Magic™ Anti-BCAR1 (Phospho S641) polyclonal antibody

Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prognosis. In accordance, TNK2 overexpression was shown to promote invasion of cancer cells - but the mechanism by which TNK2 mediates these effects is unresolved. TNK2 was suggested to regulate Cdc42-driven migration by activation of breast cancer antioestrogen resistance 1 (BCAR1); however, distinct from this effect is evidence for a role of TNK2 in the regulation of epidermal growth factor receptor (EGFR) endocytosis and degradation. In the present study we sought to investigate whether negative targeting of TNK2 by siRNA could be used to inhibit cancer cell invasion, to establish the contribution of its effect on the EGFR and to consequently attempt to resolve the issue of TNK2's mechanism of action. Methods We used siRNA to knockdown expression of TNK2 and its proposed effector BCAR1 in order to analyse the effect of this knockdown on cancer cell behaviour in vitro. We examined morphological changes using phase-contrast microscopy and immunohistochemistry. Functional parameters examined included apoptosis, proliferation, migration and invasion. We also performed flow cytometry analysis to examine EGFR cell surface expression and carried out western blot to examine the total EGFR levels. Results We observed that targeting of TNK2 by siRNA in breast cancer cells resulted in distinct morphological changes characterised by a stellate appearance and an absence of protrusions at membrane edges. These changes were not recapitulated upon siRNA targeting of BCAR1. We thus hypothesised that a component of the effects induced by TNK2 may be independent of BCAR1. Consistent with the idea of an alternative mechanism for TNK2, we observed that TNK2 associates with activated EGFR in breast cancer cells in a TNK2-kinase-independent manner. Furthermore, we demonstrated that TNK2 functions to maintain EGFRs on the cell surface. We could demonstrate that the main functional effect of activating these surface EGFRs in breast cancer cells is stimulation of migration. In accordance, TNK2 silencing by siRNA led to a significant reduction in cell surface EGFR and to a concomitant decrease in the migratory and invasive capacity of breast cancer cells. Conclusion Our data suggest that TNK2 can enhance migration and invasion of breast cancer cells via preservation of EGFR expression, notwithstanding its previously reported signalling via BCAR1, explaining its oncogenic behaviour in vitro and correlation with metastatic human breast cancer in vivo.

Ovarian follicles develop in groups yet individual follicles follow different growth trajectories. This growth and development are regulated by endocrine and locally produced growth factors that use a myriad of receptors and signal transduction pathways to exert their effects on theca and granulosa cells. We hypothesize that differential growth may be due to differences in hormonal responsiveness that is partially mediated by differences in expression of genes involved in signal transduction. We used the bovine dominant follicle model, microarrays, quantitative real-time PCR and RNA interference to examine this. We identified 83 genes coding for signal transduction molecules and validated a subset of them associated with different stages of the follicle wave. We suggest important roles for CAM kinase-1 and EphA4 in theca cells and BCAR1 in granulosa cells for the development of dominant follicles and for betaglycan and FIBP in granulosa cells of regressing subordinate follicles. Inhibition of genes for betaglycan and FIBP in granulosa cells in vitro suggests that they inhibit estradiol production in regressing subordinate follicles.