Astrocytomas are primary malignant brain tumors that originate from astrocytes. Grade IV astrocytoma or glioblastoma is a highly invasive tumor that occur within the brain parenchyma. The Rho family of small GTPases, which includes Rac1, Cdc42, and RhoA, is an important family whose members are key regulators of the invasion and migration of glioblastoma cells. In this review, we describe the role played by the Rho family of GTPases in the regulation of the invasion and migration of glioblastoma cells. Specifically, we focus on the role played by RhoA, Rac1, RhoG, and Cdc42 in cell migration through rearrangement of actin cytoskeleton, cell adhesion, and invasion. Finally, we highlight the importance of potentially targeting Rho GTPases in the treatment of glioblastoma.
Primary brain tumors are a mixture of benign and malignant tumors originating from the brain parenchyma and its surrounding microenvironment [
The invasive nature of GBM illustrates the molecular phenotype of cancer cells in general, which includes the constitutive activation of proinvasive pathways. The focus of this article is to discuss the role of Rho GTPase family proteins along with their upstream regulators and downstream effectors, which regulate the invasive and aggressive behavior of GBM through actin cytoskeleton rearrangement, cell adhesion, and invasion. Moreover, the potential ways through which Rho GTPases and associated proteins can be targeted for improved therapy will be further discussed.
Rho GTPases are involved in the regulation of cell growth, differentiation, cytoskeleton rearrangement, apoptosis, and cell motility [
Briefly, Cdc42 relays environmental cues to effector proteins, setting the orientation of the cell. Rac and Rho are spatially regulated in opposite ways, which reflects their role in cell motility; at the front of the cell, Rac stimulates the formation of the leading edge while Rho governs the appearance and organization of contractile structures at the rear of the cell [
RhoA, RhoB, and RhoC are Rho GTPase homologues that show an 88% similarity in the amino acid sequence. Each isoform has a distinct effect on the cell’s structural and migration properties by activating different downstream effector proteins including enzymes and adaptor proteins [
RhoA is a key regulator of cancer cell proliferation, progression, and metastasis. Several studies have shown that its activation leads to the formation of actin stress fibers and focal adhesions through RhoA-Rho-associated protein kinase (ROCK) signaling pathway [
RhoA is a well-established Rho GTPase that plays an important role in cell motility and invasion of glioblastoma and other tumor types. RhoA is activated upon binding of glioblastoma cells to the extracellular matrix (ECM) [
RhoA induces the formation of focal adhesion (FA) by activating a serine/threonine kinase Rho effector p160ROCK. ROCK phosphorylates myosine light chain (MLC) by inhibiting the MLC phosphatase. The coordination between ROCK and the mammalian homolog of diaphanous formins (mDia), a RhoA effector, stimulates the formation of stress fibers and focal adhesion, enhancing cellular adhesion [
RhoA activation leads to integrin clustering which promotes the activation of focal adhesion kinase (FAK) through phosphorylation at tyrosine 397. FAK signaling cascade regulates cell migration by controlling the focal complex’s assembly and disassembly at the leading edge and the focal adhesions’ (FA) disassembly at the trailing edge of the cell [
For the cancer cell to remodel the ECM and facilitate invasion, metastasis, and survival, it needs to utilize matrix metalloproteinases (MMPs), a family of extracellular zinc-dependent proteinases [
Moreover, RhoA is required in Rac1-induced lamellipodium formation at the leading edge of the invading cell [
RhoB plays a role in delivering receptors and signaling proteins to the plasma membrane through the process of endosomal trafficking [
Even though RhoA and RhoC have high sequence similarity, they regulate cellular migration differently. RhoC has been shown to induce cell migration and invasion by using a distinct signaling pathway from RhoA. A study done on prostate cancer showed that knocking down FMNL3, a protein found downstream of the RhoC signaling pathway, leads to the broadening of lamellipodia, depolarization of morphology, and inhibition of cellular invasion. Moreover, FMNL3 selectively binds to activated RhoC and not RhoA. This study also showed that RhoC regulates lamellipodium broadening by restricting Rac1 activation along the cellular membrane [
The genetic profile of glioblastoma cells shows that most of them have more than one copy of chromosome 7, on which the Rac1 gene is located [
The increased activity of Rac is the result of multiple factors including the deregulation of upstream regulators. Constitutive signaling through epithelial growth factor receptor (EGFR) and other receptor tyrosine kinases such as PDGFR
Another protein upstream of Rac1 is the TWEAK receptor Fn14, TWEAK being a TNF-like weak inducer of apoptosis. It was shown that TWEAK can play the role of a chemotactic signal by binding Fn14 and inducing cell movement in a Rac1-dependent manner [
The other members of the Rac subfamily of Rho GTPases are not as well characterized in the context of glioblastoma and other brain tumors but can potentially be playing an important, yet still undeciphered, role in controlling migration and invasion of GBM cells. For example, in addition to Rac1, Rac2 and Rac3 knockdown in glioblastoma stem cells showed a dramatic decrease in their invasive and migrative capabilities, confirming that Rac proteins in general do play a crucial and necessary role in the invasion of GBM cells [
RhoG, a Rac superfamily member, regulates cell polarity, migration, and invasion. It has been reported that RhoG, activated by EGF and hepatocyte growth factor (HGF), contributes to the formation of lamellipodia and invadopodia and promotes glioblastoma cell migration and invasion [
Findings regarding the role of Rac1 in glioma cell migration and invasion parallel those of Cdc42, for both, promote these two processes, bind to similar domains, are activated by common GEFs, and have the same intracellular location (at the front edge of the cell). High expression of Cdc42 in GBM is correlated with lower survival rates, since it was found to promote the highly invasive characteristics of GBM in vivo [
A close inspection of its subcellular localization reveals several structures and proteins with which this Rho GTPase possibly interacts. One such protein is IQGAP1, a scaffold protein which binds both Rac1 and Cdc42 and helps reorganize actin cytoskeletal structures [
Cdc42 is also believed to be a mediator of the X-ray promoted invasion of glioblastoma, a complication that occurs following radiotherapy. Cathepsin L was found to be a strong promoter of X-ray-induced glioma cell invasion by regulating Cdc42-mediated cytoskeletal remodeling, a process which was abolished once Cathepsin L was knocked down and Cdc42 levels decreased [
Another important aspect of the implication of Cdc42 in the malignancy of GBM is the interaction between GBM cells and other cells in the tumor environment, specifically pericytes. In this context, Cdc42 was found to promote the development of previously unnamed structures now called flectopodia (due to their significant differences from “classical” invadopodia) which establish communication with pericytes, turning the tumor-suppressive nature of these regulatory cells into tumor promoting and clearing the way for cancer cell invasion [
The levels of Rac1 and Cdc42 are not the same throughout the cell. According to a biosensor assay done on glioblastoma cells, low Rac1 and low Cdc42 are found on the trailing end while high Rac1 and high Cdc42 levels are found on the leading edge of the cells [
Rho GEFs promote the GDP to GTP switch which activates Rho GTPases. In agreement of our understanding of the role of Rho GTPases is the fact that Rho GEFs are involved in glioma cell migration and invasion. This also provides a theoretical basis for targeting the Rho/Rho GEF interaction as a way to inhibit GBM cell migration.
The expression of the three GEFs Ect2, Trio, and Vav3 is elevated in the glioblastoma as compared to lower-grade glioma, and the depletion of any of the three is sufficient to reduce migration and invasion of glioblastoma [
Another Rho GEF is SWAP-70 which is overexpressed in human high-grade glioma tissues and high-grade glioma patients [
Recently, novel Rho GEFs were discovered and they have been implicated in the aggressive phenotype of GBM. Overexpression of PDZ-Rho GEF in TROY-expressing glioblastoma cells mediates RhoA and RhoC activation downstream of the TROY receptor [
The loss or dysregulation of certain tumor suppressors is associated with the Rho-mediated aggressive behavior. We will briefly reflect on a couple of them. One of these proteins is phosphatase and tensin homolog (PTEN), which suppresses phosphatidylinositol-3,4,5-trisphosphate (PIP3) level cells. Loss or mutations of PTEN correlate with poor patient outcome in GBM [
Rho GAPs are Rho-negative regulators that inhibit Rho activity and abolish glioma cell migration and invasion. Neurofibromin 1 (NF1) is a GAP specific for Ras GTP, which occupies a key signaling node of the Ras/Raf/MAPK oncogenic pathway as well as crosstalks with the Pi3K pathway upstream of Rho proteins [
After studying the role of Rho GTPases in glioblastoma, therapy focuses on the search for druggable proteins and strategies that could target Rho GTPase interaction with Rho GEFs, their downstream effectors, or even their degradation. Most of the following drugs have antiproliferative effects, but in this review the major focus is on cell migration.
NSC23766 is a Rac1 inhibitor that synergistically inhibits glioma cell migration and invasion when used with anti-EGFR drugs [
Another possible way through which new drugs can act is through the downstream effectors of Rho GTPases. In this regard, resveratrol was found to suppress glioblastoma cell migration and invasion by increased activation of the RhoA/ROCK pathway [
Another way to modulate the levels of Rho GTPase is targeting them for ubiquitination. In this context, luteolin was found to facilitate the proteasomal degradation of Cdc42, leading to decreased glioblastoma migration and invasion in vitro [
Although major limitations persist from the perspective of drug delivery, we hypothesize that future development and usage of Rho GTPase signaling inhibitors in combination with standard therapy has the potential to improve outcome for patients with glioblastoma.
The aggressive nature of glioblastoma impairs most of the attempts aimed at curbing disease progression in patients. Research has enabled the understanding of the function of Rho GTPases in cancer and the interaction of cancers with their surroundings. Given the involvement of Rho GTPases in GBM migration, invasion, and aggressiveness, Rho GTPases, as well as their upstream regulators and downstream effectors are emerging as strong candidates for cancer treatment.
The authors declare that they have no conflicts of interest.