HFE Expression Research Articles

β2-microglobulin induced apoptosis of tumor cells via the ERK signaling pathway by directly interacting with HFE in HER2-overexpressing breast cancer

BackgroundOur previous study demonstrated that β2-microglobulin (β2M) promoted ER+/HER2– breast cancer survival via the SGK1/Bcl-2 signaling pathway. However, the role of β2M has not been investigated in ER–/HER2+ breast cancer. Here, we aimed to determine the role of β2M in ER–/HER2+ breast cancer.MethodsThe interaction between β2M and HFE was confirmed by co-immunoprecipitation, mass spectrometry, yeast two-hybrid screening, and His pull-down. The knockdown and overexpression of β2M or HFE were performed in MDA-MB-453 cells, and ERK signaling pathway was subsequently analyzed via western blotting. Apoptotic cells were detected using flow cytometer. β2M, HFE, and p-ERK1/2 were examined in tumor and paired adjacent tissues via immunohistochemistry.ResultsHFE was found to be an interacting protein of β2M in ER–/HER2+ breast cancer cells MDA-MB-453 by co-immunoprecipitation and mass spectrometry. A yeast two-hybrid system and His-pull down experiments verified that β2M directly interacted with HFE. β2M and HFE as a complex were mainly located in the cytoplasm, with some on the cytomembrane of MDA-MB-453 cells. In addition to breast cancer cells BT474, endogenous β2M directly interacted with HFE in breast cancer cells MDA-MB-453, MDA-MB-231, and MCF-7. β2M activated the ERK signaling pathway by interacting with HFE and induced apoptosis of MDA-MB-453 cells. The expression of HFE and p-ERK1/2 showed significantly high levels in HER2-overexpressing breast cancer tumor tissue compared with adjacent normal tissue, consistent with the results obtained from the cell experiments.Conclusionsβ2M induced apoptosis of tumor cells via activation of the ERK signal pathway by directly interacting with HFE in HER2-overexpressing breast cancer.

Homeostatic iron regulatory protein drives glioblastoma growth via tumor cell-intrinsic and sex-specific responses.

Glioblastoma (GBM) displays alterations in iron that drive proliferation and tumor growth. Iron regulation is complex and involves many regulatory mechanisms, including the homeostatic iron regulator (HFE) gene, which encodes the homeostatic iron regulatory protein. While HFE is upregulated in GBM and correlates with poor survival outcomes, the function of HFE in GBM remains unclear. We interrogated the impact of cell-intrinsic Hfe expression on proliferation and survival of intracranially implanted animals through genetic gain- and loss-of-function approaches in syngeneic mouse glioma models, along with in vivo immune assessments. We also determined the expression of iron-associated genes and their relationship to survival in GBM using public data sets and used transcriptional profiling to identify differentially expressed pathways in control compared to Hfe-knockdown cells. Overexpression of Hfe accelerated GBM proliferation and reduced animal survival, whereas suppression of Hfe induced apoptotic cell death and extended survival, which was more pronounced in females and associated with attenuation of natural killer cells and CD8+ T cell activity. Analysis of iron gene signatures in Hfe-knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Further analysis of differentially expressed pathways revealed oxidative stress as the top pathway upregulated following Hfe loss. Hfe knockdown indeed resulted in enhanced 55Fe uptake and generation of reactive oxygen species. These findings reveal an essential function for HFE in GBM cell growth and survival, as well as a sex-specific interaction with the immune response.

Abstract 2430: The role of cellular iron and the homeostatic iron regulator (HFE) in high-grade brain tumor cell migration

Abstract High-grade brain tumors such as grade III astrocytoma and glioblastoma represent among the most difficult to manage malignancies facing oncological practice. Diffuse migration and invasion into adjacent healthy brain tissue yields complete surgical resection of these tumors unfeasible. As a result, despite receiving maximal surgical resection and an aggressive course of chemoradiation, the majority of high-grade brain tumor patients suffer from recurrent disease. Increased expression levels of the homeostatic iron regulator gene (HFE) in high-grade brain tumors have been correlated with poorer outcomes. HFE is known to influence cellular iron metabolism by inhibiting transferrin-mediated iron uptake yet little is known regarding how HFE or iron impact the migratory capabilities of high-grade brain tumor cells. In order to better understand how HFE expression and cellular iron metabolism influence cell migration in high grade brain tumors, we utilized brain tumor cell lines that had been genetically manipulated to express different levels of HFE. We observed that knocking down HFE in KR158 or LN229 glioma cell lines resulted in significantly decreased migratory capacity. Since HFE is known to inhibit transferrin mediated iron uptake, we studied how directly modulating the iron status of glioma cells impacted their ability to migrate. Treatment of a panel of glioma cell lines: LN229, T98G, U87, KR158, with iron in the form of hemin or ferric ammonium citrate resulted in significantly reduced migration. Furthermore, the iron-induced reduction in migration could be rescued by the addition of deferoxamine, an iron chelator. Cell viability in response to the iron treatments was assayed and found to not be significantly altered - suggesting that cellular iron status was influencing migratory capacity independent of cell viability. To gain mechanistic insights into HFE and iron-induced effects on cell migration, we analyzed the Chinese Glioma Genome Atlas (CGGA) for correlations between HFE and the Rho GTPases RHOA, RAC1, and CDC42 – genes which are known to play a crucial role in determining the migratory capacity of cancer cells. Interestingly, we found statistically significant correlations between the Rho GTPases RHOA, RAC1, CDC42 and HFE in both grade III astrocytoma and glioblastoma patient cohorts. Immunoblotting of iron treated glioma cell lines demonstrated that expression of RhoA and Cdc42 was reduced suggesting that alterations in Rho GTPase expression and signaling may play a role in iron-induced effects on cell migration. Our results demonstrate that targeting cancer cell iron metabolism as an addition to existing treatment regimens may be a promising avenue for further investigation. Citation Format: Ganesh Shenoy, Katie Troike, Kondaiah Palsa, Madison Kuhn, Quinn Wade, Becky Slagle-Webb, Amanda Snyder, Chachrit Khunsriraksakul, Justin D. Lathia, Dhimant Desai, Hong-Gang Wang, Elizabeth Proctor, James R. Connor. The role of cellular iron and the homeostatic iron regulator (HFE) in high-grade brain tumor cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2430.

Heme oxygenase-1 & 2 and their potential contribution in heme induced colorectal carcinogenesis

BackgroundLiterature suggests Heme oxygenase (HO) system to be a double-edged sword which can promote both cytoprotection as well as carcinogenicity. The aim of this study was to investigate the role of heme in HO-1 and HO-2 induced colorectal carcinogenesis and the clinicopathological significance of their expressions in patients with colorectal carcinoma (CRC). MethodsHO-1 and HO-2 expression alterations in normal colonic epithelial (FHC) and colon cancer cells (SW480) were explored following treatment with 0 µM, 25 µM, 100 µM and 250 µM concentrations of hemin, using qPCR. Fifty paired CRC and adjacent non-neoplastic samples were subjected to qPCR to determine the HO-1 and HO-2 expression. Clinicopathological associations of HO-1 and HO-2 expression levels were determined. ResultsLow concentrations of hemin caused upregulation and high concentration caused downregulation of HO-1 expression, whereas HO-2 expression was significantly downregulated with all hemin concentrations in FHC. HO-1 expression in SW480 was increased with all hemin concentrations and HO-2 expression was downregulated at the highest hemin concentration. HO-1 and HO-2 expressions in adjacent non-neoplastic tissue was significantly higher than that of CRC. Expression of HO-1 was significantly higher than HO-2, in both CRC and adjacent non-neoplastic tissue. Sex, HFE expression and lymph-vascular invasion were significantly correlated with HO-1 expression. HO-2 expression showed significant associations with staging, local spread and recurrence of tumour. ConclusionHO-1 and HO-2 expression is respectively induced and repressed by exogenous hemin in normal colon and colon cancer cells. HO-1 and HO-2 expression profiles in CRC are correlated with the assessed clinicopathological features of CRC, suggesting the possible implications of HO expression status in CRC pathogenesis.

CBIO-06. CELL INTRINSIC HFE DRIVES SEX-SPECIFIC GLIOBLASTOMA GROWTH

Abstract Iron is an essential element required for a number of cellular processes and can contribute to malignant transformation and tumor expansion. In glioblastoma (GBM), tumor cells have been shown to modulate expression of iron-associated proteins to enhance iron uptake from the surrounding microenvironment, driving proliferation and tumor growth. The homeostatic iron regulatory (HFE) gene encodes a transmembrane glycoprotein that aids in iron homeostasis by modulating iron uptake and release. HFE is upregulated in GBM tumors compared to non-tumor brain and expression of HFE increases with tumor grade. Furthermore, HFE mRNA expression is associated with significantly reduced survival specifically in female patients with GBM. However, it is unclear how HFE impacts sex-specific GBM growth. To interrogate the underlying mechanism of HFE-mediated sex differences, we employed genetic loss and gain of function approaches using syngeneic mouse glioma models. We observed significant alterations in the expression of several iron-associated genes with Hfe knockdown or overexpression, suggesting global disruption of iron homeostasis. We found that knockdown of Hfe decreased cell number and increased apoptosis in vitro and led to a significant impairment of tumor growth in vivo, with a more pronounced effect seen in female mice. Conversely, overexpression of Hfe increased cell number and significantly decreased survival only in female animals. These findings support the hypothesis that Hfe is a critical regulator of cellular iron status and contributes to tumor aggression in a sex-dependent manner. These data also suggest an unexplored link between cell intrinsic iron signaling and sex-specific microenvironmental and immune responses, which is the focus of ongoing studies.

TAMI-42. THE ROLE OF HFE AND IRON IN CELL ADHESION AND MIGRATION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) remains one of the most difficult to treat malignancies facing modern medicine. The strong migratory and invasive capacity of GBM cells allows for diffuse invasion into neighboring healthy brain which presents a significant hurdle for complete surgical resection of these tumors. Unsurprisingly, even after receiving maximal surgical resection, radiation and chemotherapy, the majority of GBM patients end up with recurrent disease. Increased expression levels of the homeostatic iron regulator gene (HFE) in brain tumors such as GBM have been associated with poorer outcomes. In order to better understand how HFE expression impacts the adhesive and migratory capacity of GBM, we utilized syngeneic mouse glioma models (KR158, CT2A) that have been transfected to either over-express or under-express HFE. We observed that knocking down HFE in the KR158 model resulted in significantly decreased migratory capacity as well as decreased adhesion to fibronectin and artificial basement membrane. Likewise, overexpressing HFE in a CT2A model resulted in increased adhesion to fibronectin or artificial basement membrane. Since HFE is known to regulate iron uptake, we studied how modulating the iron status of GBM cells impacted their ability to migrate and adhere. We found that increasing the iron pool of these mouse glioma models by exposure to exogenous iron compounds decreased migratory capacity. To better understand mechanistically how HFE and iron status impacted migration and adhesion, we probed how expression of integrins and their downstream signaling molecules, the Rho GTPases were altered in response to iron. We found that exposure to iron decreased levels of the Rho GTPases Cdc42 and RhoA. Furthermore, cells that overexpressed HFE were found to have increased expression of integrin β1 and integrin α5 suggesting that HFE and iron may impact integrins and their downstream signaling pathways to alter migration of GBM cells.

HFE variants in colorectal cancer and their clinicopathological correlations

The study aimed to screen mutation of human homeostatic iron regulator (HFE) in colorectal carcinoma (CRC) and detect their associations with clinicopathological parameters. Expression of HFE was determined by quantitative polymerase chain reaction in matched CRC and non neoplastic colorectal mucosal tissue of 76 patients. Genomic DNA extracted were subjected to high high-resolution melt curve analysis and Sanger sequencing to detect mutations in HFE. The associations of the identified mutations with a variety of clinical features were determined. Approximately 60% of CRC showed low HFE expression. Of the ten 10 mutations identified in exons 2 and 4, c.187C>G (H63D), c845G>A (C282Y), c.193A>T (S65C), g.3828T>C, g.5795T>C, and g.5728G>A were known mutations. Four novel mutations were discovered; : c.184G>A, c.220T>G, c.322A>C, and c.324T>C. Heterozygous H63D and C282Y mutations were seen in 71% and 49% of cancer tissue, respectively. Tumour site (p=0.048) and gender (p=0.039) were significantly associated with H63D and C282Y mutation status, respectively. Local spread of cancer was significantly associated with C282Y mutations in CRC cancer and adjacent non-neoplastic tissue (p=0.029 & and p=0.004, respectively). There was a statistically significant association between H63D and C282Y negativity in matched non-neoplastic colorectal mucosa tissue and pathological staging of cancer (p=0.047 & and p=0.001, respectively). Patients with H63D and C282Y mutations in cancer tissue tend to have higher survival rates. Hence HFE mutations are common in CRC and are associated with clinicopathological parameters, implying the potential clinical significance of HFE mutations in colorectal carcinogenesis.

ESAT-6 Protein of Mycobacterium tuberculosis Increases Holotransferrin-Mediated Iron Uptake in Macrophages by Downregulating Surface Hemochromatosis Protein HFE.

Iron is an essential element for Mycobacterium tuberculosis; it has at least 40 enzymes that require iron as a cofactor. Accessibility of iron at the phagosomal surface inside macrophage is crucial for survival and virulence of M. tuberculosis ESAT-6, a 6-kDa-secreted protein of region of difference 1, is known to play a crucial role in virulence and pathogenesis of M. tuberculosis In our earlier study, we demonstrated that ESAT-6 protein interacts with β-2-microglobulin (β2M) and affects class I Ag presentation through sequestration of β2M inside endoplasmic reticulum, which contributes toward inhibition of MHC class I:β2M:peptide complex formation. The 6 aa at C-terminal region of ESAT-6 are essential for ESAT6:β2M interaction. β2M is essential for proper folding of HFE, CD1, and MHC class I and their surface expression. It is known that M. tuberculosis recruit holotransferrin at the surface of the phagosome. But the upstream mechanism by which it modulates holotransferrin-mediated iron uptake at the surface of macrophage is not well understood. In the current study, we report that interaction of the ESAT-6 protein with β2M causes downregulation of surface HFE, a protein regulating iron homeostasis via interacting with transferrin receptor 1 (TFR1). We found that ESAT-6:β2M interaction leads to sequestration of HFE in endoplasmic reticulum, causing poorer surface expression of HFE and HFE:TFR1 complex (nonfunctional TFR1) in peritoneal macrophages from C57BL/6 mice, resulting in increased holotransferrin-mediated iron uptake in these macrophages. These studies suggest that M. tuberculosis probably targets the ESAT-6 protein to increase iron uptake.

TAMI-17. RELATIONSHIP BETWEEN IRON METABOLISM, IMMUNE CELL INFILTRATION AND SEX-BASED SURVIVAL DIFFERENCES IN GLIOMAS

Abstract Iron plays a central role in cellular metabolism, both in normal cellular functioning and in tumorigenesis. Recent evidence has shown sex-based survival differences in glioblastoma (GBM) may be related to differential expression of metabolism genes. We previously reported the iron regulating gene, HFE, was shown to have a sex-based survival impact in both low-grade gliomas and GBM. We additionally found that females with low HFE expressing tumors have significantly higher survival than males in GBM. To evaluate the relationship between iron gene expression and sex-based survival differences in GBM, we analyzed TCGA GBM gene expression and clinical data. We first analyzed the impact of iron genes on sex-based survival. In addition to HFE, FTL, TFRC, TF, and SLC39A8 (ZIP8), also showed sex-based survival differences. We then compared correlations of HFE and other iron genes to identify whether male and female GBMs differ in iron regulation and metabolism. HFE expression is significantly positively correlated with HMOX1, SLC25A28, SLC11A2, FTH1, HAMP, and TFR2 only in females. Alternatively, HFE expression is negatively correlated with ACO2 (mitochondrial aconitase) in males and ACO1 (cytoplasmic aconitase) in females. We noted that the expression of certain iron genes was highly associated with immune cell infiltration based on sex. TFR2, LRP1, and XIST expression were negatively correlated with low immune cell infiltration in females, but not males. Alternatively, in males, SLC11A2, ACO2, FOXO1, HIF1a, and HAMP genes were negatively correlated with immune infiltration. This suggests that differences in iron regulation between males and females may be contributing to differences in immune function and subsequent survival in GBM. These data suggest that the iron signature of a tumor reflects and possibly drives the metabolic and immune landscape of the tumor microenvironment thereby directly impacting survival differences between male and female GBMs.

CBIO-10. REDUCED IRON EXPORT FUNCTIONS IN A CELL INTRINSIC MANNER TO DRIVE GLIOBLASTOMA GROWTH

Abstract Glioblastoma (GBM), the most common primary malignant brain tumor in adults, is characterized by invasive growth and poor prognosis. Iron is a critical regulator of many cellular processes, and GBM tumor cells have been shown to modulate expression of iron-associated proteins to enhance iron uptake from the surrounding microenvironment, driving tumor initiation and growth. While iron uptake has been the central focus of previous investigations, additional mechanisms of iron regulation, such as compensatory iron efflux, have not been explored in the context of GBM. The hemochromatosis (HFE) gene encodes a transmembrane glycoprotein that aids in iron homeostasis by limiting cellular iron release, resulting in a sequestration phenotype. We find that HFE is upregulated in GBM tumors compared to non-tumor brain and that expression of HFE increases with tumor grade. Furthermore, HFE mRNA expression is associated with significantly reduced survival specifically in female patients with GBM. Based on these findings, we hypothesize that GBM tumor cells upregulate HFE expression to augment cellular iron loading and drive proliferation, ultimately leading to reduced survival of female patients. To test this hypothesis, we generated Hfe knockdown and overexpressing mouse glioma cell lines. We observed significant alterations in the expression of several iron handling genes with Hfe knockdown or overexpression, suggesting global disruption of iron homeostasis. Additionally, we show that knockdown of Hfe in these cells increases apoptosis and leads to a significant impairment of tumor growth in vivo. These findings support the hypothesis that Hfe is a critical regulator of cellular iron status and contributes to tumor aggression. Future work will include further exploration of the mechanisms that contribute to these phenotypes as well as interactions with the tumor microenvironment. Elucidating the mechanisms by which iron effulx contributes to GBM may inform the development of next-generation targeted therapies.

Cell-specific expression of Hfe determines the outcome of Salmonella enterica serovar Typhimurium infection in mice

Mutations in HFE cause hereditary hemochromatosis type I hallmarked by increased iron absorption, iron accumulation in hepatocytes and iron deficiency in myeloid cells. HFE encodes an MHC-I like molecule, but its function in immune responses to infection remains incompletely understood. Here, we investigated putative roles of Hfe in myeloid cells and hepatocytes, separately, upon infection with Salmonella Typhimurium, an intracellular bacterium with iron-dependent virulence. We found that constitutive and macrophage-specific deletion of Hfe protected infected mice. The propagation of Salmonella in macrophages was reduced due to limited intramacrophage iron availability for bacterial growth and increased expression of the anti-microbial enzyme nitric oxide synthase-2. By contrast, mice with hepatocyte-specific deletion of Hfe succumbed earlier to Salmonella infection because of unrestricted extracellular bacterial replication associated with high iron availability in the serum and impaired expression of essential host defense molecules such as interleukin- 6, interferon-g and nitric oxide synthase-2. Wild-type mice subjected to dietary iron overload phenocopied hepatocyte-specific Hfe deficiency suggesting that increased iron availability in the serum is deleterious in Salmonella infection and underlies impaired host immune responses. Moreover, the macrophage-specific effect is dominant over hepatocytespecific Hfe-depletion, as Hfe knockout mice have increased survival despite the higher parenchymal iron load associated with systemic loss of Hfe. We conclude that cell-specific expression of Hfe in hepatocytes and macrophages differentially affects the course of infections with specific pathogens by determining bacterial iron access and the efficacy of antimicrobial immune effector pathways. This may explain the high frequency and evolutionary conservation of human HFE mutations.

H63D variant of the homeostatic iron regulator (HFE) gene alters α-synuclein expression, aggregation, and toxicity.

Pathological features of Parkinson's disease include the formation of Lewy bodies containing α-synuclein and the accumulation of iron in the substantia nigra. Previous studies have suggested that iron accumulation contributes to the Parkinson's disease pathology through reactive oxygen species production and accelerated α-synuclein aggregation. This study examines the effects of commonly occurring H63D variant of the homeostatic iron regulatory (HFE) gene on α-synuclein pathology in cell culture and animal models. H63D HFE expression in SH-SY5Y cells lowered endogenous α-synuclein levels and significantly decreased pre-formed fibril-induced α-synuclein aggregation. H63D HFE cells were also protected from pre-formed fibril-induced apoptosis. Autophagic flux, a major pathway for α-synuclein clearance, was increased in H63D HFE cells. Expression of REDD1 was elevated and rapamycin treatment was unable to further induce autophagy, indicating mTORC1 inhibition as the main mechanism of autophagy induction. Moreover, siRNA knockdown of REDD1 in H63D HFE cells decreased autophagic flux and increased the sensitivity to PFF-mediated toxicity. While iron chelator (deferiprone) treatment rescued WT HFE cells from pre-formed fibril toxicity, it exacerbated or was unable to rescue H63D HFE cells. In the in vivo pre-formed fibril intracranial injection model, H67D Hfe (mouse homolog of the human H63D HFE variant) C57BL/6J×129 mice showed less α-synuclein aggregation and less decline in motor function compared to WT Hfe. Collectively, this study suggests that H63D HFE variant modifies α-synuclein pathology through the induction of autophagy and has the potential to impact the pathogenesis and treatment response in Parkinson's disease.

Sexually dimorphic impact of the iron-regulating gene, HFE, on survival in glioblastoma.

BackgroundThe median survival for patients with glioblastoma (GBM), the most common primary malignant brain tumor in adults, has remained approximately 1 year for more than 2 decades. Recent advances in the field have identified GBM as a sexually dimorphic disease. It is less prevalent in females and they have better survival compared to males. The molecular mechanism of this difference has not yet been established. Iron is essential for many biological processes supporting tumor growth and its regulation is impacted by sex. Therefore, we interrogated the expression of a key component of cellular iron regulation, the HFE (homeostatic iron regulatory) gene, on sexually dimorphic survival in GBM.MethodsWe analyzed TCGA microarray gene expression and clinical data of all primary GBM patients (IDH-wild type) to compare tumor mRNA expression of HFE with overall survival, stratified by sex.ResultsIn low HFE expressing tumors (below median expression, n = 220), survival is modulated by both sex and MGMT status, with the combination of female sex and MGMT methylation resulting in over a 10-month survival advantage (P < .0001) over the other groups. Alternatively, expression of HFE above the median (high HFE, n = 240) is associated with significantly worse overall survival in GBM, regardless of MGMT methylation status or patient sex. Gene expression analysis uncovered a correlation between high HFE expression and expression of genes associated with immune function.ConclusionsThe level of HFE expression in GBM has a sexually dimorphic impact on survival. Whereas HFE expression below the median imparts a survival benefit to females, high HFE expression is associated with significantly worse overall survival regardless of established prognostic factors such as sex or MGMT methylation.

Impact of HFE variants and sex in lung cancer.

The homeostatic iron regulator protein HFE is involved in regulation of iron acquisition for cells. The prevalence of two common HFE gene variants (H63D, C282Y) has been studied in many cancer types; however, the impact of HFE variants, sex and HFE gene expression in lung cancer has not been studied. We determined the prevalence of HFE variants and their impact on cancer phenotypes in lung cancer cell lines, in lung cancer patient specimens, and using The Cancer Genome Atlas (TCGA) database. We found that seven out of ten human lung cancer cell lines carry the H63D or C282Y HFE variant. Analysis of lung cancer specimens from our institute (Penn State Hershey Medical Center) revealed a sex and genotype interaction risk for metastasis in lung adenocarcinoma (LUAD) patients; H63D HFE is associated with less metastasis in males compared to wild type (WT) HFE; however, females with the H63D HFE variant tend to develop more metastatic tumors than WT female patients. In the TCGA LUAD dataset, the H63D HFE variant was associated with poorer survival in females compared to females with WT HFE. The frequency of C282Y HFE is higher in female lung squamous cell carcinoma (LUSC) patients of TCGA than males, however the C282Y HFE variant did not impact the survival of LUSC patients. In the TCGA LUSC dataset, C282Y HFE patients (especially females) had poorer survival than WT HFE patients. HFE expression level was not affected by HFE genotype status and did not impact patient’s survival, regardless of sex. In summary, these data suggest that there is a sexually dimorphic effect of HFE polymorphisms in the survival and metastatic disease in lung cancer.

TMIC-53. IMPACT OF HFE EXPRESSION AND SEX ON THE TUMOR IMMUNE MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Despite aggressive treatment, the median survival for patients with GBM remains approximately 1 year. Recent evidence demonstrates GBM is a sexually dimorphic disease, and that females have greater overall survival. We previously showed expression levels of HFE, an iron-regulating gene, significantly impacts survival in GBM. Moreover, this effect is sex-specific: females with low HFE expressing tumors have significantly longer survival than males with similar HFE expression. Moreover, HFE expression and survival appeared to correlate with markers of immune activity within the tumors. To further explore the impact of HFE expression on the immune infiltration of GBM, we utilized available TCGA GBM data for analysis by EstimateMe and DeconvuluteMe. These platforms utilize expression data to estimate the magnitude and cellular composition of immune cell infiltrates in tumors, respectively. We sought to correlate these computational measures with HFE expression levels and survival. We confirmed that immune and stromal cell infiltration of tumors is negatively correlated to survival. Consistent with our findings that high HFE levels have a negative impact on survival, high HFE expressing tumors possessed higher levels of both immune and stromal cell infiltration. Additionally, sex of the patient, but not MGMT methylation status, was significantly correlated with immune and stromal infiltration, with males displaying higher levels of infiltration. High HFE tumors were composed of greater numbers of all immune cell types than low HFE tumors. In both HFE groups, monocytes and macrophages comprised the greatest fraction of cells. At low HFE and positive MGMT methylation (where we previously noted the greatest survival differences), we find females have lower monocyte and M2 macrophage markers compared to males with similar features. Collectively, these data suggest that an HFE-immune cell infiltrate axis contributes to sex differences in GBM survival.

EPID-01. SEX DIFFERENCE IN EXPRESSION OF IRON-RELATED GENES AND SURVIVAL IN GLIOBLASTOMA PATIENTS

Abstract Sex impacts the clinical outcome of glioblastoma (GBM) patients. Iron is a key metabolic driver in glioma biology impacting both the neoplastic cells directly and tumor progression through the immune system. We previously reported the expression of HFE (iron homeostatic) gene has a sexually dimorphic impact on survival in GBM patients. To further interrogate the relationship of iron and sexual dimorphism in GBMs, we expanded our analysis to include the ferrome (profile of genes and proteins involved in iron regulation). These genes were identified using IronChip and then interrogated for sexually dimorphic expression and survival in primary GBM patients using The Cancer Genome Atlas GBM database. Biological pathway analysis indicated that the Toll receptor signaling and immune system signaling pathways are most strongly correlated to sex difference and GBM patient survival. Toll like receptor 4 (TLR4) expression in the bottom quartile is associated with a modest survival benefit for female GBM patients compared to male, but in the top quartile of expression of this gene the survival for males is dramatically greater than that for females (18 months versus 3.6 months). Males expressing top quartile of Dual specificity phosphatases (DUSP1) also known as MKP1 survive 24.2 months whereas females survive 5.8 months. There is no sex effect of DUSP1 in the bottom quartile of expression. These data suggest that top quartile expression of TLR4/DUSP1 are both a benefit for males (extending their survival) and a detriment to females (shortening their survival). Both TLR4 and DUSP1 are richly expressed in macrophages. The data suggest that macrophage infiltration into the tumor could be particularly negatively impactful in females; a finding consistent with our previous reports for the HFE gene. In summary, iron related gene expression is linked to sexual dimorphism in survival and may provide insights on sex specific tumor biology.

Do pregnancies reduce iron overload in HFE hemochromatosis women? results from an observational prospective study

BackgroundHFE hemochromatosis is an inborn error of iron metabolism linked to a defect in the regulation of hepcidin synthesis. This autosomal recessive disease typically manifests later in women than men. Although it is commonly stated that pregnancy is, with menses, one of the factors that offsets iron accumulation in women, no epidemiological study has yet supported this hypothesis. The aim of our study was to evaluate the influence of pregnancy on expression of the predominant HFE p.[Cys282Tyr];[Cys282Tyr] genotype.MethodsOne hundred and forty p.Cys282Tyr homozygous women enrolled in a phlebotomy program between 2004 and 2011 at a blood centre in western Brittany (France) were included in the study. After checking whether the disease expression was delayed in women than in men in our study, the association between pregnancy and iron overload was assessed using multivariable regression analysis.ResultsOur study confirms that women with HFE hemochromatosis were diagnosed later than men cared for during the same period (52.6 vs. 47.4 y., P < 0.001). Compared to no pregnancy, having at least one pregnancy was not associated with lower iron markers. In contrast, the amount of iron removed by phlebotomies appeared significantly higher in women who had at least one pregnancy (eβ = 1.50, P = 0.047). This relationship disappeared after adjustment for confounding factors (eβ = 1.35, P = 0.088).ConclusionsOur study shows that pregnancy status has no impact on iron markers level, and is not in favour of pregnancy being a protective factor in progressive iron accumulation. Our results are consistent with recent experimental data suggesting that the difference in disease expression observed between men and women may be explained by other factors such as hormones.

Abstract 4065: Association study between iron and cholesterol for the treatment of drug resistant neuroblastoma

Abstract Neuroblastoma is the most common childhood extracranial solid malignancy. Although cancer cells need more iron and lipids for their active cell metabolism, possible links between iron, mutations in genes involved in iron metabolism (e.g., HFE) and lipid metabolism have not been studied well. We determined the HFE genotype in human neuroblastoma cells as well as patients. Neuroblastoma cells and patients have C282Y HFE mutation rates (~12%) that are similar to those of the general Caucasian population. While cultured cells derived from drug resistant neuroblastomas had less cholesterol, they expressed more ferritin, and contained more iron, than cells derived from drug susceptible tumors. In drug resistant neuroblastoma CHLA-171 cells, siRNA induced alteration of HFE expression had no effect on their iron levels but decreased their cholesterol. Results of in vitro experiments indicated that simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR), was cytotoxic to drug resistant cells as was the iron chelator di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). In vivo, the anti-tumor effect of simvastatin administered orally was minimal. In summary, we found i) an inverse association between ferritin and HMG-CoAR expression in neuroblastoma cells that was reflected in their iron and cholesterol levels, and ii) that the most cytotoxic cholesterol inhibitor of neuroblastoma cells was simvastatin, while the most cytotoxic iron chelation agent was DpC. Our results indicate that it may be possible to use HFE genotype to develop a precision medicine approach to use certain iron chelators and/or cholesterol decreasing agents in the treatment of drug resistant neuroblastomas. Our results also indicate that alterations in iron and cholesterol metabolism are part of the cell’s drug resistance mechanism in neuroblastomas. Citation Format: Sang Y. Lee, Becky Slagle-Webb, Cara-Lynne Schengrund, James Connor. Association study between iron and cholesterol for the treatment of drug resistant neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4065. doi:10.1158/1538-7445.AM2017-4065

Analysis of single nucleotide variants of HFE gene and association to survival in The Cancer Genome Atlas GBM data.

Human hemochromatosis protein (HFE) is involved in iron metabolism. Two major HFE polymorphisms, H63D and C282Y, have been associated with an increased risk of cancers. Previously, we reported decreased gender effects in overall survival based on H63D or C282Y HFE polymorphisms patients with glioblastoma multiforme (GBM). However, the effect of other single nucleotide variation (SNV) in the HFE gene on the cancer development and progression has not been systematically studied. To expand our finding in a larger sample, and to identify other HFE SNV, we analyzed the frequency of somatic SNV in HFE gene and its relationship to survival in GBM patients using The Cancer Genome Atlas (TCGA) GBM (Caucasian only) database. We found 9 SNVs with increased frequency in blood normal of TCGA GBM patients compared to the 1000Genome. Among 9 SNVs, 7 SNVs were located in the intron and 2 SNVs (i.e., H63D, C282Y) in the exon of HFE gene. The statistical analysis demonstrated that blood normal samples of TCGA GBM have more H63D (p = 0.0002, 95% Confidence interval (CI): 0.2119–0.3223) or C282Y (p = 0.0129, 95% CI: 0.0474–0.1159) HFE polymorphisms than 1000Genome. The Kaplan-Meier survival curve for the 264 GBM samples revealed no difference between wild type (WT) HFE and H63D, and WT HFE and C282Y GBM patients. In addition, there was no difference in the survival of male/female GBM patients based on HFE genotype. There was no correlation between HFE expression and survival. In conclusion, the current results suggest that somatic HFE polymorphisms do not impact GBM patients’ survival in the TCGA data set of GBM.

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.