Is There a Malignant Progression Associated with a Linear Change in Protein Expression Levels from Normal Canine Mammary Gland to Metastatic Mammary Tumors?

Klose, P.; Weise, Christoph; Bondzio, Angelika; Multhaup, Gerd; Einspanier, Ralf; Gruber, Achim D.; Klopfleisch, Robert

doi:10.1021/pr200112q

Cited by 30 publications

(16 citation statements)

References 36 publications

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“…PIP bead-binding proteins were identified by matrix-assisted laser desorption ionisation-time of flight mass spectrometry-based peptide mass fingerprinting as described previously [65]. …”

Section: Methodsmentioning

confidence: 99%

BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5β recruitment at the cytocortex

et al. 2014

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BackgroundBMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex.ResultsWe unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5β to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55γ or LL5β or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses.ConclusionsOur results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3-LL5β signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2–induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts.

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“…PIP bead-binding proteins were identified by matrix-assisted laser desorption ionisation-time of flight mass spectrometry-based peptide mass fingerprinting as described previously [65]. …”

Section: Methodsmentioning

confidence: 99%

BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5β recruitment at the cytocortex

et al. 2014

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“…The second-dimension molecular weight separation was carried out using an Ettan DALTsix Electrophoresis Unit (GE Healthcare, Freiburg, Germany). Running parameters used were 60 mA for 1 hour, 240 mA for 1 hour and 300 mA for 5 hours [31]. …”

Section: Methodsmentioning

confidence: 99%

Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

et al. 2012

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BackgroundCanine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition.ResultsTranscriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1.ConclusionsTranscriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect.

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“…Furthermore, tear fluid analysis found changes in the amount of actin, albumin and an unidentified protein in dogs with different kinds of cancer (de Freitas Campos et al, 2008). MS-based proteome analysis has also been applied to solid tumour lysates, including canine lymphomas, MCTs and mammary tumours (McCaw et al, 2007;Klopfleisch et al, 2010b;Klose et al, 2011;Schlieben et al, 2012Schlieben et al, , 2013Gorges and Pantel, 2013). The results of studies on serum or solid tumours have, however, not been translated into clinically relevant assays.…”

Section: Personalized Medicine In Veterinary Oncology: Where Are We?mentioning

confidence: 99%

Personalised medicine in veterinary oncology: One to cure just one

Klopfleisch

2015

The Veterinary Journal

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Is There a Malignant Progression Associated with a Linear Change in Protein Expression Levels from Normal Canine Mammary Gland to Metastatic Mammary Tumors?

Cited by 30 publications

References 36 publications

BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5β recruitment at the cytocortex

BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5β recruitment at the cytocortex

Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

Personalised medicine in veterinary oncology: One to cure just one

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