Marc Vooijs

Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells

The self-renewing epithelium of the small intestine is ordered into stem/progenitor crypt compartments and differentiated villus compartments. Recent evidence indicates that the Wnt cascade is the dominant force in controlling cell fate along the crypt–villus axis. Here we show a rapid, massive conversion of proliferative crypt cells into post-mitotic goblet cells after conditional removal of the common Notch pathway transcription factor CSL/RBP-J (ref. 2). We obtained a similar phenotype by blocking the Notch cascade with a γ-secretase inhibitor. The inhibitor also induced goblet cell differentiation in adenomas in mice carrying a mutation of the Apc tumour suppressor gene. Thus, maintenance of undifferentiated, proliferative cells in crypts and adenomas requires the concerted activation of the Notch and Wnt cascades. Our data indicate that γ-secretase inhibitors, developed for Alzheimers disease, might be of therapeutic benefit in colorectal neoplastic disease.

Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells

The use of Cre/loxP recombination in mammalian cells has expanded rapidly. We describe here that Cre expression in cultured mammalian cells may result in a markedly reduced proliferation and that this effect is dependent on the endonuclease activity of Cre. Chromosome analysis after Cre expression revealed numerous chromosomal aberrations and an increased number of sister chromatid exchanges. Titration experiments in mouse embryo fibroblasts with a ligand-regulatable Cre-ERT show that toxicity is dependent on the level of Cre activity. Prolonged, low levels of Cre activity permit recombination without concomitant toxicity. This urges for a careful titration of Cre activity in conditional gene modification in mammalian cells.

Metalloprotease ADAM10 Is Required for Notch1 Site 2 Cleavage

Notch signaling is controlled by ligand binding, which unfolds a negative control region to induce proteolytic cleavage of the receptor. First, a membrane-proximal cleavage is executed by a metalloprotease, removing the extracellular domain. This allows γ-secretase to execute a second cleavage within the Notch transmembrane domain, which releases the intracellular domain to enter the nucleus. Here we show that the ADAM10 metalloprotease Kuzbanian, but not ADAM17/tumor necrosis factor α-converting enzyme, plays an essential role in executing ligand-induced extracellular cleavage at site 2 (S2) in cells and localizes this step to the plasma membrane. Importantly, genetic or pharmacological inhibition of metalloproteases still allowed extracellular cleavage of Notch, indicating the presence of unknown proteases with the ability to cleave at S2. Gain of function mutations identified in human cancers and in model organisms that map to the negative control region alleviate the requirement for ligand binding for extracellular cleavage to occur. Because cancer-causing Notch1 mutations also depend on (rate-limiting) S2 proteolysis, the identity of these alternative proteases has important implications for understanding Notch activation in normal and cancer cells.

Flp-mediated tissue-specific inactivation of the retinoblastoma tumor suppressor gene in the mouse

The yeast-derived Flp-frt site-specific DNA recombination system was used to achieve pituitary-specific inactivation of the retinoblastoma (Rb) tumor suppressor gene. Whereas mice carrying only frt sites in both alleles of Rb remain tumor free, tumorigenesis ensues when the Flp recombinase is expressed. The rate of tumorigenesis in these mice depends both on the expression level of the Flp recombinase and on the presence of frt sites in one or both Rb alleles. This permitted a more accurate definition of the consecutive steps in pituitary tumorigenesis. Our study illustrates the potential of this approach for studying sporadic cancer in a defined mouse model.

Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis.

The retinoblastoma gene product, pRb, plays a crucial role in cell cycle regulation, differentiation and inhibition of oncogenic transformation. pRb and its closely related family members p107 and p130 perform exclusive and overlapping functions during mouse development. The embryonic lethality of Rb-null animals restricts the phenotypic analysis of these mice to mid-gestation embryogenesis. We employed the Cre/loxP system to study the function of Rb in adult mouse stratified epithelium. RbF19/F19;K14cre mice displayed hyperplasia and hyperkeratosis in the epidermis with increased proliferation and aberrant expression of differentiation markers. In vitro, pRb is essential for the maintainance of the postmitotic state of terminally differentiated keratinocytes, preventing cell cycle re-entry. However, p107 compensates for the effects of Rb loss as the phenotypic abnormalities of RbF19/F19;K14cre keratinocytes in vivo and in vitro become more severe with the concurrent loss of p107 alleles. p107 alone appears to be dispensable for all these phenotypic changes, as the presence of a single Rb allele in a p107-null background rescues all these alterations. Luciferase reporter experiments indicate that these phenotypic alterations might be mediated by increased E2F activity. Our findings support a model in which pRb in conjunction with p107 plays a central role in regulating epidermal homeostasis.

Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE

The four highly conserved Notch receptors receive short-range signals that control many biological processes during development and in adult vertebrate tissues. The involvement of Notch1 signaling in tissue self-renewal is less clear, however. We developed a novel genetic approach N1IP-CRE (Notch1 Intramembrane Proteolysis) to follow, at high resolution, the descendents of cells experiencing Notch1 activation in the mouse. By combining N1IP-CRE with loss-of-function analysis, Notch activation patterns were correlated with function during development, self-renewal and malignancy in selected tissues. Identification of many known functions of Notch1 throughout development validated the utility of this approach. Importantly, novel roles for Notch1 signaling were identified in heart, vasculature, retina and in the stem cell compartments of self-renewing epithelia. We find that the probability of Notch1 activation in different tissues does not always indicate a requirement for this receptor and that gradients of Notch1 activation are evident within one organ. These findings highlight an underappreciated layer of complexity of Notch signaling in vivo. Moreover, NIP-CRE represents a general strategy applicable for monitoring proteolysis-dependent signaling in vivo.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2α

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFα protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFα) and aha-1 (HIFβ), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2α-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2α, but not HIF-1α. These results identify TWIST1 as a direct target gene of HIF-2α, which may provide insight into the acquired metastatic capacity of hypoxic tumors.

The hypoxia-inducible microRNA cluster miR-199a∼214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation.

Peroxisome proliferator-activated receptor δ (PPARδ) is a critical regulator of energy metabolism in the heart. Here, we propose a mechanism that integrates two deleterious characteristics of heart failure, hypoxia and a metabolic shift toward glycolysis, involving the microRNA cluster miR-199a∼214 and PPARδ. We demonstrate that under hemodynamic stress, cardiac hypoxia activates DNM3os, a noncoding transcript that harbors the microRNA cluster miR-199a∼214, which shares PPARδ as common target. To address the significance of miR-199a∼214 induction and concomitant PPARδ repression, we performed antagomir-based silencing of both microRNAs and subjected mice to biomechanical stress to induce heart failure. Remarkably, antagomir-treated animals displayed improved cardiac function and restored mitochondrial fatty acid oxidation. Taken together, our data suggest a mechanism whereby miR-199a∼214 actively represses cardiac PPARδ expression, facilitating a metabolic shift from predominant reliance on fatty acid utilization in the healthy myocardium toward increased reliance on glucose metabolism at the onset of heart failure.

Hypoxic regulation of metastasis via hypoxia-inducible factors.

Metastases formation is a major factor in disease progression and accounts for the majority of cancer deaths. The molecular mechanisms controlling invasion, dissemination to blood or lymphatic systems and spread of tumor cells to distant organs are still poorly understood. Recent observations indicate that the meta-static phenotype may already be present during the angiogenic switch of tumors. Intratumoral hypoxia correlates with poor prognosis and enhanced metastases formation. The Hypoxia Inducible Factors (HIFs) are key molecules in the hypoxic response and play critical roles during tumor cell expansion by regulating energy metabolism and the induction of angiogenesis. Increasing evidence implicates HIF function in metastatic cell characteristics, like epithelial to mesenchymal transition, cell detachment, invasion and tumor cell seeding. Here, we review the link between tumor cell hypoxia and the acquisition of metastatic behavior. We hypothesize that polyclonal tumor selection by hypoxia enhances metastatic capacity by transcriptional control of key regulators of metastasis. This polyclonal hypoxic gene profile potentially develops into a metastatic profile, driving metastasis formation. The hypoxic gene profile in primary tumors may therefore provide a prognostic indicator in clinical decision-making.

E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells

BACKGROUND AND PURPOSE Hypoxia is a hallmark of solid cancers and associated with metastases and treatment failure. During tumor progression epithelial cells often acquire mesenchymal features, a phenomenon known as epithelial-to-mesenchymal transition (EMT). Intratumoral hypoxia has been linked to EMT induction. We hypothesized that signals from the tumor microenvironment such as growth factors and tumor oxygenation collaborate to promote EMT and thereby contribute to radioresistance. MATERIALS AND METHODS Gene expression changes under hypoxia were analyzed using microarray and validated by qRT-PCR. Conversion of epithelial phenotype upon hypoxic exposure, TGFβ addition or oncogene activation was investigated by Western blot and immunofluorescence. Cell survival following ionizing radiation was assayed using clonogenic survival. RESULTS Upon hypoxia, TGFβ addition or EGFRvIII expression, MCF7, A549 and NMuMG epithelial cells acquired a spindle shape and lost cell-cell contacts. Expression of epithelial markers such as E-cadherin decreased, whereas mesenchymal markers such as vimentin and N-cadherin increased. Combining hypoxia with TGFβ or EGFRvIII expression, lead to more rapid and pronounced EMT-like phenotype. Interestingly, E-cadherin expression and the mesenchymal appearance were reversible upon reoxygenation. Mesenchymal conversion and E-cadherin loss were associated with radioresistance. CONCLUSIONS Our findings describe a mechanism by which the tumor microenvironment may contribute to tumor radioresistance via E-cadherin loss and EMT.