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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

CXCL12 Induces Phosphorylation of PDCD4 at Ser457.A) Bar graph depicting the spectral counts of PDCD4 phosphopeptides observed in the LC-MS/MS analysis at time points of CXCL12 stimulation. B) Western blot of PDCD4 phosphorylation over time course of 0 to 60 min CXCL12 stimulation (30 nM) from CLL A patient cells. β-actin served as a loading control. C) Top panel: Representative western blot of PDCD4 phosphorylation in CLL cells from 2 different CLL patients not used in LC-MS/MS analysis over 30 nM CXCL12 stimulation time course. β-actin served as a loading control. Bottom panel: Densitometry analysis of PDCD4 phosphorylation levels CXCL12 stimulation (30 nM) time points relative to unstimulated controls and averaged from 10 separate CLL patient cells. Error bars represent standard error of the mean (SEM). D) Western blot of PDCD4 phosphorylation in unstimulated/untreated CLL cells or 3 min CXCL12 stimulations (30 nM) in the presence (+) or absence (−) of preincubation (1 h) with AMD3100 (40 µM) or Pertussis toxin (PTx) (200 ng/ml). NLC lysate represents CLL cells cultured in presence of NLCs with no further stimulation or treatment. CLL cells were removed from the adherent NLCs and lysed. β-actin served as a loading control. E) Top panel: Representative western blot detecting total levels of PDCD4 in CLL cells following 0, 4, 10 or 24 h of 30 nM CXCL12 stimulation. β-actin served as a loading control. Bottom panel: Bar graph quantifying total PDCD4 levels over 24 h time course of 30 nM CXCL12 stimulation compared to 0 h unstimulated controls and normalized to β-actin levels by densitometry analysis of western blots. Data represented are mean +/− SD of 3 separate CLL patients' cells. F) Western blot stripped and reprobed from Figure 4B for p70S6K phosphorylation (Thr389) over time course of 30 nM CXCL12 stimulation from CLL A patient cells (Top panel) and 2 other representative CLL patients' cells (bottom panel). β-actin served as a loading control. G) Diagram of PDCD4 signaling showing known upstream regulators as well as downstream targets. Akt and p70S6K are known to phosphorylate PDCD4, thereby inhibiting its function in repressing eIF4A translational activity and AP-1 transcription.
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pone-0011716-g005: CXCL12 Induces Phosphorylation of PDCD4 at Ser457.A) Bar graph depicting the spectral counts of PDCD4 phosphopeptides observed in the LC-MS/MS analysis at time points of CXCL12 stimulation. B) Western blot of PDCD4 phosphorylation over time course of 0 to 60 min CXCL12 stimulation (30 nM) from CLL A patient cells. β-actin served as a loading control. C) Top panel: Representative western blot of PDCD4 phosphorylation in CLL cells from 2 different CLL patients not used in LC-MS/MS analysis over 30 nM CXCL12 stimulation time course. β-actin served as a loading control. Bottom panel: Densitometry analysis of PDCD4 phosphorylation levels CXCL12 stimulation (30 nM) time points relative to unstimulated controls and averaged from 10 separate CLL patient cells. Error bars represent standard error of the mean (SEM). D) Western blot of PDCD4 phosphorylation in unstimulated/untreated CLL cells or 3 min CXCL12 stimulations (30 nM) in the presence (+) or absence (−) of preincubation (1 h) with AMD3100 (40 µM) or Pertussis toxin (PTx) (200 ng/ml). NLC lysate represents CLL cells cultured in presence of NLCs with no further stimulation or treatment. CLL cells were removed from the adherent NLCs and lysed. β-actin served as a loading control. E) Top panel: Representative western blot detecting total levels of PDCD4 in CLL cells following 0, 4, 10 or 24 h of 30 nM CXCL12 stimulation. β-actin served as a loading control. Bottom panel: Bar graph quantifying total PDCD4 levels over 24 h time course of 30 nM CXCL12 stimulation compared to 0 h unstimulated controls and normalized to β-actin levels by densitometry analysis of western blots. Data represented are mean +/− SD of 3 separate CLL patients' cells. F) Western blot stripped and reprobed from Figure 4B for p70S6K phosphorylation (Thr389) over time course of 30 nM CXCL12 stimulation from CLL A patient cells (Top panel) and 2 other representative CLL patients' cells (bottom panel). β-actin served as a loading control. G) Diagram of PDCD4 signaling showing known upstream regulators as well as downstream targets. Akt and p70S6K are known to phosphorylate PDCD4, thereby inhibiting its function in repressing eIF4A translational activity and AP-1 transcription.

Mentions: PDCD4 is one of the phosphoproteins that appeared to be induced by CXCL12 stimulation based on spectral counts (Figure 5A and Table 2). It is a known tumor suppressor, and downstream phosphorylation target of Akt, which is known to be activated by CXCL12 in CLL cells [9]. A phospho-specific antibody is also commercially available, making it attractive for follow-up studies [23], [24]. As a tumor suppressor protein, PDCD4 has been implicated in a number of cancers where it is often inhibited and/or downregulated, disrupting its ability to inhibit eIF4A translational and AP-1 transcriptional activity, processes that are important for cell growth and survival (Figure 5G). Phosphorylation of PDCD4 is known to occur by Akt and p70 S6Kinase (p70S6K) which inhibits its activity and leads to its ubiquitination and proteosomal degradation [23], [24], [25], [26].


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)

CXCL12 Induces Phosphorylation of PDCD4 at Ser457.A) Bar graph depicting the spectral counts of PDCD4 phosphopeptides observed in the LC-MS/MS analysis at time points of CXCL12 stimulation. B) Western blot of PDCD4 phosphorylation over time course of 0 to 60 min CXCL12 stimulation (30 nM) from CLL A patient cells. β-actin served as a loading control. C) Top panel: Representative western blot of PDCD4 phosphorylation in CLL cells from 2 different CLL patients not used in LC-MS/MS analysis over 30 nM CXCL12 stimulation time course. β-actin served as a loading control. Bottom panel: Densitometry analysis of PDCD4 phosphorylation levels CXCL12 stimulation (30 nM) time points relative to unstimulated controls and averaged from 10 separate CLL patient cells. Error bars represent standard error of the mean (SEM). D) Western blot of PDCD4 phosphorylation in unstimulated/untreated CLL cells or 3 min CXCL12 stimulations (30 nM) in the presence (+) or absence (−) of preincubation (1 h) with AMD3100 (40 µM) or Pertussis toxin (PTx) (200 ng/ml). NLC lysate represents CLL cells cultured in presence of NLCs with no further stimulation or treatment. CLL cells were removed from the adherent NLCs and lysed. β-actin served as a loading control. E) Top panel: Representative western blot detecting total levels of PDCD4 in CLL cells following 0, 4, 10 or 24 h of 30 nM CXCL12 stimulation. β-actin served as a loading control. Bottom panel: Bar graph quantifying total PDCD4 levels over 24 h time course of 30 nM CXCL12 stimulation compared to 0 h unstimulated controls and normalized to β-actin levels by densitometry analysis of western blots. Data represented are mean +/− SD of 3 separate CLL patients' cells. F) Western blot stripped and reprobed from Figure 4B for p70S6K phosphorylation (Thr389) over time course of 30 nM CXCL12 stimulation from CLL A patient cells (Top panel) and 2 other representative CLL patients' cells (bottom panel). β-actin served as a loading control. G) Diagram of PDCD4 signaling showing known upstream regulators as well as downstream targets. Akt and p70S6K are known to phosphorylate PDCD4, thereby inhibiting its function in repressing eIF4A translational activity and AP-1 transcription.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011716-g005: CXCL12 Induces Phosphorylation of PDCD4 at Ser457.A) Bar graph depicting the spectral counts of PDCD4 phosphopeptides observed in the LC-MS/MS analysis at time points of CXCL12 stimulation. B) Western blot of PDCD4 phosphorylation over time course of 0 to 60 min CXCL12 stimulation (30 nM) from CLL A patient cells. β-actin served as a loading control. C) Top panel: Representative western blot of PDCD4 phosphorylation in CLL cells from 2 different CLL patients not used in LC-MS/MS analysis over 30 nM CXCL12 stimulation time course. β-actin served as a loading control. Bottom panel: Densitometry analysis of PDCD4 phosphorylation levels CXCL12 stimulation (30 nM) time points relative to unstimulated controls and averaged from 10 separate CLL patient cells. Error bars represent standard error of the mean (SEM). D) Western blot of PDCD4 phosphorylation in unstimulated/untreated CLL cells or 3 min CXCL12 stimulations (30 nM) in the presence (+) or absence (−) of preincubation (1 h) with AMD3100 (40 µM) or Pertussis toxin (PTx) (200 ng/ml). NLC lysate represents CLL cells cultured in presence of NLCs with no further stimulation or treatment. CLL cells were removed from the adherent NLCs and lysed. β-actin served as a loading control. E) Top panel: Representative western blot detecting total levels of PDCD4 in CLL cells following 0, 4, 10 or 24 h of 30 nM CXCL12 stimulation. β-actin served as a loading control. Bottom panel: Bar graph quantifying total PDCD4 levels over 24 h time course of 30 nM CXCL12 stimulation compared to 0 h unstimulated controls and normalized to β-actin levels by densitometry analysis of western blots. Data represented are mean +/− SD of 3 separate CLL patients' cells. F) Western blot stripped and reprobed from Figure 4B for p70S6K phosphorylation (Thr389) over time course of 30 nM CXCL12 stimulation from CLL A patient cells (Top panel) and 2 other representative CLL patients' cells (bottom panel). β-actin served as a loading control. G) Diagram of PDCD4 signaling showing known upstream regulators as well as downstream targets. Akt and p70S6K are known to phosphorylate PDCD4, thereby inhibiting its function in repressing eIF4A translational activity and AP-1 transcription.
Mentions: PDCD4 is one of the phosphoproteins that appeared to be induced by CXCL12 stimulation based on spectral counts (Figure 5A and Table 2). It is a known tumor suppressor, and downstream phosphorylation target of Akt, which is known to be activated by CXCL12 in CLL cells [9]. A phospho-specific antibody is also commercially available, making it attractive for follow-up studies [23], [24]. As a tumor suppressor protein, PDCD4 has been implicated in a number of cancers where it is often inhibited and/or downregulated, disrupting its ability to inhibit eIF4A translational and AP-1 transcriptional activity, processes that are important for cell growth and survival (Figure 5G). Phosphorylation of PDCD4 is known to occur by Akt and p70 S6Kinase (p70S6K) which inhibits its activity and leads to its ubiquitination and proteosomal degradation [23], [24], [25], [26].

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