L1CAM mediates neuroendocrine phenotype acquisition in prostate cancer cells.

BACKGROUND: A specific type of prostate cancer (PC) that exhibits neuroendocrine (NE) differentiation is known as NEPC. NEPC has little to no response to androgen deprivation therapy and is associated with the development of metastatic castration-resistant PC (CRPC), which has an extremely poor prognosis. Our understanding of genetic drivers and activated pathways in NEPC is limited, which hinders precision medicine approaches. L1 cell adhesion molecule (L1CAM) is known to play an oncogenic role in metastatic cancers, including CRPC. However, the impact of L1CAM on NEPC progression remains elusive. METHODS: L1CAM expression level was investigated using public gene expression databases of PC cohorts and patient-derived xenograft models. L1CAM knockdown was performed in different PC cells to study in vitro cell functions. A subline of CRPC cell line CWR22Rv1 was established after long-term exposure to abiraterone to induce NE differentiation. The androgen receptor-negative cell line PC3 was cultured under the tumor sphere-forming condition to enrich cancer stemness features. Several oxidative stress inducers were tested on PC cells to observe L1CAM-mediated gene expression and cell death. RESULTS: L1CAM expression was remarkably high in NEPC compared to CRPC or adenocarcinoma tumors. L1CAM was also correlated with NE marker expressions and associated with the adenocarcinoma-to-NEPC progression in gene expression databases and CRPC cells with NE differentiation. L1CAM also promoted cancer stemness and NE phenotypes in PC3 cells under cancer stemness enrichment. L1CAM was also identified as a reactive oxygen species-induced gene, by which L1CAM counteracted CRPC cell death triggered by ionizing radiation. CONCLUSIONS: Our results unveiled a new role of L1CAM in the acquisition of the NE phenotype in PC, contributing to the NE differentiation-related therapeutic resistance of CRPC.

Enhancements of the production of bilirubin and the expression of ß-globin by carbon monoxide during erythroid differentiation.

Heme is degraded by heme oxygenase to form iron, carbon monoxide (CO), and biliverdin. However, information about the catabolism of heme in erythroid cells is limited. In this study, we showed the production and export of bilirubin in murine erythroleukemia (MEL) cells. The production of bilirubin by MEL cells was enhanced when heme synthesis was induced. When mouse bone marrow cells were induced with erythropoietin to differentiate into erythroid cells, the synthesis of bilirubin increased. The expression of ß-globin was enhanced by CO at the transcriptional level. These results indicate that constant production of CO from heme regulates erythropoiesis.

Continuous de novo biosynthesis of haem and its rapid turnover to bilirubin are necessary for cytoprotection against cell damage.

It is well known that haem serves as the prosthetic group of various haemoproteins that function in oxygen transport, respiratory chain, and drug metabolism. However, much less is known about the functions of the catabolites of haem in mammalian cells. Haem is enzymatically degraded to iron, carbon monoxide (CO), and biliverdin, which is then converted to bilirubin. Owing to difficulties in measuring bilirubin, however, the generation and transport of this end product remain unclear despite its clinical importance. Here, we used UnaG, the recently identified bilirubin-binding fluorescent protein, to analyse bilirubin production in a variety of human cell lines. We detected a significant amount of bilirubin with many non-blood cell types, which was sensitive to inhibitors of haem metabolism. These results suggest that there is a basal level of haem synthesis and its conversion into bilirubin. Remarkably, substantial changes were observed in the bilirubin generation when cells were exposed to stress insults. Since the stress-induced cell damage was exacerbated by the pharmacological blockade of haem metabolism but was ameliorated by the addition of biliverdin and bilirubin, it is likely that the de novo synthesis of haem and subsequent conversion to bilirubin play indispensable cytoprotective roles against cell damage.

p53 directly regulates the transcription of the human frataxin gene and its lack of regulation in tumor cells decreases the utilization of mitochondrial iron.

Mitochondrial frataxin functions in iron homeostasis, biogenesis of iron-sulfur clusters, protection from oxidative stress and apoptosis, and as a tumor suppressor protein. We examined regulation of the expression of the human frataxin by p53. Pifithrin-α, an inhibitor of p53 function, and knockdown of p53 decreased the level of frataxin mRNA in human kidney HEK 293T cells. The transcriptional activity of the human frataxin gene is enhanced by the proximal promoter containing the p53-responsive element (p53RE) on the gene. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay confirmed the binding of p53 to the human frataxin p53RE. The expression of wild-type p53 in human cancer HeLa cells increased the reporter activity carrying p53RE at the region of -209 to -200bp of the frataxin promoter. Finally, when the HeLa cells overexpressing frataxin were treated with 5-aminolevulinic acid (ALA), there was less accumulation of protoporphyrin than HeLa control cells, and it was sharply decreased by the addition of iron citrate, suggesting that the utilization of mitochondrial iron for heme biosynthesis can be dependent on the level of frataxin. Alternatively, the low expression of frataxin not regulated by p53 in tumor cells lowers the utilization of iron in mitochondria, causing the tumor-specific ALA-induced accumulation of protoporphyrin.

Imaging of heme/hemeproteins in nucleus of the living cells expressing heme-binding nuclear receptors.

Several factors involved in the core circadian rhythm are PAS domain proteins, one of which, neuronal PAS2 (NPAS2), contains a heme-binding motif. It is thought that heme controls the transcriptional activity of core circadian factors BMAL1-NPAS2, and that the heme-binding nuclear receptor REV-erbα negatively regulates the expression of BMAL1. To examine the role of heme in the nucleus, we expressed nuclear hemeproteins including the nuclear localization signal-added cytoglobin, NPAS2 and REV-erbα. Then, the living cells expressing these proteins were treated with 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). The fluorescent signal derived from DCFH-DA was observed in the nucleus. When the cells were cultured with hemin, the signal of heme in the nucleus increased. Considering that DCFH-DA reacted with heme, we propose that the use of DCFH-DA could be useful in detection of the heme moiety of hemeprotein in vivo.