Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R(2) = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

Hepcidin targets ferroportin for degradation in hepatocytes.

Hepcidin, a circulating regulatory hormone peptide produced by hepatocytes, functions as the master regulator of cellular iron export by controlling the amount of ferroportin, an iron exporter present on the basolateral surface of intestinal enterocytes and macrophages. Hepcidin binding to ferroportin induces its internalization and degradation, resulting in cellular iron retention and decreased iron export. Whether hepatocytes express ferroportin that could be targeted by hepcidin has remained a subject of debate. Here, we describe a hepatocyte culture system expressing high levels of ferroportin, and demonstrate that both endogenously secreted and synthetic hepcidin are fully active in down-regulating membrane-associated ferroportin. In agreement with this result, ferroportin is stabilized in liver hepatocytes of hepcidin-deficient mice and accumulates in periportal areas, supporting the centrolobular iron deposition observed in these mice. In conclusion, we show that hepcidin can trigger ferroportin degradation in hepatocytes, which must be taken into account when considering hepcidin therapeutics.

Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes.

BACKGROUND: The circulating hormone hepcidin plays a central role in iron homeostasis. Our goal was to establish an ex vivo iron-sensing model and to characterize the molecular mechanisms linking iron to hepcidin. DESIGN AND METHODS: Murine hepatocytes were isolated by the collagenase method, either from wild type or HFE knockout mice, and cultured 42 h without serum before treatments. RESULTS: After 42 h of serum-free culture, hepcidin gene expression was undetectable in the hepatocytes. Hepcidin gene expression could, however, be re-activated by an additional 24 h of incubation with 10% serum. Interestingly, addition of 30 microM holotransferrin consistently increased serum-dependent hepcidin levels 3- to 5-fold. The effects of serum and serum+holotransferrin were direct, transcriptional, independent of de novo protein synthesis and required the presence of bone morphogenetic protein. Transferrin receptor-2 activation by its ligand holotransferrin led to extracellular signal regulated kinase (ERK)/mitogen activated protein kinase pathway stimulation and the ERK specific inhibitor U0-126 blunted holotransferrin-mediated induction of hepcidin. ERK activation by holotransferrin provoked increased levels of phospho-Smad1/5/8 highlighting cross-talk between the bone morphogenetic protein/hemojuvelin and ERK1/2 pathways. Finally, we demonstrated, using hepatocytes isolated from Hfe(-/-) mice, that HFE was not critical for the hepcidin response to holotransferrin but important for basal hepcidin expression. CONCLUSIONS: We demonstrate that hepatocytes are liver iron-sensor cells and that transferrin receptor-2, by signaling through the ERK1/2 pathway, and bone morphogenetic protein/hemojuvelin, by signaling through the Smad pathways, coordinately regulate the iron-sensing machinery linking holotransferrin to hepcidin.

Haptoglobin is degraded by iron in C57BL/6 mice: a possible link with endoplasmic reticulum stress.

BACKGROUND: Haptoglobin is a glycoprotein produced mainly by the liver and secreted into the circulation. Haptoglobin, by virtue of its high affinity for hemoglobin, protects the tissues against hemoglobin-induced oxidative damage and allows heme iron recycling. Haptoglobin synthesis is controlled by various effectors, however, little is known concerning its regulation by iron. Haptoglobin regulation in C57BL/6 and 129sv mice fed on an iron-rich diet for 3 weeks was thus undertaken. RESULTS: Iron induced a dramatic post-transcriptional decrease of liver and serum haptoglobin in C57BL/6 mice. In contrast, no alteration of haptoglobin expression was detected in 129sv mice. We assumed that the oxidative stress induced by iron in C57BL/6 mice altered the endoplasmic reticulum (ER) environment, leading to the incorrect folding of haptoglobin and its subsequent degradation. To test this hypothesis, the levels of the RE chaperone GRP78 were measured. This chaperone is known to assist protein folding in the RE during pathophysiological conditions. Interestingly, we found that the mRNA and protein levels of GRP78 were decreased in iron-fed C57BL/6 mice, while they were unchanged in iron-fed 129sv mice. These results suggest that the correct processing of haptoglobin (glycosylation, disulfide linkage, folding, and assembly) might be sensitive to ER stress and that, in the absence of GRP78-mediated assistance, Hp is degraded. CONCLUSION: Our data demonstrate that iron regulates haptoglobin synthesis in C57BL/6 mice and suggest a possible link with iron-induced ER stress.

Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis.

We previously reported that mice made deficient for the transcriptional factor USF2 fail to express hepcidin 1 and hepcidin 2 genes as a consequence of targeted disruption of the Usf2 gene lying just upstream in the locus. These mice developed an iron overload phenotype with excess iron deposition in parenchymal cells and decreased reticuloendothelial iron. At that time, although the role of USF2 was still confounding, we proposed for the first time the role of hepcidin as a negative regulator of iron absorption and iron release from macrophages. Accordingly, we subsequently demonstrated that hyperexpression of hepcidin 1, but not hepcidin 2, resulted in a profound hyposideremic anemia. To analyze the consequences of hepcidin 1 deletion on iron metabolism without any disturbance due to USF2 deficiency, we disrupted the hepcidin 1 gene by targeting almost all the coding region. Confirming our prior results, Hepc1(-/-) mice developed early and severe multivisceral iron overload, with sparing of the spleen macrophages, and demonstrated increased serum iron and ferritin levels as compared with their controls.