Hepatic failure is commonly associated with anemia, which may result from gastrointestinal bleeding, vitamin deficiency, or liver-damaging diseases, such as infection and alcohol intoxication. At least in theory, anemia during hepatic failure may result from accelerated clearance of circulating erythrocytes. Here we show that bile duct ligation (BDL) in mice leads to severe anemia despite increased reticulocyte numbers. Bilirubin stimulated suicidal death of human erythrocytes. Mechanistically, bilirubin triggered rapid Ca2+ influx, sphingomyelinase activation, formation of ceramide, and subsequent translocation of phosphatidylserine to the erythrocyte surface. Consistent with our in vitro and in vivo findings, incubation of erythrocytes in serum from patients with liver disease induced suicidal death of erythrocytes in relation to their plasma bilirubin concentration. Consistently, patients with hyperbilirubinemia had significantly lower erythrocyte and significantly higher reticulocyte counts compared to patients with low bilirubin levels. Conclusion: Bilirubin triggers suicidal erythrocyte death, thus contributing to anemia during liver disease. (Hepatology 2015;61:275–284)

Background: As a novel class of noncoding RNAs, circRNAs have been recently identified to regulate tumorigenesis and aggressiveness. However, the function of circRNAs in colorectal cancer (CRC) metastasis remains unclear. We aimed to identify circRNAs that are upregulated in CRC tissues from patients and study their function in CRC metastasis. Methods: We compared six pairs of CRC tissues and their matched adjacent non-tumor tissues by using circRNA microarray. We first evaluated the expression of circPTK2 (hsa_circ_0005273) in fresh tissues from CRC tumors and corresponding adjacent tissues by qPCR analysis. CircPTK2 expression levels in the tissue microarray with 5 years of survival information were determined by RNA-ISH analysis. Meanwhile, the expression levels of circulating circPTK2 were further analyzed according to the patients' clinical features. We analyzed cell apoptosis, colony formation, migration, and invasion in CRC cells. To further elucidate the effect of circPTK2 in CRC metastasis, we also conducted a colon cancer hepatic and pulmonary metastasis experiment. We used RNA biotin-labeled pull down and mass spectrometry to identify the target of circPTK2. We established a PDTX model to evaluate the effect of shRNA specifically targeting circPTK2 on tumor metastasis. Results: We identified a novel circRNA, circPTK2, which is back-spliced of three exons (exons 27, 28 and 29) of PTK2 by using circRNA microarray, bioinformatics and functional studies. CircPTK2 was elevated in CRC tissues and positively associated with tumor growth and metastasis. CRC patients with increased circPTK2 expression were positively correlated with poorer survival rates. Furthermore, our studies showed that circPTK2 could promote EMT of CRC cells in vitro and in vivo by binding to vimentin protein on sites Ser38, Ser55 and Ser82. We further demonstrated the interaction of circPTK2 and vimentin mediated the regulation of CRC by knockdown or overexpression of vimentin. In addition, we revealed that tail vein injection of shRNA specifically targeting circPTK2 blunt tumor metastasis in a patient-derived CRC xenograft model. Conclusions: Collectively, these results demonstrate that circPTK2 exerts critical roles in CRC growth and metastasis and may serve as a potential therapeutic target for CRC metastasis, and also a promising biomarker for early diagnosis of metastasis.

Mechanisms underlying therapy resistance of tumor cells include protein kinase Akt. Putative Akt targets include store-operated Ca2+-entry (SOCE) accomplished by pore forming ion channel unit Orai1 and its regulator STIM1. We explored whether therapy resistant (A2780cis) differ from therapy sensitive (A2780) ovary carcinoma cells in Akt, Orai1, and STIM1 expression, Ca2+-signaling and cell survival following cisplatin (100μM) treatment. Transcript levels were quantified with RT-PCR, protein abundance with Western blotting, cytosolic Ca2+-activity ([Ca2+]i) with Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+-readdition after Ca2+-store depletion, and apoptosis utilizing flow cytometry. Transcript levels of Orai1 and STIM1, protein expression of Orai1, STIM1, and phosphorylated Akt, as well as SOCE were significantly higher in A2780cis than A2780 cells. SOCE was decreased by Akt inhibitor III (SH-6, 10μM) in A2780cis but not A2780 cells and decreased in both cell lines by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-ABP, 50μM). Phosphatidylserine exposure and late apoptosis following cisplatin treatment were significantly lower in A2780cis than A2780 cells, a difference virtually abolished by SH-6 or 2-ABP. In conclusion, Orai1/STIM1 expression and function are increased in therapy resistant ovary carcinoma cells, a property at least in part due to enhanced Akt activity and contributing to therapy resistance in those cells.

Background: The serum and glucocorticoid inducible kinase SGK1, which was originally cloned from mammary tumor cells, is highly expressed in some but not all tumors. SGK1 confers survival to several tumor cells. Along those lines, the number of colonic tumors following chemical carcinogenesis was decreased in SGK1 knockout mice. Recently, a highly selective SGK inhibitor (EMD638683) has been developed. The present study explored whether EMD638683 affects survival of colon carcinoma cells in vitro and impacts on development of colonic tumors in vivo. Methods: Colon carcinoma (Caco-2) cells were exposed to EMD638683 with or without exposure to radiation (3 Gray) and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, mitochondrial potential from JC-9 fluorescence, caspase 3 activity from CaspGlow Fluorescein staining, DNA degradation from propidium iodide staining as well as late apoptosis from annexin-V FITC and propidium iodide double staining. In vivo tumor growth was determined in wild type mice subjected to chemical carcinogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium in drinking water for 7 days). Results: EMD638683 treatment significantly augmented the radiation-induced decrease of forward scatter, increase of phosphatidylserine exposure, decrease of mitochondrial potential, increase of caspase 3 activity, increase of DNA fragmentation and increase of late apoptosis. The in vivo development of tumors following chemical carcinogenesis was significantly blunted by treatment with EMD638683. Conclusions: EMD638683 promotes radiation-induced suicidal death of colon tumor cells in vitro and decreases the number of colonic tumors following chemical carcinogenesis in vivo.

Background and Aims Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related deaths worldwide, hence a major public health threat. Pleomorphic adenoma gene like‐2 (PLAGL2) has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. Approach and Results In this study, we demonstrated that PLAGL2 was up‐regulated in HCC compared with that of adjacent nontumorous tissues and also correlated with overall survival times. We further showed that PLAGL2 promoted HCC cell proliferation, migration, and invasion both in vitro and in vivo. PLAGL2 expression was positively correlated with epidermal growth factor receptor (EGFR) expression. Mechanistically, this study demonstrated that PLAGL2 functions as a transcriptional regulator of EGFR and promotes HCC cell proliferation, migration, and invasion through the EGFR‐AKT pathway. Moreover, hypoxia was found to significantly induce high expression of PLAGL2, which promoted hypoxia inducible factor 1/2 alpha subunit (HIF1/2A) expression through EGFR. Therefore, this study demonstrated that a PLAGL2‐EGFR‐HIF1/2A signaling loop promotes HCC progression. More importantly, PLAGL2 expression reduced hepatoma cells’ response to the anti‐EGFR drug erlotinib. PLAGL2 knockdown enhanced the response to erlotinib. Conclusions This study reveals the pivotal role of PLAGL2 in HCC cell proliferation, metastasis, and erlotinib insensitivity. This suggests that PLAGL2 can be a potential therapeutic target of HCC.