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CXCR4 expression heterogeneity in neuroblastoma cells due to ligand-independent regulation.

Carlisle AJ, Lyttle CA, Carlisle RY, Maris JM - Mol. Cancer (2009)

Bottom Line: Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism.Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Oncology, Abramson Research Center, Children's Hospital of Philadelphia, ARC-907A, 3615 Civic Center Blvd, Philadelphia, Pennsylvania 19104-4399, USA. carlisle@email.chop.edu

ABSTRACT

Background: CXCR4, the receptor for the chemokine stromal-derived factor 1 (SDF-1), has been shown to mediate many of the processes essential for cancer progression such as tumor cell proliferation, metastasis, and angiogenesis. To understand the role of CXCR4 in the biology of neuroblastoma, a disease that presents with wide spread metastases in over 50% of patients, we screened ten patient derived-neuroblastoma cell-lines for basal CXCR4 expression and sought to identify characteristics that correlate with tumor cell phenotype.

Results: All cell lines expressed CXCR4 mRNA at variable levels, that correlated well with three distinct classes of CXCR4 surface expression (low, moderate, or high) as defined by flow cytometry. Analysis of the kinetics of CXCR4 surface expression on moderate and high expressing cell lines showed a time-dependent down-regulation of the receptor that directly correlated with cell confluency, and was independent of SDF1. Cell lysates showed the presence of multiple CXCR4 isoforms with three major species of approximately 87, 67 and 55 kDa associating with high surface expression, and two distinct species of 45 and 38 kDa correlating with low to surface expression. Western blot analysis of CXCR4 immunoprecipitates showed that the 87 and 67 kDa forms were ubiquitinated, while the others were not. Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.

Conclusions: Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism. Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.

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Analysis of CXCR4 Structural Heterogeneity in Neuroblastoma Cells. A) Western Blot Detection of CXCR4 in Neuroblastoma Cell Lysates. Cell lysates were generated by solubilizing neuroblastoma cells in 1% triton X-100. Detergent soluble proteins were resolved on 4-20% Bis-Tris SDS gels and transferred to nitrocellulose membranes. CXCR4 was detected by probing with a rabbit anti-human CXCR4 polyclonal primary antibody (ab2090), followed by a horse radish peroxidase conjugated donkey anti-rabbit secondary antibody, and then chemiluminescent substrate. CXCR4 was visualized using enhanced chemiluminescent (ECL). The samples represented are as follows: Lane 1- CHP-134, Lane 2-KCN, Lane 3-KCNR, Lane 4-NB-69, Lane 5-NGP, Lane 6-SK-N-FI, Lane 7-SK-N-AS, Lane 8-SK-N-ASΔ3*, Lane 9-LAN-5, Lane 10-SK-N-SH, Lane 11-SH-SY5Y. Cell lines are color coded by surface expression class: Red = Low, Blue = Medium, Green = High, Black = Not Conclusively Determined (N.D.). *SK-N-ASΔ3 is a derivative cell line of SK-N-AS that has been transfected with an expression vector containing the coding sequence for an E3 ubiquitin ligase component Cullin-5 (CUL-5). All samples were loaded equally as determined by comparing actin levels in each lane (Data not shown). B) Western Blot Detection of Ubiquitin in CXCR4 Immunoprecipitates. Cell lysates were adsorbed with rabbit anti-human CXCR4 antibodies followed by precipitation with protein G Agarose. Precipitated CXCR4 Immune complexes were resolved on 4-15% Bis-Tris SDS gels and transferred to nitrocellulose membranes. Membranes were probed with a mouse anti-human Ubiquitin monoclonal antibody followed by an HRP-conjugated donkey anti-mouse secondary antibody, and then addition of chemiluminescent substrate. Ubiquitin was visualized by measuring ECL. Sample order is the same as in panel A.
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Figure 7: Analysis of CXCR4 Structural Heterogeneity in Neuroblastoma Cells. A) Western Blot Detection of CXCR4 in Neuroblastoma Cell Lysates. Cell lysates were generated by solubilizing neuroblastoma cells in 1% triton X-100. Detergent soluble proteins were resolved on 4-20% Bis-Tris SDS gels and transferred to nitrocellulose membranes. CXCR4 was detected by probing with a rabbit anti-human CXCR4 polyclonal primary antibody (ab2090), followed by a horse radish peroxidase conjugated donkey anti-rabbit secondary antibody, and then chemiluminescent substrate. CXCR4 was visualized using enhanced chemiluminescent (ECL). The samples represented are as follows: Lane 1- CHP-134, Lane 2-KCN, Lane 3-KCNR, Lane 4-NB-69, Lane 5-NGP, Lane 6-SK-N-FI, Lane 7-SK-N-AS, Lane 8-SK-N-ASΔ3*, Lane 9-LAN-5, Lane 10-SK-N-SH, Lane 11-SH-SY5Y. Cell lines are color coded by surface expression class: Red = Low, Blue = Medium, Green = High, Black = Not Conclusively Determined (N.D.). *SK-N-ASΔ3 is a derivative cell line of SK-N-AS that has been transfected with an expression vector containing the coding sequence for an E3 ubiquitin ligase component Cullin-5 (CUL-5). All samples were loaded equally as determined by comparing actin levels in each lane (Data not shown). B) Western Blot Detection of Ubiquitin in CXCR4 Immunoprecipitates. Cell lysates were adsorbed with rabbit anti-human CXCR4 antibodies followed by precipitation with protein G Agarose. Precipitated CXCR4 Immune complexes were resolved on 4-15% Bis-Tris SDS gels and transferred to nitrocellulose membranes. Membranes were probed with a mouse anti-human Ubiquitin monoclonal antibody followed by an HRP-conjugated donkey anti-mouse secondary antibody, and then addition of chemiluminescent substrate. Ubiquitin was visualized by measuring ECL. Sample order is the same as in panel A.

Mentions: CXCR4 has been shown to display significant structural heterogeneity, [22] yet to date no extensive analysis of CXCR4 secondary structure in neuroblastoma has been performed. Western blot analysis of CXCR4 was performed on all the cells in our panel in an attempt to identify any structural heterogeneity of CXCR4 that might further refine our stratification of CXCR4 surface expression. Fig. 7A shows a significant degree of structural heterogeneity for CXCR4 with several isoforms being differentially represented across the three classes of surface expressing lines. Of the various isoforms present five appeared to differentially segregate between the high and low surface expressing classes, and served to help further characterize and distinguish between these two classes. Two major forms of approximately 38 and 45 kilodaltons, corresponded well to reported masses for native and glycosylated forms of CXCR4 respectively [22,23], and were present exclusively in the low CXCR4 surface expressing cell lines. Three major forms of approximately 55, 67, and 87 kilodaltons and equal stoichiometry appeared to primarily associate with the high surface expressing class. It should be noted that to varying degrees these isoforms were also present in the moderate class of surface expressing cell lines, however without the same stoichiometry. The presence of a large diffuse component spanning the 67-87 kDa regions in four of the five low surface expressing cell lines was observed. This material did not appear as a discrete band and was seen on immunoblots with anti-CXCR4 (Fig. 7A), as well as with isotype control immunoblots and on Ponceau S stained membranes prior to immunoblotting (data not shown). CXCR4 has been shown to be ubiquitinated, in a basal and agonist-induced manner, contributing to the degree of heterogeneity frequently observed upon secondary structural analyses [23,24]. To determine if any of the isoforms we observed were ubiquitinated, CXCR4 was immunoprecipitated from cell lysates followed by western blotting with anti-ubiquitin monoclonal antibody. There were two bands of approximately 68 and 72 kilodaltons present to varying degrees in all of the cell lines (Fig. 7B); The 68 kilodalton ubiquitinated molecule appears to correspond to the 67 kilodalton isoform described earlier as one of the three associated with high surface expressing cell lines (Fig. 7A). The 72 kilodalton ubiquitinated molecule is also believed to be a CXCR4 isoform observed in cell lines from both the high and moderate surface expressing lines, albeit not to the same extent. Attachment of ubiquitin serves as a primary signal to identify proteins targeted for turnover by the proteasome. SK-N-AS is known to be deleted at Chromosome 11q23 [25], a region which contains the gene for Cul 5, a component of the E3 ubiquitin ligase complex that mediates proteasomal degradation [26]. Comparison between SK-N-AS and SK-N-AS 3, a derivative cell line transfected with a Cul 5 expression vector, shows a shift occurred from the low surface expressing to high surface expressing CXCR4 structural profile respectively (Fig. 7A); this conversion appears to be associated with changes in Cul 5 expression levels (Fig. 8A). In an effort to further evaluate proteasomal-mediated targeting of CXCR4 as a possible mechanism for regulating its surface expression, neuroblastoma cells were treated with the irreversible proteasome inhibiting agent Lactacystin. Fig. 8B shows treatment of the high CXCR4 surface expressing cell line SK-N-SH with this compound resulted in a significant decrease in surface levels of the receptor relative to untreated cells (P < 0.005, paired t-test). The low surface expressing SH-SY5Y showed no significant changes in CXCR4 levels. These observations were similar for the other high and low surface expressing cells in our panel respectively (Data not shown).


CXCR4 expression heterogeneity in neuroblastoma cells due to ligand-independent regulation.

Carlisle AJ, Lyttle CA, Carlisle RY, Maris JM - Mol. Cancer (2009)

Analysis of CXCR4 Structural Heterogeneity in Neuroblastoma Cells. A) Western Blot Detection of CXCR4 in Neuroblastoma Cell Lysates. Cell lysates were generated by solubilizing neuroblastoma cells in 1% triton X-100. Detergent soluble proteins were resolved on 4-20% Bis-Tris SDS gels and transferred to nitrocellulose membranes. CXCR4 was detected by probing with a rabbit anti-human CXCR4 polyclonal primary antibody (ab2090), followed by a horse radish peroxidase conjugated donkey anti-rabbit secondary antibody, and then chemiluminescent substrate. CXCR4 was visualized using enhanced chemiluminescent (ECL). The samples represented are as follows: Lane 1- CHP-134, Lane 2-KCN, Lane 3-KCNR, Lane 4-NB-69, Lane 5-NGP, Lane 6-SK-N-FI, Lane 7-SK-N-AS, Lane 8-SK-N-ASΔ3*, Lane 9-LAN-5, Lane 10-SK-N-SH, Lane 11-SH-SY5Y. Cell lines are color coded by surface expression class: Red = Low, Blue = Medium, Green = High, Black = Not Conclusively Determined (N.D.). *SK-N-ASΔ3 is a derivative cell line of SK-N-AS that has been transfected with an expression vector containing the coding sequence for an E3 ubiquitin ligase component Cullin-5 (CUL-5). All samples were loaded equally as determined by comparing actin levels in each lane (Data not shown). B) Western Blot Detection of Ubiquitin in CXCR4 Immunoprecipitates. Cell lysates were adsorbed with rabbit anti-human CXCR4 antibodies followed by precipitation with protein G Agarose. Precipitated CXCR4 Immune complexes were resolved on 4-15% Bis-Tris SDS gels and transferred to nitrocellulose membranes. Membranes were probed with a mouse anti-human Ubiquitin monoclonal antibody followed by an HRP-conjugated donkey anti-mouse secondary antibody, and then addition of chemiluminescent substrate. Ubiquitin was visualized by measuring ECL. Sample order is the same as in panel A.
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Figure 7: Analysis of CXCR4 Structural Heterogeneity in Neuroblastoma Cells. A) Western Blot Detection of CXCR4 in Neuroblastoma Cell Lysates. Cell lysates were generated by solubilizing neuroblastoma cells in 1% triton X-100. Detergent soluble proteins were resolved on 4-20% Bis-Tris SDS gels and transferred to nitrocellulose membranes. CXCR4 was detected by probing with a rabbit anti-human CXCR4 polyclonal primary antibody (ab2090), followed by a horse radish peroxidase conjugated donkey anti-rabbit secondary antibody, and then chemiluminescent substrate. CXCR4 was visualized using enhanced chemiluminescent (ECL). The samples represented are as follows: Lane 1- CHP-134, Lane 2-KCN, Lane 3-KCNR, Lane 4-NB-69, Lane 5-NGP, Lane 6-SK-N-FI, Lane 7-SK-N-AS, Lane 8-SK-N-ASΔ3*, Lane 9-LAN-5, Lane 10-SK-N-SH, Lane 11-SH-SY5Y. Cell lines are color coded by surface expression class: Red = Low, Blue = Medium, Green = High, Black = Not Conclusively Determined (N.D.). *SK-N-ASΔ3 is a derivative cell line of SK-N-AS that has been transfected with an expression vector containing the coding sequence for an E3 ubiquitin ligase component Cullin-5 (CUL-5). All samples were loaded equally as determined by comparing actin levels in each lane (Data not shown). B) Western Blot Detection of Ubiquitin in CXCR4 Immunoprecipitates. Cell lysates were adsorbed with rabbit anti-human CXCR4 antibodies followed by precipitation with protein G Agarose. Precipitated CXCR4 Immune complexes were resolved on 4-15% Bis-Tris SDS gels and transferred to nitrocellulose membranes. Membranes were probed with a mouse anti-human Ubiquitin monoclonal antibody followed by an HRP-conjugated donkey anti-mouse secondary antibody, and then addition of chemiluminescent substrate. Ubiquitin was visualized by measuring ECL. Sample order is the same as in panel A.
Mentions: CXCR4 has been shown to display significant structural heterogeneity, [22] yet to date no extensive analysis of CXCR4 secondary structure in neuroblastoma has been performed. Western blot analysis of CXCR4 was performed on all the cells in our panel in an attempt to identify any structural heterogeneity of CXCR4 that might further refine our stratification of CXCR4 surface expression. Fig. 7A shows a significant degree of structural heterogeneity for CXCR4 with several isoforms being differentially represented across the three classes of surface expressing lines. Of the various isoforms present five appeared to differentially segregate between the high and low surface expressing classes, and served to help further characterize and distinguish between these two classes. Two major forms of approximately 38 and 45 kilodaltons, corresponded well to reported masses for native and glycosylated forms of CXCR4 respectively [22,23], and were present exclusively in the low CXCR4 surface expressing cell lines. Three major forms of approximately 55, 67, and 87 kilodaltons and equal stoichiometry appeared to primarily associate with the high surface expressing class. It should be noted that to varying degrees these isoforms were also present in the moderate class of surface expressing cell lines, however without the same stoichiometry. The presence of a large diffuse component spanning the 67-87 kDa regions in four of the five low surface expressing cell lines was observed. This material did not appear as a discrete band and was seen on immunoblots with anti-CXCR4 (Fig. 7A), as well as with isotype control immunoblots and on Ponceau S stained membranes prior to immunoblotting (data not shown). CXCR4 has been shown to be ubiquitinated, in a basal and agonist-induced manner, contributing to the degree of heterogeneity frequently observed upon secondary structural analyses [23,24]. To determine if any of the isoforms we observed were ubiquitinated, CXCR4 was immunoprecipitated from cell lysates followed by western blotting with anti-ubiquitin monoclonal antibody. There were two bands of approximately 68 and 72 kilodaltons present to varying degrees in all of the cell lines (Fig. 7B); The 68 kilodalton ubiquitinated molecule appears to correspond to the 67 kilodalton isoform described earlier as one of the three associated with high surface expressing cell lines (Fig. 7A). The 72 kilodalton ubiquitinated molecule is also believed to be a CXCR4 isoform observed in cell lines from both the high and moderate surface expressing lines, albeit not to the same extent. Attachment of ubiquitin serves as a primary signal to identify proteins targeted for turnover by the proteasome. SK-N-AS is known to be deleted at Chromosome 11q23 [25], a region which contains the gene for Cul 5, a component of the E3 ubiquitin ligase complex that mediates proteasomal degradation [26]. Comparison between SK-N-AS and SK-N-AS 3, a derivative cell line transfected with a Cul 5 expression vector, shows a shift occurred from the low surface expressing to high surface expressing CXCR4 structural profile respectively (Fig. 7A); this conversion appears to be associated with changes in Cul 5 expression levels (Fig. 8A). In an effort to further evaluate proteasomal-mediated targeting of CXCR4 as a possible mechanism for regulating its surface expression, neuroblastoma cells were treated with the irreversible proteasome inhibiting agent Lactacystin. Fig. 8B shows treatment of the high CXCR4 surface expressing cell line SK-N-SH with this compound resulted in a significant decrease in surface levels of the receptor relative to untreated cells (P < 0.005, paired t-test). The low surface expressing SH-SY5Y showed no significant changes in CXCR4 levels. These observations were similar for the other high and low surface expressing cells in our panel respectively (Data not shown).

Bottom Line: Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism.Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Oncology, Abramson Research Center, Children's Hospital of Philadelphia, ARC-907A, 3615 Civic Center Blvd, Philadelphia, Pennsylvania 19104-4399, USA. carlisle@email.chop.edu

ABSTRACT

Background: CXCR4, the receptor for the chemokine stromal-derived factor 1 (SDF-1), has been shown to mediate many of the processes essential for cancer progression such as tumor cell proliferation, metastasis, and angiogenesis. To understand the role of CXCR4 in the biology of neuroblastoma, a disease that presents with wide spread metastases in over 50% of patients, we screened ten patient derived-neuroblastoma cell-lines for basal CXCR4 expression and sought to identify characteristics that correlate with tumor cell phenotype.

Results: All cell lines expressed CXCR4 mRNA at variable levels, that correlated well with three distinct classes of CXCR4 surface expression (low, moderate, or high) as defined by flow cytometry. Analysis of the kinetics of CXCR4 surface expression on moderate and high expressing cell lines showed a time-dependent down-regulation of the receptor that directly correlated with cell confluency, and was independent of SDF1. Cell lysates showed the presence of multiple CXCR4 isoforms with three major species of approximately 87, 67 and 55 kDa associating with high surface expression, and two distinct species of 45 and 38 kDa correlating with low to surface expression. Western blot analysis of CXCR4 immunoprecipitates showed that the 87 and 67 kDa forms were ubiquitinated, while the others were not. Finally, treatment of cells with a proteasome inhibitor resulted in down regulation of CXCR4 surface expression.

Conclusions: Taken together, these data show that regulation of CXCR4 surface expression in neuroblastoma cells can occur independently of SDF-1 contribution arguing against an autocrine mechanism. Additionally these data suggest that post-translational modifications of CXCR4, in part through direct ubiquitination, can influence trafficking of CXCR4 to the surface of neuroblastoma cells in a ligand-independent manner.

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Related in: MedlinePlus