THE OBJECTIVES OF ONCASYM

The objective of ONCASYM was to find markers to identify adult stem cells in Humans in normal conditions and in conditions of tumorigenesis. This allows us to find and eventually eliminate the cancer stem cells. It also opens the possibility to diagnose tumours based on stemness signature which may help to know their prognoses and potential response to different therapies. The project also aims at finding the factors involved in the asymmetric division of the adult stem cells. The strategy of the project was to employ Drosophila as a gene discovery platform and use two different mammalian systems, the intestinal crypts and the mammary gland, in mouse and humans to test Drosophila and mammalian candidates to find in the adult tissue the stem cells and study them. To achieve this, an important, ambitious goal was to be able to visualize the divisions of the stem cells in situ. These reagents and techniques will allow us to understand the molecular and cellular mechanisms behind the asymmetric division of adult stem cells and how they go wrong generating a tumor in cancer conditions.

WHAT WE ACHIEVED

The ONCASYM consortium has achieved its goals by far:

i) to discover ACD markers to identify the normal and cancer stem cells,

ii) to study the asymmetric cell division of stem cells during normal and tumorigenic growth i

iiI) to use the markers to establish a stem cell signature as a tool in cancer diagnosis and therapy. In particular ONCASYM made six major achievements with a high impact in the field as reflected by 17 publications in top journals such as Cell, Nature and Science:

1. We performed the four Drosophila screens that yielded hundreds of new candidates in ACD and tumor suppression. We generated a top quality list with 17 genes. This list uncovers three novel biological processes in ACD and tumor suppression: targeted motility of Sara signaling endosomes, Ubiquitin-dependent role of the Anaphase Promoting Complex (APC) in ACD, centrosomal-dependent ACD. The genome-wide RNAi screen is published as an Article in Nature, the role of the Sara endosomes as papers in Science and Nature, the role of Brat as a paper in Nature and the role of the centrosomes in ACD and tumor suppression in Developmental Cell, Nature Genetics, Current Biology and JCB.

2. We discovered a pan-stem cell marker, Lgr5 which labels most stem cells in the adult. Furthermore, Lgr5 labels the overproliferating cells in adenoma tumors. Before this discovery, the existence and identity of stem cells, and in particular the cancer stem cells, was based on indirect criteria and soft operational definitions. This discovery made the field of cancer stem cells a very solid one and opened a plethora of experimental possibilities. This was published as articles in Nature (2), Science, Cell Stem Cells and Gastroenterology.

3. We visualized the divisions of the stem cells in the crypt and studied there the lineage and differentiation of the progenitor cells.

4. We established an assay for the labelling, visualization and purification of mammary stem cells. In particular, we imaged by video microscopy the asymmetric division of the mammary stem cells. This was published in two publications in Cell.

5. We identified Numb as a key factor in the asymmetric division of mammary stem cells reported in two publications in Cell.

6. We established a stemness molecular signature for mammary stem cells, published in Cell.

7. We successfully used this molecular signature to stratify breast cancers by tumour grade, clinical prognosis and, in part, molecular subtype. We also identified genes in the signature that can be used as biomarkers for diagnostic, prognostic and therapeutic use in the clinical setting. These results were published in Cell

The successful outcome of this project, according to the points outlined above, has important implications for a number of still unmet clinical needs in the management of breast cancer patients. In fact, based on the unifying concept of our proposal that cancer stem cells (CSCs) are responsible for the development of breast cancer, as well as for therapy failure and disease recurrence, it was evident that stem cells also hold the key for the cure of breast cancer. Therefore, by highlighting crucial molecular traits of stem cell biology we have paved the way for the development, in the forthcoming future, of suitable strategies to exploit CSCs as the weak point of carcinogenesis, in the perspective of a definitive eradication of breast tumors. One such stem cell property, which was fundamental for the maintainance of stem cell homeostasis was the asymmetric kinetics of division, which was the focus of the proposal. This capacity was at the basis of the stem cell self-renewal ability and its subversion leads to expansion of cancer stem cells. This was an aspect of the utmost importance since, in the execution of this project, we have provided knowledge that the intrinsic content of CSCs in tumors, which was increased in biologically aggressive and poor prognosis breast tumors, was predictive of their clinical and molecular characteristics.

The possibility to stratify breast cancers for their pathological, molecular and clinical features based on the degree of molecular resemblance to the stem cell state vis a vis the identification of suitable biomarkers for the identification of CSCs in tissues has immediate clinical applications, which was also one of the declared aims of the original proposal. For instance, based on the positive outcome of this project, a number of retrospective clinical trials are being designed at the IEO, to extensively validate in defined cohorts of breast cancer patients a number of selected stem cell markers for their ability to correlate with the natural evolution of breast cancer, with clinical/pathological parameters and responsiveness to therapies. This is expected to allow the refinement of currently available algorithms for diagnosis, prognosis and prediction of treatment responsiveness through the introduction of novel CSC biomarkers in the routine, and therefore to guide more accurately tailored clinical choices in the clinical management of the breast cancer neoplastic disease.

Several results obtained during the ONCASYM project have, potentially, very important clinical implications. For instance, by characterizing the molecular identity of mammary stem cells (MSCs), Partners 3 and 4 have identified a number of novel stem cell markers (e.g. TOP2A, DLL-1, DNER, Numb), which besides being of great value to research in the MSC field, are also extremely interesting from a clinical perspective (see WP2, 3 and 5). Partners 3 and 4 demonstrated that these markers can also recognize CSCs in breast tumours and, using these markers, they established that aggressive breast tumours (G3) were enriched in CSCs compared with less aggressive tumours (G1). Moreover, they showed that breast cancers can be stratified (for tumour grade, prognosis and tumour subtype) based on their degree of similarity to the MSC molecular phenotype. These studies point to a potential clinical application of these biomarkers in the diagnosis and prognosis of breast cancer.

Another interesting finding from the ONCASYM project, which has important implications for the treatment of breast cancer, was the discovery of Numb as a key regulator of asymmetric cell division in MSCs and normal mammary gland morphogenesis by Partners 3 and 4 (see WP2 and 5). Indeed, Numb ablation in MSCs promotes symmetric (instead of asymmetric) cell division, causes expansion of the stem cell compartment, and results in aberrant mammary gland morphogenesis. Previously, Partners 3 and 4 demonstrated that loss-of-Numb expression occurs in ~50% of breast cancers and correlates with increased activity of the Notch oncogene, as well as high tumour grade and poor prognosis. Taken together, these results suggest that Numb might be critical in the formation of CSCs in Numb-defective tumours. Morover, they point to the potential use of targeted therapies against the Numb-Notch system, such as gamma-secretase inhibitors (GSIs) that block Notch activation, for the treatment of breast cancer.