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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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Phosphorylation of HSP27 in Subset of CLL Patients.A) Bar graph depicting the spectral counts of HSP27 phosphopeptides (Ser82) observed in the LC-MS/MS analysis after CXCL12 stimulation. B) Western blot detecting phosphorylation of HSP27 and the upstream p38-MAPK, and total HSP27 over time course of 0 to 60 min CXCL12 stimulation (30 nM) from CLL A patient cells and 2 other representative CLL patients' cells. β-actin was run as a loading control. C) Signaling diagram of HSP27, which can protect from apoptosis, and its upstream regulation by p38-MAPK and MAPKAPK2.
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pone-0011716-g006: Phosphorylation of HSP27 in Subset of CLL Patients.A) Bar graph depicting the spectral counts of HSP27 phosphopeptides (Ser82) observed in the LC-MS/MS analysis after CXCL12 stimulation. B) Western blot detecting phosphorylation of HSP27 and the upstream p38-MAPK, and total HSP27 over time course of 0 to 60 min CXCL12 stimulation (30 nM) from CLL A patient cells and 2 other representative CLL patients' cells. β-actin was run as a loading control. C) Signaling diagram of HSP27, which can protect from apoptosis, and its upstream regulation by p38-MAPK and MAPKAPK2.

Mentions: The other phosphoprotein investigated further was HSP27 (phosphopeptide: R.QLSphosSGVEIR.H, Ser82) (mass spectrum shown in Figure S2). HSP27 was also selected due to spectral counts indicative of phosphorylation upon CXCL12 activation of CXCR4 (Figure 6A), its implications in cancer and protection from apoptosis, and the availability of a phospho-specific antibody at the Ser82 phosphorylation site [27], [28], [29]. Additionally, HSP27 is an interesting target since it is downstream of p38-MAPK signaling [27] which has not received much attention in association with CLL and therefore represents a pathway with potentially novel implications in CLL survival.


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)

Phosphorylation of HSP27 in Subset of CLL Patients.A) Bar graph depicting the spectral counts of HSP27 phosphopeptides (Ser82) observed in the LC-MS/MS analysis after CXCL12 stimulation. B) Western blot detecting phosphorylation of HSP27 and the upstream p38-MAPK, and total HSP27 over time course of 0 to 60 min CXCL12 stimulation (30 nM) from CLL A patient cells and 2 other representative CLL patients' cells. β-actin was run as a loading control. C) Signaling diagram of HSP27, which can protect from apoptosis, and its upstream regulation by p38-MAPK and MAPKAPK2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011716-g006: Phosphorylation of HSP27 in Subset of CLL Patients.A) Bar graph depicting the spectral counts of HSP27 phosphopeptides (Ser82) observed in the LC-MS/MS analysis after CXCL12 stimulation. B) Western blot detecting phosphorylation of HSP27 and the upstream p38-MAPK, and total HSP27 over time course of 0 to 60 min CXCL12 stimulation (30 nM) from CLL A patient cells and 2 other representative CLL patients' cells. β-actin was run as a loading control. C) Signaling diagram of HSP27, which can protect from apoptosis, and its upstream regulation by p38-MAPK and MAPKAPK2.
Mentions: The other phosphoprotein investigated further was HSP27 (phosphopeptide: R.QLSphosSGVEIR.H, Ser82) (mass spectrum shown in Figure S2). HSP27 was also selected due to spectral counts indicative of phosphorylation upon CXCL12 activation of CXCR4 (Figure 6A), its implications in cancer and protection from apoptosis, and the availability of a phospho-specific antibody at the Ser82 phosphorylation site [27], [28], [29]. Additionally, HSP27 is an interesting target since it is downstream of p38-MAPK signaling [27] which has not received much attention in association with CLL and therefore represents a pathway with potentially novel implications in CLL survival.

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