Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunohistochemical (IHC) Staining of Prostate Tissues for CXCR4.A, A human prostate tissue array, ranging from normal to high-grade prostate cancer, was evaluated by IHC for CXCR4 expression using standard methods. Samples were evaluated at magnification 40X, using a Q-Imaging camera of Olympus BX51 Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate tissues demonstrated slightly weak or undetectable brown staining for CXCR4 (positive cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate tissue (grade 2, stage II, T2N0M0, adenocarcinoma) demonstrated random/focal positive staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4, stage IV, T4N1M1, adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. B, CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4 protein by western blot analysis in PC3 (CXCR4 positive) or 293T (CXCR4 ) cell lines. C, CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. D, CXCR4 IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by western blot analysis. Beta-actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585330&req=5

pone-0057194-g001: Immunohistochemical (IHC) Staining of Prostate Tissues for CXCR4.A, A human prostate tissue array, ranging from normal to high-grade prostate cancer, was evaluated by IHC for CXCR4 expression using standard methods. Samples were evaluated at magnification 40X, using a Q-Imaging camera of Olympus BX51 Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate tissues demonstrated slightly weak or undetectable brown staining for CXCR4 (positive cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate tissue (grade 2, stage II, T2N0M0, adenocarcinoma) demonstrated random/focal positive staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4, stage IV, T4N1M1, adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. B, CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4 protein by western blot analysis in PC3 (CXCR4 positive) or 293T (CXCR4 ) cell lines. C, CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. D, CXCR4 IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by western blot analysis. Beta-actin was used as a loading control.

Mentions: Previous domain analysis of CXCR4 suggested that CXCR4 contains a nuclear targeting signal between amino acids 90–170 [73]. A bioinformatics analysis using the PSORT II NLS prediction software (http://psort.ims.u-tokyo.ac.jp/) revealed a putative nuclear localization sequence, ‘RPRK’ [72], [74], [75], [76] between amino acids 146–149 within CXCR4 (Table 1). Additionally, a HomoloGene/NCBI database search for the NLS within CXCR4 revealed that ‘RPRK’ is been conserved among species, including chicken, mouse, chimpanzee and others (Table 2). Moreover, CXCR4 has been detected in the nucleus of several cancer tissues [68], [70]. Based on these data, we tested whether CXCR4 protein could be detected within the nucleus of prostate tissues. Using a prostate tissue microarray ranging from normal to high-grade metastatic lesions, we detected positive immunoreactivity for CXCR4 in prostate samples. Positive immunoreactivity was detected as sporadic (CXCR4 positive cells <5%), focal (CXCR4 positive cells >11%, but less than 50%), or diffuse (CXCR4 positive cells >50%), compared to the average total density of positive cells for CXCR4 (DAB stained). Samples with immunohistochemical scores of negative, weak or moderate staining, with sporadic to focal distributions, were considered to have ‘low’ expression, whereas diffuse distributions of staining were considered to have ‘high’ expression for CXCR4. Staining intensity was sporadic to focal in the nucleus of low grade prostate tissues (Fig. 1A), while high grade malignant tissues (Fig. 1A), displayed an increased staining intensity and diffuse expression of CXCR4 throughout the tissue compared to low grade. In both low and high grade tumors, a fraction of CXCR4 clearly co-localized with the nucleus. Staining intensity for CXCR4 was weak, or even , in normal tissues (Fig. 1A).


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)

Immunohistochemical (IHC) Staining of Prostate Tissues for CXCR4.A, A human prostate tissue array, ranging from normal to high-grade prostate cancer, was evaluated by IHC for CXCR4 expression using standard methods. Samples were evaluated at magnification 40X, using a Q-Imaging camera of Olympus BX51 Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate tissues demonstrated slightly weak or undetectable brown staining for CXCR4 (positive cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate tissue (grade 2, stage II, T2N0M0, adenocarcinoma) demonstrated random/focal positive staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4, stage IV, T4N1M1, adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. B, CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4 protein by western blot analysis in PC3 (CXCR4 positive) or 293T (CXCR4 ) cell lines. C, CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. D, CXCR4 IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by western blot analysis. Beta-actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585330&req=5

pone-0057194-g001: Immunohistochemical (IHC) Staining of Prostate Tissues for CXCR4.A, A human prostate tissue array, ranging from normal to high-grade prostate cancer, was evaluated by IHC for CXCR4 expression using standard methods. Samples were evaluated at magnification 40X, using a Q-Imaging camera of Olympus BX51 Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate tissues demonstrated slightly weak or undetectable brown staining for CXCR4 (positive cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate tissue (grade 2, stage II, T2N0M0, adenocarcinoma) demonstrated random/focal positive staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4, stage IV, T4N1M1, adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. B, CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4 protein by western blot analysis in PC3 (CXCR4 positive) or 293T (CXCR4 ) cell lines. C, CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. D, CXCR4 IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by western blot analysis. Beta-actin was used as a loading control.
Mentions: Previous domain analysis of CXCR4 suggested that CXCR4 contains a nuclear targeting signal between amino acids 90–170 [73]. A bioinformatics analysis using the PSORT II NLS prediction software (http://psort.ims.u-tokyo.ac.jp/) revealed a putative nuclear localization sequence, ‘RPRK’ [72], [74], [75], [76] between amino acids 146–149 within CXCR4 (Table 1). Additionally, a HomoloGene/NCBI database search for the NLS within CXCR4 revealed that ‘RPRK’ is been conserved among species, including chicken, mouse, chimpanzee and others (Table 2). Moreover, CXCR4 has been detected in the nucleus of several cancer tissues [68], [70]. Based on these data, we tested whether CXCR4 protein could be detected within the nucleus of prostate tissues. Using a prostate tissue microarray ranging from normal to high-grade metastatic lesions, we detected positive immunoreactivity for CXCR4 in prostate samples. Positive immunoreactivity was detected as sporadic (CXCR4 positive cells <5%), focal (CXCR4 positive cells >11%, but less than 50%), or diffuse (CXCR4 positive cells >50%), compared to the average total density of positive cells for CXCR4 (DAB stained). Samples with immunohistochemical scores of negative, weak or moderate staining, with sporadic to focal distributions, were considered to have ‘low’ expression, whereas diffuse distributions of staining were considered to have ‘high’ expression for CXCR4. Staining intensity was sporadic to focal in the nucleus of low grade prostate tissues (Fig. 1A), while high grade malignant tissues (Fig. 1A), displayed an increased staining intensity and diffuse expression of CXCR4 throughout the tissue compared to low grade. In both low and high grade tumors, a fraction of CXCR4 clearly co-localized with the nucleus. Staining intensity for CXCR4 was weak, or even , in normal tissues (Fig. 1A).

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