The atypical 7-transmembrane chemokine receptor, CXCR7, transactivates the EGFR leading to increased tumor growth in several tumor types. However, the molecular mechanism of CXCR7 ligand–independent EGFR transactivation is unknown. We used cDNA knock-in, RNAi and analysis of mitogenic signaling components in both normal prostate epithelial cells and prostate cancer cells to decipher the proliferation-inducing mechanism of the CXCR7–EGFR interaction. The data demonstrate that CXCR7-induced EGFR transactivation is independent of both the release of cryptic EGFR ligands (e.g., AREG/amphiregulin) and G-protein–coupled receptor signaling. An alternate signaling mechanism involving β-arrestin-2 (ARRB2/β-AR2) was examined by manipulating the levels of β-AR2 and analyzing changes in LNCaP cell growth and phosphorylation of EGFR, ERK1/2, Src, and Akt. Depletion of β-AR2 in LNCaP cells increased proliferation/colony formation and significantly increased activation of Src, phosphorylation of EGFR at Tyr-1110, and phosphorylation/activation of ERK1/2 compared with that with control shRNA. Moreover, β-AR2 depletion downregulated the proliferation suppressor p21. Stimulation of β-AR2–expressing cells with EGF resulted in rapid nuclear translocation of phosphorylated/activated EGFR. Downregulation of β-AR2 enhanced this nuclear translocation. These results demonstrate that β-AR2 is a negative regulator of CXCR7/Src/EGFR–mediated mitogenic signaling.
Implications: This study reveals that β-AR2 functions as a tumor suppressor, underscoring its clinical importance in regulating CXCR7/EGFR–mediated tumor cell proliferation. Mol Cancer Res; 14(5); 493–503. ©2016 AACR.
This article is featured in Highlights of This Issue, p. 409
Chemokine receptors are members of the seven-transmembrane guanine nucleotide-binding protein (G protein)–coupled receptor (GPCR) superfamily. The intracellular signaling of chemokine receptors is mainly dependent on the binding of heterotrimeric G proteins on intracellular loop epitopes and the carboxyl-terminal tail of these receptors (1). The CXC-chemokine receptor 7 (CXCR7) is a unique receptor that can be engaged by the chemokines CXCL11 and CXCL12/SDF-1 (2). CXCR7 is overexpressed in many cancer cell types and elevated expression of CXCR7 promotes tumor cell proliferation and tumor growth (3–6). Unlike other classical chemokine receptors, CXCR7 does not mobilize intracellular calcium, does not elicit motility in normal cells, and preferentially signals through the β-arrestin-2 (β-AR2) pathway upon binding to its ligands (6, 7).
Arrestins belong to a family of proteins that consists of four members. Visual (arrestin 1) and cone arrestins (arrestin 4) are exclusively expressed in the retina, whereas β-arrestin-1 (arrestin 2) and β-AR2 (arrestin 3) are ubiquitously expressed in most tissues (8, 9). β-Arrestins are well-known negative regulators of GPCR signaling. β-arrestin binding to GPCRs both uncouples receptors from heterotrimeric G proteins and targets them to clathrin-coated pits for endocytosis. Recent evidence, however, demonstrates that β-arrestins can function as adaptor molecules that mediate G-protein–independent signaling by serving as scaffolds that link signaling networks and regulate signaling molecules such as the mitogen-activated protein kinases ERK, JNK, and p38 as well as Akt and PI3K (10). Importantly, while β-arrestin-1 was shown to mediate metastatic growth in breast cancer cells (11), the role of β-AR2 in tumor progression is more enigmatic and needs further clarification. Some reports suggest that β-AR2 may induce cancer cell progression (12); however, there is also evidence that depletion of β-AR2 promotes tumor growth in a murine model of lung cancer, indicating that β-AR2 might function as a tumor suppressor (13).
We reported previously that CXCR7 increases cell proliferation independent of its ligands (CXC11 and CXCL12/SDF-1α). Furthermore, alteration in CXCR7 expression levels directly affects phosphorylation of EGFR at Tyrosine1110. In addition, we have reported direct coupling of EGFR and CXCR7 in prostate cancer and breast cancer cells both in vitro and in tumors in vivo (6, 14). Although CXCR7 is an atypical GPCR, the mechanism of transactivation of EGFR by unstimulated CXCR7 is unknown. In the current work, we aimed to evaluate the role of β-AR2 in CXCR7-mediated EGFR transactivation in prostate cancer cells. We also elucidated the signaling pathways that are regulated by β-AR2, including Src activation and downstream mitogenic signaling (e.g., ERK1/2 and the PI3K/Akt) pathway, and the role of β-AR2 in regulating prostate tumor cell proliferation.
Materials and Methods
SMARTpool siRNA for various cellular targets including β-AR2 and nontarget control siRNA, and Dharmafect-2 transfection reagent were purchased from Dharmacon Inc. (Dharmacon, GE Life Sciences). Recombinant chemokines and growth factors (Human SDF-1α, CXCL11, IL8, EGF, and amphiregulin) were purchased from R&D Systems. Full-length CXCR7 cDNA was obtained from Origene and was cloned in Ampicillin vectors for stable transfection in RWPE-1 cells. CXCR7-GFP and β-AR2 expression vectors were a gift from Dr. Kathleen Luker, University of Michigan (Ann Arbor, MI). HuSH/shRNA plasmid panels for transient or stable downregulation of β-AR2 [GI326261 (1), GI326262 (2), GI326263 (3), GI326264 (4)] and scrambled shRNA [TR30013 (13)] were purchased from Origene.
Cell culture and transfection
Nonmalignant prostate epithelial cells (RWPE-1) and prostate cancer cell lines (LNCaP, C4-2B, and PC-3) were obtained from ATCC and were used within 10 passages of cell authenticity confirmation by genetic profiling (Genetica DNA Laboratory Inc.). FBS was from Atlanta Biologicals, antibiotics (gentamycin) and cell culture media were purchased from Invitrogen or Sigma-Aldrich. LNCaP, C4-2B, and PC-3 cells were grown in RPMI1640 containing 10% FBS. RWPE-1 cells were grown in keratinocyte serum-free medium supplemented with bovine pituitary extract and EGF (Invitrogen). For stable transfections, RWPE-1 cells were grown to 50%–60% confluence and transfected with 0.6 to 1.0 μg of DNA using Lipofectamine 2000 (Life Technologies). Stable clones (RWCX7) were selected and maintained in G418 (250 μg/mL). Lipofectamine 2000 was also used for cotransient transfection of RWCX7 cells with the β-AR2 cDNA constructs. For stable downregulation of β-AR2, LNCaP cells were seeded in 12-well plates (2 × 105 cells/well) in the absence of antibiotics. Twenty-four hours later, cells were transfected with HuSH/shRNA plasmid panels (500 ng/well for LNCaP) using Lipofectamine 2000 (2.125 μL/well for LNCaP). Stable clones were created through puromycin selection (2 μg/mL).
Forty-eight hours after transfection, cells were serum starved for 16 hours and then stimulated with EGF (10 ng/mL, 2 or 5 minutes). Cell lysates were subjected to Western blot analysis as described previously (6). Please see Supplementary Table S1 for details of primary antibodies, sources, and dilutions used. In parallel experiments, cells were cultured in growth factor depleted (1% FBS) medium for 24 hours, pretreated with Pertussis toxin, Amphiregulin, or neu-ab (Amphiregulin-neutralizing Ab) at concentrations and times indicated in the figure legends, followed by stimulation with EGF (10 ng/mL, 2 or 5 minutes). Pretreatment with PP2 or AG148 was used to block the activation of endogenous or stimulated EGFR as indicated in figures legends.
Coimmunoprecipitation assays were performed in LNCaP cells to visualize HSP90 association with CXCR7. Cells were lysed following incubation for 72 hours and cell lysates were cleared through incubation with Protein A-agarose beads. Lysates were incubated with anti-HSP90 antibody overnight followed by incubation with Protein A-agarose beads (Sigma-Aldrich) and immunoprecipitated CXCR7 was detected by Western blot analysis. Whole cell lysates were used as input controls. Anti-EGFR antibodies were used as positive controls for immunoprecipitation of CXCR7 (6).
We evaluated CXCR7 and HSP90 colocalization in prostate cancer cells by immunofluorescence staining. PC-3 cells were seeded and cultured in multichamber slides (Thermo Fisher Scientific) until they were semiconfluent. Cells were fixed in situ with 3% paraformaldehyde and permeabilized with 0.25% Triton X-100 in PBS. Cells were incubated with anti-CXCR7 and anti-HSP90 primary antibodies and, following washing with PBS, incubated with fluorophore (Alexa Fluor 488 or Alexa Fluor 555) labeled secondary antibodies (Thermo Fisher Scientific). Nuclei were stained with DAPI. Labeled cells were observed under a confocal microscope (Zeiss LSM 700) and images were analyzed by Zeiss imaging software.
Proximity ligations assay/in-cell coimmunoprecipitation assay
The proximity ligations assay (PLA) was performed to visualize and quantify HSP90/CXCR7 colocalization. PLA was performed using a kit and the contained instructions (Duolink In Situ, Olink Bioscience, Sigma) as described previously (14). Combination of a primary rabbit anti-CXCR7 antibody and a nonspecific mouse isotype antibody was used as a negative control. Combination of a primary rabbit anti-CXCR7 antibody and a primary mouse anti-CXCR4 antibody were used as positive controls.
Chemotactic motility assay
Chemotactic motility of LNCaP cells and the sublines created in this study was evaluated using 8-μm pore size Boyden chambers (Transwell plates, Corning/Costar Inc.; ref. 15). Cells were harvested, resuspended in culture medium containing 1% FBS, and deposited (2 × 105 cells/filter) in the top wells of the Transwell plates. The bottom well contained 10% FBS as a chemoattractant (500 μL/well). Percent of cells in each treatment group that migrated through the filter in 48 hours was determined by MTT assay as described previously (15, 16). Migratory/chemotactic activity (% migration) was defined as the ratio of optical density (OD) from the bottom wells (including the cells from the bottom side of the filters) to that of the total OD (OD of bottom plus top wells), multiplied by 100. Assays were repeated at least three times.
Multisite-directed mutagenesis for generation of mutant EGFR
EGFR-wt (without stop codon) was blunt-end inserted into the HindIII site of the pLPCX Vector (Clontech). The MYC epitope tag (with stop codon) was blunt-end cloned into the EcoRI site. The primers for the mutagenesis assays were designed using QuikChange Primer Design software (Stratagene) to introduce a nucleotide substitution within the EGFR-hybridizing sequence to generate a tyrosine to phenylalanine substitution at position 1110 (Y1110 to F1110). Mutagenesis reaction was carried out using the Stratagene Multisite-Directed Mutagenesis Kit (Agilent Technologies). DNA from five transformed bacterial colonies was isolated using Wizard Plus SV DNA Miniprep Kit (Promega) and sequenced to verify the presence of the designed mutation. The plasmid thus generated was transfected into LNCaP cells and stable lines were selected with puromycin, as described above. Selected lines were tested for pEGFRY1110 by anti-pEGFRY1110 rabbit monoclonal IgG.
All quantitative data shown, except the Western blot quantification, were from three separate experiments; each data point represents the mean of the triplicate results. Western blots have been conducted twice. Significance of data was analyzed with the GraphPad Prism software (GraphPad Inc.).
G-protein signaling is not involved in CXCR7-mediated EGFR phosphorylation
Previous studies have demonstrated the inability of CXCR7 to generate typical G-protein responses such as an increase in intracellular calcium after stimulation with either SDF-1α or CXCL11 (3, 17–19). We attempted to investigate the involvement of G protein, if any, in the CXCR7-mediated increase in EGF-induced EGFR phosphorylation at EGFRY1110. We treated nontumorigenic prostate epithelial cells, RWPE-1- and CXCR7-overexpressing RWPE-1 (RWCX7) cells for 2 hours with pertussis toxin (PTX, 0.5 μg/mL), a known inhibitor of GPCR-mediated G-protein coupling. We stimulated EGFR phosphorylation with 10 nmol/L EGF for 2 to 5 minutes. We did not observe any detectable change in EGFR phosphorylation in PTX-treated RWPE-1 or RWCX7 cells compared with that of cells treated with EGF alone (Fig. 1A and B). Similar results were observed in LNCaP cells (Fig. 1C). This observation suggested that G-protein signaling does not affect CXCR7-mediated EGFR phosphorylation.
CXCR7-mediated phosphorylation of EGFR is not associated with EGF-like ligands
Several previous studies have shown a role of EGF-like ligands such as amphiregulin (Amp) and heparin-bound EGF in chemokine receptor (e.g., CXCR4) induced EGFR transactivation (20, 21). These cryptic EGF-like compounds sequestered in the extracellular matrix (ECM) are unmasked and released as active EGF by CXC-chemokine–mediated extracellular proteolysis by matrix metallopeptidase 9 (MMP9). We hypothesized such a scenario with CXCR7-enhanced phosphorylation of EGFR. RWCX7 or LNCaP cells were treated with recombinant Amp, EGF, or both and phosphorylation of EGFR was analyzed by Western blotting. EGF stimulation resulted in rapid phosphorylation of EGFR (pEGFRY1110) which was almost completely blocked by the EGFR kinase inhibitor, tyrphostin (AG1478; Fig. 1D and E). In contrast, stimulation with Amp did not affect the pEGFRY1110 level in RWCX7 cells. Similarly, treatment of RWCX7 cells with neutralizing anti-Amp-antibody (Amp-Neu) prior to stimulation with EGF did not affect pEGFRY1110 levels, indicating Amp does not contribute to increased pEGFRY1110 in RWCX7 and LNCaP cells.
We also observed increased activation of the mitogenic signaling pathway (phosphorylation of ERK1/2) upon treatment with EGF which is consistent with increased cell proliferation relative to growth factor–starved cells (Fig. 1D and E).
β-AR-2 is a negative regulator of EGFR phosphorylation/transactivation
We have previously shown that CXCR7 mediates EGFR phosphorylation/transactivation in a CXCR7 ligand–independent fashion (6, 14). Rajagopal and colleagues (7) observed recruitment of β-AR2 by CXCR7 in human embryonic kidney-29 (HEK29) cells that are overexpressing CXCR7. This resulted in phosphorylation of ERK1/2. Attempts by us to stimulate LNCaP or RWPE-1 cells with SDF-1α did not result in redistribution of β-AR2 or increase ERK1/2 phosphorylation (data not shown). However, stimulation with EGF alone, results in an increased pEGFRY1110/total EGFR ratio in RWCX7 cells when compared with vector-only transfected RWPE-1 cells (Fig. 1A and B). In addition, stimulation of LNCaP cells overexpressing β-AR2-EGFP with SDF-1α did not alter the distribution of β-AR2-EGFP. Stimulating these cells with EGF-triggered redistribution of β-AR2-EGFP into the cytoplasm and nucleus, indicating dynamic β-AR2-EGFP relocalization following EGFR phosphorylation (Supplementary Fig. S3).
We next attempted to dissect the role of β-AR2 in CXCR7-induced EGFR transactivation when CXCR7 is signaling independently of its ligands. First, we depleted β-AR2 in LNCaP cells through transient transfection with β-AR2 shRNA (construct 1, 2, 3, or 4) and evaluated the phosphorylation status of EGFR by Western blot analysis following stimulation with EGF. Furthermore, we examined the effects of shRNA-mediated β-AR2 downregulation on ERK1/2 activation and the PI3K/Akt pathway. In parallel experiments, we overexpressed β-AR2 in LNCaP cells to evaluate its effect on EGFR phosphorylation. The shRNA-mediated β-AR2 depletion resulted in an increase of EGFR phosphorylation at the tyrosine1068 and tyrosine1110 (Y1068 and Y1110) residues. Transient depletion of β-AR2 in LNCaP cells increased the levels of phosphorylated ERK1/2 and Akt (Fig. 2A). These results were also confirmed in C4-2B cells, a castration-resistant LNCaP subline (Fig. 2C). In contrast, overexpression of β-AR2 resulted in a decrease of phosphorylated EGFR in LNCaP cells (Fig. 2B). Taken together, our results show the phosphorylation status of EGFR inversely correlates with the expression of β-AR2 in prostate cancer cells.
We next generated LNCaP cells with stably downregulated β-AR2 (β-AR2shRNA clones 1, 2, 3) and evaluated the effects on phosphorylation of EGFR, ERK1/2, and Akt, as well as the expression of the cell-cycle regulator p21. Stable downregulation of β-AR2 (LNCaP-1, 2, 3) resulted in an increase of phosphorylated EGFR, Akt, and ERK1/2 in EGF stimulated cells, whereas the level of p21 was dramatically decreased (Fig. 2D). Importantly, when β-AR2 was transiently overexpressed in the stable β-AR2 shRNA-expressing clone, LNCaP-2, the above described effects were reversed (Fig. 2E).
Src mediates CXCR7-induced EGFR transactivation
The inability of externally added CXCR7 cognate ligands to enhance EGFR phosphorylation, and the absence of activation of other cryptic EGFR ligands in CXCR7-overexpressing cells, made us inquire about the role of Src kinase in CXCR7-mediated EGFR activation. RWPE-1 and RWCX7 cells were pretreated with the Src kinase inhibitor PP2 with or without EGF and the relative increase in p-EGFR was determined by Western blot analysis. In RWPE-1 cells [Fig. 3A(i)], constitutive p-EGFR was undetectable even when we loaded twice the amount of total cell lysates as that of RWCX7 cells [Fig. 3A(ii), lane 1]. Following addition of EGF, we observed a rapid increase in p-EGFR in both the cell lines; this increase was significantly inhibited in the presence of PP2. PP2 alone was also able to inhibit basal level of pEGFRY1110 in RWCX7 cells. In a parallel approach, we investigated the effect of PP2 on EGFR phosphorylation in LNCaP cells (Fig. 3B). Pretreatment with PP2 remarkably downregulated EGF-mediated EGFRY1110 phosphorylation. These results indicate that Src is upstream of EGFR in the CXCR7–EGFR axis, and is required for EGFR phosphorylation at Y1110. Furthermore, in the absence of PP2, overexpression of β-AR2, similar to the downregulation of CXCR7, resulted in a prominent decrease of pEGFRY1110. Addition of PP2 to these cells did not further reduce the level of pEGFRY1110.
The results presented above suggest that β-AR2 is a negative regulator of Src. Therefore, we evaluated the activation of Src in LNCaP cells with stable β-AR2 depletion (Fig. 3C). Consistently, depletion of β-AR2 resulted in an induction of phosphorylated/activated Src. Moreover, overexpression of CXCR7 further induced Src activation (Fig. 3C, right). Taken together, these data demonstrate that a Src-kinase–mediated mechanism is responsible for CXCR7-induced EGFR phosphorylation at Y1110 and that this mechanism is negatively regulated by β-AR2.
β-AR-2 regulates nuclear translocation of EGFR in prostate cancer cells
Accumulating evidence suggests that there is a nuclear translocation of EGFR in cancer cells (22–26). In the nucleus, EGFR may drive expression of genes required for proliferation, such as cyclin D1 and Myc (24). We aimed to evaluate the regulatory roles of both CXCR7 and β-AR2 in the nuclear translocation of EGFR in prostate cancer cells. To first elucidate how phosphorylation regulates the translocation of EGFR to the nucleus, LNCaP cells were transfected with either wild type (WT) EGFR, mutant (MT) EGFR [with a nonphosphorylatable amino acid substitution: tyrosyl (Y) to phenylalanine (F; Y1110 to F1110)], or backbone plasmid. Cells were stimulated with EGF and nuclear and cytoplasmic protein fractions were collected and subjected to Western blot analysis for expression of pEGFR. Stimulation of LNCaP cells with EGF resulted in a rapid nuclear translocation of pEGFR (Fig. 4A). Translocation of pEGFR to the nucleus occurred independent of phosphorylation at Y1110 in LNCaP cells as shown by strongly increased pEGFRY1068 in the nuclear fraction of both EGF-stimulated WT-EGFR– and MT-EGFR–transfected LNCaP cells (Fig. 4B). Downregulation of CXCR7 significantly inhibited nuclear translocation of EGFR (Fig. 4C). Furthermore, we evaluated nuclear and cytoplasmic extracts in LNCaP cells with stable β-AR2 depletion (LNCaP-2 and LNCaP-3) compared with control shRNA-transfected LNCaP (LNCaP-13). LNCaP-3 cells show a higher level of nuclear pEGFRY1110 compared with LNCaP-13 cells (CO; Fig. 4D). Similar results were observed for PC-3 cells (Fig. 4E). In summary, depletion of β-AR2 via shRNA results in an increase of nuclear phosphorylated EGFR, suggesting that β-AR2 negatively regulates nuclear translocation of EGFR and nuclear-EGFR signaling.
Hsp90 interacts with the CXCR7/EGFR/arrestin axis and protects it from proteasome-mediated degradation
Hsp90 is a molecular chaperone that is constitutively expressed in eukaryotic cells and can be further stimulated in its expression level by physiologic stress. It participates in stabilizing over 200 proteins, many of which are essential for constitutive cell signaling (and adaptive responses to stress). As such, we were interested in evaluating whether Hsp90 stabilizes CXCR7.
Immunofluorescence staining and PLA (Duolink) were performed to evaluate colocalization of CXCR7 and Hsp90 in LNCaP cells. Coimmunoprecipitation assays were performed in LNCaP cells for examination of the Hsp90–CXCR7 interaction. Our results show that Hsp90 associates with CXCR7 in LNCaP and PC-3 cells (Fig. 5A–C). Hsp90 and CXCR7 show a predominantly cytoplasmic and perinuclear colocalization (Fig. 5A and B). Treating cells with inhibitors of Hsp90 (e.g., Geldanamycin/17-AAG) resulted in decreased CXCR7/Hsp90 colocalization.
Furthermore, we performed Western blot analysis of nuclear, cytoplasmic, and total protein extracts in the presence or absence of Geldanamycin/17-AAG to evaluate the role of Hsp90 in the stabilization of CXCR7. Kinetic analysis showed that treatment with Geldanamycin/17-AAG resulted in a decrease of CXCR7 total protein level (Fig. 5D and Supplementary Fig. S2A).
Moreover, we show that in the presence of the proteasome inhibitor MG132, Geldanamycin/17-AAG–mediated CXCR7 degradation was rescued in LNCaP and PC-3 cells, suggesting Hsp90 interacts with CXCR7 and protects it from proteasome-mediated degradation (Fig. 5E and Supplementary Fig. S2B). Furthermore, treatment with Geldanamycin resulted in downregulation of β-AR2 and EGFR (Supplementary Fig. S2C and S2D).
β-AR-2 negatively regulates proliferation and migration of prostate cancer cells
As our data suggest activation mitogenic signaling (e.g., activation of Akt and ERK1/2) in cells with stable shRNA-mediated β-AR2 depletion, we aimed to evaluate proliferation in those cell lines compared with control shRNA-transfected cells. We performed colony assays to investigate the replicative potential of β-AR2-shRNA LNCaP compared with control-shRNA LNCaP cells. All three β-AR2 shRNA LNCaP cell lines (LNCaP-1, -2, and -3) showed increased proliferation and colony formation potential compared with control cells (LNCaP-13; Fig. 6A and B). Importantly, LNCaP-3 cells had significantly increased motility compared with LNCaP-13 (Fig. 6C). A model is presented (Fig. 6D) to simplify the complex signaling mechanism involved in CXCR7, EGFR, HSP90, and β-AR2–mediated regulation of prostate cancer cell proliferation.
The data presented above demonstrate the unique mechanism of CXCR7- and β-AR2–mediated growth signaling in prostate cancer cells. A novel finding in this study is the regulation of Src-mediated phosphorylation of tyrosyl1110 in EGFR and its effects on mitogenic signaling. Although Src kinase phosphorylates EGFR at tyrosyl1173, its ability to phosphorylate EGFRY1110 is seldom reported. Another interesting finding in this study is the tumor suppressor function of β-AR2 in prostate cancer that was not reported before. Our findings strongly support the hypothesis that β-AR2 negatively regulates tumor cell proliferation and, more importantly, their chemotactic motility. LNCaP cells have poor motility relative to more aggressive prostate cancer tumor cells, such as PC-3 which express a lower level of β-AR2 (unpublished data).
Previously, we reported that the chemokine receptor CXCR7 mediates EGFR phosphorylation/activation in a CXCR7 ligand–independent fashion and plays a crucial role in proliferation of prostate and breast cancer cells (6, 14). In the current work, we show for the first time that β-AR2 negatively regulates CXCR7-mediated phosphorylation/activation of EGFR in prostate cancer cells. Depletion of β-AR2 in LNCaP cells resulted in a significant increase of phosphorylated/activated Src, EGFR, and ERK1/2 and a dramatic decrease of p21 protein level along with an induction of proliferation and clonogenic potential. Although CXCR7 heterodimerizes with a related GPCR, CXCR4, our data show that the two molecules independently interact with other molecules in the cell, such as EGFR and Hsp90. CXCR7 associates with HSP90 as shown by the PLA to demonstrate colocalization.
As previous work has shown that GPCR-mediated activation of EGFR may require activation of MMPs and subsequent cleavage of membrane-bound RTK/EGFR ligands (20), we investigated this pathway in prostate cancer cells. Interestingly, phosphorylation of EGFR in prostate cancer cells seems to be independent of ECM-bound EGFR ligands. In a further attempt, we investigated whether the abovementioned pathways can be bypassed by GPCR-induced Src activation of EGFR. Indeed, treatment of LNCaP cells with the Src inhibitor PP2 resulted in a significant decrease of pEGFRY1110 compared with that of untreated cells. Similar effects were observed when CXCR7 was depleted via siRNA; however, combined treatment with CXCR7 siRNA and PP2 did not further decrease levels of pEGFRY1110 compared with cells treated with CXCR7 siRNA or PP2 alone. These results strongly suggest that Src plays a key role in CXCR7-mediated phosphorylation/activation of EGFR and downstream proliferation signaling.
As CXCR7 is shown to signal through β-AR2 in its ligand-dependent manner (7), we investigated the role of β-AR2 in CXCR7 ligand–independent transactivation of EGFR. Surprisingly, downregulation of β-AR2 resulted in a significant increase of pSrcY416 and pEGFR as well as activation of mitogenic signaling (ERK, Akt), and an increase in proliferation and clonogenic potential. Consistently, β-AR2 overexpression resulted in a decrease of pEGFR and cell proliferation. These results demonstrate that β-AR2 is a negative regulator of CXCR7-mediated EGFR activation and subsequent cell proliferation. It is to be noted that β-arrestin has two isoforms (β-arrestin-1 and β-AR2). While β-arrestin-1 promotes GPCR-mediated Src activation and Erk1/2 phosphorylation, the opposing effects have been observed for β-AR2 in HEK 293 cells (27). Our results in prostate cancer cells show a similar mechanism of β-AR2.
A novel finding in our work is that depletion of β-AR2 resulted in an increase of nuclear EGFR. Multisite-directed mutagenesis experiments indicated that this nuclear translocation is independent of EGFR phosphorylation at Y1110. Nuclear EGFR is associated with disease progression and increased resistance to chemotherapy and anti-EGFR therapies (25). Our results show that β-AR2 negatively regulates the nuclear translocation of EGFR, underlining the potential role of β-AR2 as a tumor suppressor and its potential as a therapeutic target.
In conclusion, we show that CXCR7 mediates EGFR transactivation through activation of Src, and that this pathway is negatively regulated by β-AR2 which inhibits activation of Src in prostate cancer cells (Fig. 6D). Moreover, we show that Hsp90 stabilizes the CXCR7/EGFR/β-AR2 axis in prostate cancer cells and protects it from proteasome-mediated degradation. Although β-arrestins have been known to mediate chemokine receptor–induced mitogenic signaling in cancer cells, the current work demonstrates that β-AR2 functions as a tumor suppressor and negatively regulates mitogenic signaling/proliferation and migration in prostate cancer cells and thus has relevant clinical significance.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: G. Kallifatidis, R.K. Singh, B.L. Lokeshwar
Development of methodology: G. Kallifatidis, R.K. Singh
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G. Kallifatidis, D. Munoz, N. Salazar, J.J. Hoy
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G. Kallifatidis, R.K. Singh, J.J. Hoy, B.L. Lokeshwar
Writing, review, and/or revision of the manuscript: G. Kallifatidis, N. Salazar, J.J. Hoy, B.L. Lokeshwar
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B.L. Lokeshwar
Study supervision: B.L. Lokeshwar
Other (PI of the laboratory that conducted these studies and overall in charge of the project): B.L. Lokeshwar
The authors thank Mr. Gabriel Gaidosh for confocal image acquisition.
This work was supported in parts by the Veterans Health Administration Merit Award (BX 001517-01; to B.L. Lokeshwar), the United States' Public Health Services Awards (NIH-1R01CA156776-01; to B.L. Lokeshwar), and F31 Fellowship (to N. Salazar).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.