45.12 ANXA2 High Expression is Associated with Poor Prognosis in Pancreatic Ductal Adenocarcinoma

Posted on January 31, 2019

H. Takahashi1, E. Katsuta1, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA

Introduction:

Annexin A2 (ANXA2) is a member of the annexin family, which is a calcium-dependent phospholipid binding protein and plays a major role in the regulation of cellular growth as well as signal transduction pathways. Intracellular ANXA2 is known to be involved in exocytosis, endocytosis, membrane trafficking, cellular cytoskeleton, and cell division. In cancer tissue, ANXA2 plays a crucial role in angiogenesis, proliferation, cell migration, invasion, and adhesion. In addition, its higher expression is associated with worse prognosis in various malignancies. However, ANXA2 role in pancreatic ductal adenocarcinoma (PDAC) remains under investigation. In the present study, we aimed to study ANXA2 expression and prognosis in patients with PDAC using publicly available large data set The Cancer Genome Atlas (TCGA).   

Methods:

Genomic and clinical data of patients with PDAC were obtained from TCGA through cBioportal. Patients were classified into two groups based on ANXA2 mRNA expression level using higher tertile. Survival analysis and gene set enrichment analysis (GSEA) were conducted between the ANXA2 high and low expression groups. 

Results:

There were 147 patients with clinical and genomic information associated with PDAC in TCGA. The mean age of the cohort was 64.7 years old, with 79 (54%) patients being male. The numbers of patient on each stage of the American Joint Committee on Cancer (AJCC) were as followings; 12 patients for Stage I, 128 for Stage II, three for Stage III, and three for Stage IV. Forty-nine and 98 patients expressed ANXA2 high and low, respectively. There was no significant difference in the ANXA2 expression level among each AJCC stage (p=0.531). However, the ANXA2 high expression group demonstrated significant worse overall survival (OS), compared to the low group (median survival time: 12.5 months vs 20.6 months, p=0.004). GSEA was conducted in order to investigate the cause of worse prognosis in ANXA2 high expressing tumors. Interestingly, the ANXA2 high group enriched metabolism related gene sets, such as glycolysis (NES: 1.80, p=0.016), pentose phosphate pathway (NES: 1.58, p=0.035), and pyrimidine metabolism (NES: 1.58, p=0.038). Furthermore, DNA repair (NES: 1.78, p=0.011) and base excision repair (NES: 1.62, p=0.036) gene sets were also enriched in the same group. These findings suggest that ANXA2 high expressing tumors have more altered metabolism and DNA damage, which result in poor prognosis.

Conclusion:

High expression of ANXA2 in PDAC is associated with worse survival. It might be due to altered metabolism and DNA damage in PDAC.

45.11 Sphingosine-1-phospate Signaling Regulates Drug Resistance Mediated by Glutathione

Posted on January 31, 2019

M. Nagahashi1, J. Tsuchida1, K. Moro1, M. Ikarashi1, M. Nakajima1, M. Abe2, T. Saito3, M. Komatsu3, T. Soga4, K. Takabe5,6, K. Sakimura2, T. Wakai1  1Niigata University Graduate School of Medical and Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Brain Research Institute, Niigata University,Department Of Animal Model Development,Niigata City, NIIGATA, Japan 3Niigata University Graduate School of Medical and Dental Sciences,Department Of Biochemistry,Niigata City, NIIGATA, Japan 4Keio University,Institute For Advanced Biosciences,Shonai City, YAMAGATA, Japan 5Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 6University at Buffalo Jacobs School of Medicine and Biomedical Sciences, the State University of New York,Department Of Surgery,Buffalo, NY, USA

Introduction:
It is known that the unique metabolisms that cancer cells develop, such as “Warburg effect”, is linked with drug resistance. However, the underlying regulatory mechanisms of those metabolisms have not been fully understood. A pleiotropic bioactive lipid mediator, sphingosine-1-phosphate (S1P), produced by sphingosine kinases (SphK1 and SphK2), regulates many physiological and pathological processes, such as cell proliferation, migration, survival, and metabolism. Previously we have demonstrated that S1P and SphK2 play important roles in liver lipid metabolism (Hepatology 2015). We have also reported that expression of SphK1 is higher in cancer than normal breast tissue (J Surg Res 2016), and it associates with lymph node metastasis and worse prognosis in breast and gastric cancer patients (J Surg Res 2016, Surgery 2018). Based on these findings, we hypothesized that S1P and SphKs regulate cancer cell-specific metabolism, which related to the cancer cell survival and drug resistance.

Methods:
We used E0771 murine breast cancer and PAN02 murine pancreatic cancer cell lines, and SphK1 or SphK2 knock-out (KO) cells were generated with a CRISPR/Cas9 gene editing system. Metabolic changes in SphK1KO, SphK2KO, and their corresponding wild type (WT) cells were analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Cytotoxic assays with chemotherapeutic drugs were performed with WST-8. Quantitative reverse transcription PCR (RT-qPCR) was used for quantification of mRNA expression in the cells.

Results:
CE-TOFMS analysis revealed that different roles of SphK1 and SphK2 in cancer cell metabolism both in E0771 and PAN02 cell lines. Metabolic pathways, including glycolysis, purine metabolism, TCA cycle, and urea cycle were altered in SphK1 or SphK2KO both in PAN02 and E0771 cells. Moreover, SphK2KO E0771 cells showed significantly higher amount of glutathione (GSH), which is known to be associated with anti-oxidative stress and drug resistance, as compared with normal control cells. Importantly, gene expression of xCT, the transporter of the cysteine, was found to be significantly higher in SphK2KO E0771 cells than in WT. Similar results were also found in SphK2KO PAN02 cells, where GSH were significantly higher than the WT cells. On the other hand, GSH was less in SphK1KO compared with WT. Finally, SphK2KO cells, which is known to have compensatory high expression of SphK1, demonstrated stronger resistance to chemotherapies compared with WT cells. Taken together, our results indicate that S1P and SphK1 are associated with drug resistance, which is mediated by xCT expression and GSH production.

Conclusion:
S1P and SphKs play an important role in regulation of cancer-specific metabolism, which strengthen resistance to oxidative stress and cancer cell survival mediated by xCT and GSH.
 

45.10 Effects of TGF-β on ARID1a expression in pancreatic cancer cell lines

Posted on January 31, 2019

M. A. Alvarez1, S. M. Husain1, P. V. Dickson1,2, J. L. Deneve1,2, D. Shibata1,2, K. W. Freeman1, E. S. Glazer1,2  1University of Tennessee Health Science Center,General Surgery,Memphis, TN, USA 2UT West Cancer Center,Memphis, TN, USA

Introduction: The loss of the tumor suppressor gene ARID1a is correlated with worse oncologic outcomes in a variety of malignancies. In pancreatic ductal adenocarcinoma (PDAC), decreased ARID1a expression has been associated with increased tumor size and worse cellular differentiation. TGF-β has paradoxical functions and can be either tumor promotive or suppressive depending on disease stage. There is little data, however, on the interaction of TGF-β and ARID1a in PDAC. We hypothesize that loss of ARID1a expression in PDAC is modulated by TGF- β and is associated with decreased survival.

Methods: The Cancer Genome Atlas (TCGA) data set was used to investigate 165 unique patients with PDAC and greater than 1-day follow-up. Patients were divided into top, median and bottom quartiles based on the level of ARID1a gene expression. Survival trends were analyzed between the three groups using Wilcoxon test. In order to investigate the effects of TGF- β on ARID1a expression, Panc-1 cells (derived from primary PDAC), gemcitabine-resistant Panc-1 cells (selected by gemcitabine exposure), or Capan-1 cells (derived from metastatic PDAC) were treated with recombinant TGF- β (10 ng/mL) vs. PBS control. Cells were treated twice over 1 week with pre- and post-treatment assessments of ARID1a protein expression by Western Blot analysis.

Results: In our TCGA analysis, patients with low ARID1a expression had the shortest median survival (MS;15.8 months), whereas those with high expression had the longest MS (23.2 months), and patients with median expression demonstrated an intermediate MS (20 months, p=0.01). In our in vitro studies, ARID1a protein expression was lowest in Capan-1 cells, highest in the Panc-1 cells, and intermediate in the gemcitabine-resistant Panc-1 cells. TGF-β treatment resulted in reduced expression of ARID1a in gemcitabine-resistant Panc-1 cells and Capan-1 cells, but not in normal Panc-1 cells.

Conclusion: High expression of ARID1a, a known tumor suppressor gene was associated with a survival advantage in PDAC patients. We have demonstrated that TGF-β downregulates ARID1a in metastatic and gemcitabine-resistant PDAC cells, but not in untreated Panc-1 cells. Our data suggest that regulation of ARID1a may contribute to the oncogenic effects of TGF-β in drug resistant and metastatic PDAC. Further work will investigate the mechanistic connection between TGF-β and ARID1a to identify potential future therapeutic targets.

45.09 Role of junction plakoglobin, filaggrin, and dystonin in the survival and growth of melanoma

Posted on January 31, 2019

K. M. Leick1,2, K. Du1, M. Parlak1, T. Abbas1, V. H. Engelhard1, C. L. Slingluff1  1University of Virginia,Charlottesville, VA, USA 2University of Iowa,Iowa City, IA, USA

Introduction:

We have previously reported that survival of patients with melanoma is reduced when melanomas express a set of genes including filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), which encode proteins that mediate mechanical barrier function in normal skin. To determine whether these genes have a causative role in melanoma progression, rather than just being markers associated with poor outcome, we evaluated their effects on melanoma growth in culture and in mice following their deletion using CRISPR/Cas9 or ectopic expression by lentivirus transduction. We hypothesized that overexpression of FLG, DST, PKP3, and JUP may directly enhance melanoma cell growth in vitro and decrease host survival in a murine model, while their deletion will decrease tumor burden and improve survival.

Methods:

Co-knockout of both FLG and DST using CRISPR/Cas9 and sgRNA and control Empty Vector (EV) was performed in human DM93 melanoma cells and in murine B16-F1 melanoma cells. Proliferation of cultured control DM93 EV and sgFLGDST was measured by MTT assay (n=1), and B16-F1 EV and sgFLGDST tumors were inoculated subcutaneously into mice (n=3). Overexpression models of PKP3 and of JUP were also generated by lentiviral transduction of murine B16-AAD melanoma cells, which were implanted intraperitoneally into mice. Tumors were harvested at day 14 and weighed (n=3) to assess tumor burden.

Results:

In vitro studies with human DM93 melanoma demonstrated decreased proliferation after FLG/DST knockout (Figure 1a), suggesting that these genes confer a tumor growth advantage for human melanoma. Similarly, FLG/DST knockout in murine B16-F1 led to significantly decreased tumor burden compared to control in vivo (p=0.02, Figure 1b). Also, overexpression of JUP in murine B16-AAD melanoma resulted in significantly greater tumor burden compared to control (p=0.0001) and compared to PKP3 overexpression (p < 0.0001); however, PKP3 overexpression did not significantly impact tumor growth (Figure 1c).

Conclusion:

FLG, DST, and JUP appear to support melanoma cell growth and survival both in vitro and in vivo. These findings raise the possibility that overexpression of these selected genes may activate pro-survival or proliferation pathways, create stronger intercellular junctions, or may support immune cell exclusion from melanomas. Therefore, FLG, DST, and JUP warrant further study to identify regulatory targets of these genes in order to intervene on their expression to confer clinical benefit.

45.08 Neutrophil Extracellular Traps Regulate Mitochondrial Quality Control in Cancer Cells to Promote Tumor Growth

Posted on January 31, 2019

H. Yazdani1, R. Simmons1, A. Tsung1, S. Tohme1  1University Of Pittsburgh,General Surgery,Pittsburgh, PA, USA

Introduction: Evidence is accumulating that neutrophil extracellular traps (NETs) are present within tumors and confer a worse outcome. The exact mechanisms of their role in tumor progression have been far from answered.

Methods: Preoperative serum and tumor tissue were obtained from colorectal cancer liver metastases (CCLM) patients. In vivo, subcutaneous and liver metastasis tumor models were done in WT and nu/nu mice. DNase injection or PAD4KO mice were used to suppress NETs. Hepatocellular and colon murine and human cancer cell lines were used. In vitro, human and mice neutrophils were isolated and stimulated with PMA to form NETs to treat multiple cancer cell lines and examine NETs' effect on their bioenergetics.

Results:Tumors from CCLM patients showed increased NETs. Preoperative serum MPO-DNA, a NET marker, was found to be significantly elevated in CCLM patients compared to healthy controls. Patients with high MPO-DNA levels had significantly shorter overall and disease-free survival than patients with low MPO-DNA. The median disease-free survival was 5.8 times shorter (5.5 vs 31 months, p<0.001). In vitro, HMGB1 and chemokines released from stressed tumor cells can attract neutrophils and induce NETs. In vivo, we compared the growth rates of tumors in the subcutaneous and liver metastases models in C57BL/6 WT and PAD4KO mice. Tumors grew much slower and were significantly smaller in PAD4 KO mice. Similarly, human cancer cell lines HCT116 and Huh7, grew slower in DNAse-treated nu/nu mice. PAD4 KO or DNAse treated tumors showed decreased proliferation and increased in apoptosis and oxidative stress. These tumors had decreased mitochondrial density and mitochondrial DNA and lesser degree of ATP production. There was also a significant decrease in mitochondrial biogenesis proteins PGC-1α, TFAM and NRF-1 in PAD4KO tumors. In vitro, cancer cells treated with NETs showed a significant upregulation in the mitochondrial biogenesis associated genes, increased mitochondrial density and increased ATP production compared to control. NETs significantly enhanced the percentage of cancer cells with reduced mitochondrial membrane potential and increased the oxygen consumption rate. Furthermore, NETs increased cancer cell’s expression of fission and fusion associated proteins DRP-1 and MFN-2 and expression of mitophagy-linked proteins, PINK1 and Parkin. All of which were decreased in PAD4KO tumors. Mechanistically, we demonstrated that neutrophil elastase (NE) released from NETs activates TLR-4 on cancer cells that in turn phosphorylates p38 and activates PGC-1α to upregulate mitochondrial biogenesis and tumor growth.

Conclusion:NETs actively recruited to the tumor microenvironment can alter the metabolic programming of cancer cells by upregulating biogenesis and maintain mitochondrial fission, fusion and mitophagy which result in increased tumor growth. NETs represent a promising therapeutic target to halt cancer progression.

 

45.07 Role Of Epigenetic Alteration In CD8+ T-cells Associated With Hepatocellular Carcinoma

Posted on January 31, 2019

T. M. Dawud1, E. Kimchi1,2, J. Kaifi1,2, G. Li1,2, D. Avella1,2, K. Staveley-O’Carroll1,2  1Department of Surgery, University Of Missouri Columbia,Columbia, MO, USA 2Ellis Fischel Cancer Center,Columbia, MO, USA

Introduction:

Background: Hepatocellular cancer (HCC) is the second leading cause of cancer-related mortality worldwide. The majority of patients are diagnosed at late-stage. For these patients, curative approaches such as surgical resection, orthotopic liver transplantation and local, percutaneous tumor ablation are unavailable. Therefore, palliative treatment options play an important role in the management of patients with HCC. Recently, immunotherapy has been approved by the US FDA to treat the patients who are resistant to sorafenib, the only FDA-approved chemotherapeutic treatment. However, objective therapeutic response is only seen in about 14% patients. Elucidating the underlying mechanisms is helpful to improve and develop effective now immunotherapy. 

Hypothesis: We hypothesize that epigenetic regulation is showing as one of mechanisms to mediate exhaustion of CD8+ T-cells in HCC by inducing genomic DNA methylation in cytokine genes. 

Methods: In terms of our established strategy, we made a clinically relevant HCC murine model by combining injection of carbon tetrachloride with splenic injection of oncogenic hepatocytes from SV40 T antigen transgenic mice. We immunized these large tumor-bearing mice and wild type mice with tumor-specific antigens. The tumor-infiltrating leukocytes were isolated from these mice with large tumors. Some cells were used to measure cytokine production in CD8+ T cells. Other cells were used to purify PD1+ CD8+ T cells and PD1– CD8+ T cells by sorting with flow cytometry. The mRNAs and genomic DNAs was extracted from these purified cells for detecting mRNA expression of IFN-γ and TNF-α as well as whole genome sequencing after bisulfite conversion. Syngeneic wild-type C57BL/6J mice were used as controls.

Results: The decreased production of IFN-γ and TNF-α was detected in CD8+ T cells from HCC-bearing mice compared to cells from wild-type mice. Whole genome bisulfite sequencing showed significant methylation difference in PD1+ CD8+ T cells between HCC-bearing mice and wild-type mice. In addition, the methylation difference was detected in PD1+ CD8+ T cell and the PD1– CD8+ T cell population isolated from HCC-bearing mice.

Conclusions And Future Directions: Tumor growth results in epigenetic modification and alteration in the genome of CD8+ T cells, suggesting the possible mechanism of epigenetic regulation in tumor-induced exhaustion of CD8+ T cells. We will treat HCC-bearing mice with demethylation agents to explore its therapeutic effect. In addition, we will evaluate if combination of demethylation will be helpful to improve other immune-based therapeutic strategies in HCC control.

45.06 Evaluation of In Vitro Grown Squamous Cell Carcinoma

Posted on January 31, 2019

S. Ananthasekar1, S. N. Banerjee2, S. Collawn3  1University of Alabama School of Medicine,Birmingham, AL, USA 2University Of Alabama at Birmingham,Department Of Biochemistry And Molecular Genetics,Birmingham, AL, USA 3University Of Alabama at Birmingham,Divison Of Plastic Surgery, Department Of Surgery,Birmingham, AL, USA

Introduction:  With over one million new cases of nonmelanoma squamous cell carcinoma (SCC) annually and no guidelines for early detection of skin cancer, there is an urgent need of in vitro modeling of these cancers to accelerate the understanding of SCC. CD44, an epithelial cell surface marker, is a trans-membrane glycoprotein involved in cell-cell interaction, cell adhesion, and migration in normal epidermis. However, its function is altered during tumor progression. There is uncertainty  on how CD44 overexpression regulates cancer cell proliferation and metastasis in vivo. In order to determine the exact impact of CD44 in skin cancer, it is first necessary to evaluate the maintenance of the tumor phenotype in an in vitro model.   

Methods:  Excised tumors were grown in 3D organotypic raft cultures (raft tumor) and harvested at 4, 6, or 8 weeks of growth. Formalin-fixed paraffin embedded original and raft tumors were cut into 4-micron thin sections for analysis. Histopathology using hematoxylin and eosin (H&E) was done on both the original and the raft tumors. Next, immunofluorescent histochemistry was done using a mouse monoclonal antibody to CD44 (Abcam) which was detected with an Alexa Fluor 488 conjugated anti-mouse antibody (Thermo Fisher). Slides were observed using a fluorescent microscope (BX 63). Images were captured with DP73 Olympus camera and CellSens software at 10X and 20X magnifications.  

Results: (1) H&E images of the original tumor and raft tumor revealed similar invasion of the tumor into the dermis with classic characteristics of SCC such as keratin pearls, nests of tumor cells, and basal cell dysplasia. (2) CD44 is expressed in areas classified as SCC based on histopathology in both original and raft tumors. 

Conclusion: SCC phenotype is maintained in vitro since both CD44 immunofluorescent histochemistry and H&E staining show similar characteristics in the in vivo and in vitro tumors. In vitro SCC models have the potential to not only study the role of CD44 in tumor growth and progression but also, cellular morphology and proliferation rate, upregulated pathways, and oncogenic factors released by the tumor.
 

45.05 Trop2 Upregulation in Mouse Pre-Neoplastic Stomach Lineages

Posted on January 31, 2019

K. M. Riera1, E. Choi1, J. R. Goldenring1  1Vanderbilt University Medical Center,Surgery,Nashville, TN, USA

Introduction: Gastric adenocarcinoma develops following parietal cell loss leading to chief cell transdifferentiation into Spasmolytic Polypeptide (TFF2) Expressing Metaplasia (SPEM) and progressing to pre-neoplastic intestinal metaplasia (IM). Ras/Raf/MEK/ERK pathway is important in gastric cancer progression, and targeting downstream mediators of Ras pathway using a MEK inhibitor, selumetinib, has been shown to reverse intestinal metaplasia to normal gastric mucosa in a transgenic mouse model. Trop2 is a transmembrane glycoprotein upregulated in adult mouse stomach after damage and overexpression predicts worse prognosis in human gastric cancer. However, the mechanistic role of Trop2 in gastric pre-neoplasia progression is not well understood. 

Methods: We studied Mist1-CreERT2;LSL-K-Ras(G12D) (Mist1-Kras) mice, which express the constitutively active form of Kras (Kras(G12D)) in gastric chief cells when treated with tamoxifen. Mist1-Kras mice developed SPEM, IM, and invasive IM at 1, 3, and 4 months post-tamoxifen treatment, respectively. Mist1-Kras mice were sacrificed at 2 month wild type (control), and 1, 2, 3, and 4 months after tamoxifen induction. To evaluate downstream Ras pathway inhibition with a MEK inhibitor, Mist1-Kras mice were treated with selumetinib for 2 weeks at 3 months post-tamoxifen treatment, and sacrificed 2 weeks following selumetinib withdrawal at 4 months. Stomachs from all mice were prepared for histology. Gastric tissue was stained for Ki67 (proliferation marker), Trop2, and Cd44v9 (SPEM/IM marker) using immunofluorescence and imaged using a digital scanner.

Results: There was no significant Trop2 staining in control, 1 (SPEM phenotype), or 2 month Mist1-Kras mice (not shown). In 3 month Mist1-Kras mice (IM phenotype), scattered Trop2 expression appeared along the gastric glands above a proliferative zone of cells staining for Ki67, and was not present in SPEM cells positive for Cd44v9 at the base of the glands (Figure 1A). In 4 month Mist1-Kras mice (invasive IM phenotype), Trop2 was markedly upregulated at and above the proliferative zone, and overexpression appeared patchy within the corpus mucosa (Figure 1B,D). Trop2 positive cells did not co-stain with Cd44v9. In 4 month selumetinib-treated Mist1-Kras mice, there was no significant Trop2 or Cd44v9 staining, while a normal proliferative zone was present (Figure 1C).

Conclusion: Trop2 is upregulated during IM progression to invasive IM in Mist1-Kras mouse stomach. Chronic Ras pathway activation may contribute to Trop2 regulation, and MEK inhibition with selumetinib shows loss of Trop2 and SPEM/IM. These results suggest that Trop2 may be upregulated at a critical transition point from benign gastric metaplasia to pre-neoplastic/dysplastic lineages.

45.04 Aberrant CDK5 and HIF1α in Pheochromocytomas: A Relationship and Potential Therapy

Posted on January 31, 2019

P. Gupta1, A. Carter1, K. Strange1, D. W. House1, H. Ghayee2, K. Pacak3, J. Bibb1, S. Reddy1  2University Of Florida,Endocrinology,Gainesville, FL, USA 3National Institutes of Health,Medical Neuroendocrinology,Bethesda, MD, USA 1University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA

Introduction: Pheochromocytomas (PC) are a rare and potentially devastating neuroendocrine neoplasm. Surgical resection can be curative, but 22% of these tumors will behave aggressively. Current systemic therapy options are limited. We have previously demonstrated CDK5's role pathogenesis of PC, and CDK5 inhibitors' efficacy treating PC cell lines in vitro. CDK5 inhibitors have not been studied sufficiently in animal PC models, and the downstream targets of CDK5 have not been identified for further therapies. Hypoxic inducing factor 1α (HIF1α) has been previously described as a potential area of study in PC. In normoxic conditions, HIF1α is rapidly degraded through the ubiquitin protease pathway after hydroxylation of proline-402. In hypoxia and some malignancies, HIF1α is stabilized, leading to activation of several downstream processes including neovascularization. In this study we sought to test the efficacy of CDK5 inhibitors in vivo, to define the importance of HIF1α in human PC tumors, and to study the potential relationship between CDK5 and HIF1α in PC models.

Methods: Murine metastatic PC models were generated through tail vein injections of MTT luciferase cells into Nu/Nu mice. After development of metastatic liver tumors, the mice were treated with CDK5 inhibitors. Lesions were quantified by bioluminescence (BLI). Human PC tumors and normal adrenal medulla were examined for phosphorylated HIF1α (p-HIF1α) at serine-687 through Western blots. PHEO cells were interrogated for the relationships between CDK5 and HIF1α. Direct phosphorylation reactions were performed in vitro between CDK5 and HIF1α.

Results: In vivo BLI demonstrated that MRT3-007 is more effective than roscovitine in metastatic murine PC models (Figure A). p-HIF1α is present in sporadic and and succinate dehydrogenase mutations (SDHB and SDHD) PC, but not in von Hipple Lindau PC or in normal adrenal medualla (FIGURE B).  In normoxia, CDK5 inibition causes loss of HIF1α (FIGURE C). This was also true in hypoxia (highlighted in the box FIGURE D), consistent with the hypothesis that aberrant CDK5 activity stabilizes HIF1α. Finally, direct phosphorylation reactions demonstrate increased levels of p-HIF1α in the presence of CDK5. The addition of a CDK5 inhibitor reversed this (FIGURE E). Therefore, CDK5 directly phosphorylates and stabilizes HIF1α.

Conclusion: CDK5 inhibitors effectively treat advanced PC in murine models, serving as the basis of future CDK5 inhibitor human trials for PC patients. HIF1α phosphorylation/stabilization is the result of aberrant CDK5 activity in PC. This process can be further examined as potential therapy for PC patients.

 

45.03 Liquid Biopsy: Novel PfeRNAS Biomarkers are Effective for the Detection of NSCLC in African Americans

Posted on January 31, 2019

N. T. Fackche1, Y. Mei1, T. Ito1, M. Garner1, P. Huang1, M. V. Brock1  1The Johns Hopkins University School Of Medicine,Surgical Oncology,Baltimore, MD, USA

Introduction:

Disparities in cancer outcomes disproportionally affect minorities and underserved populations, a fact traditionally ascribed to social determinants of health. Recent literature, however, points to ethnicity-driven differential expression of cancer genes as a contributing factor. The advent of liquid biopsy tools such as Protein Functional Effector RNAs (pfeRNA) could be a key to facilitate non-invasive, cost-effective cancer detection and risk-stratification especially in resource-constrained areas of the world. PfeRNAs are small non-coding RNAs that are abundant in plasma, and have known roles in lung cancer tumorigenesis. There is recent evidence that pfeRNA genes also may be differentially expressed based on ethnicity. Our objective is to identify pfeRNAs biomarkers with discriminative potential for clinical risk-stratification in African Americans (AA) with Non-Small Cell Lung Cancer(NSCLC).  
 

Methods:
The expression levels of 28 target pfeRNAs genes previously identified through next-generation gene sequencing were quantified in the plasma sample of 37 AA. The cohort comprised 28 cancers and nine controls from a prospectively maintained biospecimen database. Clinical characteristics were collected via a retrospective chart review. A statistical algorithm was used to design a composite biomarker by combining select pfeRNAs levels and clinical factors.

Results:
Seven of the 28 targets were clinically relevant (A-D, A-E, C-D, C-H, E-H, F-G, and G-H). PfeRNA A-E performed best as a single marker to differentiate cancer from controls with a sensitivity of 90%, a specificity of 80%, and AUC of 94.6% (P= 0.004). A computer-generated composite biomarker [formula: 3.29*Age -78.96*male+ (-38.00)*A.D+ 16.45*C.H+ 5.94*E.H -24.88*F.G+ 16.25*G.H+ (-123.08)*(A.E>=4.562104) +40] was derived and performed with an accuracy of 100% in the detection of cancer (P = 0). PfeRNA C-D demonstrated a statistically significant correlation with overall survival in the cancer subgroup N(28). Among 12 patients with C-D< (-6.69), no one died during the 12 years of  follow-up. However, six of sixteen with C-D >= (-6.69) died within the first five years. 

Conclusion:
PfeRNAs demonstrate excellent potential as both clinically relevant diagnostic and prognostic tools for AAs with NSCLC.These findings improve the prospect of developing population-specific clinical assays to help guide treatment strategies in impoverished areas of the world with poor access to CT scanning as well as for economically advanced countries enrolling smokers to lung cancer screening programs. While these results are promising, the small sample size precludes a definitive conclusion. Subsequent studies in larger cohorts are therefore needed to validate these findings

45.02 MicroRNA200a enhances antitumor effects in combination with doxorubicin in Hepatocellular carcinoma (HCC)

Posted on January 31, 2019

X. CUI1,2, Q. Du1, D. Zhou2, D. Geller1  1University Of Pittsburg,Department Of Surgery,Pittsburgh, PA, USA 2The second hospital of Anhui University,Department Of General Surgery,Hefei, ANHUI, China

Introduction:
 

The inflammatory reaction in hepatocellular carcinoma (HCC) tumor microenvironment correlates with recurrence and poor prognosis after liver resection surgery. MicroRNA-200a (miR-200a) negatively regulates epithelial-mesenchymal transition (EMT) and promotes immunomodulatory function in tumors. However, the effects of miR-200a in response to HCC tumors undergoing chemotherapy are unknown. Here, we investigated the effects of miR-200a on cell growth and autophagy in HCC tumor cells treated with doxorubicin.

Methods:
 

qPCR for miR-200a was performed in tumor and adjacent non-tumor tissues from 30 patients with HCC. Proliferation assay and half-inhibitory concentration (IC 50) were employed in Huh-7 liver tumor cells with/without doxorubicin. We stably expressed miR-200a in Huh-7 cells using lentivirus transduction, and down-regulated endogenous miR200a using shRNA. Effect of miR-200a on cell growth and autophagy with/without doxorubicin treatment was determined. Informed consent was obtained from the patients involved in this research.

Results:
 

MiR-200a level in HCC tumors was lower than the adjacent non-tumor tissue (Fig. 1A, p<0.01). Huh-7 human liver tumor cells were successfully transfected with Lv-miR-200a, and shRNA targeting miR-200a (Lv-anti-miR-200a) silenced miR-200a expression (Fig.1B). MiR-200a expression was negatively associated with tumor differentiation and liver fibrosis (p=0.030; p=0.032, data not shown). Lentiviral miR-200a over-expression significantly inhibited HCC cell proliferation (Fig. 1C) and reduced the IC50 value of doxorubicin (Fig.1D), while inhibition of endogenous miR200a had the opposite effect. Autophagy with autolysosomes (red dots) and autophagosomes (yellow dots) was increased in HCC cells overexpressing miR-200a, while silencing of endogenous miR-200a decreased basal autophagy (Fig.1E, 1F). Rapamycin was used as positive control to induce autophagy, and the miR-200a – induced autophagy was abrogated with the autophagy inhibitor 3-methyladenine (3-MA).

Conclusion:

These data show a novel role of miR-200a in HCC tumor biology. Endogenous miR-200a expression is down-regulated in human HCC tissues, while overexpression of miR-200a exerts anti-tumor effects, increases autophagy, and enhances doxorubicin effects.

45.01 Nicotine Induces IL-8 Secretion in Pancreatic Cancer Stroma: Implications for Cancer Cachexia

Posted on January 31, 2019

P. W. Underwood1, D. Zhang1, A. Delitto1, M. H. Gerber1, D. Delitto1, S. J. Hughes1, A. R. Judge1, J. G. Trevino1  1University Of Florida,Gainesville, FL, USA

Introduction:
Nicotine promotes pancreatic cancer (PC) progression and chemoresistance. Interleukin 8 (IL-8) contributes to metastasis and chemoresistance in PC cells and promotes cachexia. The relationship between nicotine and the tumor microenvironment is poorly understood. We hypothesize that nicotine induces IL-8 secretion through effects on the tumor microenvironment in PC. 

Methods:
Patient-derived tumor associated stroma (TAS) and primary pancreatic ductal adenocarcinoma cancer cells (PCC) were treated with varying doses of nicotine or control (PBS). Cell lysates and mRNA were collected at different time points and ELISA and quantitative PCR was performed to assess IL-8 levels. PCR was used to assess for nicotinic acetylcholine receptors (nAChR) and IL-8 receptors on cells. Western blot was performed to assess for Src tyrosine kinase, Akt, and ERK activation. Cells were treated with ERK inhibitor UO126 to assess expression of IL-8 levels. To confirm the effects of IL-8 on cachexia, mice were then injected with 50 µg/kg of recombinant IL-8 for a week and assessed for body weight, tibialis anterior and gastrocnemius weight, and epididymal fat weight. Patient-derived xenografts (PDX) were implanted in NSG mice and treated with nicotine or control (PBS). Tumors were harvested and IL-8 expression was assessed.

Results:
nAChR receptors (α3, α5, α7, α9, β2) are present on TAS cells.  Nicotine induces increased expression of these receptors on TAS cells. IL-8 levels were significantly higher in TAS cells treated with nicotine than control (18.8 ng/ml ± 4.3 vs. 7.6 ng/ml ± 1.3, p < 0.001). Increased exposure of nicotine induced higher levels of IL-8 expression.  IL-8 levels were significantly higher in nicotine treated TAS compared to PCC (4.7 ng/ml ± 0.4 vs. 0.4 ng/ml ± 0.22, p <0.001). Nicotine induces activation of ERK MAPK but not Src tyrosine kinase or Akt.  Pharmacologic inhibition of ERK significantly reduced IL-8 levels in nicotine treated TAS cells. Relative IL-8 receptor expression was 10 fold greater on PCC cells compared to TAS before treatment with nicotine (p < 0.001). IL-8 receptor expression increased to > 20 fold higher on PCC after nicotine exposure (p < 0.001). Treatment of mice with IL-8 resulted in a significant decrease in body weight (11%), tibialis anterior weight (17%), gastrocnemius weight (15%), and epididymal fat weight (34%). In nicotine treated PDX mice, relative IL-8 expression was increased four-fold in tumor tissue when compared to control (p = 0.009).

Conclusion:
Nicotine significantly increases the expression of IL-8. TAS cells, rather than PCC, seem to be the primary source of IL-8. The pathway appears to be ERK dependent and may contribute to a cachectic phenotype in PC patients.  Further investigations into molecular targets that contribute to cancer cachexia are warranted.
 

44.20 Cartilage Oligomeric Matrix Protein (COMP): A Potential Biomarker in Early Onset Colon Cancer.

Posted on January 31, 2019

N. P. Omesiete1, J. Jandova1, K. Memeh1, V. Nfonsam1  1University of Arizona,Surgery,Tucson, AZ, USA 2University of Arizona,Surgery,Tucson, AZ, USA

Introduction:
There has been a steady rise in the incidence of early onset colon cancer (EOCC); age <50. Recent studies suggest that EOCC may be a biologically distinct disease from late onset CC (LOCC). Our previous study showed COMP is upregulated in EOCC compared to LOCC patients and its elevation is associated with tumor aggressiveness and poor survival. The aim of this study was to evaluate COMP as a potential plasma biomarker and to assess its correlation with carcinoembryonic antigen (CEA) level in patients with EOCC.

Methods:

Fresh frozen plasma samples were obtained from our Colorectal biorepository belonging to 16 patients with early and late onset CC (8 patients in each cohort). The samples were matched for stage of disease. An ELISA assay using Human COMP Quantikine ELISA Kit purchased from R&D Systems was performed using 50 ul of 100X diluted plasma sample according to the manufacturer’s protocol. COMP was detected in all the samples of patients with colon cancer.  A quantitative analysis was performed to determine the levels of COMP in each sample by normalizing the COMP protein to total protein. This was accomplished using A Pierce BCA protein assay kit from Thermo scientific. Analysis was performed to compare the levels of COMP between both groups and their CEA levels pre-treatment.

Results:

There were a total of 16 patients (8 males and 8 females). Mean age was 42 years for EOCC and 70 years for LOCC. The patients were propensity matched for stages. There was 1 stage I; 2 stage II; 3 stage III and 2 stage IV in each cohort. COMP was detected in all 16 serum samples. Mean COMP levels was 140 pg of COMP/ug of total protein in EOCC and 20 pg of COMP/ug of total protein in LOCC, indicating a seven fold difference between the cohorts, p <0.005. There was also a positive correlation between COMP and the pre-treatment CEA levels for each stage of disease in each cohort. This was however not statistically significant. 

Conclusion:
Our findings suggest that COMP is differentially and significantly elevated in EOCC with good correlation with an established biomarker CEA. COMP may be useful as a biomarker in EOCC.

44.19 Anti-Claudin-1 Conjugated to a Near Infrared Fluorophore Targets Colon Cancer in Nude Mouse Models

Posted on January 31, 2019

H. M. Hollandsworth1,2,5, T. M. Lwin1,2,5, S. Amirfakhri1,2,5, F. Filemoni1,2,5, D. Stupack2, S. K. Batra3, R. M. Hoffman1,2,4,5, P. Dhawan3, M. Bouvet1,2,5  1University Of California – San Diego,Department Of Surgery,San Diego, CA, USA 2University Of California – San Diego,Moores Cancer Center,San Diego, CA, USA 3University of Nebraska Medical Center,Department Of Biochemistry,Omaha, NE, USA 4AntiCancer, Inc.,San Diego, CA, USA 5VA San Diego Healthcare System,Surgical Services,San Diego, CA, USA

Introduction:
Colorectal cancer remains one of the most prevalent cancers in the United States and is the third leading cause of cancer related death. Claudins are tight junction proteins which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated in the development of colon cancer. Tumor cells express a significantly higher amount of Claudin when compared with normal colonic mucosa.   We postulated that Claudin-1 may be a useful target in near infrared imaging and fluorescence guided surgery for colorectal cancers.

Methods:
We conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 different human colon cancer lysates was performed with Claudin-1-IRDye800CW. Animal imaging was initially performed with LI-COR Pearl Trilogy imaging system on subcutaneous and orthotopic colon tumors with 75 mcg Claudin-1-IRDye800CW 72 hours after injection. A dose-response study was then completed on subcutaneous cell line models. Subcutaneous implantation of LS174T on the bilateral flanks of nude mice was performed. Three groups of mice were administered increasing doses of Claudin-1-IRDye800CW: 12.5, 25, and 50 micrograms reconstituted in 100 microliters PBS, via tail vein injection. 3 mice were used as controls (no treatment, antibody alone, and 1 dye alone). In vivo imaging was performed at 24, 48, and 72 hours after administration of the conjugated dye. The mice were then euthanized and laparotomy was performed to assess for enhancement of internal organs.

Results:
Western blotting revealed that 9 out of 10 colon cancer xenografts expressed varying amounts of Claudin-1.  All tumors demonstrated strong and specific fluorescence labeling at 800 nm wavelength, even with the lowest dose of 12.5 micrograms. A representative image of mouse with an orthotopic LS174T colon tumor treated with 75 mcg of Claudin-1-IRDye800CW is shown in the Figure below. The control mice that did not receive treatment and received Claudin-1 antibody alone did not have any signal on the colon or tumor. The mouse that received IRDye800CW alone had diffuse signal that did not localize to tumors.

Conclusion:
Claudin-1 is a useful target for near infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.  
 

44.17 Intestinal Alkaline Phosphatase Protects Against Radiation Induced Gut Barrier Dysfunction

Posted on January 31, 2019

P. Cavallaro1, F. Adiliaghdam1, Y. Liu1, M. Kaleko1, C. Furlan Freguia1, R. Hodin1  1Massachusetts General Hospital,General Surgery,Boston, MA, USA

Introduction: Radiation injury induces gut barrier dysfunction and local intestinal inflammation, a clinical entity known as radiation enteritis/colitis. The brush border enzyme intestinal alkaline phosphatase (IAP) has been shown to maintain the gut mucosal barrier and attenuate local and systemic inflammation in multiple mouse models of colitis. We wanted to determine if supplemental IAP can prevent radiation induced gut barrier dysfunction and local inflammation, and therefore be a potential therapy for radiation enteritis/colitis.

 

Methods: Adult WT C57BL/6J mice were subjected to whole body irradiation (IR) to a total of 850 or 1400 rads. Mice were administered either 100 U/mL IAP or vehicle in their drinking water starting 4 days prior to IR injury and then continued until mice were sacrificed 4 days after injury. Control “sham” mice received no IR injury. Gut barrier function, tissue and systemic inflammation were measured.

 

Results:There were no difference in food and water intake among any of the groups. Gut barrier function was first measured by FITC-dextran flux from intestinal lumen to systemic blood. Vehicle treated IR mice had a radiation dose dependent increase in FITC flux across the gut barrier compared to sham (10-fold increase in 850 rad group, 30-fold increase in 1400 rad group). Permeability to FITC was almost completely attenuated in IAP treated mice in both groups (p < 0.001). Quantitative PCR of tight junction proteins demonstrated a significant decrease in ileal ZO1, ZO3, and occludin that was partially restored by IAP supplementation in both IR groups. Quantitative PCR also showed a significant decrease in colonic ZO1, ZO3, and occludin, again partially restored by IAP. Similarly, Western blot analysis showed a significant decrease in TJPs in IR treated mice, restored with IAP therapy. Next, local inflammation was assessed by measuring cytokine expression. Relative ileal and colonic TNF-α expression was increased 10-fold in the 850 rad group and 30-fold in the 1400 rad group compared to sham mice and was decreased by 50-70% when treated with IAP (p <0.001). Systemic inflammation was measured by serum ELISA which showed a two-fold increase in TNF-α and IL-6. Again, this response was attenuated by IAP. Interestingly, there was no difference in serum LPS between groups.

 

Conclusion:Radiation-induced gut barrier dysfunction and both local and systemic inflammation can be attenuated with supplemental IAP. This may be a novel approach to the treatment and prevention of radiation enteritis/colitis.

44.16 Characterizing Ileal and Colonic Barrier Permeability in Farnesoid X Receptor Knock Out Mice

Posted on January 31, 2019

M. Mallicote1, C. Gayer1  1Children’s Hospital Los Angeles,Los Angeles, CA, USA

Introduction: Activation of the farnesoid X receptor (FXR) has been reported to decrease gut permeability in colonic murine injury models like DSS colitis, protecting against intestinal bacterial translocation. However, we have shown that FXR knock out (FXR-KO) mice have attenuated barrier dysfunction when challenged with LPS, which affects the small bowel much more than the colon. These observations suggest that FXR may function differently in the small versus large bowel. We hypothesize that FXR-KO animals will have increased intestinal barrier permeability in the colon while less barrier permeability in the small intestine.

 

Methods: Four cm segments of terminal ileum and proximal colon were collected from WT and FXR-KO mice. Luminal contents were gently flushed out and replaced with a fluorescein isothiocyanate-dextran (FITC) solution with or without 2 mg/ml LPS. Both ends of the intestinal segments were then sealed with 2-0 silk ties. Each sealed intestinal segment was then placed in 2 ml of phosphate-buffered saline and barrier leakage was assessed by measuring FITC levels in PBS at 0, 1, 2, 4, 8, 16, and 24-hour time intervals. Two-way repeated measures ANOVA was performed as appropriate.

 

Results: Ileal barrier permeability in FXR-KO mice was significantly increased at 24 hours compared to WT mice at baseline. However, when challenged with LPS, the ileal barrier permeability was attenuated in FXR-KO mice but was worsened in WT mice at 24 hours. No difference was seen in colonic barrier permeability in FXR-KO mice when compared to WT controls. When treated with LPS, WT animals showed significantly increased barrier permeability at 8 hours, while FXR-KO animals did not show a change in barrier function

 

Conclusions: These data suggest that FXR affects the intestinal barrier differently in the small intestine versus the colon, both at baseline and in response to inflammatory stimuli. Understanding these differential effects is crucial if we are to target FXR in diseases such as Crohn’s disease that affects both the small bowel and colon.

44.15 Genetic sub-clones in rectal cancer respond differentially to neoadjuvant therapy

Posted on January 31, 2019

L. Frydrych1, L. H. Maguire2, P. Ulintz3, A. Bankhead4,8, J. K. Greenson5, C. J. Sifuentes3, E. Fearon6,7, K. M. Hardiman2  1University Of Michigan,Department of Surgery,Ann Arbor, MI, USA 2University Of Michigan,Colorectal Surgery,Ann Arbor, MI, USA 3University Of Michigan,Bioinformatics Core,Ann Arbor, MI, USA 4University Of Michigan,Biostatistics,Ann Arbor, MI, USA 5University Of Michigan,Pathology,Ann Arbor, MI, USA 6University Of Michigan,Internal Medicine,Ann Arbor, MI, USA 7University Of Michigan,Human Genetics,Ann Arbor, MI, USA 8University Of Michigan,Computational Medicine And Bioinformatics,Ann Arbor, MI, USA

Introduction:  Recommendations for treatment of locally advanced rectal cancer include chemotherapy, radiation and surgery. Response to neo-adjuvant therapy is variable. We have previously shown rectal cancers are made up of multiple genetically distinct sub-clones.  Unique sub-clones within primary tumors may harbor mutations which contribute to treatment resistance, relapse, and inter-patient variations in response to standard-of-care neo-adjuvant therapy. Analysis of the influence of neoadjuvant therapy on the molecular evolution of rectal cancer may provide insight about mechanisms of resistance and highlight new therapeutic approaches for patients.

Methods: Rectal cancer patients with an indication for neo-adjuvant chemoradiotherapy were identified at a tertiary referral center. Primary tumor biopsies from multiple discrete geographic locations were obtained prior to treatment. At the time of surgical intervention after standard-of-care chemoradiotherapy, tissue from the treated tumor was obtained and analyzed. Pre- and post- treatment specimens were subjected to whole exome sequencing followed by confirmatory deep sequencing to define somatic mutations. Copy number variation was assessed in all samples using Oncoscan SNP arrays. Genomic data were analyzed using Pyclone to identify sub-clonal tumor populations that differed in frequency after neo-adjuvant chemoradiotherapy. Recurrent drivers of resistance were identified in the sub-clones across tumors. Using Hotnet2, pathway analysis was performed on integrated resistant gene mutation and copy number alteration data.

Results: 32 samples were obtained from 10 patients. Pyclone identified 2-9 individual subclonal populations per tumor. Neoadjuvant therapy resulted in substantial change in the relative proportions of individual sub-clones within the primary tumor. Resistant sub-clones recurrently (>30%) contained mutations in TP53, APC, ABCA13, ITIH5, MUC16, PTEN, THSD4, and TNS1. Recurrent copy number variations for selected chromosome regions were seen in cancers after therapy. Pathway analysis revealed significantly altered pathways in resistant tumor specimens associated with RNA splicing and processing, regulation of transcription, APC-mediated pathways, mTOR signaling, fatty acid biosynthesis, and histone modification.

Conclusion: Intra-tumoral heterogeneity is evident in pre-treatment rectal cancer and sub-clonal populations are selectively modified by treatment. Resistant sub-clones demonstrate high frequencies of somatic mutation in multiple known tumorigenesis driver genes, and intra-tumoral heterogeneity persists post-treatment. Our analyses reveal complex and dynamic genomic architectures in pre- and post-treatment rectal cancers. These data indicate that novel treatment strategies must take into account a changing and heterogeneous tumor mutational profile when attempting to target rectal cancers.
 

44.14 Penicillin Prevents Rat Colonic Ischemia, Validating its Use in Ischemic Gastrointestinal Disease

Posted on January 31, 2019

T. M. Gisinger1,2, V. M. Baratta2, M. Barahona2, J. Ollodart2, D. Mulligan2, J. P. Geibel2,3  1Paracelsus Medical University,Department Of Medicine,Salzburg, SALZBURG, Austria 2Yale University School of Medicine,Department Of Surgery,New Haven, CT, USA 3Yale University School of Medicine,Department Of Cellular And Molecular Physiology,New Haven, CT, USA

Introduction: Ischemic colitis (IC) is the most common type of intestinal ischemia and arises when the colonic blood supply does not meet cellular metabolic demands. Though clinical evidence is lacking, many patients with IC are nonoperatively managed with empiric antibiotics. It has been proposed that antibiotics mitigate ischemia by reducing bacterial translocation and preventing breakdown of the epithelial barrier. In this study, we demonstrate that colonic tissue perfused with Penicillin G is more resilient to ischemic injury than tissues not exposed to the drug. These findings suggest a new clinical management paradigm of preventing ischemic conditions of the gastrointestinal tract.

Methods: Colon segments from rats were obtained and perfused with an ex-vivo intestinal perfusion device. The perfusion device consists of concentric chambers that contain the bowel segments perfused at 37°C. FITC-Inulin, fluorescein isothiocyanate, was used to assess the ischemic conditions of the intestinal grafts in real-time; a drop in fluorescence (FITC-Inulin concentration) is indicative of cellular ischemic injury. Intestinal segments were perfused with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES-Ringer. To create an ischemic environment, the HEPES-Ringer was pre-saturated with 100% N2 and perfused on extraluminal surface of all rat colonic segments. The intraluminal components of experimental colons were perfused with 5 mM Penicillin G, whereas control segments were not.  

Results: Control (without Penicillin G) distal colon samples showed a significant decrease in FITC-inulin fluorescence compared with experimental (with Penicillin G) distal colons, (26.98 ±  5.035 μM FITC vs 43.62 ± 1.569 μM FITC, respectively p 0.0083, Figure 1). This indicates that Penicillin G minimizes colonic fluid secretion, which is a marker for cell death. A similar trend was seen with proximal colon rat segments (42.7 ± 1.984 μM vs. 33.28 ± 3.455 μM FITC, p 0.0356).

Conclusion: Patients with ischemic colitis are often clinically treated with antibiotics, though the pathophysiological basis of their use is not well-proven. Our study shows that Penicillin G exposure prolongs colonic viability under ischemic conditions. This result will help further guide clinical management of ischemia in the gastrointestinal tract. Further investigation of the precise mechanism by which Penicillin G mitigates ischemia needs to be conducted.

 

44.13 Hernia Repair Outcomes Enhanced with Both Doxycycline and Antioxidant Therapy

Posted on January 31, 2019

C. Totten1, J. Tharappel1, J. S. Roth1  1University Of Kentucky,General Surgery/Surgery/Medicine,Lexington, KY, USA

Introduction: Incisional hernia is one of the most common complications of abdominal surgery and repairs are associated with significant recurrence rates. Mesh repairs are associated with the best outcomes, but failures are not uncommon. Doxycycline, an antibiotic with antioxidant properties, has been demonstrated to enhance mesh hernia repair outcomes with associated increases in Collagen deposition and improved tensio-metric strength.  This study compares the outcomes of incisional hernia repair with doxycycline administration and the antioxidant tempol.

Methods: 28 male Sprague Dawley rats were assigned to 4 groups: Control (C), Doxycycline (D), Tempol (T), and Doxycyline/Tempol (D/T). Animals underwent midline laparotomy with excision of 1x4cm strip of the midline fascia and repair with a 5 x 6 cm polypropylene mesh as an intraperitoneal underlay. Animals were administered saline, doxycycline (30mg/kg), tempol (20mg/kg), or both beginning one day prior to operation and then daily for 8 weeks.  Abdominal wall was harvested at 8 weeks with analysis of tensio-metric strength and biochemical analysis.

Results:Animals were survived for 8 weeks.  The tensiometric strength of the mesh to fascial interface was increased in the experiemental groups compared to control. (C-15.29, D-24.45, T-24.35, D/T-19.36 ) [Fig 1.].  Collagen 1 deposition was increased and Collagen 3 deposition was decreased in each of the experimental groups relative to control. MMP-2 and MMP-9 was decreased in doxycycline treatment groups.

Conclusion:The strength of a polypropylene hernia repair is enhanced with the administration of doxycycline and tempol.  Dual therapy with doxycycline and tempol provided no benefit over treatment with a single agent.  Treatment with these agents is associated with increased Collagen 1/3 ratios.  The benefits of antioxidant treatment following hernia repair are similar to treatment with doxycycline. The mechanism of antioxidant mediated collagen-1 increases are not well understood and require further study.  In light of the high frequency of incisional hernia repair failures, this study has implications for improving outcomes following ventral hernia repair through the use of either doxycycline or antioxidant therapy.
 

44.12 Penicillin’s Protective Effect on Colonic Ischemia is Mediated by H,KATPase

Posted on January 31, 2019

T. M. Gisinger1,2, V. M. Baratta1, M. J. Barahona1, J. Ollodart1, D. Mulligan1, J. P. Geibel1,3  1Yale University School Of Medicine,Department of Surgery,New Haven, CT, USA 2Paracelsus Medical University,Department of Medicine,Salzburg, SALZBURG, Austria 3Yale University School Of Medicine,Department of Cellular and Molecular Physiology,New Haven, CT, USA

Introduction: Ischemic colitis is one of the most common types of intestinal ischemia. Currently, many patients with ischemic colitis are treated with empiric antibiotics, even though the mechanism of action is not fully understood. Previously, we established that Penicillin G has a protective effect from ischemia in the rat colon. In this study, we demonstrate that the protective effect is mediated through stimulation of the colonic H,KATPase, independent of the Nitric Oxide (NO) pathway.

Methods:  The colonic segments were harvested from Sprague Dawley male rats and perfused with an ex-vivo intestinal perfusion device. Each colon was maintained at 37°C and perfused both from the luminal and basolateral side. FITC-Inulin, fluorescein isothiocyanate, was used to assess the ischemic conditions of the colonic grafts in real-time. Colonic segments were perfused with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES-Ringer solution. To create an ischemic environment, HEPES-Ringer was pre-saturated with 100% N2 and exposed to the extraluminal components of all colonic segments. For the experimental colonic tissues, the intraluminal compartments were perfused with 5 mM Penicillin G and 10 μM SCH-28080, a known colonic H,KATPase inhibitor. The intraluminal components of the control group were exposed to 5 mM Penicillin G. To test the NO-dependent mechanism, we used L-NAME, N(ω)-nitro-L-arginine methyl ester, a NO synthesis inhibitor. The intraluminal compartments of the experimental tissue were exposed to 30 μM L-NAME with 5 mM Penicillin G, while control tissues were exposed to 5 mM Penicillin G.

Results: The colon samples exposed to Penicillin G and SCH-28080 exhibited a significant decrease in FITC-Inulin fluorescence, compared with the control colonic tissue exposed to Penicillin G, (36.39 ± 2.721 μM FITC-Inulin vs 43.62 ± 1.569 μM FITC-Inulin, respectively, p 0.0401, Figure 1). We observed no statistically significant difference in the FITC-Inulin concentration between tissues exposed to L-NAME with Penicillin G versus tissues exposed to only Penicillin G.

Conclusion: Our study unveils the mechanism of Penicillin G’s protective effect from ischemia. Our results indicate that Penicillin G’s protective effect against ischemia acts by stimulating the colonic H,KATPase and is not NO-dependent. Therefore, Penicillin G not only has its well-known antimicrobial properties, but also appears to modulate a transport protein on the colonic cell membrane. A better understanding of Penicillin G’s effects on colonic tissue may help further guide the clinical management of ischemia in the gastrointestinal tract.

 

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