Background
Glioma is the most common primary malignant tumor of the central nervous system (CNS) with poor prognosis. Many vessel-related pathological signs were observed in glioma and aberrant microvasculature usually appears as “glomeruloid tufts” consisting of multilayered, mitotically active endothelial cells and perivascular cells [
1,
2]. With the increasing accumulation of knowledge regarding angiogenesis, anti-angiogenic therapy has also been developed and considered as an optimistic strategy for glioma patients [
3,
4]. Bevacizumab, a recombinant monoclonal antibody targeted against VEGF-A, has been received a conditional approval for the treatment of recurrent high-grade gliomas. In addition, clinical trials have also been performed in patients with newly diagnosed glioblastoma multiforme (GBM). Improved progression-free survival and maintenance of baseline quality of life and performance status were observed in GBM patients received combined treatment of bevacizumab and radiotherapy–temozolomide [
5‐
10]. The anti-angiogenic property of bevacizumab is generally considered as a critical contributor to its anti-tumor activity. With the angiogenesis-targeted therapy widely accepted, inevitable recurrence and unexpected tumor resistance, especially increased ability of glioma cell invasion were also observed after anti-VEGF treatment [
11‐
16]. Because the disruption of VEGF autocrine loop after anti-VEGF therapy, it was also thought to be important for the glioma cell phenotypic change [
13,
14]. Unexpected tumor resistance to bevacizumab aroused the additional investigation on direct effects of anti-VEGF therapy on tumor cell migration and invasion. However, the exact mechanism underlying tumor resistance to bevacizumab remains to be elucidated.
Glioma invasion is a complicated process including cell interactions with extracellular matrix (ECM) and adjacent cells, and cell migration. Many factors are involved in this process, such as cadherins, intracellular adhesion molecules, matrix metalloproteinase, myosin II, and so on [
17]. It is well known that cell-matrix and cell-cell junction cross-talk together, and these two junctions cooperatively regulate cell adhesion, polarization, and movement [
18]. Integrins are one of the classic cell adhesion molecules that mediate cell attachment to ECM [
17], which is a critical process in tumor invasion. The binding of integrins to ECM leads to the recruitment of focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase (Pyk2) to the newly formed focal adhesion sites. Activation of FAK and Pyk2 is followed by phosphorylation of a variety of downstream effectors, resulting in cell migration, proliferation, and angiogenesis [
19,
20]. Previous studies have also demonstrated that the expression of FAK and Pyk2 was significantly correlated with the malignant grade of astrocytic tumors [
21], and that down-regulation of FAK expression inhibited glioma cell proliferation and induced apoptosis [
22]. Our previous study also showed an association of FAK and Pyk2 protein level with VEGF expression and angiogenesis in human glioma [
23]. Recently, it was reported that hypoxia contributed to up-regulation of β1 integrin and its downstream effector FAK during bevacizumab therapy, thus promoting mechanisms of survival and evasion [
24]. However, little is known about the role of FAK and Pyk2 in glioma cell invasion after anti-VEGF treatment.
The aim of this study was to determine whether FAK and/or Pyk2 are involved in glioma cell invasion induced by anti-VEGF therapy. We investigated protein levels and their activation of FAK and Pyk2 in glioma cells after anti-VEGF treatment, and then analyzed the correlation of these proteins with glioma cell invasion both in vitro and in vivo. Our results showed that the phosphorylation of Pyk2, but not of FAK, was increased after anti-VEGF treatment. In addition, increased Pyk2 phosphorylation was involved in the promotion of glioma cell invasion after anti-VEGF treatment. The present study underlines the need to combine anti-angiogenic treatment in glioma with drugs capable of specifically targeting Pyk2 to direct more effective therapy.
Discussion
Tumor cell invasion into the normal extracellular matrix is a key feature of malignant gliomas. It was considered as a limiting factor in the treatment, and as a critical factor in the clinical course of glioma because of its role in tumor recurrence. As angiogenesis-dependent invasion exists in glioma [
17], anti-angiogenic therapy might theoretically decrease tumor cell invasion, yet clinical observations are not always consistent with this expectation. Some patients for whom anti-angiogenic treatment fails had an uncharacteristic pattern of tumor progression and an apparent phenotypic shift to a predominantly infiltrative pattern of tumor progression after bevacizumab treatment was observed [
14]. Similarly, other recent studies showed that treatment with anti-VEGFR specific monoclonal antibody caused a striking increase in tumor cell invasion and metastasis [
16,
29,
30]. In accord with these studies, we also observed the increased cell migration and invasion in bevacizumab-treated C6 cells, both
in vitro and
in vivo. These studies provide evidence supporting the notion that glioma cells can directly be affected by anti-VEGF or anti-VEGFR treatment and disruption of VEGF-VEGFR autocrine loop in tumor cells maybe result in glioma cell phenotypic change. These findings will help to explain the resistance to anti-angiogenic therapies observed in clinic, and raise the question of how to elicit tumor cell invasion with anti-angiogenic therapies.
The difference between experiment-driven hypothesis and actual clinical practice indicates that glioma progression is governed by complex mechanisms, which are still not clearly understood. Invasion of tumor cells into normal tissue is thought to be a multifactorial process, consisting of cellular interactions with ECM and adjacent cells, and accompanying biochemical processes supportive of active cell movement. Glioma cell invasion requires four distinct processes including detachment of invading cells from the primary tumor mass, adhesion to the ECM, degradation of the ECM by proteases, and cell motility and intracellular contractility [
17,
31]. During these processes, focal adhesion formation regulated by FAK and Pyk2 is a key step for cell invasion.
Many malignant human tumors exhibit increased FAK expression and tyrosine phosphorylation, which are both correlated with the acquisition of an invasive cellular phenotype and increased tumor metastasis [
32]. Although Pyk2 shares a number of functionally important residues with FAK, Pyk2 has a more limited tissue expression than FAK. Particularly, Pyk2 is highly enriched in the CNS [
33]. Moreover, Pyk2 expression occurred much more frequently and with higher expression scores within the different world health organization (WHO) grades of astrocytic tumors, although significant co-expression of FAK and Pyk2 in astrocytomas has been demonstrated [
21]. Whether these two tyrosine kinases are involved in glioma cell invasion that was induced by bevacizumab therapy remains unclear. In present study, we first investigated the changes in total and active FAK and Pyk2 protein levels after bevacizumab treatment. Compared with IgG control treatment, the phosphorylation level of Pyk2 at Tyr402 significantly increased after bevacizumab treatment, although the total levels of Pyk2 protein were similar. This suggested that the invasion potential of C6 glioma cells induced by bevacizumab might be correlated with the level of activated Pyk2, but not as a consequence of increased levels of the total amount of Pyk2 protein. Furthermore, inhibition of Pyk2 phosphorylation partially reversed the invasive ability of glioma cells that was induced by bevacizumab treatment both
in vivo and
in vitro. These results indicated that activation of Pyk2 might be involved in the promotion of glioma cell invasion by anti-VEGF treatment.
Interestingly, neither the total amount of FAK protein nor the phosphorylated FAK at Tyr397 changed after bevacizumab treatment, suggesting that anti-VEGF therapy might have different effects on the activation of FAK and Pyk2. It also implicated that FAK and Pyk2 might play differential roles in regulating the biological behavior of glioma cells. Lipinski et al. [
34] investigated the role of FAK and Pyk2 in the phenotypic determination of four different human glioblastoma cell-lines (U118, G112, SF767 and T98G). Their results showed that increased FAK activity correlated with high proliferation and low migratory rates, while Pyk2 activity was significantly increased in migratory cell-lines (SF767 or T98G) and was dampened in the proliferative cell-lines (U118 or G112). Overexpression of Pyk2 stimulated migration, whereas FAK overexpression inhibited cell migration and instead stimulated cellular proliferation. In contrast, other studies showed that FAK expression was associated with melanoma metastases [
35] and FAK phosphorylation regulated U-87 glioma cell migration and invasion [
28]. These data suggests that both FAK and Pyk2 function as important signaling effectors in glioma, but their differential regulation might be a deterministic factor in the temporal development of proliferative or migrational phenotypes. In our study, the discrepant changes in FAK and Pyk2 activity after bevacizumab treatment also provided evidence that favored differential roles for FAK and Pyk2 on glioma cell migration and proliferation. Recently, a potential novel role for FAK as a nonlinear, dose-dependent regulator of angiogenesis was demonstrated and stromal-FAK heterozygosity was showed to be sufficient to enhance tumor growth and tumor angiogenesis. FAK-heterozygous endothelial cells displayed an imbalance in FAK phosphorylation at Tyr397 and Tyr861 without changes in the activity of Pyk2 or Erk1/2. Cell proliferation and microvessel sprouting, but not migration, were increased in serum-stimulated FAK-heterozygous endothelial cells [
36]. It’s implicated that FAK and/or Pyk2 may possess different biological function in different cell types including tumor cell and intratumor endothelial cell. Taken together, despite significant sequence homology and biological similarity between FAK and Pyk2, these varying outcomes with regard to the role of FAK in tumor cell migration and invasion suggest that more research is required to better understand the function of FAK and Pyk2.
Although inhibition of Pyk2 decreased glioma cell invasion, there was no difference in the median survival duration of rat with intracranial xenograft between bevacizumab treatment group and bevacizumab plus PP1 treatment group, suggesting that the development of glioma is a complex process. It should be difficult to block tumor progress by single inhibitors against only one set of proteins. Indeed, our knowledge about the therapeutic action of bevacizumab and its resistance is constantly being updated. Regression of GBM after bevacizumab treatment, also known as “magic radiological disappearance” effect of bevacizumab, is now verified to be associated with its modification of vascular permeability to gadolinium. Therefore, previous radiological criteria to evaluate the response of GBM to bevacizumab have been modified [
37,
38]. According to these new evalution criteria, recent data revealed that recurrence pattern after bevacizumab treatment in naive-GBM patients was not different between groups of other therapy. However, it is critical that many patients progress after an initial response to this drug [
10]. Mechanisms of tumor resistant and recurrence are appealing and different hypothesis are noted. Our and other findings support the notion that anti-VEGF treatment can directly promote glioma cell invasive ability by regulating the activity of some molecules [
13,
14]. The other widely realized mechanism is that hypoxic microenvironment caused by anti-VEGF treatment leads to the changes of related gene expression and then enhances tumor cell invasion [
24,
26]. All these findings suggest that the invasive process in vivo is highly complicated. Therefore, the role of VEGF receptors and the hypoxic microenvironment should be investigated in further studies about the effect of FAK and/or Pyk2 on glioma cell invasion after anti-VEGF treatment. In addition, a limitation of the current study is that we did not observe the effect of combining bevacizumab with temozolomide and/or radiotherapy, which is used as clinical therapeutic protocol. So, relative studies should be performed in future in order to reflect a true clinical practice.
Acknowledgments
This work was supported by grants from the Natural Science Foundation of Hubei Province, China (2010CDB05507), the project of Wuhan Science and Technology bureau, China (2014070404010223), and the Young Teacher Foundation of Wuhan University, China (4101018). We thank Dr. Lance Michael Ranek from Sanford School of Medicine, the University of South Dakota (USA), for proofreading the manuscript.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LMD, CSX and ZFW participated in the design of the study, performed the statistical analysis and drafted the manuscript. CSX and ZFW revised the manuscript critically for important intellectual content. SHC and LLG participated in the experiments. ZQL conceived of the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.