Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors

Background

Adenocarcinomas of the tongue are rare and represent the minority (20 to 25%) of salivary gland tumors affecting the tongue. We investigated the utility of massively parallel sequencing to characterize an adenocarcinoma of the tongue, before and after treatment.

Results

In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain. There were 1,078 genes that exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations, administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A recurring metastasis possessed 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumors and 9 new somatic protein coding mutations. The observed mutations and amplifications were consistent with therapeutic resistance arising through activation of the MAPK and AKT pathways.

Conclusions

We conclude that complete genomic characterization of a rare tumor has the potential to aid in clinical decision making and identifying therapeutic approaches where no established treatment protocols exist. These results also provide direct in vivo genomic evidence for mutational evolution within a tumor under drug selection and potential mechanisms of drug resistance accrual.     

Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction

Making use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (. Proc. Natl. Acad. Sci. 77:7209-7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (. Biophys. J. 77:000-000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca(2+) concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca(2+) concentrations. At all Ca(2+) concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca(2+) is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca(2+)-dependent equilibration among different states of the thin filament can fully account for the Ca(2+) dependence of force redevelopment and the fluorescence changes described in this study.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.     

Adverse environmental conditions influence age-related innate immune responsiveness

Background-  

The innate immune system plays an important role in the recognition and induction of protective responses against infectious pathogens, whilst there is increasing evidence for a role in mediating chronic inflammatory diseases at older age. Despite indications that environmental conditions can influence the senescence process of the adaptive immune system, it is not known whether the same holds true for the innate immune system. Therefore we studied whether age-related innate immune responses are similar or differ between populations living under very diverse environmental conditions.     

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

Background

The photorespiratory nitrogen cycle in C₃ plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO₂. Because of the loss of CO₂ and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C₃ plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.

Results

When grown in ambient air, but not in elevated CO₂, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.

Conclusions

Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.     

A GWAS assessment of the contribution of genomic imprinting to the variation of body mass index in mice

Background

Genomic imprinting is an epigenetic mechanism that can lead to differential gene expression depending on the parent-of-origin of a received allele. While most studies on imprinting address its underlying molecular mechanisms or attempt at discovering genomic regions that might be subject to imprinting, few have focused on the amount of phenotypic variation contributed by such epigenetic process. In this report, we give a brief review of a one-locus imprinting model in a quantitative genetics framework, and provide a decomposition of the genetic variance according to this model. Analytical deductions from the proposed imprinting model indicated a non-negligible contribution of imprinting to genetic variation of complex traits. Also, we performed a whole-genome scan analysis on mouse body mass index (BMI) aiming at revealing potential consequences when existing imprinting effects are ignored in genetic analysis.

Results

10,021 SNP markers were used to perform a whole-genome single marker regression on mouse BMI using an additive and an imprinting model. Markers significant for imprinting indicated that BMI is subject to imprinting. Marked variance changed from 1.218 ×10⁻⁴ to 1.842 ×10⁻⁴ when imprinting was considered in the analysis, implying that one third of marked variance would be lost if existing imprinting effects were not accounted for. When both marker and pedigree information were used, estimated heritability increased from 0.176 to 0.195 when imprinting was considered.

Conclusions

When a complex trait is subject to imprinting, using an additive model that ignores this phenomenon may result in an underestimate of additive variability, potentially leading to wrong inferences about the underlying genetic architecture of that trait. This could be a possible factor explaining part of the missing heritability commonly observed in genome-wide association studies (GWAS).     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

An improved formulation of the zirconyl hematoxylin stain for acidic mucins

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.