Ferritin Heavy Chain Binds Peroxiredoxin 6 and Inhibits Cell Proliferation and Migration.

The H Ferritin subunit (FTH1), as well as regulating the homeostasis of intracellular iron, is involved in complex pathways that might promote or inhibit carcinogenesis. This function may be mediated by its ability to interact with different molecules. To gain insight into the FTH1 interacting molecules, we analyzed its interactome in HEK293T cells. Fifty-one proteins have been identified, and among them, we focused our attention on a member of the peroxiredoxin family (PRDX6), an antioxidant enzyme that plays an important role in cell proliferation and in malignancy development. The FTH1/PRDX6 interaction was further supported by co-immunoprecipitation, in HEK293T and H460 cell lines and by means of computational methods. Next, we demonstrated that FTH1 could inhibit PRDX6-mediated proliferation and migration. Then, the results so far obtained suggested that the interaction between FTH1/PRDX6 in cancer cells might alter cell proliferation and migration, leading to a less invasive phenotype.

FTH1 Pseudogenes in Cancer and Cell Metabolism.

Ferritin, the principal intracellular iron-storage protein localized in the cytoplasm, nucleus, and mitochondria, plays a major role in iron metabolism. The encoding ferritin genes are members of a multigene family that includes some pseudogenes. Even though pseudogenes have been initially considered as relics of ancient genes or junk DNA devoid of function, their role in controlling gene expression in normal and transformed cells has recently been re-evaluated. Numerous studies have revealed that some pseudogenes compete with their parental gene for binding to the microRNAs (miRNAs), while others generate small interference RNAs (siRNAs) to decrease functional gene expression, and still others encode functional mutated proteins. Consequently, pseudogenes can be considered as actual master regulators of numerous biological processes. Here, we provide a detailed classification and description of the structural features of the ferritin pseudogenes known to date and review the recent evidence on their mutual interrelation within the complex regulatory network of the ferritin gene family.

FtH-Mediated ROS Dysregulation Promotes CXCL12/CXCR4 Axis Activation and EMT-Like Trans-Differentiation in Erythroleukemia K562 Cells.

The cell-microenvironment communication is essential for homing of hematopoietic stem cells in stromal niches. Recent evidences support the involvement of epithelial-to-mesenchymal (EMT) process in hematopoietic stem cell homeostasis as well as in leukemia cells invasiveness and migration capability. Here, we demonstrate that the alteration of iron homeostasis and the consequent increase of redox metabolism, mediated by the stable knock down of ferritin heavy chain (FtH), enhances the expression of CXCR4 in K562 erythroleukemia cells, thus promoting CXCL12-mediated motility. Indeed, addition of the CXCR4 receptor antagonist AMD3100 reverts this effect. Upon FtH knock down K562 cells also acquire an "EMT-like" phenotype, characterized by the increase of Snail, Slug and Vimentin with the parallel loss of E-cadherin. By using fibronectin as substrate, the cell adhesion assay further shows a reduction of cell adhesion capability in FtH-silenced K562 cells. Accordingly, confocal microscopy shows that adherent K562 control cells display a variety of protrusions while FtH-silenced K562 cells remain roundish. These phenomena are largely due to the reactive oxygen species (ROS)-mediated up-regulation of HIF-1α/CXCR4 axis which, in turn, promotes the activation of NF-κB and the enhancement of EMT features. These data are confirmed by treatments with either N-acetylcysteine (NAC) or AMD3100 or NF-κB inhibitor IκB-alpha which revert the FtH-silenced K562 invasive phenotype. Overall, our findings demonstrate the existence of a direct relationship among iron metabolism, redox homeostasis and EMT in the hematological malignancies. The effects of FtH dysregulation on CXCR4/CXCL12-mediated K562 cell motility extend the meaning of iron homeostasis in the leukemia cell microenvironment.

Iron and Ferritin Modulate MHC Class I Expression and NK Cell Recognition.

The ability of pathogens to sequester iron from their host cells and proteins affects their virulence. Moreover, iron is required for various innate host defense mechanisms as well as for acquired immune responses. Therefore, intracellular iron concentration may influence the interplay between pathogens and immune system. Here, we investigated whether changes in iron concentrations and intracellular ferritin heavy chain (FTH) abundance may modulate the expression of Major Histocompatibility Complex molecules (MHC), and susceptibility to Natural Killer (NK) cell cytotoxicity. FTH downregulation, either by shRNA transfection or iron chelation, led to MHC surface reduction in primary cancer cells and macrophages. On the contrary, mouse embryonic fibroblasts (MEFs) from NCOA4 null mice accumulated FTH for ferritinophagy impairment and displayed MHC class I cell surface overexpression. Low iron concentration, but not FTH, interfered with IFN-γ receptor signaling, preventing the increase of MHC-class I molecules on the membrane by obstructing STAT1 phosphorylation and nuclear translocation. Finally, iron depletion and FTH downregulation increased the target susceptibility of both primary cancer cells and macrophages to NK cell recognition. In conclusion, the reduction of iron and FTH may influence the expression of MHC class I molecules leading to NK cells activation.

Chemoresistance in H-Ferritin Silenced Cells: The Role of NF-κB.

Nuclear Factor-κB (NF-κB) is frequently activated in tumor cells contributing to aggressive tumor growth and resistance to chemotherapy. Here we demonstrate that Ferritin Heavy Chain (FHC) protein expression inversely correlates with NF-κB activation in cancer cell lines. In fact, FHC silencing in K562 and SKOV3 cancer cell lines induced p65 nuclear accumulation, whereas FHC overexpression correlated with p65 nuclear depletion in the same cell lines. In FHC-silenced cells, the p65 nuclear accumulation was reverted by treatment with the reactive oxygen species (ROS) scavenger, indicating that NF-κB activation was an indirect effect of FHC on redox metabolism. Finally, FHC knock-down in K562 and SKOV3 cancer cell lines resulted in an improved cell viability following doxorubicin or cisplatin treatment, being counteracted by the transient expression of inhibitory of NF-κB, IκBα. Our results provide an additional layer of information on the complex interplay of FHC with cellular metabolism, and highlight a novel scenario of NF-κB-mediated chemoresistance triggered by the downregulation of FHC with potential therapeutic implications.

DJ-1 is a reliable serum biomarker for discriminating high-risk endometrial cancer.

New reliable approaches to stratify patients with endometrial cancer into risk categories are highly needed. We have recently demonstrated that DJ-1 is overexpressed in endometrial cancer, showing significantly higher levels both in serum and tissue of patients with high-risk endometrial cancer compared with low-risk endometrial cancer. In this experimental study, we further extended our observation, evaluating the role of DJ-1 as an accurate serum biomarker for high-risk endometrial cancer. A total of 101 endometrial cancer patients and 44 healthy subjects were prospectively recruited. DJ-1 serum levels were evaluated comparing cases and controls and, among endometrial cancer patients, between high- and low-risk patients. The results demonstrate that DJ-1 levels are significantly higher in cases versus controls and in high- versus low-risk patients. The receiver operating characteristic curve analysis shows that DJ-1 has a very good diagnostic accuracy in discriminating endometrial cancer patients versus controls and an excellent accuracy in distinguishing, among endometrial cancer patients, low- from high-risk cases. DJ-1 sensitivity and specificity are the highest when high- and low-risk patients are compared, reaching the value of 95% and 99%, respectively. Moreover, DJ-1 serum levels seem to be correlated with worsening of the endometrial cancer grade and histotype, making it a reliable tool in the preoperative decision-making process.

Role of serum ferritin level on overall survival in patients with myelodysplastic syndromes: Results of a meta-analysis of observational studies.

BACKGROUND: The role of serum ferritin (SF) as a prognostic factor has been analyzed in patients with myelodysplastic syndromes (MDS) who have undergone hematopoietic stem cell transplantation (HSCT), but the prognostic role of elevated SF levels is still controversial in lower risk MDS patients. Therefore, we performed a meta-analysis of all available published literature to evaluate whether elevated SF levels are associated with a worse overall survival (OS) among patients with low risk MDS. MATERIAL AND METHODS: A systematic bibliographic search of relevant studies was undertaken in accordance with guidelines for meta-analysis of observational studies in epidemiology. Electronic databases were searched through July 2016 for studies examining the level of SF as a prognostic factor in the adults affected by MDS. RESULTS: Six articles were included in the meta-analysis. A significant association between OS and SF was achieved for the threshold of SF≥1000 ng/mL, when the only study that used SF cut-off ≥2000 ng/mL was not included in the meta-analysis (RR = 1.33; 95% CI = 1.06-1.67). The estimated risk was 2.58 (95% CI = 1.41-4.74) when a SF cut-off≥500 ng/mL was considered. CONCLUSIONS: Our findings underlined a worse survival in patients with MDS who had higher SF levels. The association was stronger and achieved statistical significance after stratification of analyses in which we excluded cut-offs of SF level considered as outliers. These results suggest that negative impact on OS already exist at SF level ≥500 ng/mL. Prospective studies, are needed to better understand this relationship and, above all, to clarify whether earlier iron chelation therapy could improve patients' OS.

FTH1P3, a Novel H-Ferritin Pseudogene Transcriptionally Active, Is Ubiquitously Expressed and Regulated during Cell Differentiation.

Ferritin, the major iron storage protein, performs its essential functions in the cytoplasm, nucleus and mitochondria. The variable assembly of 24 subunits of the Heavy (H) and Light (L) type composes the cytoplasmic molecule. In humans, two distinct genes code these subunits, both belonging to complex multigene families. Until now, one H gene has been identified with the coding sequence interrupted by three introns and more than 20 intronless copies widely dispersed on different chromosomes. Two of the intronless genes are actively transcribed in a tissue-specific manner. Herein, we report that FTH1P3, another intronless pseudogene, is transcribed. FTH1P3 transcript was detected in several cell lines and tissues, suggesting that its transcription is ubiquitary, as it happens for the parental ferritin H gene. Moreover, FTH1P3 expression is positively regulated during the cell differentiation process.

H ferritin silencing induces protein misfolding in K562 cells: A Raman analysis.

The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation.

H-ferritin-regulated microRNAs modulate gene expression in K562 cells.

In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC) comparing it with K562 transduced with scrambled RNA (K562shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, "Cell Death and Survival, Hematological System Development and Function, Hematopoiesis", is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.

Identification of H ferritin-dependent and independent genes in K562 differentiating cells by targeted gene silencing and expression profiling.

Ferritin is best known as the key molecule in intracellular iron storage, and is involved in several metabolic processes such as cell proliferation, differentiation and neoplastic transformation. We have recently demonstrated that the shRNA silencing of the ferritin heavy subunit (FHC) in a melanoma cell line is accompanied by a consistent modification of gene expression pattern leading to a reduced potential in terms of proliferation, invasiveness, and adhesion ability of the silenced cells. In this study we sought to define the repertoire of genes whose expression might be affected by FHC during the hemin-induced differentiation of the erythromyeloid cell line K562. To this aim, gene expression profiling was performed in four different sets of cells: i) wild type K562; ii) sh-RNA FHC-silenced K562; iii) hemin-treated wild-type K562; and iv) hemin-treated FHC-silenced K562. Statistical analysis of the gene expression data, performed by two-factor ANOVA, identified three distinct classes of transcripts: a) Class 1, including 657 mRNAs whose expression is modified exclusively during hemin-induced differentiation of K562 cells, independently from the FHC relative amounts; b) Class 2, containing a set of 70 mRNAs which are consistently modified by hemin and FHC-silencing; and c) Class 3, including 128 transcripts modified by FHC-silencing but not by hemin. Our data indicate that FHC may function as a modulator of gene expression during erythroid differentiation and add new findings to the knowledge of the complex gene network modulated during erythroid differentiation.

Identification of prognosis-related proteins in gingival squamous cell carcinoma by twodimensional gel electrophoresis and mass spectrometry-based proteomics.

OBJECTIVES: The aim of this study was to identify differentially expressed proteins in oral squamous carcinoma cells that could be potential prognosis-related cancer biomarkers. MATERIALS AND METHODS: We compared protein expression patterns from gingival squamous cellc carcinoma (GSCC) tissues and adjacent non-cancerous matched tissues by proteomic analysis using two-dimensional gel electrophoresis coupled to mass spectrometry (2D-PAGE/MS). RESULTS: Seventeen protein spots were found to be over-expressed and eight were under-expressed in cancerous tissue compared to the normal counterpart. Of these, annexin A2 and ezrin were validated by Western blot. We also demonstrated by immunohistochemistry that POSTN is highly expressed in the neoplastic tissues examined. Among the differentially expressed proteins, we focused our attention on Chloride intracellular channel 1 (CLIC1). CONCLUSION: The 2D-PAGE/MS-based proteomics appears an efficient approach in detecting and identifying differentially expressed proteins that might function as potential biomarkers and/or molecular targets for early cancer diagnosis and prognosis and that might contribute to a innovative therapeutic strategies in GSCC. However, further validation and functional studies are needed to confirm and to support these promising, still preliminary data. KEY WORDS: Cancer biomarkers, Oral squamous cell carcinoma, Proteomics.

H ferritin gene silencing in a human metastatic melanoma cell line: a proteomic analysis.

Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular the H subunit (FHC), is involved in different biological events such as cell differentiation and pathologic states (i.e., neurodegeneration and cancer). This study is aimed at investigating the whole-cell proteome of FHC-expressing and sh-RNA-silenced human metastatic melanoma cells (MM07(m)) in the attempt to identify and classify the highest number of proteins directly or indirectly controlled by the FHC. We identified about 200 differentially expressed proteins and classified them in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration, and proliferation processes. Some of them have captured our attention because of their involvement in metabolic pathways related to tumor progression and metastasis. In vitro assays confirmed that the FHC-silenced MM07(m) cells are characterized by a decreased growth activity, a reduced invasiveness, and a reduced cell adhesion capability. Moreover, nude mice (CD1 nu/nu), subcutaneously injected with FHC-silenced MM07(m) cells, showed a remarkable 4-fold reduction of their tumor growth capacity compared to those who received the FHC-unsilenced MM07(m) counterpart. In conclusion, these data indicate that gene silencing technology, coupled to proteomic analysis, is a powerful tool for a better understanding of H ferritin signaling pathways and lend support to the hypothesis that specific targeting of this gene might be an attractive and potentially effective strategy for the management of metastatic melanoma.

BRCA1 is required for hMLH1 stabilization following doxorubicin-induced DNA damage.

Human DNA mismatch repair (MMR) is involved in the removal of DNA base mismatches that arise either during DNA replication or are caused by DNA damage. In this study, we show that the activation of the MMR component hMLH1 in response to doxorubicin (DOX) treatment requires the presence of BRCA1 and that this phenomenon is mediated by an ATM/ATR dependent phosphorylation of the hMLH1 Ser-406 residue. BRCA1 is an oncosuppressor protein with a central role in the DNA damage response and it is a critical component of the ATM/ATR mediated checkpoint signaling. Starting from a previous finding in which we demonstrated that hMLH1 is able to bind to BRCA1, in this study we asked whether BRCA1 might be the bridge for ATM/ATR dependent phosphorylation of the hMLH1 molecular partner. We found that: (i) the negative modulation of BRCA1 expression is able to produce a remarkable reversal of hMLH1 stabilization, (ii) BRCA1 is required for post-translational modification produced by DOX treatment on hMLH1 which is, in turn, attributed to the ATM/ATR activity, (iii) the serine 406 phosphorylatable residue is critical for hMLH1 activation by ATM/ATR via BRCA1. Taken together, our data lend support to the hypothesis suggesting an important role of this oncosuppressor as a scaffold or bridging protein in DNA-damage response signaling via downstream phosphorylation of the ATM/ATR substrate hMLH1.

Bilateral cataract in a subject carrying a C to A transition in the L ferritin promoter region.

OBJECTIVES: The aim of this study is to evaluate the potential impact of mutations in the promoter region of the L ferritin gene on its transcriptional activity. DESIGN AND METHODS: To search for the presence of mutations in the promoter of the L gene, we amplified by PCR a DNA region of about 385 n.t. in 100 healthy subjects from Southern Italy. RESULTS: A subject carrying a C to A transition in position -216 was identified. This transition causes an increased transcriptional activity in vitro. This finding was substantiated by Real Time Quantitative PCR, which showed increased levels of L ferritin mRNA. CONCLUSIONS: A previously unidentified mutation in the promoter region of the L ferritin gene was detected in an individual. Interestingly, this subject is affected by bilateral cataract, a disease that has been correlated, in a subset of patients, with high levels of circulating ferritin. We hypothesize that changes in the expression of the L ferritin might be linked, at least to a certain extent, to the pathogenesis of this rare eye disease.

BRCA1 5083del19 mutant allele selectively up-regulates periostin expression in vitro and in vivo.

PURPOSE: The aim of this study was to explore the gene expression pattern produced by the cancer-associated BRCA1 5083del19 founder mutation by using a microarray analysis. Such a mutation, identified in a subset of familial breast cancer patients, involves a deletion at the 3' end of the BRCA1 messenger leading, in the mature protein, to the ablation of the BRCT tandem domain. EXPERIMENTAL DESIGN: We generated HeLa cells stably expressing both exogenous wild-type (HeLa/(wt)BRCA1), used as a control, and 5083del19 BRCA1 (HeLa/(5083del19)BRCA1) alleles; gene chips were then used to investigate any changes in the transcription profile induced by the 5083del19 BRCA1 mutant compared with controls. RESULTS: Among the genes showing perturbation of their expression, periostin was found to be up-regulated in HeLa/(5083del19)BRCA1 cells to an extent of 72-fold versus HeLa/(pcDNA3.1/empty) and 76-fold versus HeLa/(wt)BRCA1 cells. This finding was validated both in vitro in breast cancer cell lines harboring mutations of BRCA1 and in vivo by immunohistochemistry of breast cancer specimens bearing the 5083del19 BRCA1 mutation as well as by Western blot analysis of sera obtained from patients and healthy carriers of the same mutation. CONCLUSIONS: Our results suggest that periostin overexpression, whose product is released from cells in the extracellular fluids, might be a potential marker for early cancer detection in a specific subset of hereditary breast carcinomas triggered by cancer-associated BRCA1 mutations that affect the BRCT tandem domain.

p53-mediated downregulation of H ferritin promoter transcriptional efficiency via NF-Y.

The tumor suppressor protein p53 triggers many of the cellular responses to DNA damage by regulating the transcription of a series of downstream target genes. p53 acts on the promoter of the target genes by interacting with the trimeric transcription factor NF-Y. H ferritin promoter activity is tightly dependent on a multiprotein complex called Bbf; on this complex NF-Y plays a major role. The aim of this work was to study the modulation of H ferritin expression levels by p53. CAT reporter assays indicate that: (i) p53 overexpression strongly downregulates the transcriptional efficiency driven by an H ferritin promoter construct containing only the NF-Y recognition sequence and that the phenomenon is reverted by p53 siRNA; (ii) the p53 C-terminal region is sufficient to elicitate this regulation and that a correct C-terminal acetylation is also required. The H ferritin promoter displays no p53-binding sites; chromatin immunoprecipitation assays indicate that p53 is recruited on this promoter by NF-Y. The p53-NF-Y interaction does not alter the NF-Y DNA-binding ability as indicated by electrophoretic mobility shift assay (EMSA) analysis. These results demonstrate that the gene coding for the H ferritin protein belongs to the family of p53-regulated genes, therefore adding a new level of complexity to the regulation of the H ferritin transcription and delineate a role for this protein in a series of cellular events triggered by p53 activation.

Missense mutations of BRCA1 gene affect the binding with p53 both in vitro and in vivo.

Women with BRCA1 gene mutations have an increased risk for breast and ovarian cancer (BOC). Classification of missense variants as neutral or disease causing is still a challenge and has major implications for genetic counseling. BRCA1 is organized in an N-terminal ring-finger domain and two BRCT (breast cancer C-terminus) domains, involved in protein-protein interaction. The integrity of the C-terminal, BRCT repeat region is also critical for BRCA1 tumor suppressor function. Several molecular partners of BRCA1 have so far been identified; among them, the tumor suppressor protein p53 seems to play a major role. This study was aimed at evaluating the impact of two missense mutations, namely the W1837R and the S1841N, previously identified in BOC patients and located in the BRCT domain of the BRCA1 gene, on the binding capacity of this protein to p53. Co-immunoprecipitation assays of E. coli-expressed wild-type and mutated BRCTs challenged with a HeLa cell extract revealed, for the S1841N variant a significant reduction in the binding activity to p53, while the W1837R mutant showed an inverse effect. Furthermore, a clonogenic soft agar growth assay performed on HeLa cells stably transfected with either wild-type or mutant BRCA1 showed a marked decrease of the growth in wild-type BRCA1-overexpressing cells and in BRCA1S1841N-transfected cells, while no significant changes were detected in the BRCA1W1837R-transfected cells. These results demonstrate that: i) distinct single nucleotide changes in the BRCT domain of BRCA1 affect binding of this protein to the tumor suppressor p53, and ii) the two missense mutations here described are likely to play a role in breast tumorigenesis. We suggest that in vitro/in vivo experiments testing the effects of unclassified BRCA1 gene variants should therefore be taken in to consideration and that increased surveillance should be adopted in individuals bearing these two BRCA1 missense alterations.