Purpose: We have previously reported that human renal cell carcinoma (HRCC) cells growing in the kidney or bone of nude mice constitutively express EGFR and activated EGFR. Systemic therapy with the EGFR tyrosine kinase inhibitor, PKI166 (in combination with cytotoxic chemotherapy), inhibited the activation of EGFR and produced therapy of orthotopically growing kidney cancer cells. Immunohistochemical (IHC) analysis revealed that tumor-associated endothelial cells (but not endothelial cells in tumor-free kidney or bone) in tumors producing TGF-α/EGF also expressed activated EGFR. The purpose of the present studies was to determine whether therapy of HRCC by PKI166 is directed against the phosphorylated EGFR on tumor cells or on tumor-associated endothelial cells. Results: The EGFR-positive/TGF-α-negative SN12-PM6 parental HRCC cells were transfected with full-length sense TGF-α cDNA or vector control. SN12-PM6 parental cells (TGF-α−), vector transfected (TGF-α−) and SN12-PM6 TGF-α transfected (TGF-α+) cells exhibited similar levels of EGFR protein. The expression level of phosphorylated EGFR correlated with production of TGF-α. The three SN12-PM6 cell lines were injected into the kidney of nude mice. Three weeks later the kidneys with tumors and the adjacent normal tissue were collected. IHC analysis demonstrated that only tumors producing TGF-α had endothelial cells expressing activated EGFR. In contrast, endothelial cells in TGF-α-negative tumors or in adjacent tissue did not express detectable levels of activated EGFR. For therapy studies, SN12-PM6 parental cells (TGF-α−) and SN12-PM6 TGF-α transfected (TGF-α+) cells were injected into the kidney of nude mice (n = 10). Seven days later when tumors were well established, treatment with saline (control) or PKI166 (50 mg/kg, oral, thrice per week) began and continued for 5 weeks. TGF-α-negative kidney tumors were relatively resistant to treatment with PKI166. In sharp contrast, PKI166 treatment against TGF-α-positive kidney tumors produced a significant decrease in tumor volume. PKI166 inhibited phosphorylation of EGFR in tumor cells in both TGF-α-negative and TGF-α-positive kidney tumors. However, inhibition of EGFR phosphorylation on endothelial cells was found only in the TGF-α-positive kidney tumors. Significant induction of apoptosis of tumor-associated endothelial cells was observed only in the TGF-α-positive kidney tumors. Conclusions: The local production of TGF-α by human kidney cancer cells leads to the activation of EGFR on tumor-associated endothelial cells that serve as an essential target for therapy with tyrosine kinase inhibitors.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]