Identification of distinct mutational patterns and new driver genes in oesophageal squamous cell carcinomas and adenocarcinomas.

OBJECTIVES: Oesophageal squamous cell carcinoma (OSCC) and adenocarcinoma (OAC) are distinct cancers in terms of a number of clinical and epidemiological characteristics, complicating the design of clinical trials and biomarker developments. We analysed 1048 oesophageal tumour-germline pairs from both subtypes, to characterise their genomic features, and biological and clinical significance. DESIGN: Previously exome-sequenced samples were re-analysed to identify significantly mutated genes (SMGs) and mutational signatures. The biological functions of novel SMGs were investigated using cell line and xenograft models. We further performed whole-genome bisulfite sequencing and chromatin immunoprecipitation (ChIP)-seq to characterise epigenetic alterations. RESULTS: OSCC and OAC displayed nearly mutually exclusive sets of driver genes, indicating that they follow independent developmental paths. The combined sample size allowed the statistical identification of a number of novel subtype-specific SMGs, mutational signatures and prognostic biomarkers. Particularly, we identified a novel mutational signature similar to Catalogue Of Somatic Mutations In Cancer (COSMIC)signature 16, which has prognostic value in OSCC. Two newly discovered SMGs, CUL3 and ZFP36L2, were validated as important tumour-suppressors specific to the OSCC subtype. We further identified their additional loss-of-function mechanisms. CUL3 was homozygously deleted specifically in OSCC and other squamous cell cancers (SCCs). Notably, ZFP36L2 is associated with super-enhancer in healthy oesophageal mucosa; DNA hypermethylation in its super-enhancer reduced active histone markers in squamous cancer cells, suggesting an epigenetic inactivation of a super-enhancer-associated SCC suppressor. CONCLUSIONS: These data comprehensively contrast differences between OSCC and OAC at both genomic and epigenomic levels, and reveal novel molecular features for further delineating the pathophysiological mechanisms and treatment strategies for these cancers.

Deep sequencing reveals a global reprogramming of lncRNA transcriptome during EMT.

Several studies have shown that long non-coding RNAs (lncRNAs) may play an essential role in Epithelial-Mesenchymal Transition (EMT), which is an important step in tumor metastasis; however, little is known about the global change of lncRNA transcriptome during EMT. To investigate how lncRNA transcriptome alterations contribute to EMT progression regulation, we deep-sequenced the whole-transcriptome of MCF10A as the cells underwent TGF-ß-induced EMT. RESULTS: Deep-sequencing results showed that the long RNA transcriptome of MCF10A had undergone global changes as early as 8h after treatment with TGF-ß. The expression of 3403 known and novel lncRNAs, and 570 known and novel circRNAs were altered during EMT. To identify the key lncRNA-regulator, we constructed the co-expression network and found all junction nodes in the network are lncRNAs. One junction node, RP6-65G23.5, was further verified as a key regulator of EMT. Intriguingly, we identified 216 clusters containing lncRNAs which were located in "gene desert" regions. The expressions of all lncRNAs in these clusters changed concurrently during EMT, strongly suggesting that these clusters might play important roles in EMT. Our study reveals a global reprogramming of lncRNAs transcriptome during EMT and provides clues for the future study of the molecular mechanism of EMT.

Iron overload in hereditary tyrosinemia type 1 induces liver injury through the Sp1/Tfr2/hepcidin axis.

BACKGROUND & AIMS: Iron is an essential metal for fundamental metabolic processes, but little is known regarding the involvement of iron in other nutritional disorders. In the present study, we investigated disordered iron metabolism in a murine model of hereditary tyrosinemia type I (HT1), a disease of the tyrosine degradation pathway. METHODS: We analysed the status of iron accumulation following NTBC withdrawal from Fah(-/-) mice, a murine model for HT1. Liver histology and serum parameters were used to assess the extent of liver injury and iron deposition. To determine the physiological significance of iron accumulation, mice were subjected to a low-iron food intake to reduce the iron accumulation. Mechanistic studies were performed on tissues and cells using immunoblotting, qRT-PCR, adenovirus transfection and other assays. RESULTS: Severe iron overload was observed in the murine model of HT1 with dramatically elevated hepatic and serum iron levels. Mechanistic studies revealed that downregulation and dysfunction of Tfr2 decreased hepcidin, leading to iron overload. The Fah(-/-) hepatocytes lost the ability of transferrin-sensitive induction of hepcidin. Forced expression of Tfr2 in the murine liver reduced the iron accumulation. Moreover, transcription factor Sp1 was downregulated and identified as a new regulator of Tfr2 here. Additionally, low-iron food intake effectively reduced the iron deposits, protected the liver and prolonged the survival in these mice. CONCLUSIONS: Iron was severely overloaded in the HT1 mice via the Sp1/Tfr2/Hepcidin axis. The iron overload induced liver injury in the HT1 mice, and reduction of the iron accumulation ameliorated liver injury. LAY SUMMARY: Primary and secondary iron overload is an abnormal status affecting millions of people worldwide. Here, we reported severe iron overload in a murine model of HT1, a disease of the tyrosine degradation pathway, and elucidated the mechanistic basis and the physiological significance of iron overload in HT1. These studies are of general interest not only with respect to secondary iron-induced liver injury in HT1 but also are important to elucidate the crosstalk between the two metabolic pathways.

Off-target effects of c-MET inhibitors on thyroid cancer cells.

Aberrantly activated c-MET signaling occurs in several cancers, promoting the development of c-MET inhibitors. In this study, we found that eight of eight thyroid cancer cell lines (including six anaplastic thyroid cell lines) have prominent expression of c-MET protein. Fifty percent of the thyroid cancer cell lines (four of eight) were growth inhibited by two small molecule c-MET inhibitors (tivantinib and crizotinib) associated with apoptosis and G(2)-M cell-cycle arrest. However, crizotinib did not inhibit 50% proliferation of thyroid cancer cells (SW1736 and TL3) at a concentration at which the drug completely inhibited ligand-stimulated c-MET phosphorylation. However, tivantinib was less potent than crizotinib at inhibiting c-MET phosphorylation, but was more potent than crizotinib at decreasing cell growth. Suppressing c-MET protein expression and phosphorylation using siRNA targeting c-MET did not induce cell-cycle arrest and apoptosis. Taken together, tivantinib and crizotinib have off-target(s) activity, contributing to their antitumor activity. In vivo study showed that crizotinib markedly inhibited the growth of thyroid cancer cells (SW1736) in immunodeficient mice. In summary, c-MET inhibitors (tivantinib and crizotinib) suppress the growth of aggressive thyroid cancer cells, and this potential therapeutic benefit results from their non-MET-targeting effects.

Allelic imbalance in sporadic parathyroid carcinoma and evidence for its de novo origins.

Parathyroid cancer is a rare, clinically aggressive cause of primary hyperparathyroidism, and whether these malignancies generally evolve from pre-existing benign adenomas or arise de novo is unclear. Furthermore, while inactivation of the CDC73 (HRPT2) tumor suppressor gene, encoding parafibromin, is a major contributor, other genes essential to parathyroid carcinogenesis remain unknown. We sought to identify genomic regions potentially harboring such oncogenes or tumor suppressor genes, and to gain insight into the origins and molecular relationship of malignant versus benign parathyroid tumors. We performed genome-wide copy-number and loss of heterozygosity analysis using Affymetrix 50K SNP mapping arrays and/or comparative genomic hybridization on 16 primary parathyroid carcinomas, local recurrences or distant metastases, and matched normal controls, from 10 individuals. Recurrent regions of allelic loss were observed on chromosomes 1p, 3, and 13q suggesting that key parathyroid tumor suppressor genes are located in these chromosomal locations. Recurrent allelic gains were seen on chromosomes 1q and 16, suggesting the presence of parathyroid oncogenes on these chromosomes. Importantly, the most common alteration in benign parathyroid adenomas, loss of 11q, was not found as a recurrent change in the malignant parathyroid tissues. Molecular allelotyping using highly polymorphic microsatellite markers provided further confirmation that the prevalence of 11q loss is markedly and significantly lower in carcinomas as compared with adenomas. Our observations provide molecular support for the concept that sporadic parathyroid cancer usually arises de novo, rather than evolving from a pre-existing typical benign adenoma. Furthermore, these results help direct future investigation to ultimately determine which of the candidate genes in these chromosomal locations make significant contributions to the molecular pathogenesis of parathyroid cancer.

miR-34a functions as a tumor suppressor modulating EGFR in glioblastoma multiforme.

Chromosome 1p36.23 is frequently deleted in glioblastoma multiforme (GBM). miR-34a localizes in this region. Our experiments found that miR-34a was often deleted and epidermal growth factor receptor (EGFR) was frequently amplified in genomic DNA of 55 GBMs using single-nucleotide polymorphism DNA microarray. Notably, we found that the mean survival time was significantly shortened for patients whose GBMs had both EGFR amplification and miR-34a deletion. Expression of miR-34a was significantly lower in GBM samples compared with normal brain tissue. Forced expression of miR-34a in GBM cells decreased their ability to migrate and profoundly decreased their levels of cyclin-A1, -B1, -D1, and -D3, as well as cyclin-dependent kinase and increased expression of cyclin kinase inhibitor proteins (p21, p27). Also, human GBM cells (U251) stable overexpressing mir-34a formed smaller tumors when growing as xenografts in immunodeficient mice compared with wild-type U251 GBM cells. Furthermore, the protein expression of EGFR decreased in the cells with forced overexpression of miR-34a. Additional studies showed that mir-34a targeted Yin Yang-1 (YY1) and YY1 is a transcription factor that can stimulate the expression of EGFR. Thus, our data suggest that miR-34a acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell-cycle proteins and EGFR. Moreover, we discovered for the first time that both deletion of miR-34a and amplification of EGFR were associated with significantly decreased overall survival of GBM patients.

Inhibition of signal transducer and activator of transcription 5 by the inhibitor of janus kinases stimulates dormant human leukemia CD34+ /CD38- cells and sensitizes them to antileukemia agents.

To verify molecular mechanisms by which leukemia stem cells (LSCs) maintain a dormant state, we explored the activity of the major prosurvival signal pathways in CD34(+) /CD38(-) compartment, supposed to contain LSCs, and CD34(+) /CD38(+) counterparts from patients with acute myelogenous leukemia (AML, n = 11) by fluorescence-activated cell sorting (FACS). CD34(+) /CD38(-) cells expressed a greater amount of p-janus kinase 2 (JAK2) and p-signal transducer and activator of transcription 5 (STAT5) than CD34(+) /CD38(+) counterparts in all patients except for one case. In addition, we found that CD34(+) /CD38(-) cells were relatively resistant to cytarabine- and the inhibitor of the fms-like tyrosine kinase 3 (FLT3)-mediated growth inhibition, as measured by the clonogenic assay. Interestingly, blockade of JAK2/STAT5 signaling by the specific JAK2 inhibitor AZ960 stimulated cell cycling in CD34(+) /CD38(-) cells in conjunction with downregulation of cyclin-dependent kinase inhibitor p21(waf1) and sensitized these cells to the growth inhibition mediated by cytarabine and the FLT3 kinase inhibitor. Moreover, exposure of CD34(+) /CD38(-) cells to AZ960 potently induced apoptosis in parallel with downregulation of antiapoptotic protein Bcl-xL, as measured by Western blot analysis. Taken together, JAK2/STAT5 signaling may be a promising molecular target to eradicate CD34(+) /CD38(-) leukemia cells in individuals with AML.

Peroxisome proliferator-activated receptor gamma ligands induce growth inhibition and apoptosis of human B lymphocytic leukemia.

This study examined the expression and structural intactness of peroxisome proliferator-activated receptor gamma (PPARgamma) in human acute lymphocytic leukemia (ALL) cells and determined the effect of PPARgamma ligands on growth and apoptosis of these cells. We noted that all lymphocytic leukemia cell lines expressed PPARgamma and no PPARgamma mutations were found in these cell lines as indicated by SSCP analysis. Effect of the PPARgamma ligands on the proliferation, differentiation and apoptosis of B type ALL cells was further examined. Treatment of these cells with the PPARgamma ligands Pioglitazone (PGZ) and 15-deoxy-delta (12,14)-prostaglandin J2 (15d-PGJ2) resulted in growth inhibition in a dose-dependent manner which was associated with a G1 to S cell cycle arrest. However, this effect appeared to be PPARgamma-independent since several PPARgamma antagonists could not reverse this effect. No differentiation was induced by this treatment. Four out of five cell lines underwent apoptosis after culture with the PPARgamma ligands. This effect was partially caspase-dependent because a pan-caspase inhibitor partially reversed this effect. In conclusion, our results suggest that PPARgamma ligands may offer a new therapeutic approach to aid in the treatment of ALL.

Elevated levels of transferrin receptor 2 mRNA, not transferrin receptor 1 mRNA, are associated with increased survival in acute myeloid leukaemia.

Transferrin receptor 1 (TfR1) is a type II membrane protein that mediates cellular iron uptake. Transferrin receptor 2(TfR2), another receptor for transferrin (Tf), has recently been cloned. We examined expression levels of TfR1, TfR2-alpha (membrane form) and TfR2-beta (non-membrane form) transcripts in cells from 67 patients with de novo acute myeloid leukaemia (AML) using reverse transcription-polymerase chain reaction (RT-PCR), and correlated the results with a variety of clinical features and disease outcomes of these patients. Significant correlations were noted between the levels of both TfR1 and TfR2-alpha (r = 0.771, P < 0.001) and TfR1 and TfR2-beta (r = 0.534, P < 0.001). Unexpectedly, initial white blood cell (WBC) counts were inversely correlated with levels of expression of either TfR1(r = -0.357, P = 0.003), TfR2-alpha (r = -0.486, P < 0.0001), or TfR2-beta (r = -0.435, P = 0.0003). Only TfR2 expression was significantly associated with either serum iron (r = -0.270, P = 0.045) or serum ferritin (r = -0.364, P = 0.008). Multivariate analyses using Cox's proportional hazard model showed that elevated TfR2-alpha, but not TfR1 or TfR2-beta mRNA levels significantly contributed to a better prognosis for AML patients. Furthermore, a group with high expression levels of both TfR2-alpha and TfR2-beta survived significantly longer than a group without high expression of both of them (P < 0.01 by log-rank). The present study suggests that (i) TfRs-independent iron uptake might have an important role in in vivo proliferation of AML cells; (ii) expression of TfR2 (especially the alpha form) is a novel prognostic factor for patients with AML.

Myelodysplastic syndromes.

Myelodysplastic syndrome (MDS) is a clonal disorder characterized by ineffective hematopoiesis, which can lead to either fatal cytopenias or acute myelogenous leukemias (AML). During the last 15 years, important progress has been made in the understanding of the biology and prognosis of MDS. Risk-adapted treatment strategies were established due to the high median age (60-75 years) of MDS patients and the individual history of the disease (number of cytopenias, cytogenetic changes, transfusion requirements). The use of allogeneic bone marrow transplantation for MDS patients currently offers the only potentially curative treatment, but this form of therapy is not available for the 'typical' MDS patient who is >60 years of age. The development of small molecules directed against specific molecular targets with minimal adverse effects is the hope for the future. Innovative uses of immunomodulatory agents and the optimizing of cytotoxic treatment should continue to help in the treatment of MDS.