Growth Of Subcutaneous Xenografts Research Articles

Telomerase inhibition abolishes the tumorigenicity of pediatric ependymoma tumor-initiating cells.

Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1327-6) contains supplementary material, which is available to authorized users.

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2−/− mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.

Anti-Cancer Activity of an Osthole Derivative, NBM-T-BMX-OS01: Targeting Vascular Endothelial Growth Factor Receptor Signaling and Angiogenesis

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.

Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma

Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell-cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes. We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011 (Novartis Oncology), a highly specific CDK4/6 inhibitor. Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nmol/L in sensitive lines). LEE011 caused cell-cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. Although our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (P = 0.01), the identification of additional clinically accessible biomarkers is of high importance. Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. Clin Cancer Res; 19(22); 6173-82. ©2013 AACR.

Notch activation stimulates migration of breast cancer cells and promotes tumor growth

IntroductionDysregulated NOTCH receptor activity has been implicated in breast cancer but the mechanisms by which NOTCH contributes to transformation are not yet clear, as it has context-dependent effects on the properties of transformed cells.MethodsWe have used various in vitro and in vivo carcinogenic models to analyze the impact of Notch signaling in the onset and progression of breast tumors.ResultsWe found that ectopic expression of the Notch1 intracellular domain (N1ICD) in MCF-7 breast adenocarcinoma cell line caused reduction and delocalization of E-CADHERIN levels and increased migratory and invasive abilities. Notch inhibition in the invasive breast cancer cell line MDA-MB-231 resulted in increased E-CADHERIN expression and a parallel reduction in their invasive capacity. The growth of subcutaneous xenografts produced with MCF-7 cells was boosted after N1ICD induction, in a cell autonomous manner. In vivo Notch1 activation in the mammary gland using the MMTV-Cre driver caused the formation of papillary tumors that showed increased Hes1 and Hey1 expression and delocalized E-cadherin staining.ConclusionsThese results confirm NOTCH1 as a signal triggering epithelial-mesenchymal transition in epithelial cancer cells, which may have implications in tumor dissemination, metastasis and proliferation in vivo. The identification of specific factors interacting with NOTCH signaling could thus be relevant to fully understanding the role of NOTCH in breast neoplasia.

Abstract 5473: Anti-EGFR monoclonal antibody nimotuzumab enhances temozolomide-induced growth suppression of mutant EGFR expressing glioma xenografts.

Abstract A mutant form of EGFR, EGFRvIII or ΔEGFR, is common in glioblastoma (GBM) conferring enhanced tumorigenic activity as well as resistance to chemotherapeutic drugs. Nimotuzumab, a humanized monoclonal EGFR antibody, has been shown to exert a modest activity in patients with GBM, but its specific activity against ΔEGFR has not been fully investigated. We treated human glioma cells overexpressing either wild-type (wt) EGFR or ΔEGFR with nimotuzumab with or without temozolomide (TMZ). Nimotuzumab treatment resulted in reduction in tyrosine phosphorylation at C-terminal of ΔEGFR more preferentially than that of wtEGFR, albeit to a lesser extent than tyrosine kinase inhibitor AG1478. Nimotuzumab-induced EGFR tyrosine dephosphorylation was associated with a decrease in AKT phosphorylation, suggesting suppression of downstream signaling pathways. In animal models, growth of subcutaneous xenografts was suppressed by TMZ, but was not affected by nimotuzumab monotreatment. However, antitumor activity was synergistically enhanced when TMZ was combined with nimotuzumab in both U87MG.ΔEGFR and LNZ308.ΔEGFR xenografts, while such an effect was limited in wtEGFR-overexpressing xenografts. Similarly, compared with nimotuzumab or TMZ monotherapy, the combination treatment of TMZ with nimotuzumab significantly elongated survival of mice bearing intracerebral xenografts derived from U87MG.ΔEGFR (p < 0.001, logrank test), and U87MG.wtEGFR to a lesser extent (p = 0.006). These anti-tumor effects by the combination treatment were associated with a decreased proliferation rate and an increase in apoptosis rate in treated xenografts. The U87MG.ΔEGFR intracerebral tumors which had re-grown after the combination treatment with TMZ and nimotuzumab (“escapers”) exhibited an increase expression of MGMT as well as a decrease in mismatch repair (MMR) proteins, MSH6 and MLH1. The reduction of MMR expression was further retained in subcultured escaper cells. These expression changes were not observed in xenografts treated with any of vehicles or monotherapies. These results suggest that nimotuzumab has antitumor activity against mutant EGFR-expressing glioma in vivo when combined with TMZ, and the resistance to this combination treatment might involve expression change of both MGMT and MMR. Citation Format: Motoo Nagane, Yusuke Nitta, Saki Shimizu, Yukiko Shishido-Hara, Yoshiaki Shiokawa. Anti-EGFR monoclonal antibody nimotuzumab enhances temozolomide-induced growth suppression of mutant EGFR expressing glioma xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5473. doi:10.1158/1538-7445.AM2013-5473 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2744: CDK4/CDK6 inhibition is potently active in a definable subset of human neuroblastomas.

Abstract Background: Neuroblastoma is a pediatric embryonal cancer for which the survival of patients with high-risk disease is less than 50% and has not dramatically changed over the last several years. Recently, a number of cell cycle genes_particularly those within the Cyclin D/CDK4/CDK6/RB network_have been identified as oncogenic vulnerabilities in neuroblastoma, suggesting that their therapeutic exploitation might improve survivability. Indeed, genomic amplifications of CDK4, CDK6, and CCND1 have been reported in primary neuroblastomas, and we have previously shown via an unbiased loss of function screen that CDK4 depletion is associated with potent anti-tumor activity (Cole, PNAS 2011). Here, we sought to translate these findings into novel therapies for children with neuroblastoma by evaluating the effect of pharmacologic Cdk4/Cdk6 inhibition on neuroblastoma viability. Methods: We analyzed the effect of combined Cdk4/6 inhibition in a comprehensive panel of human-derived neuroblastoma cell lines using LEE011, a highly specific Cdk4/6 small molecule inhibitor. Anti-tumor activity was also determined in vivo in three neuroblastoma xenograft models, and integrative genomics was used to identify biomarkers of drug sensitivity. Results: Treatment with LEE011 significantly inhibited proliferation in 10 of 15 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 361 ± 97 nM, considering sensitive lines only), as evidenced by significant cell cycle arrest and senescence that were likely attributed to dose-dependent decreases in phosphorylated RB and FOXM1. In addition, responsiveness of neuroblastoma xenografts to LEE011 was reflective of in vitro data in that there was a direct correlation of IC50 values with degree of subcutaneous xenograft growth delay, with the most sensitive lines in vitro showing profound growth inhibition in vivo. While our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (p= 0.04, student's t test), a supervised hierarchical clustering of gene expression data identified several potential gene signatures that could explain the observed differential sensitivity to Cdk4/6 inhibition. Conclusions: Our data show that LEE011 is highly active in a large subset of neuroblastoma cell lines and xenograft models, and therefore support the clinical development of LEE011 as a therapy for neuroblastoma as well as efforts to validate biomarkers of drug activity. Citation Format: JulieAnn Rader, Lori Hart, Mike Russell, Michael Nakazawa, Lili Belcastro, Daniel Martinez, Erica Carpenter, Sunkyu Kim, Sudha Parasuraman, Giordano Caponigro, Robert Schnepp, Andrew Wood, Bruce Pawel, Deborah Watson, Patrick Warren, Kristina Cole, John Maris. CDK4/CDK6 inhibition is potently active in a definable subset of human neuroblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2744. doi:10.1158/1538-7445.AM2013-2744

Combined temozolomide and sunitinib treatment leads to better tumour control but increased vascular resistance in O6-methylguanine methyltransferase-methylated gliomas

IntroductionCombined antiangiogenic and cytotoxic treatment represents an appealing treatment approach for malignant glioma. In this study we characterised the antitumoural and microvascular consequences of sunitinib (Su) and temozolomide (TMZ) therapy and verified the ideal treatment protocol, with special focus on a potential therapeutic window for combined scheduling. Materials and MethodsO6-Methylguanine methyltransferase (MGMT) status was analysed by pyrosequencing. Tumour growth of subcutaneous xenografts was assessed under different treatment protocols (TMZ, SU, SU followed by TMZ, TMZ followed by SU, combined TMZ/SU). Intravital microscopy (dorsal skinfold chamber model) assessed microvascular consequences. Immunohistochemistry included tumour and endothelial cell proliferation, apoptosis and vascular pericyte coverage. Real-time polymerase chain reaction (RT-PCR) analysed the expression of angiogenesis-related pathways in response to therapy. ResultsCombined TMZ/SU resulted in significantly reduced tumour growth compared to either monotreatment (TMZ: 106±13mm3; SU: 114±53mm3; TMZ/SU: 34±7mm3) by additional antiangiogenic effects and synergistic induction of apoptosis versus TMZ monotreatment. Sequential treatment protocols did not show additive antitumour responses. TMZ/SU aggravated vascular resistance mechanisms characterised by significantly higher blood flow rate (TMZ: 74±34μl/s; SU: 164±36μl/s; TMZ/SU: 254±95μl/s), reduced permeability (TMZ: 1.05±0.02; SU: 0.99±0.07; TMZ/SU: 0.89±0.05) and recovery of pericyte–endothelial interactions (TMZ: 89±7%; SU: 67±9%, TMZ/SU:80±10%) versus either monotreatment. Vascular resistance was paralleled by an increase in Ang-1 and Tie-2 and by the downregulation of Dll4. ConclusionSequential application of TMZ and SU in the angiogenic window does not add antitumour efficacy to monotherapy. Simultaneous application yields beneficial tumour control due to additive antiangiogenic and proapoptotic effects. Combined treatment may aggravate pericyte-mediated vascular resistance mechanisms by altering Ang-1–Tie-2 and Dll4/Notch pathways.

Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon, and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT, and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway—involving AKT, CREB, Bcl-xL, survivin, and Bcl-2—downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that shRNA knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl play complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.

C1B domain peptide of protein kinase Cγ significantly suppresses growth of human colon cancer cells in vitro and in an in vivo mouse xenograft model through induction of cell cycle arrest and apoptosis

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101–112) and C1B5 (residues 141–151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60–70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/β inhibitor, Ro-32–0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G2 arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.

Abstract 1941: Development of novel NSAID conjugated molecules for lung cancer therapy

Abstract Background: Many promising chemopreventive Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) such as Flurbiprofen have acquired anti-cyclooxygenase 2 (COX2) effects with anti-cancer activities. Recently several phosphorylated-NSAID compounds were shown to improve the therapeutic potency over the parental NSAID molecules (JPET 111.183533, 2011) by 10 to 30 folds. Previously, we also reported several novel and potent molecules by the incorporation of NSAIDs such as aspirin, indomethancin, ketorolac, ketoprofen, and sulindac into the aclyting N-terminal of our lead mimetic molecules B317 (F5C-OC2Y-Atmp) (AACR 45: 476, 2004). Here we report a new series of highly potent molecules by replacing the N-terminal F5C with a flurbiprofen (Fbp). The resultant molecules effectively inhibit many cancer cell lines in vitro and in vivo, with efficacy much better than the unmodified parental NSAID molecules or those phosphorylated NSAID analogs. Methods: Standard MTT assays were used to access growth inhibitory effects of the parental and NSAID conjugated molecules. Effects of drugs on cell cycle and apoptosis were analyzed by flow cytometry. In vivo efficacy was evaluated in subcutaneous lung xenografts in athymic nude mice. Results: 32 lung cancer cell lines including 24 NSCLC and 8 SCLC cell lines were treated with various concentrations of some selected conjugated drugs for 4 days. The best compound had an average IC 50 value of 0.5 uM, with a range of 0.1 to 2 uM, while parental Flurbiprofen had IC50 value of 500 uM, with a range of 200 uM to 800uM. Cell cycle analysis showed that exposure of lung cancer cells to these new conjugates resulted in a significant shift of cells from S and G2 phases into G1 arrest, resulting in high percentage of apoptotic cell deaths in a dose- and time-dependent manner. Finally, our in vivo efficacy studies demonstrated that new NSAID conjugates at 5 mg/kg by daily ip injections significantly suppressed growth of subcutaneous xenografts of human lung adenocarcinoma in athymic nude mice without causing weight loss. Conclusion: Our new NSAID conjugate is a novel class of therapeutic agent with anti-growth effects against lung cancer cells in vitro and in vivo, with a 1000 fold increase in potency than the parental Flurbiprofen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1941. doi:1538-7445.AM2012-1941

Combined bioluminescence and fluorescence imaging visualizing orthotopic lung adenocarcinoma xenograft <italic>in vivo</italic>

Lung adenocarcinoma is the most common type of lung cancer. A close monitor of in vivo tumor development may help to better understand the pathogenesis and pathological processes of this disease. A bimodal imaging strategy has been developed, which is a very important tool to investigate the growth and metastasis of lung adenocarcinoma. In the present study, we used a combined labeling strategy in p53RE-luc-A549 cells via transfecting the reporter gene EGFP. In order to unambiguously identify the growth and metastasis of transfected A549 tumor cells, we established and observed subcutaneous and orthotopic xenografts in nude mice by in vivo bioluminescence and fluorescence imaging, which was verified by our post-mortem histological analysis. In vivo bioluminescence signal was observed for the progression of both subcutaneous and orthotopic xenografts in EGFP-p53RE-luc-A549 cells; in vivo fluorescence was only observed for the growth of subcutaneous xenograft of EGFP-p53RE-luc-A549 cells. Moreover, EGFP-p53RE-luc-A549 cells allow for the improved identification of implanted cells within host tissue during histological analysis. In conclusion, we presented a combined labeling strategy for bimodal A549 cell imaging which leads to improved detection of cellular grafts.

Adenovirus-mediated p53 gene therapy reverses resistance of breast cancer cells to adriamycin

The aim of this study was to determine whether adenovirus-mediated p53 gene (Ad-p53) transfection can enhance adriamycin cytotoxicity and reverse adriamycin resistance in human breast cancer cells and explore its effect on the expression of MDR1 gene and permeability-glycoprotein (P-gp). Human breast cancer cell lines, MCF-7 and MCF-7/ADR, were used in in-vitro studies. After infection with Ad-p53, the cytotoxicity of adriamycin was evaluated using the Cell Counting Kit-8 assay. The expression of MDR1 mRNA was detected by quantitative real-time PCR. The expression of P-gp was analyzed using western blotting. In in-vivo studies, MCF-7/ADR tumor cells were inoculated subcutaneously in athymic nude mice. After 14 days of inoculation, tumor size was measured. Apoptosis and expression of P-gp in the tumor tissue were analyzed by fluorescence activated cell sorting and western blotting. After transfection with a multiplicity of infection of 50 for Ad-p53, chemosensitivity of MCF-7/ADR cells increased by 18.1 times (P=0.001), and 50% inhibitory concentration (IC50) of adriamycin decreased from 4.54 ± 0.91 to 0.26 ± 0.11 mg/l. Real-time PCR showed that MDR1 mRNA decreased from 1.32 to 0.85 (P=0.001). Western blotting analysis showed that P-gp also decreased. In in-vivo studies, Ad-p53 combined with adriamycin dramatically inhibited the growth of subcutaneous xenograft of MCF-7/ADR. The fluorescence activated cell sorting assay showed that there were more apoptotic cells in tumor tissues treated with Ad-p53 and adriamycin. The expression of P-gp was significantly decreased in tumor tissues. This study suggests that Ad-p53 can reverse MCF-7/MDR cell resistance to adriamycin. The reversal effect was associated with inhibition of P-gp expression and induction of apoptosis.

399 DEVELOPMENT AND CHARACTERIZATION OF CLINICALLY RELEVANT TUMOR MODELS FROM PATIENTS WITH RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011399 DEVELOPMENT AND CHARACTERIZATION OF CLINICALLY RELEVANT TUMOR MODELS FROM PATIENTS WITH RENAL CELL CARCINOMA Jose Karam, Xiu-Ying Zhang, Pheroze Tamboli, Vitaly Margulis, Hua Wang, E. Jason Abel, Stephen Culp, and Christopher Wood Jose KaramJose Karam Houston, TX More articles by this author , Xiu-Ying ZhangXiu-Ying Zhang Houston, TX More articles by this author , Pheroze TamboliPheroze Tamboli Houston, TX More articles by this author , Vitaly MargulisVitaly Margulis Dallas, TX More articles by this author , Hua WangHua Wang Houston, TX More articles by this author , E. Jason AbelE. Jason Abel Houston, TX More articles by this author , Stephen CulpStephen Culp Houston, TX More articles by this author , and Christopher WoodChristopher Wood Houston, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.487AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Animal models are instrumental in understanding disease pathophysiology and mechanisms of therapy action and resistance in vivo. We sought to establish and characterize a panel of mouse models of renal cell carcinoma (RCC) derived from patients undergoing radical nephrectomy. METHODS Renal cell carcinoma mouse models were established from four patients with distinct histologies of renal cell carcinoma. Tumor tissues obtained during surgery were implanted into subcutaneous space of female BALB/c nude mice and serially passaged into new mice. Tumors obtained directly from patients and those derived from serial passages in nude mice were characterized by histology, Short Tandem Repeat (STR) fingerprinting, VHL gene sequencing, and Single Nucleotide Polymorphism (SNP) analysis for each of the four histologies. Tumor bearing mice were treated with sunitinib or everolimus and growth was compared between different histologies and different tumor passages. Primary cell cultures were also derived from patient tumors and were transfected with a lentiviral vector carrying the luciferase gene. Bioluminescence imaging (BLI) of mice was performed after intraperitoneal injection of luciferin in 2 of the models. RESULTS Four subcutaneous xenograft mouse models were developed: MDA−RCC−48, MDA−RCC−55, MDA−RCC−62, and MDA−RCC−80, representing papillary type 1, papillary type 2, clear cell, and clear cell with sarcomatoid features renal cell carcinoma, respectively. Tumor growth was dependent on the histologic type, size of implanted tumor chip, and possibly the passage number. We confirmed that the mouse tumors accurately and faithfully represent their respective original patient tumors as STR fingerprints were matching, histology on H&E staining was comparable, and SNP profiles and VHL mutation status were conserved with multiple passages. BLI results were commensurate with the subcutaneous xenograft growth patterns and enabled sequential and non−invasive monitoring of growth in an orthotopic implantation setting. Mice treated with sunitinib and everolimus exhibited an initial response to these agents, followed by a later stage of resistance to these agents, which mimics the clinical observations in patients with RCC. CONCLUSIONS We developed four mouse xenograft models of RCC with clear cell and papillary histologies, with stable histologic and molecular characteristics. We are currently using these models to identify mechanisms of action, molecular correlates of response, and resistance to novel targeted therapies. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e161 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jose Karam Houston, TX More articles by this author Xiu-Ying Zhang Houston, TX More articles by this author Pheroze Tamboli Houston, TX More articles by this author Vitaly Margulis Dallas, TX More articles by this author Hua Wang Houston, TX More articles by this author E. Jason Abel Houston, TX More articles by this author Stephen Culp Houston, TX More articles by this author Christopher Wood Houston, TX More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Development and Characterization of Clinically Relevant Tumor Models From Patients With Renal Cell Carcinoma

BackgroundAnimal models are instrumental in understanding disease pathophysiology and mechanisms of therapy action and resistance in vivo. ObjectiveTo establish and characterize a panel of mouse models of renal cell carcinoma (RCC) derived from patients undergoing radical nephrectomy. Design, setting, and participantsIn vivo and in vitro animal experiments. MeasurementsTumor tissues obtained during surgery were implanted into the subcutaneous space of female BALB/c nude mice and serially passaged into new mice. Tumors were characterized by histology, short tandem repeat (STR) fingerprinting, von Hippel-Lindau (VHL) gene sequencing, and single nucleotide polymorphism (SNP) analysis. Tumor-bearing mice were treated with sunitinib or everolimus. Primary cell cultures were derived from patient tumors and transfected with a lentivirus carrying the luciferase gene. Four subcutaneous xenograft mouse models were developed, representing papillary type 1, papillary type 2, clear cell, and clear cell with sarcomatoid features RCC. Results and limitationsRCC mouse models were established from four patients with distinct histologies of RCC. Tumor growth was dependent on histologic type, the size of the implanted tumor chip, and the passage number. Mouse tumors accurately represented their respective original patient tumors, as STR fingerprints were matching, histology was comparable, and SNP profiles and VHL mutation status were conserved with multiple passages. Bioluminescence imaging results were commensurate with subcutaneous xenograft growth patterns. Mice treated with sunitinib and everolimus exhibited an initial response, followed by a later stage of resistance to these agents, which mimics the clinical observations in patients with RCC. ConclusionsWe developed four mouse xenograft models of RCC with clear-cell and papillary histologies, with stable histologic and molecular characteristics. These models can be used to understand the basic biology of RCC as well as response and resistance to therapy.

Suppression of glycogen synthase kinase 3 activity reduces tumor growth of prostate cancer in vivo

Glycogen synthase kinase 3 (GSK-3) has been regarded as a potential therapeutic target for multiple human cancers. We previously reported that suppression of GSK-3 activity with lithium chloride (LiCl) or small chemical inhibitors impaired cellular DNA synthesis and reduced cell proliferation in prostate cancer cells. Therefore, in this study, we extended this in vitro findings to in vivo settings in order to establish a proof of concept that inhibition of GSK-3 activity is feasible in suppressing tumor growth of prostate cancer in vivo. In this study, we used three GSK-3 inhibitors, LiCl, TDZD-8, and L803-mts, which are structurally unrelated and non-ATP competitive. Human prostate cancer cell lines PC-3 and C4-2 were used for nude mouse xenograft models. The autochthonous transgenic prostate cancer TRAMP mice were used for testing GSK-3 inhibitor's effect on tumor development. Anti-Ki-67 and BrdU immunohistochemistry was used to determine cell proliferation. The pE2F-TA-LUC (E2F-LUC) luciferase reporter assay and gene specific small interferencing RNA technique were used to examine C/EBP involvement in GSK-3 inhibitor-induced E2F-1 suppression. Using mouse xenograft models, we demonstrated that LiCl and TDZD-8 significantly suppressed tumor development and growth of subcutaneous xenografts derived from human prostate cancer cells. Similarly, in the TRAMP mice, TDZD-8 and L803-mts reduced the incidence and tumor burden in the prostate lobes. Consistent with our previous in vitro findings, GSK-3 inhibitors significantly reduced BrdU incorporation and Ki67-positive cells in xenograft tumors and mouse cancerous prostates compared to the control. Further analysis revealed that following GSK-3 inhibition, C/EBPα, a negative cell cycle regulator, was remarkably accumulated in xenograft tumors or in cultured prostate cancer cells. Meanwhile, knocking down C/EBPα expression abolished GSK-3 inhibition-induced suppression of E2F1 transactivation, suggesting that C/EBPα accumulation is involved in GSK-3 inhibition-induced anti-tumor effect. Taken together, these results suggest that GSK-3 inhibition has the potential as a therapeutic strategy for prostate cancer intervention, although further pre-clinical and clinical testing are desirable.

PI3Kα Inhibitors That Inhibit Metastasis

Previous genetic analyses have suggested that mutations of the genes encoding PI3Kα facilitate invasion and metastasis but have less effect on primary tumor growth. These findings have major implications for therapeutics but have not been factored into pre-clinical drug development designs. Here we show that the inhibition of PI3Kα by newly designed small molecule inhibitors prevented metastasis formation in mice but had much less effect on the growth of subcutaneous xenografts or primary intra-abdominal tumors. These data support the idea that PI3Kα plays an important role in the metastatic process and suggest a more informed strategy for selecting drugs worthy of further development for clinical application.

Glutamate Receptor GRIA3—Target of CUX1 and Mediator of Tumor Progression in Pancreatic Cancer

Previously, we identified the transcription factor CUX1 as an important modulator of invasion and resistance to apoptosis. Expression profiles suggested that CUX1 regulates a complex transcriptional program mediating tumor progression. We aimed to identify functionally relevant targets of CUX1 by using RNA interference (RNAi)-based loss-of-function screens. Therefore, we generated an RNAi library containing putative transcriptional targets of CUX1 identified by microarrays and performed cell viability screens. Using this approach, several CUX1 targets with effect on tumor cell viability were identified, including the glutamate receptor GRIA3, which was validated in detail for its effects on proliferation, apoptosis, and cell migration using RNAi knock-down and overexpression strategies in vitro, as well as xenograft models in vivo. The expression of GRIA3 was evaluated in human pancreatic cancer tissues. We found that knock-down of GRIA3 significantly reduced proliferation and migration and enhanced apoptosis. In contrast, overexpression of GRIA3 significantly reduced apoptosis and enhanced both proliferation and tumor cell migration. GRIA3 could be confirmed as a downstream effector of CUX1 and was expressed in pancreatic cancer tissues. In vivo, GRIA3 significantly enhanced the growth of subcutaneous xenografts. Inhibitors of glutamate receptors such as GYKI52466 and SYM2206 significantly decreased survival of pancreatic cancer cells, suggesting the presence of glutamate signaling in pancreatic cancer. In conclusion, GRIA3 plays a role as a mediator of tumor progression in pancreatic cancer downstream CUX1. To our knowledge, this is the first report to identify a glutamate receptor as a modulator of tumor progression in a solid cancer outside the brain.

A water soluble parthenolide analog suppresses in vivo tumor growth of two tobacco‐associated cancers, lung and bladder cancer, by targeting NF‐κB and generating reactive oxygen species

Dimethylaminoparthenolide (DMAPT) is a water soluble parthenolide analog with preclinical activity in hematologic malignancies. Using non-small lung cancer (NSCLC) cell lines (A549 and H522) and an immortalized human bronchial epithelial cell line (BEAS2B) and TCC cell lines (UMUC-3, HT-1197 and HT-1376) and a bladder papilloma (RT-4), we aimed to characterize DMAPT's anticancer activity in tobacco-associated neoplasms. Flow cytometric, electrophoretic mobility gel shift assays (EMSA), and Western blot studies measured generation of reactive oxygen species (ROS), inhibition of NFκB DNA binding, and changes in cell cycle distribution and apoptotic proteins. DMAPT generated ROS with subsequent JNK activation and also decreased NFκB DNA binding and antiapoptotic proteins, TRAF-2 and XIAP. DMAPT-induced apoptotic cell death and altered cell cycle distribution with upregulation of p21 and p73 levels in a cell type-dependent manner. DMAPT suppressed cyclin D1 in BEAS2B. DMAPT retained NFκB and cell cycle inhibitory activity in the presence of the tobacco carcinogen nitrosamine ketone, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Using a BrdU accumulation assay, 5-20 μM of DMAPT was shown to inhibit cellular proliferation of all cell lines by more than 95%. Oral dosing of DMAPT suppressed in vivo A549 and UMUC-3 subcutaneous xenograft growth by 54% (p = 0.015) and 63% (p < 0.01), respectively, and A549 lung metastatic volume by 28% (p = 0.043). In total, this data demonstrates DMAPT's novel anticancer properties in both early and late stage tobacco-associated neoplasms as well as its significant in vivo activity. The data provides support for the conduct of clinical trials in TCC and NSCLC.

Improved efficacy of α‐particle–targeted radiation therapy

Human epidermal growth factor receptor-2 (HER-2) and tumor-associated glycoprotein 72 (TAG-72) have proven to be excellent molecular targets for cancer imaging and therapy. Trastuzumab, which binds to HER-2, is effective in the treatment of disseminated intraperitoneal disease when labeled with (213)Bi or (212)Pb. (213)Bi-humanized CC49 monoclonal antibody (HuCC49DeltaCH2), which binds to TAG-72, inhibits the growth of subcutaneous xenografts. A next logical step to improve therapeutic benefit would be to target tumors with both molecules simultaneously. Athymic mice bearing intraperitoneal human colon carcinoma xenografts were treated with a combination of trastuzumab and HuCC49DeltaCH2 labeled with (213)Bi administered through an intraperitoneal route. The sequence of administration also was examined. Before combining the 2 monoclonal antibodies, the effective doses of (213)Bi-CC49DeltaCH2 and (213)Bi-trastuzumab for the treatment of peritoneal disease were determined to be 500 muCi for each labeled antibody. Treatment with (213)Bi-HuCC49DeltaCH2 resulted in a median survival of 45 days and was comparable to the median survival achieved with (213)Bi-trastuzumab. Each combination provided greater therapeutic efficacy than either of the agents given alone. However, the greatest therapeutic benefit was achieved when (213)Bi-HuCC49DeltaCH2 and (213)Bi-trastuzumab were coinjected, and a median survival of 147 days was obtained. Dual targeting of 2 distinct molecules in tumors such as TAG-72 and HER-2 with alpha-particle radiation resulted in an enhanced, additive, therapeutic benefit. The authors also observed that this radioimmunotherapeutic strategy was well tolerated.