58.15 Antioxidant Inhibition Of Steady-State Reactive Oxygen Species And Cell Growth In Neuroblastoma

Posted on December 22, 2014

B. T. Craig1, Y. Zhu2, E. J. Rellinger1, D. R. Gius2, J. Qiao1, D. H. Chung1  1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA 2Northwestern University,Radiation Oncology,Chicago, IL, USA

Introduction:  High-risk neuroblastoma (NB) is an aggressive pediatric cancer characterized by metastasis and disease relapse despite aggressive multimodality treatment protocols, which include chemo- and radiation therapy. Only a subset of high-risk NB patients will respond to treatment, and some will even progress while on treatment. Alteration of the redox homeostasis toward elevated reactive oxygen species (ROS) is a significant mechanism of action for many chemotherapeutic agents and radiation. However, the underlying ROS levels in NB and how this may contribute to treatment responses remain poorly understood. The purpose of this study was to define the steady-state ROS level in high-risk NB and to test the hypothesis that aggressive cellular behavior, which may predict treatment failure, is regulated by ROS.

Methods:  Undifferentiated (high-risk) and differentiated (low-risk) patient NB sections were assessed for steady-state ROS levels by immunohistochemical staining for 4-hydroxynonenal (4HNE), which detects intracellular lipid peroxidation, a byproduct of elevated ROS. Three well-established human NB cell lines, MYCN-amplified (BE(2)-C) and MYCN-non-amplified (SK-N-AS and SK-N-SH), subsets of high-risk, metastatic disease were examined for our study. To assess for the steady-state ROS levels, superoxide and hydroperoxide oxidation products were detected by staining for DHE and CDCFH2, using the oxidation-insensitive analog CDCF as a negative control. Cells were treated with N-acetylcysteine (NAC; 10 mM), a thiol antioxidant, daily for 5 d and assessed for cell growth (population doubling time, Td=0.693/ln(Nt/N0), Nt=cell number at time t, N0=cell number at time 0) and anchorage-independent growth (growth on 0.4%/0.8% liquid/solid agarose soft agar, 3 wk incubation, 0.05% crystal violet staining for quantification of colony formation).

Results: 4HNE expression, an indicator of lipid peroxidation, was higher in undifferentiated (high-risk) patient tumor sections as compared to differentiated (low-risk) tumor sections. Interestingly, different levels of steady-state ROS were observed between the three high-risk in vitro cell lines (Fig. A). Treatment with antioxidant NAC inhibited growth of the aggressive BE(2)-C cells (Fig. B), and significantly attenuated anchorage-independent colony growth in soft agar (Fig. C), a well-established in vitro measure of tumorigenicity.

Conclusion: We report a novel observation that different steady-state ROS levels exist among high-risk NB, and that shifting the redox balance affects aggressive cellular behavior. Our data suggest that differences in ROS level could contribute to treatment resistance in high-risk NB.

 

58.16 Rhodiola Crenulata Extract Alters HIF-1 Activity in Neuroblastoma

Posted on December 22, 2014

K. E. Wong2,4, M. C. Mora2,4, S. S. Schneider4, K. P. Moriarty1, R. B. Arenas2,4, M. V. Tirabassi1  1Baystate Children’s Hospital,Department Of Pediatric Surgery,Springfield, MA, USA 2Baystate Medical Center,Department Of Surgery,Springfield, MA, USA 4Pioneer Valley Life Sciences Institute,Springfield, MA, USA

Introduction:  Extracts derived from Rhodiola crenulata (RC), an adaptogenic plant from Tibet, is known to have anti-neoplastic properties in a variety of cancers. We have observed that RC exerts a striking cytotoxic and anti-proliferative effect on neuroblastoma cells in vitro and these effects are augmented in MYCN amplified neuroblastoma cells. The purpose of this study is to evaluate a possible mechanism by which RC causes cell death in neuroblastoma.

Methods:  NB-1691 MYCN amplified neuroblastoma cells were transfected with a DNA vector containing a hypoxia inducible factor-1 (HIF-1) promoter on a firefly luciferase reporter gene. Following stable transfection, cells were treated with either 100ug/mL RC or ethanol vehicle control for 48 hours and a dual-luciferase reporting assay was performed to evaluate HIF-1 promoter activity. Quantitative RT-PCR was performed on RNA extracted from NB-1691 cells following 24 hour treatment with RC or ethanol to evaluate the expression of HIF-1α and its’ target genes including HK2, VEGF, MCT-1 and GLUT-1. Gene expression was normalized to GAPDH. 

Results: HIF-1 transfected NB-1691 cells treated with RC exhibited an almost 50% increase in HIF-1 activity (p=0.009, figure 1A). HIF-1α mRNA level was observed to be significantly up-regulated upon treatment with RC treatment (p=0.019, figure 1B) in agreement with HIF-1 transfection results. While VEGF was also noted to be significantly up-regulated (p=0.023), VEGFa165, an inhibitory variant of VEGF, exhibited an upward trend as well. MCT-1, a cellular transporter exhibited a significant increase in expression (p<0.001, figure 1B), while other cellular transporters, including GLUT-1 exhibited no change and SLC7A-11 exhibited a downward trend. PKM2, involved in cellular glucose metabolism exhibited a signficant increase in expression (p=0.034, figure 1B) while HK2 exhibited a downward trend in activity. Of note, GAPDH amplification was noted to be slightly reduced upon treatment with RC.

Conclusion: We have observed an increase in both HIF-1 promoter activity and mRNA in response to treatment with RC suggestive of starvation of oxygen or an increase in glucose uptake.  While several HIF1α targets were up-regulated with RC treatment, the notable exceptions were genes responsible for uptake of the nutrients needed to drive glycolysis. Taken together we hypothesize that the increased activity of HIF-1 reflects a starvation response  which is unable to rescue the tumor cells due to lack of nutrients and  leads to the rapid cell death we have observed previously.  Future studies will evaluate the nutrient uptake, respiratory and glycolytic function in RC treated neuroblastoma cells. 

 

58.17 The Macrophage Inhibitor CNI-1493 Prevents Ewing’s Sarcoma Tumor Cell Extravasation

Posted on December 22, 2014

A. J. Hesketh1,2,3, C. A. Behr1,2,4, M. Edelman1,4, R. D. Glick1,4, Y. J. Al-Abed2,3, M. J. Symons2,3, B. M. Steinberg2,3,4, S. Z. Soffer1,2,4  1Cohen Children’s Medical Center,Division Of Pediatric Surgery,New Hyde Park, NY, USA 2Feinstein Institute For Medical Research,Manhasset, NY, USA 3Elmezzi Graduate School Of Molecular Medicine,Manhasset, NY, USA 4Hofstra North Shore-LIJ School Of Medicine,Hempstead, NY, USA

Introduction: Metastatic Ewing’s Sarcoma (ES) carries a poor prognosis. Recent evidence demonstrates that tumor-associated macrophages in ES are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. Preliminary in vitro and animal model studies demonstrate that the macrophage inhibitor CNI-1493 prevents ES tumor cell invasion and lung metastases. We hypothesized that CNI-1493 inhibits M2 macrophage-stimulated ES tumor cell extravasation into the lung parenchyma, a key step in the establishment of viable metastatic foci.

 

Methods: To simulate the architecture of the lung extravasation process, in vitro extravasation assays were constructed using basement membrane-coated cell culture inserts featuring an opaque polyethylene membrane containing 8μm pores. A monolayer of primary human pulmonary microvascular endothelial cells (HPMECs) was plated on the surface of the basement membrane and impermeability was verified with Evans blue. Primary macrophages isolated from human blood were polarized to express the M2 phenotype by treatment with M-CSF followed by activation with IL-4. Fluorescently tagged human ES cells (SK-NEP) were grown in monoculture or in co-culture with M2 macrophages in the upper chamber of the cell culture insert. Alternatively, M2 macrophages were plated on the undersurface of the cell culture insert with SK-NEP cells in the upper chamber. After 48 hours, tumor cells that had invaded through the endothelial monolayer, basement membrane and culture insert into the lower chamber were quantified using fluorescence microscopy.

 

Results: Tumor cell extravasation was increased 3.4-fold in the presence of M2 macrophages (p<0.05) with no significant difference between SK-NEP/M2 co-cultures and cultures separated by the assay insert (Table 1).  CNI-1493 significantly decreased the quantity of invading tumor cells stimulated by M2 macrophages (p<0.01), and the degree of this inhibition was similar whether an HPMEC monolayer was present or not (54% and 58% reduction, respectively).

 

Conclusion: M2 macrophages promote tumor cell extravasation in vitro. This interaction is independent of direct tumor cell contact, suggesting soluble mediators. CNI-1493 decreases M2-induced tumor cell extravasation independent of the presence of an endothelium, indicating a direct effect on macrophages expressing pro-metastatic phenotypes. CNI-1493 may be useful as a novel adjunct in the treatment or prevention of metastatic ES.

58.18 Evaluating Glycogen Synthase Kinase-3 isoforms as a Therapeutic target in Neuroblastoma

Posted on December 22, 2014

M. Balamurugan1, S. Kunnimalaiyaan1, T. C. Gamblin1, M. Kunnimalaiyaan1  1Medical College Of Wisconsin,Division Of Surgical Oncology,Milwaukee, WI, USA

Introduction: Neuroblastoma (NB) is a highly malignant neuroendocrine tumor (NET) with high recurrence. NB is very rare in adults but usually present in early childhood.  NETs express high levels of achaete-scute complex-like1 (ASCL1) protein and chromogranin A (CgA) peptide, two important NET markers. Despite recent advances, about 60% of patients with high-risk NB will have a recurrence and treatment options for these patients are limited. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target, as this pathway has been shown to be crucial in the management of other NETs. Recently, we have reported that treatment with GSK-3 inhibitor reduced growth of NB and it is associated with significant reduction in phosphorylation of GSK-3 alpha compared to GSK-3 beta.  However, the role of GSK-3 isoforms on growth remains unclear. To understand the role of GSK-3 isoforms, we investigated the growth regulatory effects by depletion of each GSK-3 isoform individually in neuroblastoma.

Methods: SK-N-AS cells were transfected with shRNA of GSK-3 beta or alpha and selected on puromycin containing media to create GSK-3 alpha knockdown (GSK-3α- KO) or GSK- beta knockdown (GSK-3β-KO).  As a control GSK-3-NC cells were created using shRNA against no-target, non-specific scrambled sequence. ShRNA mediated knockdown of GSK-3α or GSK-3β were confirmed at both mRNA and proteins levels by quantitative real time polymerase chain reaction (qPCR) and Western analysis respectively. Cellular proliferation of GSK-3α-KO or GSK-3β-KO cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, the effect of GSK-3 depletion in SK-N-AS cells on the levels of NET markers (ASCL1 and CgA), as well as cell cycle regulatory protein (cyclinD1) were determined by western blot.

Results: SK-N-AS cells transfected with shRNA against GSK-3 isoform (GSK-3α-KO or GSK-3β-KO) showed significant reduction in GSK-3 beta or alpha mRNA and protein. Cellular proliferation assay on GSK-3α-KO or GSK-3β-KO showed differential growth reduction compared to no-target, non-specific scrambled sequence (GSK-3-NC).  The growth suppression effect by knockdown of GSK-3 is associated with decrease in cyclinD1.  Importantly, knockdown of GSK-3 activity resulted in attenuation of NET markers ASCL1 and CgA.

Conclusion:Knockdown of GSK-3 beta in SK-N-AS cells partially inhibited the growth compared to GSK-3 alpha knockdown suggesting that GSK-3 alpha may play a role in cellular proliferation. Therefore, our findings warrants further investigation on the role of GSK-3 alpha in neuroblastoma.

 

58.01 Enhanced Chemosensitivity of Ovarian Cancer Cells via EZH2 Knockdown and Down-regulation of mTOR

Posted on December 22, 2014

R. B. Batchu1,2, O. V. Gruzdyn1,2, A. M. Qazi1,2, J. Kaur1,2, P. Konka1,2, D. W. Weaver1, S. A. Gruber1,2  1Wayne State University,Surgery,Detroit, MI, USA 2John D. Dingell VA Medical Center,Detroit, MI, USA

Introduction:  Although combination chemotherapy often achieves an initial clinical response, tumor relapse with chemoresistance still remains a major problem in epithelial ovarian cancer (EOC). EZH2, a tumor-promoting methyltransferase, is abnormally elevated in EOC, and mTOR is known to be responsible for chemoresistance. PRAS40 is a regulatory protein which, in its active, un-phosphorylated form, binds to and inhibits the mTOR enzymatic complex (mTORC1). We have previously demonstrated a direct correlation between EZH2 over-expression and chemoresistance in EOC. Here we further elucidate the mechanisms involved in this process by evaluating whether knockdown of EZH2 by RNA interference inhibits mTOR signaling via PRAS40, hypothesizing that this will increase the chemosensitivity of EOC cells. 

Methods:  Standard protocols were followed for propagation of the SKOV-3 EOC cell line, as well as for Western blot, co-immunoprecipitation, and gene-profile analysis. 

Results: Either 1 mm paclitaxel exposure or EZH2-shRNA transfection resulted in a 60% decrease by day 3 (both p<0.02) and 80% decrease by day 5 (both p<0.01) in the number of live EOC cells vs. control, while combined treatment resulted in further significant decreases when compared with either treatment alone on both days (all p<0.02). EZH2 knockdown did not change the levels of mTOR, phosphorylated mTOR (p-mTOR), or PRAS40 by both Western blot and gene- profile analysis (Fig. 1). However, phosphorylation of PRAS40 at its threonine 246 residue was significantly reduced, thereby facilitating interaction with, and inhibition of, mTORC1. This interaction was further confirmed by co-immunoprecipitation experiments demonstrating low levels of unbound, phosphorylated PRAS40. 

Conclusion: Our results demonstrate that knockdown of EZH2 enhances the chemosensitivity of paclitaxel, and enhanced interaction of mTOR and PRAS40 results in inhibition of the former, contributing to tumor cell apoptosis. This work provides further insight into the anti-tumor effects of EZH2 knockdown, a potential therapeutic intervention for EOC. 

 

58.02 SPATA20 Expression is Associated with Rectal Cancer Pathologic Response to Neoadjuvant Chemoradiation

Posted on December 22, 2014

L. C. Duraes1,2, G. Gantt1,2, J. DeVecchio2, A. Mace1,2, G. Karagkounis1,2, L. Thai1,2, M. F. Kalady1,2  1Cleveland Clinic,Colorectal Department,Cleveland, OH, USA 2Cleveland Clinic,Department Of Stem Cell Biology And Regenerative Medicine – Lerner Research Institute,Cleveland, OH, USA

Introduction:

Patients with a pathologic complete response (pCR) to neoadjuvant chemoradiation have improved oncologic outcomes.  Unfortunately only about 20% of patients achieve pCR and ways to improve this number remain elusive.  Our laboratory studies genetic factors that may provide insight to the biology underlying response to treatment.  Preliminary work by our group has suggested that spermatogenesis associated 20 (SPATA20), which is a gene important for cell differentiation, multicellular organismal development, and spermatogenesis, may be associated with poor outcome in rectal cancer.  The purpose of this study is to determine the association between SPATA20 expression and response to neoadjuvant treatment in rectal cancer patients.

Methods:

Thirty-three rectal adenocarcinoma patients treated with neoadjuvant chemoradiation had pretreatment tumor biopsies snap frozen according to an IRB-approved protocol. Total tumor mRNA was extracted from the biopsies and gene expression was determined using high-throughput microarrays on an Illumina platform. Chemoradiation response was evaluated based on American Joint Committee on Cancer (AJCC) criteria (0 – complete response; 1 – small group of tumor cells; 2 – residual cancer outgrown by fibrosis; 3 – minimal tumor kill) and correlated with gene expression levels.  Gene expression levels as determined by microarray were validated using quantitative real-time PCR (qPCR). Protein expression was analyzed using immunofluorescence. Statistical analysis was performed and p<0.05 was considered significant.

Results:

SPATA20 expression was significantly decreased in complete responders (AJCC 0) compared to partial and non-responders (AJCC 1-3); and increased in non-responders (AJCC 3) compared to partial and complete responders (AJCC0-2) on microarray analysis. These results were further validated by qPCR (p<0.05) (figure).  Furthermore, SPATA20 protein levels were also decreased in complete responders and most elevated in non-responders, as measured by protein immunofluorescence.

Conclusion:

SPATA20 may serve as a novel biomarker in predicting rectal cancer response to chemoradiation.  Further prospective validation and exploration into the biological mechanism of how it may contribute to treatment resistance is warranted.

58.03 The Clonal Evolution of Metastatic Colorectal Cancer

Posted on December 22, 2014

J. G. Grossman1, C. Maher2,4, B. S. White2,4, A. C. Lockhart3,4, T. Fleming1,3, K. Lim3,4, B. Goetz3, E. Pittman1, S. M. Strasberg1,3, D. C. Linehan1,3, W. Hawkins1,3, S. P. Goedegebuure1,3, E. Mardis2,5, R. K. Wilson2,3,5, T. Ley2,4,5, R. C. Fields1,3  1Washington University,Department Of General Surgery,St. Louis, MO, USA 2Washington University,The Genome Institute,Saint Louis, MO, USA 3Alvin J. Siteman Cancer Center,Saint Louis, MO, USA 4Washington University,Department Of Medicine, Oncology Division,Saint Louis, MO, USA 5Washington University,Department Of Genetics,Saint Louis, MO, USA

Introduction:
Colorectal cancer is the second leading cause of cancer mortality in the United States, and death from CRC occurs via sequelae of metastases.  Our lack of understanding of mechanisms of metastasis formation has prevented the identification and direct targeting of pathways necessary for growth and survival of metastaseis.   Next-generation sequencing gives us the capability to better study the evolutionary biology of metastasis, however a comprehensive analysis comparing matched primary and metastatic colorectal tumors has not been performed to date.  Our group in collaboration with the Washington University Genome Institute is currently analyzing 10 patients’ primary and metastatic tumors by whole genome and transcriptome sequencing.  We present the evolution of the clonal relationships of primary and metastatic tumors from three cases completed to date. 

Methods:
Patients with metastatic colorectal cancer were consented, and primary tumor and liver metastases were procured during operative resection. Additionally, uninvolved colon, uninvolved liver, and peripheral blood were collected (germline controls). If necessary, tissue from prior resections was obtained from paraffin blocks.  Deep exome and WGS were used to calculate variant allele frequency (VAFs) for the somatic single nucleotide variants. We chose to incorporate deep exome sequencing thereby enabling us to more accurately calculate VAFs, which in turn improves our ability to reconstruct the clonal architecture. To accomplish this, we used SciClone, a tool developed at Washington University Genome Institute for identifying VAF clusters via variational Bayesian Beta mixture modeling.

Results:
Among the primary tumors prevalent somatically altered genes were APC, TP53, KRAS, PIK3CA, and SMAD4. Upon evaluating the clonal evolution from primary to metastases, phylogenetic trees were generated for each patient illustrating tumor similarities and differences to each other and in relation to normal tissue. Additionally, the clonal evolution from primary to metastases was mapped and clearly shows every tumors’ subclonal makeup and how they relate to one another. Every patient had a dominant clone originating from the primary tumor that was present in all of its corresponding metastases. However, unique subclones appear to arise in all metastatic samples. In some cases, these subclones are shared among various metastases of the same patient, and these similarities may be due to the spatial or temporal proximity  of the tumors.

Conclusion:
Exploring the clonal evolution from primary tumor to metastasis provides a greater understanding of cancer biology, and further elucidates the importance of tumor heterogeneity.  Continued investigation is necessary to evaluate which subclones may be biologically relevant to disease progression and treatment  resistance. In addition, future identification of altered genes associated with metastatic clones may lead to targeted cancer therapies. 
 

58.04 NPTX2 Downregulation is Associated with Sensitivity to Chemoradiation in Rectal Cancer

Posted on December 22, 2014

G. Karagkounis1,2, J. DeVecchio2, L. Thai1,2, L. C. Duraes1,2, G. A. Gantt1,2, M. F. Kalady1,2  1Cleveland Clinic,Colorectal Surgery,Cleveland, OH, USA 2Cleveland Clinic,Stem Cell Biology And Regenerative Medicine,Cleveland, OH, USA

Introduction:  Neoadjuvant chemoradiation (CRT) is the standard of care for locally advanced rectal cancer. Response is highly variable, from complete pathologic response to no treatment effect. The mechanisms behind CRT resistance remain unclear and the paucity of pretreatment predictors of response leads to a significant proportion of patients undergoing therapies from which they may derive minimal or no benefit. The goal of this study was to identify gene expression profiles associated with rectal cancer resistance to CRT.

Methods:  Freshly frozen pretreatment rectal adenocarcinoma biopsies were collected according to an established IRB-approved protocol. Thirty-three patients who underwent standard long course neoadjuvant treatment including 5-FU and external beam radiation were identified. Post-treatment resection specimens were evaluated for response based on American Joint Committee on Cancer (AJCC) criteria. Total tumor mRNA was extracted from pretreatment biopsies and gene expression levels were determined using high-throughput microarrays on an Illumina platform. Gene expression levels between complete responders (AJCC 0) to partial and non-responders (AJCC 1-3) were compared using non-parametric Wilcoxon test. Real-time quantitative PCR (RT-qPCR) was used to validate microarray gene expression levels in the same sample set.

Results: Neuronal pentraxin 2 (NPTX2), a gene normally involved in neuronal development and recently implicated in renal cell carcinoma progression, was found to be significantly downregulated among complete responders (AJCC 0) compared to partial and non-responders (AJCC 1-3) by microarray analysis (fold change 29.8, p=0.02). NPTX2 downregulation among complete responders was confirmed by RT-qPCR (p=0.012), with gradually increasing NPTX2 expression levels through the different AJCC grades (p=0.029).

Conclusion: NPTX2 is relatively under expressed in human rectal adenocarcinomas that are sensitive to neoadjuvant CRT. As response to CRT is a strong predictor of oncological outcomes, NPTX2 expression may serve as an early prognostic biomarker and could serve as a potential target for CRT sensitization. These findings provide an opportunity for further studies to elucidate its biological role in rectal cancer.

58.05 The H1047R Mutation in p110 Alpha Increases Filopodia Formation of Human Colon HCT116 Cancer Cells

Posted on December 22, 2014

A. Rajput1, G. Wan1, A. Rajput1  1University Of New Mexico HSC,Surgical Oncology/Surgery,Albuquerque, NM, USA

Introduction:
Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase through which a number of receptor tyrosine kinases signal. Gain of function mutations in the catalytic subunit p110α (PIK3CA) of Class 1A PI3K occur in up to one-third of human colorectal cancers (CRC),  and result in dysregulation of cell signaling.  Our group has previously shown that in in vivo orthotopic models, that human colon cancer cell lines bearing the H1047R point mutation in p110α are more metastatic than cells carrying wild type p110α. The purpose of this study was to compare changes in cell morphology in wild type and mutant PI3K bearing cells. We hypothesized that the H1047R mutation in PI3K would result in rearrangement of the cytoskeleton and thus morphology and cell motility.

Methods:
1) HCT116 cells with either the WT or the MUT PIK3CA allele alone were grown and fixed on coverslips. Following permeablization, F-actin was labeled by AlexaFluorR 488 phalloidin and confocal images of F-actin labeled WT and MUT cells were acquired. The cell morphology was analyzed by Image J and the degree of difference between the structures of actin filopodia in WT and MUT cells was assessed using a custom Matlab script. The actual length of the cell border between the start and end points of these line segments was then measured. The ratio between the sum of the lengths of the 5µm line segments and the total cell border length, R (eq 1), is a measure of the surface roughness of the cell, which corresponds to the number of filopodia present. Therefore, an R-value close to 1 indicates a smooth surface, whereas an R-value close to 0 is a rougher surface. 2) F-actin level was measured by Flow Cytometer and 3) cell motility was tested by wound healing assay.

Results:
HCT116 H1047R MUT bearing cells compared to WT PIK3CA cells demonstrated increased levels of PIP3 which is reflective of gain of PI3K function. There was also a decrease in F-actin levels in MUT vs. WT bearing cells. MUT bearing cells also demonstrated significantly increased numbers of cell filopodia as shown in Figure 1. Wound healing assay demonstrated increased cell motility in MUT as compared to WT cells.

Conclusion:
Our findings indicate that the H1047R mutation reorganizes actin structure; generates higher PIP3 levels and decreases F-actin levels. This function possibly contributes to the enhanced migratory capacity of HCT116 MUT cells. Our results further confirmed that the accumulation of PIP3 and change in the appearance of cytoskeleton of cells are important aspects in regulating cell motility and therefore possibly metastasis. Thus the PI3K pathway remains a desirable therapeutic target for patients with colorectal cancer.
 

58.06 Conditional Mouse Model of Colon Cancer Using Adenoviral Delivery with Murine Colonoscopy

Posted on December 22, 2014

J. W. Harris1, P. Rychahou1, M. Evers1  1University Of Kentucky,Department Of General Surgery,Lexington, KY, USA

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer death in the US. The K-ras gene is mutated in 30-50% of patients with CRC; mutations in the gene encoding p53 acquire oncogenic properties that enable them to promote invasion, metastasis, proliferation and cell survival and are present in up to 50% of CRCs.  The purpose of this study was to develop a novel model of de novo CRC through activation of oncogenic K-ras and the loss of function of p53 in combination with the intracolonic delivery of adenovirus.
 

Methods: K-ras/p53 mutant mice (3 male, 3 female) received an intracolonic submucosal injection of 30 µL (3×107) Ad-CMV-Cre adenovirus under endoscopic guidance using a high resolution mouse video endoscopic system. The mice underwent surveillance endoscopy and were sacrificed 8 wks after initial adenovirus injection.  Endoscopy video and images were recorded for technical evaluation and confirmation of tumor formation. Colonic tissues were sectioned and stained with H&E to confirm histologic presence of tumor.
 

Results: K-ras/p53 mutant mice do not spontaneously develop de novo CRCs without exposure to an oncogenic vector, nor do wild type mice typically develop CRCs when exposed to adenovirus. De novo CRC was noted in all male mice (n=3); one mouse developed a colo-cutaneous fistula, one developed an obstructing tumor, and the last mouse had a nearly obstructing tumor. H&E staining confirmed the presence of CRC in the male mice. In contrast, none of the female mice developed tumors.

Conclusion: Our technique using adenovirus to establish a primary intracolonic mucosal CRC is novel and reproducible. This conditional orthotopic model is important to better investigate genetically induced tumors and targeted therapeutics in their natural environment. Further research is required to better address the impact of genetic mutations and gender differences of CRC development in animal and human studies.

 

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