Abstract 3959: Depleting macrophage or blocking cell fusion machinery ameliorates intestine fibrosis after radiation

Abstract Purpose Delayed radiation enteritis is a frequent side effect of abdominal radiotherapy. Recent studies reveal bone marrow (BM) cells repopulate damaged tissue and contribute to repair in non-hematopoietic tissues. Fusion between BM and somatic cells is enhanced by chronic inflammation. Furthermore, BM cells (BMC) can trigger fibrotic response in damaged organ. The role of cell fusion in the development of chronic intestine fibrosis after radiotherapy needs further investigation. Materials and Methods To evaluate cell fusion and fibrosis, gender-mismatched BM transplantation (BMT) were used. Immunoblots of macrophage fusion receptor (MFR), CD47 and fibronectin, connective tissue growth factor (CCN2) were measured. BMT with CD11b (+), CD3 (+) or B220 (+) enriched BMC were applied. The mice were treated with macrophage depleting agents, clodronate liposome, after radiotherapy and BMT. In vitro co-culture study of human intestine stromal and mouse BMC was established. Furthermore, we used Rac 1 inhibitor, MMP9 neutralizing antibody and siRNA of MFR within co-culture system and animal model. Results Using triple stains of Y chromosome fluorescence in situ hybridization, GFP and hematopoietic cell markers including F4/80, CD3, B220, we found BM derived macrophages contributed majorly to cell fusion (63.4%, 47.5% and 9.6%, respectively, p=0.033) within intestine of mice after radiotherapy and BMT. The fusion within intestine decreased 57% after treatment with clodronate liposome. BMT from CD11b(+), CD3(+) or B220(+) enriched donor BMC also revealed prominent fusion only in mice receiving CD11b(+) one. The level of intestine fibrosis was decreased by clodronate liposome or BMT of CD11b(-) BMC. In vitro study showed the rate of fusion decreased from 12.8% to 6.8% between intestine stromal cells with CD11b (-) compared to that with whole mouse BMCs. Fusion rate could be increased by increasing CD11b(+) BMCs (3.5% to 19.7%). Using Rac1 inhibitor, MMP9 antibody or MFR siRNA, the fusion rate decreased (19.7% to 4.6%; 11.8% to 5.6%; 31% to 19.6%, respectively). The expression of fibronectin, CCN2, MFR and CD47 paralleled the findings of fusion. Using MMP9 neutralizing antibody in mice receiving radiotherapy and BMT, we found decreased cell fusion and fibrosis within intestine. Conclusions BM derived CD11b(+) myelomonocytic cells or macrophage contribute majorly to cell fusion and fibrosis within intestine of mice after radiotherapy Depleting macrophage or targeting cell fusion machinery of macrophage suppresses intestine fibrosis after radiotherapy. Our study impacts not only for developing novel therapies to prevent radiation enteritis but also to avoid fibrosis after BM cell therapy in organ dysfunction. Citation Format: Ya-Hui Chang, Hui-Ju Ch'ang, Li-Mei Lin. Depleting macrophage or blocking cell fusion machinery ameliorates intestine fibrosis after radiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3959. doi:10.1158/1538-7445.AM2014-3959</jats:p

Radiation sensitization of tumor cells induced by shear stress: The roles of integrins and FAK

AbstractRecent studies revealed that the interstitial fluid flow in and around tumor tissue not only played an important role in delivering anticancer agents, but also affected the microenvironment, mostly hypoxia, in modulating tumor radio-sensitivity. The current study investigated the hypoxia-independent mechanisms of flow-induced shear stress in sensitizing tumors to radiation.Colon cancer cells were seeded onto glass slides pre-coated with fibronectin. A parallel-plate flow chamber system was used to impose fluid shear stress. Cell proliferation, apoptosis and colony assays were measured after shear stress and/or radiation. Cell cycle analysis and immunoblots of cell adhesion signal molecules were evaluated. The effect of shear stress was reversed by modulating integrin β1 or FAK.Shear stress of 12dyne/cm2 for 24h, but not 3h, enhanced the radiation induced cytotoxicity to colon cancer cells. Protein expression of FAK was significantly down-regulated but not transcriptionally suppressed. By modulating integrin β1 and FAK expression, we demonstrated that shear stress enhanced tumor radiosensitivity by regulating integrin β1/FAK/Akt as well as integrin β1/FAK/cortactin pathways. Shear stress in combination with radiation might regulate integrins signaling by recruiting and activating caspases 3/8 for FAK cleavage followed by ubiquitin-mediated proteasomal degradation.Shear stress enhanced the radiation toxicity to colon cancer cells through suppression of integrin signaling and protein degradation of FAK. The results of our study provide a strong rationale for cancer treatment that combines between radiation and strategy in modulating tumor interstitial fluid flow

Abstract 569: Shear stress radiatiosensitizes colon cancer cells by downregulating FAK via integrin β1

Abstract Introduction: Interstitial fluid flow plays important role in delivering anticancer agents to tumor tissue. Recent studies suggested that anti-angiogenic or vasculature disrupting agents may enhance the therapeutic effect of radiotherapy by regulating tumor blood flow and interstitial fluid pressure. Although the importance of interstitial fluid flow in modulating tumor microenvironment has been studied, the molecular mechanisms of shear flow on tumor cell survival and sensitivity to therapeutic agents have not been much explored. We would like to investigate the interaction of shear stress with irradiation on tumor cells. Materials and methods: Human T84 and SW480 colon adenocarcinoma cells were used in this study. Cells were seeded onto glass slides pre-coated with fibronectin, and then subjected to 0-12 dyne/cm2 laminar shear stress in parallel plate flow chamber (PPFC) system for 0-24 hours. Cells were then exposed to 0-8Gy irradiation (IR) by a RS2000 X-ray irradiator. MTT and clonogenic assays were used to study cell survival. Tumor cell cycle and apoptosis were evaluated by flow cytometry and Western blot. Candidate signal molecules were measured by real time PCR and immunoblots with or without pretreatment with cyclohexamide or MG132. Results: MTT and clonogenic assays revealed a synergistic effect of 24 hour- shear stress and IR on tumor cytotoxicity. Shear stress downregulates FAK, phosphor-FAK, AKT and phosphor-AKT expression but not ILK and GSK3B through integrin β1. Decreased protein expression was mainly through proteosomal degradation but not transcriptional suppression. By enhancing G2/M phase cell accumulation, shear stress increases tumor apoptosis after IR. The above phenomenon was not observed in IR pretreated with 3 hour-shear stress. Conclusion: The above observation suggests that shear stress may enhance colon cancer cell radiation sensitivity by inducing G2/M arrest, and FAK protein degradation via integrin β1 signaling pathway Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 569. doi:10.1158/1538-7445.AM2011-569</jats:p

Destabilization of KLF10, a tumor suppressor, relies on thr93 phosphorylation and isomerase association

AbstractKLF10 is now classified as a member of the Krüppel-like transcription factor family and acts as a tumor suppressor. Although KLF10 is originally named as TGF-β-inducible early gene-1 and mimicking the anti-proliferative effect of TGF-β in various carcinoma cells, the transcriptional upregulatory function of KLF10 has been described for a variety of cytokines and in many diseases. Through in vivo and in vitro phosphorylation assays, we identified that KLF10 is a phosphorylated protein in cells. Using yeast-two hybrid screening and site direct mutagenesis, we also identified PIN1 as a novel KLF10 associated protein. PIN1 is a peptidyl-prolyl isomerase enzyme belonging to the parvulin family, which specifically recognizes phosphorylated Ser/Thr-Pro containing substrates. Through protein–protein interaction assays, we showed that the Pro-directed Ser/Thr-Pro motif at Thr-93 in the KLF10 N-terminal region is essential for the interaction between KLF10 and PIN1. More importantly, PIN1 interacts with KLF10 in a phosphorylation-dependent manner and this interaction promotes KLF10 protein degradation in cells. Therefore, KLF10 shows shorter protein stability compared with mutant KLF10 that lacks PIN1 binding ability after cycloheximide treatments. The reversely correlated expression profile between KLF10 and PIN1 as observed in cell lines was also shown in clinic pancreatic cancer specimen. Using in vitro kinase assays and depletion assays, we were able to show that RAF-1 phosphorylates the Thr-93 of KLF10 and affects the KLF10 expression level in cells. Thus these findings as a whole indicate that RAF-1 phosphorylation and PIN1 isomerization together regulate KLF10 stability and further affect the role of KLF10 in tumor progression

Characterization of a transgenic mouse model exhibiting spontaneous lung adenocarcinomas with a metastatic phenotype.

Developing lung cancer in mouse models that display similarities of both phenotype and genotype will undoubtedly provide further and better insights into lung tumor biology. Moreover, a high degree of pathophysiological similarity between lung tumors from mouse models and their human counterparts will make it possible to use these mouse models for preclinical tests. Ovine pulmonary adenocarcinomas (OPAs) present the same symptoms as adenocarcinomas in humans and are caused by a betaretrovirus. OPAs have served as an exquisite model of carcinogenesis for human lung adenocarcinomas. In this study, we characterized the histopathology and transcriptome profiles of a jaagsiekte sheep retrovirus (JSRV)-envelope protein (Env) transgenic mouse model with spontaneous lung tumors, and associations of the transcriptome profiles with tumor invasion/metastasis, especially the phenomenon of the epithelial-mesenchymal transition (EMT). Genetic information obtained from an expression array was analyzed using an ingenuity pathways analysis (IPA) and human disease database (MalaCards). By careful examination, several novel EMT-related genes were identified from tumor cells using RT-qPCR, and these genes also scored high in MalaCards. We concluded that the JSRV-Env mouse model could serve as a spontaneous lung adenocarcinoma model with a metastatic phenotype, which will benefit the study of early-onset and progression of lung adenocarcinoma. In addition, it can also be a valuable tool for biomarkers and drug screening, which will be helpful in developing intervention therapies

Abstract 2001: Krupple like factor 10 modulates stem cell phenotype of pancreatic adenocarcinoma via transcriptional regulation of Notch signal pathway

Abstract Background: Pancreatic adenocarcinoma (PDAC) is known for its deregulated TGFβ signal pathway. Therapeutic strategy targeting TGFβ is controversial due to the dual role of TGFβ with anti-proliferation in early phase and pro-metastatic in late phase of cancer progression. KLF10, an early response gene of TGFβ, positively feedback the antiproliferative effect in cancer. Recent studies revealed KLF10 expression were suppressed epigenetically in various cancers including PDAC. In contrary to TGFβ, KLF10 transcriptionally suppress Slug and Sirtuin 6 to prevent metastasis in advanced PDAC. The role of KLF10 in regulating tumorigenesis and stem cell phenotype is still unknown. Materials and Methods: From 105 patients of resectable PDAC, KLF10 expression level was evaluated to be reduced in 62% of tumor specimens. Low KLF10 expression correlated with larger tumor size and rapid loco-regional recurrence. In murine model of Kras mutation (KC mice), additional depletion of KLF10 induced 57% of PDAC compared to 0% at 18 to 24 wk of age. PDAC cells were genetically manipulated with KLF10mRNA silencing (PDACshKLF10) or inducible overexpression (PDACoeKLF10). Using limiting dilution assay of tumor growth, and orthotopic tumor implantation, we found higher tumorigenic ability of PDACshKlf10 compared to that of wild type. The stem cell phenotypes of PDACshKLF10 and PDACoeKLF10 were confirmed by sphere formation assay and FACS analysis of surface markers including CD24/CD44, ESA/c-Met, CD47 and CD133. Results: Notch signal pathway was found to be significantly enhanced from microarray analysis of wild type versus PDACshKLF10 cells. The increased Notch signal molecules were confirmed in RNA and protein level in genetically manipulated PDAC cells as well as clinical tissue specimens. Using Chip-PCR and luciferase promoter assay, KLF10 was demonstrated to bind to the promoter of Notch receptor 1, 3, and 4. DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), a Notch inhibitor, suppressed in vitro spheroid formation, cell surface expression of stem cell markers, and tumor growth of PDACshKLF10 in orthotopic murine model. We conclude that KLF10 modulates stem cell phenotype of PDAC by transcriptionally suppressing Notch signal pathway. Loss of KLF10 in PDAC patients leads to Notch signal activation, development of stem cell phenotype and tumor progression. Notch inhibition may reverse malignant growth of PDAC with reduced KLF10 expression. Conflict of interest statement: nothing to declare Citation Format: Yi-Chih Tsai, Su-Liang Chen, Shu-Ling Peng, Kuang-Hung Cheng, Shih-Sheng Jiang, Shuang-En Chuang, Ch'ang Hui-Ju. Krupple like factor 10 modulates stem cell phenotype of pancreatic adenocarcinoma via transcriptional regulation of Notch signal pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2001.</jats:p