Abstract
4409
Treatment of tumors with monoclonal antibodies or small molecule kinase inhibitors targeting the EGF receptor family reveal clinical response rates of 10-20% and little progress has been made towards better selection of patients for these agents. Our quantitave assays of HER 1-4 message levels in 100 colorectal carcinomas revealed that mean HER2 and HER3 levels doubled when compared to matched normal mucosa, prompting us to study the effects of inhibition of these receptors in 11 human colorectal cell lines. We used GW572016, a 6-thiazolyquinazoline reversible kinase inhibitor of both EGFR and HER2 as well as OSI774, an anilinoquinazoline derivative which more selectively inhibits EGFR at low doses. Methods: 1- Real-time fluorescent quantitative PCR assays for HER 1-4 mRNA 2- 72 hour growth inhibition using MTS tetrazolium colorimetric assay (Promega) 3- Soft agar growth in 1 and 5uM inhibitor concentration 4- Flow cytometric cell cycle analysis using Propidium Iodide and APO-BRDU (Phoenix) staining 5- Phospho-specific blots for AKT, MAPK, JNK 6- Immunoprecipitation and p-tyr immunoblots for EGFR, HER2, HER3 Results: Micro molar concentrations of GW572016 or OSI774 growth inhibited 7 of 11 lines studied. Further experiments focused on: LS174T sensitive to both inhibitors; HT29 which is 7x more responsive to GW572016; and conversely DLD-1 which is 14x more sensitive to OSI774. The HT-29 and LS174T lines express the highest sums of HER2 + HER3 mRNA copies. GW572016 inhibits soft agar growth in both lines, induces apoptosis, and blocks EGF induced phosphorylation of AKT, MAPK and JNK. OSI774 has an identical pattern of AKT, MAPK and JNK inhibition, but requires a higher IC50 for growth inhibition. Furthermore, unlike GW572016, OSI774 could not inhibit soft agar growth of HT-29, nor could it induce apoptosis in either line. GW572016 and OSI774 inhibition of EGF and heregulin dependent activation of Akt and MAPK often but not always correlates with growth inhibition of these lines. Summary: The HER2/HER3 heterodimer is increasingly recognized as a powerful oncogenic unit and may play a role in colorectal cancer, as evidenced by our mRNA analysis of 100 human tumors and the inhibition of tumor cell soft agar growth by EGF receptor family inhibitors. Further pathway dissection may help us understand combinatorial signaling and eventually selection of individual patients for therapy.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]
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