Phosphorylated epidermal growth factor receptor on tumor-associated endothelial cells in human renal cell carcinoma is a primary target for therapy by tyrosine kinase inhibitors

Abstract

We determined whether therapy for human renal cell carcinoma (HRCC) that grows in the kidney of nude mice by the specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, PKI166, is directed against phosphorylated EGFR on tumor cells or on tumor-associated endothelial cells. EGFR+/transforming growth factor alpha (TGF-alpha)- SN12-PM6 HRCC cells were transfected with full-length sense TGF-alpha cDNA or vector control. SN12-PM6 cells expressing low or high levels of TGF-alpha were implanted into the kidney of nude mice. Only tumors produced by TGF-alpha+ HRCC cells contained tumor-associated endothelial cells expressing activated EGFR. Oral administration of PKI166 produced significant therapy only in TGF-alpha+ tumors, which correlated with apoptosis of tumor-associated endothelial cells. These data suggest that the production of TGF-alpha by HRCC cells leads to the activation of EGFR on tumor-associated endothelial cells that serve as an essential target for therapy with tyrosine kinase inhibitors.

Figure 1

In vitro expression of TGF-α, EGFR, and pEGFR. (A) Northern blot analysis of mRNA for TGF-α in kidney cancer SN12-PM6 and SN12-PM6 (TGF-α) cell lines. GAPDH served as the control for loading. SN12-PM6 (TGF-α) expressed high levels of TGF-α-specific mRNA, whereas SN12-PM6 (parental and empty vectors) did not express detectable levels of TGF-α-specific mRNA. This is one of three representative experiments. Production of TGF-α by SN12-PM6 (TGF-α) cells, as determined by ELISA, significantly increased to 1 ng/ml. (B). Adherent SN12-PM6 and SN12-PM6 (TGF-α) were cultured for 24 hours, and the levels of EGFR protein (170 kDa) and pEGFR (170 kDa) were determined by Western blot analysis. The expression of EGFR did not differ between groups. The activation of pEGFR was dependent on the production of TGF-α. (C) EGFR surface expression, as detected by FACS, illustrated a similar right shift in fluorescent intensity for both SN12-PM6 (blue line) and SN12-PM6 (TGF-α) (green line) cells compared with control cells stained with secondary antibody only (red and orange, respectively).

Figure 2

IHC determination of TGF-α, EGFR, activated EGFR, and CD31/pEGFR in kidney cancers growing in the kidney of nude mice. Note the positive staining for CD31/pEGFR only in TGF-α⁺ tumors. Endothelial cells in TGF-α^- tumors or adjacent tissues did not express detectable levels of activated EGFR.

Figure 3

IHC analysis of MVD, activated EGFR, and TUNEL (apoptosis) in kidney tumors from mice treated with vehicle-control or PKI166. Kidney tumors were harvested after 4 weeks of treatment, and sections were stained for CD31/PECAM-1 (brown) or were double-labeled for CD31 (green) and pEGFR (red), or CD31 (red) and TUNEL (green). Only endothelial cells in TGF-α⁺ tumors expressed activated EGFR. Treatment with PKI166 inhibited the phosphorylation of EGFR in tumor cells that did or did not express TGF-α. Inhibition of pEGFR on endothelial cells was only found in TGF-α⁺ tumors. Treatment with PKI166 induced endothelial cell apoptosis only in TGF-α⁺ tumors, which correlated with reduction in MVD.