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

Dickkopf-1最广为人知的作用是作为一种Wnt抑制物，通过抑制成骨细胞功能来促进溶骨性骨损伤。我们的数据表明，DKK-1在建立乳腺癌继发的骨转移的步骤中具有多效性、上下文依赖性的作用。虽然DKK-1基因敲除后观察到了直接的抗肿瘤作用，但转移过程中的关键步骤，如迁移或形成克隆的可能性没有改变，甚至增加。在拟议的项目中，我们的目标是详细破译这些效应，解释潜在的分子机制，并帮助确定DKK-1在骨转移的复杂过程中的治疗和预后潜力，涵盖从归巢到显性骨损伤表现的各个步骤。我们的初步数据显示，其中许多过程明显受到DKK-1调制的影响。此外，在骨骼微环境中发现的高水平的DKK-1可能会影响我们的研究结果，目前还不清楚。这些实验将通过应用可诱导的DKK-1基因敲除来扩大，该基因敲除仅在骨转移已确定的情况下应用。500名乳腺癌患者将接受DKK-1检测。对于这些患者，有关于骨髓中存在播散性肿瘤细胞(DTC)的完整数据，以及完整的临床随访。DKK-1对骨转移的发生、存活率和其他临床结果参数的预后价值将在DTC存在的情况下进行评估。此外，我们的合作者将提供播散性乳腺癌细胞的单细胞深度测序数据，并对其DKK-1水平进行评估。总之，使用上述方法，我们的目标是破译DKK-1在乳腺癌骨转移中的复杂和多效性作用。

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.