Orthotopic Cancer Model Research Articles

H-prune affects anaplastic thyroid cancer invasion and metastasis.

Anaplastic thyroid cancer is one of the most aggressive human malignancies and is resistant to multimodal treatments. The expression of h-prune, the human homologue of Drosophila prune, has been reported to be correlated with progression and aggressiveness in various cancers including breast, colorectal and pancreatic cancers. We examined the role of h-prune in anaplastic thyroid cancer cell migration, invasion and metastasis. Immunohistochemical analysis of h-prune was performed with 15 surgically resected specimens of anaplastic thyroid cancers. To investigate cell motility, Boyden chamber, wound healing and matrigel invasion assays were performed using cells from anaplastic thyroid cancer cell lines. A murine orthotopic thyroid cancer model was used to investigate metastatic ability. In the immunohistochemical analysis, only weak focal or no staining of h-prune was observed in non-tumor tissue. In contrast, diffuse staining of h-prune was observed in anaplastic thyroid cancer and lymph node metastasis samples. Both inhibition of h-prune phosphodiesterase activity with dipyridamole and small interfering RNA for h-prune suppressed 8505C and KTC-3 cell motility. In addition, treatment with dipyridamole and decreased expression of h-prune suppressed tumor invasion and pulmonary metastasis in a NOD/Shi-scid, IL-2Rγnull (NOG) mouse orthotopic thyroid cancer model. In conclusion, h-prune is frequently expressed in anaplastic thyroid cancer cells and lymph nodes metastasis, and promotes migration and invasion of anaplastic thyroid cancer cells and metastasis in an anaplastic thyroid cancer model. Thus, h-prune shows promise as a targeting candidate against anaplastic thyroid cancer.

Targeting Interleukin-11 Receptor-α Impairs Human Endometrial Cancer Cell Proliferation and Invasion In Vitro and Reduces Tumor Growth and Metastasis In Vivo.

Endometrial cancer contributes to significant morbidity and mortality in women with advanced stage or recurrent disease. IL11 is a cytokine that regulates cell cycle, invasion, and migration, all hallmarks of cancer. IL11 is elevated in endometrial tumors and uterine lavage fluid in women with endometrial cancer, and alters endometrial epithelial cancer cell adhesion and migration in vitro, but its role in endometrial tumorigenesis in vivo is unknown. We injected mice subcutaneously with human-derived Ishikawa or HEC1A endometrial epithelial cancer cells (ectopic), or HEC1A cells into the uterus (orthotopic) to develop endometrial cancer mouse models. Administration of anti-human IL11 receptor (R) α blocking antibody dramatically reduced HEC1A-derived tumor growth in both models and reduced peritoneal metastatic lesion spread in the orthotopic model, compared with IgG. Anti-human IL11Rα retained a well-differentiated, endometrial epithelial phenotype in the HEC1A ectopic mice, suggesting it prevented epithelial-to-mesenchymal transition. Blockade of mouse IL11Rα with anti-mouse IL11Rα antibody did not alter tumor growth, suggesting that cancer epithelial cell IL11 signaling is required for tumor progression. In vitro, anti-human IL11Rα antibody significantly reduced Ishikawa and HEC1A cell proliferation and invasion and promoted apoptosis. Anti-human, but not anti-mouse, IL11Rα antibody reduced STAT3, but not ERK, activation in HEC1A cells in vitro and in endometrial tumors in xenograft mice. We demonstrated that targeted blockade of endometrial cancer epithelial cell IL11 signaling reduced primary tumor growth and impaired metastasis in ectopic and orthotopic endometrial cancer models in vivo Our data suggest that therapeutically targeting IL11Rα could inhibit endometrial cancer growth and dissemination. Mol Cancer Ther; 15(4); 720-30. ©2016 AACR.

Establishment of thFree human pancreatic cancer orthotopic xenograft nude mice models and serum metabolomics

Objective To analyze the metabolic profile in serum between normal and orthotopic xenograft nude mice burdened with three human pancreatic cancer cell lines, which were differentiated differently. Methods Human pancreatic cancer lines SW1990, BxPC-3 and Panc-1 were subcutaneously injected into the nude mice, respectively. When the tumor volume reached 1.0 cm3, the nude mice were euthanized and the tumor tissues were removed and implanted to the pancreas to establish the orthotopic xenograft mice model. The serum from three orthotopic xenograft tumor nude mice and the normal controls were collected and then analyzed by 1H nuclear magnetic resonance spectroscopy. Results The three orthotopic xenograft nude mice models were successfully established. In SW1990, BxPC-3 and Panc-1 group, the orthotopic xenograft tumor formation rate was 79% (11/14), 93% (13/14) and 86% (12/14), while the mortality was 7% (1/14), 0 and 7% (1/14), respectively. Compared with control group, the content of metabolites in the serum of orthotopic xenograft tumor nude mice was increased including creatine, alanine, glutamine, 1-methylhistidine, isoleucine, lactate, phenylalanine, tryptophan and valine, but the glycerolphosphocholine (GPC) and glucose levels were reduced. As the tumors progressed to be more malignant, the content of valine and isoleucine tended to increase. Conclusions The establishment of the orthotopic implantation tumor nude mice model was stable and reliable with high tumor formation rate. Obvious metabolic differences of glucose, lipid and amino acids were observed between normal and human pancreatic cancer tumor burdening nude mice models. The common metabolic features identified in all three nude mice models burdened with human pancreatic cancer could be used as the potential markers for diagnosing human pancreatic cancer. Key words: Pancreatic cancer; Metabolomics; H nuclear magnetic resonance; Animal model

SiRNA targeting YAP gene inhibits gastric carcinoma growth and tumor metastasis in SCID mice.

Yes-associated protein (YAP) is constitutively activated in numerous types of cancer, including gastric carcinoma. The aim of the present study was to investigate the effects of YAP silencing on proliferation, apoptosis, metastasis and angiogenesis in a gastric orthotopic implantation cancer model of severe combined immunodeficiency mice. Small-hairpin RNA (shRNA) targeting the YAP gene was employed to inhibit YAP expression. SGC7901 cells transfected with YAP shRNA demonstrated significantly decreased gastric cancer growth and metastasis in the orthotopic implantation mouse model. Silencing of YAP additionally promoted tumor cell apoptosis, and inhibited tumor cell proliferation and angiogenesis. Notably, YAP shRNA also downregulated the expression of TEA domain family member 1, cyclinD1, vascular endothelial growth factor and fibroblast growth factor-2. The results of the present study suggested that YAP may have a significant role in the proliferation, metastasis and angiogenesis of gastric cancer. RNA interference-mediated silencing of YAP may provide an opportunity to develop a novel treatment strategy for gastric cancer.

Characterization of human follicular thyroid cancer cell lines in preclinical mouse models.

Follicular thyroid cancer (FTC) is the second most common type of thyroid cancers. In order to develop more effective personalized therapies, it is necessary to thoroughly evaluate patient-derived cell lines in in vivo preclinical models before using them to test new, targeted therapies. This study evaluates the tumorigenic and metastatic potential of a panel of three human FTC cell lines (WRO, FTC-238, and TT1609-CO2) with defined genetic mutations in two in vivo murine models: an orthotopic thyroid cancer model to study tumor progression and a tail vein injection model to study metastasis. All cell lines developed tumors in the orthotopic model, with take rates of 100%. Notably, WRO-derived tumors grew two to four times faster than tumors arising from the FTC-238 and TT2609-CO2 cell lines. These results mirrored those of a tail vein injection model for lung metastasis: one hundred percent of mice injected with WRO cells in the tail vein exhibited aggressive growth of bilateral lung metastases within 35 days. In contrast, tail vein injection of FTC-238 or TT2609-CO2 cells did not result in lung metastasis. Together, our work demonstrates that these human FTC cell lines display highly varied tumorigenic and metastatic potential in vivo with WRO being the most aggressive cell line in both orthotopic and lung metastasis models. This information will be valuable when selecting cell lines for preclinical drug testing.

Anionic clay as the drug delivery vehicle: tumor targeting function of layered double hydroxide-methotrexate nanohybrid in C33A orthotopic cervical cancer model.

Methotrexate (MTX), an anticancer agent, was successfully intercalated into the anionic clay, layered double hydroxides to form a new nanohybrid drug. The coprecipitation and subsequent hydrothermal method were used to prepare chemically, structurally, and morphologically well-defined two-dimensional drug-clay nanohybrid. The resulting two-dimensional drug-clay nanohybrid showed excellent colloidal stability not only in deionized water but also in an electrolyte solution of Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum, in which the average particle size in colloid and the polydispersity index were determined to be around 100 and 0.250 nm, respectively. The targeting property of the nanohybrid drug was confirmed by evaluating the tumor-to-blood and tumor-to-liver ratios of the MTX with anionic clay carrier, and these ratios were compared to those of free MTX in the C33A orthotopic cervical cancer model. The biodistribution studies indicated that the mice treated with the former showed 3.5-fold higher tumor-to-liver ratio and fivefold higher tumor-to-blood ratio of MTX than those treated with the latter at 30 minutes postinjection.

Abstract B136: Development of PD-L1/L2 over-expressing mouse liver cancer models to test the efficacy of anti-PD1 therapy for hepatocellular carcinoma

Abstract Anti-PD1 therapy has shown promising anti-tumor activities in multiple cancer types, including hepatocellular carcinoma (HCC), and PD-L1 expression in tumor cells was considered a potential predictive marker for treatment efficacy. The present study explored whether PD-L1/L2 expression may affect treatment efficacy of anti-PD1 therapy for HCC. PD-L1 (MR203953; Origene Technologies, Rockville, MD) or PD-L2 (MR222499; Origene Technologies) was transfected into the BNL-MEA murine liver cancer cell lines and stable clones were selected. Effects of PDL1/L2 over-expression were confirmed by Western blotting and immunofluorescence assay. Effects of PDL1/L2 over-expression on in vitro growth characteristics were compared by MTT, colony formation, and 3-D culture assays. Orthotopic liver cancer models were established by sub-capsular injection of parental or transfected cancer cells into the left lobe of mouse liver. The anti-tumor efficacy of anti-mouse PD1 therapy (clone 4H2, Ono Pharmaceutical Co), 0.2 mg by intraperitoneal injection on day 5, 8, 11, 14, and 17 after tumor implantation, was measured by change in tumor volume using the orthotopic liver cancer models. Distribution of T-cell sub-populations in tumor-infiltrating lymphocytes (TILs) after treatment were measured by flow cytometry. The growth characteristics of parental and PD-L1/L2-expressing BNL-MEA cells did not differ significantly, as measured by MTT, colony formation, and 3-D culture assays. Survival of animals bearing the orthotopic liver tumor with or without PD-L1/L2 over-expression was also similar. Anti-PD1 therapy appeared more efficacious in PD-L1-over-expressing liver cancer models than the parental or PD-L2-over-expressing models. Distinctive TILs distribution patterns between parental and PD-L1/L2 over-expressing liver cancer models were found after anti-PD1 therapy. Conclusions: The PD-L1/L2 expressing liver cancer models may help evaluate the efficacy and mechanisms of action of anti-PD1 therapy for HCC. Citation Format: Da-Liang Ou, Shu-Ching Chen, Yu-Chia Su, Ann-Lii Cheng, Chiun Hsu. Development of PD-L1/L2 over-expressing mouse liver cancer models to test the efficacy of anti-PD1 therapy for hepatocellular carcinoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B136.

EXPRESS: BDNF Signaling Contributes to Oral Cancer Pain in a Preclinical Orthotopic Rodent Model.

The majority of patients with oral cancer report intense pain that is only partially managed by current analgesics. Thus, there is a strong need to study mechanisms as well as develop novel analgesics for oral cancer pain. Current study employed an orthotopic tongue cancer model with molecular and non-reflexive behavioral assays to determine possible mechanisms of oral cancer pain. Human oral squamous cell carcinoma cells line, HSC2, was injected into the tongue of male athymic mice and tumor growth was observed by day 6. Immunohistological analyses revealed a well-differentiated tumor with a localized immune response and pronounced sensory and sympathetic innervation and vascularization. The tumor expressed TMPRSS2, a protein previously reported with oral squamous cell carcinoma. ATF3 expression in trigeminal ganglia was not altered by tumor growth. Molecular characterization of the model demonstrated altered expression of several pain-related genes, out of which up-regulation of BDNF was most striking. Moreover, BDNF protein expression in trigeminal ganglia neurons was increased and inhibition of BDNF signaling with a tyrosine kinase B antagonist, ANA-12, reversed pain-like behaviors induced by the oral tumor. Oral squamous cell carcinoma tumor growth was also associated with a reduction in feeding, mechanical hypersensitivity in the face, as well as spontaneous pain behaviors as measured by the conditioned place preference test, all of which were reversed by analgesics. Interestingly, injection of HSC2 into the hindpaw did not reproduce this spectrum of pain behaviors; nor did injection of a colonic cancer cell line into the tongue. Taken together, this orthotopic oral cancer pain model reproduces the spectrum of pain reported by oral cancer patients, including higher order cognitive changes, and demonstrates that BDNF signaling constitutes a novel mechanism by which oral squamous cell carcinoma induces pain. Identification of the key role of tyrosine kinase B signaling in oral cancer pain may serve as a novel target for drug development.

An Advanced Orthotopic Ovarian Cancer Model in Mice for Therapeutic Trials.

A nude mouse received subcutaneous injection of human ovarian cancer cells HO-8910PM to form a tumor, and then the tumor fragment was surgically transplanted to the ovary of a recipient mouse to establish an orthotopic cancer model. Tumors occurred in 100% of animals. A mouse displayed an ovarian mass, ascites, intraperitoneal spread, and lung metastasis at natural death. The mean survival time was 34.1 ± 17.2 days, with median survival time of 28.5 days. The findings indicated that the present mouse model can reflect the biological behavior of advanced human ovarian cancers. This in vivo model can be used to explore therapeutic means against chemoresistance and metastasis, and an effective treatment would prolong the survival time.

Abstract A191: Augmenting the therapeutic efficacy of oncolytic LCMV-GP pseudotyped vesicular stomatitis virus via modulation of the innate immune system

Abstract Introduction: Vesicular stomatitis virus (VSV) is a promising oncolytic virus candidate due to its fast mode of action, high titer production, absence of pre-existing anti-VSV immunity and broad tumor tropism. However, potential neurotoxicity of wild-type VSV and a rapid induction of neutralizing antibodies have been a hindrance for further clinical advancement. Our group previously reported that pseudotyping VSV with the LCMV glycoprotein (VSV-GP) results in complete abrogation of neurotoxicity and the absence of neutralizing antibody induction [1]. Here we addressed to what extent the innate immune response to VSV-GP limits its oncolytic efficacy and analyze means to counter these inhibiting factors. Methods: MTT cell viability and TCID50 viral replication assays were used to quantify oncolytic activity in vitro. VSV-GP oncolytic efficiency was also tested in vivo using both xenograft and syngeneic mouse models. Results: VSV-GP exhibited high oncolytic efficiency in vitro, achieving 80-100% killing at 48-72 h post infection in most ovarian, melanoma, and lung cancer cell lines. Further analysis of resistant cell lines showed that these cells were able to mount an interferon (IFN-I) induced antiviral response. VSV-GP efficacy was also tested for ovarian cancer in vivo using s.c xenograft models and in an orthotopic model. Intratumoral injection of VSV-GP into ovarian s.c. xenografts led to tumor remission in all animals. Relapse was observed and relapsing tumors remained sensitive for successive VSV-GP treatment. Significantly prolonged survival was also observed in an ovarian cancer orthotopic model using IVIS in vivo imaging. Oncolytic potency of VSV-GP was enhanced with combination treatment with Jak1/2 inhibitors. Conclusion: These results confirm that LCMV GP-pseudotyped VSV exhibits a highly beneficial toxicity and efficacy profile. However, tumors escaping complete oncolyse via innate immune system activation require coapplication of targeted immune modulators. Reference: [1] - Muik, A. et al. Re-engineering Vesicular Stomatitis Virus to Abrogate Neurotoxicity, Circumvent Humoral Immunity, and Enhance Oncolytic Potency. Cancer Res 74, 3567-3578, (2014). Citation Format: Carles Rodriguez Urbiola, Catherine Dold, Janine Kimpel, Christian Marth, Frederic Santer, Zoran Culig, Alexander Muik, Tanja Knapp, Ira Winkler, Guido Wollmann, Dorothee Von Laer, Patrik Erlmann. Augmenting the therapeutic efficacy of oncolytic LCMV-GP pseudotyped vesicular stomatitis virus via modulation of the innate immune system. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A191.

Potent efficacy of metronomic topotecan and pazopanib combination therapy in preclinical models of primary or late stage metastatic triple-negative breast cancer.

Metronomic chemotherapy has shown promising activity in numerous preclinical studies and also some phase II clinical studies involving various tumor types, and is currently undergoing phase III trial evaluation. Triple-negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options and very poor prognosis following progression after standard chemotherapeutic regimens. Herein, we evaluated the potential therapeutic impact and molecular mechanisms of topotecan administered in a continuous low-dose metronomic (LDM) manner, alone or in concurrent combination with pazopanib, an antiangiogenic tyrosine kinase inhibitor (TKI), in a triple-negative, primary and metastatic breast cancer orthotopic model; potential molecular mechanisms of efficacy were also studied, especially the impact of hypoxic conditions. The combination of metronomic topotecan and pazopanib significantly enhanced antitumor activity compared to monotherapy with either drug and prolonged survival, even in the advanced metastatic survival setting, with a marked decrease in tumor vascularity, proliferative index, and the induction of apoptosis. Significant changes in tumor angiogenesis, cancer cell proliferation, apoptosis, HIF1α levels, HIF-1 target genes and ABCG2 were found both in vitro and in tumor tissue. Notably, the pazopanib and metronomic topotecan combination treatment inhibited expression of HIF1α and ABCG2 genes in cells grown under hypoxic conditions, and this was associated with an increased intracellular concentration of the active form of topotecan. Our results suggest a potential novel therapeutic option for the treatment of metastatic triple-negative breast cancer patients.

Porphysomes: Multimodal Nanoparticle for Primary Tumor Delineation and Lymphatic Metastasis Mapping in a Head-and-Neck Cancer Rabbit Model (Adv. Healthcare Mater. 14/2015).

On page 2164, J. Chen, J.C. Irish, G. Zheng, and co-workers show how 64 Cu-porphysome nanoparticles enable superior delineation of neoplastic tissues, metastatic lymph nodes, and vascular drainage on a head and neck cancer orthotopic rabbit model by PET imaging. Their selective fluorescence activation in tumor and metastatic lymph nodes permits intraoperative fluorescence guided surgeries.

Flagellin is a strong vaginal adjuvant of a therapeutic vaccine for genital cancer

ABSTRACTCervical cancer is a high-incidence female cancer most commonly caused by human papilloma virus (HPV) infection of the genital mucosa. Immunotherapy targeting HPV-derived tumor antigens (TAs) has been widely studied in animal models and in patients. Because the female genital tract is a portal for the entry of HPV and a highly compartmentalized system, the development of topical vaginal immunotherapy in an orthotopic cancer model would provide an ideal therapeutic. Thus, we examined whether flagellin, a potent mucosal immunomodulator, could be used as an adjuvant for a topical therapeutic vaccine for female genital cancer. Intravaginal (IVAG) co-administration of the E6/E7 peptides with flagellin resulted in tumor suppression and long-term survival of tumor-bearing mice. In contrast to IVAG vaccination, intranasal (IN) or subcutaneous (SC) immunization did not induce significant tumor suppression in the same model. The vaginal adjuvant effect of the flagellin was completely abolished in Toll-like receptor-5 (TLR5) knock-out mice. IVAG immunization with the E6/E7 peptides plus flagellin induced the accumulation of CD4+ and CD8+ cells and the expression of T cell activation-related genes in the draining genital lymph nodes (gLNs). The co-administered flagellin elicited antigen-specific IFNγ production in the gLNs and spleen. The intravaginally administered flagellin was found in association with CD11c+ cells in the gLNs. Moreover, after immunization with a flagellin and the E6/E7 peptides, the TLR5 expression in gLN cells was significantly upregulated. These results suggest that flagellin serves as a potent vaginal adjuvant for a therapeutic peptide cancer vaccine through the activation of TLR5 signaling.

Multimodal Nanoparticle for Primary Tumor Delineation and Lymphatic Metastasis Mapping in a Head-and-Neck Cancer Rabbit Model.

64 Cu-porphysome nanoparticles enable superior delineation of neoplastic tissues, metastatic lymph nodes, and vascular drainage on head and neck cancer orthotopic rabbit model using positron emission tomography imaging. Additionally, the nanoparticles exhibit selective fluorescence activation in tumor and metastatic lymph nodes, which permits intraoperative real-time visualization of disease tissues to precisely define surgical margins and prevents collateral damage during surgeries.

Multimodal Imaging of Orthotopic Mouse Model of Endometrial Carcinoma.

BackgroundOrthotopic endometrial cancer models provide a unique tool for studies of tumour growth and metastatic spread. Novel preclinical imaging methods also have the potential to quantify functional tumour characteristics in vivo, with potential relevance for monitoring response to therapy.MethodsAfter orthotopic injection with luc-expressing endometrial cancer cells, eleven mice developed disease detected by weekly bioluminescence imaging (BLI). In parallel the same mice underwent positron emission tomography–computed tomography (PET-CT) and magnetic resonance imaging (MRI) employing 18F-fluorodeoxyglocose (18F-FDG) or 18F- fluorothymidine (18F-FLT) and contrast reagent, respectively. The mice were sacrificed when moribund, and post-mortem examination included macroscopic and microscopic examination for validation of growth of primary uterine tumours and metastases. PET-CT was also performed on a patient derived model (PDX) generated from a patient with grade 3 endometrioid endometrial cancer.ResultsIncreased BLI signal during tumour growth was accompanied by increasing metabolic tumour volume (MTV) and increasing MTV x mean standard uptake value of the tumour (SUVmean) in 18F-FDG and 18F-FLT PET-CT, and MRI conspicuously depicted the uterine tumour. At necropsy 82% (9/11) of the mice developed metastases detected by the applied imaging methods. 18F-FDG PET proved to be a good imaging method for detection of patient derived tumour tissue.ConclusionsWe demonstrate that all imaging modalities enable monitoring of tumour growth and metastatic spread in an orthotopic mouse model of endometrial carcinoma. Both PET tracers, 18F-FDG and 18F-FLT, appear to be equally feasible for detecting tumour development and represent, together with MRI, promising imaging tools for monitoring of patient-derived xenograft (PDX) cancer models.

Abstract 3934: Restoration of NOTCH signaling in head and neck squamous cell carcinoma inhibits tumor growth and cell migratory abilities

Abstract Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common malignancy worldwide, with roughly 300,000 cancer related deaths occurring globally each year. The survival of patients with HNSCC hasn't changed significantly over the past decade leading investigators to search for promising molecular targets. To identify new treatment targets and biomarkers that could better guide therapy, we previously characterized the genomic alterations from primary HNSCC patient samples. We were among the first to discover that NOTCH1 is one of the most frequently mutated genes in this cancer type. The spectrum of inactivating NOTCH1 mutations in HNSCC suggested a tumor suppressive role for this protein, however, the mechanism of its function is currently unknown. Procedure: We used Sanger sequencing and immunoblotting to characterize 50 well- established HNSCC cell lines as being wild-type or mutant for NOTCH1. We cloned the full length NOTCH1 receptor to restore the NOTCH signaling function and activated this pathway by culturing cells on immobilized NOTCH ligand, Jagged1, coated plates. Clonogenic assays and competitive cell proliferation assays was used to evaluate cell growth and proliferation. Transwell Boyden chambers was used to investigate migration/invasion. Tumorigenicty was evaluated in vivo in a mice orthotopic model of oral cancer. An unbiased gene expression analysis was performed to identify potential downstream targets of the NOTCH pathway. Results: We show here that restoration of NOTCH signaling in mutant HNSCC cell lines significantly decreases cell growth and migratory abilities. Activation of NOTCH1 led to decreased tumorigenicity in a mice orthotopic oral cancer model. Conversely, abrogation of the NOTCH pathway using the dominant negative form of the NOTCH co-activator, MAML1, enhanced tumor growth in a weakly tumorigenic HNSCC cell line harboring wild-type NOTCH1. To evaluate the mechanism of NOTCH induced inhibition of growth and migration, we performed an unbiased microarray analysis and observed suppression of two proto-oncogenes, AXL and CTNNAL1 in the presence of NOTCH1, but not in its absence. Restoration of NOTCH1 significantly decreases protein expression levels of AXL and CTNNAL1 which then results in decreased cell growth and migratory abilities. Furthermore, we show here a novel link between NOTCH1, AXL and CTNNAL1 via HES5, one of the canonical NOTCH downstream targets. We suggest that NOTCH1 activates HES5 which then downregulates AXL and CTNNAL1, thus inhibiting cell growth and migratory abilities. Conclusions: We have data linking NOTCH1 reactivation to suppression of proto-oncogenes, AXL and CTNNAL1 along with decreased growth and migration of HNSCC cells. This underscores the need to understand the clinical significance of the NOTCH pathway and its downstream targets which can then be exploited for potential therapeutic strategies. Citation Format: Shhyam Moorthy, Rami Saade, Curtis Pickering, David Neskey, Noriaki Tanaka, Patrick Zweidler-McKay, Mitchell Frederick, Jeffrey Myers. Restoration of NOTCH signaling in head and neck squamous cell carcinoma inhibits tumor growth and cell migratory abilities. [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 3934. doi:10.1158/1538-7445.AM2015-3934

Nidogen 1 and Nuclear Protein 1: novel targets of ETV5 transcription factor involved in endometrial cancer invasion

Endometrial cancer is the most frequent malignancy of the female genital tract in western countries. Our group has previously characterized the upregulation of the transcription factor ETV5 in endometrial cancer with a specific and significant increase in those tumor stages associated with myometrial invasion. We have shown that ETV5 overexpression in Hec1A endometrial cancer cells induces epithelial to mesenchymal transition resulting in the acquisition of migratory and invasive capabilities. In the present work, we have identified Nidogen 1 (NID1) and Nuclear Protein 1 (NUPR1) as direct transcriptional targets of ETV5 in endometrial cancer cells. Inhibition of NID1 and NUPR1 in ETV5 overexpressing cells reduced cell migration and invasion in vitro and reduced tumor growth and dissemination in an orthotopic endometrial cancer model. Importantly, we confirmed a significant increase of NUPR1 and NID1 protein expression in the invasion front of the tumor compared to their paired superficial zone, concomitant to ETV5 overexpression. Altogether, we conclude that NID1 and NUPR1 are novel targets of ETV5 and are actively cooperating with ETV5 at the invasion front of the tumor in the acquisition of an invasive phenotype to jointly drive endometrial cancer invasion.

Characterization of thyroid cancer cell lines in murine orthotopic and intracardiac metastasis models.

Thyroid cancer incidence has been increasing over time, and it is estimated that ∼1950 advanced thyroid cancer patients will die of their disease in 2015. To combat this disease, an enhanced understanding of thyroid cancer development and progression as well as the development of efficacious, targeted therapies are needed. In vitro and in vivo studies utilizing thyroid cancer cell lines and animal models are critically important to these research efforts. In this report, we detail our studies with a panel of authenticated human anaplastic and papillary thyroid cancer (ATC and PTC) cell lines engineered to express firefly luciferase in two in vivo murine cancer models-an orthotopic thyroid cancer model as well as an intracardiac injection metastasis model. In these models, primary tumor growth in the orthotopic model and the establishment and growth of metastases in the intracardiac injection model are followed in vivo using an IVIS imaging system. In the orthotopic model, the ATC cell lines 8505C and T238 and the PTC cell lines K1/GLAG-66 and BCPAP had take rates >90 % with final tumor volumes ranging 84-214 mm(3) over 4-5 weeks. In the intracardiac model, metastasis establishment was successful in the ATC cell lines HTh74, HTh7, 8505C, THJ-16T, and Cal62 with take rates ≥70 %. Only one of the PTC cell lines tested (BCPAP) was successful in the intracardiac model with a take rate of 30 %. These data will be beneficial to inform the choice of cell line and model system for the design of future thyroid cancer studies.

Circumvention of cisplatin resistance in ovarian cancer by combination of cyclosporin A and low-intensity ultrasound

Cisplatin resistance is a challenge in the treatment of ovarian cancer. The aim of this study was to explore if ultrasound can overcome chemoresistance and enhance chemosensitization due to cyclosporin A. Ultrasound and/or cyclosporin A were employed to overcome cisplatin resistance in human ovarian cancer cell line COC1/DDP. Mechanisms were explored from the perspective of: DNA damage, intracellular platinum level, detoxification, and genes related to drug efflux and DNA repair. In vivo therapeutic efficacy was validated in a short-term model (subrenal cell-clot transplantation) in mice and the survival benefit was investigated in an orthotopic cancer model in mice using HO-8910PM cells. The findings were: (i) ultrasound enhanced the effect of cisplatin leading to a lower cell-survival rate (IC50 decreased from 3.19 to 0.35μg/ml); (ii) ultrasound enhanced cisplatin via direct (increasing the intercellular level of active platinum) and indirect (decreasing the glutathione level, and expression of LRP and ERCC1 genes) mechanisms that intensified cisplatin-induced DNA damage, thus enhancing cell apoptosis and necrosis; (iii) cisplatin followed by ultrasound led to small tumor sizes in the short-term model without exacerbation of the systemic toxicity, and prolonged the survival times in the orthotopic model; and (iv) ultrasound synergized the sensitization due to cyclosporin A in vitro and in vivo. These data demonstrated that ultrasound combined with cyclosporin A overcame cisplatin resistance in ovarian cancer.

Ruthenium complex Λ-WH0402 induces hepatocellular carcinoma LM6 (HCCLM6) cell death by triggering the Beclin-1-dependent autophagy pathway.

To evaluate the anticancer mechanism of the new ruthenium complex-Λ-WH0402 at the cellular level, the in vitro cytotoxicity of Λ-WH0402 was investigated on 10 human tumor cell lines. Λ-WH0402 was found to have higher anticancer activity than cisplatin toward human liver cancer HCCLM6 cells that have high tumor metastatic characteristics. Meanwhile, Λ-WH0402 showed an antimetastatic effect on HCCLM6 cells in vitro, mostly through its effect on cell adhesion, invasion and migration. In addition, Λ-WH0402 significantly reduced tumor metastasis to the lungs in orthotopic mouse hepatocellular cancer (HCC) models induced by HCCLM6 cells. Furthermore, Λ-WH0402 exerted an inhibitory effect on tumor cell growth and proliferation and induced dose-dependent cell cycle arrest in the S phase in HCCLM6 cells. Immunoblotting analysis showed that Λ-WH0402 not only decreased the expression of antiapoptotic protein Bcl-2 and nutrient-deprivation autophagy factor-1 (NAF-1), but also significantly increased the expression of Beclin-1 in HCCLM6 cells. More importantly, we identified that Λ-WH0402 treatment reduced the interaction between Bcl-2 and Beclin-1, and increased the expression of autophagic activation marker LC3B-II in HCCLM6 cells. On the whole, our results suggested that the anitcancer activity of Λ-WH0402 is mediated through promoting the Beclin-1-dependent autophagy pathway in HCCLM6 cells.