Douglas J. E. Elder

Apoptosis induction and cyclooxygenase-2 regulation in human colorectal adenoma and carcinoma cell lines by the cyclooxygenase-2-selective non-steroidal anti-inflammatory drug NS-398.

We determined the effect of the highly selective cyclooxygenase‐2 (COX‐2) inhibitor NS‐398 on proliferation, apoptosis and COX‐2 regulation in 3 pre‐malignant human colorectal adenoma cell lines (RG/C2, AA/C1, RR/C1) and compared its effect on 3 colorectal carcinoma cell lines (HT29, KS, JW2). COX‐2 protein was expressed in each cell line derived from an adenoma, thus providing evidence that COX‐2 is expressed in the tumour cells themselves at an early stage in human colorectal adenoma formation. NS‐398 (20 to 100 μM for 96 h) induced apoptosis and inhibited the proliferation of the adenoma cell lines. Of the 3 carcinoma lines, only HT29 expressed COX‐2 protein, yet each line was similarly sensitive to NS‐398. There was a positive correlation between overall sensitivity of the cell lines (determined by the attached cell yield) and sensitivity to NS‐398‐induced apoptosis, suggesting that apoptosis is the dominant anti‐proliferative effect of NS‐398. Two of the 3 adenoma cell lines (RG/C2, AA/C1) were less sensitive than the carcinoma cell lines. NS‐398 up‐regulated COX‐2 protein expression in the HT29 and adenoma cell lines. This was studied further in HT29 cultures, where treatment with NS‐398 inhibited COX‐2 activity, reducing prostaglandin E2 secretion. Here, neither the increase in COX‐2 protein expression nor the anti‐proliferative and apoptosis‐inducing effect of NS‐398 was prevented by addition of exogenous prostaglandin E2. Apoptosis appears to be the dominant anti‐proliferative effect of NS‐398 and, in COX‐2 expressing cells, may be mechanistically linked to the observed induction of COX‐2 protein expression upon treatment with NS‐398. Int. J. Cancer 86:553–560, 2000.

Akt phosphorylation on Thr308 but not on Ser473 correlates with Akt protein kinase activity in human non-small cell lung cancer

Background:The activity of the protein kinase Akt is frequently dysregulated in cancer and is an important factor in the growth and survival of tumour cells. Akt activation involves the phosphorylation of two residues: threonine 308 (Thr308) in the activation loop and serine 473 (Ser473) in the C-terminal hydrophobic motif. Phosphorylation of Ser473 has been extensively studied in tumour samples as a correlate for Akt activity, yet the phosphorylation of Thr308 or of downstream Akt substrates is rarely assessed.Methods:The phosphorylation status of Thr308 and Ser473 was compared with that of three separate Akt substrates – PRAS40, TSC2 and TBC1D4 – in fresh frozen samples of early-stage human non-small cell lung cancer (NSCLC).Results:Akt Thr308 phosphorylation correlated with the phosphorylation of each Akt substrate tested, whereas Akt Ser473 phosphorylation did not correlate with the phosphorylation of any of the substrates examined.Conclusion:The phosphorylation of Thr308 is a more reliable biomarker for the protein kinase activity of Akt in tumour samples than Ser473. Any evaluation of the link between Akt phosphorylation or activity in tumour samples and the prediction or prognosis of disease should, therefore, focus on measuring the phosphorylation of Akt on Thr308 and/or at least one downstream Akt substrate, rather than Akt Ser473 phosphorylation alone.

The MEK/ERK pathway mediates COX-2-selective nsaid-induced apoptosis and induced COX-2 protein expression in colorectal carcinoma cells

Nonsteroidal antiinflammatory drugs (NSAIDs) can prevent colorectal tumorigenesis in humans and in rodents. In vitro and in vivo studies indicate that one of their principal antineoplastic avenues is the induction of apoptosis. We have shown previously that NS‐398, which selectively inhibits cyclooxygenase‐2 (COX‐2) over cyclooxygenase‐1, induces apoptosis of colorectal tumour cells and elevates COX‐2 protein expression. Here, we have determined that the extracellular signal‐regulated kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) pathway mediates these effects of NS‐398. Treatment of HT29 colorectal carcinoma cells with 75 μM NS‐398 caused activation of ERK‐1/‐2 but not of the p38 and c‐Jun N‐terminal kinase (JNK) mitogen‐activated protein kinases. This was apparent at 24 hr and maintained at 72 hr. U0126, a specific inhibitor of the ERK‐activating kinases MEK‐1/‐2, prevented the activation of ERK induced by NS‐398 and blocked the increase in COX‐2 protein expression seen when HT29 cells were treated with NS‐398 alone. The activation of ERK by NS‐398 preceded and accompanied a decrease in attached cell yield and an increase in apoptosis. U0126 dose‐dependently protected HT29 cells from these antiproliferative effects of NS‐398, indicating an antiproliferative role for sustained ERK‐1/‐2 activation in response to this NSAID. These results point to a key role for the MEK/ERK signalling pathway in mediating the effects of a COX‐2‐selective NSAID on colorectal carcinoma cells.

Human colorectal adenomas demonstrate a size-dependent increase in epithelial cyclooxygenase-2 expression

Non‐steroidal anti‐inflammatory drugs are chemopreventive for colorectal cancer. This effect is due in part to their ability to inhibit the inducible isoform of cyclooxygenase (COX‐2). However, the cellular expression and role of COX‐2 in the premalignant stages of colorectal tumourigenesis is unclear. COX‐2 expression was assessed in 35 human colorectal adenomas and 38 sporadic invasive colorectal adenocarcinomas. Adenomas were classified as small (<5 mm in diameter), medium (5–10 mm), and large (>10 mm). All tissues were paraffin‐embedded and formalin‐fixed. COX‐2 protein expression was determined using immunohistochemistry. COX‐2 was detected in the epithelial cells in 35 of 38 carcinomas (92%) and in 8 of 8 (100%) lymph node metastases. All of the epithelial cells expressed COX‐2 in 30 of 35 (86%) carcinomas and in 100% of the lymph node metastases. Twenty‐three of 35 (66%) adenomas expressed COX‐2 in the tumour epithelium. With an increase in the size of adenoma (<5 mm, 5–10 mm, >10 mm), there was an increase in (i) the proportion of adenomas with immunoreactive COX‐2 in the epithelium (p = 0.036)—this was 38% in small adenomas and 82% in large adenomas; (ii) the extent of epithelial COX‐2 staining within a given tumour (p = 0.003)—100% of epithelial cells were COX‐2‐positive in 15% of small adenomas and in 73% of large adenomas; and (iii) the intensity of epithelial COX‐2 staining (p = 0.009)—strong COX‐2 staining occurred in 8% of small adenomas and in 36% of large adenomas. COX‐2 immunoreactivity was not detected in adjacent normal epithelium but was apparent in fibroblasts and inflammatory mononuclear cells of adjacent normal, adenoma, and carcinoma tissue. These results suggest that epithelial COX‐2 activity is important for the growth and/or survival of adenomatous epithelial cells from an adenoma diameter of less than 5 mm and that there is a selective advantage for adenoma epithelial cells expressing higher levels of COX‐2. Copyright

Targeting non-small cell lung cancer cells by dual inhibition of the insulin receptor and the insulin-like growth factor-1 receptor

Phase III trials of the anti-insulin-like growth factor-1 receptor (IGF1R) antibody figitumumab in non-small cell lung cancer (NSCLC) patients have been discontinued owing to lack of survival benefit. We investigated whether inhibition of the highly homologous insulin receptor (IR) in addition to the IGF1R would be more effective than inhibition of the IGF1R alone at preventing the proliferation of NSCLC cells. Signalling through IGF1R and IR in the NSCLC cell lines A549 and Hcc193 was stimulated by a combination of IGF1, IGF2 and insulin. It was inhibited by antibodies that block ligand binding, αIR3 (IGF1R) and IR47-9 (IR), and by the ATP-competitive small molecule tyrosine kinase inhibitors AZ12253801 and NVPAWD742 which inhibit both IGF1R and IR tyrosine kinases. The effect of inhibitors was determined by an anchorage-independent proliferation assay and by analysis of Akt phosphorylation. In Hcc193 cells the reduction in cell proliferation and Akt phosphorylation due to anti-IGF1R antibody was enhanced by antibody-mediated inhibition of the IR whereas in A549 cells, with a relatively low IR:IGF1R expression ratio, it was not. In each cell line proliferation and Akt phosphorylation were more effectively inhibited by AZ12253801 and NVPAWD742 than by combined αIR3 and IR47-9. When the IGF1R alone is inhibited, unencumbered signalling through the IR can contribute to continued NSCLC cell proliferation. We conclude that small molecule inhibitors targeting both the IR and IGF1R more effectively reduce NSCLC cell proliferation in a manner independent of the IR:IGF1R expression ratio, providing a therapeutic rationale for the treatment of this disease.

Glycogen synthase kinase 3 protein kinase activity is frequently elevated in human non-small cell lung carcinoma and supports tumour cell proliferation

Background Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC. Methods The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture. Results Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes. Conclusion NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC.