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Study of Moesin regulation and effects in breast cancer cells
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 305 Establishment of three-dimensional culture of cholangiocarcinoma cells S. Sukphokkit1 , P. Kiatwuthinon2 , S. Kumkate3 , T. Janvilisri1 . 1 Faculty of Science- Mahidol University, Biochemistry, Bangkok, Thailand, 2 Faculty of Science- Kasetsart University, Biochemistry, Bangkok, Thailand, 3 Faculty of Science- Mahidol University, Biology, Bangkok, Thailand Cholangiocarcinoma (CCA) is a bile duct cancer associated with liver fluke infection, predominantly found in Thailand. The mortality rate of CCA is high due to the difficulty in the diagnosis as CCA conceals the symptoms until the late stage of progression. At present, the in vitro three-dimensional culture overwhelms the traditional culture because it is similar to in vivo than twodimensional culture. Hence, we compared the characteristics of CCA cell lines between three-dimensional and two-dimensional cultures including cell viability, organization, and migration activity. CCA cells including KKU-100 and KKU-M213 were cultured in Matrigel® to produce multicellular tumor spheroids. We found that the three-dimensional culture of CCA cell lines exhibited the compact clusters of cells and the growth rate was drastically slower than two-dimensional culture. The migration activities of KKU-100 in both culture systems were lower than that of KKU-M213. We successfully established the three-dimensional culture of CCA cells, which will certainly be useful for drug screening for CCA. No conflict of interest. 306 Study of Moesin regulation and effects in breast cancer cells W.M. Abdel-Rahman1 , F. Alam1 , F. Mezhal1 , M.S. Ayad2 , A. El-Serafi2 , H. El Hassasna3 , J. Thiery4 , Z. Noman4 , S. Chouaib4 . 1 University of Sharjah, Medical Lab Sciences, Sharjah, U.A.E., 2 University of Sharjah, Medicine, Sharjah, U.A.E., 3 University of Sharjah, SIMR, Sharjah, U.A.E., 4 Gustave Roussy Cancer Campus, INSERM U1186 formerly U753, Villejuif, France Introduction: p53 is the most significant tumor suppressor protein in the cell. It regulates a number of micro RNAs (miRNAs) which in turn regulate carcinogenesis. This study aimed to analyze the expression of miRNAs in relation to p53 status in breast cancer cells and to delineate the role of the target gene Moesin. Material and Methods: We used isogenic breast carcinoma cell lines MCF7 (with wildtype p53), 1001 (MCF7 with mutated p53), and MCF-E6 (MCF7 in which p53 function was disrupted by transfection with the human papilloma virus type-16 E6 gene). Western blot analysis was performed to confirm the status of p53 protein in each cell line. High quality RNA was extracted by the trizol method and submitted to miRNA microarray in duplicates, using the Agilent 8x60K v16 chips. Results and Discussion: Taking a 2-fold change (2 FC) as a cut-off level, the 1001 clone with mutant p53 showed 21 upregulated and 25 downregulated miRNAs (2 FC). The predicted targets of these 46 miRNAs were 384 human genes belonging to interesting functional groups such as stem cell development and maintenance. The two most significantly down regulated miRNAs were miR141 and miR200c. Here, we focused on miR200c which targets many transcripts that are involved in Epithelial to Mesenchymal Transition (EMT) from which we chose to explore the target protein “Moesin”. Western blotting showed that Moesin was differentially expressed in our model where it was expressed in p53-mutant cell line but not in wild type cell line consistent with the observed mesenchymal features in the p53 mutant cell (vimentin positivity, E-cadherin negativity, ZEB1 positivity, and mesenchymal morphology). This prompted exploring the potential role of Moesin in the EMT phenotype observed in our model. To analyze the functions of Moesin, we silenced its expression in p53-mutant cells by siRNA transfection. Western blotting confirmed about 90% efficiency of the siRNA transfection. Then we looked at the effects of Moesin silencing on the cell morphology by confocal microscopy which showed that after Moesin silencing, the p53-mutant cells changed from mesenchymal phenotype to epithelial phenotype. Moesin silencing did not significantly alter migration and invasion, and did not affect the expression of E-cadherin and vimentin, with loss of expression of ZEB1 and insignificant reduction of SNAIL expression. Interestingly, Moesin silencing restored the 1001 sensitivity to standard chemotherapeutic agent Doxorubicin. Conclusion: The results indicate that loss of miR200c as a consequence of p53 mutation relieves significant target oncogenes from degradation and hence promotes carcinogenesis. Moesin may play a role in metastasis and drug resistance of breast cancer cells. These findings might have diagnostic and therapeutic applications. No conflict of interest. 307 Assessment of WT1 expression as a marker of treatment outcome in karyotype normal acute myeloid leukemia patients in Pakistan Z. Ahmed1 . 1 Aga Khan University Hospital-Karachi-Pakistan, Pathology and Laboratory Medicine, Karachi, Pakistan Background: Currently, there is an effort to predict relapse by follow-up monitoring of MRD and subsequently to begin the treatment of the patients during their clinical and hematological remission prior to overt hematological
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relapse. Expression of WT1 in AML is known to be independently associated with significant inferior response to therapy and short survival outcome. Followup monitoring of WT1 gene expression during or after therapy would be a valuable predictive marker for early recurrence or relapse of AML disease. Objective: To demonstrate prognostic relevance of WT1 expression levels monitored at diagnosis and during follow up of chemotherapy for predicting relapse in AML patients. Methods: For this study, five AML patients registered at the Oncology Clinics of the Aga Khan University Hospital, Karachi were recruited. Blood was collected from these patients and it used to isolate RNA from purified PBMCs. The RNA was reverse transcribed to make cDNA which served as template for quantitative RT-PCR analysis. Results: The WT1 levels were quantified by RT-PCR in five male patients with AML. Relatively higher level of WT1 mRNA (4000–9500 copies/ml) was detected in treatment na¨ıve patients, whereas only (100–250 copies/ml) were seen in two patients (one was receiving induction and the other patient was on consolidation therapy). In addition, one patient who received induction therapy showed raised WT1 mRNA levels (7700 copies/ml). Conclusion: High WT1 burden (>5000 copies/ml) in two patients is indicative of early recurrence of the disease along with shorter disease free and overall survival. The low WT1 expression (<200 copies/ml) in two patients after induction and consolidation therapy respectively is suggestive of better prognosis. No conflict of interest. 308 Influence of autophagy inhibitors on chemotherapeutic efficacy in oral squamous cell carcinoma A. Anderson1 , J. O’Sullivan1 . 1 Trinity College Dublin, Dental Science, Dublin, Ireland Background: Oral squamous cell carcinoma (OSCC) is the most common form of oral cancer and often manifests itself as ulceration with erythroplastic, and often leukoplakic, changes in the surrounding area. Autophagy has been implicated in most cancers and has been referred to as a double edged sword as dysregulation of this pathway can be considered tumourgenic or tumour suppressive. Autophagy activation can result from the high levels of metabolic stress chemotherapeutic agents put on the cell, which in turn can mediate an acquired resistance to these compounds during treatment. Potentially, autophagy inhibition may prevent resistance occurring in this way and in parallel prevent tumours from utilising the autophagic pathway for cancer cell survival. Materials and Methods: Two OSCC cell lines, Ca9.22 and TR146 were used along with a dysplastic cell line, DOK. Cisplatin, carboplatin, 5-fluorouracil and docetaxel, were used in combination with five autophagy inhibitors, chloroquine, LY294002, Dbeq, E64d and 3-methyladenine. IC50 values for the chemotherapeutic drugs were determined by the Alamar Blue assay after exposure to the drugs for 72 or 96 h. Indicative levels of autophagy were determined by identifying levels of acidic vesicle expression using acridine orange staining and relative levels were quantitatively determined. FACS analysis of apoptosis levels were determined by staining with propidium. Protein expression changes within autophagic and apoptotic pathways were investigated by western blot. Results and Discussion: Determined IC50 values for cisplatin, carboplatin, docetaxel and 5-fluorouracil for Ca9.22, TR146 and DOK cell lines ranged from 200 pM to 50 mM and were used as the concentration for subsequent experiments. The levels of acidic vesicles increased in the presence of a number of the chemotherapeutics. The quantity of acidic vesicles varied under combination treatment with different autophagic inhibitors. Combining drug treatment with autophagy inhibitors altered the efficiency of their apoptosis inducing capability within cells. Modulation of autophagic flux, levels of light chain 3-II, poly ADP-ribose polymerase, and Atg4 were confirmed in these cell lines by western blot analysis. Conclusion: Investigating the impacts of chemotherapeutics on programmed cell death OSCC has provided insights into the mechanisms by which these compounds affect OSCC and how resistance can develop. Examination of cell lines displaying different underlying pathologies facilitated the further understanding of the alteration, by OSCC, of cellular mechanisms and demonstrated the significance of the cancer source and stage. Inhibition of key stages of the authophagy pathway revealed stages of the pathway to targeted by chemotherapeutics resulting in the modulation of the cell death pathways in response to chemotherapeutic treatment. No conflict of interest. 309 Analysis of murine stromal components in patient derived xenograft (PDX) models of pancreatic cancer 1 , J. Hoffmann1 . 1 EPO − ¨ D. Behrens1 , U. Pfohl1 , C. Hallas2 , B. Buttner Experimental Pharmacology & Oncology Berlin-Buch GmbH, D82, Berlin, ¨ Germany, 2 Institut fur ¨ Hamatopathology Hamburg, Molecular Pathology, Hamburg, Germany
Background: Pancreatic cancer remains a lethal disease with only 3−8% of patients surviving 5 years after diagnosis of the tumor (WHO, 2012). Reasons
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relapse. Expression of WT1 in AML is known to be independently associated with significant inferior response to therapy and short survival outcome. Followup monitoring of WT1 gene expression during or after therapy would be a valuable predictive marker for early recurrence or relapse of AML disease. Objective: To demonstrate prognostic relevance of WT1 expression levels monitored at diagnosis and during follow up of chemotherapy for predicting relapse in AML patients. Methods: For this study, five AML patients registered at the Oncology Clinics of the Aga Khan University Hospital, Karachi were recruited. Blood was collected from these patients and it used to isolate RNA from purified PBMCs. The RNA was reverse transcribed to make cDNA which served as template for quantitative RT-PCR analysis. Results: The WT1 levels were quantified by RT-PCR in five male patients with AML. Relatively higher level of WT1 mRNA (4000–9500 copies/ml) was detected in treatment na¨ıve patients, whereas only (100–250 copies/ml) were seen in two patients (one was receiving induction and the other patient was on consolidation therapy). In addition, one patient who received induction therapy showed raised WT1 mRNA levels (7700 copies/ml). Conclusion: High WT1 burden (>5000 copies/ml) in two patients is indicative of early recurrence of the disease along with shorter disease free and overall survival. The low WT1 expression (<200 copies/ml) in two patients after induction and consolidation therapy respectively is suggestive of better prognosis. No conflict of interest. 308 Influence of autophagy inhibitors on chemotherapeutic efficacy in oral squamous cell carcinoma A. Anderson1 , J. O’Sullivan1 . 1 Trinity College Dublin, Dental Science, Dublin, Ireland Background: Oral squamous cell carcinoma (OSCC) is the most common form of oral cancer and often manifests itself as ulceration with erythroplastic, and often leukoplakic, changes in the surrounding area. Autophagy has been implicated in most cancers and has been referred to as a double edged sword as dysregulation of this pathway can be considered tumourgenic or tumour suppressive. Autophagy activation can result from the high levels of metabolic stress chemotherapeutic agents put on the cell, which in turn can mediate an acquired resistance to these compounds during treatment. Potentially, autophagy inhibition may prevent resistance occurring in this way and in parallel prevent tumours from utilising the autophagic pathway for cancer cell survival. Materials and Methods: Two OSCC cell lines, Ca9.22 and TR146 were used along with a dysplastic cell line, DOK. Cisplatin, carboplatin, 5-fluorouracil and docetaxel, were used in combination with five autophagy inhibitors, chloroquine, LY294002, Dbeq, E64d and 3-methyladenine. IC50 values for the chemotherapeutic drugs were determined by the Alamar Blue assay after exposure to the drugs for 72 or 96 h. Indicative levels of autophagy were determined by identifying levels of acidic vesicle expression using acridine orange staining and relative levels were quantitatively determined. FACS analysis of apoptosis levels were determined by staining with propidium. Protein expression changes within autophagic and apoptotic pathways were investigated by western blot. Results and Discussion: Determined IC50 values for cisplatin, carboplatin, docetaxel and 5-fluorouracil for Ca9.22, TR146 and DOK cell lines ranged from 200 pM to 50 mM and were used as the concentration for subsequent experiments. The levels of acidic vesicles increased in the presence of a number of the chemotherapeutics. The quantity of acidic vesicles varied under combination treatment with different autophagic inhibitors. Combining drug treatment with autophagy inhibitors altered the efficiency of their apoptosis inducing capability within cells. Modulation of autophagic flux, levels of light chain 3-II, poly ADP-ribose polymerase, and Atg4 were confirmed in these cell lines by western blot analysis. Conclusion: Investigating the impacts of chemotherapeutics on programmed cell death OSCC has provided insights into the mechanisms by which these compounds affect OSCC and how resistance can develop. Examination of cell lines displaying different underlying pathologies facilitated the further understanding of the alteration, by OSCC, of cellular mechanisms and demonstrated the significance of the cancer source and stage. Inhibition of key stages of the authophagy pathway revealed stages of the pathway to targeted by chemotherapeutics resulting in the modulation of the cell death pathways in response to chemotherapeutic treatment. No conflict of interest. 309 Analysis of murine stromal components in patient derived xenograft (PDX) models of pancreatic cancer 1 , J. Hoffmann1 . 1 EPO − ¨ D. Behrens1 , U. Pfohl1 , C. Hallas2 , B. Buttner Experimental Pharmacology & Oncology Berlin-Buch GmbH, D82, Berlin, ¨ Germany, 2 Institut fur ¨ Hamatopathology Hamburg, Molecular Pathology, Hamburg, Germany
Background: Pancreatic cancer remains a lethal disease with only 3−8% of patients surviving 5 years after diagnosis of the tumor (WHO, 2012). Reasons