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Elucidating the CXCL12/CXCR4 signaling network in chronic lymphocytic leukemia through phosphoproteomics analysis.

O'Hayre M, Salanga CL, Kipps TJ, Messmer D, Dorrestein PC, Handel TM - PLoS ONE (2010)

Bottom Line: To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells.In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4), is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed.Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT

Background: Chronic Lymphocytic Leukemia (CLL) pathogenesis has been linked to the prolonged survival and/or apoptotic resistance of leukemic B cells in vivo, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy.

Methodology/principal findings: To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4), is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27), which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (approximately 25%) of CLL patients cells examined.

Conclusions/significance: Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a previously unknown signaling target of chemokine receptors; therefore, these observations increase our understanding of alternative pathways to migration that may be activated or inhibited by chemokines in the context of cancer cell survival.

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Related in: MedlinePlus

Flow Chart of CLL Phosphoproteomics Analysis.Lysates were prepared from primary CLL B cells that had been stimulated over an hour time course with 30 nM CXCL12. Lysates were denatured, reduced and alkylated in preparation for trypsin digest. Tryptic peptides were then enriched for phosphopeptides by IMAC and LC-MS/MS was performed. Data was analyzed using the InsPecT database search algorithm for phosphorylations on Ser, Thr, or Tyr. A decoy database and manual validation of the spectra were used as quality control. Spectral count comparisons were made as a qualitative assessment of CXCL12 stimulation response and interesting target proteins were selected for follow-up studies if antibodies were available.
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pone-0011716-g003: Flow Chart of CLL Phosphoproteomics Analysis.Lysates were prepared from primary CLL B cells that had been stimulated over an hour time course with 30 nM CXCL12. Lysates were denatured, reduced and alkylated in preparation for trypsin digest. Tryptic peptides were then enriched for phosphopeptides by IMAC and LC-MS/MS was performed. Data was analyzed using the InsPecT database search algorithm for phosphorylations on Ser, Thr, or Tyr. A decoy database and manual validation of the spectra were used as quality control. Spectral count comparisons were made as a qualitative assessment of CXCL12 stimulation response and interesting target proteins were selected for follow-up studies if antibodies were available.

Mentions: Protein lysates from the CLL cells were trypsin digested and enriched for phosphopeptides via immobilized metal affinity chromatography (IMAC) to yield a highly enriched population of phosphorylated peptides. Phosphopeptides were analyzed by LC-MS/MS using a linear iontrap (LTQ) mass spectrometer. Data was processed using the InsPecT database search algorithm and the spectra were manually inspected for validation (see flow diagram Figure 3).


Elucidating the CXCL12/CXCR4 signaling network in chronic lymphocytic leukemia through phosphoproteomics analysis.

O'Hayre M, Salanga CL, Kipps TJ, Messmer D, Dorrestein PC, Handel TM - PLoS ONE (2010)

Flow Chart of CLL Phosphoproteomics Analysis.Lysates were prepared from primary CLL B cells that had been stimulated over an hour time course with 30 nM CXCL12. Lysates were denatured, reduced and alkylated in preparation for trypsin digest. Tryptic peptides were then enriched for phosphopeptides by IMAC and LC-MS/MS was performed. Data was analyzed using the InsPecT database search algorithm for phosphorylations on Ser, Thr, or Tyr. A decoy database and manual validation of the spectra were used as quality control. Spectral count comparisons were made as a qualitative assessment of CXCL12 stimulation response and interesting target proteins were selected for follow-up studies if antibodies were available.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908618&req=5

pone-0011716-g003: Flow Chart of CLL Phosphoproteomics Analysis.Lysates were prepared from primary CLL B cells that had been stimulated over an hour time course with 30 nM CXCL12. Lysates were denatured, reduced and alkylated in preparation for trypsin digest. Tryptic peptides were then enriched for phosphopeptides by IMAC and LC-MS/MS was performed. Data was analyzed using the InsPecT database search algorithm for phosphorylations on Ser, Thr, or Tyr. A decoy database and manual validation of the spectra were used as quality control. Spectral count comparisons were made as a qualitative assessment of CXCL12 stimulation response and interesting target proteins were selected for follow-up studies if antibodies were available.
Mentions: Protein lysates from the CLL cells were trypsin digested and enriched for phosphopeptides via immobilized metal affinity chromatography (IMAC) to yield a highly enriched population of phosphorylated peptides. Phosphopeptides were analyzed by LC-MS/MS using a linear iontrap (LTQ) mass spectrometer. Data was processed using the InsPecT database search algorithm and the spectra were manually inspected for validation (see flow diagram Figure 3).

Bottom Line: To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells.In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4), is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed.Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT

Background: Chronic Lymphocytic Leukemia (CLL) pathogenesis has been linked to the prolonged survival and/or apoptotic resistance of leukemic B cells in vivo, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy.

Methodology/principal findings: To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4), is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27), which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (approximately 25%) of CLL patients cells examined.

Conclusions/significance: Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a previously unknown signaling target of chemokine receptors; therefore, these observations increase our understanding of alternative pathways to migration that may be activated or inhibited by chemokines in the context of cancer cell survival.

Show MeSH
Related in: MedlinePlus