Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets.

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

Gene expression analysis and microdialysis suggest hypothalamic triiodothyronine (T3) gates daily torpor in Djungarian hamsters (Phodopus sungorus).

Thyroid hormones play an important role in regulating seasonal adaptations of mammals. Several studies suggested that reduced availability of 3,3',5-triiodothyronine (T3) in the hypothalamus is required for the physiological adaptation to winter in Djungarian hamsters. We have previously shown that T3 is involved in the regulation of daily torpor, but it remains unclear, whether T3 affects torpor by central or peripheral mechanisms. To determine the effect of T3 concentrations within the hypothalamus in regulating daily torpor, we tested the hypothesis that low hypothalamic T3 metabolism would favour torpor and high T3 concentrations would not. In experiment 1 gene expression in torpid hamsters was assessed for transporters carrying thyroid hormones between cerebrospinal fluid and hypothalamic cells and for deiodinases enzymes, activating or inactivating T3 within hypothalamic cells. Gene expression analysis suggests reduced T3 in hypothalamic cells during torpor. In experiment 2, hypothalamic T3 concentrations were altered via microdialysis and torpor behaviour was continuously monitored by implanted body temperature transmitters. Increased T3 concentrations in the hypothalamus reduced expression of torpor as well as torpor bout duration and depth. Subsequent analysis of gene expression in the ependymal layer of the third ventricle showed clear up-regulation of T3 inactivating deiodinase 3 but no changes in several other genes related to photoperiodic adaptations in hamsters. Finally, serum analysis revealed that increased total T3 serum concentrations were not necessary to inhibit torpor expression. Taken together, our results are consistent with the hypothesis that T3 availability within the hypothalamus significantly contributes to the regulation of daily torpor via a central pathway.

Transcriptome Analysis of Hypothalamic Gene Expression during Daily Torpor in Djungarian Hamsters (Phodopus sungorus).

Animals living at high or temperate latitudes are challenged by extensive changes in environmental conditions over seasons. Djungarian hamsters (Phodopus sungorus) are able to cope with extremely cold ambient temperatures and food scarcity in winter by expressing spontaneous daily torpor. Daily torpor is a circadian controlled voluntary reduction of metabolism that can reduce energy expenditure by up to 65% when used frequently. In the past decades it has become more and more apparent, that the hypothalamus is likely to play a key role in regulating induction and maintenance of daily torpor, but the molecular signals, which lead to the initiation of daily torpor, are still unknown. Here we present the first transcriptomic study of hypothalamic gene expression patterns in Djungarian hamsters during torpor entrance. Based on Illumina sequencing we were able to identify a total number of 284 differentially expressed genes, whereby 181 genes were up- and 103 genes down regulated during torpor entrance. The 20 most up regulated group contained eight genes coding for structure proteins, including five collagen genes, dnha2 and myo15a, as well as the procoagulation factor vwf. In a proximate approach we investigated these genes by quantitative real-time PCR (qPCR) analysis over the circadian cycle in torpid and normothermic animals at times of torpor entrance, mid torpor, arousal and post-torpor. These qPCR data confirmed up regulation of dnah2, myo15a, and vwf during torpor entrance, but a decreased mRNA level for all other investigated time points. This suggests that gene expression of structure genes as well as the procoagulation factor are specifically initiated during the early state of torpor and provides evidence for protective molecular adaptions in the hypothalamus of Djungarian hamsters including changes in structure, transport of biomolecules and coagulation.

Thyroid hormone status affects expression of daily torpor and gene transcription in Djungarian hamsters (Phodopus sungorus).

Thyroid hormones (TH) play a key role in regulation of seasonal as well as acute changes in metabolism. Djungarian hamsters (Phodopus sungorus) adapt to winter by multiple changes in behaviour and physiology including spontaneous daily torpor, a state of hypometabolism and hypothermia. We investigated effects of systemic TH administration and ablation on the torpor behaviour in Djungarian hamsters adapted to short photoperiod. Hyperthyroidism was induced by giving T4 or T3 and hypothyroidism by giving methimazole (MMI) and sodium perchlorate via drinking water. T3 treatment increased water, food intake and body mass, whereas MMI had the opposite effect. Continuous recording of body temperature revealed that low T3 serum concentrations increased torpor incidence, lowered Tb and duration, whereas high T3 serum concentrations inhibited torpor expression. Gene expression of deiodinases (dio) and uncoupling proteins (ucp) were analysed by qPCR in hypothalamus, brown adipose tissue (BAT) and skeletal muscle. Expression of dio2, the enzyme generating T3 by deiodination of T4, and ucps, involved in thermoregulation, indicated a tissue specific response to treatment. Torpor per se decreased dio2 expression irrespective of treatment or tissue, suggesting low intracellular T3 concentrations during torpor. Down regulation of ucp1 and ucp3 during torpor might be a factor for the inhibition of BAT thermogenesis. Hypothalamic gene expression of neuropeptide Y, propopiomelanocortin and somatostatin, involved in feeding behaviour and energy balance, were not affected by treatment. Taken together our data indicate a strong effect of thyroid hormones on torpor, suggesting that lowered intracellular T3 concentrations in peripheral tissues promote torpor.