CXCR4 function in cancer and normal cells differs. For example, CXCL12 induces apoptosis in primary cultures of neonatal astrocytes but promotes growth through enhanced survival in cultures of astrocytoma cells [7
]. Similarly, neuroblastoma [86
] and melanoma [87
] cells exhibit proliferation in response CXCL12 while growth of their normal counterparts is not known to do so. In each of these neuroectodermal cancers the level of CXCR4 expression has prognostic significance suggesting that the acquisition of this novel cancer-promoting effect is related, in part, to increased receptor numbers and/or an increase in the strength of receptor signals [87
]. However, under normal circumstances excessive activation of CXCR4 is stringently counter-regulated and increased receptor activation alone may not be enough to drive abnormal CXCL12-induced cancer growth. In this final section I will consider whether dysregulation of CXCR4 signaling results in the genesis of novel biological effects. The importance of recognizing and understanding dysregulated CXCR4 signals lies in the therapeutic opportunity it affords. As extended inhibition of CXCR4 signaling has thus far proved toxic [8
], it may be that normalization, rather than inhibition of CXCR4 would provide therapeutic benefit with fewer side effects.
The work of multiple investigators has led to the hypothesis that the CXCL12-CXCR4 pathway plays a significant role in astrocytoma biology [7
]. An important validation of this hypothesis is the prognostic significance of CXCL12 and CXCR4 expression. Bian at al.
found that patients with CXCR4-positive high-grade gliomas had significantly reduced survival compared to patients with gliomas that did not express CXCR4 [89
]. Similarly, Calatozzolo, et al, found that CXCL12 expression was a more powerful predictor of time-to-progression than histological subtype in oligodendroglioma or oligoastrocytoma [91
Among the potential mechanisms whereby increased CXCR4 expression could alter the nature of cellular responses is by modifying the relative abundance of CXCR4 homo- and hetero-dimers. The presence of homo- and hetero-dimers of chemokine receptors at baseline suggests that “normal” chemokine effects could result from the balance of signals initiated from both dimer species. Alterations in the numbers of CXCR4 molecules could therefore shift the balance towards homodimers or result in the formation of rare, low-affinity heterodimers with receptor types that do not normally pair with CXCR4 when receptor numbers are lower.
Changes in the balance of homo- and hetero-dimers as well as the formation of novel heterodimers could have significant effects on signaling and biology. Heterodimers formed between CXCR4 and CCR2 and between CXCR4 and δ-opioid receptor are present under basal conditions in primary isolates of T lymphocytes and blood mononuclear cells, respectively [94
]. In both instances the presence of heterodimers has a significant effect on response to each ligand. While the presence of CXCR4 - δ-opioid receptor heterodimers had no effect on response to CXCL12 alone, co-stimulation with the enkephalin δ-opioid agonist DPDPE blocked all CXCL12-induced signaling and inhibited chemotaxis in vivo
as measured by the accumulation of mononuclear cells in peritoneal fluid after intraperitoneal injection of the combined receptor agonists [95
]. This was not due to heterologous desensitization as no increase in CXCR4 phosphorylation was detected in MonoMac-1 cells after DRDPE treatment. Rather, it appeared that co-stimulation of the heterodimer resulted in a silent receptor. Similarly, Mellado et al.
showed that co-stimulation of CCR2-CCR5 heterodimers with CCL5 and CCL2 led to novel Gαq/11
activation that was relatively resistant to regulation by desensitization [96
]. This was in contrast to Gαi
mediated signals which originated from homodimers of CCR2 and CCR5, which were readily desensitized. Thus changes in the level of CXCR4 expression alone, or in combination with changes in other chemokine receptors could alter the profile of receptor homo- and hetero-dimers. This in turn could alter signaling in more than one chemokine dependent pathway and might even result in the acquisition of novel signals not possessed by any of the component receptors when functioning as homodimers.
Even in the absence of an altered receptor dimer profile, the opportunity to distort CXCR4 functions exists through modulation of the counter-regulatory mechanisms. Consistent with the known role of Gi-dependent signals in chemotaxis, loss of RGS expression or function generally results in increased chemotactic responses. This was demonstrated in lymphocytes and could have implications in leukemia and lymphoma (see above). RGS4 is expressed in neuroblastoma cells where it inhibits signaling through Gαi
]. RGS2 is expressed in pheochromocytoma derived PC12 cells where it interacts with microtubules and promotes neurite outgrowth in response to nerve growth factor [98
]. Loss of RGS attenuates the differentiating effects of NGF, a function that could be relevant to transformation within the neuroectoderm. Unexpectedly, increased RGS 3 and 4 expression enhanced U373 glioma cell adhesion and migration [99
] suggesting that the role of CXCR4 in cancer may not be a simple matter of more or less CXCR4 signaling.
Astrocytes, pre-cancerous astrocytes and astrocytoma cells
The activation of CXCR4 in the setting of RGS deficiency can still be counter-regulated through the process of desensitization. To determine whether such dysregulation of CXCR4 signaling was important to tumorigenesis, Warrington et al.
examined a spectrum of cells, from normal to malignant, derived from the astrocytoma lineage. As an intermediate between normal and malignant they utilized astrocytes with complete loss of the tumor suppressor neurofibromin. Neurofibromin is mutated in the autosomal dominant disorder neurofibromatosis Type 1, which predisposes affected patients to multiple cancers including benign and malignant astrocytomas [100
Significant differences in wildtype and Nf1
−/− astrocyte responses to CXCL12 were evident in in vitro
studies. Primary cultures of wildtype neonatal astrocytes exhibited an increase in apoptosis while Nf1
−/− astrocytes displayed a decrease in apoptosis when treated with CXCL12 [30
]. These opposed effects were dependent upon differences in CXCL12-induced cAMP suppression. Loss of neurofibromin resulted in diminished CXCR4 desensitization and prolonged AC inhibition. The molecular basis for altered desensitization was increased Erk-mediated phosphorylation and inhibition of GRK2 in Nf1
−/− astrocytes. This resulted in a decrease in CXCL12-induced CXCR4 phosphorylation. Inhibition of Erk activation with the MEK inhibitor PD98059 led to re-activation of GRK2 and normalization of CXCL12-induced CXCR4 phosphorylation and cAMP responses. These data were consistent with studies that identified abnormal suppression of cAMP as the basis for CXCL12-induced medulloblastoma and glioblastoma growth [7
]. Together these results suggest that altered CXCR4 counter-regulation gives rise to novel CXCR4 signals and functions, productive of abnormal growth promoting effects.
Alterations in GPCR desensitization play a pathophysiological role in other diseases and cancers. Beautiful work done by Balabanian et al.
demonstrated that mutation of the carboxy-tail of CXCR4 alters its internalization without abrogating its interaction with arrestins. This leads to aberrant activation of arrestin from the cell surface and enhanced chemotaxis, which contributes to the disease [83