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RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser236 in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP236 show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser236 by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.
Following publication of the data presented by von Minckwitz and colleagues it has been brought to our attention that some patients should be scored differently. Stable disease was seen in three of the eighteen patients instead of two of the eighteen patients: one patient with transitional cell carcinoma treated at 4 µg/kg scFv(FRP5)-ETA per day, and two breast cancer patients treated at 4 and 12.5 µg/kg scFv(FRP5)-ETA per day. Disease progression occured in 9 of the eighteen patients evaluated (see corrected Table 2 overleaf). This does not affect the conclusions of our study. In addition we would like to correct the following errors: patient IDs for patients U01 and U02 in the original Table 2 were interchanged. In addition, patient N03 had a grade 3 elevation of gamma-glutamyl transferase, and not grade 2 (see corrected Table 2 overleaf).
Introduction: ScFv(FRP5)-ETA is a recombinant antibody toxin with binding specificity for ErbB2 (HER2). It consists of an N-terminal single-chain antibody fragment (scFv), genetically linked to truncated Pseudomonas exotoxin A (ETA). Potent antitumoral activity of scFv(FRP5)-ETA against ErbB2-overexpressing tumor cells was previously demonstrated in vitro and in animal models. Here we report the first systemic application of scFv(FRP5)-ETA in human cancer patients.
Methods: We have performed a phase I dose-finding study, with the objective to assess the maximum tolerated dose and the dose-limiting toxicity of intravenously injected scFv(FRP5)-ETA. Eighteen patients suffering from ErbB2-expressing metastatic breast cancers, prostate cancers, head and neck cancer, non small cell lung cancer, or transitional cell carcinoma were treated. Dose levels of 2, 4, 10, 12.5, and 20 μg/kg scFv(FRP5)-ETA were administered as five daily infusions each for two consecutive weeks.
Results: No hematologic, renal, and/or cardiovascular toxicities were noted in any of the patients treated. However, transient elevation of liver enzymes was observed, and considered dose limiting, in one of six patients at the maximum tolerated dose of 12.5 μg/kg, and in two of three patients at 20 μg/kg. Fifteen minutes after injection, peak concentrations of more than 100 ng/ml scFv(FRP5)-ETA were obtained at a dose of 10 μg/kg, indicating that predicted therapeutic levels of the recombinant protein can be applied without inducing toxic side effects. Induction of antibodies against scFv(FRP5)-ETA was observed 8 days after initiation of therapy in 13 patients investigated, but only in five of these patients could neutralizing activity be detected. Two patients showed stable disease and in three patients clinical signs of activity in terms of signs and symptoms were observed (all treated at doses ≥ 10 μg/kg). Disease progression occurred in 11 of the patients.
Conclusion: Our results demonstrate that systemic therapy with scFv(FRP5)-ETA can be safely administered up to a maximum tolerated dose of 12.5 μg/kg in patients with ErbB2-expressing tumors, justifying further clinical development.