GCT Cell Research Articles

MiR-217 regulates autophagy through OPG/RANKL/RANK in giant cell tumors

BackgroundIncreasing evidence suggests that microRNAs (miRNAs) play a crucial role in cancer development and progression. Our previous study showed remarkably lower levels of miR-217 in GCT cells and tissues, and miR-217 re-expression inhibited the occurrence and development of GCT in vitro; however, the associated mechanisms remain unknown. Thus, this study aimed to explore the mechanisms underlying the proliferation inhibitory effect of miR-217 in GCT cells.MethodsThe proliferative potential of the GCT cells was measured with an MTT assay and BrdU straining. Changes in GCT cell migration and invasion was assessed by a transwell assay. Finally, Western blot and RT-PCR assays were employed to evaluate OPG/RANKL/RANK signaling pathway-related protein expression.ResultsThe excessive upregulation of miR-217 markedly suppressed GCT cell proliferation and tumorigenesis both in vitro and in vivo. miR-217 overexpression could inhibit the OPG/RANKL/RANK signaling pathway in vitro and in vivo. Furthermore, ALP activity was significantly decreased in GCT cells following miR-217 treatment. Importantly, miR-217 could inhibit autophagy-related protein expression and autophagosome/autolysosome formation in GCT cells and tissues.ConclusionThese results suggest that miR-217 upregulation could inhibit the occurrence and development of GCT by blocking autophagy. These findings offer an effective therapeutic target to improve the survival rates of patients with CGT in the future.

Abstract 1617: TGFBR1 as a novel therapeutic target in adult granulosa cell tumors

Abstract Adult granulosa cell tumor (AGCT) is a subtype of sex-cord stromal tumors and accounts for ~5% of all ovarian neoplasms. Nearly 100% of AGCT cases are caused by an oncogenic point mutation in the Forkhead Box L2 (FOXL2) transcription factor. Weis-Banke et al. found this gain-of-function mutation (FOXL2C134W) allows FOXL2 to hijack the nuclear SMAD2/3/4 complex, the downstream effector of transforming growth factor- ß (TGF-ß), and redirect to novel transcription sites, inducing transcription of epithelial to mesenchymal transition (EMT) and other oncogenes. We hypothesized that FOXL2 mutant AGCT would be sensitive to TGF-ß inhibition. To test this hypothesis, we treated two GCT cell lines with TP-6379, an orally available, investigational small molecule kinase inhibitor of TGFBR1 that has been shown to block the phosphorylation and nuclear translocation of SMAD2 and SMAD3 in cells. TP-6379 was tested in the KGN cell line, derived from an AGCT patient and heterozygous for the FOXL2C134W mutation, and the COV434 cell line, derived from a juvenile GCT patient, which is FOXL2 wild type (WT). KGN cells (IC50 = 135 nM) were observed to be more than 70-fold more sensitive to TP-6379 treatment than COV434 (IC50 = >10,000 nM), after a 6- and 7- day treatment, respectively. KGN cells that were edited to remove the WT FOXL2 or both the WT and FOXL2C134W alleles were observed to be 1.6-fold more sensitive to TP-6379 and 22-fold less sensitive than the parental KGN cells. In vivo testing using KGN cells is ongoing. Viably cryopreserved dissociated tumor cells (DTCs) from two AGCT patients and one JGCT patient, which contain a mixture of tumor, immune, endothelial, and other stromal cells, were also tested in proliferation assays with TP-6379. All three ex vivo samples were positive for the FOXL2C134W mutation as detected by a qPCR genotyping assay and were sensitive to TP-6379 (IC50 = 555-1600 nM) after 6-day treatment. Xenograft models using these patient samples are currently under development. TGF-ß signaling is also a master regulator of the tumor microenvironment (TME) and immune evasion by modulating deposition of extracellular matrix and suppression of immune cells. We performed an immunophenotyping assay in tissue microarrays of thin-needle biopsy cores of multiple cancer types by looking at the distribution of CD8 T cells within tumor and stroma. AGCT showed the strongest excluded and desert phenotype among the tested cancer types, where CD8 T cells were confined to the stroma or absent entirely. TP-6379 treatment was observed to increase expression and reverse TGF-ß induced suppression of HLA class I in KGN cells. These data suggest that TGF-ß may play a significant role in the TME of AGCT. In conclusion, preclinical data shows inhibition of TGFß signaling with TP-6379 in FOXL2C134W mutant AGCT is active at blocking cell growth and may prove to be a potential therapy in this rare disease. Citation Format: Curtis A. Allred, Richard E. Heinz, Yuta Matsumura, Tetyana V. Forostyan, David Kircher, Salah Sommakia, Thomas Welte, Veena Vuttaradhi, Jason M. Foulks, Steven L. Warner, R Tyler Hillman. TGFBR1 as a novel therapeutic target in adult granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1617.

The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors

BackgroundGerm cell tumors (GCTs) are developmental cancers, tightly linked to embryogenesis and germ cell development. The recent and expanding field of RNA modifications is being increasingly implicated in such molecular events, as well as in tumor progression and resistance to therapy, but still rarely explored in GCTs. In this work, and as a follow-up of our recent study on this topic in TGCT tissue samples, we aim to investigate the role of N6-methyladenosine (m6A), the most abundant of such modifications in mRNA, in in vitro and in vivo models representative of such tumors.MethodsFour cell lines representative of GCTs (three testicular and one mediastinal), including an isogenic cisplatin resistant subline, were used. CRISPR/Cas9-mediated knockdown of VIRMA was established and the chorioallantoic membrane assay was used to study its phenotypic effect in vivo.ResultsWe demonstrated the differential expression of the various m6A writers, readers and erasers in GCT cell lines representative of the major classes of these tumors, seminomas and non-seminomas, and we evidenced changes occurring upon differentiation with all-trans retinoic acid treatment. We showed differential expression also among cells sensitive and resistant to cisplatin treatment, implicating these players in acquisition of cisplatin resistant phenotype. Knockdown of VIRMA led to disruption of the remaining methyltransferase complex and decrease in m6A abundance, as well as overall reduced tumor aggressiveness (with decreased cell viability, tumor cell proliferation, migration, and invasion) and increased sensitivity to cisplatin treatment, both in vitro and confirmed in vivo. Enhanced response to cisplatin after VIRMA knockdown was related to significant increase in DNA damage (with higher γH2AX and GADD45B levels) and downregulation of XLF and MRE11.ConclusionsVIRMA has an oncogenic role in GCTs confirming our previous tissue-based study and is further involved in response to cisplatin by interfering with DNA repair. These data contribute to our better understanding of the emergence of cisplatin resistance in GCTs and support recent attempts to therapeutically target elements of the m6A writer complex.

GCT-22. PROTEIN DEUBIQUITINATION PATHWAY IS A NOVEL THERAPEUTIC TARGET AGAINST MALIGNANT NON-GERMINOMATOUS CNS GERM CELL TUMORS

Central nervous system germ cell tumors (CNSGCTs) are rare intracranial neoplasm usually developed in adolescents and young adults. However, in East Asia including Japan, incidence of CNSGCTs is considerably higher compare with other regions of the world. Whereas germinomas generally respond to chemo-radiotherapy well, malignant subtypes of non-germinomatous germ cell tumors (NGGCT) are refractory, and development of novel therapy against NGGCTs is urgently needed. To develop a new therapeutic strategy against aggressive NGGCTs, we have investigated novel molecular targets for NGGCT treatment. We screened a total of 120 CNSGCT tumor tissues (including 55 NGGCT), which were registered to the Intracranial Germ Cell Tumor Consortium (iGCT), and discovered multiple mutations of a molecule that regulates protein ubiquitination and degradation specifically in NGGCT cases (5 of 55 cases; 1 immature teratoma, 3 mixed gem cell tumors, and 1 embryonal carcinoma). An in vitro ubiquitination assay revealed the mutations of this molecule discovered in NGGCT cases were loss of function mutations. Reduced expression of this molecule by knockdown in an established human seminoma cell line Tcam2 or a human yolk sac tumor cell line YST1, which was recently established in our institute, resulted in enhanced proliferation as well as upregulation of MEK-ERK activation. Importantly, treatment of these two GCT cell lines with reduced expression of this molecule by MEK inhibitor trametinib suppressed augmented proliferation of these cells. Taken together, these results suggest that protein ubiquitination-related pathways as well as MEK-ERK cascade may serve as a novel therapeutic target against NGGCTs.

CBMS-11 PROTEIN DEUBIQUITINATION PATHWAY IS A NOVEL THERAPEUTIC TARGET AGAINST MALIGNANT CNS NON-GERMINOMATOUS GERM CELL TUMORS

Central nervous system germ cell tumors (CNSGCTs) are rare intracranial neoplasm usually developed in adolescents and young adults. However, in East Asia including Japan, incidence of CNSGCTs is considerably higher compare with other regions of the world. Whereas germinomas generally respond to chemo-radiotherapy well, malignant subtypes of non-germinomatous germ cell tumors (NGGCT) are refractory, and development of novel therapy against NGGCTs is urgently needed. To develop a new therapeutic strategy against aggressive NGGCTs, we have investigated novel molecular targets for NGGCT treatment. We screened a total of 120 CNSGCT tumor tissues (including 55 NGGCT), which were registered to the Intracranial Germ Cell Tumor Consortium (iGCT), and discovered multiple mutations of a molecule that regulates protein ubiquitination and degradation specifically in NGGCT cases (5 of 55 cases; 1 immature teratoma, 3 mixed gem cell tumors, and 1 embryonal carcinoma). An in vitro ubiquitination assay revealed the mutations of this molecule discovered in NGGCT cases were loss of function mutations. Reduced expression of this molecule by knockdown in an established human seminoma cell line Tcam2 or a human yolk sac tumor cell line YST1, which was recently established in our institute, resulted in enhanced proliferation as well as upregulation of MEK-ERK activation. Importantly, treatment of these two GCT cell lines with reduced expression of this molecule by MEK inhibitor trametinib suppressed augmented proliferation of these cells. Taken together, these results suggest that protein ubiquitination-related pathways as well as MEK-ERK cascade may serve as a novel therapeutic target against NGGCTs.

Efficacy of HDAC Inhibitors Belinostat and Panobinostat against Cisplatin-Sensitive and Cisplatin-Resistant Testicular Germ Cell Tumors.

Novel treatment options are needed for testicular germ cell tumor (TGCT) patients, particularly important for those showing or developing cisplatin resistance, the major cause of cancer-related deaths. As TGCTs pathobiology is highly related to epigenetic (de)regulation, epidrugs are potentially effective therapies. Hence, we sought to explore, for the first time, the effect of the two most recently FDA-approved HDAC inhibitors (HDACis), belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. In silico results were validated in 261 patient samples and differential expression of HDACs was also observed across cell lines. Belinostat and panobinostat reduced cell viability in both cisplatin-sensitive cells (NCCIT-P, 2102Ep-P, and NT2-P) and, importantly, also in matched cisplatin-resistant subclones (NCCIT-R, 2102Ep-R, and NT2-R), with IC50s in the low nanomolar range for all cell lines. Treatment of NCCIT-R with both drugs increased acetylation, induced cell cycle arrest, reduced proliferation, decreased Ki67 index, and increased p21, while increasing cell death by apoptosis, with upregulation of cleaved caspase 3. These findings support the effectiveness of HDACis for treating TGCT patients in general, including those developing cisplatin resistance. Future studies should explore them as single or combination agents.

CDK4/6 inhibition presents as a therapeutic option for paediatric and adult germ cell tumours and induces cell cycle arrest and apoptosis via canonical and non-canonical mechanisms

BackgroundGerm cell tumours (GCTs) are the most common solid malignancies in young men. Although high cure rates can be achieved, metastases, resistance to cisplatin-based therapy and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In this study, we analysed the potential of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors palbociclib and ribociclib (PaRi) as molecular drugs to treat cisplatin-resistant and -sensitive paediatric and adult GCTs.MethodsTen GCT cell lines, including cisplatin-resistant subclones and non-malignant controls, were treated with PaRi and screened for changes in viability (triphenyl tetrazolium chloride (XTT) assay), apoptosis rates (flow cytometry, caspase assay), the cell cycle (flow cytometry), the transcriptome (RNA-sequencing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and on protein level (western blot). Expression profiling was performed on paediatric and adult GCT tissues (expression microarrays, qRT-PCR, immunohistochemistry, ‘The Cancer Genome Atlas’ database).ResultsWe demonstrate that adult GCTs highly express CDK4, while paediatric GCTs strongly express CDK6 instead. Thus, both GCT types are potentially treatable by PaRi. GCTs presented as highly sensitive towards PaRi, which caused a decrease in viability, cell cycle arrest and apoptosis. Although GCTs mainly arrested in the G1/G0 phase, some embryonal carcinoma cell lines were able to bypass the G1/S checkpoint and progressed to the G2/M phase. We found that upregulation of CDK3 and downregulation of many mitosis regulation factors, like the HAUS genes, might be responsible for bypassing the G1/S checkpoint and termination of mitosis, respectively. We postulate that GCT cells do not tolerate these alterations in the cell cycle and eventually induce apoptosis.ConclusionOur study highlights PaRi as therapeutic options for cisplatin-resistant and -sensitive paediatric and adult GCTs.

Increased levels of XPA might be the basis of cisplatin resistance in germ cell tumours

BackgroundGerm cell tumours (GCTs) represent a highly curable malignity as they respond well to cisplatin (CDDP)-based chemotherapy. Nevertheless, a small proportion of GCT patients relapse or do not respond to therapy. As this might be caused by an increased capacity to repair CDDP-induced DNA damage, identification of DNA repair biomarkers predicting inadequate or aberrant response to CDDP, and thus poor prognosis for GCT patients, poses a challenge. The objective of this study is to examine the expression levels of the key nucleotide excision repair (NER) factors, XPA, ERCC1 and XPF, in GCT patients and cell lines.MethodsTwo hundred seven GCT patients’ specimens with sufficient follow-up clinical-pathological data and pairwise combinations of CDDP-resistant and -sensitive GCT cell lines were included. Immunohistochemistry was used to detect the ERCC1, XPF and XPA protein expression levels in GCT patients’ specimen and Western blot and qRT-PCR examined the protein and mRNA expression levels in GCT cell lines.ResultsGCT patients with low XPA expression had significantly better overall survival than patients with high expression (hazard ratio = 0.38, 95% confidence interval: 0.12–1.23, p = 0.0228). In addition, XPA expression was increased in the non-seminomatous histological subtype, IGCCCG poor prognosis group, increasing S stage, as well as the presence of lung, liver and non-pulmonary visceral metastases. Importantly, a correlation between inadequate or aberrant CDDP response and XPA expression found in GCT patients was also seen in GCT cell lines.ConclusionsXPA expression is an additional independent prognostic biomarker for stratifying GCT patients, allowing for improvements in decision-making on treatment for those at high risk of refractoriness or relapse. In addition, it could represent a novel therapeutic target in GCTs.

SPDR-06 PROTEIN DEUBIQUITINATION PATHWAY IS A NOVEL THERAPEUTIC TARGET AGAINST MALIGNANT CNS NON-GERMINOMATOUS GERM CELL TUMORS

Central nervous system germ cell tumors (CNSGCTs) are rare intracranial neoplasm usually developed in adolescents and young adults. However, in East Asia including Japan, incidence of CNSGCTs is considerably higher compare with other regions of the world. Whereas germinomas generally respond to chemo-radiotherapy well, malignant subtypes of non-germinomatous germ cell tumors (NGGCT) are refractory, and development of novel therapy against NGGCTs is urgently needed. To develop a new therapeutic strategy against aggressive NGGCTs, we have investigated novel molecular targets for NGGCT treatment. We screened a total of 120 CNSGCT tumor tissues (including 55 NGGCT), which were registered to the Intracranial Germ Cell Tumor Consortium (iGCT), and discovered multiple mutations of a molecule that regulates protein ubiquitination and degradation specifically in NGGCT cases (5 of 55 cases; 1 immature teratoma, 3 mixed gem cell tumors, and 1 embryonal carcinoma). An in vitro ubiquitination assay revealed the mutations of this molecule discovered in NGGCT cases were loss of function mutations. Reduced expression of this molecule by knockdown in an established human seminoma cell line Tcam2 or a human yolk sac tumor cell line YST1, which was recently established in our institute, resulted in enhanced proliferation as well as upregulation of MEK-ERK activation. Importantly, treatment of these two GCT cell lines with reduced expression of this molecule by MEK inhibitor trametinib suppressed augmented proliferation of these cells. Taken together, these results suggest that protein ubiquitination-related pathways as well as MEK-ERK cascade may serve as a novel therapeutic target against NGGCTs.

XIST-Promoter Demethylation as Tissue Biomarker for Testicular Germ Cell Tumors and Spermatogenesis Quality.

Background: The event of X chromosome inactivation induced by XIST, which is physiologically observed in females, is retained in testicular germ cell tumors (TGCTs), as a result of a supernumerary X chromosome constitution. X chromosome inactivation also occurs in male germline, specifically during spermatogenesis. We aimed to analyze the promoter methylation status of XIST in a series of TGCT tissues, representative cell lines, and testicular parenchyma. Methods: Two independent cohorts were included, comprising a total of 413 TGCT samples, four (T)GCT cell lines, and 86 testicular parenchyma samples. The relative amount of methylated and demethylated XIST promoter fragments was assessed by quantitative methylation-specific PCR (qMSP) and more sensitive high-resolution melting (HRM) methylation analyses. Results: Seminomas showed a lower amount of methylated XIST fragments as compared to non-seminomas or normal testis (p < 0.0001), allowing for a good discrimination among these groups (area under the curve 0.83 and 0.81, respectively). Seminomas showed a significantly higher content of demethylated XIST as compared to non-seminomas. The percentage of demethylated XIST fragment in cell lines reflected their chromosomal constitution (number of extra X chromosomes). A novel and strong positive correlation between the Johnsen’s score and XIST demethylation was identified (r = 0.75, p < 0.0001). Conclusions: The X chromosome inactivation event and demethylated XIST promoter are promising biomarkers for TGCTs and for assessing spermatogenesis quality.

Inhibitor of apoptosis proteins are potential targets for treatment of granulosa cell tumors \u2013 implications from studies in KGN

BackgroundGranulosa cell tumors (GCTs) are derived from proliferating granulosa cells of the ovarian follicle. They are known for their late recurrence and most patients with an aggressive form die from their disease. There are no treatment options for this slowly proliferating tumor besides surgery and chemotherapy. In a number of tumors, analogs of the second mitochondria-derived activator of caspases (SMAC), alone or in combination with other molecules, such as TNFα, are evolving as new treatment options. SMAC mimetics block inhibitor of apoptosis proteins (IAPs), which bind caspases (e.g. XIAP), or activate the pro-survival NF-κB pathway (e.g. cIAP1/2). Expression of IAPs by GCTs is yet not fully elucidated but recently XIAP and its inhibition by SMAC mimetics in a combination therapy was described to induce apoptosis in a GCT cell line, KGN. We evaluated the expression of cIAP1 in GCTs and elucidated the effects of the SMAC mimetic BV-6 using KGN as a model.ResultsEmploying immunohistochemistry, we observed cIAP1 expression in a tissue microarray (TMA) of 42 GCT samples. RT-PCR confirmed expression of cIAP1/2, as well as XIAP, in primary, patient-derived GCTs and in KGN. We therefore tested the ability of the bivalent SMAC mimetic BV-6, which is known to inhibit cIAP1/2 and XIAP, to induce cell death in KGN. A dose response study indicated an EC50 ≈ 8 μM for both, early (< 8) and advanced (> 80) passages, which differ in growth rate and presumably aggressiveness. Quantitative RT-PCR showed upregulation of NF-κB regulated genes in BV-6 stimulated cells. Blocking experiments with the pan-caspase inhibitor Z-VAD-FMK indicated caspase-dependence. A concentration of 20 μM Z-VAD-FMK was sufficient to significantly reduce apoptosis. This cell death was further substantiated by results of Western Blot studies. Cleaved caspase 3 and cleaved PARP became evident in the BV-6 treated group.ConclusionsTaken together, the results show that BV-6 is able to induce apoptosis in KGN cells. This approach may therefore offer a promising therapeutic avenue to treat GCTs.

RUNX3 Promotes the Tumorigenic Phenotype in KGN, a Human Granulosa Cell Tumor-Derived Cell Line.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.

Retrospective analysis of 51 intralesionally treated cases with progressed giant cell tumor of the bone: local adjuvant use of hydrogen peroxide reduces the risk for tumor recurrence

BackgroundGiant cell tumor of the bone (GCT) has high local recurrence rates and the prognosis is hard to predict. We therefore retrospectively analyzed clinical outcome and recurrences of 51 GCT cases focusing on the effects of adjuvant local use of hydrogen peroxide.MethodsThe series enclosed 51 advanced GCT cases of the upper and lower extremities (n = 27 Campanacci grade III; n = 24 grade II; n = 39 surgery at our institution, n = 12 elsewhere). Mean follow-up was 88.3 (± 62.0) months. Surgical details, histology, metastases, recurrences, and interview-based data on satisfaction and function including the Musculoskeletal Tumor Society (MSTS) score were evaluated. It was investigated whether hydrogen peroxide was additionally used or not to clean the tumor cavity after curettage as we hypothesized influence on recurrences. To analyze the underlying mechanisms, GCT-derived stromal cell lines were cultured in vitro and tested for cell viability and apoptosis after treatment with hydrogen peroxide. Statistical analysis was performed with Student’s t tests, analysis of variance (ANOVA) with post hoc testing, Mann-Whitney U tests, chi-square tests, Kaplan-Meier analysis, and multivariate Cox regression analysis.ResultsThe whole series had 21 recurrences (41%). Eleven recurrences were found (28%) after surgery at our institution. Kaplan-Meier analysis of cumulative recurrence-free survival revealed at 2 years follow-up 69% (72%, only our institution) and at 10 years follow-up 54% (68%, only our institution). Intralesional resection was performed by vigorous curettage, burring, and defect filling with either polymethylmethacrylate bone cement (n = 45) or cancellous bone from the iliac crest (n = 6). Univariate chi-square analysis showed significantly lower recurrence rate after bone cement filling (2.3-fold, p = 0.024). Cleaning of the lesion cavity with hydrogen peroxide significantly reduced recurrence rate (whole collective 2.9-fold, p = 0.004; our institution 2.8-fold, p = 0.04) and significantly increased cumulative recurrence-free survival rate (whole collective at 10 years follow-up 74% versus 31%, p = 0.002; our institution 79% versus 48%, p = 0.02) compared to cases without hydrogen peroxide treatment. In multivariate analysis, significant risk factors for recurrence were pathological fracture (hazard ratio 3.7; p = 0.04), high mitosis rate (hazard ratio 15.6; p = 0.01), and lack of hydrogen peroxide use (hazard ratio 6.0; p = 0.02). In vitro cell culture analyses found apoptotic nature of hydrogen peroxide induced GCT cell death.ConclusionsThe present series proved for the first time that additional cleaning of the tumor cavity with hydrogen peroxide before defect filling significantly reduced recurrence rate and significantly increased recurrence-free survival in advanced but intralesionally treated GCT cases.

Abstract A39: Characterizing the epigenetic effects of the histone 3.3 G34W mutation in giant cell tumors of bone

Abstract Introduction: Pediatric glioblastomas (pGBM) are malignant brain tumors associated with a dismal prognosis. A subset of pGBMs carry mutations of either the Lysine 27 or Glycine 34 (G34) amino acid residues of histone 3 variant genes. The same G34 residue is also mutated in 85-95% of giant cell tumors of bone (GCTs), albeit to Tryptophan (G34W) in GCTs rather than to Arginine or Valine (G34R/V) as in pGBMs. The G34 mutation is predicted to impede access of histone methyltransferases like SetD2 to the nearby Lysine 36 residue, thereby altering the epigenome and transcriptome. Methods: To elucidate the tumorigenic effect of G34 mutations, we used the gene-editing technology CRISPR/Cas9 to correct the G34W mutation to wild-type in 2 GCT cell lines. We then investigated CRISPR-edited cell lines using functional assays, proteomic, epigenomic, and transcriptomic analyses. Results: Correction of the G34W mutation to wild-type in CRISPR-edited GCT cells results in phenotypic and functional changes suggestive of reduced tumorigenicity. By mass spectrometry, G34W-mutant GCT cell lines display decreased level of Lysine 36 trimethylation (H3K36me3) on the mutant G34-peptide, similar to G34-mutated pGBM cell lines. However, unlike pGBMs, GCTs display increased levels of Lysine 36 dimethylation (H3K36me2) on the mutant G34W-peptide. Ongoing Experiments and Analyses: We are currently comparing the level and distribution of multiple histone marks by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) of CRISPR-edited wild-type clones relative to the parent G34W-mutant GCT line. We are also assessing differential gene expression by RNA-Seq and characterizing the methylation signature by 850K DNA methylation array of G34W-mutant GCTs and edited clones. Conclusion: The G34W-mutation clearly has an impact on tumorigenic potential, as evidenced by in vitro functional assays. The G34W-mutant peptide of GCT cell lines features a distinct profile of post-translational histone modifications compared to G34R- or G34V-mutant peptides of pGBM cell lines. Further investigation could elucidate the epigenetic mechanism(s) through which the G34W mutation confers its tumorigenic properties. Citation Format: Shriya Deshmukh, Sima Khazaei, Dylan Marchione, Ashot Harutyunyan, Benjamin Garcia, Nada Jabado. Characterizing the epigenetic effects of the histone 3.3 G34W mutation in giant cell tumors of bone [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A39.

Endogenous c-Jun N-terminal kinase (JNK) activity marks the boundary between normal and malignant granulosa cells

Granulosa cell tumor of the ovary (GCT) is a very rare tumor, accounting for only 2% of all ovarian tumors. It originates from sex cords in the ovary and can be divided into adult (95%) and juvenile (5%) types based on histologic findings. To date, no clear etiologic process has been identified other than a missense point mutation in the FOXL2 gene. Our previous works showed that c-Jun N-terminal kinase (JNK) pathway plays critical role in cell cycle progression and mitosis of normal and immortalized granulosa cells and follicle growth in rodent ovaries. These findings led us to investigate the role of JNK pathway in the granulosa cell tumor of the ovary. We used two different GCT cell lines (COV434 and KGN) and fresh GCT samples of adult and juvenile types obtained from the patients during surgery. We have discovered that endogenous kinase activity of JNK is markedly enhanced in the GCT samples and cell lines, whereas it was almost undetectable in mitotic non-malignant human granulosa cells. The inhibition of JNK pathway in GCT cell lines with two different pharmacologic inhibitors (SP600125 and AS601245) or siRNA resulted in a dose-dependent reduction in in vitro cell growth, increased apoptosis and diminished estradiol and AMH productions. JNK inhibition was also associated with a decrease in the number of cells positive for mitosis marker phospho-histone H3Ser 10 in the asynchronous cells; and diminished EdU uptake during S phase and cell cycle arrest at G2/M-phase transition in the synchronized cells. Ex vivo treatment of patient-derived GCT samples with JNK inhibitors for 24 h significantly decreased their in vitro growth and estradiol and AMH productions. Furthermore, in human GCT xenograft model, in vivo tumor growth was significantly reduced and plasma AMH levels were significantly decreased in SCID mice after administration of JNK inhibitors and siRNA. These findings suggest that targeting JNK pathway may provide therapeutic benefit in the treatment of granulosa cell tumors for which currently no curative therapy exists beyond surgery.

TRAIL-R1 as a novel surface marker for circulating giant cell tumor of bone.

Giant cell tumor of bone (GCT), which frequently occurs in the patients’ spine, is relatively prevalent in Chinese population. A group of GCT invades into vessels and appears to be circulating tumor cells (CTCs) responsible for the distal metastasis of the primary tumor. So far the cell surface markers of GCT have not been determined. In the current study, we aimed to identify a novel CTC marker with higher specificity in GCT. TRAIL-R1+ cells were purified from GCT cell lines. The TRAIL-R1+ cells were compared with total GCT cells for tumor sphere formation, chemo-resistance, tumor formation in nude mice, and frequency of developing distal metastases. We found that TRAIL-R1+ GCT cells appeared to be highly enriched for CTCs in GCT. Compared to total GCT cells, TRAIL-R1+ GCT cells generated significantly more tumor spheres in culture, were higher chemo-resistant, and had a higher frequency of being detected in the circulation after subcutaneous transplantation as well as development of distal metastases. Thus, we conclude that TRAIL-R1+ may be a novel CTC marker in GCT. Selective elimination of TRAIL-R1+ GCT cells may improve the current GCT therapy.

Targeting the PI3K/PTEN/AKT/mTOR Pathway in Treatment of Sarcoma Cell Lines.

Sarcoma carries a poor prognosis prompting the need for targeted therapies aimed at deregulated signaling pathways. These include the PI3K/Akt/mTOR pathway commonly up-regulated in malignancies attributed to loss of PTEN expression. However, PTEN status and activation state of PI3K/Akt/mTOR pathway have not been comprehensively studied in sarcoma. The aims of this study were to characterise PTEN and Akt expression in a panel of sarcoma cell lines and then to examine mTOR inhibition using ridaforolimus. PTEN genomic expression was analyzed using Sanger sequencing. PTEN, total Akt (tAkt) and phosphorylated Akt (pAkt) expression were quantified with western blot analysis. Antiproliferative effects of treatment regimens were designed using Chou & Talalay's isobologram and determined with crystal violet assay. Four cell lines had wild-type PTEN (exons 2 to 8), with normal protein expression. The GCT cell line had a missense mutation in exon 6 (C>T), associated with loss of PTEN protein expression. Increased pAkt expression was found in all cell lines following epidermal growth factor (EGF) stimulation, indicating that wild-type PTEN expression in four cell lines did not inhibit constitutive activation of PI3K/Akt/mTOR pathway. Nonetheless, all cell lines demonstrated sensitivity to ridaforolimus within a clinically relevant dose-range (half-maximal inhibitory concentration (IC50)=0.7-10 nM). PTEN mutation is rare in sarcoma cell lines and constitutive activation of PI3K/Akt/mTOR is independent of PTEN status.

Differential expression of filamin B splice variants in giant cell tumor cells.

Giant cell tumor of bone (GCT) is the most commonly reported non-malignant bone tumor in Hong Kong. This kind of tumor usually affects people aged 20–40 years. Also, it is well known for recurrence locally, especially when the tumor cannot be removed completely. Filamins are actin-binding proteins which contain three family members, filamin A, B and C. They are the products of three different genes, FLNA, FLNB and FLNC, which can generate various transcript variants in different cell types. In this study, we focused on the effects of FLNBv2 and FLNBv4 toward GCT cells. The only difference between FLNBv2 and FLNBv4 is that FLNBv4 does not contain hinge 1 region. We found that the relative abundance of FLNBv4 varies among different GCT cell lines while the expression level of FLNBv4 in normal osteoblasts was only marginally detectable. In the functional aspect, overexpression of FLNBv4 led to upregulation of RANKL, OCN, OPG and RUNX2, which are closely related to GCT cell survival and differentiation. Moreover, FLNBv4 can have a negative effect on cell viability of GCT cells when compare with FLNBv2. In conclusion, splicing variants of FLNB are differentially expressed in GCT cells and may play a role in the proliferation and differentiation of tumor cells.

Carbon-Coated Gold Nanorods: A Facile Route to Biocompatible Materials for Photothermal Applications

Gold nanorods and their core-shell nanocomposites have been widely studied because of their well-defined anisotropy and unique optical properties and applications. This study demonstrates a facile hydrothermal synthesis strategy for generating carbon coating on gold nanorods (AuNRs@C) under mild conditions (<200 °C), where the carbon shell is composed of polymerized sugar molecules (glucose). The structure and composition of the produced core-shell nanocomposites were characterized using advanced microscopic and spectroscopic techniques. The functional properties, particularly the photothermal and biocompatibility properties of the produced AuNRs@C, were quantified to assess their potential in photothermal hyperthermia. These AuNRs@C were tested in vitro (under representative treatment conditions) using near-infrared (NIR) light irradiation. It was found that the AuNRs produced here exhibit exemplary heat generation capability. Temperature changes of 10.5, 9, and 8 °C for AuNRs@C were observed with carbon shell thicknesses of 10, 17, and 25 nm, respectively, at a concentration of 50 μM, after 600 s of irradiation with a laser power of 0.17 W/cm(2). In addition, the synthesized AuNRs@C also exhibit good biocompatibility toward two soft tissue sarcoma cell lines (HT1080, a fibrosarcoma; and GCT, a fibrous histiocytoma). The cell viability study shows that AuNRs@C (at a concentration of <0.1 mg/mL) core-shell particles induce significantly lower cytotoxicity on both HT1080 and GCT cell lines, as compared with cetyltrimethylammonium bromide (CTAB)-capped AuNRs. Furthermore, similar to PEG-modified AuNRs, they are also safe to both HT1080 and GCT cell lines. This biocompatibility results from a surface full of -OH or -COH groups, which are suitable for linking and are nontoxic Therefore, the AuNRs@C represent a viable alternative to PEG-coated AuNRs for facile synthesis and improved photothermal conversion. Overall, these findings open up a new class of carbon-coated nanostructures that are biocompatible and could potentially be employed in a wide range of biomedical applications.

The Role of TWIST in Angiogenesis and Cell Migration in Giant Cell Tumor of Bone

Giant cell tumor of bone (GCT) is a bone tumor consisting of numerous multinucleated osteoclastic giant cells involved in bone resorption and neoplastic osteoblast-like stromal cells responsible for tumor growth. The tumor occasionally metastasizes to the lung; however, factors leading to metastasis in this tumor are unknown. The TWIST-1 protein (also referred to as TWIST) has been suggested to be involved in epithelial-mesenchymal transition (EMT) and tumor progression in some cancers. In this study we investigated the functional role of TWIST in GCT cell angiogenesis and migration. Overexpression of TWIST in neoplastic GCT stromal cells significantly increased mRNA and protein expression of VEGF and VEGFR1 in vitro, whereas knockdown of TWIST resulted in decreased VEGF and VEGFR1 expression. A stable cell line with TWIST overexpression resulted in features of EMT including increased cell migration and downregulation of E-cadherin. The results of our study indicate that TWIST may play an important role in angiogenesis and cell migration in GCT.