Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel-Lindau (VHL) protein and high, homogeneous expression of the G250 MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIX G250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250 MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250 MN promoter constructs revealed strong promoter activity in G250 MN -positive RCC cell lines, but no activity in G250 MN -negative cell lines. DNase-I footprint and bandshift analysis demonstrated that Sp1 and HIF-1a proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1a expression and G250 MN expression was observed. In contrast, no relationship between HIF-2a expression and G250 MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1a and Sp1, in combination with CBP/p300, are crucial elements for G250 MN expression in ccRCC, and CAIX G250 can be regarded as a unique HIF-1a target gene in ccRCC.
Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of metastatic clear cell renal cell carcinoma (RCC). Although TKIs have demonstrated good clinical efficacy, the lack of complete responses, the chronic nature of the treatment, and the side effects are clear disadvantages. An interesting new approach in the treatment of clear cell RCC is antibody-mediated therapy with the chimeric anti-carbonic anhydrase IX (CAIX) antibody girentuximab (cG250). As the results of several girentuximab trials become available, the question arises of whether TKI treatment can be combined with girentuximabbased therapy. In this study, we assessed the effect of the widely used TKI sorafenib on the tumor-targeting potential of 111 In-labeled girentuximab. Methods: 111 In-girentuximab imaging was performed on 15 patients suspected of having a renal malignancy, with surgery being part of their treatment plan. Of these, 10 patients were treated in a neoadjuvant setting with sorafenib (400 mg orally twice daily). Five patients received treatment during 1 wk, and 5 patients received treatment during 4 wk. In both sorafenib-treated groups, baseline and posttreatment tumor targeting of 111 In-girentuximab were compared. Surgery was performed 3 d after the last image acquisition. Five additional patients were included as a control group and had only a single 111 In-girentuximab injection and scintigraphy without any treatment. Distribution of 111 In-girentuximab was determined scintigraphically ex vivo in a 1-cm lamella of the resected tumorous kidney. Expression of CAIX and of the vascular marker CD31 was determined immunohistochemically on specimens of both tumor and normal kidney tissue. Results: Treatment with sorafenib resulted in a marked decrease of 111 In-girentuximab uptake in the tumor in clear cell RCC patients, especially in the group treated for 4 wk (mean change in both sorafenib-treated groups, 238.4%; range, 19.1% to 279.4%). Immunohistochemical analysis showed markedly reduced CD31 expression and vessel density in the sorafenib-treated groups but no differences in CAIX expression between the sorafenib-treated groups and the nontreated patients. Conclusion: Treatment with sorafenib resulted in a treatment duration-dependent significantly decreased uptake of 111 In-girentumab in clear cell RCC lesions. These results indicate that the efficacy of antibody-mediated treatment or diagnosis modalities is hampered by TKI treatment.
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