Circulating biomarker panels for targeted therapy in brain tumors

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10.2217/FON.14.238 © 2015 Future Medicine Ltd

Review

Circulating biomarker panels for targeted therapy in brain tumors

Cristiana Tanase*,1, Radu Albulescu1,2, Elena Codrici1, Ionela Daniela Popescu1, Simona Mihai1, Ana Maria Enciu1,3, Maria Linda Cruceru3, Adrian Claudiu Popa3, Ana Iulia Neagu1,4, Laura Georgiana Necula1,4, Cristina Mambet1,4 & Monica Neagu1

1Victor Babes National Institute of Pathology, Biochemistry-Proteomics Department, no 99–101 Splaiul Independentei, 050096 Sector

5 Bucharest, Romania 2National Institute for Chemical Pharmaceutical R&D, 112 Calea Vitan, 031299 Sector 3, Bucharest, Romania 3Carol Davila University of Medicine & Pharmacy, Cell Biology & Histology Department, no 8 B-dul Eroilor Sanitari, 050474 Sector 5

Bucharest, Romania 4Stefan S Nicolau Institute of Virology, Bucharest, Romania

*Author for correspondence: [email protected]

Summary An important goal of oncology is the development of cancer risk-identifier biomarkers that aid early detection and target therapy. High-throughput profiling represents a major concern for cancer research, including brain tumors. A promising approach for efficacious monitoring of disease progression and therapy could be circulating biomarker panels using molecular proteomic patterns. Tailoring treatment by targeting specific protein–protein interactions and signaling networks, microRNA and cancer stem cell signaling in accordance with tumor phenotype or patient clustering based on biomarker panels represents the future of personalized medicine for brain tumors. Gathering current data regarding biomarker candidates, we address the major challenges surrounding the biomarker field of this devastating tumor type, exploring potential perspectives for the development of more effective predictive biomarker panels.

KeywordS • biomarker panels • brain tumors • circulating biomarkers • proteomics • targeted therapy

The most common and lethal primary type of brain tumors is represented by high-grade gliomas; they present one of the highest rates of occurrence within the tumors of the CNS. Grade IV glioma – glioblastoma (GBM) – represents the most common and aggressive type of brain tumor in adults with a poor prognosis of a 1–2-year survival rate after diagnosis [1].

The treatment of GBM is a significant therapeutic challenge; it is obvious that new approaches regarding classical and new therapeutic targets involving angiogenic signals, signaling pathways, protein–protein interactions, stem cell targets and crosstalk between all of them are still an unmet need in this disease.

Due to the high complexity and heterogeneity of this type of cancer, conventional biochemical methods can fail to notice important components and/or proteomic profiles of this disease. One of the main goals of oncology is represented by biomarkers development, since it presents the potential to identify cancer risks, as well as to improve early detection and targeted therapy. We are confident that a biomarker panel would provide superior identifiers and/or predictors of a patient’s clinical outcome. High-throughput proteomic profiling and multiplex analysis, such as xMAP and protein arrays technologies, are becoming important approaches in GBM research. These technologies can supply simultaneous analyses of biomarkers in a panel for improved diagnosis, patient stratification, prognosis and drug screening. Biomarker discovery for brain tumors is an ongoing pursuit and the search for the best molecule or combination of molecules is still unfolding [2]. In the last 5 years, proteomics topics were a major presence in the literature, having a bulk of more than 5000 papers

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focusing on proteomics in cancer; around half of these papers report biomarker candidates in intracranial tumors. In spite of this abundance, clinicians still do not count on a specific tumor marker for brain tumors.

Therefore, this review, along with our hands-on experience, emphasizes the latest knowledge in circulatory biomarkers, the tumor-derived blood-based biomarkers, circulating tumor cells, circulating nucleic acids and circulating proteins in patients with malignant gliomas.

Molecular classification of GBMHistological and molecular analysis of CNS tumors can identify approximately 120 sub-types. Peripheral blood scans for predictive biomarkers in CNS indicated that certain brain tumors are indeed associated with distinct pro-files of circulating factors, such as proteins (e.g., glial fibrillary acidic protein), DNA fragments [3] or miRNAs (e.g., miRNA-21) [4].

So far, the only subclassification of GBM that also inf luences the therapeutic deci-sion is the detection of O-methylguanine-DNA methyltransferase (MGMT ) promoter hypermethylation.

Recent progress in the molecular research of GBM has led to the identification of other mark-ers useful in either the diagnosis or prognosis of this fatal disease. Mutation in isocitrate dehy-drogenase genes, notably for isoform 1 (IDH1), seems to bear favorable prognostic value for GBM patients [5]. Furthermore, The Cancer Genome Atlas Research Network showed evidence for a CpG island methylator phenotype in GBMs, associated with IDH1 mutations [6]. The newly emerged data involving IDH mutations and epi-genetic modifications prompted further inquir-ies regarding the molecular mechanisms that occur up- and down-stream from these events. RBP1 promoter hypermethylation is found in nearly all IDH1 and IDH2 mutant gliomas, and is associated with improved patient survival [7]. TET enzymes that catalyze oxidation of 5mC to 5hmC and epigenetically modify gene transcrip-tion are mutated in several types of cancer, affect-ing their activity and likely altering genomic 5hmC and 5mC patterns [8]. Modified expres-sion of TET genes seems to have a significance in risk stratification of GBM patients, along with APOBEC deaminase genes [9]. Promoter hyper-methylation of p16INK4, p14ARF, RB, PTEN and p53 were also reported to play a role in the epigenetic mechanisms of GBM pathology [10].

Loss of heterozygosity (LOH) on chromosome 1p/19q and chromosome 10 may help to predict patient outcomes [11].

The assessment of these specific alterations has been encumbered by the scarceness of biotic material; therefore, their detection in the cir-culation would decrease diagnostic time and expedite the therapeutic decision. Apart from genetic circulating tumor DNA analysis, which emulates tumor assessment for genetic and epi-genetic aberrations, serum profiling for protein biomarkers has also been proven useful as a diag-nostic and/or prognostic tool for GBM. It is now commonly accepted that a panel of biomarkers shows better predictive power over a single bio-marker, and recently, due to the development of proteomic techniques, panel attainment has become a feasible task [12]. Hence, a number of proteins has been identified as potential diag-nostic markers (A2M, factor VII, MDC, SCF [13] and staging biomarkers [haptoglobin, plas-minogen precursor, apolipoprotein A-1 and M, and transthyretin]) [14]. A systematic review of multiple independent proteomic analyses of glioma, carried out by Deighton et al., demon-strated alterations in 99 different proteins, ten of which were repeatedly reported (PHB, Hsp20, serum albumin, EGF receptor, EA-15, RhoGDI, APOA1, GFAP, HSP70 and PDIA3) [15].

Circulating tumor cells in malignant gliomasCirculating tumor cells (CTCs), originating both from primary and metastatic lesions, can be detected in the peripheral blood of patients with different solid tumors [16]. Unlike normal blood cells, CTCs are present in very low counts, up to a few hundred per milliliter, depending on the CTC definition and the methods used for their detection and isolation [17].

Several clinical studies performed in patients with metastatic breast, prostate, colorectal and non-small-cell lung cancer (NSCLC) showed a correlation between CTC count and clinical outcome, and suggested that CTC enumera-tion may represent a noninvasive adjuvant tool for prognosis, recurrence and therapy response monitoring [18].

However, it will be very useful in clinical practice to detect CTCs in patients with early-stage cancer in order to benefit from more adequate risk stratification and personalized therapy. This approach is limited by the fact that most of the current methods are not able to

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accurately establish the CTC numbers in early-stage tumors, and further investigation is needed for this purpose.

Regarding malignant gliomas, although this type of tumor is thought to rarely disseminate outside the brain, there are reports in the lit-erature of lung, liver, lymph node and bone metastasis occurring in patients with GBM multiforme [19]. Moreover, some cases of GBM transmitted through organ transplantation from affected donors are well documented [20]. These data provide indirect evidence that CTCs are present in patients with malignant gliomas, although thus far they have not been successfully detected [21].

Due to the fact that brain tumors, such as GBM, tend to lack cancer cell surface biomark-ers such as EpCAM on which current methods of CTC detection are based, in a very recent paper Macarthur KM et al. proposed a new strategy to detect CTCs. They used a telomer-ase-specific adenoviral agent to assess telomerase activity, which is increased in nearly all tumor cells, but not in normal cells. The assay proved to successfully detect CTCs in patients with brain tumors, thus providing a promising tool for monitoring treatment response [22].

High-grade gliomas are characterized by increased neovascularization and recruitment of endothelial progenitor cells (EPCs); this rep-resents one of the mechanisms involved in new vessel formation. Related to this topic, Corsini E et al. studied the effect of surgical and post-surgical treatment at the level of EPCs in glioma patients and their correlation with VEGF. They found significantly decreased levels of EPCs in all treated patients compared with untreated ones. VEGF registered decreased levels only after surgery, but not after chemotherapy; no correlation was found between VEGF and EPCs levels. Further studies are needed to assess the usefulness of EPCs as markers of angiogenesis monitoring in high-grade gliomas [23].

Oncosomes as future circulating biomarkers carriersThe complex interactions between CNS cell types and distinctive cellular milieu, gener-ates a particular biology for brain tumors, which impacts on their sensitivity to treatment. Oncosomes, or membrane-derived extracellu-lar vesicles (EVs) secreted by cancer cells, can transport proteins and nucleic acids from one cell to the other, thus being active participants

in the oncogenic transformation of various cells. It has been recently noted that oncosomes can contain several types of molecules involved in pathogenesis pathways, such as tumor prolif-eration, angiogenesis and invasion. Oncosomes can contain proteins, transcripts, DNA and miRNAs that ‘hijack’ the recipient cell’s physi-ology and cell microenvironment. Oncogenic EGF receptor (EGFRvIII), tumor suppressors (PTEN) and oncomirs (miR-520g) were found in the oncosomes identified in blood circulation and the cerebrospinal fluid (CSF) of patients diagnosed with brain tumors. These new ‘cir-culatory’ biomarkers could be useful biomark-ers in patient stratification and/or therapeutic efficacy predictors [24]. As we are focusing on circulatory biomarker candidates, the role of exosomes/oncosomes in cancer, hence a circula-tory particle capable of transferring tumor cell-derived genetic material and signaling proteins from one cell to another and thus linking tumo-rigenesis, angiogenesis and metastasis, is even more intriguing. In 2013 it was reported that internalization of exosomes was derived from GBM cells where the protein CAV1 negatively regulates the uptake of exosomes. Moreover, exosomes can induce activation of ERK1/2 and HSP27, opening new opportunities for therapy targets [25].

Thus, oncosomes have come into view in brain tumor research domain as important vehi-cles of intercellular communication, providing a new field of molecular biomarkers investigation.

miRNAs as molecules linking tissue & circulating biomarkersThe discovery of miRNA in tissues, as well as in body fluids together with their altered pro-file in various pathological conditions, offers a new perspective on the use of extracellular miRNAs as informative biomarkers of disease [26,27]. Similarly to other types of tumors, GBM multiforme was associated with numerous miR-NAs. Such miRNAs display either oncogenic or tumor-suppressive properties, and are therefore attractive therapeutic targets for future miRNA-based therapies. Moreover, some studies have suggested that miRNAs may be used as nonin-vasive diagnostic and prognostic biomarkers due to their release in the circulation [28].

In our previous paper, we also highlighted the fact that miRNA expression profiles in GBM are better predictors of clinical outcome than mRNA profiles [29].

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In order to select candidate miRNA biomark-ers for malignant astrocytomas, Yang et al. per-formed genome-wide miRNA screening of serum samples from astrocytoma patients and matched controls. After subsequent validation of the obtained results, seven miRNAs were found to be significantly decreased in patients versus controls: miR-15b, miR-23a, miR-133a, miR-150, miR-197, miR-497 and miR-548b-5p. Moreover, these identified miRNAs exhibited a marked increase after surgery. As a conclusion, this miRNA pro-file may be a potential noninvasive biomarker panel for malignant astrocytoma [30].

Different studies have demonstrated abnor-mal miRNA expression profiles in GBM tis-sues; some miRNAs were found to be upreg-ulated (e.g., miR-17, miR-21, miR-93 and miR-221/222), while others were downregulated (miR-7, miR-34a, miR-128 and miR-137) [31].

Lu et al. found an overexpression of miRNA-17 in glioma tissues that was associated with advanced pathological stage and poor overall survival [32], while in a previous study conducted on glioma cell lines, Malzkorn et al. showed that miR-17 inhibition reduces cell viability and increases apoptotic activity [33].

In vivo and in vitro studies on glioma cells pointed out that miR-93-overexpressing cells promoted endothelial cell spread, growth, migration and tube formation supporting neo-angiogenesis and tumor growth. This effect is at least partly explained by the fact that miRNA-93 targets and silences integrin-β8, whose expres-sion is associated with decreased cell growth [34].

Significant overexpression of miR-221/222 was detected in high-grade gliomas compared with low-grade gliomas, related to increased cell invasion and poor prognosis by directly targeting TIMP3 [35].

Recent work reported decreased expression of miR-128, which correlates with aggressive human glioma subtypes; miRNA-128 exerts tumor suppression effects by targeting RTK signaling [36]. Although miRNA-128 was found to be downregulated in GBM tissue compared with normal brain tissue, Roth et al. detected increased expression levels of miRNA-128 in blood cells of GBM patients [37].

Kefas B et al. found a significantly reduced expression of miR-7 in human GBM tissue and GBM cell lines, showing that miR-7 directly inhibits EGFR expression and suppresses Akt pathway activation, and thus decreases the viability and invasiveness of GBM cells [38].

Another miRNA found to be downregulated in GBM is miR-34a; its expression has an anti-tumor effect suppressing cell proliferation, G1/S cell cycle progression, cell survival, cell migra-tion and cell invasion in glioma cell lines. These properties are due to inhibition of c-Met, Notch-1 and Notch-2 [39].

Proteomic profiling for circulating biomarkers discoveryDuring the last decade, a wide range of tech-nologies has been employed in molecular and genetic profiling studies for biomarker discovery in brain tumors. However, a limited number of biomarkers has been identified and none has yet attained broad application for use in clinical gliomas prognosis, therapeutic target selection or molecular classification [40].

Proteomic profiling offers large-scale analysis of protein expression, post-translational modi-fication and protein–protein interactions. The ideal platforms for biomarker discovery include genomic and metabolomic analyses, and have the ultimate goal of unifying the information into protein networks [41].

While advanced proteomics, based on combi-nations of techniques such as 2D or 2D-DIGE and mass spectrometry, are the solution of choice for detailed studies that involve sequencing, high-throughput techniques, such as SELDI-TOF and MALDI-TOF, are used for differen-tial protein signatures. The major advantage of SELDI-TOF mainly lies in the fast output of molecular signatures, yet it has to be usually fol-lowed by more in-depth studies (based on more accurate MS/MS technology) in order to more accurately identify the proteins with modified expression [42].

Identification of potential neoplasia diagnos-tic, prognostic, predictive or treatment-assessing biomarkers could allow differential proteomic profiling of brain tumor versus disease-free state for the detection and monitoring of pathology-related changes. Specifically, proteomic analysis of different fluids is less invasive than biopsy and could provide potentially informative causes of origin and progression of brain tumor pathologies [40].

A number of studies have reported different individual serum biomarkers for GBM, such as YKL-40 [43], GFAP [44], MMP-9 [45], EGFR [46] and CD14 [47].

Reyens et al. reported elevation of several inflammatory proteins (C-reactive protein, IL-6,




TNF-α and sialic acid), coagulation factors (fibrinogen, endogen thrombin generation, pro-thrombin fragments 1 and 2, and tissue factor) and angiogenic factors (VEGF, soluble VEGF receptor 1 and thrombospondin-1) in the plasma of GBM patients [48].

SELDI-TOF MS technology has been utilized in the protein profiling analysis of a variety of specimens derived from patients with brain tumors for the discovery of biomarkers that facilitate early diagnosis, establish tumor grade and predict therapeutic outcome.

Kumar et al., based on a combination 2-DE/MS approach, observed ten differentially expressed proteins in the sera of patients with GBM and validated haptoglobin a2 as serum marker associated with tumor growth and migration in GBM [49].

In another study based on SELDI-TOF MS, Petrik et al. discovered and validated AHSG as a predictive and prognostic biomarker for GBM. Thus, low serum levels of AHSG were correlated with a short median survival rate (<3 months), with normal levels in serum being associated with prolonged survival (>2 years) [50].

In a preliminary study [51] using SELDI-TOF MS on serum samples, we identified a number of 11 clusters with significant differences between GBM and controls (p < 0.05) out of a total of 152 clusters with m/z values 2–55 kDa; six clus-ters were overexpressed and five underexpressed in GBM patients compared with control.

Gautam et al. [52], using an iTRAQ-based LC-MS/MS approach in GBM patients plasma, have identified a total of 296 proteins, out of which 61 exhibited increased levels in the patient group. Altered levels of FTL, S100A9 and CNDP1 were validated by ELISA. These proteins may form useful starting points for the development of plasma-based biomarker panels in clinical investigations for GBM [52].

Soluble biomarkers – main actors in panel set upWhile mass spectrometry-based approaches are undoubtedly some of the most effective tools in the de novo discovery of biomarkers, other approaches are available for sorting out biomark-ers from known molecules, such as cytokines, chemokines or growth factors via protein arrays or, more effective, xMAP multiplex technology.

While protein arrays are generally more pow-erful in terms of the total number of proteins that can be simultaneously detected, xMAP

arrays are more suitable to perform quantitative analysis in high-throughput conditions [2].

Reliable circulating biomarkers could sup-port the management of gliomas by facilitating neuroradiological differential diagnosis at initial presentation, planning of surgical interventions or monitoring of the disease course.

Tumor initiation and progression represent a complex process involving genomic mutations, microenvironmental factors and inflammatory mediators. An active role in determining the malignancy phenotype is owned by the tumor microenvironment. Both host cells and cancer cells crosstalk via a large variety of soluble fac-tors, whose effects determine the final outcome of the tumorigenic process [53].

Inflammatory cells, cytokines/chemokines and their receptors existing in tumors contrib-ute to tumor growth, progression, metastasis and immunosuppression. They regulate tumor growth either directly by transformation, sur-vival, proliferation and migration of cancer cells, or indirectly by enhancing angiogenesis or recruiting leukocytes [54]. The “match that lights the fire” of cancer is genetic damage; in this context, some types of inflammation may provide the “fuel that feeds the flames” [55,56].

Matrix metalloproteases (MMPs), important components of the tumor microenvironment and secreted by glioma cell lines, are responsi-ble for sVE release. Soluble VE-cadherin in the blood might reflect VEGF activity at the tumor site. Analysis of glioma patient sera confirmed the presence of sVE in the bloodstream, and sVE levels were significantly predictive of overall sur-vival, irrespective of the histopathological grade of tumors [57].

Plasma MMP-2 and VEGF were identified in the study of Xu et al. as potential biomarkers that accurately distinguished high-grade glioma patients from controls [13]. MMP-2 and VEGF have also been investigated in urine samples as diagnostic biomarkers for brain tumors [58]. MMP-2 promotes cell invasion, angiogenesis, activation of growth factors, and is involved in the development of human glioma microves-sels, facilitating glioma cell invasion [59]. VEGF can promote tumorigenesis and angiogenesis of human GBM stem cells, and VEGF expression is significantly increased in high-grade astrocyto-mas compared with low-grade astrocytomas [13].

Serum S100B and NSE were considered markers of CNS damage, yet further studies were required to establish whether S100B is an

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independent prognostic biomarker in glioma patients [60]. Subsequent studies have showen that biomarkers such as S100B, NPY and SCGN were markedly elevated in plasma of patients with malignant glioma 1 year before clinical manifestation [61].

Changes in serum levels of PlGF were associ-ated with overall survival in patients with recur-rent GBM [62]. Plasma concentrations for PlGF were correlated to neuropathological or neuro-radiological features. In addition, IL-8, BDNF and GDNF were detectable in different concen-tration levels in serum/plasma. Measurement of circulating GFAP and PlGF are potentially use-ful as clinical biomarkers that may support dif-ferential diagnosis of GBM versus intracerebral metastasis [63].

Serum IL-10 levels have been described as significantly increased in high-grade glioma patients compared with nontumor control patients [55,64]. Increased levels of IL-10 in GBM patients are consistent with the findings that Th2 cytokines are elevated in patients with advanced tumors [13]. IL-10 was identified as a serum biomarker that accurately differentiates GBM patients from controls [13].

A2M has been upregulated in migrating human glioma cells compared with nonmigrat-ing glioma cells [65,66]. Neuronal and glioma-derived SCF can induce angiogenesis within the brain [67]. Expression of tissue factor, the cell surface receptor for factor VII, correlates with histologic grade of human glioma malignancy and vascularity [68]. In addition to the accurate classification of malignant glioma patients, cir-culating factor VII was also found to have prog-nostic significance. MDC was underexpressed in plasma/serum samples from high-grade glioma patients. MDC and its receptor CCR4 both show decreased expression in human grade III astrocytoma and grade IV glioma cells [69]. Knowing that these biomarkers have differential expression levels in high-grade glioma tissues, they were also identified in glioma patients’ blood using multiplex assay [13]. Thus, A2M, fac-tor VII, MDC and SCF were identified as bio-markers in patients with malignant glioma, both in plasma and serum specimens, with signifi-cant differences in the protein expression levels between patients and controls. These molecules represent protein biomarkers that may identify patients with malignant glioma in independent cohorts with similar accuracy in either plasma or serum specimens [13].

An important issue regarding antiangio-genic treatment discontinuation and patient morbidity remains the toxicity derived from antiangiogenic therapy, thus serum/plasma bio-marker panels that correlate with drug toxicity and changes in cytokine and angiogenic fac-tors would be of great value. Changes in IL-13 from baseline to 24 h predicted on-target tox-icities. Changes in IL-6, IL-10 and IL-13 were frequently correlated with toxicity. Profiling of IL-13 as a surrogate for endothelial dysfunc-tion could individualize patients at risk during antiangiogenic therapy [70].

In our previous study [2], a panel of cytokines with modified expression was detected in GBM patients’ sera using multiplex assay. Our results indicate significant dysregulation in serum lev-els of cytokines and angiogenic factors, three-fold upregulation for IL-6, IL-1β, TNF-α and IL-10, and twofold upregulation of VEGF, FGF-2, IL-8, IL-2 and GM-CSF were noticed (Figure 1). Cytokines expression was strongly cor-related with tumor grade, proliferation markers and clinical aggressiveness in GBMs [2].

A circulating biomarker-based signature is an important approach for the clinical assessment of malignant glioma, with the abovementioned biomarkers facilitating accurate diagnosis and therapy monitoring, and assessing survival rate prognosis [40].

Assessing circulating biomarkers through the rapid and efficient method represented by array technology can be a reliable tool for the diagno-sis of brain tumors and for the discovery of new potential therapeutic targets or therapy monitor-ing. Biomarker profiling, obtained via proteomic high-throughput technologies, could offer major support to identify unique differences within an individual tumor or between tumors of the same histological grade [71].

Signaling molecules profile as potential biomarkers & therapy targetsAs stated in the previous sections, the aggres-siveness and therapeutic resistance of GBM also represents the base for the search for signaling dysregulation.

The intracellular pathways and the molecules that append to these pathways could assume both the biomarker and future therapy target role [72]. GBM shows dysregulation in various signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of RTK signaling [73].


Figure 1. Modulation of serum cytokine levels in glioblastoma patients. The data represent group averages of fold modification versus controls.

0 1

IL-6

IL-4

IL-12

IL-1β

TNF-α

IFN-γ

VEGF

FGF-2

IL-8

IL-2

GM-CSF

2 3 4 5 6Fold modification versus control

Modulation of serum cytokine levelsin glioblastoma patients



Dysregulations of PI3K/Akt/mTOR signaling were reported in GBM and induce RTK overac-tivity (EGFR, PDGFR, mesenchymal–epithelial transition factor), mutated PI3K subunits and/or loss of tumor suppressor activity, namely PTEN [74]. Indeed, 40% of GBM have a loss of PTEN, triggering excessive PI3K signaling, while other GBMs exhibit post-translational modifications inducing inactivation of PTEN. A therapeuti-cally wise approach and more specific to person-alized medicine can benefit from the identifica-tion of signaling dysregulation and the definition of novel targets [75]. The PI3K signaling pathway has been investigated as an important target for the treatment of GBM [76], seeking PI3K inhibi-tors as therapeutic agents in GBM [77]. According to one of our recently published papers, by inhibiting the PI3K pathway in patient-derived GBM cells, the expression of signaling proteins belonging to various pathways can be altered, and hence their tumor cell proliferation capacity can be reduced. Our recent study has revealed that treating GBM cell cultures isolated from patient tumors with PI3K inhibitors induced a significant decrease in the expression level of several key signaling molecules involved in cell survival (p38), proliferation (ERK1/2, IκBα, p38 MAPK CREB), differentiation (ERK1/2, CREB), migration (ERK1/2, CREB) and apop-tosis (ERK1/2, P70S6K, IκBα, JNK, CREB) [2]. We found that PI3K inhibitors reduced tumor cell proliferation, as in similar reports for other types of tumors [77]. The maximal efficacy of

PI3K inhibitor we have recorded upon cellular responses is coherent with other experimen-tal results pointing out the major relevance of PI3K signaling in GBM and furthermore that PI3K inhibitors can add to, or even increase, the limited success of EGFR inhibitors in clinical trials [78]. P70S6K, a serine/threonine kinase, can be activated by both the PI3K and ERK pathways. This multiple involvement supports the idea of the simultaneous usage of PI3K and MAPK inhibitors [79]. In accordance with our results, a similar interference between PI3K and MEK/ERK signaling pathways was reported by Sunayama et al. [80].

Pan PI3K inhibitor 2-(4-morpholinyl)-8-phe-nyl-4H-1 benzopyran-4-one (LY294002) can induce important antitumoral overall effects in experimental models, but the LY294002 com-pound has poor pharmacologic variables of insol-ubility and a short half-life. Starting from this structure, a novel RGDS-conjugated LY294002, named SF1126, was designed. The goal was to exhibit increased solubility and bind to specific intratumor integrins, thus an cause enhanced delivery of the active compound. Using the U87MG GBM cell line SF1126 had an enhanced efficacy due to the RGDS integrin (alpha v beta 3/alpha 5 beta 1) binding component. SF1126 has both antitumor and antiangiogenic activity, recommending it as a viable pan PI3K inhibitor for Phase I clinical trials [81].

In GBM tumor samples the axonal guidance protein, netrin-1, is overexpressed. Last year it

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was reported that, using tandem affinity puri-fication and mass spectrometry technologies, netrin-1 forms a complex with Notch2 and Jagged1. This co-localization was present on the cell surface, while in intracellular vesicles netrin-1 was present only with Jagged1, but not with Notch2. Netrin-1 induced Notch signaling and consequent GBM invasion. Interestingly, besides a candidate biomarker netri-1 can also be a therapeutic target since the central domain of netrin-1 counteracts the effects of netrin-1, meaning that it inhibits GBM cell invasion and Notch activation. This astonishing finding was that Notch signaling complex remained at the cell surface [82]. This year, a series of early-phase clinical trials were reported where Notch sign-aling was the therapy target using agents that either obstructed Notch receptor cleavages such as γ-secretase inhibitors (GSIs) or interfered with the Notch ligand–receptor interaction, which occurs in several solid tumors, including intrac-ranial ones [83,84]. This report again underlies the urgent need for efficacy biomarkers to sup-port the development of this class of drugs [85]. Niclosamide induced the cytostatic, cytotoxic and antimigratory effects of GBM in in vitro and in vivo models, and moreover reduced the capacity of multipotent/self-renewing cells in vitro. By analyzing the pathways triggered by this drug, simultaneous inhibition of WNT/CTNNB1-, NOTCH-, mTOR- and NF-κB cascades are revealed. The authors report that in GBM biological samples a heterozygous deletion of the NFκBIA locus was found, thus besides a possible genomic biomarker, this could serve as an explanation for predicting the synergistic activity of niclosamide with temozolomide, the current standard in GBM [75].

RTK-targeted therapy was investigated using imatinib, sunitinib and cediranib in GBM mod-els. In a panel of ten GBM cell lines, cediranib proved to have the most potent antitumoral effect, while cediranib and sunitinib sensitizes the cells to the classic temozolomide. Cediranib inhibited MAPK and AKT pathways, but the authors did not find any correlation between KIT, PDGFRA and VEGFR2 expression, and therapy responses to any of the RTK inhibitors. RTK therapy can rely upon future biomarkers for therapy response in GBM [86].

As already stated in the previous section, circulating exosomes can induce the activa-tion of ERK1/2 and HSP27 via CAV1; this recent report opens new possibilities for therapy

targets through CAV1 expression and ERK1/2 signaling [87].

Intracellular signaling molecules that basi-cally depict the molecular heterogeneity of the tumor are certainly important biomarkers in predicting responsiveness and disease outcome upon therapy. This assertion was sustained by less favorable clinical trial results when mono-therapy was used, therapy overlooking the com-plexity of intracellular network of individual tumors. Thus, intracellular key signaling mol-ecules and the actual panel of these molecules are to become important target therapies and valu-able biomarkers, especially for therapy efficacy monitoring.

The simultaneous detection of several mol-ecules involved in various signaling pathways has not been previously reported in GBM. Further studies are needed in order to assess whether this regulation is transcriptional or post-tran-scriptional. Keeping in mind that personalized medicine should be tailored to the patient’s tumor particularities, screening cell behav-ior and expression levels of signaling proteins within tumor cells can highlight the important differences between primary tumors of similar histological type, and/or allow comparisons of primary and relapse tumor samples.

Cancer stem cells as potential biomarkers for GBM aggressivenessCancer stem cells (CSCs), identified in a myriad of human solid tumors including brain tumors [88], have generated a new field of research where a clear distinction should be made between can-cer-initiating cells and cells that allow propaga-tion of the tumor [89]. Nowadays, genomics and epigenetic techniques have proven that cancer takes control of specific genetic and epigenetic programs from embryonic development circuits. Two recognized models regarding the genera-tion and function of GBM CSCs (GCSCs), the stochastic and the hierarchical models, are currently recognized (Figure 2).

According to the stochastic model, differenti-ated or committed cells acquire genetic muta-tions towards immortal proliferative capacity, leading to cancer stem cells (GCSC in our case), in contrast with the hierarchical model where a stem cell or a glial precursor undergoes a neoplastic transformation, leading to glioma initiation and development [90]. Properties such as extensive self-renewal ability and multipotency are common for neural stem cells (NSCs) and


Figure 2. Acknowledged theories regarding cancer stem cell generation – stochastic and hierarchical theories. (A) Normal neural stem cells (NSCs) give rise to progenitors cells (PCs) with limited proliferative capacity that further leads to differentiated normal cells (DCs). CSCs can form as a result of abnormal differentiation of a normal NSC or neural progenitor cell (PC). CSC can develop cancer progenitor cells (CPCs) that further are able to develop the actual tumor (T). (B) CSCs can result from terminally differentiated cells, which acquire several genetic mutations.

T

CSC

CPC

Several mutation events

PCNSC

DC

Mutation

Mutation

Mutation

NSC CSC



GCSCs, and have been included in the defini-tion of cancer stem cells. It has recently been shown that chemokine CXCL12 and its recep-tor CXCR4 can control proliferation, invasion and angiogenesis in GBM cell lines and primary cultures. In GCSCs an increased CXCR4 expres-sion was found, as well as release of CXCL12 in vitro. As also found by us, there is a clear het-erogeneity of GBMs that induce different levels of both expression and secretion in individual cultures, again an important argument for per-sonalized therapy. CXCL12 activation-induced Akt-mediated reduced apoptosis and self-renewal activities, but less proliferation, while CXCR4 antagonist AMD3100 reduced self-renewal and survival. In in vitro differentiated cells derived from the same GBMs, the abovementioned inhi-bition through AMD3100 was not obtained, this finding being a strong argument for the coupled CXCL12/CXCR4 interactions that are specific for CSC in GBM mediating survival and self-renewal, therefore representing a promising future for therapeutic targets [91]. We point out that this finding can be also extrapolated to cir-culatory biomarkers, as CXCL12 is secreted by GCSCs, enhancing the possibility to find this biomarker in patients’ circulation.

As mentioned in the previous section, the Notch pathway is highly activated in this type of solid tumor. Moreover, it has recently been shown in an animal model that mice constitutively expressing the activated intracellular domain of Notch2 display neurogenic niche hyperpla-sia and reduced neuronal lineage entry. When authors isolated neurospheres from this model, an increased proliferation, survival and resistance to apoptosis was obtained. In human GBM cell lines, the Notch2 pathway induces an increased proliferation and resistance to apoptosis. When assessing gene expression in GBM patient tumor samples, a positive correlation of Notch2 tran-scripts with the transcripts that control antia-poptotic processes, stemness and astrocyte fate was found. In the meantime, Notch2 transcripts were negatively correlated with gene transcripts controlling the proapoptotic process and oligo-dendrocyte fate. The Notch2 pathway in NSCs can drive to GCSCs and can induce astrocytic lineage entry and brain tumor development [92].

GCSC autophagy was reported to be induced by suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, as an effect of late-phase apoptosis. In vivo xenografts have shown that SAHA reduced tumor growth and

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determined autophagy. Actually, the authors report that SAHA’s action is induced by down-regulation of AKT/mTOR signaling. Taken together, this recent study shows that in GCSC therapeutic approaches, SAHA can be a capable agent for autophagy induction [93].

This year, in brain tumor initiating cells (BTICs), neurotrophin receptors (p75NTR,

TrkA, TrkB and TrkC) and their ligands (NGF, BDNF and NT3) were reported. Moreover, BTICs secrete NGF and, thus, exogenouous NGF induced BTIC proliferation. The authors note that the intracellular domain of p75NTR is to be found in GBM biological specimens, sug-gesting that the receptor is activated and cleaved in patient tumors. These results open a new

executive SummaryMolecular classification of glioblastoma

● Histologic and molecular analysis of CNS tumors can identify approximately 120 subtypes.

● Recent progress in molecular research of GBM has led to the identification of novel biomarkers useful in the diagnosis and prognosis of this fatal disease.

Circulating tumor cells in malignant gliomas

● Circulating tumor cells originating from both primary and metastatic lesions can be detected in the peripheral blood of patients with different solid tumors.

● Several clinical studies performed in patients with different types of tumors, including brain tumors, showed a correlation between CTCs and clinical outcome, and suggested that CTC detection may represent a noninvasive adjuvant tool for evaluating prognostic value, recurrence capacity and therapy response monitoring.

Oncosomes as a future circulating biomarkers carrier

● Oncosomes, or membrane-derived extracellular vesicles (EVs), secreted by cancer cells can transport proteins and nucleic acids from one cell to the other, thus being active participants in the oncogenic transformation of various cells.

● Oncosomes come into view in brain tumor research domains as an important vehicle of intercellular communication, providing a new field of molecular biomarkers investigation.

miRNAs as molecules linking tissue & circulating biomarkers

● Tumor-specific miRNAs are promising tools for the early detection of cancer and the development of personalized therapies.

Proteomic profiling for circulating biomarkers discovery

● A wide range of technologies have been employed in molecular profiling studies for biomarker discovery in brain tumors. Proteomic profiling offers large-scale analysis of protein expression, post-translational modification and protein–protein interactions.

● SELDI-TOF-MS technology has been utilized in protein profiling analysis for the discovery of biomarkers facilitating diagnosis, predicting therapeutic response and establishing tumor grade.

Signaling molecule profile with biomarker potency & future therapy targets

● GBM shows dysregulation in various signaling pathways including the G1/S cell cycle checkpoint and the MAPK and PI3K effectors of RTK signaling.

● Deregulations of PI3K/Akt/mTOR signaling were reported in GBM and induce RTK overactivity (EGFR, PDGFR), mutated PI3K subunits and/or loss of tumor suppressor activity, namely PTEN.

Cancer stem cells as potent biomarkers for GBM aggressiveness

● Cancer stem cells, identified in a myriad of human solid tumors including brain tumors, have generated a new field of research where a clear distinction should be made between cancer-initiating cells and cells that allow propagation of the tumor.

● Several molecules have been identified as candidate biomarkers for glioblastoma, but a more suitable approach for efficacious therapy would rely on the use of personalized proteomic profiles.




door in the BTIC research domain for a novel potential clinical target [94].

Conclusion & future perspectiveAlthough there are many proteins described as ‘potential biomarkers’ and although predic-tive biomarkers have not been yet validated for patients with GBM, a useful serum marker panel of brain tumors is urgently needed. Discovering a single biomarker that would be both sensi-tive and specific for cancer might be more dif-ficult than discovering a panel of biomarkers. Although some molecules have been proposed as potential GBM biomarkers, it is the current opinion that a more adequate approach would be to compare the proteomic profile of an indi-vidual with the values recorded in healthy indi-viduals (panel of biomarkers).

We believe that in the very near future a panel comprising cytokines, chemokines and angio-genic circulating biomarkers can offer an insight regarding the diagnostic and invasive potential of GBM. This front panel will be followed by the identification of CTC along with miRNA panels that depict disease progression, relapse and therapy monitoring. In the long term, it is a probability that intracellular signaling path-ways elucidation accompanied by circulating oncosomes will be developments in the bio-markers field, providing this pathology with new therapy targets.

GBM patients will benefit more from person-alized therapy by tailoring their treatment and aiming specific protein–protein interactions and signaling networks according to tumor pheno-type or patient clustering based on biomarker panels. New, promising fields for GBM personal-ized medicine are microRNA and CSC signaling.

Future therapies could emerge from the coopera-tion between cancer stem cells and their niches, which will improve the maintenance of their characteristic features and behavior, with the concomitant activation of differentiation signal-ing pathways, such as RTKs–Akt, Notch, BMPs, Hedgehog, Wnt-β-catenin, and so on.

New protocols based on a combination of chemotherapy and immunotherapy are probably a new approach for achieving therapeutic syn-ergy and more suitability for brain tumors. The cancer stem cell paradigm might become the cornerstone for novel cancer research approaches in the following years.

In this review, we have addressed the major challenges surrounding biomarker development in this particular type of devastating tumors; by gathering the current data regarding biomarker candidates, we can explore potential routes for the development of a more effective predictive biomarkers panel.

Financial & competing interests disclosureThis paper is partly supported by the Sectorial Operational Programme Human Resources Development (SOPHRD), and financed by the European Social Fund and the Romanian Government under the contract number POSDRU 141531. This work was partially supported by grants PN 09.33-04.15, PN 09.33-03.10 and PN 09.33-01.01. The authors would like to thank Irina Radu, certified translator in Medicine – Pharmacy, certificate credentials: series E no. 0048, for professional linguistic assistance. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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Circulating biomarker panels for targeted therapy in brain tumors

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