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.

Influence of the HER receptor ligand system on sensitivity to cetuximab and trastuzumab in gastric cancer cell lines.

PURPOSE: Gastric cancer remains a major health concern, and improvement of the therapeutic options is crucial. Treatment with targeted therapeutics such as the EGFR-targeting antibody cetuximab or the HER2-targeting antibody trastuzumab is either ineffective or moderately effective in this disease, respectively. In this study, we analysed the involvement of the HER receptor ligands amphiregulin (AREG), epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF) and transforming growth factor alpha (TGFα) in the responsiveness of gastric cancer cell lines to cetuximab and trastuzumab. METHODS: A panel of 11 gastric cancer cell lines was characterized for cetuximab and trastuzumab sensitivity, ligand secretion and expression and activation of the HER receptors using WST-1 cell proliferation assays, ELISAs and Western blot analyses. We further investigated the effects of an exogenous ligand application on the cetuximab and trastuzumab sensitivity. RESULTS: We found no correlation between TGFα secretion and the sensitivity to cetuximab or trastuzumab. For AREG, we confirmed previous results indicating that this ligand is a positive predictor of cetuximab sensitivity. Exogenous HB-EGF was effective in rescuing sensitive cell lines from inhibition of cell proliferation by both, cetuximab and trastuzumab. CONCLUSIONS: Our data indicate that HB-EGF may be a useful marker for the prediction of trastuzumab sensitivity in gastric cancer.

Associating approximate paths and temporal sequences of noisy detections: Application to the recovery of spatio-temporal cancer cell trajectories.

In this paper we address the problem of recovering spatio-temporal trajectories of cancer cells in phase contrast video-microscopy where the user provides the paths on which the cells are moving. The paths are purely spatial, without temporal information. To recover the temporal information associated to a given path we propose an approach based on automatic cell detection and on a graph-based shortest path search. The nodes in the graph consist of the projections of the cell detections onto the geometrical cell path. The edges relate nodes which correspond to different frames of the sequence and potentially to the same cell and trajectory. In this directed graph we search for the shortest path and use it to define a temporal parametrization of the corresponding geometrical cell path. An evaluation based on 286 paths of 7 phase contrast microscopy videos shows that our algorithm allows to recover 92% of trajectory points with respect to the associated ground truth. We compare our method with a state-of-the-art algorithm for semi-automated cell tracking in phase contrast microscopy which requires interactively placed starting points for the cells to track. The comparison shows that supporting geometrical paths in combination with our algorithm allow us to obtain more reliable cell trajectories.

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.

OWL1: an Arabidopsis J-domain protein involved in perception of very low light fluences.

To sense ambient light conditions in order to optimize their growth and development, plants employ a battery of photoreceptors responsive to light quality and quantity. Essential for the sensing of red and far-red (FR) light is the phytochrome family of photoreceptors. Among them, phytochrome A is special because it mediates responses to different light conditions, including both very low fluences (very low fluence response [VLFR]) and high irradiances (high irradiance response [HIR]). In contrast with the FR-HIR signaling pathway, in which several intermediates of the signaling pathway have been identified, specific components of the VLFR pathway remain unknown. Here, we describe owl1 (for orientation under very low fluences of light), a mutant that is specific for the VLFR, suggesting that VLFR and HIR pathways are genetically distinct, although some common mechanisms can be observed. OWL1 codes for a ubiquitous J-domain protein essential for germination, cotyledon opening, hypocotyl elongation, and deviation of the direction of hypocotyl growth from the vertical under very low light conditions. Additionally, we observed a flowering phenotype suggesting a role for the VLFR during the whole life cycle of a plant. OWL1 interacts with the basic helix-loop-helix HFR1 (LONG HYPOCOTYL IN FAR-RED) transcription factor, previously characterized as a component of the FR-HIR pathway. Both proteins are involved in the agravitropic response under FR light. We propose a central function of OWL1 in the VLFR pathway, which is essential for plant survival under unfavorable light conditions.

A rice phytochrome A in Arabidopsis: The Role of the N-terminus under red and far-red light.

The phytochrome (phy)A and phyB photoreceptors mediate three photobiological response modes in plants; whereas phyA can mediate the very-low-fluence response (VLFR), the high-irradiance response (HIR) and, to some extent, the low fluence response (LFR), phyB and other type II phytochromes only mediate the LFR. To investigate to what level a rice phyA can complement for Arabidopsis phyA or phyB function and to evaluate the role of the serine residues in the first 20 amino acids of the N-terminus of phyA, we examined VLFR, LFR, and HIR responses in phyB and phyAphyB mutant plants transformed with rice PHYA cDNA or a mutant rice PHYA cDNA in which the first 10 serine residues were mutated to alanines (phyA SA). Utilizing mutants without endogenous phyB allowed the evaluation of red-light-derived responses sensed by the rice phyA. In summary, the WT rice phyA could complement VLFR and LFR responses such as inhibition of hypocotyl elongation under pulses of FR or continuous R light, induction of flowering and leaf expansion, whereas the phyA SA was more specific for HIR responses (e.g. inhibition of hypocotyl elongation and anthocyanin accumulation under continuous far-red light). As the N-terminal serines can no longer be phosphorylated in the phyA SA mutant, this suggests a role for phosphorylation discriminating between the different phyA-dependent responses. The efficacy of the rice phyA expressed in Arabidopsis was dependent upon the developmental age of the plants analyzed and on the physiological response, suggesting a stage-dependent downstream modulation of phytochrome signaling.