Characteristics and transcriptional regulators of spontaneous epithelial-mesenchymal transition in genetically unperturbed patient-derived non-spindled breast carcinoma.

Although tumor cells undergoing epithelial-mesenchymal transition (EMT) typically exhibit spindle morphology in experimental models, such histomorphological evidence of EMT has predominantly been observed in rare primary spindle carcinomas. The characteristics and transcriptional regulators of spontaneous EMT in genetically unperturbed non-spindled carcinomas remain underexplored. We used primary culture combined with RNA sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), and in situ RNA-seq to explore the characteristics and transcription factors (TFs) associated with potential spontaneous EMT in non-spindled breast carcinoma. Our primary culture revealed carcinoma cells expressing diverse epithelial-mesenchymal traits, consistent with epithelial-mesenchymal plasticity. Importantly, carcinoma cells undergoing spontaneous EMT did not necessarily exhibit spindle morphology, even when undergoing complete EMT. EMT was a favored process, whereas mesenchymal-epithelial transition appeared to be crucial for secondary tumor growth. Through scRNA-seq, we identified TFs that were sequentially and significantly upregulated as carcinoma cells progressed through the EMT process, which correlated with increasing VIM expression. Once upregulated, the TFs remained active throughout the EMT process. ZEB1 was a key initiator and sustainer of EMT, as indicated by its earliest significant upregulation in the EMT process, its exact correlation with VIM expression, and the reversal of EMT and downregulation of EMT-upregulated TFs upon ZEB1 knockdown. The correlation between ZEB1 and vimentin expression in triple-negative breast cancer and metaplastic breast carcinoma tumor cohorts further highlighted its role. The immediate upregulation of ZEB2 following that of ZEB1, along with the observation that the knockdown of ZEB1 or ZEB2 downregulates both ZEB1 and ZEB2 concomitant with the reversal of EMT, suggests their functional cooperation in EMT. This finding, together with that of a lack of correlation of SNAI1, SNAI2, and TWIST1 expression with the mesenchymal phenotype, indicated EMT-TFs have a context-dependent role in EMT. Upregulation of EMT-related gene signatures during EMT correlated with poor patient outcomes, highlighting the biological importance of the model. Elevated EMT gene signatures and increased ZEB1 and ZEB2 expression in vimentin-positive compared to vimentin-negative carcinoma cells within the corresponding primary tumor tissue confirmed ZEB1 and ZEB2 as intrinsic, instead of microenvironmentally-induced, EMT regulators, and vimentin as an in vivo indicator of EMT. Our findings provide insights into the characteristics and transcriptional regulators of spontaneous EMT in primary non-spindled carcinoma.

Lien HC ，Yu HC ，Yu WH ，Lin SF ，Chen TW ，Chen IC ，Hsiao LP ，Yeh LC ，Li YC ，Lo C ，Lu YS ... -  《-》

Functional Hierarchy and Cooperation of EMT Master Transcription Factors in Breast Cancer Metastasis.

Several master transcription factors (TF) can activate the epithelial-to-mesenchymal transition (EMT). However, their individual and combinatorial contributions to EMT in breast cancer are not defined. We show that overexpression of EMT-TFs individually in epithelial cells upregulated endogenous SNAI2, ZEB1/2, TCF4, and TWIST1/2 as a result of positive feedback mediated in part by suppression of their negative regulator miRNAs miR200s/203/205. We identified TCF4 as a potential new target of miR200s. Expression of ZEB1/2 strongly correlated with the mesenchymal phenotype in breast cancer cells, with the CD24-/CD44+ stemness profile, and with lower expression of core epithelial genes in human breast tumors. Knockdown of EMT-TFs identified the key role of ZEB1 and its functional cooperation with other EMT-TFs in the maintenance of the mesenchymal state. Inducible ZEB1+2 knockdown in xenograft models inhibited pulmonary metastasis, emphasizing their critical role in dissemination from primary site and in extravasation. However, ZEB1+2 depletion one-week after intravenous injection did not inhibit lung colonization, suggesting that ZEB1/2 and EMT are not essential for macrometastatic outgrowth. These results provide strong evidence that EMT is orchestrated by coordinated expression of several EMT-TFs and establish ZEB1 as a key master regulator of EMT and metastasis in breast cancer. IMPLICATIONS: The EMT program is orchestrated by coordinated expression of multiple EMT transcription factors, whereas ZEB1 integrates the EMT master regulatory network and plays the major role in promoting EMT and metastasis.

Addison JB ，Voronkova MA ，Fugett JH ，Lin CC ，Linville NC ，Trinh B ，Livengood RH ，Smolkin MB ，Schaller MD ，Ruppert JM ，Pugacheva EN ，Creighton CJ ，Ivanov AV ... -  《-》

Epithelial-mesenchymal transition associated markers in sarcomatoid transformation of clear cell renal cell carcinoma.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development and progression of many cancers. Partial EMT (pEMT) could represent a critical step in tumor migration and dissemination. Sarcomatoid renal cell carcinoma (sRCC) is an aggressive form of renal cell carcinoma (RCC) composed of a carcinomatous (sRCC-Ca) and sarcomatous (sRCC-Sa) component. The role of (p)EMT in the progression of RCC to sRCC remains unclear. The aim of this study was to investigate the involvement of (p)EMT in RCC and sRCC. Tissue samples from 10 patients with clear cell RCC (ccRCC) and 10 patients with sRCC were selected. The expression of main EMT markers (miR-200 family, miR-205, SNAI1/2, TWIST1/2, ZEB1/2, CDH1/2, VIM) was analyzed by qPCR in ccRCC, sRCC-Ca, and sRCC-Sa and compared to non-neoplastic tissue and between both groups. Expression of E-cadherin, N-cadherin, vimentin and ZEB2 was analyzed using immunohistochemistry. miR-200c was downregulated in sRCC-Ca compared to ccRCC, while miR-200a was downregulated in sRCC-Sa compared to ccRCC. CDH1 was downregulated in sRCC-Sa when compared to any other group. ZEB2 was downregulated in ccRCC and sRCC compared to corresponding non-neoplastic kidney. A positive correlation was observed between CDH1 expression and miR-200a/b/c. Our results suggest that full EMT is not present in sRCC. Instead, discreet molecular differences exist between ccRCC, sRCC-Ca, and sRCC-Sa, possibly representing distinct intermediary states undergoing pEMT.

Čugura T ，Boštjančič E ，Uhan S ，Hauptman N ，Jeruc J ... -  《-》

UBE2C, Directly Targeted by miR-548e-5p, Increases the Cellular Growth and Invasive Abilities of Cancer Cells Interacting with the EMT Marker Protein Zinc Finger E-box Binding Homeobox 1/2 in NSCLC.

Jin D ，Guo J ，Wu Y ，Du J ，Wang X ，An J ，Hu B ，Kong L ，Di W ，Wang W ... -  《Theranostics》

ZEB1-regulated inflammatory phenotype in breast cancer cells.

Zinc finger E-box binding protein 1 (ZEB1) and ZEB2 induce epithelial-mesenchymal transition (EMT) and enhance cancer progression. However, the global view of transcriptional regulation by ZEB1 and ZEB2 is yet to be elucidated. Here, we identified a ZEB1-regulated inflammatory phenotype in breast cancer cells using chromatin immunoprecipitation sequencing and RNA sequencing, followed by gene set enrichment analysis (GSEA) of ZEB1-bound genes. Knockdown of ZEB1 and/or ZEB2 resulted in the downregulation of genes encoding inflammatory cytokines related to poor prognosis in patients with cancer, including IL6 and IL8, therefore suggesting that ZEB1 and ZEB2 have similar functions in terms of the regulation of production of inflammatory cytokines. Antibody array and ELISA experiments confirmed that ZEB1 controlled the production of the IL-6 and IL-8 proteins. The secretory proteins regulated by ZEB1 enhanced breast cancer cell proliferation and tumor growth. ZEB1 expression in breast cancer cells also affected the growth of fibroblasts in cell culture, and the accumulation of myeloid-derived suppressor cells in tumors in vivo. These findings provide insight into the role of ZEB1 in the progression of cancer, mediated by inflammatory cytokines, along with the initiation of EMT.

Katsura A ，Tamura Y ，Hokari S ，Harada M ，Morikawa M ，Sakurai T ，Takahashi K ，Mizutani A ，Nishida J ，Yokoyama Y ，Morishita Y ，Murakami T ，Ehata S ，Miyazono K ，Koinuma D ... -  《-》