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，来自 原发肿瘤以循环肿瘤细胞(CTCs)的形式进入血流。CTC前往遥远的器官以 建立继发性肿瘤。与形成簇的CTC相比，形成簇状CTC的转移潜能急剧增加 到单一的CTC。虽然先前的研究已将E-钙粘附素描述为CTC簇中黏附的关键介质 而转移，许多乳腺肿瘤，包括肿瘤的高转移性三阴性乳腺癌 (TNBC)，几乎不表达E-钙粘附素。因此，必须存在一种替代的、不依赖于E-钙粘素的机制来 调节CTC之间的聚集，从而促进转移和疾病进展。调查CTC 聚集机制，我们开发了一种体外肿瘤细胞聚集实验，它结合了生理学 剪切力和模拟活体条件。利用这项检测，我们发现E-钙粘附素阴性的转移性乳房 癌细胞可以形成细胞间的相互作用，其特征类似于细胞黏附连接。推测说 肿瘤细胞的细胞外基质(ECM)成分可能有助于CTC的聚集，我们对ECM进行了分析 按乳腺癌亚型分类的成分。我们发现透明质酸合成酶2(HAS2)是最主要的 负责乳腺癌细胞产生透明质酸(HA)的酶在 TNBC。我们进一步观察到，HA介导了TNBC肿瘤细胞之间的聚集，并赋予它们 克服血液中存在的侮辱的能力，包括剪切力。重要的是，我们检测到HA 在TNBC患者血液标本中相互作用的CTCs在细胞-细胞交界处的浓缩。从机械上讲，我们的 初步结果表明，转移的TNBC细胞通过染色质环路上调HAS2的表达 PRDM6是一种转录调节因子，可能是组蛋白赖氨酸甲基转移酶。 总而言之，这些初步发现引导我们假设，在侵袭性的TNBC中，高水平的HA 通过相邻细胞之间依赖HA的粘附性相互作用增强CTC聚集性。我们进一步 假设PRDM6通过表观遗传调节HAS2表达上调HA水平， 包括染色质循环相互作用。我们建议通过两个具体目标来研究我们的假设：1) 确定透明质酸在TNBC肿瘤细胞聚集和转移中的作用；2)阐明表观遗传调控 影响乳腺肿瘤细胞聚集的HAS2基因。为了验证我们的假设，我们将使用新的体外实验。 聚类分析和体内小鼠模型与电子显微镜一起揭示结构 四氯化碳集群的特点。此外，我们将利用综合多组学数据分析来阐明 共调控网络在CTC聚集性中控制HAS2的表达。重要的是，我们的结果将是 在转移性TNBC患者的血液样本中得到广泛验证。因此，成功完成 我们提出的工作不仅将确定一种新的机制，介导强烈的细胞-细胞相互作用，而且还 为确定抑制CTC聚集的新的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.