Research Description:
Overexpression of epidermal growth factor receptor (EGFR) is common in a variety of tumor types, including sarcomas, and overexpression frequently correlates with poor clinical outcome. Clinical trials suggest EGFR-targeted therapy might benefit cancer patients. Unfortunately, the prognostic value of EGFR expression is problematic because clinical data fail to support a relationship between EGFR expression and response to EGFR-targeted compounds. One strategy proposed to enhance the effectiveness of anti-EGFR therapy is to develop predictive tools capable of making a more rational selection of patients based on the likelihood of a favorable response to anti-EGFR therapy. Implementation of this initiative might enhance success rates, and will spare refractory patients the expense and pain of potentially toxic treatments, but this approach is not a realistic solution for a majority of cancer patients who will be told they do not qualify for anti-EGFR the
rapy according to the established guidelines. Instead, we propose to explore why EGFR overexpression is not predictive in sarcomas. We hypothesize that the EGFR ligand, epidermal growth factor (EGF), can signal through receptors other than EGFR, when the EGFR pathway has been compromised. This would explain the inconsistencies reported for anti-EGFR therapies applied in the clinic.
The results from recent studies completed in our laboratory that evaluated EGF/EGFR signaling in desmoid cell lines support the alternative EGF receptor hypothesis. We now propose an in vitro inquiry which will test the alternative EGF receptor hypothesis in a commercially available chondrosarcoma cell line (SW1353). Our specific aim is to demonstrate complete inhibition of the EGFR following application of the intracellular tyrosine kinase inhibitor AG1478. We must demonstrate complete inhibition of EGFR coupled with the continued transcription of EGF-induced genes in order to justify our alternative receptor hypothesis. We will accomplish this task by monitoring levels of EGFR transphosphorylation before and after inhibitor application (phosphorylation equates with activity), and by quantifying changes in transcription of the EGF-inducible genes IL-8, CXCL1, and LCN6. In order to demonstrate complete inhibition of the EGFR, We will use a cell-based EGFR (activated) ELIS
A sampler kit (RayBiotech, Inc., Norcross, GA) to monitor EGFR transphosphorylation in treated and control SW1353 cell cultures. The ELISA can be used to screen EGFR inhibition by quantifying the relative amounts of phosphorylated Tyr845, Tyr992, or Tyr1068, or the amount of total phosphorylated EGFR in cells. Cell treatments will consist of: (a) untreated control cells (48 h of serum-free medium only), (b) cells exposed for 48 h to AG1478 at predefined inhibitor concentrations, (c) cells stimulated with 200 ng/mL rhEGF for the final 24 h of the 48 h treatment period, and (d) cells inhibited with AG1478 for the first 24 h, followed by a 24 h stimulation with 200 ng/mL rhEGF coupled with receptor inhibitor. To demonstrate retention of transcription following EGFR inhibition, SW1353 cells seeded into 25-cm2 tissue culture flasks will be treated with the same four protocols listed under objective #1. Following stimulation/inhibition, total RNA will be extracted and purified using RNeasy kits supplied by Qiagen (Germantown, MD) and RNA quality evaluated using an Agilent 2100 Bioanalyzer (Santa Clara, CA). Cell mRNA content will be quantified by real time RT-PCR using TaqMan® Gene Expression Assays and the ABI PRISM® 7900 HT Sequence Detection System with related software (Applied Biosystems, Foster City, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as the reference standard. Treated and control samples will be screened for the EGF-inducible genes IL-8, CXCL1, and LCN6.
FUTURE DIRECTIONS: At this stage of our research, it is almost impossible to predict whether the findings regarding EGF signaling will eventually contribute to man’s understanding of the molecular genetics of cancer cells, or cells in general, or whether the results will be viewed as an interesting novelty, but with little direct application. Although traditionally EGF has not been associated with cells of mesenchymal origin, we have documented in previous studies an apparent direct relationship between EGF and the expression of selected tumor-associated genes, at least at the transcriptional level. We also have suggested that the activities of other growth factors, such as TGF1 and CTGF, may be regulated (or at least initiated) by EGF in mesenchymal cells. If our hypotheses are correct, or even partially correct, this would be viewed as a significant contribution to science. If, on the other hand, our hypotheses are incorrect, there must be valid explanations for
our findings and typically in science, falsifying one hypothesis typically generates new hypotheses to test.
Does this research involve human subjects or animals? No
If yes, what is the protocol number?
10/2009
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