Evaluation of epidermal growth factor receptor signaling effects in gastric cancer cell lines by detailed motility-focused phenotypic characterization linked with molecular analysis.

BACKGROUND: Gastric cancers frequently overexpress the epidermal growth factor receptor (EGFR), which has been implicated in pathological processes including tumor cell motility, invasion and metastasis. Targeting EGFR with the inhibitory antibody cetuximab may affect the motile and invasive behavior of tumor cells. Here, we evaluated the effects of EGFR signaling in gastric cancer cell lines to link the phenotypic behavior of the cells with their molecular characteristics. METHODS: Phenotypic effects were analyzed in four gastric cancer cell lines (AGS, Hs746T, LMSU and MKN1) by time-lapse microscopy and transwell invasion assay. Effects on EGFR signaling were detected using Western blot and proteome profiler analyses. A network was constructed linking EGFR signaling to the regulation of cellular motility. RESULTS: The analysis of the effects of treatment with epidermal growth factor (EGF) and cetuximab revealed that only one cell line (MKN1) was sensitive to cetuximab treatment in all phenotypic assays, whereas the other cell lines were either not responsive (Hs746T, LMSU) or sensitive only in certain tests (AGS). Cetuximab inhibited EGFR, MAPK and AKT activity and associated components of the EGFR signaling pathway to different degrees in cetuximab-sensitive MKN1 cells. In contrast, no such changes were observed in Hs746T cells. Thus, the different phenotypic behaviors of the cells were linked to their molecular response to treatment. Genetic alterations had different associations with response to treatment: while PIK3CA mutations and KRAS mutation or amplification were not obstructive, the MET mutation was associated with non-response. CONCLUSION: These results identify components of the EGFR signaling network as important regulators of the phenotypic and molecular response to cetuximab treatment.

Inhalation device requirements for patients' inhalation maneuvers.

BACKGROUND: Inhaled drugs are the mainstay of treatment for lung diseases such as asthma and chronic obstructive pulmonary disease. However, failure to use inhalation devices correctly can lead to a poorly controlled status. A vast number of inhalation devices exist and each device has specific requirements to achieve optimum inhalation of the drug. Currently, there is no overview of inhalation requirements considering all devices. This article presents a review of the literature on different inhalation device requirements and incorporates the data into a new inhalation flow algorithm. METHODS: Data from literature on commercially available inhalation devices were evaluated and parameters, such as inhalation flow rate, flow acceleration, inhalation volume, and inspiration time assessed for the required inhalation maneuver specific to the device. All agreed upon data points were used to develop an inhalation flow algorithm. RESULTS: The literature analysis revealed availability of robust data for the required inhalation flow characteristics for most devices and thus for the development of an algorithm. For those devices for which these parameters are not published, the minimum required flow criteria were defined based on published data regarding individual aspects of aerosol quality. CONCLUSIONS: This review provides an overview of inhalation devices available on the market regarding requirements for an acceptable inhalation maneuver and shows which goals should be achieved in terms of inhalation flows. The presented algorithm can be used to develop a new computer based measurement system which could help to test and train patients' individual inhalation maneuvers with their inhalation devices.

Association of amphiregulin with the cetuximab sensitivity of gastric cancer cell lines.

The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated in clinical studies. Reliable biomarkers for the identification of patients who are likely to benefit from this treatment are not available. In this study, we assessed the activity of cetuximab in five gastric cancer cell lines (AGS, AZ521, Hs746T, LMSU and MKN1). The viability of two of these cell lines, AZ521 and MKN1, was significantly reduced by cetuximab treatment. High expression and secretion levels of the EGFR-binding ligand, amphiregulin (AREG), were associated with cetuximab responsiveness. MET activation and mutations in Kirsten-Ras gene (KRAS) were associated with cetuximab resistance. By introducing a hierarchy between these markers, we established a model that facilitated the correct classification of all five gastric cancer cell lines as cetuximab responsive or non-responsive. The highest priority was allocated to activating KRAS mutations, followed by MET activation and finally by the levels of secreted AREG. In order to validate these results, we used three additional human gastric cancer cell lines (KATOIII, MKN28 and MKN45). In conclusion, we propose that our model allows the response of gastric cancer cell lines to cetuximab treatment to be predicted.

Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines.

PURPOSE: The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line. METHODS: EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods. RESULTS: We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance. CONCLUSION: These data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.

Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity.

Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs, we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently, the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated, stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast, ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results.

E-cadherin mutations and cell motility: a genotype-phenotype correlation.

E-cadherin has a determinant role in tumour progression, acting as an invasion and metastasis suppressor. Germline mutations of E-cadherin gene (CDH1) occur in 30% of families with Hereditary Diffuse Gastric Cancer (HDGC); of these 23% are missense mutations. The CDH1 missense mutations described to date span the entire gene and some lead to significant functional consequences. In this study, we explored the hypothesis that mutations affecting different E-cadherin protein domains have distinct effects on cell motility. To accomplish our objective we characterized the effect of eleven HDGC CDH1 germline missense mutations (T118R, L214P, G239R, A298T, T340A, P373L, R749W, E757K, E781D, P799R and V832M) on cell motility. Further, we studied their effect on the activation of signalling pathways known to be relevant for cell motility such as the EGFR, Src kinase and MAPKs. CDH1 mutations localized on the extracellular and juxtamembrane domains, both affecting the integrity of the extracellular domain, led to increased cell motility accompanied by increased EGFR activation. Moreover, we observed that cells expressing extracellular mutants exhibit increased activation of Src kinase and p38 MAPK. Our results allowed the identification of the E-cadherin domains pivotal for cell motility, further demonstrated a genotype-phenotype correlation, and defined a subset of HDGC cases which may benefit from EGFR inhibitors.

hGPR87 contributes to viability of human tumor cells.

Emerging in vitro and in vivo data underline the crucial role of G-protein-coupled receptors (GPCRs) in tumorigenesis. Here, we report the contribution of hGPR87, a predicted member of the P2Y subfamily of GPCRs, to proliferation and survival of human tumor cell lines. hGPR87 mRNA transcript was found to be preferentially overexpressed in squamous cell carcinomas (SCCs) of different locations and in their lymph node metastases. Up-regulation of both, transcript and protein, was detected in samples of SCC of the lung, cervix, skin and head and neck (pharynx, larynx and epiglottis). In addition to the expression of hGPR87 in tumors which originate from stratified epithelia, we identified other hGPR87-positive tumor types including subsets of large cell and adenocarcinomas of the lung and transitional cell carcinomas of the urinary bladder. Loss of function studies using siRNA in human cancer cell lines lead to antiproliferative effects and induction of apoptosis. Like other known P2Y receptors, hGPR87 was found to be mainly located on the cell surface. The overexpression of hGPR87 preferentially in SCCs together with our functional data suggests a common molecular mechanism for SCC tumorigenesis and may provide a novel intervention site for mechanism-based antitumor therapies.