Integrated signalling group

ò(I+m+E)

Serhiy Souchelnytskyi

Group Leader

PKC-dependent phosphorylation of Smad3 promotes loss of contact inhibition
Cells stably transfected with Smad3 constructs. Foci formation assay
(Yakymovych et al. FASEB J.)

Smad2 inhibited cell growth in tumor plugs.Tumorigenesis assay
(Sjöblom, Yakymovych et al. Eur J. Cancer)

TGFbeta/Smad3 counteracts BRCA1-dependent DNA damage repair. Comet assay (Dubrovska et al., Oncogene)

Development of inhibitors of TGFb receptor kinase. Active centre of the kinase with indication of the sites of interest (Yakymovych et al., JBC, 2004; Biochemistry, 2002)

Phosphoproteomics of human breast epithelial cells. Fe-IMAC gel of 32P-labeled proteins (Dubrovska et al., Proteomics, 2005)

HMVEC proteome
(Lomnytska et al. Proteomics)

Mv1Lu proteome
(Kanamoto et al.
EMBO J.)

BMPR-II interacting proteins
(Hassel et al. Proteomics)

Identification of markers of breast and ovarian cancer in plasma
(Lomnytska et al., Int.J.Cancer)

Phosphoproteomics of TGFbeta signalling
TFII phosphorylation
Regulation of cell cycle
(Stasyk, Dubrovska et al., Mol. Biol. Cell)

Our group is concentrated on exploration of human breast cancer. We study carcinogenic transformation of breast epithelial cells using proteomics, and investigate TGFß signalling in normal and malignant cells. Understaning of how intracellular signalling processes integrate in cells is our ultimate goal.

* Carcinogenesis is a complex process of acquiring by normal cells ability to uncontrolled and unlimited proliferation, escaping death-inducing signals, and developing capacities to spread in and conquer the body. Proteomics, being unbiased and comprehensive technology, is an efficient tool to unveil novel mechanisms of carcinogenic transformation. Directed studies, on the other side, allow in-depth explorations of selected regulatory mechanisms.
* We use proteomics for profiling of carcinogenic transformation of human breast epithelial cells. Proteomics is also used for profiling of TGFß signalling in human breast epithelial cells: protein expression, turnover and phosphorylation are studied.
* Systems biology is used to explore mechanisms of tumorigenic transformation of breast epithelial cells.
* In our directed studies, we explore how TGFß signalling works. Of particular interest are cross-talk between TGFß signalling components and tumor suppressors and oncogenes, with emphasis on mechanisms regulating DNA damage repair and DNA damage-induced signalling.

Proteomics in study of breast cancer

Less than 10 % of human cancers can be linked to malfunction of one gene. In other more than 90 % of cases, a number of genes and their products are involved in tumorigenesis. Therefore, global analysis of proteome of cells undergoing tumorigenic transformation may provide insights into molecular mechanisms of cancer. Proteomics may also lead to discovery of novel tumor suppressors and oncogenes. Moreover, more than 90 % of drug targets are proteins, and novel drugs targeting specific signalling pathways are also targeting proteins.

We use proteomics to explore cancer-related changes in proteomes of human breast epithelial cells. We concentrate on studies of carcinogenic transformation. For these studies we use human breast tumors and non-cancerous tissues, primary human breast epithelial cells and cultured cells of variuos degree of transformation.

We also use proteomics technique to elucidate the role of TGFß signalling in human breast carcinogenesis. We analyze protein expression and post-translational modifications, with emphasis on O-phosphorylation and O-glycosylation. We also analyze proteins forming complexes with components of TGFß signalling, e.g. with Smads and receptors. In various proteomics-based projects, we have identified more than 500 proteins affected by TGFß. These targets unveiled novel regulatory mechanisms of TGFß signalling. As an example, TGFß-dependent regulation of DNA damage repair has been explored.

Integration of proteomics and systems biology
The data generated are being used for developing of model(s) of carcinogenic transformation of breast epithelial cells and of integrated signalling with participation of TGFß pathway(s). We use proteomics data and results of in-depth studies as primary datasets, analyse them for functional and time-dependent clusters to define architecture of a model, and simulate behaviour of the defined model.

Below: GoMiner-based presentation of functional clustering of TGFbeta-regulated proteins in human breast epithelial cells is shown below. Clustering tree of proteins involved in regulation of cellular physiological processes is shown.

Below: Simulation of TFII-I- and Smad3-dependent regulation of gene transcription allows prediction of the treshold in gene expression (see also Stasyk, Dubrovska et al., Mol.Biol.Cell., 2005)

The integration of proteomics and large-scale modeling is required for the description of complex biological systems. Methods for such integration are now under development, and propose the most efficient design for proteomics experiments (see the figure below). First, proteomics experiments describe a number of proteins of interest for a selected biological process. These protein species are annotated using dedicated software and are represented in XML format, with quantitative descriptions of PTMs and interacting partners. Information about the localization of proteins is recorded, and all data are collected in real-time dynamics. This dataset is used to build a model to unveil dependencies between protein species. Then, the model describing these dependencies is interrogated in biological experiments with the modulation of expression and activities of identified key proteins. This validates the architecture of the model, and defines the levels of robustness and sensitivity of the studied biological process. Knowledge of crucial components of a system will lead to development of drugs which will target these components, and will achieve high efficiency of treatment with negligible side effects (Souchelnytskyi, Proteomics, 2005).

TGFß signalling in cancer

TGFß superfamily members regulate cell growth,differentiation, motility and apoptosis. Failure or dysregulation of the TGFß signalling is involved in development of several diseases, such as cancer, haemorragic telangiectasia, fibrotic disorders, impaired wound healing, neurodegenerative conditions, developmental disorders.

TGFß family members bind to receptors with intrinsic serine/threonine kinase activity. Upon ligand binding the receptors form an active heterotetrameric complex which triggers down-stream intracellular signalling. Smad proteins were shown to be a direct link between the activated receptors and genes to be regulated.

We concentrate our efforts on studying of mechanisms of receptor activation, Smad-dependent and Smad-independent signalling down-stream of the receptors. These studies aim to elucidate the role of TGFß signalling in carcinogenesis.

We investigate how activity of type I and type II receptors is regulated. The subject of our studies is search for antagonists and agonists of the receptor kinases.
Discovery of the Smad proteins provided a break-through in understanding of signal transduction down-stream of the receptors. We showed importance of the receptor-dependent sequential phosphorylation of R-Smads for the triggering of the signalling. However, Smad proteins are substrates not only of the receptors, but also of other kinases. These kinases and their impact on the TGFß and BMP signalling are under investigation.

Beside the phosphorylation, Smad proteins may have other type(s) of post-translational modifications (our observation), what is the subject of investigations. Direct targets of Smads are explored with use of the Proteomics technique; multiple ligand and/or Smad dependent changes have been found. Identification of Smad interacting proteins is in the scope of our interest as well.
Investigation of the Smad-independent pathway(s) down-stream of the receptors is ongoing in our lab. Regulatory pathways related to the progression of tumorigenesis, are of special interest.

Interaction of the TGFß signalling with pathways shown to play an important role in development of breast cancers is another high priority aim of our group.We explore TGFß role in regulation of DNA damage repair. The cross-talks of TGFß signalling with BRCA1 and BRCA2 are of particular interest.

- Hassel S., Eichner A., Yakymovych M., Hellman U., Knaus P., Souchelnytskyi S. (2004) Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by two-dimensional gel electrophoresis and mass spectrometry. PROTEOMICS, 4, 1346-1358.
- Lomnytska M., Lukiyanchuk V., Hellman U., Souchelnytskyi S. (2004) Functional TGFß1-regulated proteins in human endothelial cells identified by two-dimensional gel electrophoresis and mass spectrometry. PROTEOMICS, 4, 995-1006.
- Sjöblom T., Yakymovych I., Heldin C-H., Östman A., Souchelnytskyi S. (2004) Smad2 suppresses growth of Mv1Lu cells subcutaneously inoculated in mice. Eur. J. Cancer, 40, 267-274..
- Stoika R., Yakymovych M., Souchelnytskyi S., Yakymovych I. (2003) Potential role of transforming growth factor beta1 in drug resistance of tumor cells. Acta Biochimica Polonica, 50, 497-508.
- Salovaara R., Roth S., Loukola A. Launonen V., Sistonen P., Aviziente E., Kristo P., Järvinen H., Souchelnytskyi S., Sarlomo-Rikala M., Aaltonen L.A. (2002) Frequent loss of Smad4/DPC4 protein in colorectal cancers. J Clin Pathol: Mol. Pathol., 55, 385-388.

- Souchelnytskyi, S. ten Dijke, P., Miyazono, K. Heldin C.-H. (1996) Phosphorylation of Ser165 in TGF-b type I receptor modulates TGF-b1-induced cellular responses. EMBO J., 15, 6231-6240.