Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia

: We investigated the constitutive activation of the MEK/ERK pathway in acute myelogenous leukemia (AML) via a flow cytometric technique to quantitate expression of phosphorylated ERK (p-ERK). A total of 42 AML samples (16 newly diagnosed, 26 relapsed/refractory) were analyzed. Normal bone marrow CD34+ cells (n = 10) had little or no expression of p-ERK, while G-CSF-mobilized CD34+ cells exhibited enhanced p-ERK levels. Markedly elevated p-ERK levels were found in 83.3% of the AML samples, with no differences observed between the newly diagnosed and relapsed/refractory samples. Treatment with a MEK inhibitor resulted in significantly decreased p-ERK levels in both the newly diagnosed and relapsed/refractory samples, which was associated with growth arrest, but not apoptosis induction. In summary, we defined conditions for the analysis of MAPK signaling in primary AML samples. Normal CD34+ cells expressed very low levels of p-ERK, and increased p-ERK levels were found in normal G-CSF-stimulated circulating CD34+ cells. Constitutively high p-ERK levels observed in the majority of AML samples suggest deregulation of this pathway that appears to be independent of disease status. The ability of ERK inhibition to promote growth arrest rather than apoptosis suggests that clinical trials of MEK/ERK inhibitors may be more effective when combined with chemotherapy

Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer

BACKGROUND: The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. METHODS: AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. RESULTS: Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a β-lactamase reporter gene, a 10–12 fold shift or 2.5–3 fold shift toward greater potency in the IC(50 )was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. CONCLUSION: EGFR pathway components were qualified as targets for inhibition of AP-1 activation using RNAi and small molecule inhibitors. The combination of these two targeted agents was shown to increase the efficacy of EGFR and MEK-1 kinase inhibitors, leading to possible implications for overcoming or preventing drug resistance, lowering effective drug doses, and providing new strategies for interrogating cellular signalling pathways

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses

BACKGROUND: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis. METHODS: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines. RESULTS: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th(1)-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th(1)-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement. CONCLUSION: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility

Protein and lipid kinase inhibitors as targeted anticancer agents of the Ras/Raf/MEK and PI3K/PKB pathways

The identification and characterization of the components of individual signal transduction cascades, and advances in our understanding on how these biological signals are integrated in cancer initiation and progression, have provided new strategies for therapeutic intervention in solid tumors and hematological malignancies. To this end, pharmaceutical efforts have been directed to target different components of the Ras/Raf/MEK and PI3K/PKB pathways. This review article covers recent salient achievements in the identification and development of Raf, MEK, and PI3K inhibitors

Expression of phospho-ERK1/2 and PI3-K in benign and malignant gallbladder lesions and its clinical and pathological correlations

Abstract Background An increasing number of studies have shown that ERK and PI3-K/AKT signaling pathways are involved in various human cancers including hepatocellular carcinoma and cholangiocarcinoma. However, few studies have examined gallbladder cancer specimens, and little is known about the clinical and pathological significance of ERK1/2 and PI3-K/AKT signaling changes in gallbladder adenocarcinoma. In this study, we examined phospho-ERK1/2 (p-ERK1/2) and PI3K expression and analyzed its clinicopathological impact in gallbladder adenocarcinoma. Methods Immunohistochemistry was used to detect and compare the frequency of p-ERK1/2 and PI3-K expression in gallbladder adenocarcinoma, peri-tumor tissues, adenomatous polyps, and chronic cholecystitis specimens. Results The positive staining for p-EKR1/2 and PI3-K were 63/108 (58.3%) and 55/108 (50.9%) in gallbladder adenocarcinoma; 14/46 (30.4%) and 5/46 (10.1%) in peri-tumor tissues; 3/15 (20%) and 3/15 (20%) in adenomatous polyps; and 4/35 (11.4%) and 3/35 (8.6%) in chronic cholecystitis. The positive rate of p-ERK1/2 or PI3-K in gallbladder adenocarcinoma was significantly higher than that in peri-tumor tissue (both, P P P P P P P P = 0.062) was associated with decreased overall survival. Multivariate Cox regression analysis showed that increased p-ERK1/2 expression was an independent prognostic predictor in gallbladder carcinoma (P = 0.028). Conclusion Increased expression of p-ERK1/2 and PI3K might contribute to gallbladder carcinogenesis. p-ERK1/2 over-expression is correlated with decreased survival and therefore may serve as an important biological marker in development of gallbladder adenocarcinoma.</p

Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040 strongly reduces growth and improves lung structure

BACKGROUND: Signaling networks promoting cell growth and proliferation are frequently deregulated in cancer. Tumors often are highly dependent on such signaling pathways and may become hypersensitive to downregulation of key components within these signaling cascades. The classical mitogenic cascade transmits stimuli from growth factor receptors via Ras, Raf, MEK and ERK to the cell nucleus and provides attractive molecular targets for cancer treatment. For example, Ras and Raf kinase inhibitors are already in a number of ongoing phase II and phase III clinical trials. In this study the effect of the Raf kinase inhibitor BAY 43-9006 and of the MEK inhibitor CI-1040 (PD184352) on a Raf dependent lung tumor mouse model was analyzed in detail. METHODS: We have generated a lung cancer mouse model by targeting constitutively active C-Raf kinase to the lung. These mice develop adenomas within 4 months of life. At this time-point they received daily intraperitoneal injections of either 100 mg/kg BAY 43-9006 or CI-1040 for additional 21 days. Thereafter, lungs were isolated and the following parameters were analyzed using histology and immunohistochemistry: overall lung structure, frequency of adenoma foci, proliferation rate, ERK activity, caspase-3 activation, and lung differentiation. RESULTS: Both inhibitors were equally effective in vitro using a sensitive Raf/MEK/ERK ELISA. In vivo, the systemic administration of the MEK inhibitor CI-1040 reduced adenoma formation to a third and significantly restored lung structure. The proliferation rate of lung cells of mice treated with CL-1040 was decreased without any obvious effects on differentiation of pneumocytes. In contrast, the Raf inhibitor BAY 43-9006 did not influence adenoma formation in vivo. CONCLUSION: The MEK inhibitor CI-1040 may be used for the treatment of Ras and/or Raf-dependent human malignancies

Modulating sensitivity to drug-induced apoptosis: the future for chemotherapy?

Drug resistance is a fundamental problem in the treatment of most common human cancers. Our understanding of the cellular mechanisms underlying death and survival has allowed the development of rational approaches to overcoming drug resistance. The mitogen activated protein kinase family of protein serine/threonine kinases has been implicated in this complex web of signalling, with some members acting to enhance death and other members to prevent it. A recent publication by MacKeigan et al is the first to demonstrate an enhancement of drug-induced cell death by simultaneous blockade of MEK-mediated survival signalling, and offers the potential for targeted adjuvant therapy as a means of overcoming drug resistance

Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors

<p>Abstract</p> <p>Background</p> <p>The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established.</p> <p>Methods</p> <p>Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells.</p> <p>Results</p> <p>We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect.</p> <p>Conclusion</p> <p>MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.</p

Proteomic identification of secreted proteins as surrogate markers for signal transduction inhibitor activity

Epidermal growth factor receptor is a potential target for cancer treatment and new small-molecule tyrosine kinase inhibitor drugs have been designed to inhibit its activity. In this work we identify potential surrogate markers of drug activity using a proteomic analysis. Two-dimensional electrophoresis was optimised to compare expression patterns of proteins secreted from the cancer cell lines A431 and A549 treated with Gefitinib (Iressa) vs untreated or vehicle-only-treated samples. Upregulated or downregulated proteins were detected using Phoretix 2D image analysis software. Several proteins were then identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. In one case, upregulation of Protein Disulphide Isomerase in response to Gefitinib was confirmed by Western blot analysis, and the response was shown to be concentration dependent. The identification of surrogate markers may be of use for the evaluation of new drugs, in preclinical models, in clinical trials and in the therapy of individual patients to give optimal biological drug doses