We have previously shown that the activation of the PI3K/Akt pathway in bone-metastatic prostate cancer cells exposed to human bone marrow aspirates depends mostly on PDGFRα signaling [11
]. Furthermore, prostate cancer cells that lack bone-metastatic potential - and were less responsive to bone marrow – could activate Akt to the same extent of bone-metastatic cells following PDGFRα overexpression [13
]. Therefore, our first series of experiments sought to ascertain whether the acellular fraction of human bone marrow would induce phosphorylation of PDGFRα - as is the case when this receptor binds, and is activated by, its proper PDGF ligands [24
]. As done for our previous studies, we employed PC3-ML cells, a sub-population of the human PC3 cell line originally derived from a skeletal metastasis in a patient affected by prostate adenocarcinoma [25
]. PC3-ML cells were originally selected for their invasiveness in vitro
in concert with their bone-metastatic potential in animal models [21
]. When these cells were exposed to bone marrow, PDGFRα phosphorylation was clearly detected and lasted at least 30 minutes (). The ability of bone marrow to phosphorylate PDGFRα could be consistently observed using aspirates withdrawn from different donors (, top
Human bone marrow induce phosphorylation of PDGFRα and activation of downstream Akt pathway in prostate cancer cells
Structural and functional studies have shown that PDGF binding induces dimerization of PDGFR; this allows the trans
-phosphorylation of several conserved tyrosine residues on the juxtaposed intracellular portion of each receptor [26
]. In addition to enhancing the catalytic activity of the kinase domain, this event creates docking sites for the binding and activation of signal transduction mediators containing SH2 domains, including members of the PI3K family [27
]. At least eight tyrosine residues on PDGFRα become phosphorylated upon binding of PDGF ligand(s) [28
], likely contributing to the strong signal observed in our experiments ().
Interestingly, the considerably lower magnitude of PDGFRα phosphorylation induced by bone marrow - as compared to that induced by PDGF ligand(s) – did not translate to a significantly lower activation of the downstream Akt pathway. In fact, remarkably close levels of Akt phopshorylation were observed after exposing PC3-ML cells to either PDGF or bone marrow (, bottom
) and as evidenced by a time-dependent kinetic (). In addition, we had previously demonstrated that the majority of this observed Akt activation is due solely to PDGFRα signaling [11
]. This suggests that the activation of PI3K, which is functionally upstream of, and mainly responsible for, Akt phosphorylation, must be almost equivalent in these two conditions. Thus, we speculate that the majority of PDGFRα phosphorylation caused by bone marrow corresponds to Tyr-731 and Tyr-742 residues, which specifically bind and activate PI3K [24
]. This would explain the lower magnitude of receptor phosphorylation as compared to stimulation by PDGF ligands. However, the phosphorylation of only select tyrosine residues could hardly be justified in the event that bone marrow induces PDGFRα dimerization. In fact, whereas dimers could be observed upon exposure of PC3-ML cells to PDGF-AA, human bone marrow consistently lacked the ability to induce dimerization of PDGFRα in these cells ().
Recruitment of PDGFRα by human bone marrow occurs in the absence of receptor dimerization
This initial evidence of an unorthodox mechanism used by bone marrow to initiate PDGFRα signaling was further confirmed by a second set of experiments in which dimerization was blocked using a monoclonal antibody directed against the extracellular portion of the receptor. The humanized antibody IMC-3G3 blocks the ligand-binding domain of PDGFRα [11
], thereby inhibiting receptor dimerization following treatment with PDGF-AA (). By blocking ligand-induced dimerization, IMC-3G3 also prevented PDGFRα trans-phosphorylation (), which normally occurs by juxtaposition of intracellular kinase domains between receptor monomers [27
]. However, no differences in PDGFRα phosphorylation were observed when cells were exposed to bone marrow alone or in the presence of IMC-3G3 (). This result provides compelling evidence that dimerization is not a pre-requisite for the activation of PDGFRα by the soluble fraction of human bone marrow in PC3-ML prostate cancer cells. If this was indeed the case, an active kinase must still phosphorylate the tyrosine residues responsible for recruitment of PI3K to the receptor, as this step should precede downstream activation of Akt by PDGFRα exposed to bone marrow. One likely possibility was the hetero-dimerization of PDGFRα with other tyrosine-kinase receptors. However, this event could be excluded by the absence of any dimer formation following treatment of cells with bone marrow and subsequent cell-surface receptor crosslinking ().
Bone marrow-mediated activation of PDGFRα occurs upon blockade of ligand-induced receptor dimerization
Thus, we hypothesize that PDGFRα is activated without recruitment of its ligand-binding domain and consequent dimerization, possibly by a soluble tyrosine-kinase that phosphorylates the monomeric form of the receptor. In fact, this idea is strongly supported by a recent study of proliferative vitreoretinopathy showing that activated Src family kinases (SFKs) phosphorylate PDGFRα in mouse embryo fibroblasts exposed to rabbit vitreous humor, the clear viscous fluid that occupies the space between the retina and lens of vertebrates’ ocular bulb [31
In these cells, growth factors outside of the PDGF family and present in the vitreous can activate their specific receptor(s), thereby increasing the levels of intracellular reactive oxygen species (ROS). This event led to the direct activation of SFKs, which then mediated the phosphorylation of PDGFRα [19
]. We have previously shown that human bone marrow activates downstream Akt despite containing negligible levels of PDGF ligands (measured at 150pg/ml by ELISA [11
]), and also induces evident PDGFRα phosphorylation (). Thus, if bone marrow were engaging PDGFRα in prostate cancer cells with a mechanism analogous to vitreous humor in mouse embryo fibroblasts, this would similarly require an increase in intracellular ROS. In this case, either anti-oxidants or inhibitors of SFKs should then block the activation of PDGFRα. To test this possibility, we treated PC3-ML cells with either 5mM hydrogen peroxide alone or bone marrow in the presence of the anti-oxidant N-Acetylcysteine (NAC, 10mM). The first condition – adopted to induce intracellular ROS production -– was unable to induce PDGFRα phosphorylation. Analogously, NAC did not prevent the activation of the receptor and downstream PI3K/Akt signaling caused by bone marrow (data not shown).
PDGF-independent but receptor kinase-dependent phosphorylation of PDGFRα induced by human bone marrow
We could also exclude the involvement of SFKs in the activation and signaling of PDGFRα induced by bone marrow by using PP2, a specific inhibitor of this family of kinases [32
]. In PC3-ML cells, PP2 did not prevent Akt phosphorylation by bone marrow (), whereas it completely blocked stimulation of this pathway by PDGF-AA. This finding is in agreement with a described role of SFKs in coordinating ligand-induced recruitment of signaling pathways by tyrosine-kinase receptors, by acting upstream of PI3K [34
Taken together, these results lead us to conclude that human bone marrow activates PDGFRα on prostate cancer cells in a manner that is distinct from the factors found in vitreous humor, supporting the idea that there may be multiple mechanisms whereby this receptor can be activated without involvement of its canonical PDGF ligands.
It is widely recognized that the activity of the kinase domain in tyrosine kinase receptors is significantly augmented upon dimerization and trans-phosphorylation [30
]. Because we observed a lack of PDGFRα dimerization upon exposure of prostate cancer cells to human bone marrow, one would predict that the kinase domain is neither necessary nor responsible for the phosphorylation of monomeric PDGFRα. To effectively investigate this idea, we used two different prostate cancer cell lines that show low levels of this receptor (PC3-N) or completely fail to express it (DU-145) [12
] and engineered them to stably express a kinase-inactive PDGFRα mutant (R627)[36
]. Surprisingly, both R627-expressing cell types failed to show receptor phosphorylation upon bone marrow exposure, in contrast to when the same cell types over-express the wild-type form of PDGFRα (). Thus, these results implicate the kinase domain in the phosphorylation and signaling of monomeric PDGFRα induced by bone marrow. This observation suggests the possibility of a receptor autophosphorylation event, independent of ligand-induced dimerization, and most likely affecting the tyrosine residues responsible for PI3K binding and activation. The identification of the mechanism and signaling mediators responsible for this event are the focus of an ongoing investigation in our laboratory.
In previous animal studies we have shown that PDGFRα can confer bone-metastatic potential to prostate cancer cells [13
]. Intriguingly, the role exerted by this receptor in the metastatic process may require its indirect activation – likely through the activity of yet unidentified factor. To further investigate this peculiarity of PDGFRα signaling, we stably transfected cancer cells with a truncated receptor mutant (αΔX) that lacks the extracellular ligand-binding domain (see Materials and Methods for details) and is therefore unable to bind or be activated by proper PDGF ligand(s) or additional molecules that could activate the receptor is a spurious fashion [22
A first set of experiments was conducted using DU-145 prostate cancer cells, which normally do not express PDGFRα [12
] and would therefore not interfere by providing additional signaling through its wild-type full form. The αΔX expressed in these cells () was clearly insensitive to PDGF-AA, as expected due to its lack of binding capability and shown by the absence of any receptor phosphorylation (, top
). However, the exposure of DU-145(αΔX) cells to human bone marrow aspirates induced strong phosphorylation of the truncated receptor, conclusively demonstrating its ligand-independent activation (, top
). This phosphorylation involved the Tyr-742 residue (, bottom
), which on PDGFRα specifically corresponds to the PI3K binding site through its SH-2 domain [30
], unequivocally linking the soluble fraction of human bone marrow to the downstream activation of the Akt signaling pathway in prostate cancer cells. This event could be observed in cells either constitutively expressing PDGFRα ( and refs. [11
]) or engineered to express the exogenous form of this receptor ().
PDGFRα activation by human bone marrow is independent of the extracellular ligand-binding domain
A second series of experiments aimed to ascertain whether the αΔX receptor could reproduce the enhanced capacity of skeletal metastases from prostate cancer cells, as we had previously shown for its full-length form [13
]. A confirmatory result would convincingly demonstrate that PDGFRα could induce a pro-metastatic phenotype, with pronounced bone-tropism, through a ligand-independent transactivation caused by the acellular fraction of bone marrow. PC3-N cells were originally selected from the parental PC3 cell line for their lack of invasiveness in vitro
]. We have previously shown that PC3-N cells express lower levels of PDGFRα and respond weakly to human bone marrow as compared to their bone-metastatic counterpart PC3-ML cells [12
]. Most importantly, PC3-N cells disseminate to the bone when inoculated in the blood circulation of SCID mice, but fail to grow into macroscopic skeletal metastases unless they are engineered to over-express full-length PDGFRα [13
]. When PC3-N cells were stably transduced with a lentiviral vector expressing PDGFR(αΔX) () and exposed to bone marrow, Akt was phosphorylated to the same extent observed in either PC3-ML cells () or PC3-N cells expressing full-length PDGFRα treated in the same fashion.
PDGFRα(ΔX) induces a bone metastatic phenotype that is indistinguishable from cells expressing full-length PDGFRα
In order to test their bone-metastatic potential, PC3-N(αΔX) cells were then inoculated via an intracardiac route into SCID mice and the skeletal metastases observed at 4-week post-inoculation were compared to those detected in mice receiving the highly bone-metastatic PC3-ML cells. When the bone tumors in these two groups of mice were compared, no significant differences in size were observed and the location and bone-destructive properties induced by the αΔX and full-length PDGFRα were nearly identical (). These results demonstrate that PDGFRα can both increase the responsiveness of human prostate cancer cells to acellular bone marrow in vitro and promote their progression in the bone microenvironment in the absence of any involvement of its ligand-binding domain.
Recognizing that PDGFRα could contribute to dissemination and metastatic progression of prostate adenocarcinoma independently of direct ligand stimulation has significant translational implications. It could be inferred that anti-cancer therapeutics that are designed to block PDGF ligand binding to PDGFRα would not prevent the activation of downstream signaling pathways in cells that have spread to the bone marrow. In contrast, therapeutic approaches that induce internalization of PDGFRα may provide a better option to inhibit downstream signaling elicited by this receptor in disseminated prostate cancer cells when exposed to the bone marrow microenvironment.