A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB.

BACKGROUND: The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear. RESULTS: The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death. CONCLUSIONS: The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 secreted by hepatic myofibroblasts promotes migration and invasion of human hepatoma cells.

The aim of our study was to investigate whether myofibroblasts and the chemokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 may play a role in hepatocellular carcinoma progression. We observed that hepatic myofibroblast LI90 cells express MCP-1/CCL2 mRNA and secrete this chemokine. Moreover, myofibroblast LI90 cell-conditioned medium (LI90-CM) induces human hepatoma Huh7 cell migration and invasion. These effects are strongly reduced when a MCP-1/CCL2-depleted LI90-CM was used. We showed that MCP-1/CCL2 induces Huh7 cell migration and invasion through its G-protein-coupled receptor CCR2 and, to a lesser extent, through CCR1 only at high MCP-1/CCL2 concentrations. MCP-1/CCL2's chemotactic activities rely on tyrosine phosphorylation of focal adhesion components and depend on matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, we observed that Huh7 cell migration and invasion induced by the chemokine are strongly inhibited by heparin, by beta-D-xyloside treatment of cells and by anti-syndecan-1 and -4 antibodies. Finally, we developed a 3-dimensional coculture model of myofibroblast LI90 and Huh7 cells and demonstrated that MCP-1/CCL2 and its membrane partners, CCR1 and CCR2, may be involved in the formation of mixed hepatoma-myofibroblast spheroids. In conclusion, our data show that human liver myofibroblasts act on hepatoma cells in a paracrine manner to increase their invasiveness and suggest that myofibroblast-derived MCP-1/CCL2 could be involved in the pathogenesis of hepatocellular carcinoma.

Liver iron, HFE gene mutations, and hepatocellular carcinoma occurrence in patients with cirrhosis.

BACKGROUND & AIMS: The influence of HFE gene mutations and liver iron overload on hepatocellular carcinoma (HCC) occurrence in patients with cirrhosis is subjected to controversial results. The aim of this work was to clarify this influence in a large cohort of prospectively followed-up cirrhotic patients classified according to the cause of their liver disease. METHODS: Three hundred one consecutive cirrhotic patients (162 alcoholics and 139 HCV-infected patients) were included at time of diagnosis of cirrhosis and followed-up. Liver iron overload on initial biopsy according to modified Deugnier's score and C282Y/H63D HFE gene mutations were assessed. RESULTS: In patients with alcoholic cirrhosis (mean iron score, 2.0 +/- 3.0; mean time of follow-up, 66.1 +/- 45.1 months), 40 (24.6%) developed HCC. Thirteen (8.02%) were heterozygotes for C282Y HFE gene mutation and had higher hepatic iron scores (3.6 +/- 3.8 vs 1.9 +/- 2.8, respectively, P = .05). In univariate analysis, liver iron overload as a continuous variable (HR, 1.23 [1.13-1.34], P < .001) or in binary coding with an optimal threshold of iron score >/=2.0 (HR, 4.1 [2.1-7.3], P < .0001) and C282Y mutation carriage (HR, 2.7 [1.2-6.3], P = .01) were risk factors for HCC. In multivariate analysis, liver iron and C282Y mutation carriage remained independent risk factors for HCC. In patients with HCV-related cirrhosis (C282Y mutation carriage, 17 [12.23%]; mean liver iron score, 0.9 +/- 1.9; mean time of follow-up, 85.5 +/- 42.1 months; HCC, 63 [45.32%] patients), C282Y mutation carriage and liver iron were not associated with HCC occurrence. CONCLUSIONS: Liver iron overload and C282Y mutation are associated with a higher risk of HCC in patients with alcoholic but not HCV-related cirrhosis.

Novel function of STAT1beta in B cells: induction of cell death by a mechanism different from that of STAT1alpha.

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.

Resistance mechanisms to osimertinib in EGFR-mutated advanced non-small-cell lung cancer: A multicentric retrospective French study.

OBJECTIVES: The understanding of histo-molecular mechanisms associated with resistance to osimertinib is a critical step to define the optimal treatment strategy in advanced EGFR-mutated Non-Small-Cell-Lung-Cancer (NSCLC). MATERIALS AND METHODS: We performed a multicentric retrospective analysis on a cohort of consecutive patients treated with osimertinib for an advanced EGFR-mutated NSCLC and collected histo-molecular data from plasma and tumor samples at the time of progression. Next-generation sequencing (NGS) was performed for all samples. Best Overall Response Rate (ORR), Progression Free Survival (PFS), Overall Survival (OS) and data on treatment post-progression efficacy were also collected. RESULTS: Two-hundred and twenty-six patients were included from 9 Academic French Hospitals between April 2015-October 2018. Osimertinib was given in second-line or more in 219 patients (97%). Best ORR was 52% and best central nervous system ORR was 56%. Median PFS and OS were 9.5 months (IQR 4.0-17.2) and 24 months (IQR 12.4-NR) respectively. At the time of analysis, 150 patients (66%) had tumor progression. Among them, 73 contributive samples (56 tumor biopsies) were available. The most frequent molecular alterations were C797S mutation (n = 9 (13%)) and MET amplification (n = 8 (11%)). Histologic transformation occurred in 5 patients (9% of tumor biopsies). In T790M + NSCLC, loss of T790 M occurred in 68% of cases. Median PFS and OS with treatment beyond progression were 6.0 months (IQR 2.0-10.4) and 15.1 months (IQR 6.7-NR) respectively and longer in case of osimertinib continuation beyond progression. CONCLUSION: We confirmed the efficacy of osimertinib in patients with advanced EGFR mutation positive NSCLC. At progression, the most frequent molecular alterations were MET amplification and C797S mutation.

Association between myeloid malignancies and acquired deficit in protein 4.1R: a retrospective analysis of six patients.

Constitutional deficit in the erythroid protein 4.1 (4.1R), a structural component of the erythrocyte membrane, is implicated in hereditary elliptocytosis. Acquired deficit in protein 4.1R have been rarely described in myelodysplastic syndromes. Here, we report a series of six patients presenting a myelodysplastic or a myeloproliferative disease in association with an elliptocytosis curve on osmotic gradient ektacytometry and a significant decrease in protein 4.1R level. We confirm that deficit in protein 4.1R is recurrent in myeloid malignancies and should be particularly investigated when deletion del (20 q) is present, since we found this chromosomal abnormality in four out of six patients.

Progressive pulmonary sarcoidosis is associated with over-expression of TYK2 and p21Waf1/Cip1.

BACKGROUND AND AIM OF THE WORK: Sarcoidosis is a multisystemic disorder of unknown aetiology with diverse clinical phenotypes characterised by granulomatous formation in involved organs. The factors controlling the evolution of pulmonary involvement -- a major point in the progression of sarcoidosis -- are poorly understood. The aim of our study was to identify alterations of gene expression associated with the progression of the granulomatous process in pulmonary sarcoidosis. METHODS: Using microarray analysis, we compared the gene expression profiles of bronchoalveolar lavage cells in three patients with progressive pulmonary sarcoidosis and in three patients with stable pulmonary sarcoidosis. Microarrays data were analysed using a non-parametric method that estimates the False positive Detection Rate for chosen thresholds of differential expression (software SAM, Significance Analysis of Microarrays). We further controlled the expression of three selected genes by semi-quantitative RT-PCR experiments. RESULTS: Fourteen genes were found significantly upregulated in the cases with progressive sarcoidosis including three genes coding for effectors of the Th1 immune response: the protein tyrosine kinase TYK2, the Interferon-gamma receptor 2 and the cell cycle inhibitor p21Waf1/Cip1. Semi-quantitative RT-PCR confirmed the increased expression of TYK2 and p21Waf1/Cip1, but not of the Interferon-gamma receptor 2. CONCLUSIONS: Our results confirm the feasibility of microarrays analysis in bronchoalveolar lavage, cells and are consistent with an involvement of p21Waf1/Cip1 and TYK2, two components of the Th1-inflammatory response pathway, in the progression of pulmonary sarcoidosis.

Incidence of hereditary spherocytosis in a population of jaundiced neonates.

As most of hereditary spherocytosis-affected individuals experience jaundice at birth, it seemed of interest to evaluate the proportion of hereditary spherocytosis in 402 severely jaundiced neonates with a bilirubinemia level prompting phototherapy. Red cell dehydration, a hallmark of spherocytosis whether constitutional or acquired, was demonstrated in 74 of them, among whom 23 disclosed a typical pattern of spherocytosis upon red cell deformability studies. Acquired spherocytosis of immune origin was diagnosed in 19/23 and hereditary spherocytosis in 4, making the proportion of hereditary spherocytosis-affected individuals among a severely jaundiced population of neonates amount to 1%, an incidence at least 30-fold that of the overall population.

STAT1-dependent IgG cell-surface expression in a human B cell line derived from a STAT1-deficient patient.

STAT1 is a key effector of cytokines involved in the resistance to pathogens; its identified transcriptional targets mediate the innate immune response involved in the defense against viruses and bacteria. Little is known about the role of STAT1 in adaptive immunity, including its impact on BCR or surface Ig expression. Analysis of this point is difficult in humans, as STAT1 deficiency is extremely rare. SD patients die early in childhood from a severe immunodeficiency. Herein, a SD B cell line obtained from a SD patient was compared with a B cell line from a STAT1-proficient subject in search of differences in surface Ig expression. In this SD B cell line, a complete absence of surface IgG was noted. The mRNA encoding the surface form of IgG was detected only in STAT1-proficient B cells; the mRNAs encoding the secreted and the surface forms were detected in SD and STAT1-proficient B cells. Re-expression of STAT1 in SD B cells restored surface IgG expression and a functional BCR. Conversely, shRNA silencing of STAT1 in B cells reduced considerably the expression of the surface IgG. Although limited to one B cell line, these results suggest that STAT1 may play an essential role in surface IgG expression in human B cells. Possible mechanisms involve regulation of mRNA splicing, transcription, or both. These observations extend the role of STAT1 further in adaptive immunity, including the regulation of BCR expression.

Abnormal glycosylation of red cell membrane band 3 in the congenital disorder of glycosylation Ig.

A description is provided of the clinical presentation in an infant of the recently described congenital disorder of glycosylation type Ig, and the changes affecting glycosylation of red cell membrane band 3, the anion exchanger. It has been shown that the condition stems from a homozygous mutation within the human ortholog of yeast ALG12 gene, which encodes a dolichol-P-mannose:Man7GlcNAc2-PP-dolichol alpha,1-6 mannosyltransferase of the endoplasmic reticulum. The clinical phenotype included prominent central and peripheral manifestations in the CNS. Although the infant studied had no anemia, band 3 abnormally separated into two fractions upon electrophoresis. The chemical composition of the glycans of both fractions was analyzed in detail. The fraction with low electrophoretic mobility was moderately hypoglycosylated (by 27%) and its mannose content was normal. The fraction with high electrophoretic mobility was deeply carbohydrate deficient (by 64%) and had 1 mol mannose in excess but only three residues of N-acetylglucosamine. Glycophorin A was hypoglycosylated with respect to O-linked glycans. Glycosphingolipids of red cells were normal. We suggest that the incomplete biosynthesis of the N-linked glycan of band 3 was largely caused by the persistence of the 3-linked mannose residue on the 6-mannose arm of the trimannosyl moiety of the glycoprotein. It is remarkable that the changes recorded in band 3 have no clinical consequences. Band 3 alteration might serve as an additional indicator (some serum N-glycoproteins of hepatic origin are also indicative) of the congenital disorder of glycosylation type Ig.

Congenital dyserythropoietic anemia, type 1, in a polynesian patient: response to interferon alpha2b.

The authors attempted to assess the utility of interferon alpha2b treatment in a Polynesian girl with a relatively severe form of congenital dyserythropoietic anemia, type 1. The diagnosis was established using routine hematologic and biochemical tests, light and electron microscopy, and electrophoresis of red cell membrane proteins. Response to the treatment was monitored using the blood count and reticulocyte count. The patient was age 14 when interferon treatment was started. Previously, she had been partially dependent on transfusions, and gallstones and iron overload had developed. The dose of interferon alpha2b was initially 3 x 10 units three times a week for 1 year and 3 x 10 units twice a week thereafter. On this treatment, hemoglobin and reticulocytes increased and transfusions became unnecessary. In keeping with a few previous reports, interferon alpha2b proved to be effective in congenital dyserythropoietic anemia, type 1. The patient became transfusion-independent. More cases need to be studied to optimize the dosage of interferon alpha2b and determine how long the treatment can be tolerated.

A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells.

The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells.

Congenital dyserythropoietic anemia type I is caused by mutations in codanin-1.

Congenital dyserythropoietic anemias (CDAs) constitute a rare group of inherited red-blood-cell disorders associated with dysplastic changes in late erythroid precursors. CDA type I (CDAI [MIM 224120], gene symbol CDAN1) is characterized by erythroid pathological features such as internuclear chromatin bridges, spongy heterochromatin, and invagination of the nuclear membrane, carrying cytoplasmic organelles into the nucleus. A cluster of 45 highly inbred Israeli Bedouin with CDAI enabled the mapping of the CDAN1 disease gene to a 2-Mb interval, now refined to 1.2 Mb, containing 15 candidate genes on human chromosome 15q15 (Tamary et al. 1998). After the characterization and exclusion of 13 of these genes, we identified the CDAN1 gene through 12 different mutations in 9 families with CDAI. This 28-exon gene, which is transcribed ubiquitously into 4738 nt mRNA, was reconstructed on the basis of gene prediction and homology searches. It encodes codanin-1, a putative o-glycosylated protein of 1,226 amino acids, with no obvious transmembrane domains. Codanin-1 has a 150-residue amino-terminal domain with sequence similarity to collagens and two shorter segments that show weak similarities to the microtubule-associated proteins, MAP1B (neuraxin) and synapsin. These findings, and the cellular phenotype, suggest that codanin-1 may be involved in nuclear envelope integrity, conceivably related to microtubule attachments. The specific mechanisms by which codanin-1 underlies normal erythropoiesis remain to be elucidated.