Funded projects

Analysis of centrosomes in ovarian cancer

2013 - R. Basto (UMR144), O. Goundiam (team Sastre, Hospital), X. Sastre (Hospital), JF. Joanny (UMR168), E. Barillot (U900), P. Hupé (U900)

Centrosomes are the main microtubule organizing centers in animal cells. They are implicated in different cellular processes such as polarity establishment and maintenance or mitosis. Centrosomes comprise of a pair of centrioles and the surrounding pericentriolar material (PCM). The PCM consists of a meshwork of several proteins and is the site of microtubule nucleation. Each centriole consists of 9 sets of microtubules which form a cylinder with a length of ~0.5 μm and a diameter of 0.2μm (Azimzadeh and Bornens, 2007; Bornens, 2002). During S-phase, centrioles, like DNA, normally duplicate in a semi-conservative fashion once per cell cycle, ensuring that each daughter cell inherits one centrosome at the end of cell division (Godinho et al., 2009). Perturbations in the centrosome duplication cycle can cause centrosome amplification, a condition that favors chromosome instability and may lead to tumorigenesis (Basto et al., 2008).

Centrosome abnormalities are more and more frequently detected in both solid and haematological human malignancies. The defects observed in human cancers may be classified into two main categories related on either structural or functional alterations (Chan, 2011). Structural defects include changes in centrosome shape, size, number, position and/or composition. Functionally defective centrosomes may be responsible for abnormal microtubule nucleation, and formation of disorganized mitotic spindles, leading to chromosomal missegregation and aneuploidy. Cells with more than two centrosomes are prone to multipolar mitoses and cell death. However, in cancer cells, extra centrosomes can be clustered into two groups to achieve a bipolar mitosis (Godinho et al., 2009). Extra centrosomes may promote directional migration of malignant cells (Ogden et al., 2012). Regulator pathways of clustering mechanism are not completely understood. Inhibition of centrosomal clustering (for example using a PARP derived inhibitor) is linked to antitumor activity in vitro and in vivo (Castiel et al., 2011; Korzeniewski et al., 2013; Ogden et al., 2012).

Many proteins that are frequently mutated in cancers participate to the maintenance of the numeral integrity of centrosomes and to the regulation of centrosome duplication. These proteins belong to one of the three following functional groups: (1) cell-cycle regulation (G1 CDK-Cyclin kinase complexes and their downstream targets such as CDK2-cyclinE), (2) DNA-damage response and/or repair (BRCA1/2), and (3) nucleocytoplasmic transport (nuclear import and export proteins). A number of studies have shown that loss or inactivation of the proteins involved in homologous recombination repair also results in centrosome amplification and in chromosome instability (Fukasawa, 2007). Besides, some kinesins possess chromatin binding activity and can modulate DNA damage response.

Even if centrosome amplification, as other types of centrosomal defects, has been described in human tumours, in epithelial ovarian cancer (EOC), a thorough and complete analysis has never been made so far. EOC is the second most common gynecologic malignancy. The treatment of this disease is based on the association of surgery and chemotherapy. Whereas most cases are sensitive to chemotherapy, a high rate of relapse is observed and only 20-25% of patients are successfully treated. In this context, a research program on ovarian cancer has been initiated in the Department of Biopathology of the Institut Curie (Xavier Sastre team), in close association with Research Department (Renata Basto team, UMR144).

This project will be divided in three main parts:

I) Analysis of centrosome structural abnormalities

Using fluorescent microscopy, we have initiated an analysis of centrosome markers in cell lines and tissues derived from EOC. So far, we have optimized the procedure in order to consider different centrosome markers such as centriole and PCM proteins. We classified ovarian carcinoma according

to centrosomal structural defects and we correlated the result to clinical parameters. We would like to further extend our analysis to a larger set of samples in order to detect other types of abnormalities. A visual charcaterisation of the centrosome from different tumour grades will be done. We will pay particular attention to parameters such as centrosome volume, size, pericentriolar and/or centriolar origin of dysfunctions, position relative to the membrane, position relative to the nucleus, behaviour during mitosis clustering, potential involvement in spindle formation, etc. In addition, we will us the expertise of the team of Jean Fracois Joanny (UMR168) to model the behaviour of extra centrosomes. Initially this will be done from a collection of movies already available from the Basto lab. In these movies, centrosome clustering and inactivation has been followed during mitosis. The analysis from a physical modeling point of view will allow us to detect the existence of a particular pattern of extra centrosome behaviour during mitosis. This type of analysis and modeling would then be extended to the analysis of centrosome behaviour in primary ovarian tumour cell lines. This type of approach will help us to understand the underlying mechanisms that regulate bipolarization when extra centrosomes are present.

II) Identification and validation of key molecules (centrosomal proteins and/or regulators)

In order to identify genomic and molecular abnormalities in ovarian tumours, 126 cases of ovarian primary tumors have been analyzed using Affymetrix 100k SNP Array (genomic profiles) and Affymetrix GeneChip Human Genome U133plus2.0 (transcriptomic profiles). From these results, we identified a list of amplified/overexpressed centrosomal molecules or molecules implicated in centrosomes regulation. We focused on five of them because:

– Unsupervised hierarchical clustering of their mRNA expression showed 3 subgroups of patients

– Survival analysis (progression free survival, overall survival) of these 3 subgroups showed significant differences according to log-rank test (Kaplan-Meier curves).

The robustness of this signature has to be confirmed using other bioinformatic tools and a larger series of tumors (for example public available data). Biological validations are in progress to check if the targets are differentially expressed in our groups at mRNA (RT-PCR technique) and proteins (western blot technique) level. Further functional studies (in vitro and preclinical) will be required to confirm this signature, to document the potential prognostic value of these genes/proteins and to define if some of them are potential therapeutic targets.

III) Link between centrosome amplification and DNA repair

We intend to analyze the link between DNA repair pathways and centrosomes structural/molecular defects in ovarian cancer. This part of the project will be in collaboration with U900 of Institut Curie (Emmanuel Barillot Team). We investigated in recent studies whether BRCA1/2 protein expression levels may represent a biomarker of survival in epithelial ovarian cancer. Our results demonstrated that patients with absence of BRCA1 protein expression have a significantly improved overall survival and progression free survival (Kaplan-Meier statistical analysis) in comparison with patients with positive BRCA1 protein expression. We would like to analyze centrosomes status in these 2 groups of patients to correlate precisely the role of DNA repair pathway in centrosomes regulation. Using tools and methodologies mastered into the team (see http://acsn.curie.fr and (Calzone et al., 2008) and a pre-existing map of DNA repair molecular signalling (Kuperstein et al, in prep), we will construct a map of key molecules implicated in centrosome regulation and DNA repair, recapitulating their interaction network from literature mining. We will then use this map to analyse the molecular profiles of the different categories of ovarian tumors, define a network-based anatomy of ovarian cancers, and link DNA repair signatures to centrosome phenotypes. In particular the network analysis should help understanding the interplay between centrosome regulation and DNA repair.

This project will be the most complete analysis of the centrosomes abnormalities in human ovarian cancer using pluridisciplinary approaches. Our study will help to highlight the potential relevance of centrosome regulation in the definition of new therapeutic approaches.

References

  • Azimzadeh, J., and Bornens, M. (2007). Structure and duplication of the centrosome. Journal of cell science 120, 2139-2142.
  • Basto, R., Brunk, K., Vinadogrova, T., Peel, N., Franz, A., Khodjakov, A., and Raff, J.W. (2008). Centrosome amplification can initiate tumorigenesis in flies. Cell 133, 1032-1042.
  • Bornens, M. (2002). Centrosome composition and microtubule anchoring mechanisms. Current opinion in cell biology 14, 25-34.
  • Calzone, L., Gelay, A., Zinovyev, A., Radvanyi, F., and Barillot, E. (2008). A comprehensive modular map of molecular interactions in RB/E2F pathway. Molecular systems biology 4, 173.
  • Castiel, A., Visochek, L., Mittelman, L., Dantzer, F., Izraeli, S., and Cohen-Armon, M. (2011). A phenanthrene derived PARP inhibitor is an extra-centrosomes de-clustering agent exclusively eradicating human cancer cells. BMC cancer 11, 412.
  • Chan, J.Y. (2011). A clinical overview of centrosome amplification in human cancers. International journal of biological sciences 7, 1122-1144.
  • Fukasawa, K. (2007). Oncogenes and tumour suppressors take on centrosomes. Nature reviews Cancer 7, 911-924.
  • Godinho, S.A., Kwon, M., and Pellman, D. (2009). Centrosomes and cancer: how cancer cells divide with too many centrosomes. Cancer metastasis reviews 28, 85-98.
  • Korzeniewski, N., Hohenfellner, M., and Duensing, S. (2013). The centrosome as potential target for cancer therapy and prevention. Expert opinion on therapeutic targets 17, 43-52.
  • Ogden, A., Rida, P.C., and Aneja, R. (2012). Heading off with the herd: how cancer cells might maneuver supernumerary centrosomes for directional migration. Cancer metastasis reviews.