Jinlong Jian

17β-Estradiol inhibits iron hormone hepcidin through an estrogen responsive element half-site.

Interaction of estrogen with iron at the systemic level is long suspected, but direct evidence linking the two is limited. In the present study, we examined the effects of 17β-estradiol (E2) on hepcidin, a key negative regulator of iron absorption from the liver. We found that transcription of hepcidin was suppressed by E2 treatment in human liver HuH7 and HepG2 cells, and this down-regulation was blocked by E2 antagonist ICI 182780. Chromatin immunoprecipitation, deletion, and EMSA detected a functional estrogen responsive element half-site that is located between -2474 and -2462 upstream from the start of transcription of the hepcidin gene. After cloning the human hepcidin promoter into the pGL3Luc-Reporter vector, luciferase activity was also down-regulated by E2 treatment in HepG2 cells. E2 reduced hepcidin mRNA in wild-type mice as well as in hemochromatosis Fe gene knockout mice. In summary, our data suggest that hepcidin inhibition by E2 is to increase iron uptake, a mechanism to compensate iron loss during menstruation. This mechanism may also contribute to increased iron stores in oral contraceptive users.

Inhibitory effects of iron on bone morphogenetic protein 2–induced osteoblastogenesis

Postmenopausal osteoporosis is characterized by an imbalance of bone resorption exceeding bone formation, resulting in a net loss of bone mineral density (BMD). Estrogen deficiency is known to promote bone resorption. However, the causative factors that impair bone formation have not been identified. Women after menopause experience not only estrogen deficiency but also iron accumulation as a result of cessation of menstruation. In this study we investigated whether increased iron plays a role in osteoporosis. By growing primary mouse osteoclast and osteoblast progenitor cells as well as immortalized cell lines in the presence of iron, we found that increased iron had minimal effects on osteoclast cell differentiation. Interestingly, iron, particularly in its inorganic form, and to a lesser extent ferritin and transferrin all suppressed alkaline phosphatase (ALP) activities in osteoblasts. Moreover, iron downregulated mRNA levels of several other osteoblastogenic markers such as Runx2, osterix, osteopontin, and osteocalcin. To further show that this in vitro finding is relevant to the in vivo condition, we demonstrated that iron‐accumulated mice with intact ovaries exhibited a significant decrease in BMD. Although iron inhibited preosteoblast cell differentiation, it did enhance preosteoblast cell proliferation, as evidenced by increased cell growth and expression of cell cycle regulator genes such as CDK4, CDK6, cyclin D1, and cyclin D3 and G2/M phase cell population. Taken together, our results suggest that increased iron could be a factor that slows down bone formation in postmenopausal women.

Effects of iron deficiency and iron overload on angiogenesis and oxidative stress-a potential dual role for iron in breast cancer.

Estrogen alone cannot explain the differences in breast cancer (BC) recurrence and incidence rates in pre- and postmenopausal women. In this study, we have tested a hypothesis that, in addition to estrogen, both iron deficiency due to menstruation and iron accumulation as a result of menstrual stop play important roles in menopause-related BC outcomes. We first tested this hypothesis in cell culture models mimicking the high-estrogen and low-iron premenopausal condition or the low-estrogen and high-iron postmenopausal condition. Subsequently, we examined this hypothesis in mice that were fed iron-deficient and iron-overloaded diets. We show that estrogen only slightly up-regulates vascular endothelial growth factor (VEGF), an angiogenic factor known to be important in BC recurrence. It is, rather, iron deficiency that significantly promotes VEGF by stabilizing hypoxia-inducible factor-1α. Conversely, high iron levels increase oxidative stress and sustain mitogen-activated protein kinase activation, which are mechanisms of known significance in BC development. Taken together, our results suggest, for the first time, that an iron-deficiency-mediated proangiogenic environment could contribute to the high recurrence of BC in young patients, and iron-accumulation-associated pro-oxidant conditions could lead to the high incidence of BC in older women.

Effects of cellular iron deficiency on the formation of vascular endothelial growth factor and angiogenesis.

BackgroundYoung women diagnosed with breast cancer are known to have a higher mortality rate from the disease than older patients. Specific risk factors leading to this poorer outcome have not been identified. In the present study, we hypothesized that iron deficiency, a common ailment in young women, contributes to the poor outcome by promoting the hypoxia inducible factor-1α (HIF-1α and vascular endothelial growth factor (VEGF) formation. This hypothesis was tested in an in vitro cell culture model system.ResultsHuman breast cancer MDA-MB-231 cells were transfected with transferrin receptor-1 (TfR1) shRNA to constitutively impair iron uptake. Cellular iron status was determined by a set of iron proteins and angiogenesis was evaluated by levels of VEGF in cells as well as by a mouse xenograft model. Significant decreases in ferritin with concomitant increases in VEGF were observed in TfR1 knockdown MDA-MB-231 cells when compared to the parental cells. TfR1 shRNA transfectants also evoked a stronger angiogenic response after the cells were injected subcutaneously into nude mice. The molecular mechanism appears that cellular iron deficiency elevates VEGF formation by stabilizing HIF-1α. This mechanism is also true in human breast cancer MCF-7 and liver cancer HepG2 cells.ConclusionsCellular iron deficiency increased HIF-1α, VEGF, and angiogenesis, suggesting that systemic iron deficiency might play an important part in the tumor angiogenesis and recurrence in this young age group of breast cancer patients.

Src regulates Tyr(20) phosphorylation of transferrin receptor-1 and potentiates breast cancer cell survival.

Background: The role of transferrin receptor-1 (TfR1) in cell signaling remains unknown. Results: Using gambogic acid, we found that TfR1 is bound to Src and its phosphorylation at Tyr20 potentiates breast cancer cell survival. Conclusion: In addition to its iron uptake function, TfR1 is involved in cell survival. Significance: High expression of TfR1 in cancer cells presents a therapeutic target for gambogic acid and Src inhibitor. Transferrin receptor 1 (TfR1) is a ubiquitous type II membrane receptor with 61 amino acids in the N-terminal cytoplasmic region. TfR1 is highly expressed in cancer cells, particularly under iron deficient conditions. Overexpression of TfR1 is thought to meet the increased requirement of iron uptake necessary for cell growth. In the present study, we used transferrin (Tf), a known ligand of TfR1, and gambogic acid (GA), an apoptosis-inducing agent and newly identified TfR1 ligand to investigate the signaling role of TfR1 in breast cancer cells. We found that GA but not Tf induced apoptosis in a TfR1-dependent manner in breast cancer MDA-MB-231 cells. Estrogen receptor-positive MCF-7 cells lack caspase-3 and were not responsive to GA treatment. GA activated the three major signaling pathways of the MAPK family, as well as caspase-3, -8, and Poly(ADP-ribose)polymerase apoptotic pathway. Interestingly, only Src inhibitor PP2 greatly sensitized the cells to GA-mediated apoptosis. Further investigations by confocal fluorescence microscopy and immunoprecipitation revealed that Src and TfR1 are constitutively bound. Using TfR1-deficient CHO TRVB cells, point mutation studies showed that Tyr20 within the 20YTRF23 motif of the cytoplasmic region of TfR1 is the phosphorylation site by Src. TfR1 Tyr20 phosphomutants were more sensitive to GA-mediated apoptosis. Our results indicate that, albeit its iron uptake function, TfR1 is a signaling molecule and tyrosine phosphorylation at position 20 by Src enhances anti-apoptosis and potentiates breast cancer cell survival.

A link between premenopausal iron deficiency and breast cancer malignancy

BackgroundYoung breast cancer (BC) patients less than 45 years old are at higher risk of dying from the disease when compared to their older counterparts. However, specific risk factors leading to this poorer outcome have not been identified.MethodsOne candidate is iron deficiency, as this is common in young women and a clinical feature of young age. In the present study, we used immuno-competent and immuno-deficient mouse xenograft models as well as hemoglobin as a marker of iron status in young BC patients to demonstrate whether host iron deficiency plays a pro-metastatic role.ResultsWe showed that mice fed an iron-deficient diet had significantly higher tumor volumes and lung metastasis compared to those fed normal iron diets. Iron deficiency mainly altered Notch but not TGF-β and Wnt signaling in the primary tumor, leading to the activation of epithelial mesenchymal transition (EMT). This was revealed by increased expression of Snai1 and decreased expression of E-cadherin. Importantly, correcting iron deficiency by iron therapy reduced primary tumor volume, lung metastasis, and reversed EMT markers in mice. Furthermore, we found that mild iron deficiency was significantly associated with lymph node invasion in young BC patients (p<0.002).ConclusionsTogether, our finding indicates that host iron deficiency could be a contributor of poor prognosis in young BC patients.

Iron sensitizes keratinocytes and fibroblasts to UVA-mediated matrix metalloproteinase-1 through TNF-α and ERK activation

Abstract:  Oestrogen deficiency is regarded as the main causative factor in postmenopausal skin ageing and photoageing. While women after menopause experience low levels of oestrogen because of cease of ovarian function, they are also exposed to high levels of iron as a result of cessation of menstruation. In this study, we investigated whether this increase in iron presents a risk to the postmenopausal skin. Because of the lack of appropriate animal models to closely mimic the low oestrogen and high iron conditions, we tested the hypothesis in a high iron and low oestrogen culture model. Here, we showed that primary human dermal fibroblasts exposed to iron did not affect the baseline levels of matrix metalloproteinase‐1 (MMP‐1) activity. However, the iron‐exposed fibroblasts were sensitized to UVA exposure, which resulted in a synergistic increase in MMP‐1. UVA activated the three members of MAPK family: ERKs, p38, and JNKs. Additional activation of ERKs by iron contributed to the synergistic increases. Primary normal human epidermal keratinocytes (NHEK) did not respond to iron or UVA exposure as measured by MMP‐1, but produced tumor necrosis factor‐alpha (TNF‐α) in the media, which then stimulated MMP‐1 in fibroblasts. Our results indicate that iron and UVA increase MMP‐1 activity in dermal fibroblasts not only directly through ERK activation but also by an indirect paracrine loop through TNF‐α released by NHEK. We conclude that in addition to oestrogen deficiency, increased iron as a result of menopause could be a novel risk factor by sensitizing postmenopausal skin to solar irradiation.

Protection against ultraviolet A-induced oxidative damage in normal human epidermal keratinocytes under post-menopausal conditions by an ultraviolet A-activated caged-iron chelator: a pilot study

Background/purpose: Human skin is constantly exposed to ultraviolet A (UVA), which can generate reactive oxygen species and cause iron release from ferritin, leading to oxidative damage in biomolecules. This is particularly true in post‐menopausal skin due to an increase in iron as a result of menopause. As iron is generally released through desquamation, the skin becomes a main portal for the release of excess iron in this age group. In the present study, we examined a strategy for controlling UVA‐ and iron‐induced oxidative stress in skin using a keratinocyte post‐menopausal cellular model system.

Abstract 3379: Iron-deficient anemia promotes breast cancer growth and lung metastasis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Young breast cancer patients <45 years old are at a higher risk of dying from the disease than older patients. Specific risk factors contributing to this high mortality rate have not been identified. Iron deficient anemia (IDA) is a common ailment in young pre-menopausal women both in developed and developing countries. In the present study, we hypothesize that IDA promotes breast cancer growth and metastasis. To test this hypothesis, Balb/c mice were fed three levels of iron diets, 3.5 ppm Fe as iron deficient, 35 ppm as normal low, and 350 ppm as normal high for 12 wks. Consistent with previous literature reports, iron levels in diets ranging from 35 to 350 ppm, which were considered normal, did not lead to significant differences in serum iron and transferrin saturation rates. However, an iron level at 3.5 ppm in the diet caused an IDA in mice. After subcutaneously inoculating 4T1 mammary cancer cells into the flanks of mice fed different iron diets, mice with IDA condition have significant higher tumor volumes as compared to those with normal iron status (2922 + 161 mm3 in 3.5 ppm Fe group vs 1636 + 86 mm3 in 35 ppm Fe group and 1453 + 90 mm3 in 350 ppm Fe group). Lung metastasis was also higher in IDA mice with 22.8 + 0.9 nodules per lung lobule in 3.5 ppm vs 13.8 + 0.8 in 35 ppm and 13.6 + 0.9 in 350 ppm Fe. To elucidate molecular mechanisms of IDA in tumor growth and metastasis, levels of epithelial mesenchymal transition (EMT) markers, such as snail, slug and zeb1, were found to be significantly higher in tumor tissues isolated from IDA mice. When investigating signal pathways regulating EMT, it was found that Notch pathway, including Notch 2, 3, 4, and Notch ligands such as Jagged1, 2, were all induced by IDA. In contrast to Notch, transforming growth factor (TGF)-β and wingless-int (WNT) signal pathways, known to stimulate EMT, were not affected, suggesting that IDA-induced EMT is Notch pathway-specific. Since anemia causes hypoxia, the expression of HIF-1α, a key transcription factor controlling hypoxic responses, was also induced by IDA. Similarly, Notch signal pathway and EMT markers were higher after transfecting HIF-1α cDNA into 4T1 cells. These data show that IDA-mediated breast cancer growth and lung metastasis is through HIF-1α accumulation and activation of Notch and EMT. Our study suggests that identification of IDA as a poor prognostic factor and its clinical improvement as a potential cancer therapy could be significant to the high mortality rate of young breast cancer patients. This work is supported by R21 CA132684. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3379. doi:10.1158/1538-7445.AM2011-3379

Abstract 1045: Src-mediated Tyr20 phosphorylation of transferrin receptor 1 (TfR1) promotes its anti-apoptotic effect: A novel function of TfR1 in cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Transferrin receptor 1 (TfR1) is highly expressed in cancer cells and its overexpression is widely thought to be a result of the increased requirement of iron uptake for cell growth. In the present study, we have found that two ligands of TfR1, gambogic acid (GA) but not holo-transferrin (Tf) induced apoptosis in breast cancer MDA-MB-231 cells and activated the JNK and p38 signal pathways. Furthermore, PP2, an inhibitor of steroid receptor coactivator (Src), a tyrosine kinase, greatly promoted GA-mediated apoptosis. These results indicate that Src is involved in GA-mediated TfR1-dependent apoptotic pathway. Following immunoprecipitation of TfR1 and immunoblotting of Src or verse versa, TfR1 was found to be associated with Src. This association was further illustrated by their co-localization with confocal fluorescence microscope and shown resulting in constitutive tyrosine phosphorylations of the TfR1. A type II membrane receptor, TfR1 contains a short intracellular N-terminal domain with 61 amino acids. 20YTRF23, the only Tyr(Y) site within the cytoplasmic domain, has been shown as the motif essential for TfR1-Tf complex internalization. To elucidate which Tyr is phosphorylated, we used a Chinese hamster ovary (CHO)-TRVb cell line that does not express TfR1. Point mutation Y20L showed that Tyr20 of TfR1 was the phosphorylation site by Src. Further treatments of wild type and point mutant revealed that it is the Tyr20 phosphorylation that enhanced the anti-apoptotic effects of TfR1 against GA. Our results indicate that in addition to the known function of iron transport, TfR1 has a novel function of regulating cell survival and antiapoptosis through its Tyr20 phosphorylation by Src. This new function is particularly significant and relevant to cancer cells since high expression of TfR1 not only fulfills the requirement of more iron as a co-factor in DNA synthesis but also enhances their survival. This work is supported by R21 CA132684. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2011-1045