SIP1/ZEB2 is a member of the δEF-1 family of two-handed zinc finger nuclear factors. The expression of these transcription factors is associated with epithelial mesenchymal transitions (EMT) during development. SIP1 is also expressed in some breast cancer cell lines and was detected in intestinal gastric carcinomas, where its expression is inversely correlated with that of E-cadherin. Here, we show that expression of SIP1 in human epithelial cells results in a clear morphological change from an epithelial to a mesenchymal phenotype. Induction of this epithelial dedifferentiation was accompanied by repression of several cell junctional proteins, with concomitant repression of their mRNA levels. Besides E-cadherin, other genes coding for crucial proteins of tight junctions, desmosomes and gap junctions were found to be transcriptionally regulated by the transcriptional repressor SIP1. Moreover, study of the promoter regions of selected genes by luciferase reporter assays and chromatin immunoprecipitation shows that repression is directly mediated by SIP1. These data indicate that, during epithelial dedifferentiation, SIP1 represses in a coordinated manner the transcription of genes coding for junctional proteins contributing to the dedifferentiated state; this repression occurs by a general mechanism mediated by Smad Interacting Protein 1 (SIP1)-binding sites.

The ZEB family of zinc finger transcription factors are essential players during normal embryonic development. One characteristic is that they induce epithelial to mesenchymal transition (EMT), a process that reorganizes epithelial cells to become migratory mesenchymal cells. E-cadherin is a major target gene of these transcriptional repressors, and this downregulation is considered a hallmark of EMT. In recent years, the involvement of the ZEB proteins in pathological contexts has been documented as well. Mutations in ZEB encoding genes cause severe syndromic malformations and evidence is mounting that links these factors to malignant tumor progression. In this review, we describe what is currently known on the molecular pathways these transcription factors are implicated in, and we highlight their roles in development and human diseases, with a focus on tumor malignancy.

Individuals who inherit one faulty von Hippel-Lindau gene (VHL) allele are predisposed to VHL disease, which is characterized by the development of cerebellar, spinal, and retinal hemangioblastoma, pheochromocytoma, and clear-cell renal cell carcinoma (CC-RCC) (29). The tumor develops upon the somatic loss of the remaining wild-type VHL allele in a susceptible cell. Importantly, biallelic loss of VHL is associated with the vast majority of sporadic CC-RCCs, establishing VHL as a critical suppressor of renal oncogenesis (29). CC-RCC is resistant to conventional radiation and chemotherapies, and approximately one-quarter of renal cancer patients present with advanced disease, including locally invasive or metastatic CC-RCC (12). Unfortunately, one-third of patients who undergo surgical removal of localized tumors have recurrence of the disease, and the median survival for patients harboring metastatic CC-RCC is 13 months (12). Moreover, the principal cause of morbidity and death of VHL patients is CC-RCC (29). Despite the need to better understand the aggressive nature of CC-RCC, the molecular pathways governing its malignant phenotype remain unresolved.The most well-characterized function of VHL is as a substrate-recognition component of the SCF (Skp1/Cdc53/F-box protein)-like E3 ubiquitin ligase complex called ECV (elongins/Cul2/VHL) that selectively ubiquitylates oxygen-dependent prolyl-hydroxylated ␣ subunits of hypoxia-inducible factor

Zinc finger transcription factors of the Snail/Slug and ZEB-1/SIP1 families control epithelial-mesenchymal transitions in development in cancer. Here, we studied SIP1-regulated mesenchymal conversion of epidermoid A431 cells. We found that concomitant with inducing invasive phenotype, SIP1 inhibited expression of cyclin D1 and induced hypophosphorylation of the Rb tumor suppressor protein. Repression of cyclin D1 was caused by direct binding of SIP1 to three sequence elements in the cyclin D1 gene promoter. By expressing exogenous cyclin D1 in A431/SIP1 cells and using RNA interference, we demonstrated that the repression of cyclin D1 gene by SIP1 was necessary and sufficient for Rb hypophosphorylation and accumulation of cells in G1 phase. A431 cells expressing SIP1 along with exogenous cyclin D1 were highly invasive, indicating that SIP1-regulated invasion is independent of attenuation of G1/S progression. However, in another epithelial-mesenchymal transition model, gradual mesenchymal conversion of A431 cells induced by a dominant negative mutant of E-cadherin produced no effect on the cell cycle. We suggest that impaired G1/S phase progression is a general feature of cells that have undergone EMT induced by transcription factors of the Snail/Slug and ZEB-1/SIP1 families.

The expression of Smad interacting protein-1 (SIP1; ZEB2) and the de novo expression of vimentin are frequently involved in epithelial-to-mesenchynial transitions (EMTs) under both normal and pathological conditions. In the present study, we investigated the potential role of SIP1 in the regulation of vimentin during the EMT associated with breast tumor cell migration and invasion. Examining several breast tumor cell Unes displaying various degrees of invasiveness, we found SIP1 and vimentin expression only in invasive cell Unes. Also, using a model of cell migration with human mammary MCF10A cells, we showed that SIP1 is induced specifically in vimentin-positive migratory cells. Furthermore, transfection of SIP1 cDNA in MCF10A cells increased their vimentin expression both at the mRNA and protein levels and enhanced their migratory abilities in Boyden Chamber assays. Inversely, inhibition of SIP1 expression by RNAi strategies in BT-549 cells and MCF10A cells decreased vimentin expression. We also showed that SIP1 transfection did not activate the TOP-FLASH reporter system, suggesting that the β-catenin/ TCF pathway is not impUcated in the regulation of vimentin by SIP1. Our results therefore impUcate SIP1 in the regulation of vimentin observed in the EMT associated with breast tumor cell migration, a pathway that may contribute to the metastatic progression of breast cancer.

BackgroundEpithelial mesenchymal transition (EMT) is tightly regulated by a network of transcription factors (EMT-TFs). Among them is the nuclear factor ZEB2, a member of the zinc-finger E-box binding homeobox family. ZEB2 nuclear localization has been identified in several cancer types, and its overexpression is correlated with the malignant progression. ZEB2 transcriptionally represses epithelial genes, such as E-cadherin (CDH1), by directly binding to the promoter of the genes it regulates and activating mesenchymal genes by a mechanism in which there is no full agreement. Recent studies showed that EMT-TFs interact with epigenetic regulatory enzymes that alter the epigenome, thereby providing another level of control. The role of epigenetic regulation on ZEB2 function is not well understood. In this study, we aimed to characterize the epigenetic effect of ZEB2 repressive function on the regulation of a small Rab GTPase RAB25.ResultsUsing cellular models with conditional ZEB2 expression, we show a clear transcriptional repression of RAB25 and CDH1. RAB25 contributes to the partial suppression of ZEB2-mediated cell migration. Furthermore, a highly significant reverse correlation between RAB25 and ZEB2 expression in several human cancer types could be identified. Mechanistically, ZEB2 binds specifically to E-box sequences on the RAB25 promoter. ZEB2 binding is associated with the local increase in DNA methylation requiring DNA methyltransferases as well as histone deacetylation (H3K9Ac) depending on the activity of SIRT1. Surprisingly, SIRT1 and DNMTs did not interact directly with ZEB2, and while SIRT1 inhibition decreased the stability of long-term repression, it did not prevent down-regulation of RAB25 and CDH1 by ZEB2.ConclusionsZEB2 expression is resulting in drastic changes at the chromatin level with both clear DNA hypermethylation and histone modifications. Here, we revealed that SIRT1-mediated H3K9 deacetylation helps to maintain gene repression but is not required for the direct ZEB2 repressive function. Targeting epigenetic enzymes to prevent EMT is an appealing approach to limit cancer dissemination, but inhibiting SIRT1 activity alone might have limited effect and will require drug combination to efficiently prevent EMT.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0239-4) contains supplementary material, which is available to authorized users.