Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Human Cancer Cells with Acquired Resistance to Antiepidermal Growth Factor Receptor Therapy

Abstract

Purpose:The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small molecule EGFR-selective tyrosine kinase inhibitor, are in advanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase.Experimental Design: The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical significance using the Student's t test. All Ps were two-sided.Results: Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80 -90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pretreated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was observed in mice re-treated with C225 or ZD1839. GEO tumors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO-ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting revealed no major change in the expression of the EGFR ligand transforming growth factor ␣ of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-activated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5-10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474.Conclusions: Long-t...

Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumours sensitive and resistant to anti-EGFR drugs

“…GEO-CR (cetuximab resistant), GEO-GR and PC3-GR (gefitinib resistant) cells were established as described previously (Ciardiello et al, 2004).…”

“…After 10 -14 days, cells were stained and colonies up to 0.05 mm were counted (Ciardiello et al, 2004).…”

“…Frequently, activation of signalling pathways downstream or alternative to EGFR may be responsible for the development of resistance to these inhibitors (Sansal and Sellers, 2004). We, and others, have previously shown that colon tumours that acquire resistance to anti-EGFR drugs cetuximab and gefitinib overexpress Akt and VEGF, which act as the escape pathways to overcome the EGFR blockade (Viloria-Petit et al, 2001;Ciardiello et al, 2004;Bianco et al, 2005). As mentioned above, the PI3K-AKT pathway connects EGFR and mTOR.…”

“…The link between HIF-1 and EGF and the reported ability of mTOR inhibitors rapamycin and everolimus to reduce VEGF secretion in vitro and in vivo, renders mTOR inhibitors potentially effective against cancer cells resistant to EGFR inhibitors (ViloriaPetit et al, 2001;Ciardiello et al, 2004;Bianco et al, 2005). In addition, targeting together mTOR and EGFR might cause a more profound effect on tumour growth control compared with a single agent (Hynes and Lane, 2005).…”

A Phase I Dose-Escalation Study of ZD6474 in Japanese Patients with Solid, Malignant Tumors

“…[17][18][19][20] As such, ZD6474 has the potential to inhibit two key pathways in tumor growth: VEGF-dependent tumor angiogenesis, and EGFR-and RET-dependent tumor cell proliferation and survival. Indeed, preclinical evaluation of ZD6474 has demonstrated potent inhibition of VEGF-dependent signaling and angiogenesis in vivo, as well as dose-dependent inhibition of tumor growth, including profound regression in established PC-3 prostate tumors.…”