Final Report Summary - TRPV2PI3KMACROPHAGE (Analysis of the roles of the TRPV2 calcium channel in inflammation)
Breast cancer is the most common cancer in women with 13. 8 million new cases in the world in 2008 (23 % of all cancer except for the non melanoma skin cancer) [1]. Although the survival rate is improving, breast cancer is still responsible for the biggest part of cancer related death in women (13. 7 %). Thus it is important to get a better understanding of the molecular events regulating the functioning of human breast epithelial cells to improve the current anti-cancer treatment.
Phosphoinositide 3-kinases (PI3K) are signalling enzymes known to regulate many central cellular processes. The class Ia PI3K are heterodimers of one catalytic subunit (p110 alpha, beta, delta) and one regulatory subunit (p85, p55 or p50) [2]. After their activation downstream of a membrane receptor these enzymes phosphorylate the phospholipids PI (4,
5) P2 into PIP3 [2]. It will then convert the initial stimulation into a cellular function through the activation of effectors like Protein Kinase B (PKB) and downstream signalling. Modifications of the PI3K pathway are frequently found in breast tumours: mutations of p110alpha (present in 20-25 % of breast tumours) or PKB, amplification of Epidermal Growth Factor (EGF) Receptor, deletion of Phosphatase and tensin homolog (PTEN). All lead to the overactivation of the system [3]. Increasing the current knowledge of the PI3K pathway and implication of its modification in breast cell represents a key step in the cancer research.
Our study aims at defining the distinct mechanisms that are induced specifically by each class I PI3K isoform as well as the effects of individual mutant expression of in human breast epithelial cells.
We are using isogenic human breast epithelial cell lines with homologously targeted modification of different PI3K signalling components (p110alpha, PTEN, PKB). PI3K activity in those cells is determined by the measurement of both PKB phosphorylation [4, 5, 6] and PIP3 levels upon EGF stimulation alone or combined with the use of a range of PI3K inhibitors. A new method was recently set up in the lab allowing both identification and quantification of the different PIP3 species in various conditions, cell types and tissues [7]. We found that PKB phosphorylation and PIP3 levels in PTEN deficient cells were greater than in the WT cells both in basal or stimulated conditions [7]. Also the EGF-induced PKB phosphorylation was p110 alpha dependant in both WT and PTEN null cells.
The effects of each stimulation, inhibition and mutations on functional outcome of the PI3K signalling are also analysed. Chemokinesis is the non directional hormone-stimulated migration of cells and is relevant to wound healing, tumour cell invasiveness and inflammation. PI3K is a central actor in the regulation of the movement of many cell types [8, 9, 10]. We analysed which PI3K isoform and effectors were involved in chemokinesis of human breast epithelial cells. We found that EGF-induced chemokinesis is p110 alpha and PTEN-dependant but does not seem to be mediated by PKB.
In correlation with previous work [10, 11, 12], our data show the importance of p110 alpha in breast cells migration and highlight its role in the regulation of motion rather than direction. They also indicate that other effectors than PKB are involved. Their identification should lead to the eventual development of new anti-cancer drugs.
[1] http://globocan. iarc. fr/factsheets/populations/factsheet. asp? uno = 900; [2] Hawkins PT et al. Bioch Soc Trans (2006) 34 (Pt 5): 647; [3] Baselga J et al. The Oncol (2011) 16: 12; [4] Stephens L et al. Science (1998) 279: 710; [5] Sarbassov DD et al. Science (2005) 307: 1098; [6] Jacinto E et al. Cell (2006) 127: 125; [7] Clark J et al. Nat Methods (2010) 8: 267; [8] Ferguson GJ et al. Nat Cell Biol (2007) 9: 86; [9] Saudemont A et Colucci F. Cell Cycle (2009) 8: 3307; [10] Meng Q et al. Cell Sig (2006) 18: 2262; [11] Hill K et al. J Biol Chem (2000) 275: 3741; [12] Yip SC et al. Cell Mot Cytosk (2004) 59: 180-188
Phosphoinositide 3-kinases (PI3K) are signalling enzymes known to regulate many central cellular processes. The class Ia PI3K are heterodimers of one catalytic subunit (p110 alpha, beta, delta) and one regulatory subunit (p85, p55 or p50) [2]. After their activation downstream of a membrane receptor these enzymes phosphorylate the phospholipids PI (4,
5) P2 into PIP3 [2]. It will then convert the initial stimulation into a cellular function through the activation of effectors like Protein Kinase B (PKB) and downstream signalling. Modifications of the PI3K pathway are frequently found in breast tumours: mutations of p110alpha (present in 20-25 % of breast tumours) or PKB, amplification of Epidermal Growth Factor (EGF) Receptor, deletion of Phosphatase and tensin homolog (PTEN). All lead to the overactivation of the system [3]. Increasing the current knowledge of the PI3K pathway and implication of its modification in breast cell represents a key step in the cancer research.
Our study aims at defining the distinct mechanisms that are induced specifically by each class I PI3K isoform as well as the effects of individual mutant expression of in human breast epithelial cells.
We are using isogenic human breast epithelial cell lines with homologously targeted modification of different PI3K signalling components (p110alpha, PTEN, PKB). PI3K activity in those cells is determined by the measurement of both PKB phosphorylation [4, 5, 6] and PIP3 levels upon EGF stimulation alone or combined with the use of a range of PI3K inhibitors. A new method was recently set up in the lab allowing both identification and quantification of the different PIP3 species in various conditions, cell types and tissues [7]. We found that PKB phosphorylation and PIP3 levels in PTEN deficient cells were greater than in the WT cells both in basal or stimulated conditions [7]. Also the EGF-induced PKB phosphorylation was p110 alpha dependant in both WT and PTEN null cells.
The effects of each stimulation, inhibition and mutations on functional outcome of the PI3K signalling are also analysed. Chemokinesis is the non directional hormone-stimulated migration of cells and is relevant to wound healing, tumour cell invasiveness and inflammation. PI3K is a central actor in the regulation of the movement of many cell types [8, 9, 10]. We analysed which PI3K isoform and effectors were involved in chemokinesis of human breast epithelial cells. We found that EGF-induced chemokinesis is p110 alpha and PTEN-dependant but does not seem to be mediated by PKB.
In correlation with previous work [10, 11, 12], our data show the importance of p110 alpha in breast cells migration and highlight its role in the regulation of motion rather than direction. They also indicate that other effectors than PKB are involved. Their identification should lead to the eventual development of new anti-cancer drugs.
[1] http://globocan. iarc. fr/factsheets/populations/factsheet. asp? uno = 900; [2] Hawkins PT et al. Bioch Soc Trans (2006) 34 (Pt 5): 647; [3] Baselga J et al. The Oncol (2011) 16: 12; [4] Stephens L et al. Science (1998) 279: 710; [5] Sarbassov DD et al. Science (2005) 307: 1098; [6] Jacinto E et al. Cell (2006) 127: 125; [7] Clark J et al. Nat Methods (2010) 8: 267; [8] Ferguson GJ et al. Nat Cell Biol (2007) 9: 86; [9] Saudemont A et Colucci F. Cell Cycle (2009) 8: 3307; [10] Meng Q et al. Cell Sig (2006) 18: 2262; [11] Hill K et al. J Biol Chem (2000) 275: 3741; [12] Yip SC et al. Cell Mot Cytosk (2004) 59: 180-188