THE PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL ROLES OF MELANOTRANSFERRIN

Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis

Iron chelator-mediated alterations in gene expression: identification of novel iron-regulated molecules that are molecular targets of hypoxia-inducible factor-1α and p53

ABSTRACT Iron deficiency affects 500 million people, yet the molecular role of iron in gene expression remains poorly characterized. In addition, the alterations in global gene expression after iron chelation remain unclear and are important to assess for understanding the molecular pathology of iron deficiency and the biological effects of chelators. Considering this, we assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone) that have markedly different permeability properties. Sixteen genes were significantly regulated by both ligands, whereas a further 50 genes were significantly regulated by either compound. Apart from ironmediated regulation of expression via hypoxia inducible factor-1␣, it was noteworthy that the transcription factor p53 was also involved in iron-regulated gene expression. Examining 16 genes regulated by both chelators in normal and neoplastic cells, five genes (APP, GDF15, CITED2, EGR1, and PNRC1) were significantly differentially expressed between the cell types. In view of their functions in tumor suppression, proliferation, and apoptosis, these findings are important for understanding the selective antiproliferative effects of chelators against neoplastic cells. Most of the genes identified have not been described previously to be iron-regulated and are important for understanding the molecular and cellular effects of iron depletion. Iron deficiency affects approximately 500 million people. However, despite the enormity of this problem, very little is understood concerning the precise molecular roles played by iron in growth, cell-cycle progression, and apoptosis. Iron plays essential roles in cells, including DNA synthesis and cell cycle control CIP1/WAF1 , GADD45, p53, cyclin D1, etc.) Iron chelators are well known therapeutics for the treatment of iron-overload disease and some of these agents show potential for cancer therap

Relationship between ovarian cancer stem cells, epithelial mesenchymal transition and tumour recurrence

Investigating the biological processes that occur to enable recurrence and the development of chemoresistance in ovarian cancer is critical to the research and development of improved treatment options for patients. The lethality of ovarian cancer is largely attributed to the recurrence of disease with acquired chemoresistance. Cancer stem cells are likely to be critical in ovarian cancer progression, contributing to tumour malignancy, metastasis and recurrence by persisting in the body despite treatment with anti-cancer drugs. Moreover, cancer stem cells are capable of mediating epithelial-to-mesenchymal transition traits and secrete extracellular vesicles to acquire therapy resistance and disease dissemination. These attributes merit in depth research to provide insight into the biological role of ovarian cancer stem cells in disease progression and chemotherapy response, leading to the development of improved biomarkers and innovative therapeutic approaches

The potent and novel thiosemicarbazone chelators Di-2-pyridylketone-4,4- dimethyl-3-thiosemicarbazone and 2-benzoylpyridine-4,4-dimethyl-3- thiosemicarbazone affect crucial thiol systems required for ribonucleotide reductase activity

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone possesses potent and selective antitumor activity. Its cytotoxicity has been attributed to iron chelation leading to inhibition of the iron-containing enzyme ribonucleotide reductase (RR). Thiosemicarbazone iron complexes have been shown to be redox-active, although their effect on cellular antioxidant systems is unclear. Using a variety of antioxidants, we found that only N-acetylcysteine significantly inhibited thiosemicarbazone-induced antiproliferative activity. Thus, we examined the effects of thiosemicarbazones on major thiol-containing systems considering their key involvement in providing reducing equivalents for RR. Thiosemicarbazones significantly (p < 0.001) elevated oxidized trimeric thioredoxin levels to 213 ± 5% (n = 3) of the control. This was most likely due to a significant (p < 0.01) decrease in thioredoxin reductase activity to 65 ± 6% (n = 4) of the control. We were surprised to find that the non-redox-active chelator desferrioxamine increased thioredoxin oxidation to a lower extent (152 ± 9%; n = 3) and inhibited thioredoxin reductase activity (62 ± 5%; n = 4), but at a 10-fold higher concentration than thiosemicarbazones. In contrast, only the thiosemicarbazones significantly (p < 0.05) reduced the glutathione/oxidized-glutathione ratio and the activity of glutaredoxin that requires glutathione as a reductant. All chelators significantly decreased RR activity, whereas the NADPH/NADP total ratio was not reduced. This was important to consider because NADPH is required for thiol reduction. Thus, thiosemicarbazones could have an additional mechanism of RR inhibition via their effects on major thiol-containing systems. Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics

Differential regulation of the Menkes and Wilson disease copper transporters by hormones: an integrated model of metal transport in the placenta

Copper (Cu) plays a critical role in the developing foetus, but virtually nothing is known concerning the regulation of its uptake and metabolism in the placenta. In this issue of the Biochemical Journal, Hardman and colleagues, using a model of placental trophoblasts in culture, identify differential hormonal regulation of two copper-transporting ATPases; namely, those responsible for Menkes disease (ATP7A; MNK) and Wilson disease (ATP7B; WND). Insulin and oestrogen, which are essential during gestation, up-regulate MNK and this leads to trafficking of the MNK protein from the Golgi to the basolateral membrane, resulting in increased Cu efflux. At the same time, insulin decreased WND levels, and this leads to intracellular sequestration of the protein to a perinuclear region that reduces apical Cu release. As such, this results in a concerted flux of Cu from the basolateral surface of the trophoblast that would potentially be used by the developing foetus. An integrated model of vectorized Cu transport is proposed, which involves co-ordinated expression of transporters, organelle interactions and probable protein–protein interactions. The findings have wider implications for considering general models of intracellular metal transport

Correction: N-myc Downstream Regulated 1 (NDRG1) Is Regulated by Eukaryotic Initiation Factor 3a (eIF3a) during Cellular Stress Caused by Iron Depletion

<p>Correction: N-myc Downstream Regulated 1 (NDRG1) Is Regulated by Eukaryotic Initiation Factor 3a (eIF3a) during Cellular Stress Caused by Iron Depletion</p

Primers for amplification of genes used in this study.

<p>Primers for amplification of genes used in this study.</p

Schematic overview of the down-stream genes regulated by eIF3a and the resultant functional effects. (A)

<p>A working model that describes eIF3a’s role in regulating NDRG1 and p27^kip1 expression. <b>(i)</b> When eIF3a is over-expressed, iron depletion up-regulates <i>NDRG1</i> transcription and eIF3a stimulates translation of nascent <i>NDRG1</i> transcripts due to its pro-translation role as an initiation factor subunit. This facilitates <i>de novo</i> NDRG1 synthesis, while translation of non-essential transcripts is suppressed during stress <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Anderson1" target="_blank">[15]</a>. Conversely, our observation that p27^kip1 protein, but not mRNA, is down-regulated by eIF3a over-expression suggests p27^kip1 transcripts may be instead recruited to stress granules, the production of which is dynamically regulated by eIF3a, thereby suppressing p27^kip1 synthesis. (<b>ii)</b> In contrast, when eIF3a is ablated, eIF3a-containing stress granules do not form and <i>p27^kip1</i> transcripts are, by default, recruited by the translational apparatus, thereby increasing p27^kip1 protein expression during iron depletion. In the absence of eIF3a, <i>NDRG1</i> transcripts continue to be directed to the translational apparatus, but are translated at a slower rate due to the loss of eIF3a. This model is consistent with the data presented in this study and with the known ability of eIF3a to negatively regulate p27^kip1 expression by a translational mechanism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong2" target="_blank">[7]</a>. <b>(B)</b> Schematic summarizing some of the functions of eIF3a, including those demonstrated in this study. First, when eIF3a is over-expressed such as in early stages of cancer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Pincheira1" target="_blank">[80]</a> there is: <b>(i)</b> up-regulation of the metastasis suppressor, NDRG1, leading to both increased differentiation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Kovacevic1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-vanBelzen1" target="_blank">[71]</a> and decreased metastasis/invasion (shown herein); <b>(ii)</b> down-regulation of the cyclin-dependent kinase inhibitor, p27^kip1, resulting in activation of the cell cycle and proliferation (shown herein and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong2" target="_blank">[7]</a>); and <b>(iii)</b> increased expression of the ribonucleotide reductase M2 subunit (RRM2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong1" target="_blank">[6]</a> allowing DNA synthesis and growth. Second, when eIF3a expression is abrogated, such as occurs in hypoxic tissues that are typical of advanced tumors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Chen2" target="_blank">[77]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dellas1" target="_blank">[78]</a>, there is: <b>(i)</b> down-regulation of NDRG1, leading to a loss of differentiation and increased metastasis/invasion (shown herein); <b>(ii)</b> up-regulation of p27^kip1, resulting in the inactivation of the cell cycle and inhibited proliferation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong2" target="_blank">[7]</a>; and <b>(iii)</b> decreased expression of RRM2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong1" target="_blank">[6]</a>, preventing DNA synthesis and growth.</p