TGF-beta signaling in carcinogenic transformation of breast epithelial cells, using proteomics
Author: Bhaskaran, Nimesh
Date: 2008-10-09
Location: Radiummhemmets föreläsningssal, Karolinska Sjukhuset
Time: 09.30
Department: Institutionen för onkologi-patologi / Department of Oncology-Pathology
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Thesis (1.044Mb)
Abstract
Transforming growth factor-beta (TGFbeta) is a key regulator of cell proliferation, death, migration and differentiation. One of the features of TGFbeta signaling is to inhibit tumor growth at the early stage of cancer but promotes tumorigenesis at the advanced stage. Such a shift may be explained by differences in expression and activities of components of intracellular signaling.
Studies in this thesis have been focused on the mechanism of TGFbeta signaling in carcinogenic transformation of human breast epithelial cells. Using comprehensive and unbiased proteomics technologies, we identified a number of novel targets of TGFbeta in human breast epithelial cells (MCF10A). The main aim was to identify proteins involved in tumourigenic transformation of these cell lines. In a systemic and network analysis, we identified a number of novel TGFbeta-dependent pathways, whose regulation is pivotal for TGFbeta signaling in breast carcinogenesis. We focused on one such pathway (PLAG1-CNK1-RASSF1A) involved in TGFbeta-dependent ERK1/2 activation.
We observed that TGFbeta1-induced regulation of these proteins was important for TGFbeta1 dependent ERK1/2 activation and TGFbeta1-dependent induction of Erk1/2 phosphorylation was detected predominantly at the endogenous levels of Plag1, CNK1 and RASSF1A. Interestingly, we found the scaffold role of CNK1 provided duality in its effect on Erk1/2 activation i.e. pro-zone effect, not previously known in TGFbeta signaling. Further we were able to show that TGFbeta1 inhibited cell growth under conditions when TGFbeta1 induced Erk1/2 activation and any alteration in expression of these proteins could contribute to the switch of TGFbeta from a tumor suppressor into a tumor promoter.
We were also interested to unravel the mechanism of cell transformation by exploring and comparing proteomes of MCF10A and 184A1 breast epithelial cells. Generation of protein expression maps, identification of functional domains and building proliferation related network lead us to a conclusion that components of mitogenic signaling such as PDGF, PI3K, Rac, Sos, MAP kinases are more represented in MCF10A cells as compared to 184A1. Moreover, we observed that down-regulation of cdk4 and cyclin D3 with specific siRNA inhibited cell proliferation in human breast epithelial cells emphasizing their role in enhancement of cell proliferation.
Many important cellular decisions occur through post translational modifications (PTMs) of proteins. We also identified novel PTMs of Smad2 proteins in human breast carcinoma cell lines using mass spectrometry. Data from peptide mass finger printing (PMF) indicated that Smad2 can be acetylated, methylated, citrullinated, nitrated and palmitoylated. Further sequencing of selected peptides confirmed methylation at Gly122 and hydroxylation at Trp18 of Smad2. We were also able to observe novel modification previously not reported on Try128 and Try151. We therefore were able to show novel modifications on Smad2.
From our proteomics profiling study, we identified XRCC3 a DNA repair protein, whose expression was inhibited by TGFbeta1. We found that XRCC3 is a negative regulator of TGFbeta1-dependent transcriptional activation. Subsequently, we demonstrated that XRCC3 was found in a complex together with Smad3 on CAGA probe. Moreover the binding of XRCC3 on CAGA was specific, as no binding was observed on scrambled CAGA probe. Furthermore, XRCC3 was shown to induce/modulate endogenous expression of TGFbeta1-responsive genes such as c-myc, ccnd1, ccne1, c-fos and cdkn1a which have an important role in cell proliferation and cell cyle. Overexpression of XRCC3 was also observed to increase cell proliferation considerably, as compared to control cells. We therefore establish a novel regulatory mechanism of TGFbeta signaling. Thus, our investigations provided insights into molecular mechanisms of TGFbeta signaling in human breast epithelial cells and in mechanisms of human breast tumorigenesis.
Studies in this thesis have been focused on the mechanism of TGFbeta signaling in carcinogenic transformation of human breast epithelial cells. Using comprehensive and unbiased proteomics technologies, we identified a number of novel targets of TGFbeta in human breast epithelial cells (MCF10A). The main aim was to identify proteins involved in tumourigenic transformation of these cell lines. In a systemic and network analysis, we identified a number of novel TGFbeta-dependent pathways, whose regulation is pivotal for TGFbeta signaling in breast carcinogenesis. We focused on one such pathway (PLAG1-CNK1-RASSF1A) involved in TGFbeta-dependent ERK1/2 activation.
We observed that TGFbeta1-induced regulation of these proteins was important for TGFbeta1 dependent ERK1/2 activation and TGFbeta1-dependent induction of Erk1/2 phosphorylation was detected predominantly at the endogenous levels of Plag1, CNK1 and RASSF1A. Interestingly, we found the scaffold role of CNK1 provided duality in its effect on Erk1/2 activation i.e. pro-zone effect, not previously known in TGFbeta signaling. Further we were able to show that TGFbeta1 inhibited cell growth under conditions when TGFbeta1 induced Erk1/2 activation and any alteration in expression of these proteins could contribute to the switch of TGFbeta from a tumor suppressor into a tumor promoter.
We were also interested to unravel the mechanism of cell transformation by exploring and comparing proteomes of MCF10A and 184A1 breast epithelial cells. Generation of protein expression maps, identification of functional domains and building proliferation related network lead us to a conclusion that components of mitogenic signaling such as PDGF, PI3K, Rac, Sos, MAP kinases are more represented in MCF10A cells as compared to 184A1. Moreover, we observed that down-regulation of cdk4 and cyclin D3 with specific siRNA inhibited cell proliferation in human breast epithelial cells emphasizing their role in enhancement of cell proliferation.
Many important cellular decisions occur through post translational modifications (PTMs) of proteins. We also identified novel PTMs of Smad2 proteins in human breast carcinoma cell lines using mass spectrometry. Data from peptide mass finger printing (PMF) indicated that Smad2 can be acetylated, methylated, citrullinated, nitrated and palmitoylated. Further sequencing of selected peptides confirmed methylation at Gly122 and hydroxylation at Trp18 of Smad2. We were also able to observe novel modification previously not reported on Try128 and Try151. We therefore were able to show novel modifications on Smad2.
From our proteomics profiling study, we identified XRCC3 a DNA repair protein, whose expression was inhibited by TGFbeta1. We found that XRCC3 is a negative regulator of TGFbeta1-dependent transcriptional activation. Subsequently, we demonstrated that XRCC3 was found in a complex together with Smad3 on CAGA probe. Moreover the binding of XRCC3 on CAGA was specific, as no binding was observed on scrambled CAGA probe. Furthermore, XRCC3 was shown to induce/modulate endogenous expression of TGFbeta1-responsive genes such as c-myc, ccnd1, ccne1, c-fos and cdkn1a which have an important role in cell proliferation and cell cyle. Overexpression of XRCC3 was also observed to increase cell proliferation considerably, as compared to control cells. We therefore establish a novel regulatory mechanism of TGFbeta signaling. Thus, our investigations provided insights into molecular mechanisms of TGFbeta signaling in human breast epithelial cells and in mechanisms of human breast tumorigenesis.
List of papers:
I. Bhaskaran N, Souchelnytskyi S (2008). Systemic analysis of TGFbeta proteomics revealed involvement of Plag1/CNK1/ RASSF1A/Src network in TGFbeta 1-dependent activation of Erk1/2 and cell proliferation. Proteomics. 8. [Accepted]
Fulltext (DOI)
Pubmed
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II. Bhaskaran N, Lin KW, Gautier, Woksepp H, Hellman U, Souchelnytskyi S (2009). Comparative proteome profiling of MCF10A and 184A1 human breast epithelial cells emphasized involvement of CDK4 and Cyclin D3 in cell proliferation. Proteomics Clinical Applications 1. [Accepted]
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Bhaskaran N, Iwahana H, Bergquist J, Hellman U, Souchelnytskyi S (2008). Novel post-translational modifications of Smad2 identified by mass spectrometry. Central European Journal of Biology. [Accepted]
Fulltext (DOI)
View record in Web of Science®
IV. Bhaskaran N, Souchelnytskyi S (2008). XRCC3 inhibits TGFbeta signaling. [Submitted]
I. Bhaskaran N, Souchelnytskyi S (2008). Systemic analysis of TGFbeta proteomics revealed involvement of Plag1/CNK1/ RASSF1A/Src network in TGFbeta 1-dependent activation of Erk1/2 and cell proliferation. Proteomics. 8. [Accepted]
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Bhaskaran N, Lin KW, Gautier, Woksepp H, Hellman U, Souchelnytskyi S (2009). Comparative proteome profiling of MCF10A and 184A1 human breast epithelial cells emphasized involvement of CDK4 and Cyclin D3 in cell proliferation. Proteomics Clinical Applications 1. [Accepted]
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Bhaskaran N, Iwahana H, Bergquist J, Hellman U, Souchelnytskyi S (2008). Novel post-translational modifications of Smad2 identified by mass spectrometry. Central European Journal of Biology. [Accepted]
Fulltext (DOI)
View record in Web of Science®
IV. Bhaskaran N, Souchelnytskyi S (2008). XRCC3 inhibits TGFbeta signaling. [Submitted]
Issue date: 2008-09-18
Rights:
Publication year: 2008
ISBN: 978-91-7409-135-9
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