Gene expression profiling has uncovered the transcription factor Sox4 with upregulated activity during TGF-β-induced epithelial-mesenchymal transition (EMT) in normal and cancerous breast epithelial cells. Sox4 is indispensable for EMT and cell survival in vitro and for primary tumor growth and metastasis in vivo. Among several EMT-relevant genes, Sox4 directly regulates the expression of Ezh2, encoding the Polycomb group histone methyltransferase that trimethylates histone 3 lysine 27 (H3K27me3) for gene repression. Ablation of Ezh2 expression prevents EMT, whereas forced expression of Ezh2 restores EMT in Sox4-deficient cells. Ezh2-mediated H3K27me3 marks associate with key EMT genes, representing an epigenetic EMT signature that predicts patient survival. Our results identify Sox4 as a master regulator of EMT by governing the expression of the epigenetic modifier Ezh2.

The epithelial-mesenchymal transition (EMT), a prerequisite for cancer progression and metastasis formation, is regulated not only at the transcriptional but also at the post-transcriptional level, including at the level of alternative pre-mRNA splicing. Several recent studies have highlighted the involvement of splicing factors, including epithelial splicing regulatory proteins (Esrps) and RNA-binding Fox protein 2 (Rbfox2), in this process. Esrps regulate epithelial-specific splicing, and their expression is downregulated during EMT. By contrast, the role of Rbfox2 is controversial because Rbfox2 regulates epithelial as well as mesenchymal splicing events. Here, we have used several established cell culture models to investigate the functions of Rbfox2 during EMT. We demonstrate that induction of an EMT upregulates the expression of Rbfox2, which correlates with an increase in Rbfox2-regulated splicing events in the cortactin (Cttn), Pard3 and dynamin 2 (Dnm2) transcripts. At the same time, however, the epithelial-specific ability to splice the Enah, Slk and Tsc2 transcripts is either reduced or lost completely by Rbfox2, which might be due, in part, to downregulation of the expression of the Esrps cooperative factors. Depletion of Rbfox2 during EMT did not prevent the activation of transforming growth factor-β signaling, the upregulation of mesenchymal markers or changes in cell morphology toward a mesenchymal phenotype. In addition, this depletion did not influence cell migration. However, depletion of Rbfox2 in cells that have completed an EMT significantly reduced their invasive potential. Taken together, our results suggest that during an EMT, Rbfox2-regulated splicing shifts from epithelial-to mesenchymal-specific events, leading to a higher degree of tissue invasiveness.

The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellular distribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis.

IntroductionIncreasing evidence supports a role of an epithelial to mesenchymal transition (EMT) process in endowing subsets of tumor cells with properties driving malignant tumor progression and resistance to cancer therapy. To advance our understanding of the underlying mechanisms, we sought to generate a transplantable cellular model system that allows defined experimental manipulation and analysis of EMT in vitro and at the same time recapitulates oncogenic EMT in vivo.Methodology/ResultsWe have established a stable murine breast cancer cell line (Py2T) from a breast tumor of an MMTV-PyMT transgenic mouse. Py2T cells display a metastable epithelial phenotype characterized by concomitant expression of luminal and basal cytokeratins and sheet migration. Exposure of Py2T cells to transforming growth factor β (TGFβ) in vitro induces reversible EMT accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers, including EMT transcription factors, and a gain in single cell motility and invasiveness. Py2T cells give rise to tumors after orthotopic injection into syngeneic FVB/N mice. Notably, transplantation of epithelial Py2T cells results in the formation of invasive primary tumors with low to absent E-cadherin expression, indicating that the cells undergo EMT-like changes in vivo. This process appears to at least in part depend on TGFβ signaling, since tumors formed by Py2T cells expressing a dominant-negative version of TGFβ receptor widely maintain their epithelial differentiation status.Conclusions/SignificanceTogether, the data demonstrate that the Py2T cell line represents a versatile model system to study the EMT process in vitro and in vivo. The observation that Py2T cells give rise to tumors and collectively undergo EMT-like changes in vivo highlights the suitability of the Py2T model system as a tool to study tumor-related EMT. In particular, Py2T cells may serve to corroborate recent findings relating EMT to cancer cell stemness, to therapy resistance and to tumor recurrence.

Acquiring resistance against transforming growth factor b (TGFb)-induced growth inhibition at early stages of carcinogenesis and shifting to TGFb's tumour-promoting functions at later stages is a pre-requisite for malignant tumour progression and metastasis. We have identified the transcription factor distal-less homeobox 2 (Dlx2) to exert critical functions during this switch. Dlx2 counteracts TGFb-induced cell-cycle arrest and apoptosis in mammary epithelial cells by at least two molecular mechanisms: Dlx2 acts as a direct transcriptional repressor of TGFb receptor II (TGFbRII) gene expression and reduces canonical, Smad-dependent TGFb signalling and expression of the cell-cycle inhibitor p21 CIP1 and increases expression of the mitogenic transcription factor c-Myc. On the other hand, Dlx2 directly induces the expression of the epidermal growth factor (EGF) family member betacellulin, which promotes cell survival by stimulating EGF receptor signalling. Finally, Dlx2 expression supports experimental tumour growth and metastasis of B16 melanoma cells and correlates with tumour malignancy in a variety of human cancer types. These results establish Dlx2 as one critical player in shifting TGFb from its tumour suppressive to its tumour-promoting functions.

Antibody-drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required.

In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

The mammalian Golgi apparatus consists of individual cisternae that are stacked in a polarized manner to form the compact zones of the Golgi. Several stacks are linked to form a ribbon via dynamic lateral bridges. The determinants required for maintaining the characteristic Golgi structure are incompletely understood. Here, we have characterized p28, a new γ-subfamily member of p24 membrane proteins. p28 localized to endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and cis Golgi and accumulated in the ERGIC upon Brefeldin A treatment, typical for a protein cycling in the early secretory pathway. p28 interacted with a subset of p24 proteins. Its depletion by small interfering RNA (siRNA) led to fragmentation of the Golgi without affecting the overall organization of microtubules but considerably reducing the amount of acetylated tubulin. The distribution of COPI and tethers, including GM130, was not affected. At the ultrastructural level, the Golgi fragments appeared as mini-stacks with apparently unchanged cis-trans topology. Golgi fragmentation did not impair anterograde or retrograde traffic. Fluorescence recovery after photobleaching (FRAP) experiments revealed that silencing p28 prevents protein exchange between Golgi stacks during reassembly after Brefeldin A-induced Golgi breakdown. These results show that the formation of a Golgi ribbon requires the structural membrane protein p28 in addition to previously identified SNAREs, coat proteins and tethers.