions. Cell proliferation was examined by Ki67 immunofluorescence. The morphological changes of cells and tumor tissues were observed by optical microscope and hematoxylin and eosin (H&E) staining. Intracellular reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and iron contents were determined by corresponding assay kit. The related protein expression levels were detected by western blot and immunohistochemistry. Transmission electron microscope was used to observe ultrastructure changes of A549 and H1299 cells.ResultsCurcumin inhibited tumor growth and cell proliferation, but promoted cell death. Characteristic changes of ferroptosis were observed in curcumin group, including iron overload, GSH depletion and lipid peroxidation. Meanwhile, the protein level of ACSL4 was higher and the levels of SLC7A11 and GPX4 were lower in curcumin group than that in control group. Incubation of ferroptosis inhibitors ferrostatin‐1 (Fer‐1) or knockdown of iron‐responsive element‐binding protein 2 (IREB2) notably weakened curcumin‐induced anti‐tumor effect and ferroptosis in A549 and H1299 cells. Further investigation suggested that curcumin induced mitochondrial membrane rupture and mitochondrial cristae decrease, increased autolysosome, increased the level of Beclin1 and LC3, and decreased the level of P62. Curcumin‐induced autophagy and subsequent ferroptosis were both alleviated with autophagy inhibitor chloroquine (CQ) or siBeclin1.ConclusionCurcumin induced ferroptosis via activating autophagy in NSCLC, which enhanced the therapeutic effect of NSCLC.

The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity.

Although iron is required for cell proliferation, iron-dependent programmed cell death serves as a critical barrier to tumor growth and metastasis. Emerging evidence suggests that iron-mediated lipid oxidation also facilitates immune eradication of cancer. However, the regulatory mechanisms of iron metabolism in cancer remain unclear. Here we identify OTUD1 as the deubiquitinase of iron-responsive element-binding protein 2 (IREB2), selectively reduced in colorectal cancer. Clinically, downregulation of OTUD1 is highly correlated with poor outcome of cancer. Mechanistically, OTUD1 promotes transferrin receptor protein 1 (TFRC)-mediated iron transportation through deubiquitinating and stabilizing IREB2, leading to increased ROS generation and ferroptosis. Moreover, the presence of OTUD1 promotes the release of damage-associated molecular patterns (DAMPs), which in turn recruits the leukocytes and strengthens host immune response. Reciprocally, depletion of OTUD1 limits tumor-reactive T-cell accumulation and exacerbates colon cancer progression. Our data demonstrate that OTUD1 plays a stimulatory role in iron transportation and highlight the importance of OTUD1-IREB2-TFRC signaling axis in host antitumor immunity.

Genetic Analysis of EGLN1 C127S Variant in Taiwanese Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder related to nigrostriatal dopaminergic neuron degeneration and iron accumulation. As a cellular oxygen sensor, prolyl hydroxylase domain containing protein 2 (PHD2, encoded by egl-9 family hypoxia inducible factor 1, EGLN1) modifies hypoxia-inducible factor alpha (HIF-α) protein for proteasomal destruction under normoxic condition. In addition, 2-oxoglutarate- (OG-) dependent dioxygenase activity of PHD2 is involved in the oxygen and iron regulation of iron-responsive element binding protein 2 (IRP2) stability. Previously increased expression of EGLN1 was found in the substantia nigra of the parkinsonian brain. We investigated the possible role of c.380 G > C (p.C127S) of EGLN1 gene in Taiwanese patients with PD. 479 patients and 435 healthy controls were recruited. Polymerase chain reaction and BsmAI restriction enzyme analysis were applied for analysis. An association between CC genotype and reduced PD risk in the recessive model (CC vs. GG + GC) was found. Our study provides a link between EGLN1 c.380 G > C SNP and the development of PD.

Deferoxamine promotes recovery of traumatic spinal cord injury by inhibiting ferroptosis.

Ferroptosis is an iron-dependent novel cell death pathway. Deferoxamine, a ferroptosis inhibitor, has been reported to promote spinal cord injury repair. It has yet to be clarified whether ferroptosis inhibition represents the mechanism of action of Deferoxamine on spinal cord injury recovery. A rat model of Deferoxamine at thoracic 10 segment was established using a modified Allen's method. Ninety 8-week-old female Wistar rats were used. Rats in the Deferoxamine group were intraperitoneally injected with 100 mg/kg Deferoxamine 30 minutes before injury. Simultaneously, the Sham and Deferoxamine groups served as controls. Drug administration was conducted for 7 consecutive days. The results were as follows: (1) Electron microscopy revealed shrunken mitochondria in the spinal cord injury group. (2) The Basso, Beattie and Bresnahan locomotor rating score showed that recovery of the hindlimb was remarkably better in the Deferoxamine group than in the spinal cord injury group. (3) The iron concentration was lower in the Deferoxamine group than in the spinal cord injury group after injury. (4) Western blot assay revealed that, compared with the spinal cord injury group, GPX4, xCT, and glutathione expression was markedly increased in the Deferoxamine group. (5) Real-time polymerase chain reaction revealed that, compared with the Deferoxamine group, mRNA levels of ferroptosis-related genes Acyl-CoA synthetase family member 2 (ACSF2) and iron-responsive element-binding protein 2 (IREB2) were up-regulated in the Deferoxamine group. (6) Deferoxamine increased survival of neurons and inhibited gliosis. These findings confirm that Deferoxamine can repair spinal cord injury by inhibiting ferroptosis. Targeting ferroptosis is therefore a promising therapeutic approach for spinal cord injury.

Strong correlation of ferrochelatase enzymatic activity with Mitoferrin-1 mRNA in lymphoblasts of patients with protoporphyria

Accumulation of protoporphyrin IX (PPIX) and Zn-PPIX, are the clinical hallmarks of protoporphyria. Phenotypic expression of protoporphyria is due to decreased activity of ferrochelatase (FECH) or to increased activity of aminolevulinic acid synthase (ALAS) in red blood cells. Other genetic defects have been shown to contribute to disease severity including loss of function mutations in the mitochondrial AAA-ATPase, CLPX and mutations in the Iron-responsive element binding protein 2 (IRP2), in mice. It is clear that multiple paths lead to a common phenotype of excess plasma PPIX that causes a phototoxic reaction on sun exposed areas. In this study we examined the association between mitochondrial iron acquisition and utilization with activity of FECH. Our data show that there is a metabolic link between the activity FECH and levels of MFRN1 mRNA. We examined the correlation between FECH activity and MFRN1 mRNA in cell lines established from patients with the classical protoporphyria, porphyria due to defects in ALAS2 mutations. Our data confirm MFRN1 message levels positively correlated with FECH enzymatic activity in all cell types.

Abstract 809: Dietary iron intake, genetic variants in microRNA related iron regulatory pathway genes, and the risk of non-small cell lung cancer

Abstract As an essential nutrient that facilitates DNA synthesis, cell proliferation and metabolism, iron is involved in carcinogenesis. While epidemiological studies suggest a J-shaped model for the relationship of serum iron level and cancer risk with both too low and too high levels associated with increased cancer risks, the effect of genetic variants related to intracellular iron regulatory pathway and dietary iron intake on non-small cell lung cancer (NSCLC) risk is little known. In this study, we evaluated a panel of miRNA related genetic variants in iron regulatory pathway genes on the risk of NSCLC. In discovery phrase, 157 miRNA related single nucleotide polymorphisms (SNPs) from 81 genes of iron regulatory pathway were genotyped in 1656 NSCLC cases and 1486 healthy controls. Eight SNPs in 6 genes were significantly associated with risk of NSCLC. A SNP in 3’ UTR of Iron-Responsive Element Binding Protein 2 (IREB2) gene at 15q25 region was subsequently replicated in dbGaP lung cancer GWAS dataset. Combining all subjects (7,352 cases, 7,296 controls), the overall P-value was 4.9 × 10−9 (Odds ratio (OR) = 0.86). eQTL analysis showed that the SNP on IREB2 alters IREB2 gene expression (P = 3.0 × 10−11). The SNP is predicted to alter a miR-29 binding site on IREB2 gene and indeed the expression of miR-29 is inversely correlated with IREB2 expression in tumor tissues. We then determined the expression of serum miR-29a in 150 early-stage NSCLC patients and 172 healthy controls and found that miR-29a was significantly associated with higher risk of cancer (OR = 1.78, P = 0.03). By analyzing dietary intake information of the discovery population, iron intake was significantly associated with risk of NSCLC in logistic regression analysis and there was also evidence of a synergistic interaction with between the SNP and iron intake in modulating NSCLC risk (P for interaction = 0.01). Taken together, SNPs at miRNA binding sites of iron regulatory pathway genes and dietary iron intake may modify risk of NSCLC individually and jointly. Citation Format: Liren Zhang, Yuanquing Ye, Huakang Tu, Michelle A.t. Hildebrandt, John V. Heymach, Jack A. Roth, Jian Gu, Xifeng Wu. Dietary iron intake, genetic variants in microRNA related iron regulatory pathway genes, and the risk of non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 809.

Association of IREB2 Gene rs2568494 Polymorphism with Risk of Chronic Obstructive Pulmonary Disease: A Meta-Analysis.

BackgroundIt is reported that the iron-responsive element-binding protein 2 (IREB2) gene rs2568494 polymorphism might be associated with COPD risk. The purpose of this meta-analysis was to collect all eligible studies to review the association between IREB2 gene rs2568494 polymorphism and susceptibility to COPD.Material/MethodsWe carried out a comprehensive document search of electronic databases of PubMed, MEDLIN, Web of Science, and included 4 eligible studies that examined the association between IREB2 rs2568494 polymorphism and COPD susceptibility. We performed a meta-analysis of these studies based on IREB2 rs2568494 genotypes.ResultsAfter meta-analysis with fixed or random effects, no significant associations were found under the heterozygote model (GG/GA; OR=0.908, 95%CI: 0.790–1.043; P=0.172), homozygote model (GG/AA; OR=0.880, 95%CI: 0.497–1.557; P=0.661), dominant model (GG/AA+GA; OR=0.941, 95%CI: 0.748–1.182; P=0.599), or allelic model (G/A; OR=0.953, 95%CI: 0.770–1.179; P=0.655). However, we found a significant correlation under the recessive model (AA/GA+GG; OR=1.384, 95%CI: 1.092–1.755; P=0.007).ConclusionsThe current results revealed that there was significant association between IREB2 gene rs2568494 polymorphism with susceptibility to COPD; the presence of allelic A might a genetic factor conferring susceptibility to COPD.

Differential expression of genes involved in Bengal macrothrombocytopenia (BMTCP)

Bengal macrothrombocytopenia (BMTCP) is a giant platelet disorder with mild to moderate thrombocytopenia, clinically characterized by mild bleeding symptoms to totally asymptomatic condition. The pathophysiological mechanism of this condition is not fully understood yet. In the present study, 5 subjects (P1–P5) with BMTCP whose platelet counts ranged between 36140X109/l and mean platelet volume (MPV)13.5–16.1fl were analyzed for differential gene expression of platelets by suppressive subtractive hybridization (SSH) technique. Four genes i.e. myotubularin related protein 9 (MTMR9), iron responsive element binding protein 2 (IREB2), alpha tubulin(TUBA) and tyrosine kinase ligand (TKL) were found to be differentially expressed in patient platelets as compared to that of normal healthy controls which was further confirmed by quantitative RT PCR analysis. The study highlights a multi-factorial etiology for BMTCP which is widely prevalent in the northeastern region of the Indian subcontinent.

Abstract 4210: Association of the 15q25 and 5p15 lung cancer susceptibility regions with gene expression in lung tumor tissue

Abstract Lung cancer is one of the most commonly diagnosed cancers worldwide, with 1.6 million new cases reported each year and ranks first in annual global cancer deaths. Genome-wide association studies have identified two independent lung cancer susceptibility loci at chromosome 15q25 and one locus at 5p15. We examined the association of genetic variants in these lung cancer susceptibility regions with gene expression in lung tumor tissue in an effort to elucidate carcinogenic mechanisms by which these variants influence lung cancer risk. We used data from two independent studies: the JBR.10 clinical trial (131 NSCLC patients) and a patient sample from the University Health Network (UHN) in Toronto (181 NSCLC patients). We genotyped seven 15q25 and five 5p15 variants and examined their association with the expression profiles of genes in the corresponding regions measured by Affymetrix HG-U133A. The minor allele (C) of a variant representing one of the two loci at 15q25 (rs2036534) was associated with increased expression of the IREB2 (iron-responsive element binding protein 2) gene in both studies (JBR.10 P=0.042; UHN P=0.002). An FDRα0.05 in the UHN sample increased our confidence in this association. The association appears to be more prominent among patients with lung adenocarcinoma. We did not detect an association between the genotype and expression profile for the other 15q25 locus (represented by rs16969968 and rs1051730). Previous studies suggest that 15q25 variants are associated with risk through an effect on smoking behaviour, indicating risk is modulated through activity of nicotinic acetylcholine receptor genes present in this region. Our results indicate there is a second mechanism through which variants at 15q25 influence risk, operating through modulation of IREB2 gene expression. This finding may have implications for future research on lung cancer chemoprevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4210. doi:1538-7445.AM2012-4210

Transcription Deregulation at the 15q25 Locus in Association with Lung Adenocarcinoma Risk

We characterized the candidacy of the six candidate genes mapping in the chromosome 15q25 locus, which was previously reported as associated with lung cancer risk, and confirmed the locus association with lung cancer risk in an Italian population of lung adenocarcinoma patients and controls. We did a quantitative analysis of mRNA levels of IREB2 (iron-responsive element-binding protein 2), LOC123688, PMSA4 [proteasome (prosome, macropain) subunit alpha type 4], CHRNB4 (cholinergic receptor nicotinic beta 4), CHRNA3 (cholinergic receptor nicotinic alpha 3), and CHRNA5 (cholinergic receptor nicotinic alpha 5) genes in paired normal lung and lung adenocarcinoma tissue, and an immunohistochemical localization of CHRNA3- and CHRNA5-encoded proteins. We also examined the association of CHRNA5 D398N polymorphism with lung cancer risk and with CHRNA5 mRNA levels in the normal lung. Expression analysis of the six candidate genes mapping in the lung cancer risk-associated chromosome 15q25 locus revealed a 30-fold up-regulation of the gene encoding the CHRNA5 subunit and a 2-fold down-regulation of the CHRNA3 subunit in lung adenocarcinoma as compared with the normal lung. The expression of the four other candidate genes resulted either unchanged or absent. The carrier status of the 398N allele at the D398N polymorphism of the CHRNA5 gene was associated with lung adenocarcinoma risk (odds ratio, 1.5; 95% confidence interval, 1.2-2.0) in a population-based series of lung adenocarcinoma patients (n=467) and healthy controls (n=739). Analysis of a family-based series of nonsmoker lung cancer cases (n=80) and healthy sib controls (n=80) indicated a similar trend. In addition, the same D398N variation correlated with CHRNA5 mRNA levels in normal lung of adenocarcinoma patients. Our results point to the candidacy of the CHRNA5 gene for the 15q25 locus.