Nicotinamide Phosphoribosyl Transferase Is Increased in Osteosarcomas and Chondrosarcomas Compared to Benign Bone and Cartilage.

BACKGROUND/AIM: Primary bone neoplasms include osteosarcomas (OS), chondrosarcomas (CS), and giant cell tumors (GCT). Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide synthesis and is increased in multiple tumor types. In malignancies, NAMPT expression often correlates positively with tumor grade, chemotherapy resistance, and metastatic potential. MATERIALS AND METHODS: Tissue microarray was used to examine NAMPT expression in benign bone and cartilage, GCTs, OS, and different CS grades. RESULTS: For the first time, we showed that NAMPT expression was increased in GCTs and OS compared to benign bone, and in CS compared to benign cartilage. Its expression also increased with higher CS grade. CONCLUSION: Our data indicate that NAMPT plays a role in bone sarcomas and GCTs, and its higher expression may contribute to increased tumor aggressiveness.

Restoration of miR-127-3p and miR-376a-3p counteracts the neoplastic phenotype of giant cell tumor of bone derived stromal cells by targeting COA1, GLE1 and PDIA6.

Although generally benign, giant cell tumors of bone (GCTB) display an aggressive behavior associated with significant bone destruction and lung metastasis in rare cases. This and the very high recurrence rate observed after surgical resection ranging from 20 to 55% necessitates the development of more effective treatment strategies. To identify valuable therapeutic targets, we screened a previously identified microRNA signature consisting of 23 microRNAs predominantly down-regulated in GCTB. We preselected eight candidate microRNAs and analyzed the impact of their restored expression on the neoplastic phenotype of GCTB stromal cells (GCTSC). A consistent and significant inhibition of cell proliferation, migration, colony formation and spheroid formation could be induced by transfection of primary GCTSC cell lines with miR-127-3p and miR-376a-3p, respectively. Genome wide expression analysis of miR-127-3p and miR-376a-3p transfected cells revealed four novel target genes for each microRNA. Luciferase reporter assays demonstrated direct interactions of miR-127-3p with COA1 and direct interaction of miR-376a-3p with GLE1 and PDIA6, suggesting a pivotal role of these genes in the molecular etiology of GTCB. Interestingly, both microRNAs are located within a chromosomal region frequently silenced in GCTB and many other types of cancers, indicating that these microRNAs and their target genes are valuable therapeutic targets for the treatment of GCTB and possibly other tumor entities.

Curcumin inhibits cell proliferation and promotes apoptosis in human osteoclastoma cell through MMP-9, NF-κB and JNK signaling pathways.

Curcumin is a polyphenol compound extracted from ginger plant, turmeric, commonly used in a variety of food coloring and flavoring additives. Curcumin has many effects such as anti-inflammatory, anti-tumor, antioxidant and anti-microbial effects. However, the mechanism underlying the anti-cancer effect of curcumin on human osteoclastoma (Giant cell tumor, GCT) cells remains unclear. The objectives of this study were to determine the efficacy of curcumin on proliferation and apoptosis of GCT cells and its related mechanisms. In our study, cell viability, cellular apoptosis and caspase-3 activity of GCT cells were analyzed using 3.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry (FCM) assay and commercial kits, respectively. Next, MMP-9 gene expression quantity, NF-κB activity and JNK protein expression of GCT cells were tested with real-time polymerase chain reaction (RT-PCR), commercial kits and western blotting assay, respectively. Firstly, MMP-9, NF-κB and JNK inhibitors were added into GCT cells and which was researched the mechanism of curcumin on human GCT cells. In this study, the efficacy of curcumin reduced cell viability, induced cellular apoptosis and increased caspase-3 activity of GCT cells. Furthermore, curcumin inhibited the MMP-9 gene expression quantity and NF-κB activity, and activated JNK protein expression in GCT cells. Meanwhile, down-regulation of MMP-9 gene expression quantity and NF-κB activity could promote the anti-cancer effect of curcumin on cell viability of GCT cells. Interesting, down-regulation of JNK protein expression could also reversed the anti-cancer effect of curcumin on cell viability of GCT cells. Taken together, our results suggest that curcumin inhibits cell proliferation and promotes apoptosis in osteoclastoma cell through suppression of MMP-9 and NF-κB, and activation JNK signaling pathways.

Isocitrate dehydrogenase mutation is frequently observed in giant cell tumor of bone.

Giant cell tumors of bone (GCTB) are benign and locally destructive tumors that include osteoclast-type multinuclear giant cells. No available treatment is definitively effective in curing GCTB, especially in surgically unresectable cases. Isocitrate dehydrogenase (IDH) mutations have been reported not only in gliomas and acute myeloid leukemias, but also in cartilaginous tumors and osteosarcomas. However, IDH mutations in GCTB have not been investigated. The IDH mutations are remarkably specific to arginine 132 (R132) in IDH1 and arginine 172 (R172) or arginine 140 (R140) in IDH2; IDH1/2 mutations are known to convert α-ketoglutarate to oncometabolite R(-)-2-hydroxyglutarate. We recently reported that the most frequent IDH mutation in osteosarcomas is IDH2-R172S, which was detected by MsMab-1, a multispecific anti-IDH1/2 mAb. Herein, we newly report the IDH mutations in GCTB, which were stained by MsMab-1 in immunohistochemistry. DNA direct sequencing and subcloning identified IDH mutations of GCTB as IDH2-R172S (16 of 20; 80%). This is the first report to describe IDH mutations in GCTB, and MsMab-1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation-bearing GCTB.

Dedifferentiated chondrosarcoma mimicking a giant cell tumor. Is this low grade dedifferentiated chondrosarcoma?

We report a very rare case of a dedifferentiated chondrosarcoma mimicking a benign giant cell tumor. A 22-year-old male was admitted to our hospital with a history of mild left wrist pain after a skiing trauma. Radiology revealed an extensive meta-epiphyseal osteolytic lesion in the distal ulna, which appeared to be a giant cell tumor. Histological examination showed a biphasic tumor comprising chondroid and non-chondroid areas with a giant cell-rich lesion resembling a conventional giant cell tumor of the bone. Immunohistochemistry showed no expression of p16(INK4a), VEGFR1, KDR (VEGFR2), VEGFR3, cKIT, MDM2 or CDK4. However, high expression of the tyrosine kinases PDGFRA and PDGFRB was observed. Molecular analysis showed no amplification of the cMYC gene and no activating mutations in the cKIT (exons 9 and 11) or PDGFRA (exon 18) genes. He has been on follow-up for ten months, with no evidence of local recurrence or metastatic disease. In summary, this report highlights a very rare case of a dedifferentiated chondrosarcoma in which the dedifferentiated component of the tumor bears histologic resemblance to a conventional giant cell tumor of bone. We suggest that this tumor might be categorized in the group of low-grade dedifferentiated chondrosarcomas.

MiR-126-5p regulates osteoclast differentiation and bone resorption in giant cell tumor through inhibition of MMP-13.

Giant cell tumor (GCT) of bone is an aggressive skeletal tumor characterized by localized bone resorption. Matrix metalloproteinase-13 (MMP-13) is the principal proteinase expressed by the stromal cells of GCT (GCTSCs) and also considered to play a crucial role in formation of the osteolytic lesion in GCT. However, the exact mechanism of the regulation of MMP-13 expression in GCTSCs was unknown. In this study, we identified miR-126-5p was significantly downregulated in GCTSCs and affect osteoclast (OC) differentiation and bone resorption by repressing MMP-13 expression at the post-transcriptional level. Thus, our studies show that miR-126-5p plays an important physiological role in multinucleated giant cell formation and osteolytic lesion in GCT.

Protease expression in giant cell tumour of bone: a comparative study on feline and human samples.

Human giant cell tumour of bone (GCTB) is a rare low grade of malignancy tumour with tendency to recur. During tumourigenesis the bone remodeling balance is subverted by the tumour cellular components that interacting with bone matrix induce release of growth factors and cytokines, promoting cell proliferation and bone resorption. The master regulators of this positive feed-back are acid and neutral proteases that destroying extracellular matrix increase osteolysis. In contrast, in cats, very few data are reported on GCTB biological activity. In this study, histological features and metalloproteinase (MMPs) and urokinase plasminogen activator system (uPA) expression were compared in human and feline GCTB and differences in distribution and intensity related to histological pattern and clinical behaviour were determined. In both species, the overexpression of these molecules suggested a strong and complex cross-talk between tumour and microenvironment.

Evidence for the role of matrix metalloproteinase-13 in bone resorption by giant cell tumor of bone.

Giant cell tumor of bone (GCT) is an aggressively osteolytic primary bone tumor that is characterized by the presence of abundant multinucleated osteoclast-like giant cells, hematopoietic monocytes, and a distinct mesenchymal stromal cell component. Previous work in our laboratory has shown that matrix metalloproteinase (MMP)-13 is the principal proteinase expressed by the stromal cells of GCT. The release of cytokines, particularly interleukin-1beta, by the giant cells of GCT acts on stromal cells to stimulate a surge in MMP-13 secretion. The purpose of this study was to determine the bone resorption capabilities of the cellular elements of GCT and the significance of the MMP-13 expression involved in GCT bone resorption. We present a 3-dimensional histomorphometric technique developed to analyze resorption pit depth and yield an accurate measurement of bone resorption with a direct physical view of lacunae on bone slices. In this study, we demonstrate that the mesenchymal stromal cells and the multinucleated giant cells of GCT are independently capable of bone resorption. However, coculture of these 2 cell fractions shows a synergistic increase in bone resorption. In addition, inhibition of MMP-13 reduces resorptive activity of the cells indicating that MMP-13 likely plays an important role in this tumor. This cell-cell cooperation involves giant cell-derived cytokine up-regulation of MMP-13 in the stromal cells, which in turn assists the giant cells in bone resorption. Future research will involve elucidation of the role of cell-cell/matrix communication pathways in bone resorption and tumorigenesis in GCT.

Upregulation of MMP-13 via Runx2 in the stromal cell of Giant Cell Tumor of bone.

Giant Cell Tumor of bone (GCT) is an aggressively osteolytic and cytokine-rich bone tumor. Previous work in our lab has shown that matrix metalloproteinase-13 (MMP-13) is the principal proteinase expressed by the mesenchymal stromal cells of GCT. The Runx2 transcription factor is known to have a binding site in the MMP-13 promoter region, and we have previously found this transcription factor to be constitutively expressed in GCT stromal cells. The purpose of this study was to determine the role of Runx2 in MMP-13 regulation in GCT stromal cells. Following in vitro stimulation of GCT stromal cells with incremental concentrations of cytokine IL-1beta or TNF-alpha, the level of MMP-13 mRNA expression increased dramatically over 100-fold with a concomitant increase in MMP-13 protein expression. Inhibition of the ERK and JNK signaling pathways inhibited the upregulation of MMP-13 in these cells. Runx2 siRNA knockdown resulted in MMP-13 knockdown, and this effect was amplified following cytokine stimulation. Our study provides the first evidence that Runx2 may play a crucial role in cytokine-mediated MMP-13 expression in GCT stromal cells.

Distribution and prognostic significance of human telomerase reverse transcriptase (hTERT) expression in giant-cell tumor of bone.

Giant-cell tumor of bone is considered a benign, locally aggressive and rarely metastasizing neoplasm of bone. Specific microscopic or radiographic findings that reliably predict behavior have remained elusive. However, recent evidence suggests that activity of the telomerase enzyme complex correlates with recurrence in giant-cell tumor, although the subset of cells with telomerase activity in these heterogeneous tumors has not been defined. In the present study, we investigated whether immunostaining for human telomerase reverse transcriptase, a component of the telomerase complex, correlates with outcome in giant-cell tumor and the distribution of telomerase reverse transcriptase staining in these tumors. We analyzed 58 cases of giant-cell tumor for the presence and pattern of telomerase reverse transcriptase immunostaining, presence of soft tissue involvement and the type of initial surgery, and correlated these findings with recurrence-free survival and metastasis-free survival. Specific staining with telomerase reverse transcriptase was present in 20 out of 58 tumors (35%) in the nuclei of mononuclear cells and, occasionally, osteoclast-like giant cells. Furthermore, positive telomerase reverse transcriptase immunohistochemistry correlated with recurrence-free survival (P=0.02), whereas the presence of soft tissue extension (P=0.3) and the type of initial surgery (P=0.2) did not. Only soft-tissue extension significantly correlated with metastasis-free survival (P=0.003). Therefore, telomerase reverse transcriptase expression may predict recurrence in giant-cell tumor insofar as positive immunostaining correlates with shorter recurrence-free survival and may be a useful prognostic marker to stratify patients to more aggressive treatment protocols.

Serum total acid phosphatase for monitoring the clinical course of giant cell tumors of bone--26 patients with 5 local recurrences.

BACKGROUND: Giant cell tumor of bone (GCT) is a bone-destroying tumor that sometimes recurs locally after treatment. A recent study showed increased levels of serum total acid phosphatase (TACP). METHODS: We assessed TACP in the serum of 26 patients with primary GCT, and in 5 of them who developed a local recurrence. RESULTS: We found a correlation between TACP level in serum and tumor size. TACP levels that were elevated preoperatively in patients with GCT became normalized after surgery, but increased in 3 of the 5 patients with local recurrence. INTERPRETATION: TACP could be used as a tumor marker for monitoring response to treatment of GCT.

[Expression of a disintegrin-like and metalloproteinase protein 8 and 12 in the giant cell lesions of jaw].

OBJECTIVE: To detect the expression of a disintegrin-like and metalloproteinase (ADAM) 8 and 12 gene in the giant cell lesions of jaw and to study their effects on the histogenesis of cells in these lesions. METHODS: ADAM8 and ADAM12 was detected by immunohistochemistry (SP) in 40 paraffin-embedded specimens of central giant cell lesions of jaw, 10 peripheral giant cell lesions, 9 cherubisms, 6 aneurysmal bone cysts. RESULTS: ADAM8 and ADAM12 were positive in the cytomembrane and cytoplasm of all multinucleated giant cells and some round mononuclear cells of the lesions; ADAM12 was positive for some spindle mononuclear stromal cells in central and peripheral giant cell lesions. CONCLUSIONS: Multinucleated giant cells probably originated from the fusion of the round mononuclear cells, and ADAM8 and ADAM12 were involved in this process. In addition, ADAM12 might play a role in the maturation of spindle mononuclear stromal cells.

Cathepsin K is the principal protease in giant cell tumor of bone.

Giant cell tumor (GCT) of bone is a neoplasm of bone characterized by a localized osteolytic lesion. The nature of GCT is an enigma and the cell type(s) and protease(s) responsible for the extensive localized clinicoradiological osteolysis remain unresolved. We evaluated protease expression and cellular distribution of the proteolytic machinery responsible for the osteolysis. mRNA profiles showed that cathepsin K, cathepsin L, and matrix metalloproteinase (MMP)-9 were the preferentially expressed collagenases. Moderate expression was found for MMP-13, MMP-14, and cathepsin S. Specific protease activity assays revealed high cathepsin K activity but showed that MMP-9 was primarily present (98%) as inactive proenzyme. Activities of MMP-13 and MMP-14 were low. Immunohistochemistry revealed a clear spatial distribution: cathepsin K, its associated proton pump V-H(+)-ATPase, and MMP-9 were exclusively expressed in osteoclast-like giant cells, whereas cathepsin L expression was confined to mononuclear cells. To explore a possible role of cathepsin L in osteolysis, GCT-derived, cathepsin L-expressing, mononuclear cells were cultured on dentine disks. No evidence of osteolysis by these cells was found. These results implicate cathepsin K as the principal protease in GCT and suggest that osteoclast-like giant cells are responsible for the osteolysis. Inhibition of cathepsin K or its associated proton-pump may provide new therapeutic opportunities for GCT.

Plasminogen activators and their inhibitor in bone tumors and tumor-like damages.

Expression of urokinase- and tissue-type plasminogen activators and their inhibitor PAI-1 in the cytosolic fraction of 20 osteosarcomas, 20 chondrosarcomas, 13 giant-cell bone tumors, 5 Ewing's sarcomas, and 7 osteochondral exostoses was studied by enzyme immunoassay. The content of urokinase-type plasminogen activator increased, while the concentration of tissue-type plasminogen activator decreased in bone tumors of various histological compositions compared to osteochondral exostoses. A positive correlation was found between PAI-1 content and the volume of osteo- and chondrosarcomas. Expression of urokinase-type plasminogen activator increased in patients with primary osteosarcomas characterized by early generalization of the pathological process.

[Expression of ADAM12 (Meltrin-alpha) gene in giant cell tumor of bone].

OBJECTIVE: To detect expression and location of ADAM12 (meltrin-alpha) gene in giant cell tumor of bone and to study its effect on the formation of multinucleated giant cells in the tumor. METHODS: ADAM12 mRNA was detected by RT-PCR and RNA in situ hybridization in 18 cases of giant cell tumors, 6 cases of cultured tumor cells, 2 specimens of embryonic muscle tissue and 5 specimens of adult muscle tissue. RESULTS: RT-PCR showed that 12/18 (67%) cases of giant cell tumor of bone expressed ADAM12 mRNA. RNA in situ hybridization demonstrated positive ADAM12 mRNA in 12 cases of the detected tumors. The ADAM 12 mRNA positive signal was localized in the cytoplasm of almost all multinucleated giant cells and mononuclear stromal cells. However, the expression of ADAM12 mRNA decreased gradually, and eventually lost with increased passages of tumor cells and the disappearance of multinucleated giant cells in the culture. CONCLUSION: Multinucleated giant cells probably originated from the fusion of mononuclear stromal cells and ADAM12 may participate in the fusion process.

[The comparative biochemical and morphological characteristics of chondrosarcoma and giant-cell tumor of the bone]. / Sravnitel'naia biokhimicheskaia i morfologicheskaia kharakteristika khondrosarkomy i gigantokletochnoi opukholi kosti.

Spectrometry has been employed to assess the levels of collagenase, catepsin D, trypsin-like proteinases and their inhibitors as well as bone acid and alkaline phosphatase both in the center and along the periphery of giant cell tumor of bone (GCTB) and chondrosarcoma. The levels of collagenase, trypsin-like proteinases and their inhibitors in the center of chondrosarcoma were much higher while those of alkaline phosphatase--lower than along tumor periphery. The catepsin D and acid phosphatase concentrations of the center and periphery of chondrosarcoma were similar. It was suggested that an extremely low concentration of trypsin-like inhibitors may contribute to degradation of the matrix in tissues adjacent to chondrosarcoma and, consequently, to tumor invasion development.

[Indices of proteolysis in evaluation of resorption in bone tumors]. / Pokazateli proteoliza v otsenke rezorbtsii pri opukholiakh kosti.

Activities of acid and alkaline phosphatases, collagenase, cathepsin D, trypsin-like proteinases, alpha(1)-proteinase inhibitor (alpha(1)-PI), alpha(2)-macroglobulin (alpha(2)-MG) were measured in blood plasma and tumor tissue of patients with giant-cell tumor of the bone (GCTB) and bone chondrosarcoma. These tumors differed by enzymatic activities. GCTB is characterized by increased activity of alkaline phosphatase, while in chondrosarcoma tissue the activities of collagenase and cathepsin D were the highest. Activities of acid phosphatase, collagenase, trypsin-like proteinases were increased in the plasma of patients with both tumors; alpha(1)-PI/alpha(2)-MG ratio was increased. Bone resorption parameters correlated with proteolysis inhibitors. Activities of collagenase and acid phosphatase were increased in tumor tissue and plasma in the presence of low activities of alpha(2)-MG and increased alpha(1)-PI/alpha(2)-MG index, which seems to require special attention during the postoperative period.

Telomerase activity in benign bone tumors and tumor-like lesions.

To assess the role and status of telomerase activity in benign bone tumors and tumor-like lesions, we performed telomerase assays in four giant cell tumors of bone, four fibrous dysplasias, three osteochondromas, three aneurysmal bone cysts, two osteoblastomas, one juvenile bone cyst and one myositis ossificans. A very sensitive non-radioactive TRAP assay was applied. Low level activity was detected in 7 of 18 tumor samples (38.9%), and high level activity was not detected in any of the cases. Telomerase activity was observed in all patients with osteochondromas, in two of the three aneurysmal bone cysts, in one of the four giant cell tumors of bone and in one of the four fibrous dysplasias, but not in osteoblastomas, juvenile bone cyst and myositis ossificans. Although the origin of this enzyme is still unclear, it might play a role in precancerous immortalization of benign bone tumors. Other possible reasons explaining the occurrence of telomerase activity, such as migrating lymphocytes or contamination of immortalized non-tumor cells, should not be ruled out. Telomerase activity, however, does exist in those samples having no malignant phenotype, for which reason telomerase assays are not always useful for the clinical and diagnostic approach in benign bone tumors. Determination of the telomerase status in benign lesions may contribute to a better understanding of the regulation mechanism of telomerase activity during progression of bone tumors.

Transcriptional regulation of MMP-9 expression in stromal cells of human giant cell tumor of bone by tumor necrosis factor-alpha.

We determined whether certain factor(s) secreted by multinucleated giant cells, which is of monocyte/macrophage lineage in giant cell tumor of bone (GCT), regulate the induction of matrix metalloproteinase (MMP)-9 expression in mononucleated stromal cells. Our data derived using enzyme linked immunosorbant assays (ELISAs) suggest that the GCT cells in primary culture produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha). Further, the MMP-9 expression in GCT primary cultures was partially abrogated by neutralizing antibody to TNF-alpha, suggesting that TNF-alpha secretion by the multinucleated giant cells may be one of the factors responsible for the production of MMP-9 by the stromal cells in vivo. In order to confirm this we examined the role of TNF-alpha on the induction of MMP-9 expression in bone GCT stromal cells. These cells express MMP-2, but not MMP-9. However, treatment of these cells with TNF-alpha induced the expression of MMP-9 in a concentration-dependent manner. Kinetic experiments revealed that the secretion of MMP-9 peaked 12 h post TNF-alpha stimulation. Immunofluorescence studies confirmed the expression of MMP-9 after stimulation of GCT stromal cells with TNF-alpha. Further, TNF-alpha-induced MMP-9 expression was completely blocked with neutralizing antibody to TNF-alpha, thereby demonstrating the specificity. In addition, the induction of MMP-9 expression by TNF-alpha was completely abrogated in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that de novo protein synthesis may be required. Nuclear run-on analysis demonstrated that treatment of GCT stromal cells significantly enhanced the MMP-9 gene transcription. Together, our data suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. These studies also suggest the existence of an essential cell-cell interaction in the regulation of MMP-9 expression in GCT.

Regulation of MMP-9 (92 kDa type IV collagenase/gelatinase B) expression in stromal cells of human giant cell tumor of bone.

Matrix metalloproteinases (MMPs) play an important regulatory role in tissue morphogenesis, cell differentiation, tumor invasion and metastasis. Several authors have reported a direct correlation between the production of 72 kDa (MMP-2) and 92 kDa (MMP-9) type IV collagenases/gelatinases and the metastatic potential of cancer cells. Recently, we have identified the expression of both MMP-2 and MMP-9 in primary cultures of human giant cell tumor (GCT) of bone in vitro, and in tissue extracts in vivo. Interestingly, MMP-9 is not secreted by late-passaged GCT cells. It is possible that the production of MMP-9 is regulated by certain factor(s) secreted by the multinucleated giant cells in the primary culture. In order to test this hypothesis, the effect of primary-culture-conditioned medium on the expression of MMP-9 by late-passaged mononuclear stromal cells was examined. Adding conditioned medium from the primary GCT culture to the late-passaged stromal cells induced MMP-9, as evidenced by the presence of lytic bands at M(r) 92,000 and 72,000 on a gelatin zymogram. These enzyme activities were inhibited by EDTA, a well-known inhibitor of the MMPs. We confirmed these results by Western blotting using specific antibodies and RT-PCR for MMP-2 and MMP-9. Immunofluorescence studies with specific antibodies to MMP-9 further confirmed its expression by the passaged stromal cells cultured in the primary-culture-conditioned medium. The data indicate that MMP-2 and MMP-9 are produced by the mononuclear stromal cells when cultured in GCT primary-culture-conditioned medium. This suggests that multinucleated giant cells in primary cultures secrete a factor(s) that stimulates stromal cells to produce MMP-9, which, in turn, may contribute to the aggressive behavior of GCT.