Estrogen receptor α regulates non-canonical autophagy that provides stress resistance to neuroblastoma and breast cancer cells and involves BAG3 function.

Breast cancer is a heterogeneous disease and approximately 70% of newly diagnosed breast cancers are estrogen receptor (ER) positive. Out of the two ER types, α and ß, ERα is the only ER that is detectable by immunohistochemistry in breast cancer biopsies and is the predominant subtype expressed in breast tumor tissue. ER-positive tumors are currently treated with anti-hormone therapy to inhibit ER signaling. It is well known that breast cancer cells can develop endocrine resistance and resistance to anti-hormone therapy and this can be facilitated via the autophagy pathway, but so far the description of a detailed autophagy expression profile of ER-positive cancer cells is missing. In the present study, we characterized tumor cell lines ectopically expressing ERα or ERß as well as the breast cancer-derived MCF-7 cell line endogenously expressing ERα but being ERß negative. We could show that ERα-expressing cells have a higher autophagic activity than cells expressing ERß and cells lacking ER expression. Additionally, for autophagy-related gene expression we describe an ERα-specific 'autophagy-footprint' that is fundamentally different to tumor cells expressing ERß or lacking ER expression. This newly described ERα-mediated and estrogen response element (ERE)-independent non-canonical autophagy pathway, which involves the function of the co-chaperone Bcl2-associated athanogene 3 (BAG3), is independent of classical mammalian target of rapamycin (mTOR) and phosphatidylinositol 3 kinase (PI3K) signaling networks and provides stress resistance in our model systems. Altogether, our study uncovers a novel non-canonical autophagy pathway that might be an interesting target for personalized medicine and treatment of ERα-positive breast cancer cells that do not respond to anti-hormone therapy and classical autophagy inhibitors.

Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors.

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

Ubiquitination and selective autophagy.

Ubiquitination has long been recognised as a key determinator of protein fate by tagging proteins for proteasomal degradation. Most recently, the ability of conjugated ubiquitin chains to confer selectivity to autophagy was demonstrated. Although autophagy was first believed to be a bulk, non-selective 'self-eating' degradative process, the molecular mechanisms of selectivity are now starting to emerge. With the discovery of autophagy receptors - which bind both ubiquitinated substrates and autophagy specific light chain 3 (LC3) modifier on the inner sheath of autophagosomes - a new pathway of selective autophagy is being unravelled. In this review, we focus on the special role of ubiquitin signals and selective autophagy receptors in sorting a variety of autophagic cargos.

Acetylsalicylic acid enhances antiproliferative effects of the EGFR inhibitor gefitinib in the absence of activating mutations in gastric cancer.

The epidermal growth factor receptor (EGFR) is highly expressed in gastric cancer indicating its suitability as a target for receptor tyrosine kinase (RTK) inhibitors. In the current study we explored the role of EGFR and its potential use as a therapeutic target in gastric cancer. First we analyzed 66 gastric cancer samples of Asian and Caucasian patients for the presence of EGFR mutations. No activating EGFR mutations were found and gefitinib alone was only weakly effective in gastric cancer cell lines. However, acetylsalicylic acid (ASA) significantly enhanced the inhibitory effects of gefitinib indicating synergistic action. Whole genome expression profiling indicated significant regulation of 120 genes in the case of co-administration of gefitinib and ASA (32 induced, 88 repressed) in gastric adenocarcinoma cells. Further analyses indicated that several important signalling pathways were effectively inhibited by simultaneous exposure to gefitinib and ASA. Our findings indicate that although gastric cancer does not seem to harbour mutations which render the cancer cells constitutively susceptible to gefitinib, the co-administration of ASA can strengthen RTK inhibitor activity in adenocarcinoma cells by EGFR activation. This is the first report of effective modulation of EGFR-inhibition activity in cancer.

[Mutations of growth factor receptor Flt3 in acute myeloid leukemia: transformation of myeloid cells by Ras-dependent and Ras-independent mechanisms]. / Mutationen des Wachstumsfaktor-Rezeptors Flt3 bei Akuter Myeloischer Leukämie - Transformation myeloischer Zellen durch Ras-abhängige und Ras-unabhängige Mechanismen

BACKGROUND AND OBJECTIVE: The tyrosine kinase receptor Flt3 mediates important functions in early hematopoietic progenitors. Recently mutations of a growth factor receptor have been identified in about 30 % of patients with acute myeloid leukemia (AML). These mutations are associated with a poor prognosis. In-vitro and animal data show their involvement in leukemic transformation. Experiments analyzing the effects of these mutations on signal transduction and gene expression patterns of myeloid cells allow for the classification of this receptor as an oncogene. Furthermore, they help to define the receptor and its signaling intermediates as therapeutic targets. METHODS: In order to analyze the signaling properties of mutated FLT3 receptors, we isolated the receptor mRNA from two patients with AML. Wild-type and mutant Flt3 isoforms were expressed in 32D cells that were subsequently analyzed for proliferation, survival, activation of signaling intermediates and gene expression levels. Also, the effects of of Ras-, MAP-Kinase and PI3-Kinase inhibition were analyzed. RESULTS: The expression of mutated Flt3 (Flt3-ITD) induced factor-independent proliferation and survival in the myeloid progenitor cell line 32D. Flt3-ITD activated Ras- and PI3-kinase-dependent signaling pathways, as well as STAT5 and STAT3. Activation of STAT proteins was followed by the induction of known STAT target genes like SOCS2, SOCS3 and CIS. Inhibition of Ras-dependent signal transduction by a dominant negative Ras construct inhibited some, but not all biological effects of Flt3-ITD. Similar results were obtained by chemical inhibition of the MAP kinases. In contrast, inhibition of PI3 kinase activity inhibited growth factor-independent growth and apoptosis resistance of 32D cells. CONCLUSIONS: Inhibition of Ras-dependent signaling pathways is not sufficient to abrogate the functional consequences of Flt3-mutations in myeloid cells. Therefore, therapeutic intervention by Ras-Inhibitors may not be sufficient to treat Flt3-driven disease.

Topical transforming growth factor-beta3 in the prevention or alleviation of chemotherapy-induced oral mucositis in patients with lymphomas or solid tumors.

Transforming growth factor (TGF)-beta3 has been hypothesized to prevent or alleviate oral mucositis (OM) in cancer patients receiving high-dose chemotherapy (CT). Two double-blind, placebo-controlled, multicenter, phase II studies of TGF-beta3 were initiated in the United States, Europe, and Argentina in patients with lymphomas or solid tumors who were receiving highly stomatotoxic CT regimens. Patients were to apply 10-mL mouthwash applications of TGF-beta3 (25 microg/mL) or placebo four times daily (or twice daily) 1 day before and all days during CT. The patients were subsequently evaluated for OM incidence, severity, and duration using National Institute of Cancer Common Toxicity Criteria (NCI-CTC) criteria and an objective scoring system (1). After the start of the trials, negative results from new preclinical studies suggesting suboptimal formulation and/or dosing led to an interim analysis of the ongoing clinical trials. One hundred fifty-two patients from the combined studies were included in the interim analysis, with 116 patients on the TGF-beta3 four times daily and placebo arms. Most (72%) patients had breast cancer, 22% had lymphomas, and 6% had other solid tumors. Although 98% (149 of 152) of patients experienced adverse events, only 14% (22 of 152) experienced events that were judged as possibly or probably related to the study drug (primarily gastrointestinal symptoms). No clinically relevant differences were seen between the treatment and placebo arms regarding safety, nor was there evidence for systemic absorption of TGF-beta3. Finally, there was no advantage of TGF-beta3 treatment regarding the incidence (TGF-beta3 four times daily versus placebo [46% versus 47%]), onset, or duration of NCI-CTC grade 3 or 4 OM. For this dose, formulation, regimen. and patient population, TGF-beta3 was not effective in the prevention or alleviation of CT-induced OM.

p27Kip1 is required for PTEN-induced G1 growth arrest.

The tumor suppressor PTEN is one of the most commonly inactivated genes in human cancer. Glioblastoma multiforme cells harboring mutant PTEN have abnormally high levels of 3' phosphoinositides and elevated protein kinase B activity. Expression of wild-type PTEN in glioma cells, containing endogenous mutant PTEN, reduces 3' phosphoinositides levels, inhibits PKB activity, and induces G1 cell cycle arrest. We investigated the mechanism of the PTEN-induced growth arrest in glioma cell lines. Expression of PTEN is associated with increased expression of p27Kip1, decreased expression of cyclins A and D3, inhibition of cdk2 activity, and dephosphorylation of pRb. Inactivation of p53, by the human papilloma virus E6 oncoprotein, does not prevent PTEN-induced G1 arrest, implying that p53 is not required for G1 arrest. In contrast, p27Kip1 antisense oligonucleotides abrogated the growth arrest induced by PTEN. Furthermore, blocking p27Kip1 expression prevented the PTEN-induced reduction of cyclin-dependent kinase 2 activity, indicating that p27Kip1 functions upstream of cyclin-dependent kinase 2 in the PTEN regulatory cascade. These results implicate p27Kip1 as a critical mediator of PTEN-induced G1 arrest.

Declining chloroquine resistance of Plasmodium falciparum in Lambaréné, Gabon from 1992 to 1998.

Plasmodium falciparum malaria continues to threaten human populations in the tropics and travellers in endemic areas. Drug resistance of the parasite is a major problem in treating this devastating disease. In a prospective trial we investigated the in vitro sensitivity of Plasmodium falciparum to chloroquine, quinine and mefloquine in the Albert Schweitzer Hospital in Lambaréné, Gabon every second year from 1992 to 1998. We used the standard WHO in vitro sensitivity assay. Parasite sensitivity to quinine and mefloquine remained stable over the years. However, parasite resistance to chloroquine decreased highly significantly with the change in local malaria treatment policy. In 1992, 100% of parasite isolates showed resistance to chloroquine, whereas in 1998 only 45% were found resistant.

Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015).

The adenovirus mutant dl1520 (ONYX-015) does not express the E1B-55K protein that binds and inactivates p53. This virus replicates in tumor cells with mutant p53, but not in normal cells with functional p53. Although intra-tumoral injection of dl1520 shows promising responses in patients with solid tumors, previous in vitro studies have not established a close correlation between p53 status and dl1520 replication. Here we identify loss of p14ARF as a mechanism that allows dl1520 replication in tumor cells retaining wild-type p53. We demonstrate that the re-introduction of p14ARF into tumor cells with wild-type p53 suppresses replication of dl1520 in a p53-dependent manner. Our study supports the therapeutic use of dl1520 in tumors with lesions within the p53 pathway other than mutation of p53.

5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells.

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

The circadian rhythm of body temperature is preserved during malarial fever.

Body temperature follows a circadian rhythm with a low around 6 a.m. and a peak about 12 hours later. The effect of fever on this endogenous oscillation is unknown. We obtained hourly measurements of the rectal temperature of 66 children with Plasmodium falciparum malaria. Even at febrile temperatures, the temperature followed a clear circadian rhythm. 33 patients (50%) had fever above 38 degrees C at 6 p.m. on the first day compared to only 9 (14%) at 6 a.m. the next morning. This considerable difference was also found on the second day of observation. Since in clinical practice antipyretics are often given above a certain fever threshold, the time of day should be taken into account when antipyretics are applied.

Effect of paracetamol on parasite clearance time in Plasmodium falciparum malaria.

BACKGROUND: Routine antipyretic therapy in children with infectious diseases has long been the source of controversy. Each year, in addition to antimalarial medication, millions of children with Plasmodium falciparum malaria receive paracetamol to reduce fever. However, the usefulness of this practice has not been proven. METHODS: In a randomised trial in Lambaréné, Gabon, 50 children with P falciparum malaria were treated with intravenous quinine, and received either mechanical antipyresis alone, or in combination with paracetamol. Rectal body temperature and parasitaemia were recorded every 6 h for 4 days. Plasma concentrations and inducible concentrations of tumour necrosis factor (TNF) and interleukin-6 were measured every 24 h. In addition, production of oxygen radicals was measured in both groups. FINDINGS: The mean fever clearance time was 32 h for children treated with paracetamol and 43 h for those who received mechanical antipyresis alone; however, this 11 h difference was not significant (95% CI -2 to 24 h; p = 0.176). Parasite clearance time was significantly prolonged in patients who received paracetamol with a difference of 16 h (8-24 h; p = 0.004). Plasma concentrations of TNF and interleukin-6 were similar in both groups during the study. However, the induced concentrations of TNF, and the production of oxygen radicals, were significantly lower in children treated with paracetamol than those who received mechanical antipyresis alone. INTERPRETATION: These data suggest that paracetamol has no antipyretic benefits over mechanical antipyresis alone in P falciparum malaria. Moreover, paracetamol prolongs parasite clearance time, possibly by decreased production of TNF and oxygen radicals.

PIP: Although paracetamol is routinely used to control fever in African children with Plasmodium falciparum, the usefulness of this treatment has not been established. In a randomized clinical trial, 50 children 2-7 years of age from Lambarene, Gabon, with P. falciparum malaria were treated with intravenous quinine and received either mechanical antipyresis alone (electric fanning, tepid sponging, and cool blankets) or in combination with paracetamol. The mean fever clearance time was 32 hours for children treated with paracetamol and 43 hours for those who received antipyresis alone--a nonsignificant difference. Parasite clearance time was significantly prolonged (by an average of 16 hours) in children who received paracetamol. Although plasma concentrations of tumor necrosis factor and interleukin-6 were similar in both groups, induced concentrations of tumor necrosis factor and the production of oxygen radicals were significantly lower in paracetamol-treated children. Overall, these findings indicate that paracetamol confers no benefits over mechanical antipyresis alone and actually prolongs parasite clearance time. Further studies are required, however, before recommendations for ancillary treatment can be changed.

[The dual role of tumor necrosis factor (TNF) during a malarial attack]. / Double rôle du tumor necrosis factor (TNF) pendant l'accès palustre.

The roles of the various factors implicated in the pathogenesis of severe malaria are not well understood. Tumor necrosis factor (TNF), a cytokine produced mainly by macrophages, seems to play a crucial role in both the host's defence against the parasite and the development of severe complications. This review investigates the dual role of TNF in acute malaria, summarizing current knowledge of the beneficial and detrimental effects of this molecule. Recent work has suggested a possible explanation for this dualism, involving a complex interaction between TNF and its soluble receptors.

Prediction of accelerated cure in Plasmodium falciparum malaria by the elevated capacity of tumor necrosis factor production.

Cytokine regulation was compared in three groups of Gabonese patients with Plasmodium falciparum malaria before and after therapy; adults with uncomplicated malaria, children with uncomplicated malaria, and children with severe malaria. Plasma levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-8, TNF receptors (TNF R), and the TNF/TNF R ratios were significantly higher in severe malaria compared with uncomplicated malaria. High plasma levels of all immunoregulatory molecules were associated with slow cure after therapy. In all patients, phytohemagglutinin-induced cytokine production was depressed on admission compared with convalescence. A significant difference was the higher TNF production capacity in patients with severe malaria on day 2 and day 5 compared with that in patients with uncomplicated malaria. In contrast to IL-6 and IL-8, a high TNF production capacity during the acute phase of malaria predicted a rapid clinical and parasitologic cure in the patients. These findings illustrate the dual role of TNF in the protection and pathology of malaria.

Malaria epidemiology in the province of Moyen Ogoov, Gabon.

In the course of epidemiological and immunological baseline studies parasitological surveys were conducted, in 1992, in three localities situated in our near rain forest in the area of Lambaréné, Gabon, western Central Africa. Anopheles gambiae s.s. and A. funestus are considered to be the main vectors of malaria. The three localities represent strata with obvious differences in the intensity of malaria transmission. The lowest parasite rates were recorded in the village around the Albert-Schweitzer-Hospital where environmental sanitation and easy access to diagnostic and therapeutic facilities afford a fair measure of malaria control. The villages of Bellevue and Tchad show a much higher prevalence of Plasmodium falciparum, followed by P. malariae and P. ovale. In all three villages parasite rates and geometric mean parasite densities of P. falciparum showed the age pattern typical for areas with stable, hyperendemic malaria. Analysis by season showed the period of the long rains to be the epidemiologically calmest while the dry season and even more the short rainy season produced an increase of parasite rates and densities. In Tchad, the most affected of the three villages, the parasite rates in female adults were significantly lower than in male adults. This was accompanied by lower parasite densities in female adults.

Drug sensitivity of Plasmodium falciparum in Gabon. Activity correlations between various antimalarials.

The sensitivity of Plasmodium falciparum to chloroquine, mefloquine, quinine, halofantrine, and sulfadoxine/pyrimethamine has been investigated at Lambaréné, in the Gabonese rain forest, between April and September 1992. WHO standard micro in vitro tests were performed. Of 43 isolates tested for response to chloroquine all were resistant to the drug with mean EC 50 and EC 90 values of 1.86 and 4.18 mumol/l blood, respectively, indicating the highest degree of resistance ever reported from Central Africa. With sulfadoxine/pyrimethamine 19 out of 27 isolates showed 90% inhibition of schizont maturation at a pyrimethamine concentration of at least 75 nmol/l blood medium mixture, indicating 30% of resistance to sulfadoxine/pyrimethamine. In contrast all isolates tested were fully inhibited by mefloquine at 3.2 mumol/l blood (40 isolates), quinine at 5.12 mumol/l blood medium mixture (41 isolates) and halofantrine at 3 nmol/l blood medium mixture (40 isolates) indicating full sensitivity to these drugs. A significant positive correlation was found between responses to quinine and mefloquine. The response to halofantrine was positively correlated with the responses to quinine and mefloquine, in the case of chloroquine and halofantrine an inverse relationship was observed. Compared with previous data from Gabon, the findings suggest a substantial increase of chloroquine resistance, in contrast to reports from neighbouring countries, which show stabilising or even declining chloroquine resistance patterns.