Liver Tumor Growth Research Articles

Abstract 5032: Acute phase liver graft injury mobilizes regulating B cells after transplantation for HCC patients through TLR4/CXCL10/CXCR3 signaling

Abstract We aim to investigate the impact of acute-phase small-for-size graft injury on mobilization of circulating regulating B cells (Bregs) in HCC patients after liver transplantation and to explore the molecular mechanism therein. Methods From May 2000 to November 2009, 115 HCC recipients were included. The intragraft gene expression profile and Bregs infiltration of the grafts greater (Group 1) and less than 60% (Group 2) of standard liver weight (SLW) were detected by RT-PCR and immunostaining. Circulating Bregs (CD19+CD24hiCD38hi) were compared by FACS analysis after transplantation. Clinical-pathological data including the incidence of tumor recurrence and metastasis were compared. The direct roles of TLR4, CXCL10 and CXCR3 on circulating Bregs mobilization were investigated in TLR4-/-, CXCL10-/- and CXCR3-/- mice models, respectively. The association of intragraft Bregs infiltration and tumor invasiveness were also examined in a rat liver transplantation for liver cancer model. The role of Bregs on liver tumor growth and invasiveness were further studied in a series of in vitro and in vivo functional experiments. Results The patients were grouped into Group 1 (> = 60% SLW, n = 37) and Group 2 (<60% SLW, n = 78). Much more patients in Group 2 developed tumor recurrence and lung metastasis [19/78(24.4%) vs 3/37(8%), p = 0.04]. Level of circulating Bregs was significantly higher in Group 2 (Week1: 7.02 vs 1.31/10^5PBMC, p = 0.03; month3: 5.7 vs 1.3/10^5PBMC, p = 0.03). There was more intragraft Bregs infiltration in group 2 indicated by CD20/IL10 staining. Intragraft gene expression of TLR4, CXCL10 and CXCR3 were significantly higher in Group 2 at early phase after transplantation. In rat liver transplantation model, more Bregs infiltration at early phase after transplantation correlated with late phase invasive tumor growth. Levels of circulating Bregs were significantly lower in the mice model with major hepatectomy and hepatic I/R injury using TLR4-/-, CXCL10-/- and CXCR3-/- mice, respectively. Bregs also promoted liver cancer cell proliferation and migration in vitro, and tumor growth in vivo. Conclusion A significantly higher population of circulating Bregs, which are mobilized by small-for-size graft injury, may lead to a higher incidence of tumor recurrence and metastasis after LDLT. TLR4/CXCL10/CXCR3 signaling may play important roles on Bregs mobilization. Citation Format: Kwan Man, Yan Shao, Chang Xian Li, Chung Mau Lo. Acute phase liver graft injury mobilizes regulating B cells after transplantation for HCC patients through TLR4/CXCL10/CXCR3 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5032. doi:10.1158/1538-7445.AM2015-5032

Abstract 461: Therapeutic effect of sorafenib is attenuated by tumor-infiltrating Ly6G+ myeloid-derived suppressor cells (MDSCs) and is restored by anti-interleukin-6 (IL-6) antibody treatment in an orthotopic mouse liver cancer model

Abstract Purpose: CD11b+Gr1+ MDSC, a subset of bone marrow cells with immune-suppressive and proangiogenic activities, has been shown to mediate resistance to antiangiogenic therapy in various cancer models. The significance of tumor-infiltrating MDSCs in mediating the resistance to sorafenib in hepatocellular carcinoma (HCC) has not been elucidated. Experimental designs: We adopted an orthotopic mouse liver cancer model by implanting BNL murine liver cancer cells in BALB/c mice, which were treated with sorafenib at the dose of 5 mg/Kg/d from day 8 after tumor cells implantation for 1 week or 3 weeks. Results: Both proportion and number of CD11b+Gr1+Ly6G+Ly6Clo MDSCs markedly increased in liver tumors, but not in spleens. This increment of tumor-infiltrating CD11b+Gr1+Ly6G+Ly6Clo MDSCs was found at one week of sorafenib treatment, when intra-tumor levels of multiple proangiogenic factors including VEGF, MCP-1 and IL-6 significantly increased, and the ratios of immune suppressors versus interferon-gamma expressing effectors tilted toward immune-suppression. CD11b+Gr1+Ly6G+Ly6Clo MDSCs isolated from liver tumors treated with sorafenib expressed higher levels of CD115, CD124, CD274 or CCR2 on cell surface and higher transcripts of pro-angiogenic factors, including VEGF, COX2, Bv8/Prok2, STAT3, S100A8, S100A9, or NOX2 than those from liver tumors treated with vehicle. Tumor-derived CD11b+Gr1+Ly6G+Ly6Clo MDSCs from sorafenib-treated mice were capable of suppressing specific tumor cell killing of CD8+ T cells and inducing the generation of immunosuppressive CD4+ T cells. These MDSCs also induced angiogenesis, as demonstrated in increased vascular density and hemoglobin. In sorafenib- treated mice, the concomitant use of anti-IL-6 antibody alleviated the early infiltration of CD11b+Gr1+Ly6G+Ly6Clo MDSCs in liver tumors, decreased the levels of anti-angiogenic factors, reduced vascular density, restored the proliferative activity of tumor-infiltrating T cells and suppressed the liver tumor growth synergistically. Conclusions: Our data indicate that intratumor infiltration of CD11b+Gr1+Ly6G+Ly6Clo MDSCs impaired the therapeutic efficacy of sorafenib through promoting immune-suppression and angiogenesis. Targeting this subset of MDSCs by anti-IL-6 antibody could improve the anti-HCC efficacy of sorafenib.(This work was supported by Grant. NSC 102-2314-B-002 -144 -) Citation Format: Chun-Jung Chang, Yao-Hsu Yang, Chiao-Juno Chiu, Bor-Luen Chiang, Xiao-yi Lee, Chih-Hung Hsu, Ann-Lii Cheng. Therapeutic effect of sorafenib is attenuated by tumor-infiltrating Ly6G+ myeloid-derived suppressor cells (MDSCs) and is restored by anti-interleukin-6 (IL-6) antibody treatment in an orthotopic mouse liver cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2015-461

Withaferin A Suppresses Liver Tumor Growth in a Nude Mouse Model by Downregulation of Cell Signaling Pathway Leading to Invasion and Angiogenesis

Purpose : To investigate the effect of withaferin A on tumor growth and metastasis in liver in a nude mouse model. Methods : Withaferin A was injected through a portal vein to the orthotopic liver tumor in a nude mice model. Xenogen in vivo imaging system was used to monitor tumor growth and metastasis. The effect of withaferin A on tumor volume, invasive growth pattern, expression of Pyk2, upregulation of BAX/P53, apoptotic signaling and ROCK/IP10/VEGF pathway along with cytoskeletal protein actin projection formation was studied. Tumor/non-tumor margin was examined under electron microscopy. In addition, the direct effect of withaferin A on liver cancer cells and endothelial cells was further investigated. Results : A significant inhibition of tumor growth and lower incidence of lung metastasis was observed after withaferin A treatment. Withaferin A treatment led to a decrease in the incidence of intrahepatic metastasis from 90 (9 of 10) to 10 % (1 of 10, p = 0.041). There was decrease in macrophage infiltration in the liver tumors and vessels. Western blot analysis revealed inhibition of expression of Pyk2, ROCK1 protein and VEGF. Electron microscopy showed tumor vascular endothelial cell damage and significant necrosis of tumor tissues. It also suppressed formation of cytoskeletal protein actin projection involved in cell migration. Conclusion : Withaferin A inhibits liver tumor invasion and angiogenesis by downregulation of cell signalling pathway leading to invasion and angiogenesis. Therefore, withaferin A is a promising candidate for the treatment of liver tumor invasion and angiogenesis. Keywords : Withaferin A, Macrophage, Lung metastasis, Angiogenesis, Vascular endothelial growth factor, Rho kinase, Withania somnifera

100. Multifunctional Drug Enhances rAAV-Mediated Transgene Expression Through Different Mechanisms in Different Cell Types

Several groups have demonstrated that a number of naturally occurring compounds can significantly enhance the therapeutic efficacy of viral vectors, especially recombinant AAV vectors (Ren et al., Neurosci Lett. 479(3): 187-91, 2010; Zhang et al., Gene Ther. 18(2): 128-34, 2011; Mitchell et al., J Virol. 87(8): 4571-83, 2013; Wang et al., J Integr Med. 12(1): 20-34, 2014). In our recently published studies (Ling et al., Hum Gene Ther. 25(12): 1023-34, 2014), we provided further evidence of enhanced efficacy of a combinatorial approach involving drug-mediated chemotherapy and rAAV vector-mediated cancer gene therapy to target human hepatocellular carcinoma. In these studies, we identified that shikonin, a naphthoquinone, inhibits human liver tumor growth as well as significantly enhances the efficacy of rAAV vectors carrying a therapeutic gene, tricosanthin (TCS), in a murine xenograft model in vivo. Although our initial mechanistic studies suggested that shikonin-mediated enhancement of rAAV vector transduction was through its proteasome inhibitory activity, which was similar to that of MG132, a specific proteasome inhibitor. However, no further increase was observed when cells were co-treated with both shikonin and MG132.Since shikonin is a multifunctional compound, we wished to evaluate its effect on rAAV vector-mediated transgene expression in additional human cell types. In a human erythroleukemia cell line, K562, frequently used as a model for hematopoietic cell transduction studies, shikonin enhanced the transduction efficiency of various rAAV serotype vectors by up to 10-fold. Surprisingly, however, MG132 had no effect in K562 cells, even at the highest concentration that did not induce cytotoxicity. Thus, in a systematic study, various proteasome and protease inhibitors were evaluated. Western blot assays for the detection of polyubiquitinated total cellular proteins were performed to confirm the effectiveness of these drugs in K562 cells. Whereas carfilzomib only had a modest effect (<2-fold enhancement), surprisingly, bortezomib significantly reduced the AAV vector-mediated transgene expression in K562 cells. E-64 and PMSF, a cysteine and a serine protease inhibitor, respectively, also had no effect. Additional studies revealed that a specific inhibitor of the reactive oxygen species (ROS) formation, blocked the enhancing effect of shikonin on rAAV vector-mediated transgene expression in K562 cells but not other cell types. Furthermore, shikonin treatment did augment rAAV vector-mediated transduction ex vivo in both mouse and human primary hematopoietic stem cells.Taken together, our studies also emphasize the need to exercise caution in interpreting rAAV vector-mediated transgene expression data among different cell types involving the use of multifunctional drugs.

MiR-21 Inhibition Reduces Liver Fibrosis and Prevents Tumor Development by Inducing Apoptosis of CD24+ Progenitor Cells.

miR-21 is upregulated in hepatocellular carcinoma and intrahepatic cholangiocarcinoma, where it is associated with poor prognosis. Here, we offer preclinical evidence that miR-21 offers a therapeutic and chemopreventive target in these liver cancers. In mice with hepatic deletion of Pten, anti-miR-21 treatment reduced liver tumor growth and prevented tumor development. These effects were accompanied with a decrease in liver fibrosis and a concomitant reduction of CD24(+) liver progenitor cells and S100A4(+) cancer-associated stromal cells. Notch2 inhibition also occurred in tumors following anti-miR-21 treatment. We further showed that miR-21 is necessary for the survival of CD24(+) progenitor cells, a cellular phenotype mediated by Notch2, osteopontin, and integrin αv. Our results identify miR-21 as a key regulator of tumor-initiating cell survival, malignant development, and growth in liver cancer, highlighting the role of CD24(+) cells in the expansion of S100A4(+) cancer-associated stromal cells and associated liver fibrosis.

Far-Red/Near-Infrared Conjugated Polymer Nanoparticles for Long-Term In Situ Monitoring of Liver Tumor Growth.

The design and synthesis is reported for a fluorescent conjugated polymer (CP), poly{[4,4,9,9‐tetrakis(4‐(octyloxy)phenyl‐4,9‐dihydro‐s‐indaceno[1,2‐b:5,6‐b′]dithiophene)]‐alt‐co‐[4,7‐di(thiophen‐2‐yl)‐2,1,3‐benzothiadiazole]} (PIDT‐DBT), with absorption and emission profiles fallen within far‐red/near infrared (FR/NIR) region and further demonstrate its application in long‐term in vitro cell tracing and in vivo imaging of liver tumor growth. PIDT‐DBT‐Tat nanoparticles (NPs) have an absorption maximum at ≈600 nm with an emission maximum at ≈720 nm in water. In vitro cell tracing studies reveal that PIDT‐DBT‐Tat NPs can trace HepG2 liver cancer cells over 8 d. In vivo imaging results indicate that PIDT‐DBT‐Tat NPs can monitor liver tumor growth for more than 27 d in a real‐time manner. Both in vitro and in vivo studies demonstrate that PIDT‐DBT‐Tat NPs are superior to commercial Qtracker 705 as fluorescent probes. This study demonstrates for the first time the feasibility for long‐term in vivo imaging of tumor growth by utilizing CP‐based fluorescent probes, which will encourage the development of NIR fluorescent CPs for in vivo bioimaging.

Anti-cancer effect of N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxamide, a novel synthetic compound.

Naphthofuran compounds have been known to regulate HNF 4α which is associated with proliferation, progression and metastasis of HCC. In this study, we investigated whether N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxamide (NHDC), a novel synthetic naphthofuran compound inhibits liver tumor growth through activation of HNF 4α. Treatment with different concentrations (1-10.8 µM) of NHDC for various periods (0-72 h) inhibited liver cancer cells (HepG2, Hep3B) growth as well as colony formation followed by induction of apoptosis in a concentration dependent manner. NHDC also induced expression of the apoptosis regulating genes as well as inhibiting the action of STAT3. These inhibitory effects were associated with enhancement of expression and DNA binding activity of HNF 4α. In vivo study confirmed that liver tumor growth was prevented with NHDC (5 mg/kg), and its effect was also related with inhibition of STAT3 pathway through enhancement of expression and DNA binding activity of HNF 4α. Moreover, siRNA of HNF 4α abolished NHDC-induced cell growth inhibition as well as DNA binding activity and phosphorylation of STAT3. Pull down assay docking prediction analysis proved that NHDC directly binds to hydrophobic fatty acid ligand binding site of HNF 4α. A novel naphthofuran compound, NHDC inhibited liver tumor growth by inactivating of STAT3 through direct biding to HNF 4α.

Plasma membrane gp96 enhances invasion and metastatic potential of liver cancer via regulation of uPAR

Targeted therapy is currently under intensive investigation due to the resistance of liver cancer to cytotoxic chemotherapies. Dissecting the molecular events that drive the progression of liver cancer and defining specific targets are urgently needed to develop efficient tailored therapies. Cell membrane gp96 (mgp96) has been implicated in tumor growth and malignancy. Here, we explored the functional and clinical relevance of mgp96 in liver cancer. We found that elevated mgp96 abundance was associated with tumor metastasis and recurrence in patients with primary liver tumors. Decreased KDELR1 levels in hepatoma cells contribute to cell membrane translocation of the normally ER-resident gp96. Urokinase-type plasminogen activator receptor (uPAR) was identified as a mgp96 client protein, and mgp96 stabilized uPAR protein. Our clinical results proved that elevated mgp96 abundance is positively correlated with uPAR expression levels in liver tumors. We further provided evidence that targeting mgp96 with siRNA or a specific mAb that blocked the mgp96-uPAR interaction led to inhibited cell growth, survival, and invasion in vitro, as well as the suppression of liver tumor growth and metastasis in vivo. mgp96 promotes liver cancer progression through increasing the protein stability and signaling of uPAR, and may be a new promising target for suppressing uPAR-mediated tumor growth and metastasis in liver cancer.

Heat shock protein gp96 decreases p53 stability by regulating Mdm2 E3 ligase activity in liver cancer

The resistance to apoptosis displayed by liver cancer plays a key role in hepatocarcinogenesis, tumor progression, and resistance to chemo- or radio-therapy. In this study, we uncovered the potential role and mechanism of heat shock protein gp96 in regulating liver tumor cell growth and apoptosis. P53 protein was identified as a gp96 client protein by profiling apoptosis-related proteins in gp96-knockdown liver cancer cells. Overexpression and knockdown studies both demonstrated that gp96 decreases p53 protein levels, and gp96 regulated cell apoptosis in a p53-dependent manner. We further provide evidence that gp96 interacts with both p53 and Mdm2 to enhance Mdm2-mediated p53 ubiquitination and degradation. Moreover, targeting gp96 with siRNA induced cell apoptosis and led to the suppression of liver tumor growth in vivo. In conclusion, we elucidated an underlying mechanism by which gp96 promotes p53 degradation via increasing Mdm2 E3 ligase activity and provided a new therapeutic strategy to target the gp96-mediated anti-apoptotic characteristic of hepatocellular carcinoma.

Asparagus polysaccharide and gum with hepatic artery embolization induces tumor growth and inhibits angiogenesis in an orthotopic hepatocellular carcinoma model.

Liver cancer is one of leading digestive malignancies with high morbidity and mortality. There is an urgent need for the development of novel therapies for this deadly disease. It has been proven that asparagus polysaccharide, one of the most active derivates from the traditional medicine asparagus, possesses notable antitumor properties. However, little is known about the efficacy of asparagus polysaccharide as an adjuvant for liver cancer chemotherapy. Herein, we reported that asparagus polysaccharide and its embolic agent form, asparagus gum, significantly inhibited liver tumor growth with transcatheter arterial chemoembolization (TACE) therapy in an orthotopic hepatocellular carcinoma (HCC) tumor model, while significantly inhibiting angiogenesis and promoting tumor cell apoptosis. Moreover, asparagine gelatinous possessed immunomodulatory functions and showed little toxicity to the host. These results highlight the chemotherapeutic potential of asparagus polysaccharide and warrant a future focus on development as novel chemotherapeutic agent for liver cancer TACE therapy.

Selective in vivo targeting of human liver tumors by optimized AAV3 vectors in a murine xenograft model.

Current challenges for recombinant adeno-associated virus (rAAV) vector-based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer.

Codelivery of doxorubicin and curcumin with lipid nanoparticles results in improved efficacy of chemotherapy in liver cancer.

Liver cancer is a leading cause of cancer deaths worldwide. The combination therapy of cytotoxic and chemosensitizing agents loaded in nanoparticles has been highlighted as an effective treatment for different cancers. However, such studies in liver cancer remain very limited. In our study, we aim to develop a novel lipid nanoparticles loaded with doxorubicin (DOX) (an effective drug for liver cancer) and curcumin (Cur) (a chemosensitizer) simultaneously, and we examined the efficacy of chemotherapy in liver cancer. DOX and Cur codelivery lipid nanoparticles (DOX/Cur-NPs) were successfully prepared using a high-pressure microfluidics technique, showing a mean particle size of around 90 nm, a polydispersity index <0.3, and a zeta potential <−10 mV. The encapsulation efficacy was >90% for both DOX and Cur. The blank lipid nanoparticles were nontoxic, as determined by a cell cytotoxicity study in human normal liver cells L02 and liver cancer cells HepG2. In vitro DOX release studies revealed a sustained-release pattern until 48 hours in DOX/Cur-NPs. We found enhanced cytotoxicity and decreased inhibitory concentration (IC)50 in HepG2 cells and reduced cytotoxicity in L02 cells treated with DOX/Cur-NPs, suggesting the synergistic effects of DOX/Cur-NPs compared with free DOX and DOX nanoparticles (NPs). The optimal weight ratio of DOX and Cur was 1:1. Annexin-V-fluorescein isothiocyanate/propidium iodide double staining showed enhanced apoptosis in HepG2 cells treated with DOX/Cur-NPs compared with free DOX and DOX-NPs. An in vivo experiment showed the synergistic effect of DOX/Cur-NPs compared with DOX-NPs on liver tumor growth inhibition. Taken together, the simultaneous delivery of DOX and Cur by DOX/Cur-NPs might be a promising treatment for liver cancer.

ZHX2 enhances the cytotoxicity of chemotherapeutic drugs in liver tumor cells by repressing MDR1 via interfering with NF-YA.

We previously reported the tumor suppressor function of Zinc-fingers and homeoboxes 2 (ZHX2) in hepatocellular carcinoma (HCC). Other studies indicate the association of increased ZHX2 expression with improved response to high dose chemotherapy in multiple myeloma. Here, we aim to test whether increased ZHX2 levels in HCC cells repress multidrug resistance 1(MDR1) expression resulting in increased sensitivity to chemotherapeutic drugs. We showed evidence that increased ZHX2 levels correlated with reduced MDR1 expression and enhanced the cytotoxicity of CDDP and ADM in different HCC cell lines. Consistently, elevated ZHX2 significantly reduced ADM efflux in HepG2 cells and greatly increased the CDDP-mediated suppression of liver tumor growth in vivo. Furthermore, immunohistochemical staining demonstrated the inverse correlation of ZHX2 and MDR1 expression in HCC tissues. Luciferase report assay showed that ZHX2 repressed the MDR1 promoter activity, while knockdown of NF-YA or mutating the NF-Y binding site eliminated this ZHX2-mediated repression of MDR1 transcription. Co-IP and ChIP assay further suggested that ZHX2 interacted with NF-YA and reduced NF-Y binding to the MDR1 promoter. Taken together, we clarify that ZHX2 represses NF-Y-mediated activation of MDR1 transcription and, in doing so, enhances the effects of chemotherapeutics in HCC cells both in vitro and in vivo.

Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry

CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue.

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine (TCM) drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus (rAAV) vectors has not been attempted. We synthesized the cDNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged cDNAs were subcloned into a rAAV plasmid vector. The protein expression was confirmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into rAAV vectors, under the control of a liver cancer-specific promoter. The liver tumor growth was then monitored following intratumor administration of the rAAV vectors. The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin (TCS) gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of rAAV vectors containing the TCS gene significantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model. Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Chemically induced mouse liver tumors are resistant to treatment with atorvastatin.

BackgroundAtorvastatin is a potent inhibitor of the mevalonate pathway and widely used as a hypolipidemic drug. Some epidemiological studies and animal experiments indicate that the long-term use of atorvastatin and structurally related drugs might be associated with a reduced risk of developing hepatocellular carcinoma (HCC), the most common hepatocellular malignancy in humans. However, the potential of atorvastatin to inhibit HCC formation is controversially discussed.MethodsHepatocellular tumors were chemically induced by treatment of C3H/He mice with 10 μg/g body weight N-nitrosodiethylamine and the ability of atorvastatin to interfere with tumor formation was investigated by treatment of mice with 0.1% atorvastatin in the diet for 6 months. Tumor size and tumor multiplicity were analyzed, as were tissue levels of cholesterol and atorvastatin.ResultsAtorvastatin treatment efficiently reduced serum cholesterol levels. However, the growth of tumors driven by activated MAPK (mitogen-activated protein kinase) signaling was not attenuated by the presence of the drug, as evidenced by a lack of reduction of tumor volume or tumor multiplicity by atorvastatin. Levels of the atorvastatin uptake transporters Oatp1a4 and Oatp1b2 were down-regulated at the mRNA and protein levels in chemically induced mouse liver tumors, but without striking effects on atorvastatin concentrations in the tumor tissue.ConclusionIn summary, the present data provide substantial evidence that atorvastatin does not beneficially influence tumor growth in mouse liver and thereby challenge the hypothesis that statin use might protect against hepatocellular cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-766) contains supplementary material, which is available to authorized users.

Abstract A43: Therapeutic importance of a long noncoding RNA in Ewing sarcoma

Abstract Background: Ewing sarcoma is a highly invasive pediatric bone malignancy with a 20% 5-year survival rate for patients with metastatic disease. The EWS-FLI1 or equivalent fusion gene is detected in 95% of these cases, and functions as a transcription factor that regulates oncogenesis-related gene expression. Various oncoproteins including EWS-FLI1 have been studied at a molecular level to explore their therapeutic potential in order to improve patient outcomes. With a similar aim, we study non-coding genes in Ewing sarcoma that can be targeted using RNA therapeutics. Methods: Total RNA from 500 tissues including over 100 primary Ewing, other cancers, normal tissues, and modified Ewing cell lines were run on Affymetrix Human Exon microarrays. RNA-seq data was generated on a pair of primary and metastatic Ewing cell lines. Data were analyzed using Genetrix®, Partek®, and Ingenuity Pathway Analysis®. Functional studies on Ewing cell lines were performed using various RNAi techniques and viral transductions followed by quantitative PCR. Chromatin immunoprecipitation (ChIP) was performed to detect direct interactions of the RNA. Tail vein injection model was developed to study tumor metastasis model in mice. Results: Expression profiling results indicated that several non-coding transcripts had a strong association with Ewing sarcoma. Molecular characterization of one such transcript, AK057037 (also known as FEZF1-AS), identified it as a multi-exonic, polyadenylated , long non-coding RNA (lncRNA) with multiple expressed isoforms. Like many other Ewing sarcoma associated biomarkers, EWS-FLI1 directly regulated this lncRNA by binding to the GGAA microsatellite region in its promoter. Increased stable expression of this transcript led to a statistically significant increase in anchorage independence. RNAi mediated decrease in its expression reduced the invasiveness of Ewing sarcoma cell lines. In vivo studies using AK057037-overexpressing cells showed increased tumor cell engraftment and growth in the mice liver. Pathway analysis of expression profiling data on the AK057037-knockdown cells identified cellular adhesion and movement pathways. RNA-seq analysis showed increased transcriptional activity in the metastatic cell line when compared to the primary. RNA-IP studies showed that AK057037 was bound to EZH2, a methyltransferase in the polycomb repressor complex 2 (PRC2). ChIP assays using histone antibodies showed changes in methylation pattern in promoter regions of genes affected by the lncRNA knockdown. H3K27me3 marks correlated with increased repression of metastatic genes on knockdown of the transcript. Conclusion: AK057037 is a lncRNA that is highly expressed in Ewing sarcoma. It is directly regulated by the chimeric oncogene, EWS-FLI1. The lncRNA functions as an oncogene and promotes metastasis in this tumor. Its association with the PRC2 complex leads to methylation changes in promoters of genes that are involved in cell migration. Finally, AK057037 is a functional lncRNA that influences Ewing sarcoma pathogenesis by its genetic and epigenetic interactions and has the potential to be developed into a therapeutic target to improve survival of patients with metastatic disease. Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Jonathan D. Buckley, William A. May, Philipp Kapranov, Robert J. Arceci, Timothy J. Triche. Therapeutic importance of a long noncoding RNA in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A43.

Long-term, low-dose exposure to microcystin toxin does not increase the risk of liver tumor development or growth in mice.

Cyanotoxins are biological toxins produced by cyanobacteria (blue green algae) that have been implicated in the pathogenesis of liver tumors. Based on acute toxicity studies, the World Health Organization has designated 1.0 μg/L of cyanotoxin-contaminated drinking water as the safe allowable limit for daily oral consumption. The aim of this study was to determine whether long-term exposure to this concentration of cyanotoxins is capable of initiating or promoting the growth of liver tumors. In the present study, four groups of adult, male CD-1 mice (n = 20/group) were exposed to either drinking water alone (water group), drinking water containing 1.0 μg/L of microcystin-LR (MC-LR group), MC-LR plus the tumor promoter thioacetamide (MC-LR/TAA group) or thioacetamide alone (TAA group). Following 28 weeks of exposure, mice were killed and the livers examined for tumor number and size. No tumors were present in the water or MC-LR alone groups while five mice in the MC-LR/TAA group and four in the TAA alone group developed liver tumors. The mean size of the tumors in the MC-LR/TAA and TAA alone groups were similar as were the results of Ki-67 staining, number of atypical mitoses and liver cancer gene expression profiles. In vitro MC-LR (0.1-1000 μg/L) exposure did not induce malignant transformation of WB-F344 hepatic stem cells or increase the proliferative activity or invasiveness of PLC/PRF/5 malignant hepatocytes. The results of this study suggest that long-term, low dose cyanotoxin exposure is unlikely to result in liver tumor development or enhance existing tumor growth.

Gα12 gep oncogene deregulation of p53-responsive microRNAs promotes epithelial-mesenchymal transition of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Gα12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Gα12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Gα12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Gα12 (Gα12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Gα12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Gα12. Decreases of miR-200a/b, -192 and -215 by Gα12 caused ZEB1 induction. The ability of Gα12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Gα12QL induced ZEB1 and other epithelial-mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Gα12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Gα12 level, a correlation existed in the comparison of relative changes of Gα12 and ZEB1. In conclusion, Gα12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial-mesenchymal transition and growth of liver tumor. These findings highlight the significance of Gα12 upregulation in liver tumor progression, implicating Gα12 as an attractive therapeutic target.

A novel small-molecule activator of procaspase-3 induces apoptosis in cancer cells and reduces tumor growth in human breast, liver and gallbladder cancer xenografts

Purpose: Procaspase-3, a proenzyme of apoptotic executioner caspase-3, is overexpressed in numerous tumors. We aimed to characterize a novel procaspase-3 activator, WF-210, which may have potential as an anticancer drug.Experimental design: The procaspase-3 activating ability, antitumor efficacy, mechanisms of action, and toxicity profiles of WF-210 were investigated in vitro and in vivo, using normal cells, cancer cells, and mouse xenograft models. The role of procaspase-3 in WF-210-induced apoptosis was explored by manipulating procaspase-3 expression in cultured cells.Results: WF-210 activated procaspase-3 with an EC50 of 0.95 μM, less than half that of its mother compound PAC-1 (2.08 μM). The mechanism involved the chelation of inhibitory zinc ions, subsequently resulting in an auto-activation of procaspase-3. WF-210 was more cytotoxic than PAC-1 to human cancer cells, but less cytotoxic to normal cells. Cancer cells with high procaspase-3 expression, like HL-60 and U-937, were particularly sensitive. WF-210-induced the apoptosis of HL-60 and U-937 cells by activating procaspases and promoting proteasome-dependent degradation of XIAP and Survivin. The level of WF-210-induced apoptosis in cultured cells was related to the level of procaspase-3 expression. Finally, WF-210 was superior to PAC-1 in retarding the in vivo growth of breast, liver and gallbladder xenograft tumors which overexpress procaspase-3, and induced no substantial weight loss or neurotoxicity. WF-210 and PAC-1 had no effect on the growth of MCF-7 xenograft tumors, which do not express procaspase-3.Conclusion: We identified WF-210 as a potent small-molecule activator of procaspase-3. The favorable antitumor activity and acceptable toxicity profile of WF-210 provide a strong rationale for its clinical evaluation in the treatment of tumors with high procaspase-3 expression.