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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

Summary of CXCL12-mediated Signaling in CLL.Signaling diagram depicting pathways activated downstream of CXLC12. Through direct or indirect mechanisms, arrows indicate factors that are activated, red lines ending with a bar indicate factors that are inhibited by the upstream factor, and lines (no arrowhead) indicate interactions. Proteins in hexagons were identified and validated herein or were previously known targets also detected in the LC-MS/MS. Proteins in rectangles are known key signaling molecules of these pathways that were not detected in this LC-MS/MS data set. Proteins in ovals with dashed lines are likely intermediates/targets of the pathways based on previous studies. Proteins in oval shape were also identified by LC-MS/MS but have yet to be validated. Much of our focus has been on the PI3K/Akt and Raf/MEK/ERK pathways due to known implications in CLL cell survival and resistance to apoptosis. Furthermore, the potential involvement of the p38-MAPK pathway in some CLL patients with activation of HSP27 and LSP1 is outlined.
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pone-0011716-g007: Summary of CXCL12-mediated Signaling in CLL.Signaling diagram depicting pathways activated downstream of CXLC12. Through direct or indirect mechanisms, arrows indicate factors that are activated, red lines ending with a bar indicate factors that are inhibited by the upstream factor, and lines (no arrowhead) indicate interactions. Proteins in hexagons were identified and validated herein or were previously known targets also detected in the LC-MS/MS. Proteins in rectangles are known key signaling molecules of these pathways that were not detected in this LC-MS/MS data set. Proteins in ovals with dashed lines are likely intermediates/targets of the pathways based on previous studies. Proteins in oval shape were also identified by LC-MS/MS but have yet to be validated. Much of our focus has been on the PI3K/Akt and Raf/MEK/ERK pathways due to known implications in CLL cell survival and resistance to apoptosis. Furthermore, the potential involvement of the p38-MAPK pathway in some CLL patients with activation of HSP27 and LSP1 is outlined.

Mentions: Herein we present follow-up data to PDCD4 and HSP27, although there are numerous other candidate phosphoprotein targets of CXCL12 signaling in CLL cells that have been proposed (Table 2). A summary of our findings from phosphoproteomics analysis combined with some previously established pathways of CXCL12 signaling in CLL are summarized in a signaling diagram (Figure 7). Overall, our data suggests that CXCL12 may preferentially activate survival signaling pathways rather than those involved in cell migration in CLL cells, although some of the pathway components (Gi, Erk, Akt) are common nodes. We have demonstrated that the use of phosphoproteomics is a feasible and informative means of evaluating signaling responses to CXCL12 in CLL, which could be employed for investigating a variety of other stimuli in these or other primary cells. Through phosphoproteomics detection and western blot validation, PDCD4 was found to be a common phosphorylation target of CXCL12-signaling in CLL while HSP27 was present in only a subset of CLL patients. Although our focus was on CXCL12 as a survival factor, it is likely that other growth and survival stimuli may synergistically activate these pathways and downstream targets. Therefore, PDCD4 and HSP27, which have previous implications in regulation of apoptosis and carcinogenesis, may represent potential therapeutic targets for treatment of CLL. In fact, small molecule stabilizers of PDCD4, that enhance its function as a tumor suppressor by inhibiting its degradation, are currently being developed due to its potential as a therapeutic target for numerous cancers. Such agents could prove to be useful agents in combination with other therapeutic modalities for the treatment of CLL [40].


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)

Summary of CXCL12-mediated Signaling in CLL.Signaling diagram depicting pathways activated downstream of CXLC12. Through direct or indirect mechanisms, arrows indicate factors that are activated, red lines ending with a bar indicate factors that are inhibited by the upstream factor, and lines (no arrowhead) indicate interactions. Proteins in hexagons were identified and validated herein or were previously known targets also detected in the LC-MS/MS. Proteins in rectangles are known key signaling molecules of these pathways that were not detected in this LC-MS/MS data set. Proteins in ovals with dashed lines are likely intermediates/targets of the pathways based on previous studies. Proteins in oval shape were also identified by LC-MS/MS but have yet to be validated. Much of our focus has been on the PI3K/Akt and Raf/MEK/ERK pathways due to known implications in CLL cell survival and resistance to apoptosis. Furthermore, the potential involvement of the p38-MAPK pathway in some CLL patients with activation of HSP27 and LSP1 is outlined.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011716-g007: Summary of CXCL12-mediated Signaling in CLL.Signaling diagram depicting pathways activated downstream of CXLC12. Through direct or indirect mechanisms, arrows indicate factors that are activated, red lines ending with a bar indicate factors that are inhibited by the upstream factor, and lines (no arrowhead) indicate interactions. Proteins in hexagons were identified and validated herein or were previously known targets also detected in the LC-MS/MS. Proteins in rectangles are known key signaling molecules of these pathways that were not detected in this LC-MS/MS data set. Proteins in ovals with dashed lines are likely intermediates/targets of the pathways based on previous studies. Proteins in oval shape were also identified by LC-MS/MS but have yet to be validated. Much of our focus has been on the PI3K/Akt and Raf/MEK/ERK pathways due to known implications in CLL cell survival and resistance to apoptosis. Furthermore, the potential involvement of the p38-MAPK pathway in some CLL patients with activation of HSP27 and LSP1 is outlined.
Mentions: Herein we present follow-up data to PDCD4 and HSP27, although there are numerous other candidate phosphoprotein targets of CXCL12 signaling in CLL cells that have been proposed (Table 2). A summary of our findings from phosphoproteomics analysis combined with some previously established pathways of CXCL12 signaling in CLL are summarized in a signaling diagram (Figure 7). Overall, our data suggests that CXCL12 may preferentially activate survival signaling pathways rather than those involved in cell migration in CLL cells, although some of the pathway components (Gi, Erk, Akt) are common nodes. We have demonstrated that the use of phosphoproteomics is a feasible and informative means of evaluating signaling responses to CXCL12 in CLL, which could be employed for investigating a variety of other stimuli in these or other primary cells. Through phosphoproteomics detection and western blot validation, PDCD4 was found to be a common phosphorylation target of CXCL12-signaling in CLL while HSP27 was present in only a subset of CLL patients. Although our focus was on CXCL12 as a survival factor, it is likely that other growth and survival stimuli may synergistically activate these pathways and downstream targets. Therefore, PDCD4 and HSP27, which have previous implications in regulation of apoptosis and carcinogenesis, may represent potential therapeutic targets for treatment of CLL. In fact, small molecule stabilizers of PDCD4, that enhance its function as a tumor suppressor by inhibiting its degradation, are currently being developed due to its potential as a therapeutic target for numerous cancers. Such agents could prove to be useful agents in combination with other therapeutic modalities for the treatment of CLL [40].

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