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Cysteine (C)-x-C receptor 4 undergoes transportin 1-dependent nuclear localization and remains functional at the nucleus of metastatic prostate cancer cells.

Don-Salu-Hewage AS, Chan SY, McAndrews KM, Chetram MA, Dawson MR, Bethea DA, Hinton CV - PLoS ONE (2013)

Bottom Line: Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues.Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function.Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA, USA.

ABSTRACT
The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), 'RPRK', within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

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Nuclear CXCR4 was Functional at the Nucleus.A, Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. B, Whole cells were treated with SDF1α prior to isolating and lysing intact nuclei. Nuclei lysates (1 mg) were immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed for Gαi (first row) or CXCR4 antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo1, nuclear) were used as markers for fractionation purity and as loading controls. C, PC3 nuclei were isolated, incubated with FluoForte dye Ca2+ probe, followed by incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDF1α for 30 min. An increase in fluorescent-bound Ca2+ was measured on a microplate reader at ex = 490 nm/em = 525 nm.
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pone-0057194-g005: Nuclear CXCR4 was Functional at the Nucleus.A, Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. B, Whole cells were treated with SDF1α prior to isolating and lysing intact nuclei. Nuclei lysates (1 mg) were immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed for Gαi (first row) or CXCR4 antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo1, nuclear) were used as markers for fractionation purity and as loading controls. C, PC3 nuclei were isolated, incubated with FluoForte dye Ca2+ probe, followed by incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDF1α for 30 min. An increase in fluorescent-bound Ca2+ was measured on a microplate reader at ex = 490 nm/em = 525 nm.

Mentions: Finally, we sought to determine whether CXCR4 receptors at the nucleus are functional. Upon ligand stimulation and activation, GPCRs, including CXCR4, dissociates from a trimer of G-proteins (Gα and Gβγ) which initiates secondary signaling pathways, such as increased cyclic AMP, increased intracellular calcium levels, and others [90]. Thus, a reduction in the G-alpha protein, Gαi, associated with CXCR4 represents an active state of a receptor. Therefore, we co-immunoprecipitated CXCR4 and Gαi and determined Gαi expression levels by Western blot analysis, to assess whether nuclear-associated CXCR4 was active. Whole cells were stimulated with SDF1α then harvested to isolate intact nuclei (Fig. 5A). Nuclei were lysed, then immunoprecipitated with anti-CXCR4, prior to immunobloting for associated Gαi. In untreated cells, we observed a basal level in Gαi expression, which decreased upon treatment with SDF1α, suggesting that nuclear-associated CXCR4 is functional and can respond to SDF1α.


Cysteine (C)-x-C receptor 4 undergoes transportin 1-dependent nuclear localization and remains functional at the nucleus of metastatic prostate cancer cells.

Don-Salu-Hewage AS, Chan SY, McAndrews KM, Chetram MA, Dawson MR, Bethea DA, Hinton CV - PLoS ONE (2013)

Nuclear CXCR4 was Functional at the Nucleus.A, Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. B, Whole cells were treated with SDF1α prior to isolating and lysing intact nuclei. Nuclei lysates (1 mg) were immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed for Gαi (first row) or CXCR4 antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo1, nuclear) were used as markers for fractionation purity and as loading controls. C, PC3 nuclei were isolated, incubated with FluoForte dye Ca2+ probe, followed by incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDF1α for 30 min. An increase in fluorescent-bound Ca2+ was measured on a microplate reader at ex = 490 nm/em = 525 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585330&req=5

pone-0057194-g005: Nuclear CXCR4 was Functional at the Nucleus.A, Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. B, Whole cells were treated with SDF1α prior to isolating and lysing intact nuclei. Nuclei lysates (1 mg) were immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed for Gαi (first row) or CXCR4 antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo1, nuclear) were used as markers for fractionation purity and as loading controls. C, PC3 nuclei were isolated, incubated with FluoForte dye Ca2+ probe, followed by incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDF1α for 30 min. An increase in fluorescent-bound Ca2+ was measured on a microplate reader at ex = 490 nm/em = 525 nm.
Mentions: Finally, we sought to determine whether CXCR4 receptors at the nucleus are functional. Upon ligand stimulation and activation, GPCRs, including CXCR4, dissociates from a trimer of G-proteins (Gα and Gβγ) which initiates secondary signaling pathways, such as increased cyclic AMP, increased intracellular calcium levels, and others [90]. Thus, a reduction in the G-alpha protein, Gαi, associated with CXCR4 represents an active state of a receptor. Therefore, we co-immunoprecipitated CXCR4 and Gαi and determined Gαi expression levels by Western blot analysis, to assess whether nuclear-associated CXCR4 was active. Whole cells were stimulated with SDF1α then harvested to isolate intact nuclei (Fig. 5A). Nuclei were lysed, then immunoprecipitated with anti-CXCR4, prior to immunobloting for associated Gαi. In untreated cells, we observed a basal level in Gαi expression, which decreased upon treatment with SDF1α, suggesting that nuclear-associated CXCR4 is functional and can respond to SDF1α.

Bottom Line: Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues.Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function.Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA, USA.

ABSTRACT
The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), 'RPRK', within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

Show MeSH
Related in: MedlinePlus