Intermolecular biparatopic trapping of ErbB2 prevents compensatory activation of PI3K/AKT via RAS-p110 crosstalk.

Compensatory mechanisms, such as relief of AKT-ErbB3-negative feedback, are known to desensitize ErbB2-dependent tumours to targeted therapy. Here we describe an adaptation mechanism leading to reactivation of the PI3K/AKT pathway during trastuzumab treatment, which occurs independently of ErbB3 re-phosphorylation. This signalling bypass of phospho-ErbB3 operates in ErbB2-overexpressing cells via RAS-PI3K crosstalk and is attributable to active ErbB2 homodimers. As demonstrated by dual blockade of ErbB2/RAS and ErbB3 by means of pharmacological inhibition, RNA interference or by specific protein binders obstructing the RAS-p110α interaction, both routes must be blocked to prevent reactivation of the PI3K/AKT pathway. Applying these general principles, we developed biparatopic designed ankyrin repeat proteins (DARPins) trapping ErbB2 in a dimerization-incompetent state, which entail pan-ErbB inhibition and a permanent OFF state in the oncogenic signalling, thereby triggering extensive apoptosis in ErbB2-addicted tumours. Thus, these novel insights into mechanisms underlying network robustness provide a guide for overcoming adaptation response to ErbB2/ErbB3-targeted therapy.

Analysis of physiological and behavioural parameters in mice after toe clipping as newborns.

In this study, we have investigated the short- and long-term impact of toe clipping, a commonly used method for marking and simultaneously taking biopsies of pups, which is controversially discussed because of its potentially negative impact on animals. Furthermore, we have analysed animal welfare aspects such as health, behaviour, development, stress and detrimental effects in young animals and in adults after toe clipping at postnatal days 3 (P3) and 7 (P7). Our findings indicate that for both P3 and P7 pups amputations at the second phalange of one toe of each paw do not have any negative effects on growth and physical development and that the clipped pups do not suffer from rejection by their mother. Our data indicate that even though at both ages no abnormalities have been detected in histology, clipping at P7 is the preferable age for an adequate marking mostly because of the small size of the toes at P3. This was also confirmed by grip tests at the age of 12 weeks where P3 animals had lower grip strength than control animals, whereas P7 pups did not show any impairment. Hotplate tests indicated that toe clipping performed at P3 and P7 did not cause hyperalgesia at the amputation stump. Serum corticosterone analysis directly performed on P7 pups after clipping indicated that major stress was provoked mainly through the handling and not because of the clipping itself. Taken together, these data lead to the conclusion that toe clipping is from a morphological, physiological and welfare point of view an acceptable method for marking and genotyping newborn mice.