The new cohort of EMT genes are activated by benzo(a)pyrene and/or SATB1 during the malignant transformation of breast epithelial cells. The new cohort of EMT genes are activated by benzo(a)pyrene and/or SATB1 during the malignant transformation of breast epithelial cells

Tumor metastasis, the migration of tumor cells from a primary site to distant organs, is the leading cause of death among cancer patients. Despite significant advancements in cancer research, much remains to be learned about how tumor cells acquire invasive properties at the molecular level. Here, we investigated the transcriptional program that enables breast epithelial cells to develop highly invasive malignant phenotypes. We focused on genes that promote epithelial-to mesenchymal transition (EMT), a process critical for both embryogenesis and cancer cell invasion, and we compiled a cohort of 339 genes associated with EMT functions with many identified in recent literature. Using an immortalized, non-malignant breast epithelial cell line, MCF10A, we analyzed changes in the transcriptional activity of these genes through Global Run-On Sequencing (GRO-seq) and RNA-seq following stable SATB1 transduction. SATB1 is a nuclear protein with genome-organizing functions that promotes metastatic cancer, including breast cancer. SATB1-expressing MCF10A cells acquire both tumorigenic and metastatic properties. We identified a total of 98 EMT promoting genes that were upregulated in SATB1-expressing MCF10A cells compared to control cells. Additionally, we investigated the effects of the chemical carcinogen benzo[a]pyrene (B[a]P), known to induce malignancy in MCF10A cells. Our findings revealed that B[a]P significantly upregulated 54 EMT genes after 3 hours. By day 3, this number increased to 72 genes, with 36 genes being common between those elevated by SATB1. Notably, the significant effects of B[a]P on EMT gene expression observed in MCF10A cells were largely diminished in SATB1 expressing MCF10A cells, suggesting that the pre-existing SATB1-regulated transcriptional program is dominant. Our results indicate that, despite the distinct causes of breast cancer, both the chemical carcinogen B[a]P and SATB1 activate many common EMT genes during the malignant transformation of breast epithelial cells. This suggests a key network of gene expression that drives the malignant phenotypes of these cells. Overall design: We investigated the changes in the transcriptional program of breast epithelial cells using the MCF10A-1 cell line by stably expressing SATB1 compared to control cells, or by exposing MCF10A-1 cells to benzo[a]pyrene (B[a]P) compared to non-treated controls. We focused on studying a particular group of genes that function in promoting epithelial-to-mesenchymal transition (EMT), which is essential during embryogenesis as well as during cancer metastasis, as it promotes cell motility. We compiled a gene set consisting of 300 EMT genes from recent literature and from gene ontology (GO) lists, and we studied which of these genes are activated or upregulated when MCF10A-1 cells undergo malignant transformation and acquire metastatic phenotypes. We employed a method called GRO-seq, which detects nascent transcripts genome-wide to capture the early transcriptional response to environmental stimuli, as well as RNA-seq, which measures accumulated levels of mRNAs.

肿瘤转移（tumor metastasis）指肿瘤细胞从原发部位迁移至远端器官的过程，是癌症患者死亡的首要诱因。尽管癌症研究已取得诸多重大进展，但人们对肿瘤细胞在分子层面如何获得侵袭特性仍有诸多未解之处。本研究中，我们探究了可使乳腺上皮细胞形成高侵袭性恶性表型的转录程序。我们将研究重点聚焦于促进上皮间质转化（epithelial-to-mesenchymal transition, EMT）的基因——该过程对胚胎发生与癌细胞侵袭均至关重要，并整理了339个与EMT功能相关的基因集，其中多数基因已在近期文献中被报道。我们使用永生化非恶性乳腺上皮细胞系MCF10A，通过稳定转导SATB1后，利用全局转录运行测序（Global Run-On Sequencing, GRO-seq）与RNA测序（RNA-seq）分析了上述基因的转录活性变化。SATB1是一种具有基因组组织功能的核蛋白，可促进包括乳腺癌在内的转移性癌症发生。表达SATB1的MCF10A细胞可同时获得致瘤性与转移特性。我们共鉴定出98个在表达SATB1的MCF10A细胞中相较于对照细胞显著上调的EMT促进基因。此外，我们还探究了化学致癌物苯并[a]芘（benzo[a]pyrene, B[a]P）的作用——该物质已被证实可诱导MCF10A细胞发生恶性转化。研究结果显示，B[a]P处理3小时后可显著上调54个EMT基因的表达；至第3天，该上调基因数量增至72个，其中36个与SATB1所上调的基因重合。值得注意的是，在MCF10A细胞中观察到的B[a]P对EMT基因表达的显著调控效应，在表达SATB1的MCF10A细胞中大幅减弱，这表明预先存在的SATB1调控转录程序占据主导地位。我们的研究结果表明，尽管乳腺癌的致病诱因各不相同，但化学致癌物B[a]P与SATB1均可在乳腺上皮细胞恶性转化过程中激活大量共有的EMT基因，这提示存在一个驱动上述细胞恶性表型的关键基因表达网络。实验整体设计：我们以MCF10A-1细胞系为研究对象，通过两种方式分别设置对照：一是将稳定表达SATB1的细胞与对照细胞进行对比，二是将经苯并[a]芘（B[a]P）处理的细胞与未处理对照组进行对比，以此探究乳腺上皮细胞转录程序的变化。我们将研究重点聚焦于一类可促进细胞迁移的上皮间质转化（EMT）相关基因——该过程在胚胎发生与癌症转移过程中均发挥关键作用。我们从近期文献与基因本体（gene ontology, GO）注释列表中整理得到包含300个EMT基因的基因集，并探究当MCF10A-1细胞发生恶性转化并获得转移表型时，其中哪些基因会被激活或上调。我们同时采用了两种测序技术：全局转录运行测序（GRO-seq）可在全基因组范围内检测新生转录本，以捕捉细胞对环境刺激的早期转录应答；RNA测序（RNA-seq）则用于检测mRNA的累积表达水平。