MicroRNA-17-92 cluster regulates pancreatic beta-cell proliferation and adaptation.

MiR-17-92 cluster contributes to the regulation of mammalian development, aging and tumorigenesis. The functional roles of miR-17-92 in pancreatic beta-cells are largely unknown. In this study, we found that conditional deletion of miR-17-92 in mouse pancreatic beta-cells (miR-17-92ßKO) significantly reduces glucose tolerance and the first phase of insulin secretion, despite normal ad libitum fed and fasting glucose levels. Proliferation is down-regulated in pancreatic beta-cells after deleting miR-17-92. MiR-17-92ßKO mice show higher phosphatase and tensin homologue (PTEN) and lower phosphorylated AKT in islets. Under high fat diet challenge for 16 weeks, miR-17-92ßKO mice lose compensation and exhibit higher glucose levels, and lower insulin secretion. Collectively, these data suggest that miR-17-92 is a critical contributor to molecular mechanisms regulating glucose-stimulated insulin secretion and pancreatic beta-cell adaptation under metabolic stress.

Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future.

Spatial prevalence and associations among respiratory diseases in Maine.

Chronic respiratory diseases rank among the leading global disease burdens. Maine's respiratory disease prevalence exceeds the US average, despite limited urbanization/industrialization. To provide insight into potential etiologic factors among this unique, rural population, we analyzed the spatial distributions of, and potential associations among asthma, COPD, pneumonia, and URI adult outpatient data (n=47,099) from all outpatient transactions (n=5,052,900) in 2009 for Maine hospitals and affiliate clinics, using spatial scan statistic, geographic weighted regression (GWR), and a Delaunay graph algorithm. Non-random high prevalence regions were identified, the majority of which (84% of the population underlying all regions) exhibited clusters for all four respiratory diseases. GWR provided further evidence of spatial correlation (R(2)=0.991) between the communicable and noncommunicable diseases under investigation, suggesting spatial interdependence in risk. Sensitivity analyses of known respiratory disease risks did not fully explain our results. Prospective epidemiology studies are needed to clarify all contributors to risk.

Aberrant overexpression of FOXM1 transcription factor plays a critical role in lung carcinogenesis induced by low doses of arsenic.

Environmental or occupational exposure to low doses of arsenic induces a series of health problems including cancer. The molecular events in arsenic-induced carcinogenicity remain to be defined. In the NuLi-1 immortalized human lung epithelial cell line with p53 and pRb deficiency, exposure to low doses of arsenic trioxide for 72 h promoted cell proliferation and upregulated the gene transcription levels of FOXM1, CDC6, CDC25A, and cyclin D1, which are both critical cell cycle regulatory genes and proto-oncogenes. Continuous in vitro exposure to 1 µM arsenic trioxide for 34 wks induced malignant cell transformation, as evidenced by enhanced anchorage-independent cell growth. The expression of FOXM1, CDC6, CDC25A, and Cyclin D1 was dynamically elevated at the gene transcription and protein levels in the process of cell transformation. The carcinogenic ability of transformed cell colonies coincides with the expression levels of FOXM1 in in vitro anchorage-independent growth assays and in vivo tumor xenograft formation assays. In reverse, the knockdown of FOXM1 in lung adenocarcinoma A549 cells or arsenic-transformed NuLi-1 cells significantly decreased anchorage-independent cell growth and tumor xenograft formation. The transformed NuLi-1 cells showed genomic instability in the form of copy number variation (CNV) at chromosome 1, 5, 6, 18, and 20, but not loss of heterozygosity (LOH). These results showed for the first time that chronic exposure to low doses of arsenic trioxide promoted lung carcinogenicity, in part by aberrantly upregulating FOXM1 and its associated oncogenes, when the tumor suppressor genes p53 and pRb were inactivated.

miR-335 inhibits small cell lung cancer bone metastases via IGF-IR and RANKL pathways.

UNLABELLED: Small cell lung cancer (SCLC) is a rapidly progressing, incurable cancer that frequently spreads to bone. New insights are needed to identify therapeutic targets to prevent or retard SCLC metastatic progression. Human SCLC SBC-5 cells in mouse xenograft models home to skeletal and nonskeletal sites, whereas human SCLC SBC-3 cells only pervade nonskeletal sites. Because microRNAs (miRNA) often act as tumor regulators, we investigated their role in preclinical models of SCLC. miRNA expression profiling revealed selective and reduced expression of miRNA (miR)-335 and miR-29a in SBC-5 cells, compared with SBC-3 cells. In SBC-5 cells, miR-335 expression correlated with bone osteolytic lesions, whereas miR-29a expression did not. Overexpression of miR-335 in SBC-5 cells significantly reduced cell migration, invasion, proliferation, colony formation, and osteoclast induction in vitro. Importantly, in miR-335 overexpressing SBC-5 cell xenografts (n = 10), there were minimal osteolytic lesions in the majority of mice and none in three mice. Expression of RANK ligand (RANKL) and insulin-like growth factor-I receptor (IGF-IR), key mediators of bone metastases, were elevated in SBC-5 as compared with SBC-3 cells. Mechanistically, overexpression of miR-335 in SBC-5 cells reduced RANKL and IGF-IR expression. In conclusion, loss of miR-335 promoted SCLC metastatic skeletal lesions via deregulation of IGF-IR and RANKL pathways and was associated with metastatic osteolytic skeletal lesions. IMPLICATIONS: These preclinical findings establish a need to pursue the role of miR-335 in human SCLC with metastatic skeletal disease.

Spatial and temporal distributions of lung cancer histopathology in the state of Maine.

Maine has among the highest rates of lung cancer in the United States (US). Maine serves as a geographical representation of US rural communities, and their associated health disparities. As the key risks of tobacco use decrease and radon abatement increases, previously obscured environmental exposures may measurably contribute to the attributable risk fraction of lung cancer. To generate hypotheses of novel environmental exposures associated with lung cancer, we investigated if there was non-random spatial distribution of lung cancer in Maine. Case data (n = 14,038) between 1995 and 2006 were obtained from the Maine Cancer Registry. Population data were obtained from the 2000 US Census. We assessed the spatial distribution of lung cancers among white cases by histopathology subtype [non-small cell lung carcinoma (NSCLC): adenocarcinoma (n = 3680), squamous cell (n = 2801) and large cell (n = 1195); and small cell lung carcinoma (SCLC) (n = 1994)], using spatial scan statistic, assuming a discrete Poisson distribution adjusted for age and population density. Because of time-dependent trends in lung cancer differential diagnostic criteria, we repeated our analyses, limiting it to 2002-2006. While SCLC rates were equivalent across the state, we identified discrete regions with elevated rates of adenocarcinoma among females and squamous cell carcinoma among males. Independent of gender, the most striking geospatial observation was elevated large cell lung cancer specifically in one of the poorest counties in the US. A selective spatial distribution of large cell lung cancer has not been previously reported. More research is needed to identify factors inducing large cell carcinoma pathology, and to determine if in rural communities health disparities are associated with increased risk for this diagnosis.

Informed consent for biobanking research: cancer patient recruitment from rural communities in Maine.

Biobanking research seeks to improve the diversity, availability, and quality of human specimens critical for translational research, including biospecimen collections from disadvantaged minorities. American rural whites are seldom represented in such initiatives as geographic isolation makes obtaining informed consent challenging. We report a case series of 83 newly diagnosed cancer patients, attending a rural community medical center, who consented to participate in cancer research. To enable pooling with population studies, we created a BioGeoBank using 2007 NCI and ISBER Best Practices, after a protocol approval by Eastern Maine Medical Center (EMMC) IRB and OHP HRPO. Informed consent forms were at Flesch-Kincaid 8th Grade reading level, supplemented by NCI educational brochures. Of 108 patients identified, 85 were eligible. Of these, 83 patients (49 lung cancer, 21 breast cancer, and 13 other cancers) consented to donate data, blood, and tissue specimens for future research, and maintained eligibility. Two years later, we executed a legacy protocol to transfer specimens to NCI's biorepository. Of the 69 surviving patients, 9 patients could not be contacted. All those contacted (60) agreed to provide additional data on environmental risks, and consented to specimen transfer. Self-organizing map analyses showed no evidence that age, education, income, familial susceptibility, or lifestyle factors were associated with consent to donate data or biospecimens. Cancer cases reported 1-3 co-morbid chronic diseases (mostly cardiovascular), near lifetime smoking and/or alcohol consumption; familial cancer risks, and many had a prior cancer history. Anecdotally, willingness to consent was based on altruistic hopes that research would generate knowledge to reduce cancer incidence. Our study shows that cancer patients from disadvantaged white rural communities with health disparities associated with geographic isolation are motivated to consent to participate and support biobank research.

Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

FOXM1 is a critical regulator of the G1/S and G2/M cell cycle transitions, as well as of the mitotic spindle assembly. Previous studies have suggested that FOXM1 regulates CDC25A gene transcription, but the mechanism remains unknown. Here, we provide evidence that FOXM1 directly regulates CDC25A gene transcription via direct promoter binding and indirect activation of E2F-dependent pathways. Prior literature reported that CDC25B and CDC25C activate CDK1/cyclinB complexes in order to enable phosphorylation of FOXM1. It was unknown if CDC25A functions in a similar manner. We report that FOXM1 transcriptional activity is synergistically enhanced when co-expressed with CDC25A. The increase is dependent upon CDK1 phosphorylation of FOXM1 at T600, T611 and T620 residues. We also report a novel protein interaction between FOXM1 and CDC25A via the C-terminus of FOXM1. We demonstrate that the phosphorylation of Thr 600 and Thr 611 residues of FOXM1 enhanced this interaction, and that the interaction is dependent upon CDC25A phosphatase activity. Our work provides novel insight into the underlying mechanisms by which FOXM1 controls the cell cycle through its association with CDC25A.

A novel animal model for bone metastasis in human lung cancer.

Metastases account for 90% of lung cancer mortalities, frequently target the skeleton and lead to rapid deterioration in quality of life. The molecular mechanism underlying bone metastases is largely unknown. Development of xenograft mouse models, such as the severe combined immunodeficient (SCID) CB-17 mouse and the non-obese diabetic (NOD)/SCID mouse, both of which lack functional B- and T-cells and are able to host allogeneic or xenogeneic tumor cells, has made great contributions in this area. However, residual natural killer (NK) cells in these models are able to significantly modify local tumor growth and metastasis. Treatment with anti-murine IL-2 receptor ß chain Ab (TM-ß1) antibody can abrogate NK cell activity in vivo; however, the antibody treatment may result in unexpected effects and the stability is hard to control. To overcome these shortcomings, we evaluated xenografts in NOD-scid IL2Rγ(null) immunodeficient mice that lacked mature T cells, B cells and functional NK cells. We compared the target tissue distribution of the human small cell lung cancer cell lines SBC-5 and SBC-3. Gross necropsy and whole skeletal X-ray film examination of the host mice were conducted 30 days post-tail vein injection. The SBC-5 cells colonized bone and formed lytic lesions. The cells also colonized liver, spleen and, less frequently, the pancreas, ovary and kidney. The SBC-3 cell xenografts formed easily visible tumor foci in the liver, pancreas, ovary/uterus and kidney, but not bone metastases. Our results showed that SBC-5 cells in NOD-scid IL2Rγ(null) immunodeficient mice provide a suitable xenograft model system for bone metastasis of human lung cancer. This novel animal model may therefore be used to study the molecular pathway of bone metastases and to evaluate targets for effective therapies.

MicroRNA-204 critically regulates carcinogenesis in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue sarcomas accounting for 3%-10% of all soft tissue sarcomas. Neurofibromatosis type 1 (NF1) is the most important known risk factor. MPNSTs are often diagnosed at an advanced stage when distant metastases have developed. Although surgical resection remains the main treatment for MPNSTs, complete surgical resection is rarely possible. The prognosis for patients with MPNSTs is poor. There is an urgent need for improved therapies. To this end, we investigated whether microRNA (miR), specifically miR-204, might be implicated in MPNSTs because it is located at a cancer-associated genomic region exhibiting high frequency of loss of heterozygosity in tumors. We show that miR-204 expression is downregulated in NF1 and non-NF1 MPNST tumor tissues and in tumor cell lines. Restoring miR-204 expression in MPNST cell lines STS26T (non-NF1), ST88-14 (NF1), and T265p21 (NF1) significantly reduces cellular proliferation, migration, and invasion in vitro. Restoring miR-204 expression in STS26T decreases tumor growth and malignant progression in vivo. We also report that miR-204 inhibits Ras signaling and expression of high mobility group gene A2. These findings support the hypothesis that miR-204 plays critical roles in MPNST development and tumor progression. miR-204 may represent a novel biomarker for diagnosis and a candidate target with which to develop effective therapies for MPNSTs.

Analyzing spatial and temporal (222)Rn trends in Maine.

Prolonged radon exposure has been linked to lung cancer. Cancer registry data indicates excess risk for age-adjusted lung cancer in Maine. Maine's mean residential radon activity exceeds the EPA maximum contaminant level (MCL). This paper describes the application of spatial autocorrelation methods to retrospective data as a means of analyzing radon activity in Maine. Retrospective air and well water radon activity data, sampled throughout Maine between 1993 and 2008, are standardized and geocoded for analysis. Three spatial autocorrelation algorithms-local Getis-Ord, local Moran, and spatial scan statistic-are used to identify spatial, temporal, and spatiotemporal radon activity clusters and/or outliers. Spatial clusters of high air- and well water-Rn activity are associated with Maine's Lucerne and Sebago granitic formations. Spatial clusters of low air- and well water-Rn activity are associated with Biddeford Granite and the metamorphic bedrock formation Silurian Ordovician Vassalboro. Space-time analysis indicates that most spatial clusters persist over the period of sampling. No significant temporal clusters are identified. Persistent spatial variations in radon may help to better understand and predict radon-related health risks associated with Maine residences.

Activation of the p38 MAPK/Akt/ERK1/2 signal pathways is required for the protein stabilization of CDC6 and cyclin D1 in low-dose arsenite-induced cell proliferation.

Arsenic trioxide (ATO) is a first-line anti-cancer agent for acute promyelocytic leukemia, and induces apoptosis in other solid cancer cell lines including breast cancer cells. However, as with arsenites found in drinking water and used as raw materials for wood preservatives, insecticides, and herbicides, low doses of ATO can induce carcinogenesis after long-term exposure. At 24 h after exposure, ATO (0.01-1 µM) significantly increased cell proliferation and promoted cell cycle progression from the G1 to S/G2 phases in the non-tumorigenic MCF10A breast epithelial cell line. The expression of 14 out of 96 cell-cycle-associated genes significantly increased, and seven of these genes including cell division cycle 6 (CDC6) and cyclin D1 (CCND1) were closely related to cell cycle progression from G1 to S phase. Low-dose ATO steadily increased gene transcript and protein levels of both CDC6 and cyclin D1 in a dose- and time-dependent manner. Low-dose ATO produced reactive oxygen species (ROS), and activated the p38 MAPK, Akt, and ERK1/2 pathways at different time points within 60 min. Small molecular inhibitors and siRNAs inhibiting the activation of p38 MAPK, Akt, and ERK1/2 decreased the ATO-increased expression of CDC6 protein. Inhibiting the activation of Akt and ERK1/2, but not p38 MAPK, decreased the ATO-induced expression of cyclin D1 protein. This study reports for the first time that p38 MAPK/Akt/ERK1/2 activation is required for the protein stabilization of CDC6 in addition to cyclin D1 in ATO-induced cell proliferation and cell cycle modulation from G1 to S phase.

MicroRNA-10b regulates tumorigenesis in neurofibromatosis type 1.

MicroRNAs (miRNAs) are frequently deregulated in human tumors, and play important roles in tumor development and progression. The pathological roles of miRNAs in neurofibromatosis type 1 (NF1) tumorigenesis are largely unknown. We demonstrated that miR-10b was up-regulated in primary Schwann cells isolated from NF1 neurofibromas and in cell lines and tumor tissues from malignant peripheral nerve sheath tumors (MPNSTs). Intriguingly, a significantly high level of miR-10b correlated with low neurofibromin expression was found in a neuroectodermal cell line: Ewing's sarcoma SK-ES-1 cells. Antisense inhibiting miR-10b in NF1 MPNST cells reduced cell proliferation, migration and invasion. Furthermore, we showed that NF1 mRNA was the target for miR-10b. Overexpression of miR-10b in 293T cells suppressed neurofibromin expression and activated RAS signaling. Antisense inhibition of miR-10b restored neurofibromin expression in SK-ES-1 cells, and decreased RAS signaling independent of neurofibromin in NF1 MPNST cells. These results suggest that miR-10b may play an important role in NF1 tumorigenesis through targeting neurofibromin and RAS signaling.

Psychosocial adjustment of siblings of children with cancer: a systematic review.

OBJECTIVES: To promote a broader understanding of the psychosocial impact of childhood cancer on siblings, a systematic review was undertaken. Directions for future research are proposed and clinical strategies are suggested for addressing the needs of these children. METHODS: Searches of Medline, PsycINFO and CINAHL revealed 65 relevant qualitative, quantitative, or mixed methods' papers published between 1997 and 2008. These papers were rated for scientific merit and findings were extracted for summary. RESULTS: Siblings of children with cancer do not experience elevated mean rates of psychiatric disorders, but a significant subset experiences post-traumatic stress symptoms, negative emotional reactions (e.g. shock, fear, worry, sadness, helplessness, anger, and guilt), and poor quality of life in emotional, family, and social domains. In general, distress is greater closer to time of diagnosis. School difficulties are also evident within 2 years of diagnosis. Qualitative studies reveal family-level themes such as loss of attention and status as well as positive outcomes including increased sibling maturity and empathy. CONCLUSIONS: Research regarding siblings of children with cancer continues to be methodologically limited. The conclusions of qualitative and quantitative studies differ considerably. We propose a research agenda to propel this field forward including greater attention to alterations in normative development (as opposed to psychiatric conditions), development of more appropriate quantitative measures, examination of potential moderators of adaptation, and use of prospective longitudinal designs. Siblings of children with cancer are a psychosocially at-risk group and should be provided with appropriate supportive services.

Skeletal abnormalities in neurofibromatosis type 1: approaches to therapeutic options.

The skeleton is frequently affected in individuals with neurofibromatosis type 1, and some of these bone manifestations can result in significant morbidity. The natural history and pathogenesis of the skeletal abnormalities of this disorder are poorly understood and consequently therapeutic options for these manifestations are currently limited. The Children's Tumor Foundation convened an International Neurofibromatosis Type 1 Bone Abnormalities Consortium to address future directions for clinical trials in skeletal abnormalities associated with this disorder. This report reviews the clinical skeletal manifestations and available preclinical mouse models and summarizes key issues that present barriers to optimal clinical management of skeletal abnormalities in neurofibromatosis type 1. These concepts should help advance optimal clinical management of the skeletal abnormalities in this disease and address major difficulties encountered for the design of clinical trials.

Proteomic characteristics of ex vivo-enriched adult human bone marrow mononuclear cells in continuous perfusion cultures.

A major challenge in developing cell therapies is reliable characterization of the cell product at the molecular level. Fresh autologous and passaged human bone marrow enriched for stem and mesenchymal stromal stem cells have been used to regenerate bone. We report the proteome of an innovative autologous human bone marrow-derived mixed cell product (BMMCP), cultured ex vivo for 12 days, in automated continuous media perfusion system to avoid passaging, and discuss reproducibility of protein composition. Each BMMCP is compared to its originating human adult bone marrow mononuclear cells (BMMNC). With the use of 2-D LC-MS/MS approach, 638 (BMMNC) and 867 (BMMCP) distinct proteins were identified including cell adhesion molecules, extracellular matrix and growth factors. Overlap of protein identifications revealed that 67% of the BMMNC proteome was retained in the BMMCP, and protein expression of selected cell lineages was enhanced. Isotope-coded affinity tags (ICAT) and MS/MS were used to identify and quantify relative changes in the proteome of BMMNC and their related BMMCP, obtained from 3 separate donors. In 3 separate ICAT experiments, 57% of proteome identified was shared between donors. Measurable and definable proteomic characterization of BMMCP will facilitate their use in clinical trials and provide insight into cell functionality needed to support multiple therapeutic indications.

IGF-I improved bone mineral density and body composition of weaver mutant mice.

Our recent report on a parallel decrease in the body weights and serum IGF-I levels of weaver mice suggests that IGF-I's endocrine function may be impaired in neurodegenerative diseases. To further understand the overall effects of IGF-I deficiency on the postnatal growth, we measured bone mineral density (BMD), bone mineral content (BMC), lean body mass (LBM) and fat mass in male and female weaver mice and wild-type littermates on D21 (prepuberty), D45 (puberty), and D60 (postpuberty) using dual-energy X-ray absorptiometry (DEXA). In both male and female weaver mice, we found that the levels of circulating IGF-I paralleled those of BMD, BMC, and LBM, but not the fat mass. Male weaver mice have normal fat mass at all three ages studied, whereas female weaver mice showed a trend to increase their fat mass as they mature. To determine whether circulating IGF-I is a determinant of body composition, we crossbred IGF-I transgenic mice with homozygous weaver mice, which resulted in a significant increase in circulating IGF-I levels in both male and female weaver mice and normalization of their BMD, BMC and body weights. In summary, our results demonstrated that normal circulating IGF-I levels are important in maintaining BMD, BMC, and body composition in neurodegenerative diseases, such as hereditary cerebellar ataxia.

RECQL4-deficient cells are hypersensitive to oxidative stress/damage: Insights for osteosarcoma prevalence and heterogeneity in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a heterogeneous disease, associated with increased prevalence of osteosarcoma in very young patients with a mutated RECQL4 gene. In this study, we tested the ability of RECQL4 deficient fibroblasts, derived from a RTS patient to recover from hydrogen peroxide (H(2)O(2))-induced oxidative stress/damage. Immunoperoxidase staining for 8-oxo-deoxyguanosine (8-oxo-dG) formation in RTS and normal human fibroblasts were compared to assess DNA damage. We determined DNA synthesis, cell growth, cell cycle distribution, and viability in RTS and normal human fibroblasts before and after H(2)O(2) treatment. H(2)O(2) induces 8-oxo-dG formation in both RTS and normal fibroblasts. In normal human fibroblasts, RECQL4 was predominantly localized to cytoplasm; nuclear translocation and foci formation occurred in response to oxidant stimulation. After recovery from oxidant exposure, viable RTS fibroblasts showed irreversible growth arrest compared to normal fibroblasts. DNA synthesis decreased significantly in treated RTS cells, with concomitant reduction of cells in the S-phase. These results suggest that enhanced oxidant sensitivity in RECQL4 deficient fibroblasts derived from RTS patients could be attributed to abnormal DNA metabolism and proliferation failure. The ramifications of these findings on osteosarcoma prevalence and heterogeneity in RTS are discussed.