- Email: [email protected]
224 Heat Shock Transcription Factor 1 (HSF1) Enhances Mobility of Mouse Melanoma B16(F10) Cells
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european journal of cancer 48, suppl. 5 (2012) S25–S288
of the most important regulatory enzyme of glycolysis phosphofructokinase 1 (PFK1). We found that silencing of PFKFB4 increased fructose-2,6biphosphate levels selectively in the prostate cancer cells, suggesting that it was mainly functioning as a fructose-2,6-biphosphatase. PFK2 isoforms can modulate the distribution of metabolites between glycolysis and the pentose phosphate pathway. Prostate cancer cells also showed lower NADPH and reduced glutathione levels after PFKFB4 silencing, resulting in enhanced oxidative stress and cell death. Moreover, inducible depletion of PFKFB4 inhibited tumor growth in a xenograft model, indicating that it is required under physiological nutrient levels. Interestingly, PFKFB4 is expressed at higher levels in metastatic prostate cancer compared to primary site tumors in public data sets from human prostate cancer. The requirement of PFKFB4 for cancer cell survival was not specific to prostate, since a subset of bone, brain, breast, colon, lung, ovaric and stomach cancer cell lines were also dependent on its expression for their survival. Conclusion: We found that the glycolytic enzyme PFKFB4 is essential for prostate cancer cell survival by maintaining the balance between the use of glucose for energy generation and the synthesis of anti-oxidants. PFKFB4 is also required to support tumor growth in vivo. Taken together, these results indicate that PFKFB4 is a potential target for the development of anti-neoplastic agents. 221 FER Kinase Promotes Breast Cancer Growth and Metastasis by Regulating Cell Adhesion and Migration I.A. Ivanova1 , J.F. Vermeulen1 , C. Ercan1 , J.M. Houthuijzen1 , F. Al Saig1 , E. van der Wall1 , P.J. van Diest1 , M. Vooijs1 , P.W.B. Derksen1 . 1 University Medical Center Utrecht, Department of Pathology, Utrecht, The Netherlands Introduction: The Feline Sarcoma (FES) and Fes-related kinase (FER) proteins are the two members of a unique family of non-receptor tyrosine kinases. FER has been shown to regulate cell cycle progression, cell adhesion and migration in multiple cell types, and has been implicated in prostate and hepatocellular cancer formation and metastasis. The aim of our study was to determine whether FER is involved in breast cancer development and progression. Methods: We examined FER expression in 485 cases of invasive breast carcinoma by immunohistochemistry and used human breast cancer cells to determine the contribution of FER to cell adhesion, migration and invasion. Finally, a mouse xenograft breast cancer model was used to investigate the role of FER in tumour growth and metastasis. Results: High FER expression was significantly correlated with tumour size, grade and mitotic activity. We found that basal and HER2-driven tumours have higher FER levels as compared to luminal carcinomas. High FER expression correlated with a significantly worse prognosis based on overall survival. Further, multivariate analysis revealed that high FER expression is an independent prognostic factor. RNAi-mediated downregulation of FER in metastatic cells inhibited invasion and migration through increased a6 and b1 integrin-dependent cell adhesion. Conversely, overexpression of FER in non-metastatic breast cancer cells induced lamellipodia formation and increased cell migration. In agreement with the pro-metastatic role of FER in vitro, we found that inducible FER downregulation in vivo inhibited tumour growth and the formation of distant metastases. Conclusions: Our data indicate that FER is highly expressed in aggressive breast carcinomas and that high FER expression is an independent predictor of decreased patient survival. Moreover, we have shown that FER regulates tumour growth and metastasis in vivo. I.A. Ivanova is a Research Fellow of the Terry Fox Foundation (Award #700041). 222 Generation and Characterization of Novel Metastatic Variants of the Human Melanoma Cell Line Mel-Juso B. Mosch1 , J. Pietzsch1 . 1 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmacy, Dresden, Germany Introduction: Melanoma is a highly metastatic tumor with early metastasis in distant organs like lymph nodes, lungs, liver and brain. In order to understand the complex process of metastasis and to identify molecules involved, suitable in vivo and in vitro models are essential. The aim of this study was to establish variants of the human melanoma cell line Mel-Juso with same genetic background but different metastatic potential. Material and Methods: Mel-Juso cells were inoculated into the tail vein of athymic nude mice. Lung metastases were harvested, pooled, cultured in vitro and injected in another set of mice. Different melanoma variants were generated by repeated cycles of in vivo passage. The obtained metastatic variants (L3 and L5) were characterized genetically and concerning the expression of a melanoma marker, certain Eph receptor tyrosine kinases, growth properties, and in vivo metastasis.
Sunday 8 − Tuesday 10 July 2012
Results and Discussion: STR DNA genotyping showed no differences between the parental cell line and two selected metastatic variants. Moreover, no differences in the expression of the melanoma-associated chondroitin sulfate proteoglycan and in Eph receptors could be detected. Interestingly, we detected a reduced proliferation in metastatic variants accompanied by reduced or even lost capability of colony formation, indicating some substantial changes in metastatic properties despite of genetic similarity. Nevertheless, these in vitro differences between Mel-Juso and the metastatic variants could not be confirmed in an in vivo metastasis assay. Therefore, we started an additional cycle of in vivo passage with preparation of metastatic variants (L6) from individual lung metastases. By now 15 individual cell clones could be established derived from lungs of 4 individual mice and are currently analyzed concerning their cellular properties. Conclusion: The generation of melanoma cell line variants with same genetic background and different metastatic potential showed no success when using pooled lung metastases. Further steps will focus on the generation of variants from individual metastases to better reflect the varying tumorigenic potential of individual melanoma cells. Using such variants would facilitate the identification of molecules involved in metastasis, which show promise to be potential targets for the diagnosis and therapy of metastatic melanoma. 223 Thrombospondin 2 Gene (THBS2) and Its Role in Ovarian Cancer A. Cortez1 , K. Kujawa1 , M. Olbryt1 , J. Kupryjanczyk2 , K. Lisowska1 . 1 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Center for Translational Research and Molecular Biology of Cancer, Gliwice, Poland, 2 Maria Sklodowska-Curie Memorial Center and Institute of Oncology, Department of Molecular Pathology, Warsaw, Poland Introduction: THBS2 is multidomain Ca-binding extracellular glycoprotein that play a role in platelet aggregation, inflammatory response and assembly of extracellular matrix. THBS2 may be expressed by activated endothelial cells and acts as a potent angiogenesis inhibitor. It’s expression is induced by NF-kB depletion. On the other side THBS2 can enhance Notch signaling. It was shown to be associated with the pathogenesis of coronary disease and myocardial infarction. Growing body of evidence shows that THBS2 could play a role in the development and progression of different neoplasms. Methods: Microarray analysis of 99 ovarian cancer samples was performed using Affymetrix HGU 133 Plus arrays. Real-time PCR was done with AGI PRISM 7900 HT System. THBS2 coding sequence was reverse transcribed from human fibroblasts and cloned in two fragments using TOPO vector than ligated into the pLNCX2 vector. Competent bacteria were transformed using several modifications of chemical method and electroporation. Results: In our microarray study THBS2 gene was found as one of the potential prognostic markers correlated with the survival rate of ovarian cancer patients. These results were confirmed by real-time PCR. In the next step we decided to check the role of the THBS2 using the in vitro model. We planned to evaluate whether THBS2 may influence chemotherapy resistance, ability to undergo apoptosis or the motility of cancer cells. We checked 5 ovarian cancer cell lines for the expression of THBS2. Only one line was able to synthesize this protein. Thus we aimed to modify 4 other cell lines to obtain overexpression of THBS2. Than the original and modified cell lines could be analyzed by different in vitro tests. However, so far our attempts to clone THBS2 cDNA in pLNCX2 vector were unsuccessful. Discussion: As a possible reasons for the cloning failure we excluded inefficient ligation and recombinant vector size (possibly to large). It seems that the last option is that THBS2 protein is toxic for bacteria. We are currently investigating this possibility. We would greatly appreciate the discussion and feedback from the conference attendants on how to overcome this problem and successfully clone the THBS2 gene. Conclusion: Preliminary results indicate that THBS2 may play an important role in ovarian cancer progression, however this must be further confirmed. Acknowledgments: *Alexander Cortez was supported by the European Social Fund (UDA − POKL-04.01.01−00–014/10−00). 224 Heat Shock Transcription Factor 1 (HSF1) Enhances Mobility of Mouse Melanoma B16(F10) Cells A. Toma1 , T. Cichon1 , R. Smolarczyk1 , W. Widlak1 , N. Vydra1 . 1 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Center for Translational Research and Molecular Biology of Cancer, Gliwice, Poland Introduction: Heat Shock Transcription Factor 1 (HSF1) is a main regulator of the heat (stress) response. It activates heat shock genes, which encode heat shock proteins (HSPs). HSPs function as molecular chaperones by folding proteins during cellular stress. Beyond the classical induction of HSPs, HSF1 binds to broad array of non-HSP genes, associated with cell signaling, cytoskeleton organization and energy production. This activity of HSF1 seems to be important for its role in carcinogenesis. Materials and Methods: To study the role of HSF1 in tumor growth, we have constructed a model of mouse B16F10 melanoma cells with an overexpression
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
of constitutively active, human HSF1, with deletion of regulatory domain (amino acids 221–315; HSF1DRD). Simultaneously we have constructed cells with down-regulation of HSF1 expression using shRNA particles specific for 3 UTR and coding sequences of mouse HSF1. Results and Discussion: The expression of HSF1DRD was confirmed in transfected cells on mRNA and protein levels. Expression of several inducible Hsp genes (Hsph1, Hspb1, Hspa1) was increased in cells expressing HSF1DRD. The decreased expression (up to 50−70%) of HSF1 and several inducible Hsp genes (Hsph1, Hspb1, Hspa1) was detected in B16F10 cells with HSF1 knockdown. We have found that expression of constitutively active HSF1 enhanced anchorage independent growth of B16F10 cells, while B16F10 cells with down-regulated expression of HSF1 has the same ability to form colonies in soft agar as nonmodified cells. Using Boyden chamber assay we showed that B16F10 expressing HSF1DRD had higher ability to migrate. To determine the expression profile of genes associated with cell motility, specific RT2 PCR array was used. We found that expression of several genes (Vin, Cav1, Capn1, Ptk2) was decreased in B16F10 cells expressing HSF1DRD. Conclusions: We can conclude that HSF1 activation might support cell mobility and enhance tumor metastasis. Acknowledgments: *A.Toma is supported by the European Social Fund (UDA − POKL-04.01.01−00–014/10−00). 225 The Long Non-coding RNA ANRIL is a Direct Transcriptional Target of E2F1 That Mediates E2F1-induced Proliferation D. Bashari1 , A. Jerbi1 , O. Feldstein1 , D. Ginsberg1 . 1 Bar-Ilan University, The Mina & Everard Goodman Faculty of Life Sciences, Ramat Gan, Israel Introduction: Long non-coding RNAs (lncRNAs) regulate gene expression by diverse mechanisms and are involved in a variety of biological functions. ANRIL (Antisense Non-coding RNA in the INK4 Locus) is a 3.8 kb long noncoding RNA transcribed from the INK4b-ARF-INK4a locus on chromosome 9p21.3, which encodes three candidate tumor suppressors. ANRIL functions in recruitment of PRC2 to this locus and silencing of the p15INK4B tumor suppressor gene. The RB/E2F pathway is a pivotal regulator of cell cycle progression and plays a role in tumorigenesis. E2F1, the most investigated member of the E2F family, can stimulate both proliferation and apoptosis. Therefore, understanding the variables that determine the outcome of E2F1 activation is pivotal for cancer research and treatment. Here, we explore the possibility that ANRIL is regulated by E2F1 and affects E2F1-induced proliferation. Material and Method: We employed Q-PCR to analyze the expression of ANRIL and p15INK4b in cells containing a conditionally active E2F1 (ER-E2F1). To activate endogenous E2F we expressed viral proteins that disrupt RB/E2F complexes. Luciferase assay was used to test activation of ANRIL promoter. Finally, S phase entry was detected using Flow cytometry. Results: ANRIL mRNA is up-regulated upon over-expression of E2F1 in SAOS, H1299 and 22RV1 cells and upon activation of endogenous E2F. Also, Human ANRIL promoter contains putative E2F1 binding sites and our data indicate that E2F1 directly activates the ANRIL promoter. p15 is a known target of ANRIL and its mRNA level is down-regulated upon E2F1 activation. Importantly, inhibition of ANRIL expression significantly hinders E2F1-induced S phase entry and elevates p15 levels. Conclusions: Our data identify ANRIL as novel transcriptional target of E2F1 that mediates E2F1-induced S phase entry. 226 CXC-chemokines as Crucial Immune Mediators in Colorectal Carcinogenesis L. Kistner1 , D. Doll1 , K.P. Janssen1 . 1 Klinikum Rechts der Isar der TU-Munchen, ¨ Surgery, Muenchen, Germany Background: The tumor microenvironment strongly influences tumor progression and aggressiveness, e.g., through components of the immune system. It has been shown that high intratumoral numbers of T-lymphocytes predict good prognosis in colorectal cancer. However, the complex interactions between cancer cells and the immune system are far from being understood. Notably, it is unclear how an infiltration of T-cells into the tumor is mediated. We have investigated here the functional role of interferon regulated CXCChemokines (CXCL9, CXCL10 and CXCL11), soluble immune factors that are able to recruit T-cells and block angiogenesis. Material and Methods: Differential expression of CXC-chemokines in colorectal cancer was shown previously by transcriptome analysis. To confirm these data, qRT-PCR was performed on an independent patient collective of 163 patients with colon cancer. Blood vessel density and T-lymphocyte subpopulations were assessed by immunohistochemistry. Furthermore, ex vivo organ culture was performed on resected tumors to investigate CXCchemokine expression in response to cytokine stimulation by qRT-PCR and ELISA. Additionally, an orthotopic mouse model was established, based on an isogenic murine rectal carcinoma cell line engineered to express CXCL10. Results and Discussion: Intratumoral expression of CXC-chemokines was an independent prognostic predictor for postoperative survival in colon cancer.
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Above-threshold expression of CXC-chemokines predicted good prognosis, and was significantly correlated with high intratumoral numbers of cytotoxic T-cells and TH1 -helper cells. However, a connection with intratumoral blood vessel density was not found. Ex vivo culture on resected human tissue specimen demonstrated that a fraction of the tumors is stimulatable by cytokines to secrete CXC-chemokines. The orthotopic mouse model confirmed that intratumoral CXCL10 expression has protective anti-tumoral effects, which could only be seen in immunocompentent mice. Intratumoral CXCL10 expression in immune-deficient Rag-KO mice had no effect, indicating that the anti-tumoral effects of CXC-chemokines are mediated by the adaptive immune system. Conclusion: Interferon-regulated CXC-chemokines emerged as excellent prognostic predictors in colorectal cancer. A high chemokine level within the tumor was correlated with a high intratumoral T-cell density. Therefore, CXCchemokines constitute promising targets for therapeutic intervention. 227 Identification of Novel Therapeutic Targets in Advanced Patient-matched Renal Cell Carcinoma Models T. Rigo-Watermeier1 , C. Klein2 , V. Vogel2 , C. Eisen2 , T. Hoefner3 , W. Weichert4 , S. Pahernik3 , M. Hohenfellner3 , M. Sprick2 , A. Trumpp1 . 1 Deutsches Krebsforschungszentrum (DKFZ), Stem Cells and Cancer/ HI-STEM, Heidelberg, Germany, 2 Heidelberg Institute for Stem Cell Technology and Experimental Medicine at the DKFZ, Experimental Oncology/ Stem Cells and Cancer, Heidelberg, Germany, 3 University Heidelberg, Department of Urology, Heidelberg, Germany, 4 University Heidelberg, Department of Pathology, Heidelberg, Germany Introduction: Clear-cell renal cell carcinoma (ccRCC) is highly resistant to conventional treatments. Pre-clinical in vitro and in vivo models accurately mimicking this disease are critical for developing more effective therapies. Since previous attempts to culture these cells were mostly unsuccessful, only few RCC cell lines are available. In addition most of them have changed during long-term FCS-culturing and do not resemble the original tumor anymore. This might explain why drugs identified by screening these cell lines cannot be successfully transferred into the clinic. Therefore the development and characterization of clinically relevant ccRCC-models is absolutely essential to improve the treatment of this malignancy. Material and Methods: Renal cancer cells isolated out of primary renal tumor specimens were propagated in a serum-free spheroid system and patient-matched xenografts were established by orthotopical injection into immunocompromised mice. The surface proteome of the tumor cells was determined by mass spectrometry and FACS flow cytometry. Results and Discussion: We established a ‘patient-matched’ serum-free spheroid culture system that can be used to propagate ccRCC cells in vitro. The success of the culture establishment was highly dependant on the tumor grade of the corresponding patient. The ccRCC cells retained their tumor-initiating potential and mimicked the human malignancy upon orthotopic injection into immunodeficient mice. Further these cells showed de novo metastasis to the lungs, the most common metastatic site for ccRCC. The established ccRCC cultures can be used as a screening platform for novel subpopulation-specific surface proteins by FACS analysis and mass spectrometry. Our analyses revealed marker heterogeneity determining different subpopulations. Further in vitro and in vivo assays showed, that the identified subpopulations also displayed distinct functional characteristics. Currently these markers are validated for their suitability as diagnostic and therapeutic targets and the subpopulations are analyzed for their role in tumorinitiation, metastasis and drug-resistance. Conclusion: We developed an advanced in vitro culture system for ccRCC that can serve as a platform for the analysis and characterization of different subpopulations. Tumors resulting out of these cell lines have stable histological and molecular characteristics and clearly resemble their corresponding primary tumor. This might contribute to the understanding of the molecular basis underlying the progression, therapy-resistance and metastasis of ccRCC. 228 Retinoid Receptors in Pancreatic Cancer − Link to the Epithelial-mesenchymal Transition and Patient Survival A. Bazhin1 , T. Bleul1 , J. Werner1 . 1 University Heidelberg, Department of General Surgery, Heidelberg, Germany Background: Pancreatic adenocarcinoma is a cancer with extremely poor prognosis and limited therapeutic options. Although preclinical experiments with retinoids showed beneficial effects of retinoid treatment, clinical studies had disappointing results. However, little quantitative data is available concerning retinoid receptor expression in healthy pancreas compared to pancreatic carcinoma. The main aim of this work was to study comprehensively the retinoid receptor expression in order to evaluate their role in pancreatic cancer and try to find reasons for the negative clinical results. Methods: 9 human pancreatic carcinoma cell lines and one healthy cell line were used. Expression of the retinoic acid receptor (RAR) and retinoic X receptor (RXR) subtypes (a, b, g) was quantified on RNA level in all
european journal of cancer 48, suppl. 5 (2012) S25–S288
of the most important regulatory enzyme of glycolysis phosphofructokinase 1 (PFK1). We found that silencing of PFKFB4 increased fructose-2,6biphosphate levels selectively in the prostate cancer cells, suggesting that it was mainly functioning as a fructose-2,6-biphosphatase. PFK2 isoforms can modulate the distribution of metabolites between glycolysis and the pentose phosphate pathway. Prostate cancer cells also showed lower NADPH and reduced glutathione levels after PFKFB4 silencing, resulting in enhanced oxidative stress and cell death. Moreover, inducible depletion of PFKFB4 inhibited tumor growth in a xenograft model, indicating that it is required under physiological nutrient levels. Interestingly, PFKFB4 is expressed at higher levels in metastatic prostate cancer compared to primary site tumors in public data sets from human prostate cancer. The requirement of PFKFB4 for cancer cell survival was not specific to prostate, since a subset of bone, brain, breast, colon, lung, ovaric and stomach cancer cell lines were also dependent on its expression for their survival. Conclusion: We found that the glycolytic enzyme PFKFB4 is essential for prostate cancer cell survival by maintaining the balance between the use of glucose for energy generation and the synthesis of anti-oxidants. PFKFB4 is also required to support tumor growth in vivo. Taken together, these results indicate that PFKFB4 is a potential target for the development of anti-neoplastic agents. 221 FER Kinase Promotes Breast Cancer Growth and Metastasis by Regulating Cell Adhesion and Migration I.A. Ivanova1 , J.F. Vermeulen1 , C. Ercan1 , J.M. Houthuijzen1 , F. Al Saig1 , E. van der Wall1 , P.J. van Diest1 , M. Vooijs1 , P.W.B. Derksen1 . 1 University Medical Center Utrecht, Department of Pathology, Utrecht, The Netherlands Introduction: The Feline Sarcoma (FES) and Fes-related kinase (FER) proteins are the two members of a unique family of non-receptor tyrosine kinases. FER has been shown to regulate cell cycle progression, cell adhesion and migration in multiple cell types, and has been implicated in prostate and hepatocellular cancer formation and metastasis. The aim of our study was to determine whether FER is involved in breast cancer development and progression. Methods: We examined FER expression in 485 cases of invasive breast carcinoma by immunohistochemistry and used human breast cancer cells to determine the contribution of FER to cell adhesion, migration and invasion. Finally, a mouse xenograft breast cancer model was used to investigate the role of FER in tumour growth and metastasis. Results: High FER expression was significantly correlated with tumour size, grade and mitotic activity. We found that basal and HER2-driven tumours have higher FER levels as compared to luminal carcinomas. High FER expression correlated with a significantly worse prognosis based on overall survival. Further, multivariate analysis revealed that high FER expression is an independent prognostic factor. RNAi-mediated downregulation of FER in metastatic cells inhibited invasion and migration through increased a6 and b1 integrin-dependent cell adhesion. Conversely, overexpression of FER in non-metastatic breast cancer cells induced lamellipodia formation and increased cell migration. In agreement with the pro-metastatic role of FER in vitro, we found that inducible FER downregulation in vivo inhibited tumour growth and the formation of distant metastases. Conclusions: Our data indicate that FER is highly expressed in aggressive breast carcinomas and that high FER expression is an independent predictor of decreased patient survival. Moreover, we have shown that FER regulates tumour growth and metastasis in vivo. I.A. Ivanova is a Research Fellow of the Terry Fox Foundation (Award #700041). 222 Generation and Characterization of Novel Metastatic Variants of the Human Melanoma Cell Line Mel-Juso B. Mosch1 , J. Pietzsch1 . 1 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmacy, Dresden, Germany Introduction: Melanoma is a highly metastatic tumor with early metastasis in distant organs like lymph nodes, lungs, liver and brain. In order to understand the complex process of metastasis and to identify molecules involved, suitable in vivo and in vitro models are essential. The aim of this study was to establish variants of the human melanoma cell line Mel-Juso with same genetic background but different metastatic potential. Material and Methods: Mel-Juso cells were inoculated into the tail vein of athymic nude mice. Lung metastases were harvested, pooled, cultured in vitro and injected in another set of mice. Different melanoma variants were generated by repeated cycles of in vivo passage. The obtained metastatic variants (L3 and L5) were characterized genetically and concerning the expression of a melanoma marker, certain Eph receptor tyrosine kinases, growth properties, and in vivo metastasis.
Sunday 8 − Tuesday 10 July 2012
Results and Discussion: STR DNA genotyping showed no differences between the parental cell line and two selected metastatic variants. Moreover, no differences in the expression of the melanoma-associated chondroitin sulfate proteoglycan and in Eph receptors could be detected. Interestingly, we detected a reduced proliferation in metastatic variants accompanied by reduced or even lost capability of colony formation, indicating some substantial changes in metastatic properties despite of genetic similarity. Nevertheless, these in vitro differences between Mel-Juso and the metastatic variants could not be confirmed in an in vivo metastasis assay. Therefore, we started an additional cycle of in vivo passage with preparation of metastatic variants (L6) from individual lung metastases. By now 15 individual cell clones could be established derived from lungs of 4 individual mice and are currently analyzed concerning their cellular properties. Conclusion: The generation of melanoma cell line variants with same genetic background and different metastatic potential showed no success when using pooled lung metastases. Further steps will focus on the generation of variants from individual metastases to better reflect the varying tumorigenic potential of individual melanoma cells. Using such variants would facilitate the identification of molecules involved in metastasis, which show promise to be potential targets for the diagnosis and therapy of metastatic melanoma. 223 Thrombospondin 2 Gene (THBS2) and Its Role in Ovarian Cancer A. Cortez1 , K. Kujawa1 , M. Olbryt1 , J. Kupryjanczyk2 , K. Lisowska1 . 1 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Center for Translational Research and Molecular Biology of Cancer, Gliwice, Poland, 2 Maria Sklodowska-Curie Memorial Center and Institute of Oncology, Department of Molecular Pathology, Warsaw, Poland Introduction: THBS2 is multidomain Ca-binding extracellular glycoprotein that play a role in platelet aggregation, inflammatory response and assembly of extracellular matrix. THBS2 may be expressed by activated endothelial cells and acts as a potent angiogenesis inhibitor. It’s expression is induced by NF-kB depletion. On the other side THBS2 can enhance Notch signaling. It was shown to be associated with the pathogenesis of coronary disease and myocardial infarction. Growing body of evidence shows that THBS2 could play a role in the development and progression of different neoplasms. Methods: Microarray analysis of 99 ovarian cancer samples was performed using Affymetrix HGU 133 Plus arrays. Real-time PCR was done with AGI PRISM 7900 HT System. THBS2 coding sequence was reverse transcribed from human fibroblasts and cloned in two fragments using TOPO vector than ligated into the pLNCX2 vector. Competent bacteria were transformed using several modifications of chemical method and electroporation. Results: In our microarray study THBS2 gene was found as one of the potential prognostic markers correlated with the survival rate of ovarian cancer patients. These results were confirmed by real-time PCR. In the next step we decided to check the role of the THBS2 using the in vitro model. We planned to evaluate whether THBS2 may influence chemotherapy resistance, ability to undergo apoptosis or the motility of cancer cells. We checked 5 ovarian cancer cell lines for the expression of THBS2. Only one line was able to synthesize this protein. Thus we aimed to modify 4 other cell lines to obtain overexpression of THBS2. Than the original and modified cell lines could be analyzed by different in vitro tests. However, so far our attempts to clone THBS2 cDNA in pLNCX2 vector were unsuccessful. Discussion: As a possible reasons for the cloning failure we excluded inefficient ligation and recombinant vector size (possibly to large). It seems that the last option is that THBS2 protein is toxic for bacteria. We are currently investigating this possibility. We would greatly appreciate the discussion and feedback from the conference attendants on how to overcome this problem and successfully clone the THBS2 gene. Conclusion: Preliminary results indicate that THBS2 may play an important role in ovarian cancer progression, however this must be further confirmed. Acknowledgments: *Alexander Cortez was supported by the European Social Fund (UDA − POKL-04.01.01−00–014/10−00). 224 Heat Shock Transcription Factor 1 (HSF1) Enhances Mobility of Mouse Melanoma B16(F10) Cells A. Toma1 , T. Cichon1 , R. Smolarczyk1 , W. Widlak1 , N. Vydra1 . 1 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Center for Translational Research and Molecular Biology of Cancer, Gliwice, Poland Introduction: Heat Shock Transcription Factor 1 (HSF1) is a main regulator of the heat (stress) response. It activates heat shock genes, which encode heat shock proteins (HSPs). HSPs function as molecular chaperones by folding proteins during cellular stress. Beyond the classical induction of HSPs, HSF1 binds to broad array of non-HSP genes, associated with cell signaling, cytoskeleton organization and energy production. This activity of HSF1 seems to be important for its role in carcinogenesis. Materials and Methods: To study the role of HSF1 in tumor growth, we have constructed a model of mouse B16F10 melanoma cells with an overexpression
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
of constitutively active, human HSF1, with deletion of regulatory domain (amino acids 221–315; HSF1DRD). Simultaneously we have constructed cells with down-regulation of HSF1 expression using shRNA particles specific for 3 UTR and coding sequences of mouse HSF1. Results and Discussion: The expression of HSF1DRD was confirmed in transfected cells on mRNA and protein levels. Expression of several inducible Hsp genes (Hsph1, Hspb1, Hspa1) was increased in cells expressing HSF1DRD. The decreased expression (up to 50−70%) of HSF1 and several inducible Hsp genes (Hsph1, Hspb1, Hspa1) was detected in B16F10 cells with HSF1 knockdown. We have found that expression of constitutively active HSF1 enhanced anchorage independent growth of B16F10 cells, while B16F10 cells with down-regulated expression of HSF1 has the same ability to form colonies in soft agar as nonmodified cells. Using Boyden chamber assay we showed that B16F10 expressing HSF1DRD had higher ability to migrate. To determine the expression profile of genes associated with cell motility, specific RT2 PCR array was used. We found that expression of several genes (Vin, Cav1, Capn1, Ptk2) was decreased in B16F10 cells expressing HSF1DRD. Conclusions: We can conclude that HSF1 activation might support cell mobility and enhance tumor metastasis. Acknowledgments: *A.Toma is supported by the European Social Fund (UDA − POKL-04.01.01−00–014/10−00). 225 The Long Non-coding RNA ANRIL is a Direct Transcriptional Target of E2F1 That Mediates E2F1-induced Proliferation D. Bashari1 , A. Jerbi1 , O. Feldstein1 , D. Ginsberg1 . 1 Bar-Ilan University, The Mina & Everard Goodman Faculty of Life Sciences, Ramat Gan, Israel Introduction: Long non-coding RNAs (lncRNAs) regulate gene expression by diverse mechanisms and are involved in a variety of biological functions. ANRIL (Antisense Non-coding RNA in the INK4 Locus) is a 3.8 kb long noncoding RNA transcribed from the INK4b-ARF-INK4a locus on chromosome 9p21.3, which encodes three candidate tumor suppressors. ANRIL functions in recruitment of PRC2 to this locus and silencing of the p15INK4B tumor suppressor gene. The RB/E2F pathway is a pivotal regulator of cell cycle progression and plays a role in tumorigenesis. E2F1, the most investigated member of the E2F family, can stimulate both proliferation and apoptosis. Therefore, understanding the variables that determine the outcome of E2F1 activation is pivotal for cancer research and treatment. Here, we explore the possibility that ANRIL is regulated by E2F1 and affects E2F1-induced proliferation. Material and Method: We employed Q-PCR to analyze the expression of ANRIL and p15INK4b in cells containing a conditionally active E2F1 (ER-E2F1). To activate endogenous E2F we expressed viral proteins that disrupt RB/E2F complexes. Luciferase assay was used to test activation of ANRIL promoter. Finally, S phase entry was detected using Flow cytometry. Results: ANRIL mRNA is up-regulated upon over-expression of E2F1 in SAOS, H1299 and 22RV1 cells and upon activation of endogenous E2F. Also, Human ANRIL promoter contains putative E2F1 binding sites and our data indicate that E2F1 directly activates the ANRIL promoter. p15 is a known target of ANRIL and its mRNA level is down-regulated upon E2F1 activation. Importantly, inhibition of ANRIL expression significantly hinders E2F1-induced S phase entry and elevates p15 levels. Conclusions: Our data identify ANRIL as novel transcriptional target of E2F1 that mediates E2F1-induced S phase entry. 226 CXC-chemokines as Crucial Immune Mediators in Colorectal Carcinogenesis L. Kistner1 , D. Doll1 , K.P. Janssen1 . 1 Klinikum Rechts der Isar der TU-Munchen, ¨ Surgery, Muenchen, Germany Background: The tumor microenvironment strongly influences tumor progression and aggressiveness, e.g., through components of the immune system. It has been shown that high intratumoral numbers of T-lymphocytes predict good prognosis in colorectal cancer. However, the complex interactions between cancer cells and the immune system are far from being understood. Notably, it is unclear how an infiltration of T-cells into the tumor is mediated. We have investigated here the functional role of interferon regulated CXCChemokines (CXCL9, CXCL10 and CXCL11), soluble immune factors that are able to recruit T-cells and block angiogenesis. Material and Methods: Differential expression of CXC-chemokines in colorectal cancer was shown previously by transcriptome analysis. To confirm these data, qRT-PCR was performed on an independent patient collective of 163 patients with colon cancer. Blood vessel density and T-lymphocyte subpopulations were assessed by immunohistochemistry. Furthermore, ex vivo organ culture was performed on resected tumors to investigate CXCchemokine expression in response to cytokine stimulation by qRT-PCR and ELISA. Additionally, an orthotopic mouse model was established, based on an isogenic murine rectal carcinoma cell line engineered to express CXCL10. Results and Discussion: Intratumoral expression of CXC-chemokines was an independent prognostic predictor for postoperative survival in colon cancer.
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Above-threshold expression of CXC-chemokines predicted good prognosis, and was significantly correlated with high intratumoral numbers of cytotoxic T-cells and TH1 -helper cells. However, a connection with intratumoral blood vessel density was not found. Ex vivo culture on resected human tissue specimen demonstrated that a fraction of the tumors is stimulatable by cytokines to secrete CXC-chemokines. The orthotopic mouse model confirmed that intratumoral CXCL10 expression has protective anti-tumoral effects, which could only be seen in immunocompentent mice. Intratumoral CXCL10 expression in immune-deficient Rag-KO mice had no effect, indicating that the anti-tumoral effects of CXC-chemokines are mediated by the adaptive immune system. Conclusion: Interferon-regulated CXC-chemokines emerged as excellent prognostic predictors in colorectal cancer. A high chemokine level within the tumor was correlated with a high intratumoral T-cell density. Therefore, CXCchemokines constitute promising targets for therapeutic intervention. 227 Identification of Novel Therapeutic Targets in Advanced Patient-matched Renal Cell Carcinoma Models T. Rigo-Watermeier1 , C. Klein2 , V. Vogel2 , C. Eisen2 , T. Hoefner3 , W. Weichert4 , S. Pahernik3 , M. Hohenfellner3 , M. Sprick2 , A. Trumpp1 . 1 Deutsches Krebsforschungszentrum (DKFZ), Stem Cells and Cancer/ HI-STEM, Heidelberg, Germany, 2 Heidelberg Institute for Stem Cell Technology and Experimental Medicine at the DKFZ, Experimental Oncology/ Stem Cells and Cancer, Heidelberg, Germany, 3 University Heidelberg, Department of Urology, Heidelberg, Germany, 4 University Heidelberg, Department of Pathology, Heidelberg, Germany Introduction: Clear-cell renal cell carcinoma (ccRCC) is highly resistant to conventional treatments. Pre-clinical in vitro and in vivo models accurately mimicking this disease are critical for developing more effective therapies. Since previous attempts to culture these cells were mostly unsuccessful, only few RCC cell lines are available. In addition most of them have changed during long-term FCS-culturing and do not resemble the original tumor anymore. This might explain why drugs identified by screening these cell lines cannot be successfully transferred into the clinic. Therefore the development and characterization of clinically relevant ccRCC-models is absolutely essential to improve the treatment of this malignancy. Material and Methods: Renal cancer cells isolated out of primary renal tumor specimens were propagated in a serum-free spheroid system and patient-matched xenografts were established by orthotopical injection into immunocompromised mice. The surface proteome of the tumor cells was determined by mass spectrometry and FACS flow cytometry. Results and Discussion: We established a ‘patient-matched’ serum-free spheroid culture system that can be used to propagate ccRCC cells in vitro. The success of the culture establishment was highly dependant on the tumor grade of the corresponding patient. The ccRCC cells retained their tumor-initiating potential and mimicked the human malignancy upon orthotopic injection into immunodeficient mice. Further these cells showed de novo metastasis to the lungs, the most common metastatic site for ccRCC. The established ccRCC cultures can be used as a screening platform for novel subpopulation-specific surface proteins by FACS analysis and mass spectrometry. Our analyses revealed marker heterogeneity determining different subpopulations. Further in vitro and in vivo assays showed, that the identified subpopulations also displayed distinct functional characteristics. Currently these markers are validated for their suitability as diagnostic and therapeutic targets and the subpopulations are analyzed for their role in tumorinitiation, metastasis and drug-resistance. Conclusion: We developed an advanced in vitro culture system for ccRCC that can serve as a platform for the analysis and characterization of different subpopulations. Tumors resulting out of these cell lines have stable histological and molecular characteristics and clearly resemble their corresponding primary tumor. This might contribute to the understanding of the molecular basis underlying the progression, therapy-resistance and metastasis of ccRCC. 228 Retinoid Receptors in Pancreatic Cancer − Link to the Epithelial-mesenchymal Transition and Patient Survival A. Bazhin1 , T. Bleul1 , J. Werner1 . 1 University Heidelberg, Department of General Surgery, Heidelberg, Germany Background: Pancreatic adenocarcinoma is a cancer with extremely poor prognosis and limited therapeutic options. Although preclinical experiments with retinoids showed beneficial effects of retinoid treatment, clinical studies had disappointing results. However, little quantitative data is available concerning retinoid receptor expression in healthy pancreas compared to pancreatic carcinoma. The main aim of this work was to study comprehensively the retinoid receptor expression in order to evaluate their role in pancreatic cancer and try to find reasons for the negative clinical results. Methods: 9 human pancreatic carcinoma cell lines and one healthy cell line were used. Expression of the retinoic acid receptor (RAR) and retinoic X receptor (RXR) subtypes (a, b, g) was quantified on RNA level in all