Noncanonical GLI1 signaling promotes stemness features and in vivo growth in lung adenocarcinoma.

Aberrant Hedgehog/GLI signaling has been implicated in a diverse spectrum of human cancers, but its role in lung adenocarcinoma (LAC) is still under debate. We show that the downstream effector of the Hedgehog pathway, GLI1, is expressed in 76% of LACs, but in roughly half of these tumors, the canonical pathway activator, Smoothened, is expressed at low levels, possibly owing to epigenetic silencing. In LAC cells including the cancer stem cell compartment, we show that GLI1 is activated noncanonically by MAPK/ERK signaling. Different mechanisms can trigger the MAPK/ERK/GLI1 cascade including KRAS mutation and stimulation of NRP2 by VEGF produced by the cancer cells themselves in an autocrine loop or by stromal cells as paracrine cross talk. Suppression of GLI1, by silencing or drug-mediated, inhibits LAC cells proliferation, attenuates their stemness and increases their susceptibility to apoptosis in vitro and in vivo. These findings provide insight into the growth of LACs and point to GLI1 as a downstream effector for oncogenic pathways. Thus, strategies involving direct inhibition of GLI1 may be useful in the treatment of LACs.

High-pressure physiological saline isotonic solution administration enhances brain NGF and NGF-receptors expression.

OBJECTIVE: Nerve growth factor (NGF) is a neurotrophin which promotes and regulates the survival of neurons in the peripheral nervous system. The aim of this study was to investigate the effect of high-pressure administration of sterile physiological saline isotonic solution (HpPSIS) into nasal cavity of laboratory animals on NGF levels and NGF-receptor expression in the olfactory bulbs and brain. MATERIALS AND METHODS: For this study we used three weeks old female Sprague Dawley SD rats (n=48). Rats were divided into two groups, the first one treated delivering physiological saline solution with a normal syringe modified at the extremity to fit the rats' nostril (5 ml) (n=24) and the second one treated spray with HpPSIS (n=24 rats). Rats were treated three times a day either for 5 consecutive days (shorth term treatment) or 10 consecutive days (longer treatment) in both nostrils of HpPSIS delivered at high pressure (pression emission level: PEL: 7 g/sec for emission time ET: 0.5 sec) with a specific forced spray erogator. Untreated rats received a similar manipulation three times a day through a syringe in the nostrils, but no HpPSIS administration. RESULTS: The results of these studies highlight the possibility that endogenous enhancement of NGF by stimulation of NGF-producing cells within the nasal cavities and also in the CNS represent a novel experimental approach to enhance the brain NGF levels with a new therapy. HpPSIS treatment further enhances the presence of NGF in the four brains examined. Indeed, a significant increase of NGF was first observed after 5 days of HpPSIS treatment, compared to HpPSIS untreated rats. The increase was over 25% in the OB, ST, HI and in CX, while 10 days after HpPSIS treatments the levels of NGF were even higher. These differences were statistically significant, p < 0.05. CONCLUSIONS: It was found that forced administration of HpPSIS enhances the presence of these neurotrophic signals, not only in the olfactory bulbs, but also in forebrain cholinergic neurons, which are known to degenerate as result of memory loss and brain aging, including Alzheimer Disease. These findings for the first time in the literature demonstrate the possibility of enhancing the endogenous NGF to protect NGF-damaged neurons. Since the enhanced expression of NGF was first observed after 5 days of treatment and higher after 10 days of treatment, a reasonable hypothesis is that longer HpPSIS treatment might further enhance the level of NGF in brain and olfactory bulbs.

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer.

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFRtyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFRtyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFRtyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFRtyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.

Elimination of quiescent/slow-proliferating cancer stem cells by Bcl-XL inhibition in non-small cell lung cancer.

Lung cancer is the most common cause of cancer-related mortality worldwide, urging the discovery of novel molecular targets and therapeutic strategies. Stem cells have been recently isolated from non-small cell lung cancer (NSCLC), thus allowing the investigation of molecular pathways specifically active in the tumorigenic population. We have found that Bcl-XL is constantly expressed by lung cancer stem cells (LCSCs) and has a prominent role in regulating LCSC survival. Whereas chemotherapeutic agents were scarcely effective against LCSC, the small molecule Bcl-2/Bcl-XL inhibitor ABT-737, but not the selective Bcl-2 inhibitor ABT-199, induced LCSC death at nanomolar concentrations. Differently from gemcitabine, which preferentially eliminated proliferating LCSC, ABT-737 had an increased cytotoxic activity in vitro towards quiescent/slow-proliferating LCSC, which expressed high levels of Bcl-XL. In vivo, ABT-737 as a single agent was able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors. Altogether, these results indicate that quiescent/slow-proliferating LCSC strongly depend on Bcl-XL for their survival and indicate Bcl-XL inhibition as a potential therapeutic avenue in NSCLC.

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy.

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Lung cancer stem cells: tools and targets to fight lung cancer.

Cancer stem cell (CSC) theory states that tumors are organized in a similar hierarchical manner as normal tissues, with a sub-population of tumorigenic stem-like cells that generate the more differentiated nontumorigenic tumor cells. CSCs are chemoresistant and seem to be responsible for tumor recurrence and formation of metastases. Therefore, the study of these cells may lead to crucial advances in the understanding of tumor biology as well as to innovative and more effective therapies. Lung cancer represents the leading cause of cancer-related mortality worldwide. Despite improvements in medical and surgical management, patient survival rates remain stable at approximately 15%, calling for innovative strategies that may contribute to improve patient outcome. The discovery of lung CSCs and the possibility to characterize their biological properties may provide powerful translational tools to improve the clinical outcome of patients with lung cancer. In this report, we review what is known about lung CSCs and discuss the diagnostic, prognostic and therapeutic prospective of these findings.

The pyridinylfuranopyrimidine inhibitor, PI-103, chemosensitizes glioblastoma cells for apoptosis by inhibiting DNA repair.

The failure of conventional therapies in glioblastoma (GBM) is largely due to an aberrant activity of survival cascades, such as PI3 kinase (PI3K)/Akt-mediated signaling. This study is the first to show that the class I PI3K inhibitor, PI-103, enhances chemotherapy-induced cell death of GBM cells. Concurrent treatment with PI-103 and DNA-damaging drugs, in particular doxorubicin, significantly increases apoptosis and reduces colony formation compared with chemotherapy treatment alone. The underlying molecular mechanism for this chemosensitization was shown by two independent approaches, that is, pharmacological and genetic inhibition of PI3K, DNA-PK and mTOR, to involve inhibition of DNA-PK-mediated DNA repair. Accordingly, blockage of PI3K or DNA-PK, but not of mTOR, significantly delays the resolution of doxorubicin-induced DNA damage and concomitantly increases apoptosis. Importantly, not only are several GBM cell lines chemosensitized by PI-103 but also GBM stem cells. Clinical relevance was further confirmed by the use of primary cultured GBM cells, which also exhibit increased cell death and reduced colony formation on combined treatment with PI-103 and doxorubicin. By identifying class I PI3K inhibitors as powerful agents in enhancing the lethality of DNA-damaging drugs, to which GBMs are usually considered unresponsive, our findings have important implications for the design of rational combination regimens in overcoming the frequent chemoresistance of GBM.

Identification and expansion of the tumorigenic lung cancer stem cell population.

Lung carcinoma is often incurable and remains the leading cancer killer in both men and women. Recent evidence indicates that tumors contain a small population of cancer stem cells that are responsible for tumor maintenance and spreading. The identification of the tumorigenic population that sustains lung cancer may contribute significantly to the development of effective therapies. Here, we found that the tumorigenic cells in small cell and non-small cell lung cancer are a rare population of undifferentiated cells expressing CD133, an antigen present in the cell membrane of normal and cancer-primitive cells of the hematopoietic, neural, endothelial and epithelial lineages. Lung cancer CD133(+) cells were able to grow indefinitely as tumor spheres in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The injection of 10(4) lung cancer CD133(+) cells in immunocompromised mice readily generated tumor xenografts phenotypically identical to the original tumor. Upon differentiation, lung cancer CD133(+) cells acquired the specific lineage markers, while loosing the tumorigenic potential together with CD133 expression. Thus, lung cancer contains a rare population of CD133(+) cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may provide valuable information to be exploited in the clinical setting.

The colorectal malignant polyp: scoping a dilemma.

Colorectal adenomas containing invasive carcinoma represent the majority of early colorectal cancers. The malignant polyp carries a significant risk of lympho-haematic metastasis and mortality due to the penetration of cancerous cells into the submucosal layer. The therapeutic dilemma is whether to perform endoscopic or surgical resection. A thorough assessment of the endoscopic, histological and clinical variables is needed to unravel the best treatment for each patient. In particular, a unique staging of such lesions, based on certain histopathological features, has been deeply implicated in the therapeutic choice. Aim of this article is to review the main endoscopic, histological and clinical features of the malignant polyp in order to propose a systematic management of this lesion.

Reduction of risk factors for cardiovascular diseases in morbid-obese patients following biliary-intestinal bypass: 3 years' follow-up.

BACKGROUND: Obese patients are often affected by hypertension, dyslipidaemia, impaired glucose metabolism, and suffer from cardiovascular disease (CVD), related to the characteristic metabolic alterations. AIM OF THE STUDY: To evaluate reduction of risk factors for CVDs in morbid-obese patients (body mass index (BMI)>40 kg/m2) after weight loss upon bariatric surgery intervention of biliary-intestinal bypass. SUBJECTS: 45 (17 men, 28 women) morbid-obese patients (age: 19-49 y, BMI>40 kg/m2). All patients were selected on the basis of medical history, physical and biochemical evaluation and of psychiatric tests, which were performed on all individuals admitted to our Day Hospital to verify the safety of surgical intervention. MEASUREMENTS: Body weight, body composition (by dual X-ray absorptiometry, DXA), blood pressure, lipid profile, fibrinogen and glucose metabolism were monitored at baseline and 1, 3, 6, 9, 12, 24 and 36 months after surgery. RESULTS: A significant and persistent weight loss was present in all patients at the end of the 3 y follow-up period (P<0.001), with a progressive reduction of total and trunk fat mass as evaluated by means of DXA. Additionally, a parallel significant reduction in systolic (P<0.001) and diastolic (P<0.001) blood pressure was observed. Total and LDL cholesterol were significantly reduced (P<0.001), while HDL showed no modifications; triglycerides declined progressively during the 3 y follow-up (P<0.001). Fibrinogen decreased from 364.5+/-82.4 to 266.4+/-45.7 mg/dl at the end of the period (P<0.001). Fasting glucose levels and glucose levels 120 min after an oral glucose tolerance test were reduced from 95.1+/-20.3 to 78.6+/-9.1 mg/dl (P<0.001) and from 116.9+/-34.7 to 77.6+/-15.5 mg/dl (P<0.001), respectively, at baseline and at the end of the study. Moreover, fasting insulin decreased from 30.0+/-20.4 to 8.6+/-2.9 microUI/ml (P<0.001) after 3 y, while insulin levels after (120 min) oral glucose load decreased from 105.5+/-61.5 to 12.0+/-6.0 microUI/ml (P<0.001). CONCLUSION: Our results show that biliary-intestinal bypass may represent a valid and alternative therapeutic approach in patients with morbid obesity since it induces a significant and stable reduction of body weight and obesity-related risk factors for CVD.

Control of erythroid cell production via caspase-mediated cleavage of transcription factor SCL/Tal-1.

SCL/Tal-1 is a helix-loop-helix (HLH) transcription factor required for blood cell development, whose abnormal expression is responsible for induction of T-cell acute lymphoblastic leukemia. We show here that SCL/Tal-1 is a key target of caspases in developing erythroblasts. SCL/Tal-1 degradation occurred rapidly after caspase activation and preceded the cleavage of the major erythroid transcription factor GATA-1. Expression of a caspase-resistant SCL/Tal-1 in erythroid progenitors was able to prevent amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis induced by growth factor deprivation or death receptor triggering. The potent proerythropoietic activity of uncleavable SCL/Tal-1 was clearly evident in the absence of erythropoietin, a condition that did not allow survival of normal erythroid cells or expansion of erythroblasts expressing caspase-resistant GATA-1. In the absence of erythropoietin, cells expressing caspase-resistant SCL/Tal-1 maintain high levels of Bcl-X(L), which inhibits amplification of the caspase cascade and mediates protection from apoptosis. Thus, SCL/TAL-1 is a survival factor for erythroid cells, whereas caspase-mediated cleavage of SCL/Tal-1 results in amplification of caspase activation, GATA-1 degradation and impaired erythropoiesis.

Patency of anastomoses after bilio-intestinal bypass: radioisotope demonstration.

BACKGROUND: Bilio-intestinal bypass (BIB) is effective for the treatment of refractory obesity. BIB permits bile flow into the non-functional jejunum, whereas food transit occurs via the remaining intestine. We used the radioisotope method of 99mTc-Hida cholescintigraphy (HC) in the follow-up of patients. METHODS: 21 patients were studied 3 months to 3 years after BIB with HC. After 3 hours acquisition, images were reviewed by two independent observers. Regions of interest (ROIs) were drawn on images: liver parenchyma, cholecysto-jejunal anastomosis (CC), choledochus (COL). Radioactivity taken up by liver was compared with radioactivity of CC and COL. % radioactivity passing through CC (%CC) and through COL (%COL) were determined. The final parameter, -COL, indicates the radioactive bile which does not pass through the choledochus. RESULTS: Anastomoses were found patent a few months to 3 years after operation. -COL showed linear correlation with the decrease in cholesterolemia and in body weight in the 1st year after BIB. CONCLUSIONS: HC shows passage of radioactive bile through anastomoses and provides semiquantitative evaluation of bile flux diversion. Bile flux towards the gallbladder and non-functional jejunal limb far exceeds flux directed towards the duodenum via the choledochus.

A program on complementary therapies for long-term care nursing assistants.

Nursing assistants (NAs) receive insufficient support in the face of heavy workloads and challenging job demands. This article describes a course on complementary therapies (CTs) intended to improve NAs' quality of life by helping them plan, develop, implement, and evaluate CTs. Future studies may examine the impact, if any, such courses have on recruitment and retention.

Plasma lipoproteins affect rate of cholesterol absorbed from bile by gallbladder: preliminary data.

BACKGROUND: The excessive accumulation of cholesterol absorbed from bile by the gallbladder impairs its contractility and favours gallstone formation. The total low plasma and high density lipoprotein cholesterol concentrations are associated with gallstone disease. AIMS: To investigate the effect of plasma lipoproteins on gallbladder cholesterol and phosphatidylcholine absorption from bile and to establish whether cholesterol absorption is Brefeldin A-sensitive. METHODS: Gallbladder mucosa lipid absorption rates were measured using: 1) in vitro isolated intra-arterially perfused pig gallbladder model with and without plasma lipoproteins perfusing the vascular tree; 2) human gallbladder fragments mounted in Ussing chambers with plasma lipoproteins at different concentrations in the serosal side; 3) pig gallbladder fragments mounted in Ussing chambers in the presence and absence of Brefeldin A. RESULTS: Total lipoproteins and high density lipoprotein significantly increased the release of biliary cholesterol and phosphatidylcholine in plasma and significantly decreased the tissue accumulation of cholesterol absorbed from bile. The scavenger effect of plasma lipoproteins on cholesterol absorbed from bile was concentration dependent. Brefeldin A did not influence gallbladder absorption of biliary cholesterol. CONCLUSIONS: Biliary cholesterol is absorbed by gallbladder mucosa via a Brefeldin-insensitive pathway and is removed by plasma lipoproteins.

Identification and characterization of a ligand-independent oligomerization domain in the extracellular region of the CD95 death receptor.

The CD95 death receptor plays an important role in several physiological and pathological apoptotic processes involving in particular the immune system. CD95 ligation leads to clustering of the receptor cytoplasmic "death domains" and recruitment of the zymogen form of caspase-8 to the cell surface. Activation of this protease through self-cleavage, followed by activation of downstream effector caspases, culminates in cleavage of a set of cellular proteins resulting in apoptosis with disassembly of the cell. It is very well known that the extracellular region of the CD95 receptor is required for CD95L interaction and that the death domain is necessary for the induction of the apoptotic signaling. Here, we identified and characterized a novel CD95 ligand- and death domain-independent oligomerization domain mapping to the NH(2)-terminal extracellular region of the CD95 receptor. In vitro and in vivo studies indicated that this domain, conserved among all soluble CD95 variants, mediates homo-oligomerization of the CD95 receptor and of the soluble CD95 proteins, as well as hetero-oligomerization of the receptor with the soluble variants. These results offer new insight into the mechanism of apoptosis inhibition mediated by the soluble CD95 proteins and suggest a role of the extracellular oligomerization domain in the regulation of the non-signaling state of the CD95 receptor.

Caspase activation without death.

Since molecular cloning of the C. elegans ced-3 gene revealed its homology with mammalian IL-1beta-converting enzyme,1 14 members of the caspase family have been identified, which have often been involved as mediators of one or more phases of the apoptotic process. 2,3 However, an over-simplified role of these proteases may be insufficient to explain the usually constitutive expression of such a large and complex family of enzymes, many of which display overlapping specificity. In addition to the well-established role of caspase-1 in the production of active IL-1beta and IL-18 in inflammation,4 an increasing number of reports has recently suggested that caspases may have a function outside of apoptosis. In this review, the situations in which cells survive despite the presence of activated caspases in their cytoplasm will be examined and discussed, with the intent to gather all recent advances in this new field that promises to be a focus for caspase research in the near future.

Negative regulation of erythropoiesis by caspase-mediated cleavage of GATA-1.

The production of red blood cells follows the sequential formation of proerythroblasts and basophilic, polychromatophilic and orthochromatic erythroblasts, and is promoted by the hormone erythropoietin (Epo) in response to tissue hypoxia. However, little is known about the negative regulation of this process. Death receptors are a family of surface molecules that trigger caspase activation and apoptosis in a variety of cell types. Here we show that immature erythroid cells express several death receptors whose ligands are produced by mature erythroblasts. Exposure of erythroid progenitors to mature erythroblasts or death-receptor ligands resulted in caspase-mediated degradation of the transcription factor GATA-1, which is associated with impaired erythroblast development. Expression of a caspase-resistant GATA-1 mutant, but not of the wild-type gene, completely restored erythroid expansion and differentiation following the triggering of death receptors, indicating that there is regulatory feedback between mature and immature erythroblasts through caspase-mediated cleavage of GATA-1. Similarly, erythropoiesis blockade following Epo deprivation was largely prevented by the expression of caspase-inhibitory proteins or caspase-resistant GATA-1 in erythroid progenitors. Caspase-mediated cleavage of GATA-1 may therefore represent an important negative control mechanism in erythropoiesis.