Arene Candide

Aguet Michel | Role of BCL9/BCL9L in regulating Wnt-mediated epithelial-mesenchymal transition, stem cell traits and drug sensitivity in Wnt-activated human cancers (KFS 2674-08-2010)

The concept of cancer stem cells (CSCs) has stimulated new directions for addressing the problem of cancer treatment. CSCs are increasingly viewed as responsible, at least in part, for the emergence of resistance to ther- apy and tumour recurrence. Therapy-refractory tumour cells from a variety of solid tumours have been reported to exhibit stem cell-related traits and may represent CSC- enriched tumour cell populations selected under thera- peutic constraint. Targeting CSCs has therefore emerged as a tempting new therapeutic strategy.

We recently described a mouse model of colon adenocar- cinoma in which a signalling branch of the Wnt pathway (BCL9) was critically involved in CSC maintenance. We showed that inactivating this branch resulted in virtual loss of stemness and other malignancy associated traits. This project aimed at validating these findings in other models, including human cancer cell lines. We were able to identify a human colon cancer cell line based upon ex- pression of intestinal stem cell markers, which appeared to consist of heterogeneous subpopulations of stem cell- like as well as more differentiated cells, pointing to a spontaneously occurring differentiation cascade. Prelimi- nary findings suggest that the same Wnt signalling branch is involved in regulating this equilibrium and that more differentiated cells lose stemness as well as associated malignancy traits.

The aim was to explore to what extent pharmacological inhibition of BCL9 proteins may be of benefit in the con- text of a combination therapy to reduce the CSC pool and the risk of tumour recurrence.

Project coordinator Prof. Dr. Michel Aguet

Institut suisse de recherche expérimentale sur le cancer (ISREC)

EPF de Lausanne CH-1011 Lausanne [email protected]

Andres Anne-Catherine | The molecular mechanisms provoking the ephrin-B2 induced deregulation of the mammary stem cell niche and leading to metastatic tumour growth (KLS 2825-08-2011)

Stem and progenitor cells are thought to represent the origin of carcinogenesis and to be responsible for meta- static tumour growth. Thus, the elaboration of efficient treatment strategies depends at least in part upon a pro- found understanding of the mechanisms controlling stem cell development. We showed previously that deregu- lated expression of the receptor EphB4 and its ligand ephrin-B2 in the mammary epithelium alters the stem cell niche and thereby contributes to metastatic tumour growth.

In this project, we investigated whether the abnormal be- haviour of the stem cells originates from the stem cells themselves or is the consequence of abnormal stimula- tion by the neighbouring cells constituting their niche. Moreover, we aimed to identify the molecular pathway transmitting the EphB4-ephrin-B2 emitted signals. To these ends, we isolated the mammary epithelial cells of transgenic mice exhibiting deregulated expression of EphB4 or ephrin-B2 and separated these cells into four distinct cell populations: luminal and basal progenitors, bi-potent progenitors, and the stem cells. From each of these cell populations we determined the gene expres- sion profile and compared these profiles to those of nor- mal mammary epithelial cells.

These experiments revealed that components of the Wnt signalling pathway and the oestrogen receptor are pre- dominantly affected by the aberrant EphB4 or ephrin-B2 expression. The oestrogen receptor is the main inducer of mammary epithelial proliferation in the adult, whereas signalling by Wnt growth factors is involved in a variety of morphogenic processes such as determination of tissue boundaries and pattern formation. Interestingly, the main discrepancies were found in the bi-potent progenitor cells and the stem cell population, which both do not express the transgenes.

These results demonstrate that EphB4 and ephrin-B2 ex- ert their effect indirectly by activating the Wnt and oes- trogen signalling cascade. Thus, our project has led to the identification of a particular aspect of the control mecha- nisms ensuring the homeostasis of the stem cell niche that might be a suitable target for stem cell specific therapeu- tic intervention.

Project coordinator

Prof. Dr. phil. nat. Anne-Catherine Andres Departement Klinische Forschung Universität Bern

CH-3010 Bern

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Sex hormones are known culprits in breast cancer. For ex- ample, women undergoing combined hormone replace- ment therapy with artificial progesterone mimics (called progestins) and oestrogen have an increased risk of get- ting breast cancer.Unfortunately, it is difficult to study the effects of these hormones on normal breasts. Fresh breast cells grown in the lab quickly lose their hormone receptors for reasons that we still do not understand. Mouse models can be useful, but mouse mammary glands are different from human mammary glands, and the animals tend to get unique types of breast cancers that are not seen in humans. We studied fresh pieces of breast tissue taken from women (with no previous history of breast cancer) undergoing breast reduction surgery and developed a tissue microstructure technique that pre- serves breast cells’ natural environment in the lab. We found that progesterone boosts cell growth in normal breasts. Cells in breast milk ducts start to produce large amounts of a secreted protein called RANKL in response to progesterone. RANKL seems to be the loudspeaker that progesterone uses to command breast cells to proliferate. If these cells are repeatedly stimulated by progesterone, breast cancer risk may increase. Inhibiting progesterone or RANKL with denosumab, a drug already used to treat bone diseases, could potentially benefit women under 50 who are at high-risk of developing breast cancer.

Project coordinator Prof. Dr. Cathrin Brisken

Institut suisse de recherche expérimentale sur le cancer (ISREC)

EPF de Lausanne CH-1015 Lausanne [email protected]

Brown Steven A. | The mechanism of cancer and circa- dian clock interactions and its usefulness in the design of therapeutic strategies (KFS 2642-08-2010)

The circadian clock and the cell cycle are two autono- mous regulatory cascades that control cellular physiol- ogy, metabolism and division. Dysfunction of either can lead to cancer. In our project, we sought to understand the mechanisms linking these two cascades, and how exploiting these connections might lead to improved therapeutic strategies.

Aims of the study

First, we sought to understand whether primary human tumours show the wide range of circadian function shown in tumour cell lines, using glioblastoma multiforme as a model. Second, we wished to understand how the pres- ence or absence of a functional circadian clock could change tumour growth and physiology.

Methods

With the help of virally delivered bioluminescent report- ers, we examined cell-autonomous circadian rhythms of gene expression in cells from different primary tumours. In a second step, we injected clock-containing and simi- lar clock-less tumours expressing the same biolumi- nescent reporters into mice and examined both tumour growth rates and circadian cell division in vivo.

perfectly functional circadian clocks and others no clock at all, even in tumours of the same grade. Surprisingly, in an experimental mouse model no differences in tumour growth rate were observed between clock-containing and clock-less tumours, and both types showed circadian cell division in vivo. Therefore, the host animal’s circadian clock gates tumour cell division systemically, even in the absence of local clock function.

Potential relevance for patients

If tumour growth is gated by host circadian function, then reduction in tumour growth rates might be achieved both by supporting host circadian function and by opti- mized timing of chemo- and radiotherapy.

Project coordinator Prof. Dr. Steven A. Brown

Institut für Pharmakologie und Toxikologie Universität Zürich

CH-8057 Zürich

[email protected]

Christofori Gerhard | The functional role of the transcription factors DIx2 and Lhx2 in epithelial- mesenchymal transition (EMT) and in malignant tumour progression (KLS 2535-02-2010)

Most cancer deaths are due to the systemic dissemination of cancer cells and the formation of secondary tumours (metastasis) in distant organs. The majority of cancers originate from epithelial tissues. To leave the primary tu- mour and to invade into the surrounding tissue, tumour cells dissolve their cell-cell contacts and adjust to a more transient, migratory and invasive mode. This temporary and reversible phenomenon is known as an epithelial-to- mesenchymal transition (EMT), a multistage process that involves a dramatic reprogramming of cancer cells and leads to metastasizing cells with stem cell-like capabili- ties.

We identified an increased expression of the transcription factors Dlx2 and Lhx2 during EMT. Gain and loss of func- tion experiments have delineated the regulatory func- tions of Dlx2 and Lhx2 during EMT in vitro and during metastasis formation in mouse models of cancer in vivo. Employing chromatin-immunoprecipitation experiments, we identified direct targets of Dlx2 and Lhx2 transcrip- tional control and analysed the functional contribution of some of these target genes to EMT and malignant tumour progression. Finally, we assessed whether the gene ex- pression signatures identified here may provide prognos- tic tools for the prediction of clinical outcome in patients. We found that Dlx2 exerts critical functions during malig- nant tumour progression by counteracting TGF -induced cell cycle arrest and apoptosis by at least two molecular mechanisms: Dlx2 acts as a transcriptional repressor of TGF receptor II expression and thus overcomes TGF - mediated cell cycle arrest. On the other hand, Dlx2 in- duces 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 and correlates with tumour malignancy in a variety of human cancer types. For Lhx2 we found that it promotes vessel maturation, primary tumour growth, tumour cell intravasation and metastasis by inducing the expression and secretion of platelet-derived growth factor (PDGF)-B by tumour cells. The data indicate that Lhx2 exerts a dual role in malignant tumour progression by pro- voking autocrine PDGF-B signalling required for meta- static dissemination and paracrine PDGF-B signalling to support blood vessel functionality and primary tumour growth.

Project coordinator

Prof. Dr. phil. Gerhard Christofori Institut für Biochemie und Genetik Departement Biomedizin

Universität Basel CH-4058 Basel

[email protected]

Constam Daniel | Role of activin signalling in metastatic melanoma (KFS 2487-08-2009) Aim of the study

Melanoma, the deadliest type of skin cancer, has a poor prognosis once it forms metastases. What triggers meta- static spread is not well understood. Activin and related secreted growth factors that regulate the development of stem cells in healthy tissues are also found in several can- cers including melanoma, especially during the progres- sion towards metastatic growth. We therefore asked whether such factors promote melanoma progression, and by what mechanism(s).

Methodology

To address this question, we assessed the effects of ac- tivin and the consequences of inhibiting activin by genetic approaches in mice that received grafts of melanoma cells. We also tested whether activin influences the tu- mour cells directly or indirectly by altering their microen- vironment in the host tissue.

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melanoma cell lines formed metastases irrespective of the presence or absence of activin but that tumour-derived activin was responsible for a dramatic loss of body weight, consistent with its known inhibitory effect on muscle stem cells. A similar weight loss was induced by activin in mice with a normal immune system that received mouse mela- noma cells. However, in this context, activin in addition dramatically accelerated the growth of primary tumours and of lung metastases, suggesting that its tumourigenic effect consists largely in suppressing an anti-tumour im- mune response. Concurring with this conclusion, direct effects of activin on melanoma cells themselves varied among cell lines and were not sufficient to promote tu- mourigenesis.

Significance

Metastatic growth and tumour-induced muscle loss are the major cause of cancer deaths, but neither process is sufficiently understood at the molecular level to design effective therapies. Another major challenge is that an ab- normal high frequency of gene mutations enables cancer cells to rapidly develop drug resistance. Our new findings suggest that existing or future activin inhibitors will likely benefit at least a subset of melanoma patients by blocking tumour-induced immune evasion. Moreover, since activin primarily acts on the host immune system rather than on the genetically unstable cancer cells, resistance to such drugs is less likely to develop.

Project coordinator Prof. Dr. Daniel Constam

Institut suisse de recherche expérimentale sur le cancer (ISREC)

EPF de Lausanne CH-1015 Lausanne [email protected]

Gönczy Pierre | Mechanisms of centrosome duplica- tion: from model organism towards therapeutic opportunities (KLS 2584-02-2010)

We focused the bulk of our study on the analysis of two proteins, HsSAS-6 and CPAP, which are necessary for centrosome duplication, a process that is essential for cell proliferation and that is often aberrant in cancer cells. Aims of the study

The centrosome contains two centrioles and is the orga- nelle that ensures microtubule organization in animal cells. Centrosome duplication occurs only in proliferating cells and is necessary for correct cell division. Cancer cells often exhibit aberrations in the number or structure of centrosomes. Therefore, centrosome duplication offers therapeutic opportunities in the fight against cancer. Methods

We combined the advantages of two experimental sys- tems, the nematode C elegans and human cells in culture, to obtain novel information regarding the mechanisms of centrosome duplication, in particular through analysis of the HsSAS-6 and CPAP proteins.

of abnormal centriole number or structure in a stem cell lineage. Moreover, we analysed in detail the way in which HsSAS-6 and CPAP contribute to the formation of centri- oles in human cells in culture. Further, using proteomic methods, we identified proteins that are associated with HsSAS-6 and that may modulate its function in normal cells, as well as in tumour cells. In the long term, we ex- pect this research to lead to the identification of new drugs that target preferentially proliferating cells harbour- ing centrosome aberrations, as is characteristic of numer- ous cancer cells.

Project coordinator Prof. Dr Pierre Gönczy

Institut suisse de recherche expérimentale sur le cancer (ISREC)

EPF de Lausanne CH-1015 Lausanne [email protected]

Grassi Fabio | Purinergic signalling in the pathophysi- ology of central nervous system infiltration in T-cell leukaemia (KFS 2445-08-2009)

Adenosine-triphosphate (ATP) is usually confined inside the cell, where it constitutes the source of chemical en- ergy for cellular functions. ATP is also released by cells as a signalling molecule. In T-cells extracellular ATP regu- lates T-cell proliferation and activity but can also promote cell death via purinergic P2 receptors.

Aim of the study

The aim of this study was to address whether pharmaco- logical inhibition of P2 receptors might be useful in T-acute lymphoblastic leukaemia (T-ALL) therapy and ameliorate central nervous system infiltration, a dramatic complication of T-ALL.

Method and procedure

We tested in vitro the effect of periodate-oxidized ATP (oATP), a P2 receptors inhibitor, on the proliferation of T-ALL cells from mice with leukaemia. Among the leukae- mic cell population, only a small subset of cancer stem cells can initiate, regenerate and maintain leukaemia af- ter transplantation. The haematopoietic niche in the bone marrow constitutes a reservoir of T-ALL stem cells. We addressed whether purinergic antagonism might control haematopoietic stem cells pool size and self-renewal in the bone marrow. Finally, we tested treatment in vivo with oATP in immunodeficient mice transplanted with T-ALL cells.

Results of the study

The treatment with oATP prominently inhibited expansion of T-ALL cells in vitro, thus suggesting that ATP contrib- utes to the expansion of T-ALL cells via P2 receptors. We showed that oATP decreased the number of proliferating cells in the haematopoietic niche of the bone marrow (BM), which is exploited by T-ALL stem cells for constant regeneration, indicating that P2 activation contributes to leukaemogenesis. Since P2 stimulation by ATP can in turn induce ATP release, self-perpetuating release of ATP by T-ALL stem cells might play an important role in control- ling their activity and constant generation of circulating

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leukaemic cells. In vivo treatment with oATP revealed the importance of BM niche as a site of T-ALL proliferation, as a prelude to central nervous system infiltration by leukaemia.

Potential benefits for patients

This research project revealed the importance of ATP in regulating leukaemogenic potential of T-ALL cells resid- ing in the BM and of BM as a prominent site for progres- sion to the neuropathological form of the disease.

Project coordinator Dr Grassi Fabio

Istituto di ricerca in biomedicina (IRB) CH-6500 Bellinzona

[email protected]

Hall Jonathan | Targeting pre-let-7 biogenesis in cancer (KFS 2648-08-2010)

Many human cancers exhibit dysregulation of microRNA expression, in which they are non-functional or over- expressed. A prominent example is the let-7 family, in which let-7 prevents normal cells from becoming tumour- igenic. In several cancers levels of let-7 are very low. This is due to the RNA-binding protein Lin28, which binds to the precursor of let-7, preventing its correct processing. New studies have shown also that the Lin28/let-7 plays an important role in maintenance of cancer stem cells. In the previous project, we synthesized short antisense oli- gonucleotides that bind to the let-7 precursor and protect it from degradation by Lin28. This leads to increased lev- els of let-7 in cells and inhibition of cancer cell growth. One goal of this project was to improve the properties of the antisense oligonucleotides through chemical modifi- cation. This should lead to a stronger effect on the precur- sor and will be examined in mouse cancer models. The results of the study have provided new insights into the mechanisms of anti-microRNA antisense oligonucleotides in living models of cancer.

Project coordinator Prof. Dr. Jonathan Hall

Institut für Pharmazeutische Wissenschaften ETH Zürich

CH-8093 Zürich

[email protected]

Hynes Nancy | Role of the bone marrow niche in breast cancer metastasis and therapy response (KFS 2743-02-2011)

Metastatic breast cancer, the major cause of breast can- cer deaths, is currently not treatable. To uncover new ap- proaches to block metastasis, we established mammary carcinoma models with bone marrow (BM) tropism using tumour cell lines representing common breast cancer sub- types. To characterize BM specific changes, metastatic cancer cells were isolated, and gene expression profiling was carried out and compared to patterns obtained from cancer cells isolated from primary mammary tumours. A number of differentially expressed genes were uncovered.

For example, in bone metastases, the expression of ID1