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Cell aggregation induces phosphorylation of PECAM-1 and Pyk2 and promotes tumor cell anchorage-independent growth.

Zhang X, Xu LH, Yu Q - Mol. Cancer (2010)

Bottom Line: Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood.Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates.We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Shanghai Institute of Materia Medica, China Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China. xingzhangg@hotmail.com

ABSTRACT

Background: Apoptosis caused by inadequate or inappropriate cell-matrix interactions is defined as anoikis. Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood. We investigated the mechanisms of anoikis resistance of tumor cells.

Results: We observed that cell aggregation in suspension promoted cell survival and proliferation. We demonstrated a correlation between tumor cell aggregation in suspension and cell growth in soft agar. Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates. We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2. Furthermore, RNA interference-mediated reduction of Pyk2 and PECAM-1 protein levels reduced cell aggregation and inhibited the growth of tumor cells in soft agar.

Conclusions: The data demonstrated that Pyk2 and PECAM-1 were critical mediators of both anchorage-independent growth and anoikis resistance in tumor cells.

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Induction of phosphorylation of Pyk2 and PECAM by cell aggregation. A, Phosphorylation of Pyk2 and PECAM in high versus low cell density cultures. Different concentrations of HBE, SK-LU-1, or H460 cells (H: 5.0 × 105 cells L: 2.5 × 105 cells) were cultured in 60-mm polyHEMA-coated dishes for 0, 6, or 15 h. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-Pyk2 (Tyr881), or anti-Pyk2 antibody (left). Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with 4G10 antibody (Right). B. Phosphorylation of Pyk2 and PECAM in regular suspension versus methyl-cellulose suspension cultures. H1792 and H460 cells were cultured in polyHEMA-coated dishes (S) or methyl-cellulose dishes (MS) for 6 or 15 h, harvested, and lysed with RIPA buffer. A, Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with anti-phosphotyrosine 4G10 antibody (Upper). Equal amounts of whole-cell extracts were immunoblotted with anti-pPyk2 (Tyr881) or anti-Pyk2 antibody (Lower).
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Figure 3: Induction of phosphorylation of Pyk2 and PECAM by cell aggregation. A, Phosphorylation of Pyk2 and PECAM in high versus low cell density cultures. Different concentrations of HBE, SK-LU-1, or H460 cells (H: 5.0 × 105 cells L: 2.5 × 105 cells) were cultured in 60-mm polyHEMA-coated dishes for 0, 6, or 15 h. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-Pyk2 (Tyr881), or anti-Pyk2 antibody (left). Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with 4G10 antibody (Right). B. Phosphorylation of Pyk2 and PECAM in regular suspension versus methyl-cellulose suspension cultures. H1792 and H460 cells were cultured in polyHEMA-coated dishes (S) or methyl-cellulose dishes (MS) for 6 or 15 h, harvested, and lysed with RIPA buffer. A, Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with anti-phosphotyrosine 4G10 antibody (Upper). Equal amounts of whole-cell extracts were immunoblotted with anti-pPyk2 (Tyr881) or anti-Pyk2 antibody (Lower).

Mentions: In order to understand the molecular nature of the cell aggregation-generated survival and growth signals, we analyzed the phosphorylation/activation of PECAM-1 and Pyk2. PECAM protein was immunoprecipitated with PECAM antibody and then immunoblotted with 4G10 antibody (anti-phosphotyrosine monoclonal antibody) to detect tyrosine phosphorylation. Interestingly, we found that the phosphorylation/activation of PECAM-1 and Pyk2 correlated with cell aggregation, although the total protein level of PECAM-1 and Pyk2 were not related to cell aggregation (Fig. 3A, B and Additional file 1 Figure S1). High- and low-density suspension cultures were established in order to study the phosphorylation status of these molecules in relation to cell aggregation. H460, SK-LU-1, and HBE cells formed larger aggregates in high-density cultures compared to low-density cultures. H460 and H1792 formed larger aggregates in polyHEMA cultures compared to methyl cellulose cultures. We observed higher levels of phosphorylated PECAM-1 and Pyk2 in large aggregates (high-density cultures, H) than in small aggregates or single cells (low-density cultures, L) (Fig. 3A)Similarly, higher levels of phosphorylated PECAM-1 and Pyk2 were seen when cells were cultured in large aggregates (polyHEMA suspension culture, S) compared to small aggregates (methyl cellulose suspension culture, MS) (Fig. 3B), suggesting that the phosphorylation/activation of PECAM-1 and Pyk2 correlated with cell aggregation.


Cell aggregation induces phosphorylation of PECAM-1 and Pyk2 and promotes tumor cell anchorage-independent growth.

Zhang X, Xu LH, Yu Q - Mol. Cancer (2010)

Induction of phosphorylation of Pyk2 and PECAM by cell aggregation. A, Phosphorylation of Pyk2 and PECAM in high versus low cell density cultures. Different concentrations of HBE, SK-LU-1, or H460 cells (H: 5.0 × 105 cells L: 2.5 × 105 cells) were cultured in 60-mm polyHEMA-coated dishes for 0, 6, or 15 h. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-Pyk2 (Tyr881), or anti-Pyk2 antibody (left). Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with 4G10 antibody (Right). B. Phosphorylation of Pyk2 and PECAM in regular suspension versus methyl-cellulose suspension cultures. H1792 and H460 cells were cultured in polyHEMA-coated dishes (S) or methyl-cellulose dishes (MS) for 6 or 15 h, harvested, and lysed with RIPA buffer. A, Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with anti-phosphotyrosine 4G10 antibody (Upper). Equal amounts of whole-cell extracts were immunoblotted with anti-pPyk2 (Tyr881) or anti-Pyk2 antibody (Lower).
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Figure 3: Induction of phosphorylation of Pyk2 and PECAM by cell aggregation. A, Phosphorylation of Pyk2 and PECAM in high versus low cell density cultures. Different concentrations of HBE, SK-LU-1, or H460 cells (H: 5.0 × 105 cells L: 2.5 × 105 cells) were cultured in 60-mm polyHEMA-coated dishes for 0, 6, or 15 h. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-Pyk2 (Tyr881), or anti-Pyk2 antibody (left). Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with 4G10 antibody (Right). B. Phosphorylation of Pyk2 and PECAM in regular suspension versus methyl-cellulose suspension cultures. H1792 and H460 cells were cultured in polyHEMA-coated dishes (S) or methyl-cellulose dishes (MS) for 6 or 15 h, harvested, and lysed with RIPA buffer. A, Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with anti-phosphotyrosine 4G10 antibody (Upper). Equal amounts of whole-cell extracts were immunoblotted with anti-pPyk2 (Tyr881) or anti-Pyk2 antibody (Lower).
Mentions: In order to understand the molecular nature of the cell aggregation-generated survival and growth signals, we analyzed the phosphorylation/activation of PECAM-1 and Pyk2. PECAM protein was immunoprecipitated with PECAM antibody and then immunoblotted with 4G10 antibody (anti-phosphotyrosine monoclonal antibody) to detect tyrosine phosphorylation. Interestingly, we found that the phosphorylation/activation of PECAM-1 and Pyk2 correlated with cell aggregation, although the total protein level of PECAM-1 and Pyk2 were not related to cell aggregation (Fig. 3A, B and Additional file 1 Figure S1). High- and low-density suspension cultures were established in order to study the phosphorylation status of these molecules in relation to cell aggregation. H460, SK-LU-1, and HBE cells formed larger aggregates in high-density cultures compared to low-density cultures. H460 and H1792 formed larger aggregates in polyHEMA cultures compared to methyl cellulose cultures. We observed higher levels of phosphorylated PECAM-1 and Pyk2 in large aggregates (high-density cultures, H) than in small aggregates or single cells (low-density cultures, L) (Fig. 3A)Similarly, higher levels of phosphorylated PECAM-1 and Pyk2 were seen when cells were cultured in large aggregates (polyHEMA suspension culture, S) compared to small aggregates (methyl cellulose suspension culture, MS) (Fig. 3B), suggesting that the phosphorylation/activation of PECAM-1 and Pyk2 correlated with cell aggregation.

Bottom Line: Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood.Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates.We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Shanghai Institute of Materia Medica, China Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China. xingzhangg@hotmail.com

ABSTRACT

Background: Apoptosis caused by inadequate or inappropriate cell-matrix interactions is defined as anoikis. Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood. We investigated the mechanisms of anoikis resistance of tumor cells.

Results: We observed that cell aggregation in suspension promoted cell survival and proliferation. We demonstrated a correlation between tumor cell aggregation in suspension and cell growth in soft agar. Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates. We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2. Furthermore, RNA interference-mediated reduction of Pyk2 and PECAM-1 protein levels reduced cell aggregation and inhibited the growth of tumor cells in soft agar.

Conclusions: The data demonstrated that Pyk2 and PECAM-1 were critical mediators of both anchorage-independent growth and anoikis resistance in tumor cells.

Show MeSH
Related in: MedlinePlus