Orthotopic Xenograft Mouse Model Research Articles

The CNS-penetrating taxane drug TPI 287 potentiates antiglioma activity of the AURKA inhibitor alisertib in vivo.

Glioblastoma (GBM) has a very poor prognosis despite current treatment. We previously found cytotoxic synergy between the AURKA inhibitor alisertib and the CNS-penetrating taxane TPI 287 against GBM tumor cells in vitro. We used an orthotopic human GBM xenograft mouse model to test if TPI 287 potentiates alisertib in vivo. Western blotting, immunohistochemistry, siRNA knockdown, annexin V binding, and 3-dimensional Matrigel invasion assays were used to investigate potential mechanisms of alisertib and TPI 287 treatment interactions. Alisertib + TPI 287 combination therapy significantly prolonged animal survival compared to vehicle (p = 0.011), but only marginally compared to alisertib alone. Alisertib, TPI 287, and combined alisertib + TPI 287 reduced animal tumor volume compared to vehicle-treated controls. This was statistically significant for the combination therapy at 4 weeks (p < 0.0001). Alisertib + TPI 287 treatment decreased anti-apoptotic Bcl-2 protein levels in vivo and in vitro. Expression of the pro-apoptotic protein Bak was significantly increased by combination treatment (p < 0.0001). Pro-apoptotic Bim and Bak knockdown by siRNA decreased apoptosis by alisertib + TPI 287 in GB9, GB30, and U87 cells (p = 0.0005 to 0.0381). Although alisertib and TPI 287 significantly reduced GBM cell invasion (p < 0.0001), their combination was no more effective than TPI 287 alone. Results suggest that apoptosis is the dominant mechanism of potentiation of GBM growth inhibition by alisertib + TPI 287, in part through effects on Bcl-2 family proteins, providing a rationale for further laboratory testing of an AURKA inhibitor plus TPI 287 as a potential therapy against GBM.

Tumor Growth Suppression of Pancreatic Cancer Orthotopic Xenograft Model by CEA-Targeting CAR-T Cells.

Chimeric antigen receptor engineered T cell (CAR-T) therapy has high therapeutic efficacy against blood cancers, but it has not shown satisfactory results in solid tumors. Therefore, we examined the therapeutic effect of CAR-T therapy targeting carcinoembryonic antigen (CEA) in pancreatic adenocarcinoma (PDAC). CEA expression levels on the cell membranes of various PDAC cell lines were evaluated using flow cytometry and the cells were divided into high, medium, and low expression groups. The relationship between CEA expression level and the antitumor effect of anti-CEA-CAR-T was evaluated using a functional assay for various PDAC cell lines; a significant correlation was observed between CEA expression level and the antitumor effect. We created orthotopic PDAC xenograft mouse models and injected with anti-CEA-CAR-T; only the cell line with high CEA expression exhibited a significant therapeutic effect. Thus, the therapeutic effect of CAR-T therapy was related to the target antigen expression level, and the further retrospective analysis of pathological findings from PDAC patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. Therefore, CEA expression levels in biopsies or surgical specimens can be clinically used as biomarkers to select PDAC patients for anti-CAR-T therapy.

Ferroportin depletes iron needed for cell cycle progression in head and neck squamous cell carcinoma.

Ferroportin (FPN), the only identified eukaryotic iron efflux channel, plays an important role in iron homeostasis and is downregulated in many cancers. To determine if iron related pathways are important for Head and Neck Squamous Cell Carcinoma (HNSCC) progression and proliferation, we utilize a model of FPN over-expression to simulate iron depletion and probe associated molecular pathways. The state of iron related proteins and ferroptosis sensitivity was assessed in a panel of metastatic HNSCC cell lines. Stable, inducible expression of FPN was confirmed in the metastatic HNSCC lines HN12 and JHU-022 as well as the non-transformed normal oral keratinocyte (NOK) cell line and the effect of FPN mediated iron depletion was assessed in these cell lines. HNSCC cells are sensitive to iron chelation and ferroptosis, but the non-transformed NOK cell line is not. We found that FPN expression inhibits HNSCC cell proliferation and colony formation but NOK cells are unaffected. Inhibition of cell proliferation is rescued by the addition of hepcidin. Decreases in proliferation are due to the disruption of iron homeostasis via loss of labile iron caused by elevated FPN levels. This in turn protects HNSCC cells from ferroptotic cell death. Expression of FPN induces DNA damage, activates p21, and reduces levels of cyclin proteins thereby inhibiting cell cycle progression of HNSCC cells, arresting cells in the S-phase. Induction of FPN severely inhibits Edu incorporation and increased β-galactosidase activity, indicating cells have entered senescence. Finally, in an oral orthotopic mouse xenograft model, FPN induction yields a significant decrease in tumor growth. Our results indicate that iron plays a role in HNSCC cell proliferation and growth and is important for cell cycle progression. Iron based interventional strategies such as ferroptosis or iron chelation may have potential therapeutic benefits in advanced HNSCC.

Total flavonoids of Litchi seed attenuate stem cell-like properties in breast cancer by regulating Notch3 signaling pathway

Ethnopharmacological relevanceBreast cancer has been the most commonly-diagnosed cancer worldwide, and the treatment and prognosis of which are often limited by breast cancer stem cells (BCSCs). Litchi seeds have shown good anti-cancer activity in various cancers including prostate cancer, lung cancer and breast cancer. However, the activity and underlying mechanism of Litchi seeds against BCSCs remain unknown. Aim of the studyTo investigate the activity and mechanism of total flavonoids of litchi seed (TFLS) against BCSCs in vitro and in vivo. Materials and methodsTwo orthotopic xenograft mouse models were established using HCC1806 cells pretreated or untreated with TFLS to determine whether TFLS could target BCSCs in vivo. Mammosphere formation and flow cytometry assays were employed to evaluate the effect of TFLS on BCSCs in vitro. The underlying mechanism was investigated using RT-qPCR, Western blot, immunohistochemistry and immunofluorescence experiments. ResultsTFLS could significantly inhibit the viability of HCC1806, MCF-7 and HCC1937 cells in vitro and suppress the growth of HCC1806 cells in vivo. TFLS attenuated stem cell-like properties of breast cancer through reducing the percentage of CD44+CD24-/low cells, inhibiting the mammospheres formation and down-regulating the mRNA and protein levels of cancer stem cells related markers (Oct4, Nanog, Sox2) in MCF-7 and HCC1806 cells. Meanwhile, TFLS suppressed the tumor-initiating ability of BCSCs via reducing the percentage of CD44+CD24-/low cells in tumor and lowering tumor incidence rate in orthotopic xenograft mice. In addition, TFLS treatments restricted the expression and nuclear translocation of Notch3, subsequently down-regulated Hes1 and Runx2 expressions. ConclusionsTFLS could suppress the growth of breast cancer and eliminate breast cancer stem cells by inhibiting the Notch3 signaling pathway.

Mesenchymal Stem-Like Cells Derived from the Ventricle More Effectively Enhance Invasiveness of Glioblastoma Than Those Derived from the Tumor.

Glioblastoma (GBM) is one of the most lethal human tumors with a highly infiltrative phenotype. Our previous studies showed that GBM originates in the subventricular zone, and that tumor-derived mesenchymal stem-like cells (tMSLCs) promote the invasiveness of GBM tumorspheres (TSs). Here, we extend these studies in terms of ventricles using several types of GBM patient-derived cells. The invasiveness of GBM TSs and ventricle spheres (VSs) were quantified via collagen-based 3D invasion assays. Gene expression profiles were obtained from microarray data. A mouse orthotopic xenograft model was used for in vivo experiments. After molecular and functional characterization of ventricle-derived mesenchymal stem-like cells (vMSLCs), we investigated the effects of these cells on the invasiveness of GBM TSs. We found that vMSLC-conditioned media (CM) significantly accelerated the invasiveness of GBM TSs and VSs, compared to the control and even tMSLC-CM. Transcriptome analyses revealed that vMSLC secreted significantly higher levels of several invasiveness-associated cytokines. Moreover, differentially expressed genes between vMSLCs and tMSLCs were enriched for migration, adhesion, and chemotaxis-related gene sets, providing a mechanistic basis for vMSLC-induced invasion of GBM TSs. In vivo experiments using a mouse orthotopic xenograft model confirmed vMSLC-induced increases in the invasiveness of GBM TSs. Although vMSLCs are non-tumorigenic, this study adds to our understanding of how GBM cells acquire infiltrative features by vMSLCs, which are present in the region where GBM genesis originates.

Targeting Autophagy With the Synergistic Combination of Chloroquine and Rapamycin as a Novel Effective Treatment for Well-differentiated Liposarcoma.

Liposarcoma is a type of soft-tissue sarcoma arising from fat tissue. It is relatively common among soft-tissue sarcomas. Chloroquine (CQ), an antimalarial drug, can inhibit autophagy and induce apoptosis in cancer cells. Rapamycin (RAPA) is an inhibitor of mTOR. The combination of RAPA and CQ is a strong inhibitor of autophagy. Previously, we showed that the combination of RAPA and CQ was effective against a de-differentiated liposarcoma patient-derived orthotopic xenograft (PDOX) mouse model. In the present study, we investigated the mechanism of efficacy of the combination of RAPA and CQ to target autophagy in a well-differentiated liposarcoma (WDLS) cell line in vitro. The human WDLS cell line 93T449 was used. The WST-8 assay was used to test the cytotoxicity of RAPA and CQ. Western blotting was used to detect microtubule-associated protein light chain 3-II (LC3-II) which is a component of autophagosomes. Immunostaining of LC3-II was also performed for autophagosome analysis. Τhe TUNEL assay was used to detect apoptotic cells, and apoptosis-positive cells were counted in three randomly selected microscopic fields for statistical validation. RAPA alone and CQ alone inhibited the viability of 93T449 cells. The combination of RAPA and CQ inhibited 93T449 cell viability significantly more than either drug alone and increased the number of autophagosomes which led to extensive apoptosis. The combination of RAPA and CQ increased the number of autophagosomes which led to apoptosis in 93T449 WDLS cells, suggesting novel effective treatment for this recalcitrant cancer by targeting autophagy.

BI-907828, a novel potent MDM2 inhibitor, inhibits glioblastoma brain tumor stem cells in vitro and prolongs survival in orthotopic xenograft mouse models.

The tumor suppressor TP53 (p53) is frequently mutated, and its downstream effectors inactivated in many cancers, including glioblastoma (GBM). In tumors with wild-type status, p53 function is frequently attenuated by alternate mechanisms including amplification and overexpression of its key negative regulator, MDM2. We investigated the efficacy of the MDM2 inhibitor, BI-907828, in GBM patient-derived brain tumor stem cells (BTSCs) with different amplification statuses of MDM2, in vitro and in orthotopic xenograft models. In vitro growth inhibition and on-target efficacy of BI-907828 were assessed by cell viability, co-immunoprecipitation assays, and western blotting. In vivo efficacy of BI-907828 treatments was assessed with qPCR, immunohistochemistry, and in intracranial xenograft models. BI-907828 decreases viability and induces cell death at picomolar concentrations in both MDM2 amplified and normal copy number TP53 wild-type BTSC lines. Restoration of p53 activity, including robust p21 expression and apoptosis induction, was observed in TP53 wild-type but not in TP53 mutant BTSCs. shRNA-mediated knock-down of TP53 in wild-type BTSCs abrogated the effect of BI-907828, confirming the specificity of the inhibitor. Pharmacokinetic-pharmacodynamic studies in orthotopic tumor-bearing severe combined immunodeficiency (SCID) mice demonstrated that a single 50mg/kg p.o. dose of BI-907828 resulted in strong activation of p53 target genes p21 and MIC1. Long-term weekly or bi-weekly treatment with BI-907828 in orthotopic BTSC xenograft models was well-tolerated and improved survival both as a single-agent and in combination with temozolomide, with dose-dependent efficacy observed in the MDM2 amplified model. BI-907828 provides a promising new therapeutic option for patients with TP53 wild-type primary brain tumors.

Hypoxia activated HGF expression in pancreatic stellate cells confers resistance of pancreatic cancer cells to EGFR inhibition.

Epidermal growth factor receptor (EGFR) is an essential target for cancer treatment. However, EGFR inhibitor erlotinib showed limited clinical benefit in pancreatic cancer therapy. Here, we showed the underlying mechanism of tumor microenvironment suppressing the sensitivity of EGFR inhibitor through the pancreatic stellate cell (PSC). The expression of alpha-smooth muscle actin (α-SMA) and hypoxia marker in human pancreatic cancer tissues were detected by immunohistochemistry, and their correlation with overall survival was evaluated. Human immortalized PSC was constructed and used to investigate the potential effect on pancreatic cancer cell lines in hypoxia and normoxia. Luciferase reporter assay and Chromatin immunoprecipitation were performed to explore the potential mechanisms invitro. The combined inhibition of EGFR and Met was evaluated in an orthotopic xenograft mouse model of pancreatic cancer. We found that high expression levels of α-SMA and hypoxia markers are associated with poor prognosis of pancreatic cancer patients. Mechanistically, we demonstrated that hypoxia induced the expression and secretion of HGF in PSC via transcription factor HIF-1α. PSC-derived HGF activates Met, the HGF receptor, suppressing the sensitivity of pancreatic cancer cells to EGFR inhibitor in a KRAS-independent manner by activating the PI3K-AKT pathway. Furthermore, we found that the combination of EGFR inhibitor and Met inhibitor significantly suppressed tumor growth in an orthotopic xenograft mouse model. Our study revealed a previously uncharacterized HIF1α-HGF-Met-PI3K-AKT signaling axis between PSC and cancer cells and indicated that EGFR inhibition plus Met inhibition might be a promising strategy for pancreatic cancer treatment. This study was supported by The National Natural Science Foundation of China.

Brusatol suppresses the tumor growth and metastasis of colorectal cancer via upregulating ARRDC4 expression through modulating PI3K/YAP1/TAZ Pathway

BackgroundColorectal cancer (CRC) is one of the most commonly diagnosed cancers with high metastasis and lethality. Arrestin domain-containing 4 (ARRDC4) is involved in inhibiting cancer glycolytic phenotypes. Brusatol (BR), extracted from Bruceae Fructus, exerts good anti-cancer effects against a number of cancers. PurposeIn the present study, we aimed to explore the efficacy of BR on inhibiting CRC metastasis and elucidate the underlying mechanisms involving the upregulation of the ARRDC4 expression. MethodsCell viability, colony formation, wound healing and transwell assay were used to detect the anti-proliferative and anti-metastatic effects of BR against CRC in vitro. Microarray analysis was performed to find out differential genes in CRC cells after treatment with BR. Analysis of the CRC patients tumor samples and GEPIA database were first conducted to identify the expression of ARRDC4 on CRC. Stable overexpression and knockdown of ARRDC4 CRC cells were established by lentiviral transfection. The role of ARRDC4 in mediating the anti-metastatic effects of BR on CRC was measured using qRT-PCR, western blotting, immunohistochemical and immunofluorescence analysis. Orthotopic xenograft and pulmonary metastasis mouse models of CRC were established to determine the anti-cancer and anti-metastatic effects of ARRDC4 and BR. ResultsBR markedly suppressed the cell proliferation, migration, invasion and inhibited tumor growth and tumor metastasis. Microarray analysis demonstrated that BR treatment markedly increased the gene expression of ARRDC4 in CRC cells. ARRDC4 was significantly repressed in CRC in the clinical samples and GEPIA analysis. ARRDC4 overexpression plus BR produced better inhibitory effects on CRC metastasis than BR treatment alone, while ARRDC4 knockdown could partially eliminate the inhibitory effects of BR against CRC metastasis. BR exerted anti-metastatic effects against CRC via upregulating ARRDC4 and inhibiting epithelial-mesenchymal transition (EMT) processing through modulating PI3K/Hippo pathway. ConclusionThis study reported for the first time that BR is a potent ARRDC4 agonist, and is worthy of further development into a new therapeutic strategy for CRC.

Guggulsterone inhibits migration and invasion through proteasomal and lysosomal degradation in human glioblastoma cells

Glioblastoma multiforme (GBM) is a deadly brain malignancy, and current therapies offer limited survival benefit. The phytosterol guggulsterone (GS) has been shown to exhibit antitumor efficacy. This study aimed to investigate the effects of GS on migration and invasion and its underlying mechanisms in human GBM cell lines. After GS treatment, the survival rate of GBM cells was reduced, and the migration and invasion abilities of GBM cells were significantly decreased. There was also concomitant decreased expression of focal adhesion complex, matrix metalloproteinase-2 (MMP2), MMP9 and cathepsin B. Furthermore, GS induced ERK phosphorylation and autophagy, with increased p62 and LC3B-II expression. Notably, treatment of in GBM cells with the proteasome inhibitor MG132 or the lysosome inhibitor NH4Cl reversed the GS-mediated inhibition of migration and invasion. In an orthotopic xenograft mouse model, immunohistochemical staining of brain tumor tissues demonstrated that MMP2 and cathepsin B expression was reduced in GS-treated mice. GS treatment inhibited GBM cell migration and invasion via proteasomal and lysosomal degradation, suggesting its therapeutic potential in clinical use in the future.

CSP I-plus modified rEndostatin inhibits hepatocellular carcinoma metastasis via down-regulation of VEGFA and integrinβ1

BackgroundIn our previous study, N end of the Circumsporozoite protein (CSP I-plus) modified recombinant human Endostatin (rEndostatin, endostar) (rES-CSP) was constructed, which had antiangiogenic capability and bound to hepatocellular carcinoma in vivo and in vitro. In this study, the inhibition of rES-CSP on hepatocellular carcinoma metastasis was verified in vivo and in vitro, and its possible mechanism was explored.MethodsFirstly, the impact of rES-CSP on the migration, adhesion of hepatoma cell HCCLM3 was identified by wound healing, transwell, and on metastasis of orthotopic xenograft model was identified in nude mouse. Then the expression of metastasis-associated molecules (MMP2, E-cadherin, integrinβ1) and angiogenesis-related factors (VEGFA) in vitro and in vivo were detected by real-time PCR, western blotting, immunohistochemistry.ResultsFinally, we found that rES-CSP could inhibit the migration and invasion of HCCLM3, and decrease tumor metastasis and growth in nude mouse orthotopic xenograft models. The tumor inhibiting rates of rES-CSP and Endostar were 42.46 ± 5.39% and 11.1 ± 1.88%. The lung metastasis rates of the control, Endostar and rES-CSP were 71, 50, and 42.8%, respectively. Compared with Endostar, rES-CSP significantly down-regulated the expression of VEGFA and integrinβ1. Heparin, a competitive inhibitor of CSP I-plus, which can be bind to the highly-sulfated heparan sulfate proteoglycans (HSPGs) over-expressed in liver and hepatocellular carcinoma, alleviated the down-regulation of VEGFA and integrinβ1.ConclusionsThese indicate that rES-CSP may play a role in inhibiting tumor growth and metastasis by down-regulating the angiogenic factor VEGF and the metastasis-related molecules or by interfering with HSPGs-mediated tumor metastasis.

Hypoxia Enhances Glioma Resistance to Sulfasalazine-Induced Ferroptosis by Upregulating SLC7A11 via PI3K/AKT/HIF-1α Axis.

Glioma is the most common primary brain tumor, with a high rate of recurrence and treatment resistance. Glioblastoma is highly invasive, infiltrating surrounding brain parenchyma, and is known to cause intracranial metastasis resulting in a dismal prognosis. Hypoxia contributes significantly to chemo- and radiotherapy resistance in cancer. Ferroptosis is a nonapoptotic oxidative cell death that has been identified as a potential anticancer mechanism. Sulfasalazine (SAS) activates ferroptosis and plays a potential role in tumor treatment. However, the relationship between hypoxia and SAS resistance has not been elucidated. This study is aimed at investigating the role of hypoxia in SAS-induced ferroptosis and the underlying mechanisms. Here, we found that hypoxia significantly suppressed SAS-induced ferroptosis by upregulating SLC7A11 expression in the U87 and U251 glioma cell lines. Hypoxia promotes SLC7A11 expression by enhancing the PI3K/AKT/HIF-1α pathway. The AKT inhibitor MK-2206 and HIF-1α inhibitor PX-478 significantly reversed this effect. In addition, under normoxia, PX-478 induced a higher lipid peroxidation level by decreasing SLC7A11 expression in the U87 and U251 cells but could not induce cell death directly; it could significantly enhance the tumor cell killing effect of SAS. In vivo, the combination of PX-478 and SAS had a coordinated synergistic effect on anticancer activity, as revealed by subcutaneous and orthotopic xenograft mouse models. In conclusion, hypoxia enhanced glioma resistance to SAS-induced ferroptosis by upregulating SLC7A11 via activating the PI3K/AKT/HIF-1α axis. Combination therapy with PX-478 and SAS may be a potential strategy against glioma.

EXTH-66. BIOENERGETIC SWITCH AND BIOLOGIC RESPONSE WITH SLC25A11 INHIBITION TARGETING MALATE-ASPARTATE SHUTTLE (MAS) ON GLIOBLASTOMA TUMORSPHERE

Abstract BACKGROUND: Cancer cells depended on cytosolic nicotinamide adenine dinucleotide (NADH) transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. KN612 (N-phenylmaleimide), a MAS inhibitor, is known to interfere with cancer growth by reducing ATP production, supported by several studies except on glioblastoma (GBM). Therefore, this study was designed to elucidate whether MAS could be an aimable target in GBM. METHODS: We compared expression levels of MAS conforming enzymes between normal and GBM samples. Gene expression profiles were analyzed using RNA-sequencing. Mitochondrial activity was measured by oxygen consumption (OCR), tetramethylrhodamine-ethylester (TMRE) staining, and liquid chromatograph-tandem mass spectrometer (LC-MS/MS). Also, biological functions were measured by cell viability, ATP levels, NADH levels, stemness, and invasiveness. In vivo efficacies were confirmed using a mouse orthotopic xenograft model. RESULTS: An analysis of the microarray database revealed that expression levels of several MAS enzymes including OGC (SLC25A11) were elevated in GBM. Through RNA sequencing, it was confirmed that KN612 accurately targeted SLC25A11 and decreased its expression. KN612-treated cells showed decreased viability, ATP production, and NADH levels compared with control cells. Under the same conditions, a significant decrease in stemness, invasion, and MMP was confirmed. In addition, KN612 confirmed remarkable therapeutic responses in a mouse orthotopic xenograft model. CONCLUSION: Our results show that KN612 effectively inhibits cancer cells both at the cellular level and at the in vivo level. This shows that targeting MAS could be a potential treatment option in addition to the currently limited standard GBM therapy.

EXTH-18. TARGETING THE ADENOSINERGIC IMMUNE SUPPRESSION PATHWAY IN HIGH GRADE GLIOMA SYNERGIZES WITH INNATE IMMUNE CHECKPOINT BLOCKADE

Abstract Immune response in the tumor microenvironment is modulated by the conversion of eATP to AMP to adenosine via the ecto-enzymes CD39 and CD73. Overexpression of CD73 in tumors leads to an increase in extracellular adenosine concentration and decrease in eATP, resulting in immune suppression. In this study, CD73 knockouts were created in adult and pediatric glioma cell lines (DIPG17, T3691, BT245) and used to quantify the effects of CD73 knockout on macrophage phagocytosis in the presence of extracellular AMP. In cell line DIPG17, CD73 knockout significantly increased phagocytosis with and without external AMP compared to the WT. In the WT condition, addition of AMP significantly reduced phagocytosis, while this decrease was not significant in the CD73-KO. The addition of anti-CD47 antibody significantly increased phagocytosis with external AMP in the WT condition. Similar results were obtained with cell line T3691. However, CD73 knockout combined with anti-CD47 treatment and external AMP showed significantly greater phagocytosis than the WT condition. This suggests that a combination of anti-CD73 and anti-CD47 treatment may be more effective in the tumor microenvironment. Lastly, in cell line BT245, CD73 knockout significantly increased phagocytosis as seen in the other cell lines. However, no significant difference was observed between WT and CD73-KO with extracellular AMP or anti-CD47. In vivo studies were conducted in orthotopic xenograft mouse models with DIPG17 CD73 knockout cells. Combination treatment with anti-CD47 antibody significantly decreased tumor burden and prolonged survival in the CD73-KO tumors compared to anti-CD47 treated DIPG17-WT tumors. We conclude that the CD73 adenosinergic pathway and the CD47-SIRPα pathway may present a target for immunotherapy in pediatric and adult gliomas.

STEM-18. EPIGENETIC AND MOLECULAR COORDINATION BETWEEN HDAC2 AND SMAD3-SKI IS REQUIRED FOR GROWTH AND STEM CELL CHARACTERISTICS OF BRAIN TUMOUR STEM CELLS

Abstract Brain tumour stem cell population in glioblastoma (GBM) display key cancer stem cell characteristics of high self-renewal and drug resistance that are maintained by the coordinated functions of epigenetic and molecular regulators. Yet, specific epigenetic mechanisms that, in collaboration with relevant molecular pathways, help maintain a stem-like state in BTSCs remain poorly understood. Here, we identify HDAC2 as a foremost epigenetic regulator in BTSCs that specifically utilizes the transforming growth factor-β (TGF-β) pathway related proteins, SMAD3-SKI, for remodelling BTSC chromatin accessibility and transcriptional programs to facilitate their stemness and tumorigenic potentials. Our initial drug screening revealed that selective inhibition of HDAC1 and 2 with romidepsin was effective in targeting BTSC viability, cell proliferation and self-renewal in vitro. Using CRISPR-cas9 knockout and shRNA knockdown strategies, we further demonstrated that loss of HDAC2 disrupts an epigenetic and molecular coordination between HDAC2 and SMAD-SKI proteins, which negatively impacts BTSC survival, cell proliferation and self-renewal in vitro and improves median survival in orthotopic xenograft mouse models. Loss of HDAC2 showed reduction in the protein abundance of transcriptional regulator, SMAD3 and negative regulator protein, SKI. However, overexpression of SMAD3 in HDAC2 deficient BTSCs could partially rescues their cell functional deficits. These findings suggest that context-specific epigenetic regulations by HDAC2 and its interaction with the critical transcriptional regulators, SMAD3-SKI, maintains the stemness and growth characteristics of BTSCs. Further HDAC2 overexpression increases cell proliferation and self-renewal abilities in normal neural stem cells (NSCs). These findings thus support the role of HDAC2 as a key epigenetic determinant of stemness in normal NSCs and of cancer stem cell characteristics and tumorigenic potential in BTSCs. Collectively, our data raises the potential that disruption of the coordinated mechanisms regulated by HDAC2-SMAD3-SKI axis may be an effective therapeutic approach for targeting GBM BTSCs.

DDDR-35. TARGETING GBM INVASION BY INHIBITING KCNA1 WITH 4-AMINOPYRIDINE: AN FDA APPROVED DRUG THAT EASILY PASS THROUGH THE BBB

Abstract Diffuse invasion is a hall mark of glioblastoma (GB) and one of the primary causes of poor clinical outcomes in GBM patients. Tumor cells migrate deep into the normal brain tissues are frequently protected by the BBB, making them particularly difficult to treat. New therapeutic targets are needed. Our previous studies using spatially dissected and functionally validated matching pairs of invasive and tumor core GBM cells identified KCNA1 as a shared gene that is selectively over-expressed in the invasive GBM cells in 6 patient derived orthotopic xenograft (PDOX) mouse models of pediatric GBM (Huang YL et al, Adv Science 2021). A subsequent analysis of adult GBM RNAseq data from IVY Atlas revealed a significantly elevated expression of KCNA1 (4.9 fold) in the invasive edges of patient GBM tumors. It is also one of the 11 core molecules identified in GEO and TCGA databases through an integrated bioinformatic analysis (Yang J, Front Onc 2021). To determine the anti-invasive activities of targeting KCNA1, we treated three highly invasive adult GBM PDOX models with 4-aminopyridine (4-AP), an old lipid soluble drug that easily penetrate the BBB, at 5 mg/kg, i.p., 5 days/week for 8 weeks acing alone or in combination with fractionated radiation (at 2 Gy/day x days). As single agent, 4-AP significantly extended median animal survival times in 1/3 GBM models (69 to 77 days, P = 0.033). Combination with XRT did not significantly improve the animal survival times in the three models. Systematic analysis of GBM invasion in mouse brains of the 3 PDOX models before, during and after 4-AP treatment revealed remarkable inhibition of tumor invasion. Our data highlighted the role of KCNA1 in promoting GBM invasion and support the fine tuning of 4-AP dose, schedule, and length of treatment to serve as a novel component of anti-GBM invasion therapies.

NPRL2 down-regulation facilitates the growth of hepatocellular carcinoma via the mTOR pathway and autophagy suppression.

Hepatocellular carcinoma (HCC) is a highly invasive malignancy. Recently, GATOR1 (Gap Activity TOward Rags 1) complexes have been shown to play an important role in regulating tumor growth. NPRL2 is a critical component of the GATOR1 complex. Therefore, this study used NPRL2 knockdown to investigate how GATORC1 regulates the prognosis and development of HCC via the mammalian target of rapamycin (mTOR) and autophagy signaling pathways. We established HepG2 cells with NPRL2 knockdown using small interfering RNA (siRNA) and short hairpin RNA (shRNA) systems. The siRNA-mediated and shRNA-mediated NPRL2 down-regulation significantly reduced the expression of NPRL2 and two other GATPOR1 complex components, NPRL3 and DEPDC5, in HepG2 cells; furthermore, the efficient down-regulation of NPRL2 protein expression by both the shRNA and siRNA systems enhanced the proliferation, migration, and colony formation in vitro. Additionally, the NPRL2 down-regulation significantly increased HCC growth in the subcutaneous and orthotopic xenograft mouse models. The NPRL2 down-regulation increased the Rag GTPases and mTOR activation and inhibited autophagy in vitro and in vivo. Moreover, the NPRL2 level in the tumors was significantly associated with mortality, recurrence, the serum alpha fetoprotein level, the tumor size, the American Joint Committee on Cancer stage, and the Barcelona Clinic Liver Cancer stage. Low NPRL2, NPRL3, DEPDC5, and LC3, and high p62 and mTOR protein expression in the tumors was significantly associated with disease-free survival and overall survival in 300 patients with HCC after surgical resection. Conclusion: The efficient down-regulation of NPRL2 significantly increased HCC proliferation, migration, and colony formation in vitro, and increased HCC growth in vivo. Low NPRL2 protein expression in the tumors was closely correlated with poorer clinical outcomes in patients with HCC. These results provide a mechanistic understanding of HCC and aid the development of treatments for HCC.

NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

Overcoming therapeutic resistance in glioblastoma (GBM) is an essential strategy for improving cancer therapy. However, cancer cells possess various evasion mechanisms, such as metabolic reprogramming, which promote cell survival and limit therapy. The diverse metabolic fuel sources that are produced by autophagy provide tumors with metabolic plasticity and are known to induce drug or radioresistance in GBM. This study determined that autophagy, a common representative cell homeostasis mechanism, was upregulated upon treatment of GBM cells with ionizing radiation (IR). Nuclear receptor binding factor 2 (NRBF2)—a positive regulator of the autophagy initiation step—was found to be upregulated in a GBM orthotopic xenograft mouse model. Furthermore, ATP production and the oxygen consumption rate (OCR) increased upon activation of NRBF2-mediated autophagy. It was also discovered that changes in metabolic state were induced by alterations in metabolite levels caused by autophagy, thereby causing radioresistance. In addition, we found that lidoflazine—a vasodilator agent discovered through drug repositioning—significantly suppressed IR-induced migration, invasion, and proliferation by inhibiting NRBF2, resulting in a reduction in autophagic flux in both in vitro models and in vivo orthotopic xenograft mouse models. In summary, we propose that the upregulation of NRBF2 levels reprograms the metabolic state of GBM cells by activating autophagy, thus establishing NRBF2 as a potential therapeutic target for regulating radioresistance of GBM during radiotherapy.

Orthotopic murine xenograft model of uveal melanoma with spontaneous liver metastasis.

Uveal melanoma is the most common intraocular malignancy in adults. Despite the effective primary treatment, up to 50% of patients with uveal melanoma will develop metastatic lesions mainly in the liver, which are resistant to conventional chemotherapy and lead to patient's death. To date, no orthotopic murine models of uveal melanoma which can develop spontaneous metastasis are available for preclinical studies. Here, we describe a spontaneous metastatic model of uveal melanoma based on the orthotopic injection of human uveal melanoma cells into the suprachoroidal space of immunodeficient NSG mice. All mice injected with bioluminescent OMM2.5 ( n = 23) or MP41 ( n = 19) cells developed a primary tumor. After eye enucleation, additional bioluminescence signals were detected in the lungs and in the liver. At necropsy, histopathological studies confirmed the presence of lung metastases in 100% of the mice. Liver metastases were assessed in 87 and in 100% of the mice that received OMM2.5 or MP41 cells, respectively. All tumors and metastatic lesions expressed melanoma markers and the signaling molecules insulin-like growth factor type I receptor and myristoylated alanine-rich C-kinase substrate, commonly activated in uveal melanoma. The novelty of this orthotopic mouse xenograft model is the development of spontaneous metastases in the liver from the primary site, reproducing the organoespecificity of metastasis observed in uveal melanoma patients. The faster growth and the high metastatic incidence may be attributed at least in part, to the severe immunodeficiency of NSG mice. This model may be useful for preclinical testing of targeted therapies with potential uveal melanoma antimetastatic activity and to study the mechanisms involved in liver metastasis.

Oral-recombinant Methioninase in Combination With Rapamycin Eradicates Osteosarcoma of the Breast in a Patient-derived Orthotopic Xenograft Mouse Model

Primary osteosarcoma of the breast is a very rare malignancy that shares histological features with osteosarcoma. It is also highly sensitive to methionine restriction due to methionine addiction. We previously established a patient-derived orthotopic xenograft (PDOX) nude-mouse model derived from tumor tissue of a patient with primary mammary osteosarcoma. In the present study, we investigated the efficacy of oral-recombinant methioninase (o-rMETase), combined with rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on a mammary osteosarcoma PDOX nude-mouse model. The PDOX mouse model was established by surgically transplanting a specimen of primary osteosarcoma of the breast into the mammary gland of nude mice. Mice implanted with tumors were randomly divided into four groups: Control group, N=5; rapamycin-treated group, N=5; o-rMETase-treated group, N=5; and a group treated with the combination of o-rMETase and rapamycin, N=5. Mice were treated for 2 weeks after transplantation, and tumor volume was measured during the treatment period. Treatment with the combination of rapamycin and o-rMETase eradicated the osteosarcoma of the breast compared to the untreated control (p=0.000008). o-rMETase alone did not significantly inhibit tumor growth, and rapamycin alone only partially inhibited the tumor (p=0.78 and p=0.018, respectively) compared to the untreated control. There was not a significant difference in mouse weight between the groups. The combination of rapamycin and o-rMETase was highly effective against primary osteosarcoma of the breast in a PDOX model, suggesting a future clinical strategy for this rare cancer type that currently has no first-line treatment.