Recherche cancer
Evolution cancer
Constam Daniel, Associate scientist e-mail pdf group members publications
Daniel Constam obtained his doctoral degree in natural sciences from the Swiss Federal Institute of Technology (ETH), Zürich, in 1993. Between 1994 and 1999, he worked in the laboratory of Elizabeth Robertson (Harvard University, Cambridge MA) to study the molecular genetics of early mammalian development. In 2000, he joined ISREC as an associate scientist. In 2004, he was nominated Tenure Track Assistant Professor in the School of Life Sciences at EPFL.
Coordination of cell proliferation and differentiation by morphogenetic signals

Our lab seeks to understand how cells in developing and regenerating tissues communicate with each other and their microenvironment to proliferate and differentiate in a coordinate manner, and how corruption of these signaling networks promotes tumorigenesis.

At the blastocyst stage, pluripotent progenitors in the inner cell mass can be isolated to derive embryonic stem (ES) cell lines. When implanted ectopically into adult tissues, these stem cells are highly tumorigenic, whereas in their native microenvironment, they differentiate in an orderly fashion and are allocated to distinct germ layers due to specific interactions with neighboring primitive endoderm and stem cells in the trophectoderm. Our work has identified the secreted proteases Furin and PACE4 as key determinants of this microenvironment since they stimulate the organizer activity of the TGFß family member Nodal in the neighboring embryonic lineage. The cleaved form of Nodal in turn induces a local source of soluble feedback inhibitors, the so-termed anterior visceral endoderm (AVE), whereas at the opposite pole it sustains a positive feedback loop mediated by the oncoprotein Cripto that is reinforced by Wnt3. Available evidence suggests that this signaling network generates a Nodal activity gradient that informs each cell about its position with respect to its neighbors, and dictates corresponding fates. However, recent studies in human melanoma suggest that tumor cells can hijack the Nodal pathway to promote an undifferentiated, metastatic phenotype.

Using transgenic mice, combined with biochemical analysis and cell-based assays, we seek to determine how TGFß ligands such as Nodal and their proprotein convertases intersect with other pathways to either inhibit or stimulate the differentiation of naive progenitor cells, and how a balance between these opposite activities might be restored to halt tumor progression.

1) Defining the role of Nodal in stem cells of the implanted blastocyst

Our previous work indicated that Furin and PACE4 activities at the onset of gastrulation are secreted by the extraembryonic ectoderm to cleave the Nodal precursor in neighboring epiblast cells. However, it was unclear how these proteases activate Nodal at the opposite pole, i.e. far away from their source, to specify DVE. Prompted by this question, we asked whether at an earlier stage, Nodal is transiently coexpressed together with a convertase locally within the visceral endoderm. While this turned out to be true for Furin (Figure 1), our analysis of early Nodal null mutants in addition revealed that Nodal is initially required as an anti-differentiation signal to sustain the expression of several determinants of pluripotency, including the transcription factors Oct4, Nanog and Foxd3. This finding is consistent with observations that Nodal signaling is also essential in cultured human embryonic stem cells for their self-renewal. We hypothesize such an anti-differentiation activity might also explain how Nodal maintains a plastic phenotype in tumor cells. In an ongoing project, we therefore elucidate the underlying molecular mechanism how Nodal during gastrulation is converted from a stem cell factor into a differentiation signal and how these mutually antagonistic activities are balanced during development and in cancer cells.

Figure 1: Expression and function of Nodal in the implanted blastocyst. Whereas Nodal and its coreceptor Cripto are coexpressed in the inner cell mass (ICM, blue), the secreted proprotein convertases Furin and PACE4 are initially produced by cells in the primitive endoderm (dark green) and trophectoderm (grey), respectively, and activate the Nodal precursor. Cleaved Nodal amplifies Nodal and Cripto expression in an autoinductive feedback loop to maintain determinants of pluripotency, whereas the columnar primitive endoderm cells are induced to differentiate into a squamous epithelium of embryonic visceral endoderm (EmVE). Consequently, Furin expression by E5.5 is confined to the extraembryonic region, whereas distal cells (yellow) upregulate the Nodal feedback antagonists Lefty-1 and Cer1 and eventually move to the future anterior pole.

2) Defining cleavage-independent functions of Nodal during embryogenesis and in cancer

Furin and PACE4 are likely to activate additional substrates besides Nodal. To directly assess the specific role of Nodal processing, we generated the knock-in allele NodalNr encoding a mutant precursor which is resistant to Furin and PACE4 due to disruption of the cleavage site. Analysis of Nr/Nr homozygotes confirmed that Nodal processing is essential in the implanted blastocyst to amplify Nodal and Cripto expression in an autoinductive feedback loop. However, during gastrulation, the Nodal precursor was induced independently of cleavage and triggered a signaling cascade involving Bmp4, and Wnt3 which we found to mediate mesoderm induction. Furthermore, the Nodal precursor also maintained the source of its own convertases Furin and PACE4 (Figure 2). To elucidate the underlying mechanism, we purified cleavage-resistant Nodal precursor, and we characterized its activity in embryo explants and receptor binding assays. The results of these experiments suggest that even uncleaved Nodal binds signaling receptor complexes and thereby induces a subset of Nodal target genes. Moreover, mathematical modeling indicated that different functions of Nodal in the embryo might not only reflect local differences in its concentration. Rather, by unknown mechanisms, cells may remember for how long they have been exposed to this protein and choose distinct fates based on differences in the Nodal signal integrated over time. In ongoing studies, we investigate why cellular responses to cleaved and uncleaved Nodal are qualitatively different. In addition, it will be interesting to define the role of cleavage-independent Nodal signaling and related activities in cancer.

Figure 2: A Nodal activity gradient established by feedback regulation coordinates germ layer differentiation. Uncleaved Nodal maintains the source of proprotein convertases and Bmp4 in trophoblast stem cells, and thereby triggers Wnt3 expression. Besides inducing mesoderm, Wnt3 acting together with mature Nodal also upregulates Cripto wat the posterior pole (P), whereas this process is blocked anteriorly (A) by the AVE-derived feedback inhibitors. When integrated over time, the Nodal signal thus is highest at the posterior pole where it drives mesoderm and endoderm formation, but low in the vicinity of the AVE.

3) Regulation of left-right asymmetry and tubular morphogenesis by monocilia

In all vertebrates, Nodal is also essential after gastrulation to establish normal left-right asymmetry of the visceral situs. To this end, Nodal signaling is restricted to the future left side by motile monocilia, which generates a directional fluid flow towards the left side of a small indentation, the so-termed node, at the base of the midline. Cilia also harbor Ca2+ channels and components of the hedgehog signal transduction machinery such as Smoothened and transcription factors of the Gli family, and they are crucial to regulate tissue polarity and prevent cyst formation in tubular epithelial cells. In the kidney, loss of cilia function has been implicated by numerous studies to result in polycystic disease and eventually kidney failure. However, how cilia control morphogenetic activities of the hedgehog and TGFß families, and the role of these pathways in hyperplastic kidneys of PKD patients remain poorly defined. Analysis of the underlying molecular mechanisms will provide important insights into how cilia govern tissue polarity to suppress hyperplastic growth, especially in tubular epithelial cells of the kidney.

Keywords

Research fields:
Cell communication in development and cancer; regulation and function of TGFß signaling; role of monicilia in the establishment of tissue polarity
Competences:
Mouse molecular genetics, Biochemistry, Cell Biology