Osteoclast-independent mechanisms of early-stage bone colonization of breast canc

描述(申请人提供)：几乎所有与乳腺癌相关的死亡都是由转移引起的。骨骼是最常受乳腺癌影响的器官。在临床有意义的阶段，骨转移是由癌细胞和破骨细胞(骨吸收细胞)之间的恶性循环驱动的。近年来，我们对这种恶性循环的了解大大增加，针对破骨细胞的治疗通常可以显著延缓疾病的进展。然而，骨转移仍然是无法治愈的。另一方面，在临床上检测到骨转移之前，往往需要几年到几十年的潜伏期，这表明残留的癌细胞可以在骨或骨髓中长期存在，而不会激活破骨细胞。我们着手于在恶性循环开始之前，发现早期骨定植中破骨细胞非依赖的机制。我们的初步数据表明，成骨细胞(造骨细胞)及其前体细胞构成了微观骨转移的微环境。癌细胞和“成骨细胞壁龛”之间的直接细胞接触对它们的增殖至关重要。进一步的研究表明，mTOR通路的重新激活是骨转移启动的一个标志。我们还获得了初步证据，表明mTOR的激活是通过在癌细胞和壁细胞之间形成黏附连接(AJ)来介导的。基于这些发现，我们假设成骨细胞小生境通过增强mTOR通路的活性，可能通过AJ复合体下游的信号，促进乳腺癌从单细胞向多细胞微转移的骨转移进展。为了验证这一假说，我们将追求两个特定的目标：1)确定介导癌细胞和成骨细胞利基之间的串扰的机制，这需要细胞与细胞的直接接触并导致mTOR信号的激活；2)确定mTOR的下游效应因子，从而驱动转移的启动。我们的工作是创新和可行的，因为它使用了一种新的技术，通过循环选择性地将癌细胞输送到后肢骨骼中。这种方法能够在单细胞分辨率下快速检查和可靠地量化骨微转移，同时避免了其他传统方法的警告。将这项技术应用于几种癌症模型，导致了模拟人类疾病的惰性或休眠的骨转移。我们将使用这种方法分别对人和小鼠癌细胞进行异种移植和同基因移植，并研究AJs、mTOR复合体及其相关信号分子在骨转移启动中的作用。此外，我们还发明了3D共培养系统，真实地概括了癌症与利基相互作用的许多特征，这将有助于剖析分子机制，并加快我们对候选介体的审查。这些目标的实现将使靶向治疗的设计能够抑制或根除潜伏的肿瘤细胞，并减少显性骨转移相关症状的发生率和死亡率。

DESCRIPTION (provided by applicant): Metastasis accounts for nearly all breast cancer-related deaths. Bone is the organ most frequently affected by breast cancer. In the clinically significant stage, bone metastasis is driven by a vicious cycle between cancer cells and osteoclasts (bone-resorbing cells). Our knowledge of this vicious cycle has vastly increased in recent years, and therapies targeting osteoclasts can often significantly delay the progression of disease. However, bone metastases still remain incurable. On the other hand, there is often a latency of years to decades before bone metastases become clinically detectable, suggesting that residual cancer cells can exist in bone or bone marrow for a protracted period of time without activating osteoclasts. We set out to discover osteoclast-independent mechanisms in early-stage bone colonization before the onset of the vicious cycle. Our preliminary data demonstrated that osteoblasts (bone-making cells) and their precursor cells constitute the microenvironment niche of microscopic bone metastases. The direct cell-cell contact between cancer cells and the "osteoblastic niche" is crucial for their proliferation. Further studies indicated that the re-activation of the mTOR pathway is a hallmark of bone metastasis initiation. We also obtained preliminary evidence suggesting that the activation of mTOR is mediated by the formation of adhesion junctions (AJs) between cancer cells and niche cells. Based on these findings, we hypothesize that the osteoblastic niche facilitates bone metastasis progression of breast cancer from single cells to multi-cell micrometastases by augmenting the activity of the mTOR pathway, possibly through signaling downstream of AJ complexes. To test this hypothesis, we will pursue two specific aims: 1) to determine the mechanism mediating the crosstalk between cancer cells and the osteo-blastic niche, which entails direct cell-cell contact and leads to the activation of mTOR signaling, and 2) to identify the downstream effectors of mTOR that drive metastasis initiation. Our work is innovative and feasible because it employs a novel technique that selectively delivers cancer cells into hind limb bones via the circulation. This approach enables swift inspection and robust quantification of bone micrometastases at a single-cell resolution, yet avoids caveats of other conventional approaches. Application of this technique to several cancer models resulted in indolent or dormant bone metastases that mimic human diseases. We will use this approach for xenograft and syngeneic transplantation of human and mouse cancer cells, respectively, and investigate the roles of AJs, the mTOR complexes, and their related signaling molecules in bone metastasis initiation. In addition, we also invented a 3D co-culture system that faithfully recapitulates many features of cancer-niche interaction, which will facilitate the dissection of molecular mechanisms and accelerate our examination of candidate mediators. The fulfillment of these aims will enable the design of targeted therapies to suppress or eradicate latent tumor cells, and reduce the incidence of overt bone metastasis-related symptoms and mortality.