The Role of Kinase Signaling in Resistance to Bevacizumab Therapy for Glioblastoma Multiforme

Introduction

Glioblastomas multiforme (GBMs, World Health Organization grade IV gliomas) are the most common malignant primary brain tumors in adults. ¹ They are complex and heterogeneous originating from either a previous lower-grade astrocytoma (secondary GBM) or precursor cells (primary GBM). ² These clinical variants possess distinct molecular profiles. Likewise, they are visibly different in prognosis and clinical outcome. Secondary GBM arises in younger people and carries a longer overall survival (OS) than primary GBM. ¹ Currently, a standard therapeutic protocol to manage the GBMs is an intensive multimodal therapy, including maximal surgical resection, concurrent chemoradiotherapy, and adjuvant chemotherapy. Notwithstanding, median survival rate of GBM patients is generally very low and most of these patients usually experience tumor relapse within 1 year of the treatment onset. ³ Despite the development of advances in the treatment of other types of tumors, no cure for GBM patients has been successful to date. It denotes the challenges and difficulties in effective diagnosis and treatment of GBM. Overall, these challenges have motivated the scientists and clinicians to improve the GBM therapy aiming to maximal efficacy with minimal toxicities. ⁴ Fortunately, the advances in intraoperative imaging and surgical techniques have maximized the capability of neurosurgeons to operate the extensive tumor safely, optimize surgical utility, ⁵ and presumably enhance the patient survival rate. ⁶ Despite this, as a supplementary therapeutic approach in neuro-oncology, chemotherapy has been used for decades. At the present time, concurrent application of the oral methylator temozolomide (TMZ) and radiation followed by adjuvant TMZ therapy in the standard practice have shown pronounced clinical benefits. ³ Nevertheless, chemoradiotherapy has not succeeded to eradicate the GBM, and the tumor relapse eventually takes place. Hence, many efforts have been dedicated to advance in neuro-oncology. To date, antiangiogenesis therapy has been hopefully proposed to achieve a detectable therapeutic success after tumor resistance to standard therapy. ⁷ Even though antiangiogenic therapies such as bevacizumab has led to an increase in progression-free survival (PFS) but not OS, both in newly diagnosed ^8,9 and in recurrent GBM, ¹⁰ tumor relapse is manifested, finally. ^{11 –13} In this review, the authors preliminarily characterized a subpopulation of GBM cells in the tumor mass involved in GBM angiogenesis and resistance. Then, they pointed out the biological and therapeutic actions of bevacizumab in GBM therapy. They also described the compensatory aspects of increased kinase expression and activity within tumor cells due to bevacizumab therapy, leading to the tumor invasion in bevacizumab-resistant GBMs. Herein, it is focused on both hypoxia-dependent and -independent underlying mechanisms promoting kinase signaling-mediated tumor invasion after bevacizumab therapy. Here, the relevant possible solutions and novel molecular therapeutic targeting are provided to sensitize the GBMs to the bevacizumab, as well. Available preclinical and clinical trial studies in this context are presented.

Glioblastoma Stem-Like Cells Participates in Tumor Formation, Angiogenesis, and Refractoriness

The evidence supports the role of glioblastoma stem-like cell (GSCs), an intractable GBM subpopulation also called tumor-initiating cells in the loss of sensitivity of this malignant tumor against GBM therapy ^14,15 especially, bevacizumab. ¹⁶ The GSCs express stem cell-specific markers ^17,18 and deregulated intracellular signaling pathways to retain their stem phenotype. ¹⁹ In addition to the contribution of GSCs in the therapeutic resistance, they take part in the tumorigenesis ^{18,20 –22} and tumor formation in secondary host. ²³ GSCs also are able to dislocate upon exposure to a hypoxic circumstance and can migrate toward a favorable microenvironment to survive. ¹⁴ Likewise, it has suggested that GSCs having aberrant gene profile innately are aggressive and can stimulate an uncontrolled tumor and vasculature growth. ²⁴ Besides, GSCs are often protected by an intact neurovascular unit that provides oxygen and nutrients to these actively proliferating tumor cells. ¹ These rebellious cells principally reside adjacent to the vascular niches. The endothelial cells (ECs) constitute the main elements of these niches. There is a reciprocal functional interaction between GSCs and ECs largely through vascular endothelial growth factor A (VEGF_A) secretion, supporting the cell survival and proliferation. ^{25 –27} This finding propels the investigators to develop the first-generation antiangiogenic agents blocking VEGF_A axis such as bevacizumab for recurrent GBM. Although bevacizumab inhibits VEGF_A signaling in ECs, it may stimulate kinase signaling in GBM cells in a compensatory manner that has been suggested to be a possible mechanism for bevacizumab failure in the clinic. ^28,29

Bevacizumab Brings About an Early Clinical Benefit in GBM Patients by Targeting VEGF_A

Today, numerous molecularly targeted therapies have been explored in the clinical trials of recurrent GBM. ^{30 –35} Basically, there is a convincing rationale for targeting the tumor vasculature, because GBMs are extremely angiogenic and susceptible to antiangiogenic agents. ³⁶ At the present time, bevacizumab (Avastin) belonging to the first-generation angiogenesis inhibitors has been approved exclusively by the US FDA for use in the patients with GBM relapse. ³⁷ However, bevacizumab at the therapeutic prescription dose represents a fast outstanding but unstable clinical profit ³⁸ with no serious adverse events for the patients. ³⁷ Since the routine clinical use of bevacizumab is extensively developed in recurrent or newly diagnosed GBMs, the authors focused on the bevacizumab therapy of GBMs in this study.

Bevacizumab, a recombinant humanized monoclonal antibody, directly neutralizes VEGF_A and causes the inhibition of the ligand-receptor interaction. It is worth noting that a vital VEGF isotype to promote ECs proliferation, survival, and migration is VEGF_A, ³⁹ abundantly secreted from GSCs on ECs in a paracrine manner. ^36,40,41 Fundamentally, VEGF_A-dependent cell growth promoting signals are mostly transmitted through VEGF receptor 2 (VEGFR2) that is a receptor tyrosine kinase (RTK). ^39,42 Active VEGFR2 on ECs stands on the top of signal transduction cascades triggering angiogenesis process and induces phosphorylation/activation of protein kinases such as AKT and extracellular signal-related kinase (ERK) that mediate the proangiogenic genes transcription and translation through their downstream effectors. ^42,43 Hence, targeting VEGF_A-VEGFR2 pathway by bevacizumab blocks VEGF_A-dependent cell growth-promoting signals on EC, leading to an antiangiogenesis response. ^44,45 Furthermore, VEGF_A secretion by GSCs on themselves ^{46 –48} through an autocrine loop and subsequently the induction of kinase activity of VEGFR2 on these cells has also played a significant role in GSCs maintenance. ^{46 –48} Hereby, blocking VEGF_A/VEGFR2 interaction on GBM cells by bevacizumab may be associated with a cytotoxic activity of drug that is thought to be mediated through the inhibition of ERK activity. ⁴⁹ In addition, targeting the VEGF-VEGFR interplay may strengthen host immunity through the inhibition of VEGF-mediated immune suppression, ⁵⁰ intensifying the effectiveness of immunotherapy. ⁵¹ Notably, inhibitors of VEGF pathway not only suppress the process of angiogenesis commonly, but also they may play a role in the normalization of the tumor vasculature and improve drug delivery, ¹³ and thereby enhance the efficacy of cytotoxic regimens. This may explain more therapeutic advantages provided by concurrent application of standard chemotherapy and bevacizumab in newly diagnosed GBMs. It has been highlighted that adding bevacizumab to first-line treatment could result in a clinical usefulness that appears to be initially related to a significant PFS improvement, while it is associated with the lack of OS benefit ^52,53 without causing a major toxicity in patients. ⁵⁴ A study has recently shown that combining the bevacizumab with standard chemoradiotherapy in 637 GBM patients could enhance the median PFS by 10.7 months more than standard therapy alone (7.3 months). However, there was no significant difference in median OS between combination group (16.1 months) and those treated with standard therapy alone (15.7 months). ⁹ In the case of patients with recurrent GBM who previously received the standard protocol, the findings of several clinical trials have demonstrated an improved clinical response in terms of median PFS and 6-month PFS percentage after the intravenous taking of bevacizumab either alone or in combination with chemotherapy. ^{30,31,33 –35,38,55 –60} A variable range of median PFS (5.8–7.6 months) and OS (7.7–11.5 months) has been found after administration of bevacizumab plus irinotecan in various studies. ^{61 –63} Data have exhibited a 6-month PFS by 63.7% in 51 bevacizumab plus irinotecan recipients having GBM. ⁶³ Based on the clinical data, a plausible mechanism of bevacizumab for the early recovery of GBM clinical symptoms is that blocking of VEGF_A brings about a fast reduction of tumor vascular permeability that allows minimizing the peritumoral vasogenic edema, the neurological comorbidities. ^13,64,65 Accordingly, the rapid reduction of contrast enhancement on brain MRI or CT scans actually are the pseudoresponses after bevacizumab therapy due to the alleviation of peritumoral edema. ³⁸ It is worth noting that the responses based on the contrast enhancement on radiographic images could not exactly represent an intrinsic antitumor activity of bevacizumab. ^11,12 It seems that the advanced techniques are demanded to accurately define real tumor responses and progression.

Altogether, bevacizumab may be able to dramatically ameliorate the GBM symptoms because of its effects on the vascular permeability and progression as well as drug delivery ^{13,38,66 –68} through blocking VEGF_A in the lumen of the capillaries of blood vessels in the brain; however, it is not able to cross the blood–brain barrier to act on the tumor site owing to its high molecular weight ⁶⁹ and also it has not a durable therapeutic advantage. ^12,70 Occasionally, clinical deterioration may have emerged after permanent usage of bevacizumab that seems to be associated with compensatory activation of alternative tumor growth and angiogenesis pathways due to the upregulation of other growth factors within tumor cells in response to the lack of VEGF_A, developing the drug resistance and tumor relapse. ^{16,28,29,71 –77} Thus, it is thought that GBM quickly adapts to anti-VEGF therapy leading to a rapid tumor propagation without a clinically significant improvement in OS rate. Furthermore, bevacizumab causes serious side-effects that limit its clinical application at higher doses. ^78,79

The Role of Kinase Signaling in the Resistance to Bevacizumab Therapy in GBM

Based on the evidence, compensatory activation or overexpression of some RTKs such as MET, a hepatocyte growth factor receptor, ²⁸ and VEGFR1²⁹ within tumor cells have been observed in response to the inhibitory effect of bevacizumab on binding VEGF_A with VEGFR2 on GBM cells. Moreover, overexpression or reactivation of some growth factors such as placental growth factor (PlGF), VEGF_B, VEGF_C, VEGF_D, and basic fibroblast growth factor (b-FGF) in GBM cells were reported after the tumor resistance to bevacizumab, leading to the tumor angiogenesis and metastasis. ^29,73,74 Given that the bevacizumab can only inhibit the VEGF_A/VEGFR2 cascade and has not any direct effect on other growth factors and RTks, it has suggested that tumor resistance against bevacizumab may be triggered by compensatory activation of PlGF/VEGFR1, ^71,72 hepatocyte growth factor (HGF)/MET, ²⁸ and VEGFc/VEGFR2⁷⁵ pathways in the absence of VEGF_A/VEGFR2 interaction. According to data obtained from several investigations, bevacizumab-induced compensatory activation of RTKs by other cytokines and VEGF family except for VEGF_A on both GBM cells and ECs can bring about the tumor invasion and reactivation of angiogenesis probably through inducing the phosphorylation of AKT and/or ERK kinases ^{29,74,75,80,81} that transmit signals stimulating the expression of matrix metalloproteinases (MMP) ^41,74,80 and tumor-derived proangiogenic factors such as epidermal growth factor, platelet-derived growth factor (PDGF), PlGF, b-FGF, HGF, and angiogenins. ^{16,41,74,76,77} Furthermore, the evidence has supported that VEGFR1 is activated by VEGF_B or PlGF secreted on both GBM cells and ECs and participates in tumor growth, angiogenesis, and metastasis ^81,82 especially when VEGF_A is not present. ⁸³

Hypoxia-Dependent Kinase Signaling Stimulated by Bevacizumab Allows Invading the Tumor

So far, many preclinical and clinical data mainly support the hypothesis of bevacizumab-induced tumor invasion as one of the mechanisms complicated in tumor resistance. ^{11,29,63,84 –86} Particularly, the role of hypoxia in tumor progression, metastasis, ⁸⁷ and resistance to antiangiogenic therapy ^{88 –90} is well established.

Actually, antiangiogenesis drugs destruct the irregular vascular network and yield a hostile hypoxic microenvironment from which tumor cells try to escape to distant metastatic sites. ^91,92 Apparently, the clinical findings revealed an increased rate of distant tumor progression in GBM patients after treatment with bevacizumab and chemotherapy in contrast to chemotherapy alone, suggesting bevacizumab-induced tumor infiltration. ⁶³ On the basis of the radiographic data, the most frequent of tumor progression pattern in GBM patients treated by bevacizumab plus standard therapy versus those received standard therapy alone, has been characterized by a complete disappearance of contrast enhancement on T1-weighted sequences during treatment and an increase in contrast enhancement at progression. Based on these results, the frequency of a tumor progression type detected by the signal increase on T2/FLAIR-weighted images and a slight decrease in signal intensity on T1-weighted images was not different between groups. Also, this tumor progression pattern was associated with the longest survival in both groups. ⁹³ However, a piece of evidence has supported that a lack of change in the contrast enhancement along with an increase in the noncontrast-enhancing infiltrative component of the tumor manifested in post-treatment follow-up brain scans of bevacizumab-resistant patients may be associated with bevacizumab-induced hypoxia. ¹¹ According to the results of histopathological studies on the post-treatment tumor biopsies or surgical specimens taken from the tumor region representing an abnormality on T2-weighted scan, it is clear that the appearance of bevacizumab-induced tumor invasion in the tumor edge happens simultaneously with the microvessel pruning within densely cellular tumor mass that expresses a high level of CA9 hypoxia biomarker as well as very high levels of MMP2 known as marker of cell migration. ¹¹ Therefore, a possible explanation for the transient responses observed in bevacizumab-treated patients is that an initial decrease in tumor burden temporarily leads to a good clinical outcome followed by bevacizumab-induced hypoxia allows the tumor invasion.

Based on preclinical data, tumor invasion after bevacizumab therapy is coupled with the mesenchymal transition ⁸⁵ and vessel co-option ⁸⁹ events. It seems that GBM cells acquire an endothelial-like behavior in response to bevacizumab leading to the tumor infiltration along perivascular spaces. ⁹⁴ Surprisingly, some studies have highlighted that bevacizumab-induced tumor aggression associated with mesenchymal phenotype is probably mediated through an RTK called MET in a hypoxia-dependent manner. ^28,85 Indeed, during the bevacizumab therapy, hypoxia induction may transcriptionally upregulate the expression of MET or HGF receptor in a subset of GBM patients. ⁹⁵ It has been found that MET transcript is increased through a transcription factor known as hypoxia-inducible factor 1α. ⁹⁶ Evidently, hypoxia-mediated MET overexpression takes part in GBM cell migration. ⁹⁶ Importantly, hypoxia can also activate Src family kinases (SFKs) belonging to a non-RTKs (nRTKs). ^97,98 SFK hyperactivity in response to bevacizumab is observed in the edge of invasive GBM tumor ⁹⁹ in which the hypoxia coupled with blood vessel pruning induced by bevacizumab. ¹¹ Hereby, it is argued that bevacizumab-activated SFK signaling may contribute to the tumor invasion in a hypoxia-dependent pathway.

Taken together, hypoxia-dependent tumor invasion induced by bevacizumab has captured the attention of many scholars, so far. ^16,100,101 In this regard, the role of MET and SFKs signaling activated in response to bevacizumab-induced hypoxia is significant. The combination of therapeutic approaches targeting MET and SFKs may, at least in part, aid in improving the bevacizumab efficacy.

Bevacizumab Directly Induces Tumor Invasion Through Hypoxia-Independent Mechanisms Activating Protein Kinases

In the case of active kinas-mediated tumor invasion caused by hypoxia-independent and direct action of bevacizumab, it is evident that bevacizumab typically neutralizes the VEGF_A but not RTKs activity. ¹⁰² It has been suggested that taking bevacizumab ultimately leads to a more aggressive state of the tumor presumably through RTKs activated by other cytokines except for VEGF_A, in a compensatory manner. ^{28,29,84,103,104} Tumor invasion-related signals triggered by active RTKs may be transmitted through protein kinase B (AKT) and ERKs ^28,80 pertaining to serine/threonine kinase family that are key cell growth-promoting signal transduction pathways. ^105,106

A piece of evidence on normoxia cultures containing GBM cells alone proposes the possibility of a hypoxia-independent cell migration in response to direct exposure of bevacizumab through overactivation of AKT and ERK kinases. ²⁹ In accordance with these findings, the proproliferative and proinvasive effects of bevacizumab may happen when a very high concentration of the drug about 1 mg/mL is used. It seems that this applied dose of drug causes a more free VEGF_A sequestration by 10-fold in the culture medium of GBM cells only expressing VEGFR1. ²⁹ However, an experiment in the authors' laboratory indicated that GSCs representing the expression of VEGFR2 but not VEGFR1 are sensitive to the bevacizumab used at a concentration of 6.5 μg/mL. The cytotoxic effect of bevacizumab appears to be associated with an intracellular ERK inactivation and a depletion of secreted VEGF_A levels by threefold in GSCs culture medium without a significant increase in the levels of active AKT kinase. ⁴⁹ It seems that these inconsistent responses may be linked to the heterogeneous nature of GBMs or applied dose of bevacizumab. However, it has detected a proinvasive response in VEGFR2-positive glioma cells treated with bevacizumab in a dose-dependent manner. ¹⁰⁷ According to aforementioned descriptions, it is thought that the excessive sequestration of secreted VEGF_A by higher concentrations of bevacizumab may activate the tumor invasion-related resistance signals probably through overexpression of some growth factors and their RTKs in the absence of VEGF_A. ^{28,29,48,84,104}

Based on the evidence, a possible mechanism of bevacizumab-induced tumor invasion may be linked to the hypoxia-independent MET activation but not an expression that is triggered by the abolition of the inhibitory effect of VEGF_A on the MET activation in presence of bevacizumab. ²⁸ It seems that the absence of VEGF_A due to bevacizumab application provides a suitable situation that allows binding the HGF and MET receptor ²⁸ triggering the molecular signaling implicated in tumor invasion. ^19,28,80,108 Conversely, in the absence of bevacizumab, VEGF_A-VEGFR2 interplay dramatically leads to VEGFR2 dimerization/activation and subsequently an increase in the recruitment of protein tyrosine phosphatase 1B to MET complex. This process negatively regulates the tumor cell invasion through MET dephosphorylation/deactivation. It is obvious that bevacizumab counteracts this process by depriving the VEGF_A-VEGFR2 of binding. Therefore, the tyrosine kinase activity of MET and consequently the activation of its downstream effectors, including the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases MAPK/ERK pathways can be restored through the VEGF_A ablation by higher concentrations of bevacizumab in GBMs expressing both MET and VEGFR2. Although VEGFR2 activation by VEGF_A stimulates PI3K/AKT signal, the MET-activated PI3K function appears to be stronger than that activated by VEGFR2 to trigger the tumor invasion process. ²⁸

Besides, it is suggested that VEGFR1 inhibits VEGFR2 activity under high concentrations of exogenous VEGF_A. ⁴⁸ Thus, considering the fact that bevacizumab does not directly block the VEGFRs, another alternative feasible mechanism of bevacizumab-induced tumor invasion especially for GBMs expressing both VEGFR1 and 2 may be that the depletion of free VEGF_A due to bevacizumab application perhaps abrogates the negative feedback effect of VEGFR1 on VEGFR2 activation in human GBM cells. Therefore, it is possible to cause the tumor invasion through autophosphorylation of VEGFR2. ¹⁰⁹

Hypoxia-independent activation of SFKs by bevacizumab in GBM has been considered as a possible mechanism of post-treatment tumor invasion that may involve the compensatory activation of some RTKs such as MET upon bevacizumab-induced VEGF_A suppression. ^28,110,111 SFK activity promotes GBM tumor cell motility and migration through phosphorylation of multiple targets disrupting cell–cell adhesion and extracellular matrix ¹¹² and gives rise to the tumor metastases. ^{98,113 –116} Evidently, a member of the SrcA subfamily pertaining SFKs called Yes constitutes a complex with the p85 regulatory subunit of PI3K to induce GBM cell invasion. ¹¹⁷

As a result, it is possible that in a monotherapy system of bevacizumab for GBMs expressing MET receptor, VEGFR1 and VEGFR2, an efficient antitumor activity of bevacizumab happens at a therapeutic dosage adjusted according to free VEGF_A levels with no activation of kinase-mediated compensatory growth pathways. This means that an optimal dose and protocol of bevacizumab administration should be well defined to adequately rescue the tumor vasculature abnormality without the compensatory activation of protein kinases promoting cell migration signals within GBM cells. Importantly, it is suggested that the differential responses of patients to the bevacizumab may be stemmed from the distinct molecular profile of GBMs. Tumoral expression of VEGFR2 appears to be an adverse prognostic biomarker of bevacizumab efficacy in GBM patients. Accordingly, identification of biomarkers predicting bevacizumab efficiency may help to identify the subgroups that likely profited from bevacizumab.

Potential Therapeutic Strategies to Overcome the Tumor Invasion Induced by Bevacizumab in Gliomas

Definitely, there is an urgent requirement to introduce alternative therapeutic approaches to effectively control the tumor propagation in bevacizumab-resistant GBMs. According to the current knowledge, application of a combination method in which the bevacizumab and pharmacological inhibitors of RTKs or PI3K/AKT or SFKs are concurrently administrated may circumvent the resistance mechanisms of bevacizumab monotherapy.

Conclusions and New Direction

Bevacizumab currently accepted as a popular antiangiogenesis agent in the routine clinical use of recurrent GBMs has not completely succeeded to cure the disease, although it has demonstrated an early substantial but temporary response after an initial administration of the drug. Inevitably, the tumor progression emerges after constant application of bevacizumab. Clinical efficacy of bevacizumab may be mediated through multiple mechanisms that are different in distinct subsets and stages of GBMs. Obviously, identification of validated biomarkers that may predict sensitivity or resistance to the bevacizumab would be mandatory for the better selection of patients who benefit from this medication. Accordingly, to validate the tissue and/or circulating-based biomarkers associated with bevacizumab response, they should be routinely utilized in clinical practice. Likewise, to gain insight into the impact of therapeutic protocol-related factors such as dose and duration of bevacizumab administration on the activation of kinase-mediated invasion signals that arise in tumor cells after bevacizumab therapy, a lot of preclinical efforts, including tumor patient-derived models and/or primary cultures of GSC, should be supplementarily carried out. It should be taken into consideration that these investigations may help to refine the bevacizumab therapy. Regarding the very high heterogeneity of GBMs in terms of molecular and tissue phenotypes, clinical manifestations in response to bevacizumab personalized medicine are still attractive therapeutic perspectives in this context. In conclusion, the identification of biomarkers well defining sensitive tumors to bevacizumab based on the individual molecular profile of GBMs is currently a serious priority.

Specifically, the thorough recognition of underlying intrinsic and adaptive mechanisms of bevacizumab failure causing GBM progression and invasion have allowed the authors to develop the novel molecular targeted therapies and combination strategies to effectively overcome the tumor resistance to bevacizumab in human malignant GBMs. Particularly, an optimal advantage would be probably obtained when bevacizumab is given in combination with chemical agents targeting PI3K/AKT or MET and VEGFRs belonging to RTKs. Therefore, it is recommended that suggested combination therapies are incorporated in a future generation of clinical trials. It appears that a tumor type-specific combination therapy needs to be done according to the specific molecular fingerprint of tumor in patients.