Katrin E. Wiese

Activation and repression by oncogenic MYC shape tumour-specific gene expression profiles

In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.

A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer.

In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.

The Role of MIZ-1 in MYC-Dependent Tumorigenesis

A hallmark of MYC-transformed cells is their aberrant response to antimitogenic signals. Key examples include the inability of MYC-transformed cells to arrest proliferation in response to antimitogenic signals such as TGF-β or DNA damage and their inability to differentiate into adipocytes in response to hormonal stimuli. Given the plethora of antimitogenic signals to which a tumor cell is exposed, it is likely that the ability to confer resistance to these signals is central to the transforming properties of MYC in vivo. At the same time, the inability of MYC-transformed cells to halt cell-cycle progression on stress may establish a dependence on mutations that impair or disable apoptosis. We propose that the interaction of MYC with the zinc finger protein MIZ-1 mediates resistance to antimitogenic signals. In contrast to other interactions of MYC, there is currently little evidence that MIZ-1 associates with MYC in normal, unperturbed cells. The functional interaction of both proteins becomes apparent at oncogenic expression levels of MYC and association with MIZ-1 mediates both oncogenic functions of MYC as well as tumor-suppressive responses to oncogenic levels of MYC.

BIM Is the Primary Mediator of MYC-Induced Apoptosis in Multiple Solid Tissues

MYC is one of the most frequently overexpressed oncogenes in human cancer, and even modestly deregulated MYC can initiate ectopic proliferation in many postmitotic cell types in vivo. Sensitization of cells to apoptosis limits MYCs oncogenic potential. However, the mechanism through which MYC induces apoptosis is controversial. Some studies implicate p19ARF-mediated stabilization of p53, followed by induction of proapoptotic BH3 proteins NOXA and PUMA, whereas others argue for direct regulation of BH3 proteins, especially BIM. Here, we use a single experimental system to systematically evaluate the roles of p19ARF and BIM during MYC-induced apoptosis, in vitro, in vivo, and in combination with a widely used chemotherapeutic, doxorubicin. We find a common specific requirement for BIM during MYC-induced apoptosis in multiple settings, which does not extend to the p53-responsive BH3 family member PUMA, and find no evidence of a role for p19ARF during MYC-induced apoptosis in the tissues examined.

Repression of SRF target genes is critical for Myc‐dependent apoptosis of epithelial cells

Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long‐lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP‐Sequencing experiments show that high levels of Myc invade target sites that lack consensus E‐boxes in a complex with Miz1 and repress transcription. Myc/Miz1‐repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum‐response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc‐induced apoptosis, and reverts a Myc‐dependent decrease in Akt phosphorylation and activity, a well‐characterized suppressor of Myc‐induced apoptosis. We propose that high levels of Myc engage Miz1 in repressive DNA binding complexes and suppress an SRF‐dependent transcriptional program that supports survival of epithelial cells.

Wnt signalling: conquering complexity

ABSTRACT The history of the Wnt pathway is an adventure that takes us from mice and flies to frogs, zebrafish and beyond, sketching the outlines of a molecular signalling cascade along the way. Here, we specifically highlight the instrumental role that developmental biology has played throughout. We take the reader on a journey, starting with developmental genetics studies that identified some of the main molecular players, through developmental model organisms that helped unravel their biochemical function and cell biological activities. Culminating in complex analyses of stem cell fate and dynamic tissue growth, these efforts beautifully illustrate how different disciplines provided missing pieces of a puzzle. Together, they have shaped our mechanistic understanding of the Wnt pathway as a conserved signalling process in development and disease. Today, researchers are still uncovering additional roles for Wnts and other members of this multifaceted signal transduction pathway, opening up promising new avenues for clinical applications. Summary: This Primer discusses how instrumental developmental biology has been in unravelling specific aspects of Wnt signalling, and its impact on current clinical research and the development of novel treatments for cancer.

The ninth ENBDC Weggis meeting: growth and in-depth characterisation of normal and neoplastic breast cells

Mammary gland biologists gathered for the ninth annual workshop of the European Network for Breast Development and Cancer (ENBDC) at Weggis on the shores of Lake Lucerne in March 2017. The main themes were oestrogen receptor alpha signalling, new techniques for mammary cell culture, CRISPR screening and proteogenomics.

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

ABSTRACT Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.

Abstract IA16: Transcriptional regulation by physiological and oncogenic Myc levels

Myc proteins bind globally to open promoters of all three RNA polymerases and in some instances globally enhance transcription. Surprisingly, Myc-driven tumor cells are characterized by specific gene expression profiles, which are defined by both activation and repression of specific target genes and which hold considerable prognostic value and biological information about tumor physiology. One mechanism that contributes to this specificity is promoter affinity and functionally distinct classes of Myc target genes vary widely in their apparent affinity to Myc. As a result, physiological and supraphysiological/oncogenic levels of Myc regulate different gene expression programs. Furthermore, oncogenic levels of Myc engage the zinc finger protein, Miz1, to repress transcription and I will briefly review what we know about the mechanism of repression. I will discuss two examples how different levels of Myc sensitize cells towards apoptosis and regulate the self-renewing potential of adult stem cells. Citation Format: Bjoern von Eyss, Elmar Wolf, Katrin Wiese, Martin Eilers. Transcriptional regulation by physiological and oncogenic Myc levels. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA16.

Abstract B19: MYC induces PLD6 to suppress YAP/TAZ-dependent self-renewal of mammary stem cells

For the maintenance of a given tissue it is absolutely critical to balance proliferation and differentiation of stem cells, progenitor cells and terminally differentiated cells. Perturbations of these finely tuned processes can lead to various diseases such as cancer. One factor that has been implicated in the transition from a stem cell to a progenitor/ transit-amplifying cell is the oncogenic transcription factor MYC. Paradoxically, despite its strong pro-tumorigenic capabilities it has been shown to promote differentiation in several tissues such as the skin or the hematopoietic system. To identify critical pathways that might explain MYC9s effect on the differentiation of epithelial stem cells we expressed MYC in mammary stem cells. Here, MYC induction led to a drastic reduction in sphere formation indicative of a decreased stem cell potential. Further RNA-Seq and bioinformatical analysis revealed that one of MYC9s main functions in mammary stem cells is the repression of YAP/TAZ target genes. YAP and its orthologue TAZ act as nuclear co-activators and are the downstream effectors of the Hippo pathway, a recently discovered pathway with key functions in adult stem cell maintenance and growth control. Using a targeted siRNA screen for MYC target genes we were able to identify a single target gene that is absolutely essential for MYC9s ability to repress YAP/TAZ, namely PLD6. PLD6 is a mitochondrial protein that promotes mitochondrial fusion, so that this MYC target gene couples a mitochondrial checkpoint to repression of YAP/TAZ. We were also able to demonstrate the relevance of this pathway in vivo using a mouse that expresses MYC in the mammary stem cell compartment. Interestingly, this pathway is also operational in human breast cancer since a high MYC signature correlates with a low YAP/TAZ signatue in breast cancer patients ultimately leading to a bad prognosis for the respective patient. In summary, we were able to identify a new pathway that couples MYC to mitochondrial biogenesis and the repression of an adult stem cell program. Citation Format: Bjoern von Eyss, Laura Annika Jaenicke, Katrin Wiese, Andreas Rosenwald, Martin Eilers. MYC induces PLD6 to suppress YAP/TAZ-dependent self-renewal of mammary stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B19.