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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 Expression in Prostate Cancer Cell Lines.A, Normal prostate epithelial (RWPE1) and PCa (PC3, DU145, 22RV1) cells were stimulated with SDF1α (100 ng/ µl) prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls. The bar graphs are quantitative results of the band density representing expression of CXCR4 in each fraction. Data were mean +S.E. from three independent experiments. *, P<0.05. B, Immunocytochemistry of PCa cell lines for CXCR4. PCa cells were stimulated with SDF1α (100 ng/ µl), fixed with methanol, blocked then incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated anti-rabbit antibody. Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclear-associated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bars represent 50 µm.
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pone-0057194-g002: Nuclear CXCR4 Expression in Prostate Cancer Cell Lines.A, Normal prostate epithelial (RWPE1) and PCa (PC3, DU145, 22RV1) cells were stimulated with SDF1α (100 ng/ µl) prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls. The bar graphs are quantitative results of the band density representing expression of CXCR4 in each fraction. Data were mean +S.E. from three independent experiments. *, P<0.05. B, Immunocytochemistry of PCa cell lines for CXCR4. PCa cells were stimulated with SDF1α (100 ng/ µl), fixed with methanol, blocked then incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated anti-rabbit antibody. Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclear-associated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bars represent 50 µm.

Mentions: We used biochemical fractionation to confirm the nuclear localization of CXCR4 detected in our tissue staining. PCa cell lines were fractionated into nuclear and non-nuclear samples for detection of CXCR4 by western blot analysis (Fig. 2A). We found that normal prostate epithelial cells (RWPE1) were for CXCR4 [77]; however, three CXCR4-expressing PCa cell lines (22RV1, DU145 and PC3) dually expressed CXCR4 in both nuclear and non-nuclear fractions independent of SDF1α stimulation (Fig. 2A). The purity of subcellular fractions was confirmed by expression of CD44, a non-nuclear marker [78], and topoisomerase 1, a nuclear marker [79], which ruled out the possibility that expression of CXCR4 observed in nuclear fractions was due to contamination.


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 Expression in Prostate Cancer Cell Lines.A, Normal prostate epithelial (RWPE1) and PCa (PC3, DU145, 22RV1) cells were stimulated with SDF1α (100 ng/ µl) prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls. The bar graphs are quantitative results of the band density representing expression of CXCR4 in each fraction. Data were mean +S.E. from three independent experiments. *, P<0.05. B, Immunocytochemistry of PCa cell lines for CXCR4. PCa cells were stimulated with SDF1α (100 ng/ µl), fixed with methanol, blocked then incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated anti-rabbit antibody. Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclear-associated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bars represent 50 µm.
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pone-0057194-g002: Nuclear CXCR4 Expression in Prostate Cancer Cell Lines.A, Normal prostate epithelial (RWPE1) and PCa (PC3, DU145, 22RV1) cells were stimulated with SDF1α (100 ng/ µl) prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls. The bar graphs are quantitative results of the band density representing expression of CXCR4 in each fraction. Data were mean +S.E. from three independent experiments. *, P<0.05. B, Immunocytochemistry of PCa cell lines for CXCR4. PCa cells were stimulated with SDF1α (100 ng/ µl), fixed with methanol, blocked then incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated anti-rabbit antibody. Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclear-associated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bars represent 50 µm.
Mentions: We used biochemical fractionation to confirm the nuclear localization of CXCR4 detected in our tissue staining. PCa cell lines were fractionated into nuclear and non-nuclear samples for detection of CXCR4 by western blot analysis (Fig. 2A). We found that normal prostate epithelial cells (RWPE1) were for CXCR4 [77]; however, three CXCR4-expressing PCa cell lines (22RV1, DU145 and PC3) dually expressed CXCR4 in both nuclear and non-nuclear fractions independent of SDF1α stimulation (Fig. 2A). The purity of subcellular fractions was confirmed by expression of CD44, a non-nuclear marker [78], and topoisomerase 1, a nuclear marker [79], which ruled out the possibility that expression of CXCR4 observed in nuclear fractions was due to contamination.

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