- Email: [email protected]
A new targeted combination therapy overcomes the acquired resistance of colorectal cancer stem cells
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 of 5 mg/kg. In this way we obtained more resistant xenografts (MNHOC124-R and MNHOC218-R). At terminal sacrifice tumors were immediately snap frozen in liquid nitrogen, and metabolites were extracted. Then, a supervised targeted metabolomic approach was performed by AbsoluteIDQ® p180 kit (BIOCRATES Life Sciences) by liquid chromatography/tandem mass spectrometry (LCMS/MS). Data were analyzed by nonparametric comparisons using Wilcoxon Method. Results: MNHOC124-R and MNHOC218-R were more resistant to cisplatin than the corresponding sensitive PDXs, as shown by the increased T/C% values from 3.8% to 30.9% and from 10.3% to 30.8%, respectively in the sensitive and resistant MNHOC124 and MNHOC218. A different metabolic profile was observed in all the sensitive and resistant xenografts, and a total of 13 metabolites (one lyso-phosphatidylcholine, 8 phosphatydilcholine, one aminoacid, 2 biogenic amine and one acylcarnitina) were found differentially regulated between the two groups. L-Lysin, alpha aminoadipic acid, and C6 acylcarnitine were the most interesting differentially regulated metabolites, showing a fold change in R versus S of −1.2, −3.2, and −2 respectively. Studies are ongoing to characterize the key enzymes in lysine metabolism in sensitive and resistant PDXs. Conclusions: The different metabolic profiles between cisplatin-sensitive and cisplatin-resistant PDXs might suggest new therapeutic combinations to prevent and/or revert the resistance to cisplatin. No conflict of interest. 252 The role of profilin I and II in glioma progression N. Svergun1,2 , U. Shahzad1 , C. Figueiredo1 , S. Agnihotri1 , B. Golbourn1 , A. Luck1 , C. Smith1 , J. Rutka1 . 1 The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumour Research Centre, Toronto, Canada, 2 National Cancer Institute, Experimental Oncology, Kyiv, Ukraine Background: The aim of our study was to examine the role of two major isoforms of profilin (PFN) in glioma progression. PFNs are considered to be important control elements for actin polymerization and have been linked to a broad spectrum of cellular functions, including cellular proliferation and migration. Despite numerous studies on profilin biology, the exact mechanism of action of PFNs on glioma progression still remains to be elucidated. Methods: Using REMBRANDT glioma data set on the Affymetrix HG U133 platform, we performed an in silico analysis to compare the PFN1 and PFN2 gene expression levels in 425 primary glioma tumours versus 20 normal brain samples. The expression levels of PFN1 and PFN2 were validated in several glioma lines, as well as normal human fetal neural stem cells (NHFNSC). siRNA-mediated knockdown was performed to examine the role of PFNs in gliomas. The effects of PFN1 and PFN2 knockdown on glioma cell invasion were studied through cell migration and cell cycle assays. Results: In all primary gliomas, we observed a statistically significant increase of PFN1 expression (P < 0.001), as well as a significant decrease of PFN2 expression (P < 0.001), compared to normal brain samples. The greatest expression level of PFN1 was evident in the most aggressive subtype of gliomas, glioblastoma multiforme (GBM). Similarly, GBM patients had the lowest expression of PFN2, compared to other subtypes of gliomas. We also observed a negative correlation (R = −0.4) between the expression levels of PFN1 and PFN2 in glioma samples. Moreover, a significant correlation was observed between high PFN1 levels and unfavourable survival in glioma patients. In a similar manner, low PFN2 expression was correlated with poor survival in glioma patients, suggesting the possible role of PFNs as biomarkers of glioma aggressiveness. The results of Western blotting, RTPCR, and immunofluorescence staining showed a robust expression of PFN1 and PFN2 in well-characterized GBM cell lines (U87, U251) as well as in NHFNSC. PFN1 primarily accumulated in the nucleus and perinuclear area of glioma cells, whereas PFN2 was evenly distributed in the cytosol. The siRNA-mediated knockdown of PFN1 did not affect the migration potential of U251 cells; however, it did inhibit cellular proliferation through a significant decrease in proportion of cells in the S-phase. Meanwhile, the knockdown of PFN2 significantly enhanced the migration activity of U251 cells, while also significantly increasing PFN1 mRNA expression level in glioma cells. Conclusion: Our results demonstrate that PFNs play an important role in glioma progression and have important implications for tumour cell migration and proliferation. PFN expression levels can be used as markers of glioma prognosis, and represent an attractive and promising target for better understanding and treatment of human gliomas. No conflict of interest.
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253 Radium-223 in the treatment of metastatic prostate cancer I.A. Marques1 , A.M. Abrantes1 , A.S. Pires1 , G. Costa2 , E. Tavares-Silva3 , M.F. Botelho1 . 1 IBILI- CIMAGO- Faculty of Medicine- University of Coimbra, Institute of Biophysics and Biomathematics, Coimbra, Portugal, 2 CHUC, Nuclear Medicine Deparment, Coimbra, Portugal, 3 CHUC- University of Coimbra, Department of Urology and Transplantation- Institue of Biophysics and Biomathematics, Coimbra, Portugal Background: Metastatic castration-resistant prostate cancer (mCRPC) is in therapeutic terms the biggest challenge of prostate cancer (PCa). The radiopharmaceutical Radium-223 (223 Ra) acts as a calcium mimetic by forming complexes with the bone mineral hydroxyapatite in areas of high bone turnover, thereby directly targeting the areas of bone metastases. It is considered the best treatment for patients with mCRPC. The radiopharmaceutical 99m Tc-HMDP is also an analog of calcium used in bone scintigraphy. Given the relative lack of information of the molecular pathways responsible for the effects of 223 Ra in cells of mCRPC, this project aims to study the effects of 223 Ra in cell lines of PCa, using as controls one osteosarcoma cell line and the uptake profile of 99m Tc-HMDP. Material and Methods: Three tumor cell lines PC3 (metastatic PCa), LNCaP (no metastatic PCa) and MNNG-HOS (osteosarcoma) were incubated with the 223 Ra radiopharmaceutical (0.5 mCi/mL) or 99m Tc-HMDP (25 mCi/mL). For uptake studies samples of 200 mL were taken at 5, 30, 60, 90, 120, 150 and 180 minutes. These were centrifuged to separate the supernatant and the pellet. The uptake percentage of 223 Ra and 99m Tc-HMDP was determined after measuring the radioactivity of both fractions in a well counter, in counts per minute. Results: Preliminary results show that, at 120 minutes, the percentage of 223 Ra uptake by PC3 is twice higher (1.6±0.1%) than by MNNGHOS (0.8±0.13%). A similar uptake profile was obtained for LNCaP cells (1.26±0.16%). The uptake of 223 Ra by MNNG-HOS is five times higher (1.0±0.1%) than the uptake of 99m Tc-HMDP (0.2±0.09%). In PC3 cells line the same is verified. By the other side, in LNCaP cell line the uptake profile is similar between the two radiopharmaceuticals. Conclusion: Preliminary results suggest that, although both radiopharmaceuticals are analogues of calcium, the uptake mechanisms may be different. This study also enhances the therapeutic potential of 223 Ra in the treatment of metastatic PCa. Acknowledgement: The authors would like to thank Foundation for Science and Technology (FCT) (Strategic Project CNC.IBILI: UID/NEU/04539/2013), COMPETE-FEDER for financial support. No conflict of interest. 255 A new targeted combination therapy overcomes the acquired resistance of colorectal cancer stem cells A. Benfante1 , L.R. Mangiapane1 , M.L. Colorito2 , M. Gaggianesi1 , A. Nicotra1 , A. Giammona1 , E. Scavo1 , A. Chinnici1 , M. Todaro3 , G. Stassi1 . 1 University of Palermo, Department of Surgical- Oncological and Stomatological Sciences, Palermo, Italy, 2 Aging Research Center Ce.S.I.- Chieti- Italy., Research Unit on Non-coding RNA and Cancer, Chieti, Italy, 3 University of Palermo, Biomedical department of internal and specialistical medicine, Palermo, Italy Background: Despite new advances in therapy of colorectal cancer (CRC), a large number of patients develops recurrences and metastasis due to the failure of current therapies. The adverse clinical outcome of metastatic CRC patients is due to a rapid evolution of clonal populations characterized by distinct acquired resistance mechanisms. This concept supports the existence of cellular hierarchies where cancer stem cells (CSCs) are the unique subpopulation responsible for tumor propagation, relapse and metastatic dissemination. Recently, we have demonstrated that CD44v6 is a functional marker of metastatic CR-CSCs subpopulation, characterized by enhanced activation of PI3K/AKT pathway. We have observed that PI3K inhibition impairs metastatic potential and survival of CD44v6+ cells. Based on these evidences we assumed that PI3K pathway could be implicated in the acquired resistance. Material and Methods: CR-CSCs obtained from human CRC tissues were characterized on the bases of different mutational profile. In vitro and in vivo experiments were performed on BRAF or KRAS with or without PI3KCA mutated CR-CSCs in order to analyze the effects of combinatorial treatments. Flow cytometry assays showed the percentage of rescued CD44v6+ cells. Western blotting and miRNAs analyses revealed the mechanisms altered in resistant cellular subpopulations. Results: The combined treatment with BRAFV600E inhibitor and EGFR antibodies induced CR-CSCs acquired resistance mediated by CD44v6 overexpression as well as high PI3K/AKT activity. Based on these observations, we demonstrated that the use of BRAF, EGFR2 and PI3K inhibitors affected cell viability of CR-CSCs. This treatment reduced tumor growth capacity of BRAF and KRAS mutated CR-CSCs enhancing the percentage of mice survival rate. Conversely, BRAF or KRAS/PI3KCA mutated CR-CSCs, treated as mentioned above, maintain the capacity to generate secondary tumors, overcoming the
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combinatorial treatment by MYC miRNA targets upregulations. The additional administration of MEK inhibitor affected MYC expression sensitizing CR-CSCs to cell death. The triple treatment with EGFR2, MEK and PI3K inhibitors prevents the onset of CSCs resistance clones, PI3KCA mutated, inhibiting their tumor growth capacity. Interestingly, this treatment spared the rescued population of BRAF or KRAS/PI3KCA mutated CR-CSCs also after a first line of treatment with BRAF, EGFR2 and PI3K inhibitors. Conclusions: These results support the use of this triple combination to avoid CSCs-mediated chemoresistance also in a second line of treatment. No conflict of interest. 256 Increasing mechanistic understanding of the role of p53 mutations in triple negative breast cancer and identifying potential roles in chemoresistance R. Steele1 , S. Eddie1 , S. McDade1 , P. Mullan1 . 1 Queen’s University Belfast, Centre for Cancer Research and Cell Biology, Belfast, United Kingdom Introduction: Breast Cancer is the most common type of Cancer in women worldwide. Despite a dramatic improvement in survival rates over the last 40 years, the triple negative breast cancer (TNBC) subtype still has the poorest survival rates of all. Triple negative tumours lack expression of estrogen and progesterone receptors and HER2 receptor amplification, consequently, there are currently no targeted therapies. FEC chemotherapy; a combination treatment, is currently given to TNBC patients, however, chemoresistance is prominent. Presently, we do not know the key driver genes responsible for TNBCs, or the high rate of chemoresistance within the subtype, but they are known to contain high rates of p53 mutation. P53 mutations are elevated in TNBC’s, with approximately 85% containing at least one p53 aberration, in comparison to 15−20% in other breast cancer subgroups. Additionally, mutant p53 is often elevated in expression in relation to the canonical wild-type protein. Many of the p53 mutations in TNBC’s not only disable p53 but switch it from an anti-cancer to a pro-cancer gene. Materials and Methods: The project is based upon the novel BirA fusion technique, to identify novel mutant p53 (R280K and R273H) interacting proteins and potential co-regulated transcriptional targets. FEM resistant cell lines have also been generated from the TNBC cell line MDA-MB-468 (R273H p53 mutant), to gain mechanistic insight into whether mutant p53 and potentially p53 isoforms play a role in chemotherapy resistance in this breast cancer subtype. Results and Discussion: We strongly believe that mutant p53 proteins may be acting as oncogenic drivers in TNBC, as research within the lab has observed that siRNA-mediated knockdown of GOF mutant p53 protein is detrimental to TNBC cell survival and proliferation within cell lines. Conclusion: Through investigating the mechanisms which mutant p53 and p53 isoforms can drive TNBC growth, we hope to improve the poor outcomes currently associated with TNBC and associated high rates of chemoresistance. No conflict of interest. 257 17b-Estradiol stimulates generation of reactive oxygen species and nitric oxide in ovarian adenocarcinoma cells M.H. Moghadasi1 . 1 Department of Medical Laboratory- Shahid Labbafinejad Medical Center- Iranian Social Security Organization Tehran- Iran, medical lab, Tehran, Iran Background: Experimental and epidemiological evidence supports a role for steroid hormones in the pathogenesis of ovarian cancer. Among steroid hormones, 17b-estradiol (E2) has the most potent effect on proliferation, apoptosis and metastasis. Objectives: In the present study, we investigated the effect of E2 on production of ROS and NO in ovarian cancer cells. Materials and Methods: Ovarian adenocarcinoma cell line (OVCAR-3) was cultured and treated with various concentrations of E2, antioxidants (N-acetyle cysteine and Ebselen) and ICI182780 as an estrogen receptor antagonist. MTT test was performed to evaluate cell viability. NO and ROS levels were measured by Griess and DCFH-DA methods, respectively. Results: ROS levels as well as NO levels were increased in OVCAR-3 cells treated with E2. The increase in ROS production was in parallel with increased cell viability which indicates that estrogen-induced ROS can participate in cancer progression. ICI182780 abolished E2-induced ROS production. Progesterone was also effective in reducing ROS and NO generation. Conclusions: NO and ROS are important molecules in signaling networks in cell. These molecules can be used as therapeutic targets for prevention and treatment of ovary cancer and other estrogen-induced malignancies. No conflict of interest.
259 Regulation mechanism of Notoginsenoside R1 on human colorectal cancer metastasis C.C. Wu1 , S. Hsieh2 , L.C. Hsieh3 , Y.H. Kuo4 , S.L. Hsieh4 . 1 Chang Jung Christian University, Department of Nutrition and Health Sciences, Tainan, Taiwan, 2 National Sun Yat-sen University, Department of Chemistry, Kaohsiung, Taiwan, 3 Kaohsiung Municipal United Hospital, Department of Dietetics, Kaohsiung, Taiwan, 4 National Kaohsiung Marine University, Department of Seafood Science, Kaohsiung, Taiwan Background: Panax notoginseng is a traditional Chinese medicine for cardiovascular disease, but it is limited on anti-metastasis study. The goal of this study was to investigate the effect of notoginseng R1 (R1), derived from Panax notoginseng, on the regulation of human colorectal cancer (CRC) metastasis. Material and Methods: The migratory, invasive, and adhesive abilities and metastasis-associated regulatory molecule expression of cultured human CRC cells (HCT-116) treated with R1 were analyzed in this study. Results: The migratory and invasive abilities of HCT-116 cells were reduced after R1 treatment. When HCT-116 cells were incubated with R1 for 24 h, MMP-9 expression was lower compared with the control group. The adhesion reaction assay results indicated that treatment with R1 led to significantly decreased HCT-116 adhesion to endothelial cells (EA.hy926 cells). In HCT116 cells, integrin-1 protein levels significantly decreased following treatment with R1. Similarly, E-selectin and intercellular adhesion molecule-1 (ICAM-1) protein levels significantly decreased when the EA.hy926 endothelial cells were treated with R1. Scanning electron microscope (SEM) examination indicated that HCT-116 cells treated with LPS combined with R1 had a less flattened and retracted shape compared with LPS-treated cells; this change in shape was a phenomenon of extravasation invasion. The transepithelial electrical resistance (TEER) of the EA.hy926 endothelial cell monolayer increased after incubation with R1 for 24 h. Conclusion: These results demonstrate the anti-metastatic properties of R1. R1 affects cells via inhibition of cell migration, invasion, and adhesion and via regulation of metastasis-associated signaling molecule expression. No conflict of interest. 261 Next generation sequencing in laryngeal cancer specimens reveals alterations of the DIAPH2 gene that can putatively contribute to the metastatic potential of squamous carcinoma M. Giefing1,2 , E. Byzia1 , N. Zemke1 , K. Kiwerska1 , M. Kostrzewska-Poczekaj1 , M. Jarmuz-Szymczak1 , M. Wierzbicka2 , G. Greczka2 , R. Grenman3,4 , K. Szyfter1,5 . 1 Institute of Human Genetics- Polish Academy of Sciences, Department of Cancer Genetics, Poznan, Poland, 2 Poznan University of Medical Sciences, Department of Otolaryngology and Laryngological Oncology, Poznan, Poland, 3 Turku University Central Hospital and Turku University, Department of Otorhinolaryngology- − Head and Neck Surgery, Turku, Finland, 4 Turku University Central Hospital and Turku University, Department of Medical Biochemistry, Turku, Finland, 5 Poznan University of Medical Sciences, Department of Phoniatrics and Audiology, Poznan, Poland Background: Recently, we performed an array-CGH screen aimed at the identification of homozygous deletions in laryngeal squamous cell carcinoma cell lines (LSCC). Despite the relatively low number of analyzed cell lines (n = 10) we reported bi-allelic losses targeting the DIAPH2 and DIAPH3 genes. Under physiological conditions these diaphanous genes (formin family) are involved in the regulation of cell movement and adhesion and therefore are potentially involved in tumor metastasis. Intrigued by this finding that two diaphanous genes are targeted in the analyzed samples and regarding their function we found it as a rationale to perform a deep sequencing screen for potential mutations of these genes and to analyze their distribution in (N(0) n = 53 and N(+) n = 42) primary LSCC specimens. Material and Methods: We pre-selected five metastases derived LSCC cell lines (UT-SCC-4, UT-SCC-6B, UT-SCC-9, UT-SCC-17, UT-SCC-58) and sequenced all coding exons of the analyzed genes (Sanger) and thereafter extended the analysis to Next Generation Sequencing of 95 primary LSCC specimens (Illumina MiSeq; paired-end sequencing, coverage for single exons: DIAPH2 range 20–6451, median 528; DIAPH3 range 20–10367, median 1436). Moreover, we analyzed the expression of the two genes in LSCC cell lines using previously published expression profiles (Affymetrix U133 plus 2.0). Results: We observed a 7.9 fold downregulation of DIAPH2 expression in LSCC cell lines and a 2.35 fold downregulation in primary specimens compared to no-tumor laryngeal controls. In line with this finding, we identified a homozygous deletion of exon 23 (NM_006729; c.3116_3240del125) in DIAPH2 in UT-SCC-17 and 4 heterozygous mutations in the 95 primary LSCC specimens. Three of them, located in highly conserved functional domains FH2 and GBD/FH3 were identified in the N(+) samples whereas the mutation in a N(0) sample was located outside functional domains. Interestingly, by combining these results with the sequencing data from the cBioPortal we show a significant enrichment of DIAPH2 mutations (p = 0.036)
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253 Radium-223 in the treatment of metastatic prostate cancer I.A. Marques1 , A.M. Abrantes1 , A.S. Pires1 , G. Costa2 , E. Tavares-Silva3 , M.F. Botelho1 . 1 IBILI- CIMAGO- Faculty of Medicine- University of Coimbra, Institute of Biophysics and Biomathematics, Coimbra, Portugal, 2 CHUC, Nuclear Medicine Deparment, Coimbra, Portugal, 3 CHUC- University of Coimbra, Department of Urology and Transplantation- Institue of Biophysics and Biomathematics, Coimbra, Portugal Background: Metastatic castration-resistant prostate cancer (mCRPC) is in therapeutic terms the biggest challenge of prostate cancer (PCa). The radiopharmaceutical Radium-223 (223 Ra) acts as a calcium mimetic by forming complexes with the bone mineral hydroxyapatite in areas of high bone turnover, thereby directly targeting the areas of bone metastases. It is considered the best treatment for patients with mCRPC. The radiopharmaceutical 99m Tc-HMDP is also an analog of calcium used in bone scintigraphy. Given the relative lack of information of the molecular pathways responsible for the effects of 223 Ra in cells of mCRPC, this project aims to study the effects of 223 Ra in cell lines of PCa, using as controls one osteosarcoma cell line and the uptake profile of 99m Tc-HMDP. Material and Methods: Three tumor cell lines PC3 (metastatic PCa), LNCaP (no metastatic PCa) and MNNG-HOS (osteosarcoma) were incubated with the 223 Ra radiopharmaceutical (0.5 mCi/mL) or 99m Tc-HMDP (25 mCi/mL). For uptake studies samples of 200 mL were taken at 5, 30, 60, 90, 120, 150 and 180 minutes. These were centrifuged to separate the supernatant and the pellet. The uptake percentage of 223 Ra and 99m Tc-HMDP was determined after measuring the radioactivity of both fractions in a well counter, in counts per minute. Results: Preliminary results show that, at 120 minutes, the percentage of 223 Ra uptake by PC3 is twice higher (1.6±0.1%) than by MNNGHOS (0.8±0.13%). A similar uptake profile was obtained for LNCaP cells (1.26±0.16%). The uptake of 223 Ra by MNNG-HOS is five times higher (1.0±0.1%) than the uptake of 99m Tc-HMDP (0.2±0.09%). In PC3 cells line the same is verified. By the other side, in LNCaP cell line the uptake profile is similar between the two radiopharmaceuticals. Conclusion: Preliminary results suggest that, although both radiopharmaceuticals are analogues of calcium, the uptake mechanisms may be different. This study also enhances the therapeutic potential of 223 Ra in the treatment of metastatic PCa. Acknowledgement: The authors would like to thank Foundation for Science and Technology (FCT) (Strategic Project CNC.IBILI: UID/NEU/04539/2013), COMPETE-FEDER for financial support. No conflict of interest. 255 A new targeted combination therapy overcomes the acquired resistance of colorectal cancer stem cells A. Benfante1 , L.R. Mangiapane1 , M.L. Colorito2 , M. Gaggianesi1 , A. Nicotra1 , A. Giammona1 , E. Scavo1 , A. Chinnici1 , M. Todaro3 , G. Stassi1 . 1 University of Palermo, Department of Surgical- Oncological and Stomatological Sciences, Palermo, Italy, 2 Aging Research Center Ce.S.I.- Chieti- Italy., Research Unit on Non-coding RNA and Cancer, Chieti, Italy, 3 University of Palermo, Biomedical department of internal and specialistical medicine, Palermo, Italy Background: Despite new advances in therapy of colorectal cancer (CRC), a large number of patients develops recurrences and metastasis due to the failure of current therapies. The adverse clinical outcome of metastatic CRC patients is due to a rapid evolution of clonal populations characterized by distinct acquired resistance mechanisms. This concept supports the existence of cellular hierarchies where cancer stem cells (CSCs) are the unique subpopulation responsible for tumor propagation, relapse and metastatic dissemination. Recently, we have demonstrated that CD44v6 is a functional marker of metastatic CR-CSCs subpopulation, characterized by enhanced activation of PI3K/AKT pathway. We have observed that PI3K inhibition impairs metastatic potential and survival of CD44v6+ cells. Based on these evidences we assumed that PI3K pathway could be implicated in the acquired resistance. Material and Methods: CR-CSCs obtained from human CRC tissues were characterized on the bases of different mutational profile. In vitro and in vivo experiments were performed on BRAF or KRAS with or without PI3KCA mutated CR-CSCs in order to analyze the effects of combinatorial treatments. Flow cytometry assays showed the percentage of rescued CD44v6+ cells. Western blotting and miRNAs analyses revealed the mechanisms altered in resistant cellular subpopulations. Results: The combined treatment with BRAFV600E inhibitor and EGFR antibodies induced CR-CSCs acquired resistance mediated by CD44v6 overexpression as well as high PI3K/AKT activity. Based on these observations, we demonstrated that the use of BRAF, EGFR2 and PI3K inhibitors affected cell viability of CR-CSCs. This treatment reduced tumor growth capacity of BRAF and KRAS mutated CR-CSCs enhancing the percentage of mice survival rate. Conversely, BRAF or KRAS/PI3KCA mutated CR-CSCs, treated as mentioned above, maintain the capacity to generate secondary tumors, overcoming the
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combinatorial treatment by MYC miRNA targets upregulations. The additional administration of MEK inhibitor affected MYC expression sensitizing CR-CSCs to cell death. The triple treatment with EGFR2, MEK and PI3K inhibitors prevents the onset of CSCs resistance clones, PI3KCA mutated, inhibiting their tumor growth capacity. Interestingly, this treatment spared the rescued population of BRAF or KRAS/PI3KCA mutated CR-CSCs also after a first line of treatment with BRAF, EGFR2 and PI3K inhibitors. Conclusions: These results support the use of this triple combination to avoid CSCs-mediated chemoresistance also in a second line of treatment. No conflict of interest. 256 Increasing mechanistic understanding of the role of p53 mutations in triple negative breast cancer and identifying potential roles in chemoresistance R. Steele1 , S. Eddie1 , S. McDade1 , P. Mullan1 . 1 Queen’s University Belfast, Centre for Cancer Research and Cell Biology, Belfast, United Kingdom Introduction: Breast Cancer is the most common type of Cancer in women worldwide. Despite a dramatic improvement in survival rates over the last 40 years, the triple negative breast cancer (TNBC) subtype still has the poorest survival rates of all. Triple negative tumours lack expression of estrogen and progesterone receptors and HER2 receptor amplification, consequently, there are currently no targeted therapies. FEC chemotherapy; a combination treatment, is currently given to TNBC patients, however, chemoresistance is prominent. Presently, we do not know the key driver genes responsible for TNBCs, or the high rate of chemoresistance within the subtype, but they are known to contain high rates of p53 mutation. P53 mutations are elevated in TNBC’s, with approximately 85% containing at least one p53 aberration, in comparison to 15−20% in other breast cancer subgroups. Additionally, mutant p53 is often elevated in expression in relation to the canonical wild-type protein. Many of the p53 mutations in TNBC’s not only disable p53 but switch it from an anti-cancer to a pro-cancer gene. Materials and Methods: The project is based upon the novel BirA fusion technique, to identify novel mutant p53 (R280K and R273H) interacting proteins and potential co-regulated transcriptional targets. FEM resistant cell lines have also been generated from the TNBC cell line MDA-MB-468 (R273H p53 mutant), to gain mechanistic insight into whether mutant p53 and potentially p53 isoforms play a role in chemotherapy resistance in this breast cancer subtype. Results and Discussion: We strongly believe that mutant p53 proteins may be acting as oncogenic drivers in TNBC, as research within the lab has observed that siRNA-mediated knockdown of GOF mutant p53 protein is detrimental to TNBC cell survival and proliferation within cell lines. Conclusion: Through investigating the mechanisms which mutant p53 and p53 isoforms can drive TNBC growth, we hope to improve the poor outcomes currently associated with TNBC and associated high rates of chemoresistance. No conflict of interest. 257 17b-Estradiol stimulates generation of reactive oxygen species and nitric oxide in ovarian adenocarcinoma cells M.H. Moghadasi1 . 1 Department of Medical Laboratory- Shahid Labbafinejad Medical Center- Iranian Social Security Organization Tehran- Iran, medical lab, Tehran, Iran Background: Experimental and epidemiological evidence supports a role for steroid hormones in the pathogenesis of ovarian cancer. Among steroid hormones, 17b-estradiol (E2) has the most potent effect on proliferation, apoptosis and metastasis. Objectives: In the present study, we investigated the effect of E2 on production of ROS and NO in ovarian cancer cells. Materials and Methods: Ovarian adenocarcinoma cell line (OVCAR-3) was cultured and treated with various concentrations of E2, antioxidants (N-acetyle cysteine and Ebselen) and ICI182780 as an estrogen receptor antagonist. MTT test was performed to evaluate cell viability. NO and ROS levels were measured by Griess and DCFH-DA methods, respectively. Results: ROS levels as well as NO levels were increased in OVCAR-3 cells treated with E2. The increase in ROS production was in parallel with increased cell viability which indicates that estrogen-induced ROS can participate in cancer progression. ICI182780 abolished E2-induced ROS production. Progesterone was also effective in reducing ROS and NO generation. Conclusions: NO and ROS are important molecules in signaling networks in cell. These molecules can be used as therapeutic targets for prevention and treatment of ovary cancer and other estrogen-induced malignancies. No conflict of interest.
259 Regulation mechanism of Notoginsenoside R1 on human colorectal cancer metastasis C.C. Wu1 , S. Hsieh2 , L.C. Hsieh3 , Y.H. Kuo4 , S.L. Hsieh4 . 1 Chang Jung Christian University, Department of Nutrition and Health Sciences, Tainan, Taiwan, 2 National Sun Yat-sen University, Department of Chemistry, Kaohsiung, Taiwan, 3 Kaohsiung Municipal United Hospital, Department of Dietetics, Kaohsiung, Taiwan, 4 National Kaohsiung Marine University, Department of Seafood Science, Kaohsiung, Taiwan Background: Panax notoginseng is a traditional Chinese medicine for cardiovascular disease, but it is limited on anti-metastasis study. The goal of this study was to investigate the effect of notoginseng R1 (R1), derived from Panax notoginseng, on the regulation of human colorectal cancer (CRC) metastasis. Material and Methods: The migratory, invasive, and adhesive abilities and metastasis-associated regulatory molecule expression of cultured human CRC cells (HCT-116) treated with R1 were analyzed in this study. Results: The migratory and invasive abilities of HCT-116 cells were reduced after R1 treatment. When HCT-116 cells were incubated with R1 for 24 h, MMP-9 expression was lower compared with the control group. The adhesion reaction assay results indicated that treatment with R1 led to significantly decreased HCT-116 adhesion to endothelial cells (EA.hy926 cells). In HCT116 cells, integrin-1 protein levels significantly decreased following treatment with R1. Similarly, E-selectin and intercellular adhesion molecule-1 (ICAM-1) protein levels significantly decreased when the EA.hy926 endothelial cells were treated with R1. Scanning electron microscope (SEM) examination indicated that HCT-116 cells treated with LPS combined with R1 had a less flattened and retracted shape compared with LPS-treated cells; this change in shape was a phenomenon of extravasation invasion. The transepithelial electrical resistance (TEER) of the EA.hy926 endothelial cell monolayer increased after incubation with R1 for 24 h. Conclusion: These results demonstrate the anti-metastatic properties of R1. R1 affects cells via inhibition of cell migration, invasion, and adhesion and via regulation of metastasis-associated signaling molecule expression. No conflict of interest. 261 Next generation sequencing in laryngeal cancer specimens reveals alterations of the DIAPH2 gene that can putatively contribute to the metastatic potential of squamous carcinoma M. Giefing1,2 , E. Byzia1 , N. Zemke1 , K. Kiwerska1 , M. Kostrzewska-Poczekaj1 , M. Jarmuz-Szymczak1 , M. Wierzbicka2 , G. Greczka2 , R. Grenman3,4 , K. Szyfter1,5 . 1 Institute of Human Genetics- Polish Academy of Sciences, Department of Cancer Genetics, Poznan, Poland, 2 Poznan University of Medical Sciences, Department of Otolaryngology and Laryngological Oncology, Poznan, Poland, 3 Turku University Central Hospital and Turku University, Department of Otorhinolaryngology- − Head and Neck Surgery, Turku, Finland, 4 Turku University Central Hospital and Turku University, Department of Medical Biochemistry, Turku, Finland, 5 Poznan University of Medical Sciences, Department of Phoniatrics and Audiology, Poznan, Poland Background: Recently, we performed an array-CGH screen aimed at the identification of homozygous deletions in laryngeal squamous cell carcinoma cell lines (LSCC). Despite the relatively low number of analyzed cell lines (n = 10) we reported bi-allelic losses targeting the DIAPH2 and DIAPH3 genes. Under physiological conditions these diaphanous genes (formin family) are involved in the regulation of cell movement and adhesion and therefore are potentially involved in tumor metastasis. Intrigued by this finding that two diaphanous genes are targeted in the analyzed samples and regarding their function we found it as a rationale to perform a deep sequencing screen for potential mutations of these genes and to analyze their distribution in (N(0) n = 53 and N(+) n = 42) primary LSCC specimens. Material and Methods: We pre-selected five metastases derived LSCC cell lines (UT-SCC-4, UT-SCC-6B, UT-SCC-9, UT-SCC-17, UT-SCC-58) and sequenced all coding exons of the analyzed genes (Sanger) and thereafter extended the analysis to Next Generation Sequencing of 95 primary LSCC specimens (Illumina MiSeq; paired-end sequencing, coverage for single exons: DIAPH2 range 20–6451, median 528; DIAPH3 range 20–10367, median 1436). Moreover, we analyzed the expression of the two genes in LSCC cell lines using previously published expression profiles (Affymetrix U133 plus 2.0). Results: We observed a 7.9 fold downregulation of DIAPH2 expression in LSCC cell lines and a 2.35 fold downregulation in primary specimens compared to no-tumor laryngeal controls. In line with this finding, we identified a homozygous deletion of exon 23 (NM_006729; c.3116_3240del125) in DIAPH2 in UT-SCC-17 and 4 heterozygous mutations in the 95 primary LSCC specimens. Three of them, located in highly conserved functional domains FH2 and GBD/FH3 were identified in the N(+) samples whereas the mutation in a N(0) sample was located outside functional domains. Interestingly, by combining these results with the sequencing data from the cBioPortal we show a significant enrichment of DIAPH2 mutations (p = 0.036)