ABSTRACT
BACKGROUND: Locally advanced skin cancers including squamous cell carcinoma (SCC) of the skin are increasing in incidence. Patients are often elderly with significant comorbidities and therapy can be difficult. New targeted therapies, such as treatment directed at the epidermal growth factor receptor (EGFR), may be effective and less toxic in these patients. However, before designing appropriate clinical trials it is necessary to characterize the expression and activation of targets such as the EGFR to evaluate the rationale of using EGFR inhibitors (EGFRIs) in the treatment of this type of cancer. OBJECTIVES: To characterize the expression and activation by phosphorylation of EGFR in SCC of the skin by quantitative Western blotting using the LiCor immunofluorescence detection system with validation by immunohistochemistry. Secondary objectives were to evaluate downstream targets of EGFR expression and activation in SCC of the skin and to examine the associations between EGFR, pathological features and clinical behaviour of these tumours. METHODS: Twenty-one mainly locally advanced skin SCCs collected in our institution and stored in our tissue bank over a 4-year period were used for the study. RESULTS: Nine of 21 (43%) tumours expressed EGFR above background. Of those nine, five expressed phosphorylated EGFR. There was no correlation with downstream activation of canonical signalling pathways, pathological features or clinical behaviour. CONCLUSIONS: EGFR is expressed in a minority of tumours and then is not always activated. These results show that, before designing a trial with a targeted agent such as an EGFRI in SCC of the skin, it is important to verify the presence of the appropriate target to maximize the best outcome.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Epidermal Growth Factor/metabolism , Neoplasm Proteins/metabolism , Skin Neoplasms/metabolism , Adolescent , Adult , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/pathology , Child , Child, Preschool , Female , Gene Expression Regulation, Neoplastic/physiology , Humans , Infant , Male , Phosphorylation/radiation effects , Retrospective Studies , Skin Neoplasms/diagnosis , Skin Neoplasms/pathology , Treatment OutcomeABSTRACT
Activation of the phosphatidylinositol 3-kinase (PI3K) plays an important role in the mitogenic response of many cell types. Recently, two serine/threonine kinases Akt and p70(S6k) have been identified as physiological targets of PI3K. Observations that expression of activated forms of Akt led to the activation of p70(S6k) implied Akt might mediate mitogenic signaling through activation of p70(S6k). To clarify the relationship between signaling through these two kinases, we have examined their regulation by various mitogenic stimuli. In this study we have focused on the role of calcium in the regulation of each kinase in Balb/c-3T3 fibroblasts. Depletion of intracellular calcium stores by EGTA pretreatment has no effect on growth factor-induced Akt activation but completely abolishes p70(S6k) stimulation. Increase of intracellular calcium induced by ionomycin or thapsigargin results in a full activation of p70(S6k), whereas little or no activation of Akt is observed. Furthermore, although PI3K in anti-phosphotyrosine immunoprecipitates is only very weakly activated by ionomycin, the calcium-induced stimulation of p70(S6k) is completely inhibited by the specific PI3K inhibitor wortmannin. We conclude Akt and p70(S6k) lie on separate signaling pathways. Activation of signaling to Akt is insufficient for the activation of p70(S6k), which can be achieved independently of Akt. p70(S6k) requires a separate calcium-dependent and wortmannin-sensitive process that is likely to be independent of type IA PI3K family members.
Subject(s)
Calcium/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction , 3T3 Cells , Androstadienes/pharmacology , Animals , Down-Regulation , Epidermal Growth Factor/metabolism , Ionomycin/pharmacology , Mice , Mice, Inbred BALB C , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-akt , Tetradecanoylphorbol Acetate/pharmacology , Vanadates/pharmacology , WortmanninABSTRACT
To identify the mechanism underlying the exaggerated hyperglycemia during exercise in the heat, six trained men were studied during 40 min of cycling exercise at a workload requiring 65% peak pulmonary oxygen uptake (VO2peak) on two occasions at least 1 wk apart. On one occasion, the ambient temperature was 20 degrees C [control (Con)], whereas on the other, it was 40 degrees C [high temperature (HT)]. Rates of glucose appearance and disappearance were measured by using a primed continuous infusion of [6,6-2H]glucose No differences in oxygen uptake during exercise were observed between trials. After 40 min of exercise, heart rate, rectal temperature, respiratory exchange ratio, and plasma lactate were all higher in HT compared with Con (P < 0.05). Plasma glucose levels were similar at rest (Con, 4.54 +/- 0.19 mmol/l; HT, 4.81 +/- 0.19 mmol/l) but increased to a greater extent during exercise in HT (6.96 +/- 0.16) compared with Con (5.45 +/- 0.18; P < 0.05). This was the result of a higher glucose rate of appearance in HT during the last 30 min of exercise. In contrast, the glucose rate of disappearance and metabolic clearance rate were not different at any time point during exercise. Plasma catecholamines were higher after 10 and 40 min of exercise in HT compared with Con (P < 0.05), whereas plasma glucagon, cortisol, and growth hormone were higher in HT after 40 min. These results indicate that the hyperglycemia observed during exercise in the heat is caused by an increase in liver glucose output without any change in whole body utilization.
