Dissecting the pleiotropic effects of Dickkopf-1 in homing and colonization of breast cancer cells to and within bone.

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Dickkopf-1 is best known for its role as a Wnt-Inhibitor that promotes osteolytic bone lesions by inhibiting osteoblast function. Our data indicate that DKK-1 has pleiotropic, context dependent effects during the steps of establishing bone metastases secondary to breast cancer. While direct anti-tumor effects were observed following DKK-1 knockout, key steps of the metastatic process like migration or the potential to form colonies were unaltered or even increased. In the proposed project, we aim to decipher these effects in detail, explain the underlying molecular mechanisms, and help to define the therapeutic and prognostic potential of DKK-1 in the complex process of bone metastases, covering the steps from homing to the manifestation of overt bone lesions. We will apply a range of in vitro methods to specifically address the effects of DKK-1 knockout or inhibition using a neutralizing antibody on vitality, migration, adhesion, invasion and colony formation. Our preliminary data show that many of these processes are distinctly influenced by DKK-1 modulation. Furthermore, it remains unclear how high DKK-1 levels, as found in the bone microenvironment, may influence our findings. Therefor the importance of osteoblast-derived DKK-1 will be specifically addressed using co-culture and supernatant experiments.These experiments will be complemented by a range of murine models. Specifically, influence of DKK-1 on homing will be assessed using breast cancer cell lines depleted of DKK-1 using CRISPR/Cas9 and compared to the potential of these cells to establish overt bone metastases. These experiments will be expanded by the application of an inducible DKK-1 knockout that will be applied only once metastases have been established in the bone. To dissect effects of bone-derived DKK-1, our osteoblast-specific DKK-1 knockout mice will be tested.In a last step, serum samples of approx. 500 breast cancer patients will be measured for DKK-1. For these patients full data with regard to the presence of disseminated tumor cells (DTCs) in the bone marrow is available as well as full clinical follow-up. The prognostic value of DKK-1 for occurrence of bone metastases, survival and other parameters of clinical outcome will be assessed in the context of DTC presence. In addition, single cell deep sequencing data of disseminated breast cancer cells will be provided by our collaborator and assessed for their DKK-1 levels.In summary, using the above described approaches we aim to decipher the complex and pleiotropic effects of DKK-1 in the context of breast cancer bone metastases.