- Email: [email protected]
Crizotinib overcomes microenvironment mediated drug resistance in Ph positive leukemia
S92
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
process by serial transient miR-135a force-expression in a precancerous cell model and examine in vitro tumor forming ability of its CD133+ subpopulation. Material and Method: NC104-E6/E7 is normal cervix epithelia cell-line immortalized by HPV E6 and E7. The transformation process was done by serial miR-135a transient force-expressions. Functional assays examined the tumorigenic properties after each miR-135a force-expression and compared to the control force-expression counterpart. Correspondingly, the CD133+ and CD133− subpopulations are isolated by CD133 antibody coated microbeads and their in vitro tumor sphere forming abilities are examined. Results and Discussion: After 3 successive miR-135a force-expressions, NC104-E6/E7-3t-135a cells exhibited higher invasiveness. Other malignant properties such as proliferation and migration were only slightly changed compared to the control group. The expression of E-cadherin was decreased accompanied by an increase of N-cadherin during the miR-135a mediated transformation process, indicating that the epithelial–mesenchymal transition (EMT) has occurred. CD133+ cells were found after 3 successive miR-135a force expressions. The CD133+ cells isolated from the NC104-E6/E7-3t-135a cells exhibited higher tumor spheres forming ability; the number and size of tumor spheres formed were larger than their CD133− counterparts. CD133+ subpopulation from the two control cell lines, NC104-E6/E7 and NC104-E6/E73t-ctrl cells failed to form tumor spheres. Conclusion: MiR-135a mediated cervical cancer malignant transformation is a multi-step process. Force expressing miR-135a favored the EMT process of precancerous cell model, which might be responsible for the stemness acquisition of cancer stem cells. This transformation process induced formation of CD133+ subpopulation. No conflict of interest. 432 DigiWest multiplex protein profiling of extracellular vesicles to decipher cancer metastasis and to identify biomarkers G. Erdmann1 , C. Sachse1 , M. Templin2 , J. Gross3 . 1 NMI TT GmbH, Pharmaservices, Berlin, Germany, 2 NMI, Protein Profiling & Assay ¨ Development, Reutlingen, Germany, 3 University Medical Center Gottingen, Haematology and Oncology, Goettingen, Germany Background: Improved diagnostic tests for cancer treatment are crucial in the clinic as well as during drug development. Technically, liquid biopsies are highly desirable, which has raised interest in CTCs and secreted extracellular vesicles. Most cell types secrete extracellular vesicles (EVs), a heterogeneous population of membrane particles, named according to their size and origin, as microvesicles (MVs) (250–1000 nm) or exosomes (50–100 nm). They have been described as a specific form of inter/extracellular communication. For example, tumor cell-derived EVs prime metastatic sites for incoming tumor stem cells (Peinado et al. 2012). In addition, a variety of signaling molecules are secreted on EVs from their sending cells, e.g. Wnt proteins, which allows them to induce Wnt signaling activity on target cells (Gross et al. 2012) and even to enhance migration and invasiveness of tumor cells (Menck et al. 2013). Hence, interrogating signaling molecules transmitted via tumor derived EVs may reveal biomarkers that might be more conclusive than circulating nucleic acids. Thus, analyzing proteins incorporated on EVs may offer important diagnostic information prior to and during treatment. Our aim is to analyze proteins traveling on extracellular vesicles and determine their utility as biomarkers. Methods: Extracellular vesicles were purified from different breast cancer cell lines as well as plasma from cancer patients by a differential centrifugation, separating exosomes and microvesicles by ultracentrifugation. To simultaneously detect a wide range of proteins traveling on the EVs, a novel protein profiling platform, DigiWest, was utilized. This method combines sizebased separation by gel electrophoresis with the Luminex bead system into a highly multiplexed “digital Western blot”. Using this approach, >90 proteins were analyzed for their presence on extracellular vesicles from as little as 10 mg of total protein. Results: Using the DigiWest assay on extracellular vesicles, we were able to identify and quantify known specific marker proteins for exosomes as well as microvesicles, respectively. Overall, we identified proteins specific for one type of vesicles or the other, but also found proteins equally present in both sample types. Furthermore, we identified a number of signaling proteins and receptors on both extracellular vesicles fractions that we further analyze for their robustness and relevance to breast cancer. Conclusion: Monitoring signaling molecules on extracellular vesicles using the DigiWest multiplex protein profiling technology is a highly promising approach for elucidation of the biological function of extracellular vesicles during tumor metastasis as well as for identification of specific biomarker candidates. Conflict of interest: Other Substantive Relationships: NMI TT Pharmaservices offers DigiWest as a service (for research use only).
433 Fasting in combination with curcumin induces a fatal energy “black out” in tumor cells L. Raffaghello1 , G. Bianchi1 , N. Bertola1 , A. Amaro2 , G. Angelini2 , L. Emionite3 , S. Ravera4 , U. Pfeffer2 . 1 G.Gaslini Children’s Hospital, Laboratory of Oncology, Genova, Italy, 2 IRCCS AOU San Martino − IST Istituto Nazionale per la Ricerca sul Cancro, Molecular Pathology, Genoa, Italy, 3 IRCCS AOU San Martino − IST Istituto Nazionale per la Ricerca sul Cancro, Animal Facility, Genoa, Italy, 4 University of Genoa, Department of Pharmacy, Genoa, Italy Introduction: The dietary polyphenol curcumin (CUR) has been described to exert anti-tumoral effects without toxicity. At present, 126 clinical studies using CUR or its derivatives are registered at clinicaltrials.gov. CUR has recently been defined as a caloric restriction mimetic (CRM), as it mimics biochemical and functional effects of calorie restriction (CR). In mice, two day cycles of fasting with only water access (short term starvation: STS) represent a peculiar example of CR that has been shown to protect normal cells but not tumor cells against cytotoxicity of chemotherapy and to render many tumors more susceptible to chemotherapy through an anti-Warburg effect. Preliminary clinical data indicate that in cancer patients fasting is feasible, not associated with major toxicity and able to reduce several side effects associated to chemotherapy. Aim of this study is to investigate the effects and mechanisms of fasting in combination with CUR on tumor metabolism. Material and Method: We tested the cytotoxicity of different concentrations of CUR +/− STS (1% FBS + 0.5 g/L glucose) on murine and human cell lines of melanoma, neuroblastoma, breast, colon cancer, and chronic lymphatic leukemia by Trypan Blue and Annexin V staining. Reactive oxygen species production was measured by 2 ,7 -dichlorodihydrofluorescein diacetate staining. Protein expression and activity of the key glycolytic enzymes, respiratory chain complexes as well as autophagy and proliferative pathways (PI3K/AKT/mTOR) were studied by Western blot and spectrophotometric assays. Results and Discussion: CUR reduces cell proliferation and induces apoptosis of various tumor cell lines, without affecting ROS production. It exerts an anti-Warburg effect by decreasing the activity of the main glycolytic enzymes. CUR inhibits ATP synthase and reduces O2 consumption leading to a dramatic drop of ATP production. STS shows similar anti-proliferative and apoptotic effects. STS-treated tumor cells exhibit a significant induction of respiratory chain complexes I, III, IV associated to a striking uncoupling between induced O2 consumption and reduced ATP synthesis. This latter event leads to a significant increase of ROS generation and apoptosis. The highest tumor cytotoxicity was observed for the combination of STS and CUR that additively reduces glycolysis and oxidative phosphorylation leading to a complete impairment of energy charge and consequent cell death. Differences between cell lines based on their basal respiratory activity are observed. The efficacy of STS+CUR in vivo is currently being tested. Conclusion: Fasting combined with CUR causes a strong suppression of tumor cell viability by inducing a fatal energy impairment. Thus, this association represents a promising strategy for the treatment of human cancer. No conflict of interest. 434 Crizotinib overcomes microenvironment mediated drug resistance in Ph positive leukemia O. Regev1 , N. Kidan1 , H. Khamisie1 , J. Mahajna1 . 1 Galilee Technology Center- Migal, Cancer Drug Discovery Program, Kiryat Shmona, Israel Background: Abl Kinase Inhibitors (AKIs) exhibiting good clinical efficacy in Ph+ leukemia. However, in one-third of patients, first-line TKI treatment will eventually become ineffective, which attributable to either non-mutational or kinase mutations in BCR/ABL. Conditions within the Bone Marrow (BM) niche contribute to reduced drug sensitivity of cancer cells residing in the BM including Ph+ leukemia. Crizotinib, a kinase inhibitor designed to target c-ALK for the treatment of a subtype of lung cancer, exhibits activity also against the ABL-kinase. Aim: Our aim is to understand BM microenvironment role in Ph+ leukemia AKI drug resistance and investigated the therapeutic potential of Crizotinib for the treatment of advanced and therapy of resistant disease. Methods: To study BM microenvironment mediated drug resistance in Ph+ leukemia; we exposed CML cells to condition media (CM) collected from mesenchymal cells. Cell proliferation was monitored by XTT assay and apoptosis inducing function of AKIs was assayed by following PARP cleavage. In addition we followed levels of STAT proteins. Results: CM collected from mesenchymal cells confers Imatinib drug resistance to CML cells. However, CM collected from ovarian or murine skin melanoma cells were not efficient in conferring drug resistance to Imatinib. Moreover, no significant change in CM mediated Imatinib drug resistance when CM were filtered with 0.4 or 0.2 micron. Exposer of CML cells to CM abolished ability of Imatinib to induce PARP cleavage. In contrasts, Crizotinib overcomes CM-mediated drug resistance. Levels of variety of soluble factors in the CM were monitored using a cytokine assay kit and revealed the presence
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 of significant amount of mIL-3. Furthermore, we showed that exposure to CM caused a significant increase in levels of pSTATs. Conclusion: These findings indicate that soluble factors found in CM contributes to Imatinib drug resistance in CML. Additionally, Crizotinib was active in overcoming CM-mediated drug resistance. No conflict of interest. 435 Impact of Aspirin on factors associated with breast cancer lymph node metastasis S. Khan1 , K. Gilligan1 , K. O’Brien1 , B. Moloney1 , I.S. Miller2 , E. Ramphul1 , T.I. Barron3 , K. Bennett2 , A.T. Byrne2 , M.J. Kerin1 , R.M. Dwyer1 . 1 Lambe Institute for Translational Research, Surgery, Galway, Ireland, 2 Royal College of Surgeons Ireland, Physiology & Medical Physics Dept, Dublin, Ireland, 3 Trinity College Dublin, School of Medicine, Dublin, Ireland Introduction: Long-term aspirin use in breast cancer patients has been associated with reduced spread to lymph nodes, potentially mediated through VEGF-C/-D. VEGF-C is a pro-angiogenic factor regulated down-steam of cyclooxygenase-2 (COX-2), which is a target of Aspirin. This study aimed to investigate circulating VEGF-C in breast cancer patients compared to healthy controls. The impact of Aspirin on VEGF-C secretion and angiogenic factors In Vitro, and on tumour recurrence In Vivo was also determined. Materials and Methods: Circulating levels of VEGF-C were measured by ELISA in serum from breast cancer patients (n = 58) and healthy controls (n = 20). Primary Normal stromal cells were derived from fresh tissue samples harvested in theatre with informed consent. Human Mesenchymal Stem Cells (MSCs) were isolated from the iliac crest of healthy volunteers. Cells were cultured either individually or with HCC-1954 breast cancer epithelial cells. Cells were exposed to Aspirin, conditioned media harvested, and secreted VEGF-C quantified. A tubule formation assay was performed to determine the impact of Aspirin on angiogenesis. Pro-angiogenic protein expression was investigated using an array. In vivo, breast tumour bearing mice were treated with Aspirin, tumours resected and disease recurrence monitored using bioluminescent and photoaccoustic (PA) imaging. Results and Discussion: Circulating VEGF-C levels were found to be elevated in breast cancer patients (5438±1270 pg/ml) compared to controls (3560±1477 pg/ml, p > 0.01). MSCs secreted highest levels of VEGF-C (305±35 pg/ml), followed by normal stromal cells (153±21 pg/ml), with no detectable levels secreted by HCC-1954 cells. Aspirin exposure resulted in loss of VEGF-C secretion from co-cultured cell populations, and reduced ability to form tubules. Decreased levels of angiogenesis related proteins were also observed following treatment with Aspirin, with the greatest decrease seen in Urokinase Plasminogen Activator (uPA, −42%). Preliminary In Vivo results revealed reduced tumour recurrence in mice treated with Aspirin. Conclusion: The data presented here highlights elevated circulating VEGF-C in breast cancer patients. Further, Aspirin was shown to have a significant impact on VEGF-C and uPA. Initial results also revealed that Aspirin may impact breast cancer recurrence in vivo. Acknowledgement: Funding: Irish Cancer Society BREAST-PREDICT (CCRC13GAL). No conflict of interest. 436 RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation E.J. Arenas Lahuerta1 , R. Gomis Cabre´ 1 , M. Morales1 . 1 IRB Barcelona, Oncology, Barcelona, Spain Despite a recent decrease, breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. In Estrogen Receptor Negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Therefore, a better understanding of the biology of breast cancer lung metastasis is required. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we show that RARRES3 is a metastatic suppressor gene in breast cancer. Using the ER− MDA-MB-231 breast cancer cell line model and lung metastatic derivatives, we functionally validated that RARRES3 loss of expression confers a selective advantage for the colonization of the lung in vivo (xenografts and syngeneic models). RARRES3 silencing engages metastasis initiating capabilities by facilitating extravasation and adhesion of the tumor cells to the lung. Furthermore, RARRES3 phospholipase A1/A2 activity contributes to tumor cell differentiation, thereby blocking lung metastasis demonstrated in 3D organotypic cultures and by a re-initiation assay in vivo. Therefore, our results show that genes selected for metastasis contribute to the different steps of this process and represent the random accumulation of traits that provide the necessary advantage for adaptation to the microenvironment of a different organ. RARRES3 restrains the lung metastatic capacity of breast cancer cells and more important, RARRES3 levels in the primary tumor are clinically relevant as may predict risk of relapse.
S93
The contribution of RARRES3 to differentiation over self-renewal suggests that reduced RARRES3 expression could be also predictive of therapy-resistant tumors, identifying patients possibly requiring new therapies designed to target breast cancer-initiating cells. Therefore, screening for compounds that activate RARRES3 may contribute to the development of new differentiation-inducing strategies to target therapy-resistant breast tumors. Main point: Functionally validate RARRES3 as a lung metastatic tumor suppressor gene in breast cancer in vitro, in vivo, and the most important, the clinical relevance in a human cohort. No conflict of interest. 437 Role of integrated TGF-b superfamily signaling in melanoma progression E. Tuncer1 , D. Zingg1 , P. Cheng2 , A. Antunes1 , J. Haeusel1 , C.X. Deng3 , I. Kleiter4 , L. Sommer1 . 1 Institute of Anatomy, University of Zurich, Zurich, Switzerland, 2 Department of Dermatology, University Hospital Zurich, ¨ Zurich, ¨ Switzerland, 3 National Institutes of Health, Genetics of Development and Diseases Branch, Bethesda- MD 20892, USA, 4 Department of Neurology, St. Josef-Hospital Ruhr-University, Bochum, Germany In various cancer types including melanoma, signalling by TGF-b superfamily has been implicated in both suppressing tumour growth and promoting metastasis. Several members of the TGF-b superfamily are expressed in melanoma and have been shown to affect tumour cell proliferation or invasiveness in a context-dependent manner. However, the role of the integrated TGF-b signalling in melanoma progression in vivo remains to be determined. Here, we show that conditional deletion of the TGF-b mediator Smad4, which abrogates all canonical TGF-b family signalling, abolishes tumourigenesis in a transgenic mouse model of melanoma. Intriguingly, this phenotype was brought about by reduced proliferation of tumour cells, thus pointing to the existence of pro-proliferative TGF-b family factors. After screening several TGF-b superfamily ligands, we identified BMP7 as a crucial ligand for melanoma cell proliferation. Moreover, BMP7 was sufficient to override the cytostatic and pro-invasive effects of TGF-b and Nodal. We next asked whether melanoma tumourigenesis could be increased by SMAD signalling activation. For that, we conditionally depleted Smad7 in a transgenic melanoma mouse model. Interestingly, Smad7 depletion resulted in massive metastasis formation in vivo. In agreement, cutaneous melanoma patients with low SMAD7 expression display reduced overall survival. Taken together, our results suggest that TGF-b signalling promotes melanoma growth, and in the context of reduced SMAD7 expression induces metastasis formation. No conflict of interest. 438 Tissue transglutaminase correlates with disease progression and the acquisition of epithelial–mesenchymal transition in colorectal cancer cells O. Ayinde1 , Z. Wang1 , C. Tselepsis2 , M. Griffin1 . 1 Aston University, School of life and health science, Birmingham, United Kingdom, 2 University of Birmingham, Institute of Cancer and Genomic Sciences, Birmingham, United Kingdom Background: Tissue transglutaminase (TG2) is a multifunctional and ubiquitous enzyme. Clinical studies indicate TG2 is elevated in various cancer cell types, however conflicting reports posit both pro and anti-tumour role for the enzyme. Previous studies using murine model showed TG2 regulates colorectal cancer cell (CRC) progression, however the role of TG2 in the human disease remains unclear. This study employs human CRCs at different disease stage to investigate the role of TG2 in tumour progression. Methods: TG2 expression was manipulated using Lentiviral particles carrying wild type TG2 or shRNA for TG2 in human CRCs RKO, SW480 and SW620. Correlation of TG2 expression with epithelial–mesenchymal transition (EMT), CRC chemo-resistance to 5 Fluorouracil (5-FU) and cancer stem cell (CSC) formation were characterized using Western blotting, XTT assay, and spheroid formation for CSCs. Results: Results show TG2 was sufficiently expressed in the metastatic cell line SW620 when compared to primary cell lines SW480 and RKO. SW620 cells exhibited higher resistance to 5FU treatment, while knockdown of TG2 using shRNA in these cells reduced cell resistance to 5FU. SW620 demonstrated EMT markers while SW480 and RKO expressed predominantly epithelial markers. TG2 over expression and TG2 shRNA treated cells showed increased and decreased expression of mesenchymal and epithelia markers respectively when compared to their corresponding parental cell types. TG2 knockdown resulted in the reduced ability of SW620 cells to form spheroids enriched with cancer stem cell phenotype in culture. Conclusions: This study has shown that TG2 correlates with in vitro tumour progression in human CRCs and that TG2 is involved in EMT, drug resistance and CSC formation. Therefore TG2 could serve as a potential biomarker for CRC tumour progression with therapeutic significance. No conflict of interest.
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
process by serial transient miR-135a force-expression in a precancerous cell model and examine in vitro tumor forming ability of its CD133+ subpopulation. Material and Method: NC104-E6/E7 is normal cervix epithelia cell-line immortalized by HPV E6 and E7. The transformation process was done by serial miR-135a transient force-expressions. Functional assays examined the tumorigenic properties after each miR-135a force-expression and compared to the control force-expression counterpart. Correspondingly, the CD133+ and CD133− subpopulations are isolated by CD133 antibody coated microbeads and their in vitro tumor sphere forming abilities are examined. Results and Discussion: After 3 successive miR-135a force-expressions, NC104-E6/E7-3t-135a cells exhibited higher invasiveness. Other malignant properties such as proliferation and migration were only slightly changed compared to the control group. The expression of E-cadherin was decreased accompanied by an increase of N-cadherin during the miR-135a mediated transformation process, indicating that the epithelial–mesenchymal transition (EMT) has occurred. CD133+ cells were found after 3 successive miR-135a force expressions. The CD133+ cells isolated from the NC104-E6/E7-3t-135a cells exhibited higher tumor spheres forming ability; the number and size of tumor spheres formed were larger than their CD133− counterparts. CD133+ subpopulation from the two control cell lines, NC104-E6/E7 and NC104-E6/E73t-ctrl cells failed to form tumor spheres. Conclusion: MiR-135a mediated cervical cancer malignant transformation is a multi-step process. Force expressing miR-135a favored the EMT process of precancerous cell model, which might be responsible for the stemness acquisition of cancer stem cells. This transformation process induced formation of CD133+ subpopulation. No conflict of interest. 432 DigiWest multiplex protein profiling of extracellular vesicles to decipher cancer metastasis and to identify biomarkers G. Erdmann1 , C. Sachse1 , M. Templin2 , J. Gross3 . 1 NMI TT GmbH, Pharmaservices, Berlin, Germany, 2 NMI, Protein Profiling & Assay ¨ Development, Reutlingen, Germany, 3 University Medical Center Gottingen, Haematology and Oncology, Goettingen, Germany Background: Improved diagnostic tests for cancer treatment are crucial in the clinic as well as during drug development. Technically, liquid biopsies are highly desirable, which has raised interest in CTCs and secreted extracellular vesicles. Most cell types secrete extracellular vesicles (EVs), a heterogeneous population of membrane particles, named according to their size and origin, as microvesicles (MVs) (250–1000 nm) or exosomes (50–100 nm). They have been described as a specific form of inter/extracellular communication. For example, tumor cell-derived EVs prime metastatic sites for incoming tumor stem cells (Peinado et al. 2012). In addition, a variety of signaling molecules are secreted on EVs from their sending cells, e.g. Wnt proteins, which allows them to induce Wnt signaling activity on target cells (Gross et al. 2012) and even to enhance migration and invasiveness of tumor cells (Menck et al. 2013). Hence, interrogating signaling molecules transmitted via tumor derived EVs may reveal biomarkers that might be more conclusive than circulating nucleic acids. Thus, analyzing proteins incorporated on EVs may offer important diagnostic information prior to and during treatment. Our aim is to analyze proteins traveling on extracellular vesicles and determine their utility as biomarkers. Methods: Extracellular vesicles were purified from different breast cancer cell lines as well as plasma from cancer patients by a differential centrifugation, separating exosomes and microvesicles by ultracentrifugation. To simultaneously detect a wide range of proteins traveling on the EVs, a novel protein profiling platform, DigiWest, was utilized. This method combines sizebased separation by gel electrophoresis with the Luminex bead system into a highly multiplexed “digital Western blot”. Using this approach, >90 proteins were analyzed for their presence on extracellular vesicles from as little as 10 mg of total protein. Results: Using the DigiWest assay on extracellular vesicles, we were able to identify and quantify known specific marker proteins for exosomes as well as microvesicles, respectively. Overall, we identified proteins specific for one type of vesicles or the other, but also found proteins equally present in both sample types. Furthermore, we identified a number of signaling proteins and receptors on both extracellular vesicles fractions that we further analyze for their robustness and relevance to breast cancer. Conclusion: Monitoring signaling molecules on extracellular vesicles using the DigiWest multiplex protein profiling technology is a highly promising approach for elucidation of the biological function of extracellular vesicles during tumor metastasis as well as for identification of specific biomarker candidates. Conflict of interest: Other Substantive Relationships: NMI TT Pharmaservices offers DigiWest as a service (for research use only).
433 Fasting in combination with curcumin induces a fatal energy “black out” in tumor cells L. Raffaghello1 , G. Bianchi1 , N. Bertola1 , A. Amaro2 , G. Angelini2 , L. Emionite3 , S. Ravera4 , U. Pfeffer2 . 1 G.Gaslini Children’s Hospital, Laboratory of Oncology, Genova, Italy, 2 IRCCS AOU San Martino − IST Istituto Nazionale per la Ricerca sul Cancro, Molecular Pathology, Genoa, Italy, 3 IRCCS AOU San Martino − IST Istituto Nazionale per la Ricerca sul Cancro, Animal Facility, Genoa, Italy, 4 University of Genoa, Department of Pharmacy, Genoa, Italy Introduction: The dietary polyphenol curcumin (CUR) has been described to exert anti-tumoral effects without toxicity. At present, 126 clinical studies using CUR or its derivatives are registered at clinicaltrials.gov. CUR has recently been defined as a caloric restriction mimetic (CRM), as it mimics biochemical and functional effects of calorie restriction (CR). In mice, two day cycles of fasting with only water access (short term starvation: STS) represent a peculiar example of CR that has been shown to protect normal cells but not tumor cells against cytotoxicity of chemotherapy and to render many tumors more susceptible to chemotherapy through an anti-Warburg effect. Preliminary clinical data indicate that in cancer patients fasting is feasible, not associated with major toxicity and able to reduce several side effects associated to chemotherapy. Aim of this study is to investigate the effects and mechanisms of fasting in combination with CUR on tumor metabolism. Material and Method: We tested the cytotoxicity of different concentrations of CUR +/− STS (1% FBS + 0.5 g/L glucose) on murine and human cell lines of melanoma, neuroblastoma, breast, colon cancer, and chronic lymphatic leukemia by Trypan Blue and Annexin V staining. Reactive oxygen species production was measured by 2 ,7 -dichlorodihydrofluorescein diacetate staining. Protein expression and activity of the key glycolytic enzymes, respiratory chain complexes as well as autophagy and proliferative pathways (PI3K/AKT/mTOR) were studied by Western blot and spectrophotometric assays. Results and Discussion: CUR reduces cell proliferation and induces apoptosis of various tumor cell lines, without affecting ROS production. It exerts an anti-Warburg effect by decreasing the activity of the main glycolytic enzymes. CUR inhibits ATP synthase and reduces O2 consumption leading to a dramatic drop of ATP production. STS shows similar anti-proliferative and apoptotic effects. STS-treated tumor cells exhibit a significant induction of respiratory chain complexes I, III, IV associated to a striking uncoupling between induced O2 consumption and reduced ATP synthesis. This latter event leads to a significant increase of ROS generation and apoptosis. The highest tumor cytotoxicity was observed for the combination of STS and CUR that additively reduces glycolysis and oxidative phosphorylation leading to a complete impairment of energy charge and consequent cell death. Differences between cell lines based on their basal respiratory activity are observed. The efficacy of STS+CUR in vivo is currently being tested. Conclusion: Fasting combined with CUR causes a strong suppression of tumor cell viability by inducing a fatal energy impairment. Thus, this association represents a promising strategy for the treatment of human cancer. No conflict of interest. 434 Crizotinib overcomes microenvironment mediated drug resistance in Ph positive leukemia O. Regev1 , N. Kidan1 , H. Khamisie1 , J. Mahajna1 . 1 Galilee Technology Center- Migal, Cancer Drug Discovery Program, Kiryat Shmona, Israel Background: Abl Kinase Inhibitors (AKIs) exhibiting good clinical efficacy in Ph+ leukemia. However, in one-third of patients, first-line TKI treatment will eventually become ineffective, which attributable to either non-mutational or kinase mutations in BCR/ABL. Conditions within the Bone Marrow (BM) niche contribute to reduced drug sensitivity of cancer cells residing in the BM including Ph+ leukemia. Crizotinib, a kinase inhibitor designed to target c-ALK for the treatment of a subtype of lung cancer, exhibits activity also against the ABL-kinase. Aim: Our aim is to understand BM microenvironment role in Ph+ leukemia AKI drug resistance and investigated the therapeutic potential of Crizotinib for the treatment of advanced and therapy of resistant disease. Methods: To study BM microenvironment mediated drug resistance in Ph+ leukemia; we exposed CML cells to condition media (CM) collected from mesenchymal cells. Cell proliferation was monitored by XTT assay and apoptosis inducing function of AKIs was assayed by following PARP cleavage. In addition we followed levels of STAT proteins. Results: CM collected from mesenchymal cells confers Imatinib drug resistance to CML cells. However, CM collected from ovarian or murine skin melanoma cells were not efficient in conferring drug resistance to Imatinib. Moreover, no significant change in CM mediated Imatinib drug resistance when CM were filtered with 0.4 or 0.2 micron. Exposer of CML cells to CM abolished ability of Imatinib to induce PARP cleavage. In contrasts, Crizotinib overcomes CM-mediated drug resistance. Levels of variety of soluble factors in the CM were monitored using a cytokine assay kit and revealed the presence
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 of significant amount of mIL-3. Furthermore, we showed that exposure to CM caused a significant increase in levels of pSTATs. Conclusion: These findings indicate that soluble factors found in CM contributes to Imatinib drug resistance in CML. Additionally, Crizotinib was active in overcoming CM-mediated drug resistance. No conflict of interest. 435 Impact of Aspirin on factors associated with breast cancer lymph node metastasis S. Khan1 , K. Gilligan1 , K. O’Brien1 , B. Moloney1 , I.S. Miller2 , E. Ramphul1 , T.I. Barron3 , K. Bennett2 , A.T. Byrne2 , M.J. Kerin1 , R.M. Dwyer1 . 1 Lambe Institute for Translational Research, Surgery, Galway, Ireland, 2 Royal College of Surgeons Ireland, Physiology & Medical Physics Dept, Dublin, Ireland, 3 Trinity College Dublin, School of Medicine, Dublin, Ireland Introduction: Long-term aspirin use in breast cancer patients has been associated with reduced spread to lymph nodes, potentially mediated through VEGF-C/-D. VEGF-C is a pro-angiogenic factor regulated down-steam of cyclooxygenase-2 (COX-2), which is a target of Aspirin. This study aimed to investigate circulating VEGF-C in breast cancer patients compared to healthy controls. The impact of Aspirin on VEGF-C secretion and angiogenic factors In Vitro, and on tumour recurrence In Vivo was also determined. Materials and Methods: Circulating levels of VEGF-C were measured by ELISA in serum from breast cancer patients (n = 58) and healthy controls (n = 20). Primary Normal stromal cells were derived from fresh tissue samples harvested in theatre with informed consent. Human Mesenchymal Stem Cells (MSCs) were isolated from the iliac crest of healthy volunteers. Cells were cultured either individually or with HCC-1954 breast cancer epithelial cells. Cells were exposed to Aspirin, conditioned media harvested, and secreted VEGF-C quantified. A tubule formation assay was performed to determine the impact of Aspirin on angiogenesis. Pro-angiogenic protein expression was investigated using an array. In vivo, breast tumour bearing mice were treated with Aspirin, tumours resected and disease recurrence monitored using bioluminescent and photoaccoustic (PA) imaging. Results and Discussion: Circulating VEGF-C levels were found to be elevated in breast cancer patients (5438±1270 pg/ml) compared to controls (3560±1477 pg/ml, p > 0.01). MSCs secreted highest levels of VEGF-C (305±35 pg/ml), followed by normal stromal cells (153±21 pg/ml), with no detectable levels secreted by HCC-1954 cells. Aspirin exposure resulted in loss of VEGF-C secretion from co-cultured cell populations, and reduced ability to form tubules. Decreased levels of angiogenesis related proteins were also observed following treatment with Aspirin, with the greatest decrease seen in Urokinase Plasminogen Activator (uPA, −42%). Preliminary In Vivo results revealed reduced tumour recurrence in mice treated with Aspirin. Conclusion: The data presented here highlights elevated circulating VEGF-C in breast cancer patients. Further, Aspirin was shown to have a significant impact on VEGF-C and uPA. Initial results also revealed that Aspirin may impact breast cancer recurrence in vivo. Acknowledgement: Funding: Irish Cancer Society BREAST-PREDICT (CCRC13GAL). No conflict of interest. 436 RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation E.J. Arenas Lahuerta1 , R. Gomis Cabre´ 1 , M. Morales1 . 1 IRB Barcelona, Oncology, Barcelona, Spain Despite a recent decrease, breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. In Estrogen Receptor Negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Therefore, a better understanding of the biology of breast cancer lung metastasis is required. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we show that RARRES3 is a metastatic suppressor gene in breast cancer. Using the ER− MDA-MB-231 breast cancer cell line model and lung metastatic derivatives, we functionally validated that RARRES3 loss of expression confers a selective advantage for the colonization of the lung in vivo (xenografts and syngeneic models). RARRES3 silencing engages metastasis initiating capabilities by facilitating extravasation and adhesion of the tumor cells to the lung. Furthermore, RARRES3 phospholipase A1/A2 activity contributes to tumor cell differentiation, thereby blocking lung metastasis demonstrated in 3D organotypic cultures and by a re-initiation assay in vivo. Therefore, our results show that genes selected for metastasis contribute to the different steps of this process and represent the random accumulation of traits that provide the necessary advantage for adaptation to the microenvironment of a different organ. RARRES3 restrains the lung metastatic capacity of breast cancer cells and more important, RARRES3 levels in the primary tumor are clinically relevant as may predict risk of relapse.
S93
The contribution of RARRES3 to differentiation over self-renewal suggests that reduced RARRES3 expression could be also predictive of therapy-resistant tumors, identifying patients possibly requiring new therapies designed to target breast cancer-initiating cells. Therefore, screening for compounds that activate RARRES3 may contribute to the development of new differentiation-inducing strategies to target therapy-resistant breast tumors. Main point: Functionally validate RARRES3 as a lung metastatic tumor suppressor gene in breast cancer in vitro, in vivo, and the most important, the clinical relevance in a human cohort. No conflict of interest. 437 Role of integrated TGF-b superfamily signaling in melanoma progression E. Tuncer1 , D. Zingg1 , P. Cheng2 , A. Antunes1 , J. Haeusel1 , C.X. Deng3 , I. Kleiter4 , L. Sommer1 . 1 Institute of Anatomy, University of Zurich, Zurich, Switzerland, 2 Department of Dermatology, University Hospital Zurich, ¨ Zurich, ¨ Switzerland, 3 National Institutes of Health, Genetics of Development and Diseases Branch, Bethesda- MD 20892, USA, 4 Department of Neurology, St. Josef-Hospital Ruhr-University, Bochum, Germany In various cancer types including melanoma, signalling by TGF-b superfamily has been implicated in both suppressing tumour growth and promoting metastasis. Several members of the TGF-b superfamily are expressed in melanoma and have been shown to affect tumour cell proliferation or invasiveness in a context-dependent manner. However, the role of the integrated TGF-b signalling in melanoma progression in vivo remains to be determined. Here, we show that conditional deletion of the TGF-b mediator Smad4, which abrogates all canonical TGF-b family signalling, abolishes tumourigenesis in a transgenic mouse model of melanoma. Intriguingly, this phenotype was brought about by reduced proliferation of tumour cells, thus pointing to the existence of pro-proliferative TGF-b family factors. After screening several TGF-b superfamily ligands, we identified BMP7 as a crucial ligand for melanoma cell proliferation. Moreover, BMP7 was sufficient to override the cytostatic and pro-invasive effects of TGF-b and Nodal. We next asked whether melanoma tumourigenesis could be increased by SMAD signalling activation. For that, we conditionally depleted Smad7 in a transgenic melanoma mouse model. Interestingly, Smad7 depletion resulted in massive metastasis formation in vivo. In agreement, cutaneous melanoma patients with low SMAD7 expression display reduced overall survival. Taken together, our results suggest that TGF-b signalling promotes melanoma growth, and in the context of reduced SMAD7 expression induces metastasis formation. No conflict of interest. 438 Tissue transglutaminase correlates with disease progression and the acquisition of epithelial–mesenchymal transition in colorectal cancer cells O. Ayinde1 , Z. Wang1 , C. Tselepsis2 , M. Griffin1 . 1 Aston University, School of life and health science, Birmingham, United Kingdom, 2 University of Birmingham, Institute of Cancer and Genomic Sciences, Birmingham, United Kingdom Background: Tissue transglutaminase (TG2) is a multifunctional and ubiquitous enzyme. Clinical studies indicate TG2 is elevated in various cancer cell types, however conflicting reports posit both pro and anti-tumour role for the enzyme. Previous studies using murine model showed TG2 regulates colorectal cancer cell (CRC) progression, however the role of TG2 in the human disease remains unclear. This study employs human CRCs at different disease stage to investigate the role of TG2 in tumour progression. Methods: TG2 expression was manipulated using Lentiviral particles carrying wild type TG2 or shRNA for TG2 in human CRCs RKO, SW480 and SW620. Correlation of TG2 expression with epithelial–mesenchymal transition (EMT), CRC chemo-resistance to 5 Fluorouracil (5-FU) and cancer stem cell (CSC) formation were characterized using Western blotting, XTT assay, and spheroid formation for CSCs. Results: Results show TG2 was sufficiently expressed in the metastatic cell line SW620 when compared to primary cell lines SW480 and RKO. SW620 cells exhibited higher resistance to 5FU treatment, while knockdown of TG2 using shRNA in these cells reduced cell resistance to 5FU. SW620 demonstrated EMT markers while SW480 and RKO expressed predominantly epithelial markers. TG2 over expression and TG2 shRNA treated cells showed increased and decreased expression of mesenchymal and epithelia markers respectively when compared to their corresponding parental cell types. TG2 knockdown resulted in the reduced ability of SW620 cells to form spheroids enriched with cancer stem cell phenotype in culture. Conclusions: This study has shown that TG2 correlates with in vitro tumour progression in human CRCs and that TG2 is involved in EMT, drug resistance and CSC formation. Therefore TG2 could serve as a potential biomarker for CRC tumour progression with therapeutic significance. No conflict of interest.