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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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CXCL12-mediated Migration of CLL B Cells and Normal B Cells.A) Representative migration assay profiles for normal B cells (solid line) compared to 2 different CLL patients' cells (dashed lines) performed in triplicate over a range of 0–100 nM CXCL12 and normalized to no chemokine. Data represents the percent of cells migrated through the transwell filter. B) Bar graph comparing the maximum percent cell migration observed in normal B cells and CLL B cells. Data represents an average of multiple transwell migration assays from separate donors, normal B cells n = 5, CLL B cells n = 7, each performed in triplicate. Differences were found to be statistically significant (p<0.0001) based on Student's t-test. C) Table depicting the maximum percent migration to CXCL12 (efficacy) and the concentration at which maximal migration is achieved (potency) observed in normal B cells compared to CLL B cells.
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pone-0011716-g002: CXCL12-mediated Migration of CLL B Cells and Normal B Cells.A) Representative migration assay profiles for normal B cells (solid line) compared to 2 different CLL patients' cells (dashed lines) performed in triplicate over a range of 0–100 nM CXCL12 and normalized to no chemokine. Data represents the percent of cells migrated through the transwell filter. B) Bar graph comparing the maximum percent cell migration observed in normal B cells and CLL B cells. Data represents an average of multiple transwell migration assays from separate donors, normal B cells n = 5, CLL B cells n = 7, each performed in triplicate. Differences were found to be statistically significant (p<0.0001) based on Student's t-test. C) Table depicting the maximum percent migration to CXCL12 (efficacy) and the concentration at which maximal migration is achieved (potency) observed in normal B cells compared to CLL B cells.

Mentions: Despite the upregulation of CXCR4 and strong Akt and ERK signaling demonstrated by CLL cells in response to CXCL12, the CLL cells actually migrate less efficiently to CXCL12 than B cells from healthy donors in a transwell migration assay (Figure 2). Thus, in CLL cells, it appears that signaling downstream of CXCL12/CXCR4 may be redirected towards survival signaling in lieu of cell migration. To better characterize the signaling responses to CXCL12 stimulation, primary CLL cells isolated from 5 patients were subjected to phosphoproteomic analysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Rather than attempting to characterize the complete phosphoproteome of CLL cells, this approach was designed to generate new hypotheses about the CXCL12/CXCR4 signaling network in CLL survival, and to identify downstream proteins that might be good therapeutic targets. While many phosphoproteins were identified in the CLL cells, comparison of spectral counts between CXCL12 stimulated and unstimulated cells allowed identification of proteins phosphorylated as a consequence of CXCL12/CXCR4 signaling. With follow-up experiments, the tumor suppressor PDCD4 was validated as a downstream phosphorylation target of CXCL12 signaling in all CLL patient cells examined (n = 10) and HSP27 was similarly validated in a subset of CLL patients (∼25%). Although these proteins have been previously linked to cancer cell survival, they have not been previously associated with CLL nor has PDCD4 been established as a downstream phosphorylation target of CXCL12 signaling. Furthermore, a number of other proteins (many of which do not have commercially available phospho-specific antibodies available) have been proposed as potential downstream phosphorylation targets of CXCL12 stimulation based on spectral count analysis of the CLL phosphoproteomics data.


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-mediated Migration of CLL B Cells and Normal B Cells.A) Representative migration assay profiles for normal B cells (solid line) compared to 2 different CLL patients' cells (dashed lines) performed in triplicate over a range of 0–100 nM CXCL12 and normalized to no chemokine. Data represents the percent of cells migrated through the transwell filter. B) Bar graph comparing the maximum percent cell migration observed in normal B cells and CLL B cells. Data represents an average of multiple transwell migration assays from separate donors, normal B cells n = 5, CLL B cells n = 7, each performed in triplicate. Differences were found to be statistically significant (p<0.0001) based on Student's t-test. C) Table depicting the maximum percent migration to CXCL12 (efficacy) and the concentration at which maximal migration is achieved (potency) observed in normal B cells compared to CLL B cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011716-g002: CXCL12-mediated Migration of CLL B Cells and Normal B Cells.A) Representative migration assay profiles for normal B cells (solid line) compared to 2 different CLL patients' cells (dashed lines) performed in triplicate over a range of 0–100 nM CXCL12 and normalized to no chemokine. Data represents the percent of cells migrated through the transwell filter. B) Bar graph comparing the maximum percent cell migration observed in normal B cells and CLL B cells. Data represents an average of multiple transwell migration assays from separate donors, normal B cells n = 5, CLL B cells n = 7, each performed in triplicate. Differences were found to be statistically significant (p<0.0001) based on Student's t-test. C) Table depicting the maximum percent migration to CXCL12 (efficacy) and the concentration at which maximal migration is achieved (potency) observed in normal B cells compared to CLL B cells.
Mentions: Despite the upregulation of CXCR4 and strong Akt and ERK signaling demonstrated by CLL cells in response to CXCL12, the CLL cells actually migrate less efficiently to CXCL12 than B cells from healthy donors in a transwell migration assay (Figure 2). Thus, in CLL cells, it appears that signaling downstream of CXCL12/CXCR4 may be redirected towards survival signaling in lieu of cell migration. To better characterize the signaling responses to CXCL12 stimulation, primary CLL cells isolated from 5 patients were subjected to phosphoproteomic analysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Rather than attempting to characterize the complete phosphoproteome of CLL cells, this approach was designed to generate new hypotheses about the CXCL12/CXCR4 signaling network in CLL survival, and to identify downstream proteins that might be good therapeutic targets. While many phosphoproteins were identified in the CLL cells, comparison of spectral counts between CXCL12 stimulated and unstimulated cells allowed identification of proteins phosphorylated as a consequence of CXCL12/CXCR4 signaling. With follow-up experiments, the tumor suppressor PDCD4 was validated as a downstream phosphorylation target of CXCL12 signaling in all CLL patient cells examined (n = 10) and HSP27 was similarly validated in a subset of CLL patients (∼25%). Although these proteins have been previously linked to cancer cell survival, they have not been previously associated with CLL nor has PDCD4 been established as a downstream phosphorylation target of CXCL12 signaling. Furthermore, a number of other proteins (many of which do not have commercially available phospho-specific antibodies available) have been proposed as potential downstream phosphorylation targets of CXCL12 stimulation based on spectral count analysis of the CLL phosphoproteomics data.

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