- Email: [email protected]
Sa1798 Expression of Bone Morphogenic Protein 4 (BMP4) in Esophageal Cancer is Regulated by Stroma-Dependent Sonic Hedgehog Signals
were 43 deregulated serum miRNAs in CCA patients. (C) There were 63 deregulated bile miRNAs in CCA patients (CCAB) (P<0.01). Figure 2. miRNAs continuously changed in both serum and bile of CCA patients. (A) There were 28 miRNAs continuously changed in both serum and bile of CCA patients.(B) Among these miRNAs, 4 miRNAs were changed in serum of PSC patients. (C)qRT-PCR verified the expression of miR-150-5p. Figure 3. Prediction of functions and signalling pathways regulated by the 28 miRNAs. (A) miRNA-GO network and (B)miRNA-KEGG-network were generated according to the relationships between significant functions and the 28 miRNAs. Figure 4. The growth curves of CCA cells. (A)HCCC-9810 and RBE (B) cells were transfected with miR-150-5p mimic (miR-150-5pM) or nonspecific mimic (NSM).
Sa1799
Introduction: Using Back-Scattered Interference Spectroscopic (BaSIS) microscopy, a nanoscale sensitive imaging technique developed in our lab, we study the role of the nanoscale organization of higher order chromatin structure (from 20-200 nm) as a key event in early carcinogenesis. Based on preliminary work, we hypothesize that the altered physical chromatin nanoenvironment facilitates (pre)cancerous cells to explore a greater genomic landscape and consequently evolve and evade chemotherapy. The chromatin nanoenvironment can be physio-chemically modulated by the use of compounds and shifted toward the normalized state, which can prevent cells from being able to evolve chemoresistance. Thus, these existing compounds can be repurposed for "chromatin protection therapy" (CPT). CPT might be used as an adjunct therapy to increase the efficacy of other existing chemotherapy compounds and could improve outcomes for patients on chemotherapy regimes. In this study, we investigate whether celecoxib and sulindac sulfide physio-chemically modulate the chromatin nanoarchitecture of cancer cells. CPTs could be used as an approach to limit the genomic exploration of cancer cells thereby preventing the potential development of chemoresistance. Methods: Modulation of the chromatin nanoenvironment was performed with low, medium, and high (75, 150, and 200 uM) celecoxib and sulindac sulfide (Sigma, St. Louis, MO). Drug concentrations for the cell lines were determined by WST-1 cell proliferation assay. Colon (HCT116 adenocarcinoma) and breast (MDA-MB-231 adenocarcinoma and MCF 10a non-tumorigenic) cell line models were used. BaSIS microscopy was performed as previously described to assess the response of the chromatin structure to the NSAIDs. Results: We found that treatment of colonic and breast cell lines with celecoxib and sulindac leads to a physio-chemical modulation of the chromatin nanoenvironment within minutes (Figure 1). These results support our hypothesis that the chromatin nanoenvironment can be physio-chemically modulated by the use of compounds. Conclusions: We demonstrate for the first time that existing compounds such as the NSAIDs celecoxib and sulindac can be repurposed for "chromatin protection therapy" (CPT) to help potentially prevent cells from evolving chemoresistance. These novel findings indicate that CPT might be used as an adjunct therapy to improve outcomes for patients on chemotherapy regimes.
Figure 5. The invasion study of CCA cells. (A-E) After transfection,cells were trypsinized and seeded in 24-well plates with Matrigel-coated membranes for invasion assays. Cells that had invaded through the Matrigel to the bottom of the insert were fixed with formalin, stained with crystal violet, and counted in 10 random view fields under inverted microscope at 24 and 48 h after incubation. Figure 6. Wound healing study. (A-J) After transfection, cells were trypsinized and seeded in 24-well plates. A line was scratched using a sterile pipette tip after the cells reached 90% confluence. The cells were observed in same 10 random views under inverted microscope and took picture at 0, 24, and 48 h after scratching . Figure7. miRNA-mRNA network and the target gene verification. (A) miRNA-mRNAnetwork was generated.(B) Target scan was used to predict the putative target genes of miR150-5p. (C) The target gene ELK1 of miR-150-5p was verified by Western blot.
Sa1798 Expression of Bone Morphogenic Protein 4 (BMP4) in Esophageal Cancer is Regulated by Stroma-Dependent Sonic Hedgehog Signals Silvia Calpe, Matthew Read, Sanne Hoefnagel, Maria del Carmen Sancho-Serra, Danielle Straub, Nicholas J. Clemons, David Liu, Wayne A. Phillips, Kausilia K. Krishnadath Bone morphogenic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor-beta (TGFb) superfamily. Amongst them, BMP4 is becoming increasingly attractive due to its crucial role in the development of many cancers. In gastrointestinal cancers BMP4 possesses tumorigenic capacities. In these cancers, BMP4 acts on tumor epithelial cells enhancing their pro-metastatic behavior and chemo-resistance. However, whether BMP4 is being secreted by the epithelial cells or by the stroma, has not been well established yet, in part due to a lack of specific anti-BMP4 antibodies. To elucidate the functional implications of the origin of BMP4 production, we made use of a novel esophageal adenocarcinoma (EAC) tumor xenograft model (1) and a newly generated Llama-derived anti-BMP4 antibody (2). Using in vivo molecular imaging techniques we find that BMP4 expression in the tumor is increased after engraftment of BMP4 negative human EAC tumor cells into immunodeficient mice. Immunohistochemical studies of the engrafted tissue reveal a human origin of BMP4 protein, suggesting that epithelial cells are involved in BMP4 secretion. This also implies that the stroma produces molecules that activate BMP4 production by the cancer epithelial cells. In vitro analysis such as co-cultures with different stromal cells revealed that Sonic Hedgehog (Shh) signaling on epithelial cells activates autocrine BMP4 secretion as well as an increase in the malignant features of EAC cancer cells, such as chemoresistance as well as invasive capacities. In sum, these studies suggest a pivotal role of the microenvironment in regulating BMP4 expression in a model of esophageal cancer. In particular, it suggests a close relationship between Shh and BMP4, as mediators of the malignant features of these cancers. As BMP4 has also been shown to be responsible for tumor progression in other gastrointestinal cancers, stromal regulation of BMP4 production might not be restricted to EAC, and might be a common feature in gastrointestinal cancers. 1. Read M, Liu D, Duong CP, Cullinane C, Murray WK, Fennell CM, et al. Intramuscular Transplantation Improves Engraftment Rates for Esophageal Patient-Derived Tumor Xenografts. Ann Surg Oncol. 2015; 2. Calpe S, Wagner K, Khattabi M El, Rutten L, Zimberlin C, Dolk E, et al. Effective inhibition of Bone Morphogenetic Protein function by highly specific llama-derived antibodies. Mol Cancer Ther. 2015;
Figure 1. Physio-chemical modulation of the chromatin nanoenvironment within 30 minutes of treatment with celecoxib. Nuclear nanostructure of MCF 10a cells before (A) and 30 min post- (B) addition of 75 uM celecoxib. Nuclear signal decreases over time indicating a physiochemical modulation of the chromatin nanoenvironment in response to treatment. Variations in mass flux before (C) and 30 minutes post-treatment (D) show a decrease in mass flux in the nucleus and cytoplasm with treatment.
Sa1800 The microRNAs Expression Profiles and Morphological Appearance of Ulcerative Colitis Associated Cancer Yoshihito Nakagawa, Yukihiro Akao, Tomomitsu Tahara, Tomoyuki Shibata, Naoki Ohmiya Aim: Accumulating data indicate that some microRNAs (miRNAs or miRs) function as tumor suppressors or oncogenes in cancer development. We previously reported that certain miRNAs (miR-143, -145, -34a, -21, and -7) are differently expressed in samples of tumors and paired non-tumorous samples taken from the same patients with colorectal tumors. On the other hand, ulcerative colitis associated colorectal cancer (UCAC) is different carcinogenesis of adenoma-carcinoma sequence. And it is difficult of early diagnosis of UCAC. In the current study, we focused on the miRNA expression profiles of ulcerative colitis associated cancer compared with adenoma-carcinoma sequence. Design: At first, we examined the miRNA expression profiles and morphological appearance of 102 sporadic colorectal adenomas, 114 sporadic colorectal cancers, 10 tumors of UCAC, and 21 tumors of familial adenomatous polyposis (FAP). We compared the miRNA expression profile and morphological appearance of these tumors. Results: The expression levels of miRs-143 and -145 were reduced in all tumors compared with the paired non-tumorous samples in the same patient. The expression levels of miR-21 were significantly up-regulated in UCAC compared with other tumors. The miRNA expression profiles and morphological appearance of UCAC were
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AGA Abstracts
Chromatin Protection Therapy: Physio-Chemical Modulation of the Chromatin Nanostructure to Prevent Chemoresistance Greta Bauer, Luay Almassalha, Alexis Kendra, Scott Gladstein, Hemant K. Roy, Vadim Backman
Sa1799
Introduction: Using Back-Scattered Interference Spectroscopic (BaSIS) microscopy, a nanoscale sensitive imaging technique developed in our lab, we study the role of the nanoscale organization of higher order chromatin structure (from 20-200 nm) as a key event in early carcinogenesis. Based on preliminary work, we hypothesize that the altered physical chromatin nanoenvironment facilitates (pre)cancerous cells to explore a greater genomic landscape and consequently evolve and evade chemotherapy. The chromatin nanoenvironment can be physio-chemically modulated by the use of compounds and shifted toward the normalized state, which can prevent cells from being able to evolve chemoresistance. Thus, these existing compounds can be repurposed for "chromatin protection therapy" (CPT). CPT might be used as an adjunct therapy to increase the efficacy of other existing chemotherapy compounds and could improve outcomes for patients on chemotherapy regimes. In this study, we investigate whether celecoxib and sulindac sulfide physio-chemically modulate the chromatin nanoarchitecture of cancer cells. CPTs could be used as an approach to limit the genomic exploration of cancer cells thereby preventing the potential development of chemoresistance. Methods: Modulation of the chromatin nanoenvironment was performed with low, medium, and high (75, 150, and 200 uM) celecoxib and sulindac sulfide (Sigma, St. Louis, MO). Drug concentrations for the cell lines were determined by WST-1 cell proliferation assay. Colon (HCT116 adenocarcinoma) and breast (MDA-MB-231 adenocarcinoma and MCF 10a non-tumorigenic) cell line models were used. BaSIS microscopy was performed as previously described to assess the response of the chromatin structure to the NSAIDs. Results: We found that treatment of colonic and breast cell lines with celecoxib and sulindac leads to a physio-chemical modulation of the chromatin nanoenvironment within minutes (Figure 1). These results support our hypothesis that the chromatin nanoenvironment can be physio-chemically modulated by the use of compounds. Conclusions: We demonstrate for the first time that existing compounds such as the NSAIDs celecoxib and sulindac can be repurposed for "chromatin protection therapy" (CPT) to help potentially prevent cells from evolving chemoresistance. These novel findings indicate that CPT might be used as an adjunct therapy to improve outcomes for patients on chemotherapy regimes.
Figure 5. The invasion study of CCA cells. (A-E) After transfection,cells were trypsinized and seeded in 24-well plates with Matrigel-coated membranes for invasion assays. Cells that had invaded through the Matrigel to the bottom of the insert were fixed with formalin, stained with crystal violet, and counted in 10 random view fields under inverted microscope at 24 and 48 h after incubation. Figure 6. Wound healing study. (A-J) After transfection, cells were trypsinized and seeded in 24-well plates. A line was scratched using a sterile pipette tip after the cells reached 90% confluence. The cells were observed in same 10 random views under inverted microscope and took picture at 0, 24, and 48 h after scratching . Figure7. miRNA-mRNA network and the target gene verification. (A) miRNA-mRNAnetwork was generated.(B) Target scan was used to predict the putative target genes of miR150-5p. (C) The target gene ELK1 of miR-150-5p was verified by Western blot.
Sa1798 Expression of Bone Morphogenic Protein 4 (BMP4) in Esophageal Cancer is Regulated by Stroma-Dependent Sonic Hedgehog Signals Silvia Calpe, Matthew Read, Sanne Hoefnagel, Maria del Carmen Sancho-Serra, Danielle Straub, Nicholas J. Clemons, David Liu, Wayne A. Phillips, Kausilia K. Krishnadath Bone morphogenic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor-beta (TGFb) superfamily. Amongst them, BMP4 is becoming increasingly attractive due to its crucial role in the development of many cancers. In gastrointestinal cancers BMP4 possesses tumorigenic capacities. In these cancers, BMP4 acts on tumor epithelial cells enhancing their pro-metastatic behavior and chemo-resistance. However, whether BMP4 is being secreted by the epithelial cells or by the stroma, has not been well established yet, in part due to a lack of specific anti-BMP4 antibodies. To elucidate the functional implications of the origin of BMP4 production, we made use of a novel esophageal adenocarcinoma (EAC) tumor xenograft model (1) and a newly generated Llama-derived anti-BMP4 antibody (2). Using in vivo molecular imaging techniques we find that BMP4 expression in the tumor is increased after engraftment of BMP4 negative human EAC tumor cells into immunodeficient mice. Immunohistochemical studies of the engrafted tissue reveal a human origin of BMP4 protein, suggesting that epithelial cells are involved in BMP4 secretion. This also implies that the stroma produces molecules that activate BMP4 production by the cancer epithelial cells. In vitro analysis such as co-cultures with different stromal cells revealed that Sonic Hedgehog (Shh) signaling on epithelial cells activates autocrine BMP4 secretion as well as an increase in the malignant features of EAC cancer cells, such as chemoresistance as well as invasive capacities. In sum, these studies suggest a pivotal role of the microenvironment in regulating BMP4 expression in a model of esophageal cancer. In particular, it suggests a close relationship between Shh and BMP4, as mediators of the malignant features of these cancers. As BMP4 has also been shown to be responsible for tumor progression in other gastrointestinal cancers, stromal regulation of BMP4 production might not be restricted to EAC, and might be a common feature in gastrointestinal cancers. 1. Read M, Liu D, Duong CP, Cullinane C, Murray WK, Fennell CM, et al. Intramuscular Transplantation Improves Engraftment Rates for Esophageal Patient-Derived Tumor Xenografts. Ann Surg Oncol. 2015; 2. Calpe S, Wagner K, Khattabi M El, Rutten L, Zimberlin C, Dolk E, et al. Effective inhibition of Bone Morphogenetic Protein function by highly specific llama-derived antibodies. Mol Cancer Ther. 2015;
Figure 1. Physio-chemical modulation of the chromatin nanoenvironment within 30 minutes of treatment with celecoxib. Nuclear nanostructure of MCF 10a cells before (A) and 30 min post- (B) addition of 75 uM celecoxib. Nuclear signal decreases over time indicating a physiochemical modulation of the chromatin nanoenvironment in response to treatment. Variations in mass flux before (C) and 30 minutes post-treatment (D) show a decrease in mass flux in the nucleus and cytoplasm with treatment.
Sa1800 The microRNAs Expression Profiles and Morphological Appearance of Ulcerative Colitis Associated Cancer Yoshihito Nakagawa, Yukihiro Akao, Tomomitsu Tahara, Tomoyuki Shibata, Naoki Ohmiya Aim: Accumulating data indicate that some microRNAs (miRNAs or miRs) function as tumor suppressors or oncogenes in cancer development. We previously reported that certain miRNAs (miR-143, -145, -34a, -21, and -7) are differently expressed in samples of tumors and paired non-tumorous samples taken from the same patients with colorectal tumors. On the other hand, ulcerative colitis associated colorectal cancer (UCAC) is different carcinogenesis of adenoma-carcinoma sequence. And it is difficult of early diagnosis of UCAC. In the current study, we focused on the miRNA expression profiles of ulcerative colitis associated cancer compared with adenoma-carcinoma sequence. Design: At first, we examined the miRNA expression profiles and morphological appearance of 102 sporadic colorectal adenomas, 114 sporadic colorectal cancers, 10 tumors of UCAC, and 21 tumors of familial adenomatous polyposis (FAP). We compared the miRNA expression profile and morphological appearance of these tumors. Results: The expression levels of miRs-143 and -145 were reduced in all tumors compared with the paired non-tumorous samples in the same patient. The expression levels of miR-21 were significantly up-regulated in UCAC compared with other tumors. The miRNA expression profiles and morphological appearance of UCAC were
S-369
X : 10052$CH01 03-28-16 00:57:27 PDFd : 10052B : o
Page 369
AGA Abstracts
AGA Abstracts
Chromatin Protection Therapy: Physio-Chemical Modulation of the Chromatin Nanostructure to Prevent Chemoresistance Greta Bauer, Luay Almassalha, Alexis Kendra, Scott Gladstein, Hemant K. Roy, Vadim Backman