Whole-animal mounts of Caenorhabditis elegans for 3D imaging using atomic force microscopy.

The 3D surface of Caenorhabditis elegans was imaged at nanometer resolution using atomic force microscopy (AFM). Oscillation of a medium stiffness silicon AFM cantilever at the upper second amplitude peak, typically 6 times above the fundamental frequency, vastly improved image quality on the moist, sticky, and soft worms. Whole-animal mounts of normal and double-headed mutants of the nematode worm were prepared and scanned. Well-preserved anatomical features including annuli, furrows, alae, and rows of never before seen nanometer-sized pores dotting the molted worm's outermost surface coat were resolved. Well-preserved anatomical features including annuli, furrows, alae, and rows of nanometer-sized pores or struts dotting the molted worm's outermost surface were resolved. This AFM method represents a simple and rapid new approach for nanometer-resolved 3D imaging and analysis of whole-animal specimens of C. elegans. FROM THE CLINICAL EDITOR: In this interesting article the authors describe a new AFM sampling method to allow better images on whole-animal mounts such as C. elegans. This method would generate more information and in the future may be useful for differentiating even individual animals with different genetic backgrounds.

Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC. METHODS: Using IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay. RESULTS: Relative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0-3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability. CONCLUSION: PAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.

Differential expression of RON in small and non-small cell lung cancers.

RON is a MET related receptor tyrosine kinase (RTK) and its natural ligand is macrophage stimulating protein (MSP). RON plays a very important role in the regulation of inflammation. Several studies have previously reported overexpression of RON in a variety of cancers including lung and identified numerous RON alternate splice forms that very likely contribute to tumor growth and metastasis. Here, we have analyzed the expression of total RON protein as well as its kinase-active form (phospho-RON) in 175 archival lung tumor FFPE (formalin fixed paraffin embedded) samples that included non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and their metastatic forms. The frequency and intensity of RON protein expression was much higher in lung tumors of neuroendocrine origin such as SCLC and in secondary tumors that metastasized to brain. In addition, the majority of the expressed RON protein was phospho-RON. We also identified 62, and 30 kDa isoforms of RON (GenBank accession numbers are JN689381 and JN689382) using RNA isolated from pooled lung cancer cell lines and RT-PCR. A majority of the NSCLC cell lines expressed a 150 kDa band that corresponded to the RON ß chain and 120 kDa band in the panel of SCLC cell lines tested. RON was expressed on the cell surface in NSCLC cell lines. Finally, knock down of RON expression resulted in a significant loss in viability as well as motility in lung cancer cells suggesting that RON is a potential therapeutic target.

Role of protein kinase C ß and vascular endothelial growth factor receptor in malignant pleural mesothelioma: Therapeutic implications and the usefulness of Caenorhabditis elegans model organism.

PURPOSE: To examine the role of both protein kinase C (PKC)-ß and vascular endothelial growth factor receptor (VEGFR)-2 in malignant pleural mesothelioma (MPM) using respective inhibitors, enzastaurin and KRN633. MATERIALS AND METHODS: MPM cell lines, control cells, and a variety of archived MPM tumor samples were used to determine the protein expression levels of PKC-ß, VEGFR-2, VEGF, and p-AKT. Effects of enzastaurin and KRN633 on phosphorylation status of key signaling molecules and viability of the mesothelioma cells were determined. The common soil nematode, Caenorhabditis elegans, was treated with enzastaurin to determine its suitability to screen for highly potent kinase inhibitors. RESULTS: PKC-ß1, PKC-ß2 and VEGFR-2/KDR were overexpressed in MPM cell lines and MPM tumor tissues. Enzastaurin treatment resulted in significant loss in viability of VEGF induced cell proliferation; however, the effect of KRN633 was much less. Enzastaurin also dramatically decreased the phosphorylation of PKC-ß, its downstream target p-AKT, and surprisingly, the upstream VEGFR-2. The combination of the two drugs at best was additive and similar results were obtained with respect to cell viability. Treatment of C. elegans with enzastaurin resulted in clear phenotypic changes and the worms were hypermotile with abnormal pattern and shape of eggs, suggesting altered fecundity. CONCLUSIONS: PKC-ß1 and VEGFR-2 are both excellent therapeutic targets in MPM. Enzastaurin was better at killing MPM cells than KRN633 and the combination lacked synergy. In addition, we show here that C. elegans can be used to screen for the next generation inhibitors as treatment with enzastaurin resulted in clear phenotypic changes that could be assayed.

C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

Tributyltin differentially promotes development of a phenotypically distinct adipocyte.

OBJECTIVE: Environmental endocrine disrupting chemicals (EDCs) are increasingly implicated in the pathogenesis of obesity. Evidence implicates various EDCs as being proadipogenic, including tributyltin (TBT), which activates the peroxisome proliferator activated receptor-γ (PPARγ). However, the conditions required for TBT-induced adipogenesis and its functional consequences are incompletely known. METHODS: The costimulatory conditions necessary for preadipocyte-to-adipocyte differentiation were compared between TBT and the pharmacological PPARγ agonist troglitazone (Trog) in the 3T3-L1 cell line; basal and insulin-stimulated glucose uptake were assessed using radiolabeled 2-deoxyglucose. RESULTS: TBT enhanced expression of the adipocyte marker C/EBPα with coexposure to either isobutylmethylxanthine or insulin in the absence of other adipogenic stimuli. Examination of several adipocyte-specific proteins revealed that TBT and Trog differentially affected protein expression despite comparable PPARγ stimulation. In particular, TBT reduced adiponectin expression upon maximal adipogenic stimulation. Under submaximal stimulation, TBT and Trog differentially promoted adipocyte-specific gene expression despite similar lipid accumulation. Moreover, TBT attenuated Trog-induced adipocyte gene expression under conditions of cotreatment. Finally, TBT-induced adipocytes exhibited altered glucose metabolism, with increased basal glucose uptake. CONCLUSIONS: TBT-induced adipocytes are functionally distinct from those generated by a pharmacological PPARγ agonist, suggesting that obesogen-induced adipogenesis may generate dysfunctional adipocytes with the capacity to deleteriously affect global energy homeostasis.

Dietary exposure to the endocrine disruptor tolylfluanid promotes global metabolic dysfunction in male mice.

Environmental endocrine disruptors are implicated as putative contributors to the burgeoning metabolic disease epidemic. Tolylfluanid (TF) is a commonly detected fungicide in Europe, and previous in vitro and ex vivo work has identified it as a potent endocrine disruptor with the capacity to promote adipocyte differentiation and induce adipocytic insulin resistance, effects likely resulting from activation of glucocorticoid receptor signaling. The present study extends these findings to an in vivo mouse model of dietary TF exposure. After 12 weeks of consumption of a normal chow diet supplemented with 100 parts per million TF, mice exhibited increased body weight gain and an increase in total fat mass, with a specific augmentation in visceral adipose depots. This increased adipose accumulation is proposed to occur through a reduction in lipolytic and fatty acid oxidation gene expression. Dietary TF exposure induced glucose intolerance, insulin resistance, and metabolic inflexibility, while also disrupting diurnal rhythms of energy expenditure and food consumption. Adipose tissue endocrine function was also impaired with a reduction in serum adiponectin levels. Moreover, adipocytes from TF-exposed mice exhibited reduced insulin sensitivity, an effect likely mediated through a specific down-regulation of insulin receptor substrate-1 expression, mirroring effects of ex vivo TF exposure. Finally, gene set enrichment analysis revealed an increase in adipose glucocorticoid receptor signaling with TF treatment. Taken together, these findings identify TF as a novel in vivo endocrine disruptor and obesogen in mice, with dietary exposure leading to alterations in energy homeostasis that recapitulate many features of the metabolic syndrome.

Critical role for the receptor tyrosine kinase EPHB4 in esophageal cancers.

Esophageal cancer incidence is increasing and has few treatment options. In studying receptor tyrosine kinases associated with esophageal cancers, we have identified EPHB4 to be robustly overexpressed in cell lines and primary tumor tissues. In total, 94 squamous cell carcinoma, 82 adenocarcinoma, 25 dysplasia, 13 Barrett esophagus, and 25 adjacent or unrelated normal esophageal tissues were evaluated by immunohistochemistry. EPHB4 expression was significantly higher in all the different histologic categories than in adjacent normal tissues. In 13 esophageal cancer cell lines, 3 of the 9 SCC cell lines and 2 of the 4 adenocarcinomas expressed very high levels of EPHB4. An increased gene copy number ranging from 4 to 20 copies was identified in a subset of the overexpressing patient samples and cell lines. We have developed a novel 4-nitroquinoline 1-oxide (4-NQO)-induced mouse model of esophageal cancer that recapitulates the EPHB4 expression in humans. A specific small-molecule inhibitor of EPHB4 decreased cell viability in a time- and dose-dependent manner in 3 of the 4 cell lines tested. The small-molecule inhibitor and an EPHB4 siRNA also decreased cell migration (12%-40% closure in treated vs. 60%-80% in untreated), with decreased phosphorylation of various tyrosyl-containing proteins, EphB4, and its downstream target p125FAK. Finally, in a xenograft tumor model, an EPHB4 inhibitor abrogated tumor growth by approximately 60% compared with untreated control. EphB4 is robustly expressed and potentially serves as a novel biomarker for targeted therapy in esophageal cancers.

RON (MST1R) is a novel prognostic marker and therapeutic target for gastroesophageal adenocarcinoma.

RON (MST1R) is one of two members of the MET receptor tyrosine kinase family, along with parent receptor MET. RON has a putative role in several cancers, but its expression and function is poorly characterized in gastroesophageal adenocarcinoma. A recognized functional role of MET tyrosine kinase in gastroesophageal cancer has led to early phase clinical trials using MET inhibitors, with unimpressive results. Therefore, the role of RON in gastroesophageal cancer, as well as its role in cooperative signaling with MET and as a mechanism of resistance to MET inhibition, was studied in gastroesophageal tissues and cell lines. By IHC, RON was highly over-expressed in 74% of gastroesophageal samples (n=94), and over-expression was prognostic of poor survival (p=0.008); RON and MET co-expression occurred in 43% of samples and was prognostic of worst survival (p=0.03). High MST1R gene copy number by quantitative polymerase chain reaction, and confirmed by fluorescence in situ hybridization and/or array comparative genomic hybridization, was seen in 35.5% (16/45) of cases. High MST1R gene copy number correlated with poor survival (p=0.01), and was associated with high MET and ERBB2 gene copy number. A novel somatic MST1R juxtamembrane mutation R1018G was found in 11% of samples. RON signaling was functional in cell lines, activating downstream effector STAT3, and resulted in increased viability over controls. RON and MET co-stimulation assays led to enhanced malignant phenotypes over stimulation of either receptor alone. Growth inhibition as evidenced by viability and apoptosis assays was optimal using novel blocking monoclonal antibodies to both RON and MET, versus either alone. SU11274, a classic MET small molecule tyrosine kinase inhibitor, blocked signaling of both receptors, and proved synergistic when combined with STAT3 inhibition (combination index < 1). These preclinical studies define RON as an important novel prognostic marker and therapeutic target for gastroesophageal cancer warranting further investigation.

CBL is frequently altered in lung cancers: its relationship to mutations in MET and EGFR tyrosine kinases.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC. METHODS AND FINDINGS: Using archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility. CONCLUSIONS: Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.