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

Dickkopf-1最为人所知的作用是作为Wnt抑制剂，通过抑制成骨细胞功能促进溶骨性骨病变。我们的数据表明，DKK-1具有多效性，在建立继发于乳腺癌的骨转移的步骤中的上下文依赖性作用。虽然在DKK-1敲除后观察到直接的抗肿瘤作用，但转移过程的关键步骤如迁移或形成集落的潜力没有改变甚至增加。在拟议的项目中，我们的目标是详细解读这些效应，解释潜在的分子机制，并帮助定义DKK-1在骨转移的复杂过程中的治疗和预后潜力，包括从归巢到明显骨病变表现的步骤。 我们将应用一系列体外方法来具体解决DKK-1敲除或使用中和抗体抑制对活力、迁移、粘附、侵袭和集落形成的影响。我们的初步数据表明，这些过程中有许多明显的DKK-1调制的影响。此外，目前还不清楚在骨微环境中发现的DKK-1水平有多高，可能会影响我们的研究结果。因此，成骨细胞衍生的DKK-1的重要性将通过共培养和上清液实验来具体说明，这些实验将通过一系列小鼠模型来补充。具体来说，将使用CRISPR/Cas9缺失DKK-1的乳腺癌细胞系来评估DKK-1对归巢的影响，并与这些细胞建立明显骨转移的潜力进行比较。这些实验将通过应用诱导型DKK-1敲除来扩展，该敲除仅在骨中建立转移后应用。为了分析骨源性DKK-1的作用，将测试我们的成骨细胞特异性DKK-1敲除小鼠。将对500名乳腺癌患者进行DKK-1检测。对于这些患者，可获得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。将在DTC存在的背景下评估DKK-1对骨转移发生、存活率和临床结果的其他参数的预后价值。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，并评估其DKK-1水平。总之，使用上述方法，我们的目标是在乳腺癌骨转移的背景下破译DKK-1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.