Mechanisms of tumor cell clustering in breast cancer metastasis

转移是乳腺癌相关发病率和死亡率的主要原因。在转移过程中， 原发性肿瘤作为循环肿瘤细胞（CTC）脱落到血流中。CTC会转移到远处的器官， 形成继发性肿瘤。形成簇的CTC表现出转移潜力的急剧增加， 单个CTC。虽然先前的研究已经描述了E-钙粘蛋白作为CTC簇中粘附的关键介质 和转移，许多乳腺肿瘤，包括高转移性三阴性乳腺癌的肿瘤， （TNBC），表达很少至不表达E-钙粘蛋白。因此，必须存在一种替代的、不依赖于E-钙粘蛋白的机制， 介导CTC之间的聚集，从而促进转移和疾病进展。调查CTC 聚类机制，我们开发了一种体外肿瘤细胞聚类分析， 剪切力和模拟体内条件。使用这种检测，我们发现E-钙粘蛋白阴性的转移性乳腺癌， 癌细胞可以形成具有类似于细胞粘附连接的特征的细胞相互作用。猜测 来自肿瘤细胞的细胞外基质（ECM）成分可能有助于CTC聚集，我们分析了ECM 乳腺癌的治疗方法我们发现，透明质酸合成酶2（HAS 2），这是主要的 在乳腺癌细胞中负责透明质酸（HA）产生的酶，在乳腺癌细胞中显著上调。 TNBC。我们进一步观察到，HA介导TNBC肿瘤细胞之间的聚集，并赋予它们与TNBC肿瘤细胞之间的相互作用。 克服血流中存在的损伤的能力，包括剪切力。重要的是，我们检测到HA 在TNBC患者血液样本中，在相互作用的CTC的细胞-细胞连接处富集。机械地，我们 初步结果表明，转移性TNBC细胞在染色质环中上调HAS 2表达， PRDM 6是一种转录调节因子，也是公认的组蛋白赖氨酸甲基转移酶。 总的来说，这些初步发现使我们假设，在侵袭性TNBC中，高水平的HA 通过相邻细胞之间的HA依赖性粘附相互作用增强CTC聚类。我们进一步 假设PRDM 6通过HAS 2表达的表观遗传调节上调HA水平， 包括染色质成环相互作用。我们建议通过两个具体目标来研究我们的假设：1） 确定HA在TNBC肿瘤细胞聚集和转移中的作用，以及2）阐明HA在TNBC肿瘤细胞聚集和转移中的表观遗传调节 影响乳腺肿瘤细胞聚集的HAS 2。为了研究我们的假设，我们将利用新的体外 聚类分析和体内小鼠模型沿着与电子显微镜，以揭示结构 CTC集群的特征。此外，我们将利用整合的多组学数据分析来阐明 在CTC聚类期间控制HAS 2表达的共调节网络。重要的是，我们的结果将是 在来自转移性TNBC患者的血液样本中得到广泛验证。因此，圆满完成 我们提出的工作不仅将确定一种新的机制，介导强细胞间的相互作用， 为确定抑制CTC聚集的新治疗方案奠定了机制基础。

Metastasis is the primary cause of breast cancer-related morbidity and mortality. During metastasis, cells from primary tumors shed into the bloodstream as circulating tumor cells (CTCs). CTCs travel to distant organs to establish secondary tumors. CTCs that form clusters exhibit a drastic increase in metastatic potential compared to single CTCs. While previous studies have described E-cadherin as a key mediator of adhesion in CTC clusters and metastasis, many breast tumors, including tumors of the highly metastatic triple negative breast cancer (TNBC), express little to no E-cadherin. Thus, an alternative, E-cadherin-independent mechanism must exist to mediate clustering between CTCs, thereby promoting metastasis and disease progression. To investigate CTC clustering mechanisms, we developed an in vitro tumor cell clustering assay that incorporates physiological shear force and mimics in vivo conditions. Using this assay, we found that E-cadherin-negative metastatic breast cancer cells can form cellular interactions with characteristics similar to cell adherens junctions. Speculating that extracellular matrix (ECM) components from tumor cells may contribute to CTC clustering, we analyzed ECM components by breast cancer subtypes. We found that hyaluronic acid synthase 2 (HAS2), which is the primary enzyme responsible for hyaluronic acid (HA) production in breast cancer cells, is significantly upregulated in TNBC. We further observed that HA mediates clustering between TNBC tumor cells and confers them with the ability to overcome insults present in the bloodstream, including shear forces. Importantly, we detected HA enrichment at the cell-cell junction of interacting CTCs in TNBC patient blood specimens. Mechanistically, our preliminary results suggest that metastatic TNBC cells upregulate HAS2 expression in a chromatin looping mechanism mediated by PRDM6, a transcriptional regulator and putative histone lysine methyltransferase. Collectively, these preliminary findings lead us to hypothesize that in aggressive TNBC, high levels of HA augment CTC clustering via HA-dependent adhesive interactions between neighboring cells. We further hypothesize that the PRDM6 upregulates HA levels through epigenetic modulation of HAS2 expression, including chromatin looping interactions. We propose to study our hypotheses through two specific aims: 1) determine the role of HA in TNBC tumor cell clustering and metastasis and 2) elucidate the epigenetic regulation of HAS2 that impacts breast tumor cell clustering. To investigate our hypotheses, we will utilize novel in vitro clustering assays and in vivo mouse models along with electron microscopy to reveal the structural characteristics of CTC clusters. In addition, we will utilize integrative multi-omics data analysis to elucidate the co-regulatory network governing HAS2 expression during CTC clustering. Importantly, our results will be extensively validated in blood specimens from metastatic TNBC patients. Consequently, successful completion of our proposed work will not only identify a novel mechanism that mediates strong cell-cell interactions, but also pave the mechanistic groundwork for identifying novel therapeutic options to suppress CTC clustering.