Abstract
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.
Glioblastoma (GBM) is the most common, but extremely malignant, brain tumor; thus, the development of novel therapeutic strategies for GBMs is imperative. Many tyrosine kinase inhibitors (TKIs) have ...been approved for various cancers, yet none has demonstrated clinical benefit against GBM. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is confirmed only during the embryonic development period in humans. In addition, various ALK gene alterations are known to act as powerful oncogenes and therapeutic targets in various tumors. The antitumor activity of various TKIs was tested against three human GBM cell lines (U87MG, LN229, and GSC23), which expressed substantially low ALK levels; second‐generation ALK inhibitors, alectinib and ceritinib, effectively induced GBM cell death. In addition, treatment with either alectinib or ceritinib modulated the activation of various molecules downstream of RTK signaling and induced caspase‐dependent/‐independent cell death mainly by inhibiting signal transducer and activator of transcription 3 activation in human GBM cells. In addition, alectinib and ceritinib also showed antitumor activity against a U87MG cell line with acquired temozolomide resistance. Finally, oral administration of alectinib and ceritinib prolonged the survival of mice harboring intracerebral GBM xenografts compared with controls. These results suggested that treatment with the second‐generation ALK inhibitors, alectinib and ceritinib, might serve as a potent therapeutic strategy against GBM.
Anaplastic lymphoma kinase (ALK) inhibitors, alectinib and ceritinib, demonstrated antitumor activity for glioblastoma (GBM) cells which expressed substantially low ALK levels in vitro and in vivo. Treatment with either alectinib or ceritinib induced cell death mainly by inhibiting signal transducer and activator of transcription 3 activation in GBM cells. Alectinib and ceritinib might serve as potent therapeutic agents against GBM.
Glioblastomas (GBM) often acquire resistance against temozolomide (TMZ) after continuous treatment and recur as TMZ‐resistant GBM (TMZ‐R‐GBM). Lomustine (CCNU) and nimustine (ACNU), which were ...previously used as standard therapeutic agents against GBM before TMZ, have occasionally been used for the salvage therapy of TMZ‐R‐GBM; however, their efficacy has not yet been thoroughly examined. Therefore, we investigated the antitumor effects of CCNU and ACNU against TMZ‐R‐GBM. As a model of TMZ‐R‐GBM, TMZ resistant clones of human GBM cell lines (U87, U251MG, and U343MG) were established (TMZ‐R‐cells) by the culture of each GBM cells under continuous TMZ treatment, and the antitumor effects of TMZ, CCNU, or ACNU against these cells were analyzed in vitro and in vivo. As a result, although growth arrest and apoptosis were triggered in all TMZ‐R‐cells after the administration of each drug, the antitumor effects of TMZ against TMZ‐R‐cells were significantly reduced compared to those of parental cells, whereas CCNU and ACNU demonstrated efficient antitumor effects on TMZ‐R‐cells as well as parental cells. It was also demonstrated that TMZ resistance of TMZ‐R‐cells was regulated at the initiation of DNA damage response. Furthermore, survival in mice was significantly prolonged by systemic treatment with CCNU or ACNU but not TMZ after implantation of TMZ‐R‐cells. These findings suggest that CCNU or ACNU may serve as a therapeutic agent in salvage treatment against TMZ‐R‐GBM.
We investigated the antitumor effects of lomustine (CCNU) and nimustine (ACNU), which were previously used as standard therapeutic agents for glioblastomas (GBM), against the model cells of human GBM cases, which gained acquired temozolomide (TMZ) resistance after continuous treatment by TMZ (TMZ‐R‐cells). We discovered that the antitumor effects of TMZ against TMZ‐R‐cells were significantly reduced compared to those of parental cells, whereas CCNU and ACNU demonstrated efficient antitumor effects on TMZ‐R‐cells as well as parental cells both in vitro and in vivo. In addition, it was also demonstrated that TMZ resistance of TMZ‐R‐cells was regulated at the level of DNA damage response initiation. These findings suggest that CCNU or ACNU may serve as a therapeutic agent in salvage treatment against GBM cases with acquired TMZ resistance.
Pineal parenchymal tumors (PPTs) are clinically rare and a biopsy is often required for a definitive diagnosis. To improve the accuracy of histological assessment of PPTs, we examined the ...proliferative capacity of PPT cells and investigated DICER1 expression and
KBTBD4
mutations. This study included 19 cases of PPTs 3 pineocytomas (PCs), 10 PPTs of intermediate differentiation (PPTID), and 6 pineoblastomas (PBs). Immunohistochemistry for Ki-67, PHH3, and DICER1, as well as Sanger sequencing analysis for
KBTBD4
mutations, was performed using formalin-fixed paraffin-embedded tissue specimens that were resected during surgery. Tumor cell proliferation was quantified using an image analysis software. For the PHH3 and MIB-1 indices, a significant difference was observed between the PPTIDs and PBs (
P
< 0.05). Loss of DICER1 was not specific for PB; 0/3 PCs (0.0%), 2/9 PPTIDs (22.2%), and 2/4 PBs (50.0%).
KBTBD4
mutations were detected in 1/3 PCs (33.3%), 6/9 PPTIDs (66.7%), and 0/4 PBs (0.0%). Thus, combined application of the proliferative marker index and
KBTBD4
mutation analysis may be useful for the differential diagnosis of PPTs. Furthermore, detection of
KBTBD4
mutations using Sanger sequencing analysis may support the diagnosis of PPTID.
Two hot spot mutations (C228T, C250T) in the telomerase reverse transcriptase (
TERT
) gene are frequently identified in glioblastoma and oligodendroglioma.
TERT
mutations predicts an aggressive ...clinical course in isocitrate dehydrogenase (
IDH
) wild-type astrocytic tumors. Therefore, it is important to accurately detect
TERT
promoter mutations in glioma. Sanger DNA sequencing is the currently standard method for analyzing
TERT
mutations. However, PCR amplification in the first step of the sequencing has proven technically difficult because of the high GC content around the
TERT
mutation. In this report, we described a novel droplet digital PCR (ddPCR) assay to evaluate
TERT
hot spot mutations in fresh frozen and formalin-fixed paraffin-embedded (FFPE) specimens of glioma and verified the difference in results from the Sanger DNA sequencing results. We obtained the mutant allele fraction for
TERT
mutations of in a single ddPCR run in all cases, including the micro-dissected FFPE sections. On the contrary, up to twice the DNA sequences were required from fresh frozen tissue to obtain the results, consistent with ddPCR assay. When FFPE specimens were used, more time was required to evaluate
TERT
mutations through DNA sequencing. DdPCR is an effective and sensitive assay compared to the conventional standard Sanger DNA sequencing.
Abstract
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.
To manage refractory and invasive glioblastomas (GBM)s, photodynamic therapy (PDT) using talaporfin sodium (NPe6) (NPe6-PDT) was recently approved in clinical practice. However, the molecular ...machineries regulating resistance against NPe6-PDT in GBMs and mechanisms underlying the changes in GBM phenotypes following NPe6-PDT remain unknown. Herein, we established an in vitro NPe6-mediated PDT model using human GBM cell lines. NPe6-PDT induced GBM cell death in a NPe6 dose-dependent manner. However, this NPe6-PDT-induced GBM cell death was not completely blocked by the pan-caspase inhibitor, suggesting NPe6-PDT induces both caspase-dependent and -independent cell death. Moreover, treatment with poly (ADP-ribose) polymerase inhibitor blocked NPe6-PDT-triggered caspase-independent GBM cell death. Next, it was also revealed resistance to re-NPe6-PDT of GBM cells and GBM stem cells survived following NPe6-PDT (NPe6-PDT-R cells), as well as migration and invasion of NPe6-PDT-R cells were enhanced. Immunoblotting of NPe6-PDT-R cells to assess the behavior of the proteins that are known to be stress-induced revealed that only ERK1/2 activation exhibited the same trend as migration. Importantly, treatment with the MEK1/2 inhibitor trametinib reversed resistance against re-NPe6-PDT and suppressed the enhanced migration and invasion of NPe6-PDT-R cells. Overall, enhanced ERK1/2 activation is suggested as a key regulator of elevated malignant phenotypes of GBM cells surviving NPe6-PDT and is therefore considered as a potential therapeutic target against GBM.
Pediatric supratentorial ependymomas often have a clear cell morphology and reveal a RELA fusion. When a clear cell neoplasm is intraoperatively diagnosed, intracytoplasmic dot‐like inclusions by ...cytology are a useful cytopathological feature of ependymoma.
Pediatric supratentorial ependymomas often have a clear cell morphology and reveal a RELA fusion. When a clear cell neoplasm is intraoperatively diagnosed, intracytoplasmic dot‐like inclusions by cytology are a useful cytopathological feature of ependymoma.
Abstract INTRODUCTION pHGGs are the most common cause of cancer-related death in children, of which the most malignant and devastating tumors include diffuse midline glioma (DMG). Its anatomical ...location in the braistem, infiltrative nature, and blood-brain barrier (BBB) limit surgical resection and the distribution of systemically administered drugs. Intranasal delivery (IND) is a noninvasive method that bypasses the BBB, by leveraging the unique anatomic connections of the olfactory and trigeminal nerve pathways with the nasal cavity. We previously demonstrated the efficacy of IND of liposomal (LS) formulation of the active metabolite of the DNA topoisomerase I inhibitor, irinotecan, LS-SN-38, in orthotopic human brainstem xenograft models. The specificity of liposomes can be enhanced by using immunoliposome (iLS) formulation, which are coated with antibodies specific to the tumor cells leading to targeted drug delivery and reduced toxicity to normal cells. Platelet-derived growth factor receptor alpha (PDGFRA) plays a role in DMG oncogenesis and is expressed in as much as 70% of DMG by immunohistochemistry. In this study, we evaluated the specificity of anti-PDGFRA monoclonal antibody conjugated immunoliposome (PDGFRA-iLS) in pHGG cell lines. METHODS PDGFRA signals were assessed in pHGG cells using western blotting. Cells with high and low expression of PDGFRA were treated with rhodamine (Rho) labeled PDGFRA-iLS and imaged with fluorescent microscope. RESULTS DIPG007, SF7761 and KNS42 expressed high levels of PDGFRA, while SF8628 and DIPG36 cells showed low expression. Fluorescence microscopy revealed high PDGFRA-rhodamine (Rho)-iLS expression in DIPG007 cells (PDGFRA high), relative to SF8628 and DIPG36 cells (PDGFRA low). CONCLUSION PDGFRA-Rho-iLS shows increase cellular uptake in DIPG cells. Work is underway to determine the biodistribution, host toxicity, and anti-tumor activity of IND of PDGFRA-iLS-SN- 38 alone and in combination with radiation therapy in pHGG xenograft models.
Abstract BACKGROUND Diffuse midline glioma (DMG) is one of the most devastating childhood cancers that limited response to radiation therapy (RT). There is a critical need for new therapeutics that ...enhance the radiation effect. We, therefore, tested the hypothesis that targeting RAD52 activity sensitizes the radiation response of DMG. METHODS Genome-wide CRISPR/Cas9 screening was performed to identify the potential therapeutic target. RAD52 inhibition was used by shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03. The protein expression, cell proliferation, DNA damage marker expression, DNA repair pathway, and transcriptional alteration were analyzed by western blotting, MTS assay, colony formation assay, immunocytochemistry, DNA repair pathway assay, and RNA sequencing. Mice with DMG patient-derived xenograft (PDX) models were treated with RAD52 inhibitor alone or combination with RT. RESULTS Genome-wide CRISPR/Cas9 screening identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition suppressed DMG cell proliferation. Importantly, RAD52 inhibition in combination with RT further increased the radiosensitivity of DMG cells. Immunocytochemistry of DNA double-strand breaks (DSB) marker γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. Western blotting also revealed that RAD52 inhibition causes a sustained level of phosphorylated Rad50 and γH2AX in irradiated DIPG cells over 24 hours. DNA repair assay showed that RAD52 inhibition suppressed homologous recombination DNA repair pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Finally, the combination therapy of RAD52 inhibitor and RT further suppressed tumor growth and increased survival of mice bearing DMG PDXs, outperforming either monotherapy. CONCLUSIONS these results highlight RAD52 inhibition as a potential radiosensitization and provide a rationale for developing combination therapy with radiation in the treatment of DMG.
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