Human gliomas are heterogeneous in their degree of malignancy, ranging from more slowly growing astrocytoma to fast growing and highly invasive GBM. This study showed that even in a well-established GBM cell line, FPR is expressed at different levels by cell subsets, and an FPR-expressing subset is more motile and forms more rapidly growing invasive tumours in nude mice as compared with its FPR-negative counterpart. The FPR-expressing clone F9 also expressed higher levels of vimentin, an astroglia precursor marker, but lower levels of the differentiation marker GFAP than the FPR-negative subset G3. This is consistent with the findings with surgically removed human glioma specimens, in which FPR expression is associated with poorly differentiated tumours, and within individual human gliomas, tumour cells frequently exhibit remarkable heterogeneity (Zhou et al, 2005
). This may explain our findings that 3 out of 14 anaplastic astrocytoma were negative for FPR and 2 out of 13 of less invasive astrocytoma are positive for FPR (Zhou et al, 2005
). The presence of highly proliferative and invasive cell populations in a given solid tumour determines the rate of tumour progression and the lethality to the host. The U87 glioblastoma cell line contains both FPR+
subpopulations: although the FPR+
subpopulation represented by F9 clone, the G3 subclone is FPR−
as reported in this study. However, both the FPR+
F9 subclone and the FPR−
G3 subclone express EGF receptor and proliferate in response to EGF. Therefore, in the heterogeneous U87 cell line, the FPR+
cells did not show apparent growth advantage over FPR−
cells in vitro
. This may explain why FPR−
cells may sustain their growth over longitudinal passaging and are uncoverable in the heterogeneous population. The FPR gene has a single copy in FPR+
U87 cells. Over a period of 15 passages, these FPR+
cells represented by the F9 clone maintained the same level of FPR expression. However, it is interesting to note that it has been reported that glioma stem-like cells isolated from U87 cell line and primary human gliomas express FPR (Yao et al, 2008b
) and such stem-like cells differentiate in vitro
to yield both FPR+
descendents. Therefore, it seems that FPR+
malignant glioma cells may represent a more poorly differentiated cell population with higher degree of malignancy.
One of the most important features of malignant tumour cells is their capacity to break the barrier of surrounding normal host tissues. This invasion process is known to depend not only on tumour cell motility, but also on tumour cell-secreted MMPs to degrade ECM. The MMPs also cleave and activate other growth factors such as TGF-β that are implicated in GBM motility and proliferation.
The expression of MMPs in tumour cells is induced by cytokines, growth factors, tumour promoters, physical stress, oncogenic transformation, and cell–matrix and cell–cell interactions. Several MMP genes are inducible by extracellular stimuli, which activate the AP1 transcription-factor complex through pathways involving MAPKs (ERK1/22, JNKs and p38) and PKC; PKC is also known to have a function in increasing MMP9 expression by malignant glioma cells (Yabkowitz et al, 1999
) through cytoskeletal changes and NF-κ
B (Chintala et al, 1998
). Activation of FPR in myeloid and GBM cells triggers these signalling events (Zhou et al, 2005
; Kam et al, 2007
); therefore, provide mechanistic basis for its ability to regulate the production and activation of MMPs. The capacity to mediate MMP production may be a common feature for chemoattractant GPCRs. For instance, an FPR variant receptor, FPRL1, in monocytic cells (Lee et al, 2005
) and a chemokine GPCR CXCR3 in colon cancer cells have also been shown to up-regulate MMP9 (Zipin-Roitman et al, 2007
). Thus, GPCRs have an important function in increasing the proteolytic processes favouring leucocyte infiltration of tissues and tumour dissemination.
Progression of malignant tumours depends on timely neovascularisation in tumour and surrounding tissues. One of the most potent angiogenic factors produced in solid tumours is VEGF, which not only acts on vascular endothelial cells, but also increases the survival, migration and invasion of many tumour cells bearing VEGF receptors. In addition, VEGF is a potent suppressor of antigen-presenting cells in tumour microenviroment, contributing to the establishment of immune privilege of tumours. Malignant gliomas, notably GBMs, are characterised by vigorous angiogenesis and the production of copious amounts of VEGF. Our earlier study showed that stimulation of FPR in human GBM cells increased cell production of VEGF (Zhou et al, 2005
) and another angiogenic chemokine IL8 (CXCL8) (Yao et al, 2008a
). This study indicates that expression of FPR alone in originally FPR-negative tumour cells is sufficient for tumour cells to produce VEGF in conventional culture conditions in the presence of FCS or HS, and stimulation with FPR agonist peptide fMLF further increased VEGF production. The more vigorous angiogenesis by FPR+
GBM cells was further shown by higher microvessel density in tumours formed by such tumour cells, indicating a pivotal function of FPR in enhancing the angiogenic and vasculogenic process in GBM. It is interesting to note that MMPs have been reported to also act as ‘angiogenic switch' in tumour neovascularisation (Bergers et al, 2000
). Thus, FPR may promote GBM angiogenesis through direct induction of angiogenic factors and through elevated MMPs in tumour microenvironment.
Our study showing FPR agonist activity in the serum and supernatants of necrotic tumour cells (Zhou et al, 2005
) strongly suggests that this receptor expressed by GBM cells may interact with host-derived agonists in vivo
. Although the precise chemical nature of FPR agonist(s) contained in serum and necrotic tumour supernatants remains to be clarified, our preliminary results suggest that the FPR agonists are of small molecule peptide nature (Zhou et al, 2005
). Plasma is the most complex human-derived proteome known to contain proteins derived from blood cells and other tissues including those released by normal or damaged cells and aberrant proteins from tumour cells. As proteins in the circulatory system are exposed to a variety of proteases and peptidases, plasma is rich in peptides of various size to constitute ‘peptidome'. In this context, some endogeneous agonists of FPR and its variant receptors have been identified, such as the neutrophil granule protein cathepsin G (Sun et al, 2004
), cleaved peptide from urokinase plasminogen activator receptor (Resnati et al, 2002
), a fragment of heme-binding proteins (F2L) (Migeotte et al, 2005
) and formylated peptides from mitochondria (Rabiet et al, 2005
). In addition, a phospholipid-binding protein annexin I, which was reported to be a ligand for FPR and its variant receptors, has been implicated in promoting the invasiveness of a human intestinal cancer cell line and a mouse melanoma cell line (Babbin et al, 2006
; Rondepierre et al, 2009
). Although the molecular nature of the FPR-stimulating activity in sera remains to be elucidated, our further studies showed that the chemotactic activity and colony-stimulating activity contained in the supernatant of necrotic GBM cells were depleted by immunoabsorption with an antibody against annexin I (Yang et al
, manuscript in preparation), suggesting that annexin I is likely a major component responsible for the FPR-stimulating activity released by necrotic GBM cells. Thus, identification and characterisation of FPR agonist(s) in human GBM tissues, which may activate the receptor on tumour cells will shed light on the mechanisms of tumour–microenvironment interaction.
In summary, malignant tumours exploit their microenvironment to favour their survival, growth, invasion of fertile ‘soil', and angiogenesis by ‘hijacking' receptors involved in physiological processes to sense agonists present in the intercellular milieu. The FPR behaves as an oncoprotein that confers a highly malignant phenotype on GBM cells with increased cell motility, adhesion and release of proteases and production of angiogenic factors. Our recent study (Huang et al, 2008b
) found that the aberrant expression of FPR in highly malignant GBM cells is closely associated with increased methylation of the p53 tumour suppressor gene promoter that reduced the capacity of p53 to repress the active transcription of FPR gene. Consequently, treatment of FPR-expressing GBM cells with methyltransferase inhibitor or transfection of the wild-type p53 reduced FPR expression and promoted GBM cell differentiation. Thus, studies of the regulation and signal transduction of FPR in GBM may yield novel molecular targets for anti-glioma therapy.