Mouse Subcutaneous Tumor Model Research Articles

DDX56 transcriptionally activates MIST1 to facilitate tumorigenesis of HCC through PTEN-AKT signaling.

Rationale: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver that is the leading cause of cancer-related mortality worldwide. However, genetic alterations and mechanisms underlying HCC development remain unclear.Methods: Tissue specimens were used to evaluate the expression of DEAD-Box 56 (DDX56) to determine its prognostic value. Colony formation, CCK8, and EdU-labelling assays were performed to assess the effects of DDX56 on HCC proliferation. The in vivo role of DDX56 was evaluated using mouse orthotopic liver xenograft and subcutaneous xenograft tumor models. Dual-luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were performed to examine the effect of DDX56 on the MIST1 promoter.Results: DDX56 expression in HCC tissues was elevated and this increase was strongly correlated with poor prognoses for HCC patients. Functionally, DDX56 promoted HCC cell proliferation both in vitro and in vivo, while mechanistically interacting with MECOM to promote HCC proliferation by mono-methylating H3K9 (H3K9me1) on the MIST1 promoter, leading to enhanced MIST1 transcription and subsequent regulation of the PTEN/AKT signaling pathway, which promotes HCC proliferation. More importantly, the PTEN agonist, Oroxin B (OB), blocked the DDX56-mediated PTEN-AKT signaling pathway, suggesting that treating HCC patients with OB may be beneficial as a therapeutic intervention. Furthermore, we observed that ZEB1 bound to DDX56 and transcriptionally activated DDX56, leading to HCC tumorigenesis.Conclusions: Our results indicated that the ZEB1-DDX56-MIST1 axis played a vital role in sustaining the malignant progression of HCC and identified DDX56 as a potential therapeutic target in HCC tumorigenesis.

Linoleic Acid Upregulates Microrna-494 to Induce Quiescence in Colorectal Cancer.

Cancer dormancy is a state characterized by the quiescence of disseminated cancer cells, and tumor recurrence occurs when such cells re-proliferate after a long incubation period. These cancer cells tend to be treatment resistant and one of the barriers to successful therapeutic intervention. We have previously reported that long-term treatment of cancer cells with linoleic acid (LA) induces a dormancy-like phenotype. However, the mechanism underpinning this effect has not yet been clarified. Here, we investigate the mechanism of LA-induced quiescence in cancer cells. We first confirmed that long-term treatment of the mouse colorectal cancer cell line CT26 with LA induced quiescence. When these cells were inoculated subcutaneously into a syngeneic mouse and fed with an LA diet, the inoculated cancer cells maintained the quiescent state and exhibited markers of dormancy. LA-treated CT26 cells showed reduced oxidative phosphorylation, glycolysis, and energy production as well as reduced expression of the regulatory factors Pgc1α and MycC. MicroRNA expression profiling revealed that LA induced an upregulation in miR-494. The expression of Pgc1α and MycC were both induced by an miR-494 mimic, and the LA-induced decrease in gene expression was abrogated by an miR-494 inhibitor. The expression of miR-494 was enhanced by the mitochondrial oxidative stress produced by LA. In a syngeneic mouse subcutaneous tumor model, growth suppression by an LA diet and growth delay by LA pretreatment + LA diet were found to have similar effects as administration of an miR-494 mimic. In contrast, the effects of LA were abrogated by an miR-494 inhibitor. Analysis of human colorectal cancer tissue revealed that miR-494 was present at low levels in non-metastatic cases and cases with simultaneous liver metastases but was expressed at high levels in cases with delayed liver metastases, which also exhibited reduced expression of PGC1α and MYCC. These results suggest that miR-494 is involved in cancer dormancy induced by high levels of LA intake and that this microRNA may be valuable in targeting dormant cancer cells.

Two Laser Treatments Can Improve Tumor Ablation Efficiency of Chemophototherapy.

Chemophototherapy is an emerging tumor ablation modality that can improve local delivery of chemotherapeutic agents. Long circulating doxorubicin (Dox) in porphyrin-phospholipid (PoP) liposomes (LC-Dox-PoP) has previously been developed as an effective chemophototherapy agent. In the present study, we observed that in mice, LC-Dox-PoP showed enhanced accumulation in human pancreatic tumor xenografts even with suboptimal light doses, as assessed by fluorometric analysis of tissue homogenates and microscopic imaging of Dox and PoP in tumor slices. A second laser treatment, at a time point in which tumors had greater drug accumulation as a result of the first laser treatment, induced potent tumor ablation. Efficacy studies were carried out in two human pancreatic cancer subcutaneous mouse tumor models; MIA PaCa-2 or low-passage patient derived pancreatic cancer xenografts. A single treatment of 3 mg/kg LC-Dox-PoP and an initial 150 J/cm2 laser treatment 1 h after drug administration, followed by second laser treatment of 50 J/cm2 8 h after drug administration, was more effective than a single laser treatment of 200 J/cm2 at either of those time points. Thus, this study presents proof-of-principle and rationale for using two discrete laser treatments to enhance the efficacy of chemophototherapy.

IM-6 HVJ-E containing PD-L1 siRNA inhibits immunosuppressive activities and elicits antitumor immune responses in glioma

Inactivated Sendai virus particle, hemagglutinating virus of Japan-envelope (HVJ-E), is a non-replicating virus-derived vector, in which the genomic RNA of Sendai virus (HVJ) has been destroyed. HVJ-E is a promising vector that enables the highly efficient and safe introduction of enclosed molecules such as RNA into target cells. Moreover, HVJ-E provokes robust antitumoral immunity by activating natural killer (NK) cells and CD8+ T lymphocytes and their induction into the tumor periphery, and by suppressing regulatory T lymphocytes (Treg) locally in the tumor. In the present study, we investigated a novel combination of antitumor immunotherapy by the antitumor immune-activating effect of HVJ-E itself with the inhibition of tumor PD-L1 molecule expression. We confirmed that intratumoral injection of HVJ-E containing siRNA targeting PD-L1 (siPDL1/HVJ-E) inhibited tumor PD-L1 protein expression in a mouse subcutaneous tumor model using TS, a mouse glioma stem-like cell. We conducted treatment experiments in the mouse brain tumor model in three groups: control group (PBS), siNC/HVJ-E group (negative control siRNA + HVJ-E), and siPDL1/HVJ-E group. We obtained a significant prolongation of overall survival in the siPDL1/HVJ-E group. Flow cytometric analyses of brain tumor models showed that the proportions of brain-infiltrating CD8+ T lymphocytes and NK cells were significantly increased after giving siPDL1/HVJ-E; in contrast, the rate of Treg/CD4+ lymphocytes was significantly decreased in HVJ-E-treated tumors (siNC/HVJ-E and siPDL1/HVJ-E). No difference was observed in the proportions of macrophages or M2 macrophages. CD8 depletion abrogated the therapeutic effect of siPDL1/HVJ-E, indicating that CD8+ T lymphocytes mainly mediated this therapeutic effect. We believe that this non-replicating immunovirotherapy may be a novel therapeutic alternative to treat patients with glioblastoma. The full article has been published (Cancer Science. 2021 Jan;112(1):81–90).

An engineered oncolytic vaccinia virus encoding a single-chain variable fragment against TIGIT induces effective antitumor immunity and synergizes with PD-1 or LAG-3 blockade

BackgroundIn addition to directly lysing tumors, oncolytic viruses also induce antitumor immunity by recruiting and activating immune cells in the local tumor microenvironment. However, the activation of the immune cells...

REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is a leading cause of global cancer-related mortality and the most common form of liver cancer. REXO4 (RNA exonuclease 4 homolog) downregulation has previously been linked to enhanced chemosensitivity in breast cancer cells. The present study sought to comprehensively clarify the functional role of REXO4 in HCC. REXO4 expression levels in HCC tumor tissues and control tissue samples were established by analyzing data from the Gene Expression Omnibus (GEO) database. The expression of REXO4 was then knocked down in HCC cell lines to explore its functional role in these cells, while a gene set enrichment analysis (GSEA) approach was used to assess the functional regulator network associated with REXO4, and the Cell type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm was used to determine the relationship between this gene and immune cell infiltration of tumor tissues. The relationship between REXO4 and metabolic pathway was analyzed by oil red O staining. Cell Counting Kit-8 assays, colony formation, wound-healing assay, and a nude mouse subcutaneous tumor model were used to evaluate the function of REXO4 in HCC. REXO4 was highly upregulated in HCC tumors and cell lines, and was an effective predictor of HCC patient prognosis. The results indicated that the knockdown of REXO4 inhibited the proliferation and progression of HCC in vitro and in vivo. GSEA approaches also revealed REXO4 to be associated with tumor progression. Furthermore, REXO4 was associated with the degree of increase of intratumoral immune cell infiltration in HCC tissues and cells, and this gene was also linked with altered lipid metabolism in HCC cells. In summary, these analyses revealed REXO4 to be upregulated in HCC and to be associated with poor patient prognosis. In addition, this gene was closely linked to key cancer hallmark pathways and was revealed to play an important role in the susceptibility of liver tumors to immune cell infiltration and activation. Thus, targeting REXO4 may be a promising approach to treating patients with HCC in the near future.

Diosgenin inhibits the proliferation of gastric cancer cells via inducing mesoderm posterior 1 down-regulation-mediated alternative reading frame expression.

Whether and how mesoderm posterior 1 (MESP1) plays a role in the proliferation of gastric cancer cells remain unclear. The expression of MESP1 was compared in 48 human gastric cancer tissues and adjacent normal tissues. Knockdown of MESP1 was performed to investigate the role of MESP1 in the proliferation and apoptosis of BGC-823 and MGC-803 gastric cancer cells. Knockdown of alternative reading frame (ARF) was performed to study the role of ARF in the inhibitory effect of MESP1 knockdown on cell proliferation in gastric cancer cells. Mouse subcutaneous xenograft tumor model bearing BGC-823 cells was used to investigate the role of MESP1 in the growth of gastric tumor in vivo. The effect of seven active ingredients from T. terrestris on MESP1 expression was tested. The anti-cancer effect of diosgenin was confirmed in gastric cancer cells. MESP1 dependence of the anti-cancer effect of diosgenin was confirmed by MESP1 knockdown. MESP1 was highly expressed in human gastric cancer tissues (p < 0.05). MESP1 knockdown induced apoptosis and up-regulated the expression of ARF in gastric cancer cells (p < 0.05). Knockdown of ARF attenuated the anti-cancer effect of MESP1 knockdown (p < 0.05). In addition, MESP1 knockdown also suppressed tumor growth in vivo (p < 0.05). Diosgenin inhibits both mRNA and protein expression of MESP1 (p < 0.05). MESP1 knockdown attenuated the anti-cancer effect of diosgenin (p < 0.05). MESP1 promotes the proliferation of gastric cancer cells via inhibiting ARF expression. Diosgenin exerts anti-cancer effect through inhibiting MESP1 expression in gastric cancer cells.

Long non-coding RNA CTSLP8 mediates ovarian cancer progression and chemotherapy resistance by modulating cellular glycolysis and regulating c-Myc expression through PKM2

PurposeLong non-coding RNAs (lncRNAs) play vital roles in tumor progression and resistance. Ovarian cancer (OC), a common gynecological cancer, is associated with poor prognosis as it can progress to peritoneal metastasis and develop resistance to chemotherapy. This study aimed to examine the role of lncRNAs in the development of chemotherapy resistance in OC.MethodsThe clinical samples were divided into chemotherapy-sensitive and chemotherapy-resistant groups based on the chemotherapy response at follow-up. The glycolysis levels in the two groups were analyzed using positron emission tomography/computed tomography (PET/CT) scanning and immunohistochemistry. GEO dataset analysis revealed the expression of CTSLP8 in chemotherapy-resistant patients with OC. Two pairs of normal and diamminodichloroplatinum (DDP)-resistant cells were transfected with CTSLP8 overexpression and knockdown constructs to examine the functions of CTSLP8 in the OC cells and elucidate the underlying mechanisms. The in vivo effect of CTSLP8 overexpression and knockdown on the chemotherapy response of tumors was examined using a mouse subcutaneous tumor model. The tissue chips were subjected to fluorescence in situ hybridization and immunohistochemical (IHC) staining to examine the correlation among CTSLP8 expression, DDP resistance, and prognosis in OC.ResultsThe dataset analysis demonstrated that CTSLP8 was upregulated in chemotherapy-resistant tumor tissues. CTSLP8 promoted the proliferation and development of DDP resistance in the OC cells. Moreover, CTSLP8 promoted c-Myc expression by facilitating the binding of PKM2 to the promoter region of c-Myc, thereby upregulating glycolysis. The analysis of tissue chips revealed that the upregulation of CTSLP8 was associated with the development of DDP resistance and poor prognosis in patients with OC.ConclusionsThese findings indicate that CTSLP8 forms a complex with PKM2 to regulate c-Myc, and this action results in the upregulation of cellular glycolysis, consequently promoting OC progression and development of chemotherapy resistance.Headlights1. CTSLP8 was upregulated in the chemotherapy-resistant tumor tissues.2. CTSLP8 promoted the proliferation and cisplatin resistance in the OC cells.3. CTSLP8 promoted glycolysis by facilitating the binding of PKM2 to the promoter region of c-Myc.4. Inhibition of CTSLP8 or the combination of c-Myc inhibitors with cisplatin were potential therapeutic strategies for chemotherapy-resistant of OC.

Knockdown of PGM1 enhances anticancer effects of orlistat in gastric cancer under glucose deprivation

BackgroundPhosphoglucomutase 1 (PGM1) acts as an important regulator in glucose metabolism. However, the role of PGM1 in gastric cancer (GC) remains unclear. This study aims to investigate the role of PGM1 and develop novel regimens based on metabolic reprogramming in GC.MethodsCorrelation and enrichment analyses of PGM1 were conducted based on The Cancer Genome Atlas database. Data derived from the Kaplan–Meier Plotter database were analyzed to evaluate correlations between PGM1 expression and survival time of GC patients. Cell counting kit-8, 5-Ethynyl-2-deoxyuridine, flow cytometry assays, generation of subcutaneous tumor and lung metastasis mouse models were used to determine growth and metastasis in vitro and in vivo. Cell glycolysis was detected by a battery of glycolytic indicators, including lactate, pyruvic acid, ATP production and glucose uptake. Fatty Acid Synthase (FASN) activity and expression levels of lipid enzymes were determined to reflect on lipid metabolism.ResultsCorrelation and enrichment analyses suggested that PGM1 was closely associated with cell viability, proliferation and metabolism. PGM1 was overexpressed in GC tissues and cell lines. High PGM1 expression served as an indicator of shorter survival for specific subpopulation of GC patients. It was also correlated with pathological tumor stage and pathological tumor node metastasis stage of GC. Under the glucose deprivation condition, knockdown of PGM1 significantly suppressed cell viability, proliferation and glycolysis, whereas lipid metabolism was enhanced. Orlistat, as a drug that was designed to inhibit FASN activity, effectively induced apoptosis and suppressed lipid metabolism in GC. However, orlistat conversely increased glycolytic levels. Orlistat exhibited more significant inhibitive effects on GC progression after knockdown of PGM1 under glucose deprivation due to combination of glycolysis and lipid metabolism both in vitro and in vivo.ConclusionsDownregulation of PGM1 expression under glucose deprivation enhanced anti-cancer effects of orlistat. This combination application may serve as a novel strategy for GC treatment.

CircRNA_103948 inhibits autophagy in colorectal cancer in a ceRNA manner.

Circular RNA (circRNA) is implicated in many types of cancer; however, the expression and role of circRNAs in colorectal cancer (CRC) remains poorly understood. In this study, a circRNA microarray assay was performed to detect abnormally expressed circRNAs in CRC, and tissue arrays were used to determine the prognosis for CRC patients. Cell counting kit-8, clone formation, wound healing, and transwell assays were used to evaluate cell functions in vitro, and a mouse subcutaneous tumor model was designed for in vivo analysis. Autophagy was observed using confocal laser scanning and transmission electron microscopy. The expression of circRNA, miRNA, and mRNA was detected using qPCR; western blot, RNA pull-down assay, RNA immunoprecipitation, and dual luciferase assessment were applied for mechanistic studies. We found that circRNA_103948 expression is upregulated in CRC tissues, compared with adjacent normal tissues, and associated with poor prognosis. Knockdown of circRNA_103948 suppressed CRC both in vitro and in vivo. Mechanistically, circRNA_103948 could directly bind to miR-1236-3p and relieve suppression of the target TPT1. Furthermore, circRNA_103948 inhibited autophagy of CRC cells. Taken together, circRNA_103948 knockdown inhibited CRC cell growth by targeting miR-1236-3p/TPT1 axis-mediated autophagy. Thus, the circRNA_103948/miR-1236-3p/TPT1 axis affects CRC progression via modulation of autophagy.

Triptolide reverses epithelial-mesenchymal transition in glioma cells via inducing autophagy.

BackgroundTo observe the effects of triptolide (TP) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of glioma cells, and to explore the possible mechanisms of phenotypic changes in EMT.MethodsThe U87 and U251 glioma cell lines were treated TP. The Cell Counting Kit-8 (CCK-8) method was used to detect the half-maximal inhibitory concentration (IC50) of TP in these two cell lines and the inhibition of cell proliferation at the IC50 concentration. The wound-healing experiment and Transwell invasion assay were used to detect the cells’ migration and invasion abilities, respectively. Using western blot protocol, the expression levels of the EMT markers were analyzed, and the levels of the autophagy markers were also detected. The pEGFP-C2-LC3B plasmid was transfected into glioma cells, and the effect of TP on autophagy was detected by immunofluorescence. A subcutaneous tumor model in nude mice was established to observe the effect of TP on cell proliferation in vivo, and immunohistochemistry (IHC) was used to detect the expression levels of EMT markers in mouse tumor tissues.ResultsTP significantly inhibited the proliferation of U87 and U251 cells in a dose- and time-dependent manner. TP had a significant inhibitory effect on the migration and invasion of U87 and U251 cells. Western blot showed that TP reversed the process of EMT in glioma cells, which was evidenced by the upregulated expression of the epithelial marker E-cadherin, and the downregulated expression of the mesenchymal markers N-cadherin, Vimentin, ZEB1, Snail, and Slug. TP increased autophagy in glioma cells, increased the LC3B II/I ratio, and upregulated Beclin-1 and Atg-7 expression. Immunofluorescence showed that the number of autophagosomes increased significantly after TP was applied to cells. In the nude mouse subcutaneous tumor model, experiments revealed an inhibitory effect of TP on glioma cell proliferation in vivo. IHC confirmed that the expression of E-cadherin was upregulated in mouse tumor tissues, while the expression levels of N-Cadherin and Vimentin were downregulated.ConclusionsTP can inhibit glioma cell proliferation, migration, and invasion, and reverse EMT progression. The possible mechanism of EMT reversal in glioma cells is that TP induces autophagy.

Abstract LB239: CryoVizTM and photoacoustic imaging for assessment of tumor vasculature in mouse models of pancreatic tumors

Abstract Introduction: Assessing the tumor vasculature in models of pancreatic tumors can improve the characterization of these models, which can then support the eventual assessments of anti-cancer treatments. In this study, we sought to relate hypoxia, vascular perfusion, and vascular patency using synergistic imaging methods. In vivo Multispectral Optoacoustic Tomography (MSOT; also known as photoacoustic imaging) can measure %sO2 a relative measurement of oxy- vs. deoxy-hemoglobin in tumors. In vivo Dynamic Contrast Enhanced (DCE) MSOT can measure vascular perfusion. CryoVizTM imaging, a serial-sectioning-and-block-face-imaging technique, provides high-resolution 3D microscopic color anatomy and sensitive fluorescence making it ideal for assessing vascular perfusion by imaging ICG uptake and vascular patency by imaging Dylight488 lectin uptake. Methods: We performed MSOT and CryoVizTM imaging with subcutaneous tumor mouse models of SU.86.86 and MIA PaCa-2 pancreatic cancer. We performed MSOT with 21% O2 and 100% O2 carrier gas, which measures %sO2 (% oxyhemoglobin/total hemoglobin) as a biomarker of hypoxia. We performed DCE MSOT with 50 nmol indocyanine green (ICG), which measures Kapp (uptake rate from blood to tumor tissue) - a biomarker of vascular perfusion. To assess microvessel patency, we injected Dylight488 lectin via retro-orbital injection into the mice 3 min before euthanasia to enable CryoVizTM visualization of functional blood vessels. Subsequently, mice were euthanized, embedded in optimal cutting temperature (OCT) gel and flash-frozen in liquid nitrogen, and then imaged on the CryoVizTM. We acquired brightfield color anatomy, Dylight488 lectin (visible) fluorescence (ex: 460-490 nm, em&amp;gt;510nm) and ICG (NIR) fluorescence images (ex: 747/11 nm, em: &amp;gt;790nm) with an in-plane resolution of 10.232µm and a section thickness of 40 µm. Results: The in vivo MSOT analysis indicated that the MIA PaCa-2 model was more hypoxic than the SU.86.86 model, based on lower %sO2 measurements with both carrier gases. The in vivo DCE MSOT analysis indicated that the MIA PaCa-2 model had greater vascular perfusion than SU.86.86, based on a higher Kapp value. CryoVizTM images showed higher levels of lectin and ICG fluorescence in MIA PaCa-2 as compared to SU.86.86, indicating greater vascular patency and perfusion in the former. A strong lectin signal was observed in the injection site (retro-orbital sinuses), and in the renal medulla. ICG was also found to be retained in the axillary lymph node in both MIA PaCa-2 and SU.86.86, and in the kidney/adrenal glands of the MIA PaCa-2 mouse. Conclusion: Together, these results confirmed that the MIA PACa-2 model had higher hypoxia, vascular perfusion, and vascular patency, demonstrating the relationship between hypoxia and angiogenesis. This study reveals the power of 3D CryoVizTM imaging as a gold standard validation tool in cancer imaging. Citation Format: Madhusudhana Gargesha, Shreya Goel, Bryan Scott, Mark D. Pagel, Debashish Roy. CryoVizTM and photoacoustic imaging for assessment of tumor vasculature in mouse models of pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB239.

MascRNA and its parent lncRNA MALAT1 promote proliferation and metastasis of hepatocellular carcinoma cells by activating ERK/MAPK signaling pathway

MALAT1-associated small cytoplasmic RNA (mascRNA) is a cytoplasmic tRNA-like small RNA derived from nucleus-located long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). While MALAT1 was extensively studied and was found to function in multiple cellular processes, including tumorigenesis and tumor progression, the role of mascRNA was largely unknown. Here we show that mascRNA is upregulated in multiple cancer cell lines and hepatocellular carcinoma (HCC) clinical samples. Using HCC cells as model, we found that mascRNA and its parent lncRNA MALAT1 can both promote cell proliferation, migration, and invasion in vitro. Correspondingly, both of them can enhance the tumor growth in mice subcutaneous tumor model and can promote metastasis by tail intravenous injection of HCC cells. Furthermore, we revealed that mascRNA and MALAT1 can both activate ERK/MAPK signaling pathway, which regulates metastasis-related genes and may contribute to the aggressive phenotype of HCC cells. Our results indicate a coordination in function and mechanism of mascRNA and MALAT1 during development and progress of HCC, and provide a paradigm for deciphering tRNA-like structures and their parent transcripts in mammalian cells.

Mechanism of circADD2 as ceRNA in Childhood Acute Lymphoblastic Leukemia.

Background: Acute lymphocytic leukemia (ALL) is the most common malignant tumor in children. Increasing evidence suggests that circular RNAs (circRNAs) play critical regulatory roles in tumor biology. However, the expression patterns and roles of circRNAs in childhood acute lymphoblastic leukemia (ALL) remain largely unknown.Methods: circADD2 was selected by microarray assay and confirmed by qRT-PCR; in vitro effects of circADD2 were determined by CCK-8 and flow cytometry; while mice subcutaneous tumor model was designed for in vivo analysis. RNA immunoprecipitation and dual-luciferase assay were applied for mechanistic study. Protein levels were examined by Western blot assay.Results: circADD2 was down-regulated in ALL tissues and cell lines. Overexpression of circADD2 inhibited cell proliferation and promoted apoptosis both in vitro and in vivo. Briefly, circADD2 could directly sponge miR-149-5p, and the level of AKT2, a target gene of miR-149-5p, was downregulated by circADD2.Conclusion: circADD2, as a tumor suppressor in ALL, can sponge miR-149-5p, and may serve as a potential biomarker for the diagnosis or treatment of ALL.

Inhibition of c-MET reverses radiation-induced malignant potential in pancreatic cancer.

As a treatment option for PDAC, radiation therapy induces good local control. However, radiation also reportedly enhances the malignant potential (e.g., invasion and migration ability) in various cancers, thus increasing the risk of distant metastasis. It remains unclear how radiation induces malignant potential, and how such enhanced malignant potential can be suppressed. In the current study, we evaluated the sequential change of c-Met expression in pancreatic cancer cells following irradiation. We found that irradiation transiently induced c-Met expression in vitro. In an in vivo subcutaneous tumor mouse model, irradiation also enhanced downstream phosphorylated Met (p-Met). Furthermore, this enhancement of p-Met protein expression was suppressed by oral administration of the c-Met inhibitor INC280. Irradiated pancreatic cancer cells with enhanced c-Met expression exhibited higher malignant potential, including invasion and migration ability, compared with cells showing low c-Met expression. Pancreatic cancer cells that overexpressed c-met also showed enhanced malignant potential, which was reversed by c-Met inhibition. Additionally, c-Met inhibitor suppressed the metastatic potential in a liver metastasis mouse model using c-met-overexpressing cells. Overall, our present results revealed that irradiation could induce c-met expression in pancreatic cancer cells, leading to enhanced malignant potential (e.g., invasion and migration ability) and thus promoting distant metastasis. Moreover, a c-Met inhibitor could reverse this enhanced malignant potential.

The Effect of Sirtuin 2 (Sirt2) Overexpressing Bone Marrow Mesenchymal Stem Cells on the Growth of Human Epidermal Growth Factor Receptor 2 (Her-2) Breast Cancer Cells and Its Mechanism

Our study investigates the effect of high expression of Sirt2 in MSCs (MSCs-Sirt2) on Her-2 breast cancer cell proliferation. A mouse subcutaneous xenograft tumor model was established and MSCssirt2 analysis was performed on nude mice. TUNEL staining, flow cytometry, western-blot, real-time PCR and immunohistochemistry were used to detect cancer cell apoptosis. The number of NK cells infiltrated by flow cytometry detected the tumor tissue of tumor-bearing mice, and its killing activity on tumor-bearing mice was detected by isotope labeling and release method. The levels of TNF-α, IFN-γ, IL-8, IL-6 and IL-10 were detected by ELISA. Caspase-3 level was decreased in the MSCs group (P &lt;0.01) while increased in the MSCs-sirt2 group (P &lt;0.001). However, PCNA expression showed an opposite profile in the Her-2 group and MSCs-sirt2 group compared to Caspase-3 level (P &lt;0.01). The tumor volume and weight in the MSCs-sirt2 group was significantly reduced (P &lt; 0.01), while increased in the MSCs group significantly (P &lt; 0.05). The number of Ki-67-positive tumor cells in MSCs-sirt2 group was significantly reduced (P &lt;0.01) and increased in MSCs group (P &lt; 0.001) with oppositive number of TUNEL-positive tumor cells in the MSCs-sirt2 group and MSCs group (P &lt;0.01). IFN-γ level showed an upward trend (P &lt;0.001). The NK cell toxicity of MSCs-Sirt2 group was significantly higher (P &lt;0.001). MSCs-Sirt2 has an inhibitory effect on Her-2 breast cancer cell growth by enhancing the local inflammatory response of NK cells.

Baicalin triggers apoptosis, inhibits migration, and enhances anti-tumor immunity in colorectal cancer via TLR4/NF-κB signaling pathway.

Aberrant activation of the nuclear factor-kappa B (NF-κB) signaling pathway is closely implicated in colorectal cancer (CRC) growth, metastasis, and immune escape. In the present study, we reported natural derived compound of baicalin (BA), an efficient inhibitor of NF-κB, with good anti-tumor effect on CRC. CCK8 and colony formation assays showed that Baicalin significantly inhibit viability and proliferation in HCT-116 and CT26 cells. Additionally, Baicalin dramatically triggers mitochondria-mediated apoptosis in both HCT-116 and CT-26 cells, which is evidenced by loss of mitochondrial membrane potential and elevated cellular reactive oxygen species level. Treatment with Baicalin suppresses migration and invasion of CT26 cells by impairing TLR4/NF-κB signaling pathway. What's more, administration of Baicalin significantly retarded tumor growth rate in a subcutaneous xenograft tumor mouse model of CT26 cells. Treatment with Baicalin could ameliorate tumor immunosuppressive environment by downregulation of PD-L1 expression and proportion of myeloid-derived suppressor cells (MDSCs) and upregulation of percent of CD4+ and CD8+ T cells in CT26 tumors, thus improving anti-tumor immunity. In conclusion, our study demonstrated that baicalin triggers apoptosis, inhibits migration, and enhances anti-tumor immunity in colorectal cancer via TLR4/NF-κB signaling pathway, suggesting it might serve as a potential candidate drug for the treatment of CRC. PRACTICAL APPLICATIONS: In the present study, we reported natural derived compound of baicalin (BA), an efficient inhibitor of NF-κB, with good anti-tumor effect on CRC. We demonstrated that baicalin triggers mitochondria-mediated apoptosis, inhibits migration, and improves anti-tumor immunity in colorectal cancer via TLR4/NF-κB signaling pathway.

Gastric cancer-secreted exosomal X26nt increases angiogenesis and vascular permeability by targeting VE-cadherin.

Angiogenesis is closely associated with tumorigenesis, invasion, and metastasis by providing oxygen and nutrients. Recently, increasing evidence indicates that cancer‐derived exosomes which contain proteins, coding, and noncoding RNAs (ncRNAs) were shown to have proangiogenic function in cancer. A 26‐nt‐long ncRNA (X26nt) is generated in the process of inositol‐requiring enzyme 1 alpha (IRE1α)‐induced unspliced XBP1 splicing. However, the role of X26nt in the angiogenesis of gastric cancer (GC) remains largely unknown. In the present study, we found that X26nt was significantly elevated in GC and GC exosomes. Then, we verified that X26nt could be delivered into human umbilical vein endothelial cells (HUVECs) via GC cell exosomes and promote the proliferation, migration, and tube formation of HUVECs. We revealed that exosomal X26nt decreased vascular endothelial cadherin (VE‐cadherin) by directly combining the 3′UTR of VE‐cadherin mRNA in HUVECs, thereby increasing vascular permeability. We further demonstrated that X26nt accelerates the tumor growth and angiogenesis in a mouse subcutaneous tumor model. Our findings investigate a unique intercellular communication mediated by cancer‐derived exosomes and reveal a novel mechanism of exosomal X26nt in the regulation of tumor vasculature.

Predicting Schwannoma Growth in a Tumor Model Using Targeted Imaging.

Vestibular schwannoma (VS) is a common pathology encountered in neurotology clinics. Many patients are observed with a "wait and scan" approach. Previous efforts to determine radiographic indicators of future growth have been unsuccessful. Using a mouse subcutaneous tumor model, we seek to determine if fluorescent imaging with directed immunotargets could be used to predict schwannoma growth rate. Anti-VEGFR2 and anti-Her2/Neu monoclonal antibodies were covalently linked to a near-infrared probe (IRDye800). Immunodeficient mice underwent subcutaneous injections with a rat-derived schwann (R3) cell line. When tumor growth was evident, either Anti-VEGFR2-IRDye800, anti-Her2/Neu-IRDye800, or Immunoglobulin G (IgG) Isotype-IRDye800 (control) were injected via tail vein. The mice were serially imaged in a closed field near-IR device. Fluorescent data were analyzed for tumor signal and correlated with tumor sie and growth rate. Heterogeneity of fluorescent tumor signal was also assessed. In both anti-VEGFR2 and anti-Her2/Neu groups, there were strong correlations between day 1 mean tumor fluorescence and eventual maximum tumor volume (p = 0.002, 0.001; r2 = 0.92, 0.86). There was also strong correlation with maximum tumor signal on day 1 and maximum tumor volume (p = 0.003, 0.008; r2 = 0.90, 0.91). There was no such correlation in the control group (p = 0.99, 0.75; r2 = 0.0002, 0.028). Given the potential morbidity in VS intervention, observation is an appropriate approach for patients with slow-growing or stagnant tumors. We seek to identify immunotargets in a murine model that show promise in predicting schwannoma growth with advanced imaging techniques. Both Her2/Neu and VEGFR2 correlated strongly wth tumor size and growth rates and are promising targets that merit further investigation.

AMD1 upregulates hepatocellular carcinoma cells stemness by FTO mediated mRNA demethylation.

BackgroundS‐adenosylmethionine decarboxylase proenzyme (AMD1) is a key enzyme involved in the synthesis of spermine (SPM) and spermidine (SPD), which are associated with multifarious cellular processes. It is also found to be an oncogene in multiple cancers and a potential target for tumor therapy. Nevertheless, the role AMD1 plays in hepatocellular carcinoma (HCC) is still unknown.MethodsHCC samples were applied to detect AMD1 expression and evaluate its associations with clinicopathological features and prognosis. Subcutaneous and orthotopic tumor mouse models were constructed to analyze the proliferation and metastasis of HCC cells after AMD1 knockdown or overexpression. Drug sensitive and tumor sphere assay were performed to investigate the effect of AMD1 on HCC cells stemness. Real‐time quantitative PCR (qRT‐PCR), western blot, immunohistochemical (IHC) and m6A‐RNA immunoprecipitation (Me‐RIP) sequencing/qPCR were applied to explore the potential mechanisms of AMD1 in HCC. Furthermore, immunofluorescence, co‐IP (Co‐IP) assays, and mass spectrometric (MS) analyses were performed to verify the proteins interacting with AMD1.ResultsAMD1 was enriched in human HCC tissues and suggested a poor prognosis. High AMD1 level could promote SRY‐box transcription factor 2 (SOX2), Kruppel like factor 4 (KLF4), and NANOG expression of HCC cells through obesity–associated protein (FTO)‐mediated mRNA demethylation. Mechanistically, high AMD1 expression increased the levels of SPD in HCC cells, which could modify the scaffold protein, Ras GTPase‐activating‐like protein 1 (IQGAP1) and enhance the interaction between IQGAP1 and FTO. This interaction could enhance the phosphorylation and decrease the ubiquitination of FTO.ConclusionsAMD1 could stabilize the interaction of IQGAP1 with FTO, which then promotes FTO expression and increases HCC stemness. AMD1 shows prospects as a prognostic predictor and a therapeutic target for HCC.