2.2.1. Sustaining Proliferative Signaling
Normal tissue growth is tightly regulated through the release of growth-promoting and growth inhibitory signals [
31]. Cancer cells, however, have developed mechanisms to proliferate independently of these regulatory signals. In GBM, alterations in RTK/Ras/PI3K enable the tumor to proliferate constantly [
40]. For instance, overexpression of the epidermal growth factor receptor (EGFR) and inactivation of phosphatase and tensin homolog (PTEN) causes downstream activation of the growth- and survival-promoting PI3K/Akt/mTOR pathway [
40,
52,
96,
97,
98].
Numerous studies support a pro-proliferative role of TSPO in GBM. High TSPO expression in the C6 rat glioma cell line is correlated to enhanced cell proliferation [
19], and modulation of TSPO activity by the TSPO ligand PK11195 either had no effect or pro-proliferative effects in patient-derived glioma cell lines [
99]. Likewise, overexpression of TSPO in C6 rat glioma cells enhanced proliferation as well as the ability to overcome contact-induced cell growth inhibition [
100]. In addition, a more recent study revealed that the transfection of TSPO into Jurkat cells increased cell proliferation and motility [
101] whereas lentiviral knockdown of TSPO reduced the proliferation rate in BV-2 mouse microglial cells [
102].
In contrast, TSPO knockdown and TSPO ligands were able to promote proliferation and migration in glioblastoma U118MG cells due to a decrease in TSPO related apoptosis [
103]. Knockout of TSPO with the CRISPR/Cas9 system in mouse GL261 glioma cells resulted in increased proliferation and viability in comparison to wild type cells [
104]. This anti-proliferative role of TSPO is supported by pharmacological studies with a variety of TSPO ligands [
22,
105]. It is noteworthy that, reported earlier, the effects of TSPO ligands on cell proliferation are context-dependent and vary with ligand concentration, resulting in pro-proliferative effects at nanomolar concentrations and anti-proliferative effects at micromolar concentrations [
19,
106,
107]. Context-dependent factors that could influence the role of TSPO are likely cell line-, species-, and signaling pathway-specific [
102,
108].
There is evidence that endogenous steroid hormones play a role in the development of gliomas. Epidemiological data suggest that female sex hormones play a tumor-suppressive role in GBM since the incidence rate of GBM is higher in men compared to women [
109]. Previous studies have also shown that steroid hormone receptors such as ERβ as well as the testosterone-estradiol converting enzyme aromatase are expressed by some gliomas and glioblastomas [
110,
111,
112]. Selective estrogen receptor modulators such as estradiol and 2-methoxyestradiol are shown to inhibit the proliferation of gliomas and induce cell death in experimental in vitro settings [
109]. In line with these findings, an increased level of testosterone has been reported in patients with GBM [
113]. In addition, androgen receptors are overexpressed in human GBM, and the genetic silencing of androgen receptors as well as their pharmacological inhibition, induce GBM cell death in vivo and in vitro [
114,
115,
116]. Furthermore, the proliferation of GBM-derived cells was increased by testosterone, an effect that was antagonized by the androgen receptor antagonist flutamide [
113]. The effects of the hormonal agonists and antagonists can either depend on classical steroid hormone receptor signaling or on alternative pathways [
109]. In view of these findings, further studies are needed to elucidate the role of TSPO as a modulator of steroid synthesis in affecting the development and proliferation of glioma. Elucidating the role of hormonal pathways in gliomagenesis could eventually lead to the design of novel, preventive therapies.
The exact mechanism by which TSPO modulates cell proliferation is still unclear. Growing information indicates that TSPO impacts the bioenergetic profile of a cell by modulating ATP production, thus providing the energy for increased proliferation [
18,
25,
101]. However, to gain a deeper understanding of the exact mechanisms and signaling pathways involved, further in-depth studies are required.
2.2.2. Evading Growth Suppressors
Apart from sustaining proliferative signals, cancer cells also have the ability to escape growth inhibition and positively regulate cell proliferation through the loss of tumor suppressor genes such as
NF2,
LKB1,
RB, and
TP53 [
31]. The prevalence of mutations in the
RB and
TP53 genes, though important drivers in many tumors, illustrates again that GBM is highly heterogeneous and depends on many distinct alterations:
RB is mutated in only 6–11% of GBM cases and 27–33.8% of GBM bear mutations in the
TP53 gene [
40,
97]. Moreover, TP53-dependent cell cycle control can also be impaired by
MDM2 and
MDM4 amplification, which is the case in 12% and 4% of glioblastomas, respectively [
117,
118]. It is worth noting that the corresponding signaling pathways are nonetheless major targets of inactivating mutations in GBM and were altered in 78–79% of GBM and 87% of GBM cases for pRB and p53, respectively [
40,
97].
RB and
TP53 and the connected pathways play crucial roles in the inhibition of proliferation, predominantly by halting cells in the G1 phase. This delays entrance into the S phase, slowing down repair of DNA damage or ultimately causing apoptosis [
52].
Changes in cell cycle regulation may enable cells to evade the control of growth suppressors. Early data suggested that TSPO is involved in the regulation of the cell cycle [
119,
120,
121]. In a bioinformatical analysis investigating drug-response associated gene expression,
TSPO has been found as a key driver gene for positive regulation of mitotic cell cycle phase transition [
122]. Another publication revealed
TSPO as a critical, differentially expressed gene in neuroblastoma with cyclin-dependent kinase (CDK) 2 silencing.
TSPO was identified as a key target gene of CDK2, and CDK2 may be involved in tumor progression via the regulation of the interaction of TSPO and CDK1 [
123]. Other publications corroborated an interaction of TSPO with cell cycle-related genes, e.g., in the U118MG glioblastoma cell line. The same authors showed that the down-regulation of TSPO expression caused an increase of cells in the S and G2/M phase and a decrease of cells in the G1/G0 phase [
124]. An independent group obtained similar results with an enhanced ratio of TSPO knockout cells in the S phase [
104].
Results with pharmacological inhibitors of TSPO also support evidence that TSPO modulates cell cycle progression. For instance, TSPO ligands inhibit cell proliferation by halting these cells in the G1/G0 phase and therefore inhibiting the progression to the S and G2/M phase [
125,
126]. Short-term treatment with PK11195 resulted in a reduction of the S and G2/M phase and a consequent increase of the G1/0 phase, whereas longer treatment periods caused a decrease of cells in the S phase and accumulation in the G2/M phase [
124]. A follow-up study confirmed that exposure of U118MG glioblastoma cells to PK11195 induced time-dependent changes in the regulation of the cell cycle and cell proliferation. These functional effects were most likely achieved by modulating the expression of immediate early genes and cell cycle regulators. These results suggest that TSPO exerts such effects as a part of the mitochondrial-to-nucleus signaling pathway that modulates nuclear gene expression [
127].
In summary, the latest evidence hints at a role for TSPO in cell cycle regulation. In light of alterations of various important cell cycle regulators and signaling pathways in GBM, it will be interesting to understand the interactions of TSPO with cell cycle checkpoint molecules and tumor suppressors in more detail.
2.2.3. Resisting Cell Death
Cell death plays an important role in suppressing cancer development and deregulation of cell death mechanisms—such as apoptosis, autophagy, and necrosis—is one of the main reasons for GBM treatment failure [
128,
129,
130]. Apoptosis can be divided into an extrinsic (mediated by death receptors) and an intrinsic (mediated by mitochondria) arm, which both lead to the activation of the executioner caspases 3 and 7 [
131]. Thereby, mitochondria play a key role in cell death signaling by initiating the caspase cascade through outer mitochondrial membrane permeabilization and subsequent release of cytochrome c [
132]. It is, therefore, reasonable to suggest that TSPO, as a mitochondrial protein, has one of its major functions here. Knockdown studies conducted over the last decade revealed that downregulation of TSPO reduced the apoptotic rate, implying a direct [
133,
134] or indirect pro-apoptotic role of TSPO, for example by reducing the pro-apoptotic effect of glutamate [
135].
An apoptosis-promoting role of TSPO is also supported by pharmacological evidence. For instance, TSPO ligands were able to induce cell death in colorectal cancer cell lines [
120], in chronic lymphocytic leukemia cells [
136], as well as in neuroblastoma cell lines in a dose-dependent manner [
126]. In addition, the exposure of several glioma and GBM cell lines to various TSPO ligands resulted in the collapse of the mitochondrial membrane potential (ΔΨm), activation of the caspase cascade, and subsequent apoptosis [
137,
138,
139].
In contrast, the TSPO ligands PK11195 and Ro5 4864 were also described to reduce apoptosis in different glioma cell lines, as well as in human monocytic cells and in a rat model of myocardial ischemia-reperfusion [
19,
22,
140,
141]. This points to a notorious problem with TSPO ligands, namely their, often not well-defined, function as agonists or antagonists as well as possible concentration-dependent effects. Results from TSPO ligand assays should always be carefully considered, as even the most advanced synthetic ligands tend to yield off target-effects, implying that a pro-apoptotic effect of higher ligand concentrations may rather depend on interactions with other targets [
107,
108,
142,
143,
144,
145].
The mechanism by which TSPO regulates apoptosis is still an enigma. In 1995, it was suggested that TSPO, together with the voltage-dependent anion channel 1 (VDAC1) and the adenine nucleotide transporter (ANT), form the mitochondrial permeability transition pore (mPTP) [
146]. Moreover, a variety of studies reported that both endogenous and synthetic TSPO ligands modulate the activity of the mPTP [
147,
148,
149]. Since TSPO is proposed as a critical regulator of this complex through modulation of VDAC1 conductance [
150], regulation of cell death may indeed be the most important function of TSPO.
The effects of TSPO include the regulation of redox stress homeostasis [
21,
151] and ΔΨm [
134,
152], which can eventually lead to apoptosis by inducing cytochrome c release, caspase activation, and DNA fragmentation [
19,
21,
22,
133]. Recently, however, the role of TSPO on mPTP function has been challenged. For instance, a study using conditional liver- and heart-specific TSPO
−/− mice revealed that TSPO does not function as a member of the mPTP. Furthermore, TSPO ligands had no impact on mPTP activity and the outer mitochondria membrane regulation of mPTP activity occurred through mechanisms independent of TSPO [
143]. In a study that used the CRISPR/Cas9 system, TSPO knockout MA-10 cells displayed a significantly reduced ΔΨm compared to control, as well as resistance to apoptosis [
153]. Another study investigating the role of TSPO in ischemia/reperfusion injury revealed that the upregulation of ROS and oxidative stress, as well as the collapse of the ΔΨm, mPTP opening, and apoptosis induced through anoxia/reoxygenation, were completely abolished by TSPO knockdown [
154]. Whether TSPO modulates the ΔΨm through the mPTP, therefore, remains controversial. Based on gene expression analysis data, it was proposed that TSPO might regulate the expression of genes associated with apoptotic processes through mechanisms such as ΔΨm collapse, ROS generation, Ca
2+ release, and ATP production, which, as a functional consequence, could potentially lead to cell death [
127]. Further, the described mechanism may well depend on the respective context in view of tissue of origin, microenvironment, and experimental conditions.
In summary, mitochondria are tightly linked to cell death through a variety of mechanisms. Considering TSPOs position in the mitochondrial membrane and according to experimental evidence, published data clearly indicate that TSPO is involved in the regulation of cell death. However, it remains open in what exact mechanistic way and in which context TSPO modulates the resistance of GBM cells to apoptotic stimuli and how this is related to other cell death mechanisms such as autophagy and necrosis.
2.2.4. Enabling Replicative Immortality
With each round of cellular replication, telomeres shorten until they finally reach a critical length that is unable to support the stable formation of shelterin protein complexes that protect telomeres from DNA damage surveillance mechanisms [
155]. In order to divide uncontrollably, cancer cells need to acquire an infinite capacity to replicate. To meet these conditions, they alter the expression of genes such as
TERT which encodes the telomerase reverse transcriptase [
156]. By extending the length of the telomeres, this enzyme actively contributes to the capacity of unlimited proliferation.
TERT mutations in GBM occur frequently with ~83% of GBM
IDH wildtype being
TERT mutated [
157]. On the other hand, GBM
IDH mutated tumors have been reported to have a lower incidence of
TERT mutation [
158], which makes
TERT status a valuable additive tool for defining GBM prognosis.
Even though TSPO might modulate the aging of cells through indirect mechanisms such as controlling the energy supply, cell death, and angiogenesis, our literature research revealed no data that could link TSPO to immortality-related mechanisms at this time.
2.2.5. Inducing Angiogenesis
Alterations in angiogenic pathways contribute largely to the aggressiveness of GBM. GBM exhibits an extensive network of abnormal vasculature to supply itself with nutrients and oxygen, and several upregulated angiogenic receptors and factors stimulate angiogenesis signaling pathways (reviewed in [
159]). Neo-vascularization in GBM is mainly mediated by vascular endothelial growth factor (VEGF), basic fibroblast growth factors (bFGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), matrix metallopeptidases (MMPs), and angiopoietins (Angs) (reviewed by [
160]). Unfortunately, the survival benefit of treatment with angiogenesis inhibitors is limited, since tumor cells can evade them through the modulation of evasive resistance pathways (reviewed in [
161]).
It has been suggested that TSPO may be involved in the angiogenesis of tumors [
162]. For instance, tumors developed from U118MG TSPO knockdown cells exhibited expanded angiogenesis in chorioallantois membranes of chicken embryos compared to tumors developed from scrambled controls [
103]. In a TSPO knockout GL261 xenograft glioma model, extensive hemorrhages were observed. Moreover, the levels of angiogenesis regulators such as HIF-1α, VEGF-A, MMP2, and IL-8 were significantly increased in TSPO knockout gliomas in comparison to wild type. The authors concluded that TSPO-deficiency triggers HIF-1α upregulation, leading to a subsequent increase in key angiogenesis regulators that fueled angiogenesis and a tumor-promoting microenvironment [
104]. Finally, a role for TSPO in the aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) was demonstrated [
18]. Briefly, overexpressing TSPO in VSMC cells had a positive effect on proliferation and migration, whereas knockdown of TSPO or modulating TSPO function with its ligands PK11195 and Ro5 4864 resulted in a significant decrease in proliferation and migration of PDGF-BB treated VSMCs [
18].
To date, only a few mechanistic studies on TSPO and angiogenesis are available and further research is needed to understand the role of TSPO in the angiogenesis of GBM. Available results suggest that high levels of TSPO counteract angiogenesis in GBM potentially through modulation of genes connected with the canonical pathway for angiogenesis [
127].
2.2.6. Activating Invasion and Metastasis
One of the major reasons for GBM recurrence is the migration and diffuse invasion of tumor cells into the surrounding brain. The background mechanism that regulates migration and invasion is an epithelial-to-mesenchymal transition (EMT) [
163,
164]. Through activation of this program, tumor cells develop the ability to detach from their primary site and invade surrounding tissue, hence, acquire an aggressive, invasive phenotype [
165]. In the context of EMT, changes in the shape of tumor cells, as well as aberrations in the attachment of cancer cells to other cells and to the extracellular matrix (ECM), are highly relevant [
31,
166,
167].
High expression of TSPO is associated with invasiveness in several cancers including GBM [
168]. In the C6 rat glioma cell line, the overexpression of TSPO increased the ability to overcome contact-dependent inhibition of cell growth. This was accompanied by an increase in the motility rate and the transmigrative phenotype of C6 cells [
100]. Wu and Gallo (2013) demonstrated, by means of transient overexpression or silencing of TSPO, that TSPO contributes to the migration of breast cancer cells [
23]. In particular, TSPO overexpression in a poorly migratory breast cancer cell line resulted in increased migration, whereas the silencing of TSPO in a highly invasive breast cancer cell line decreased migratory capabilities [
23]. In contrast, knockdown of TSPO in U118MG resulted in decreased adhesion to extracellular matrix proteins such as collagen I and IV, fibronectin, laminin I, and fibrinogen as well as in an increase in migratory capability [
103]. In addition, treatment of U118MG with PK11195 resulted in an upregulation of genes related to migration, which was confirmed by microscopic observations showing the congregation and segregation of cells [
127].
Adhesion molecules are involved in cell-cell interactions and contact to the ECM, and are therefore important actors in migration and invasion. Early studies have observed that vascular cell adhesion molecule-1 (VCAM-1) is aberrantly expressed in several cancers, including GBM [
169,
170]. Interestingly, studies in vascular endothelial cells revealed that TSPO modulates VCAM-1 and ICAM-1 expression. Overexpression of TSPO inhibited TNFα-induced, as well as phorbol 12-myristate 13-acetate (PMA)-induced, VCAM-1 and ICAM-1 expression in a dose-dependent manner [
171].
In view of these rather scarce results, the underlying mechanisms whereby TSPO influences cell migration are unknown at this time. To our knowledge, a link between TSPO, VCAM-1, and the infiltrative phenotype of GBM has not been established yet. However, it is conceivable that TSPO regulates migration and invasion through modulating the expression of adhesion molecules and/or affects the cellular energy production necessary for migration [
23,
127]. Identification of the underlying molecular mechanisms may provide a deeper understanding.