- Email: [email protected]
368: ErbB3 in the nucleus – mechanisms of nuclear entry
S88
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
367 Elucidating the synthetic lethal effect of CDK inhibition in triple negative breast cancer J. Rohrberg1 , A. Corella1 , L. Starnes1 , A. Goga1 . 1 Tissue and Cell Biology, Medicine, San Francisco, USA Background: Triple negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer and to date, targeted therapeutics for this subtype are lacking. The MYC oncogene is elevated in those cancers and an activated MYC pathway is associated with poor prognosis. We recently showed that MYC overexpressing cancers regress upon CDK inhibition. Normal cells are not sensitive to such killing, a phenomenon referred to as synthetic lethality. We aim to understand the biology of this synthetic lethality to develop more effective therapeutics. Material and Methods: Using The Cancer Genome Atlas (TCGA) database we compared the mRNA expression levels of cyclin-dependent kinase (CDK) substrates in triple negative versus other subtypes of breast cancer. CDKs promote cell cycle progression and cell division through the orderly phosphorylation of downstream target proteins. We asked, which of these candidate molecules participate in the synthetic lethal effect. We used RNAi knock down to compare the effect on death of normal versus MYC overexpressing cells. Results: We found 20 CDK1 substrates being differentially expressed in TNBC. The knock down of some CDK1 substrate can induce cell death or growth arrest in cells overexpressing MYC while sparing normal cells. To date ~100 substrates for CDK1 have been identified. As this collection of substrates is unlikely to be comprehensive, we aim to identify new CDK target proteins on a proteome-wide scale. Due to the structural similarity of CDKs no specific inhibitors are available that distinguish different CDKs. To inhibit CDK1 specifically we will make use of the analog-sensitive approach. Stable Isotope Labeling of Amino Acids in Culture (SILAC) and quantitative mass spectrometry will be used to identify new CDK1 substrate proteins. Conclusions: To understand why inhibition of CDK activity selectively kills cells that overexpress MYC we checked if CDK substrate proteins are disregulated in TNBC. We found 20 CDK1 substrate proteins being deregulated in those cancers and could identify those which mediate the synthetic lethality effect. No conflict of interest. 368 ErbB3 in the nucleus − mechanisms of nuclear entry 2 1 R. Reif1 , A. Adawy2 , G. Gunther ¨ . IfaDo, Dortmund, Germany, 2 IfaDo, LivTox, Dortmund, Germany
Background: Members of the receptor tyrosine kinase family (RTK) are often overexpressed in tumors. In contrast to healthy tissue, RTKs are frequently located in the nuclei of tumor cells. This translocation correlates with increased tumor proliferation and poor survival in cancer. In the current study, we focus on the nuclear translocation of ErbB3. Material and Methods: We were investigating ErbB3 trafficking using classical biochemical methods like subcellular fractionation, immune precipitation, biotinylation and immunostainings of breast cancer cell lines. Result: We demonstrated that ErbB3’s natural ligand, heregulin and stress conditions within the tumor microenvironment effectively stimulate ErbB3 nuclear translocation. This nuclear transfer is strictly dependent on functional ErbB2/ErbB3 heterodimerization and subsequent ErbB3 phosphorylation. Investigation of different endocytic pathways in T-47D and BT474 breast cancer cells provides evidence that both Arf6 and clathrin contribute to ErbB3 translocation. Moreover, ErbB3 was found to interact with importin b1 which drives the internalized receptor through the nuclear pore complex into the nucleus. Conclusion: Beside the other family members ErbB3 is under stress conditions transported to the nucleus. Once in the nucleus, ErbB3 is shown to interact with chromatin, suggesting a potential role of ErbB3 in transcriptional regulation, which remains to be elucidated. No conflict of interest. 369 An in vivo shRNA screen to identify novel drivers and therapeutic targets of breast cancer metastasis A. Van Weverwijk1 , M. Ashenden1 , N. Murugaesu1 , M. Iravani1 , C.M. Isacke1 . 1 Institute of Cancer Research, Breast Cancer Research, London, United Kingdom Introduction: Despite major improvements in understanding the underlying mechanisms of cancer progression and therapy resistance, metastatic disease is still the leading cause of breast cancer mortality in women. Consequently, there is an urgent need to identify novel modulators of breast cancer metastasis and therapeutic targets to suppress secondary disease. RNA interference based genetic screens have been used as a high-throughput, unbiased approach to identify cancer associated genes. Cell based screens have been successful in identifying genes involved in tumour cell survival, proliferation and therapy resistance. However, in vitro approaches lack the ability to interrogate all aspects of the metastatic cascade, in particular tumour
cell dissemination, tumour cell extravasation, colonization of distant organs and the role of the tumour microenvironment in modulating therapeutic response. In this project, an in vivo shRNA screen was peformed with the goal of identifying novel modulators of breast cancer metastasis. Materials and Methods: An in vivo shRNA screen using the Cancer 1000 mouse shRNA retroviral library was conducted. 4T1-Luc cells, derived from a spontaneously arisen mammary tumour in a female BALB/cfC3H mouse, were infected with the Cancer 1000 library in subpools and each subpool injected intravenously into syngeneic recipient mice. 21 days later the lungs of the mice were collected at necroscopy. gDNA was extracted from the combined plasmid pool, combined infected cells, and lung samples. DNA samples were PCR amplified and the shRNAs were subjected to next generation sequencing. Results and Discussion: The effect of each shRNA on metastasis is assessed using paired statistics. shRNAs with a Z score −2 considered as significantly enriched and shRNAs with a Z score <−2 considered as significantly depleted. In the first instance, we investigated enriched shRNAs and identified the sialyltransferase ST6GalNAc2 as a novel metastasis suppressor (Murugaesu et al., 2014. Cancer Discovery). Currently we are investigating the depletion of shRNAs targeting metastasis enhancers. Among the shRNAs that showed significant depletion are shRNAs targeting the known metastasis enhancer Met and a number of anti-apoptotic genes. Novel putative metastasis enhancers are currently being validated in in vivo functional analysis. Potential novel therapies targeting identified metastatic modulators will be explored and the clinical relevance of obtained results determined. Conclusion: An in vivo shRNA screen combined with next generation sequencing is proving to be an effective tool in identifying novel breast cancer metastasis suppressors and enhancers. No conflict of interest. 370 Lymphotoxin signalling alters the vasculature to increase tumour cell metastasis N. Simonavicius1 , B. Seubert2 , L. Borsig3 , D. Wohlleber4 , J. Browning5 , 2 , M. Heikenwalder1 . 1 Helmholtz Zentrum Munchen, ¨ Institute A. Kruger ¨ ¨ Munchen, of Virology, Muenchen, Germany, 2 Technische Universitat ¨ 1Institute for Experimental Oncology and Therapy Research/Institute of Molecular Immunology, Muenchen, Germany, 3 University of Zurich, Institute of Physiology, Zurich, Switzerland, 4 University Hospital Bonn, Institutes of Molecular Medicine and Experimental Immunology, Bonn, Germany, 5 Boston University School of Medicine, Department of Microbiology and Section of Rheumatology, Boston, USA The importance of an inflammatory microenvironment in affecting carcinogenesis and metastasis is becoming increasingly evident. One of the key steps of tumour cell extravasation to secondary sites is the transmigration through the blood vessel, which consists of a barrier of endothelial cells and closely associated pericytes. The impact of an inflammatory microenvironment to the vascular wall and the consequences for metastasis remain elusive. Here, we investigate the role of the pro-inflammatory cytokine lymphotoxin (LT) − either provided systemically or produced by the tumour cells − in altering the vasculature to facilitate tumour cell extravasation. In an in vivo experimental metastasis assay we observed that the T-lymphoma cells express the LT ligands to enable their own extravasation through the liver vasculature as both the knockdown of LT as well as a blocking antibody decreased the number of T-lymphoma metastases in the liver. Congruently, preconditioning of the liver with an agonistic lymphotoxin b receptor (LTbR) antibody, mimicking a pro-carcinogenic environment, leads to an increased number of T-lymphoma cells. The effects are neither specific to lymphoma cells nor the liver as experimental metastasis assays using melanoma or colon carcinoma cells also showed increased lung metastases after LTbR agonisation. Importantly, the stimulation of the LTbR pathway led to vascular disintegration as monitored by Evans Blue leakage. In vivo the liver vasculature responds to LTbR agonisation via induction of target genes, such as matrix metalloproteinases, that may be responsible for the increase in vascular permeability and metastases. Interestingly, neither macrophages nor the LTbR on hepatocytes mediate the increased vascular permeability after LTbR agonisation. In conclusion, we elucidate a mechanism how the presence of proinflammatory LTs either administered systemically or via expression by the tumour cells themselves changes the vasculature to enhance tumour cell metastasis. This not only provides insight into the link of inflammation and metastasis but may also provide novel therapeutic opportunities. No conflict of interest.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
367 Elucidating the synthetic lethal effect of CDK inhibition in triple negative breast cancer J. Rohrberg1 , A. Corella1 , L. Starnes1 , A. Goga1 . 1 Tissue and Cell Biology, Medicine, San Francisco, USA Background: Triple negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer and to date, targeted therapeutics for this subtype are lacking. The MYC oncogene is elevated in those cancers and an activated MYC pathway is associated with poor prognosis. We recently showed that MYC overexpressing cancers regress upon CDK inhibition. Normal cells are not sensitive to such killing, a phenomenon referred to as synthetic lethality. We aim to understand the biology of this synthetic lethality to develop more effective therapeutics. Material and Methods: Using The Cancer Genome Atlas (TCGA) database we compared the mRNA expression levels of cyclin-dependent kinase (CDK) substrates in triple negative versus other subtypes of breast cancer. CDKs promote cell cycle progression and cell division through the orderly phosphorylation of downstream target proteins. We asked, which of these candidate molecules participate in the synthetic lethal effect. We used RNAi knock down to compare the effect on death of normal versus MYC overexpressing cells. Results: We found 20 CDK1 substrates being differentially expressed in TNBC. The knock down of some CDK1 substrate can induce cell death or growth arrest in cells overexpressing MYC while sparing normal cells. To date ~100 substrates for CDK1 have been identified. As this collection of substrates is unlikely to be comprehensive, we aim to identify new CDK target proteins on a proteome-wide scale. Due to the structural similarity of CDKs no specific inhibitors are available that distinguish different CDKs. To inhibit CDK1 specifically we will make use of the analog-sensitive approach. Stable Isotope Labeling of Amino Acids in Culture (SILAC) and quantitative mass spectrometry will be used to identify new CDK1 substrate proteins. Conclusions: To understand why inhibition of CDK activity selectively kills cells that overexpress MYC we checked if CDK substrate proteins are disregulated in TNBC. We found 20 CDK1 substrate proteins being deregulated in those cancers and could identify those which mediate the synthetic lethality effect. No conflict of interest. 368 ErbB3 in the nucleus − mechanisms of nuclear entry 2 1 R. Reif1 , A. Adawy2 , G. Gunther ¨ . IfaDo, Dortmund, Germany, 2 IfaDo, LivTox, Dortmund, Germany
Background: Members of the receptor tyrosine kinase family (RTK) are often overexpressed in tumors. In contrast to healthy tissue, RTKs are frequently located in the nuclei of tumor cells. This translocation correlates with increased tumor proliferation and poor survival in cancer. In the current study, we focus on the nuclear translocation of ErbB3. Material and Methods: We were investigating ErbB3 trafficking using classical biochemical methods like subcellular fractionation, immune precipitation, biotinylation and immunostainings of breast cancer cell lines. Result: We demonstrated that ErbB3’s natural ligand, heregulin and stress conditions within the tumor microenvironment effectively stimulate ErbB3 nuclear translocation. This nuclear transfer is strictly dependent on functional ErbB2/ErbB3 heterodimerization and subsequent ErbB3 phosphorylation. Investigation of different endocytic pathways in T-47D and BT474 breast cancer cells provides evidence that both Arf6 and clathrin contribute to ErbB3 translocation. Moreover, ErbB3 was found to interact with importin b1 which drives the internalized receptor through the nuclear pore complex into the nucleus. Conclusion: Beside the other family members ErbB3 is under stress conditions transported to the nucleus. Once in the nucleus, ErbB3 is shown to interact with chromatin, suggesting a potential role of ErbB3 in transcriptional regulation, which remains to be elucidated. No conflict of interest. 369 An in vivo shRNA screen to identify novel drivers and therapeutic targets of breast cancer metastasis A. Van Weverwijk1 , M. Ashenden1 , N. Murugaesu1 , M. Iravani1 , C.M. Isacke1 . 1 Institute of Cancer Research, Breast Cancer Research, London, United Kingdom Introduction: Despite major improvements in understanding the underlying mechanisms of cancer progression and therapy resistance, metastatic disease is still the leading cause of breast cancer mortality in women. Consequently, there is an urgent need to identify novel modulators of breast cancer metastasis and therapeutic targets to suppress secondary disease. RNA interference based genetic screens have been used as a high-throughput, unbiased approach to identify cancer associated genes. Cell based screens have been successful in identifying genes involved in tumour cell survival, proliferation and therapy resistance. However, in vitro approaches lack the ability to interrogate all aspects of the metastatic cascade, in particular tumour
cell dissemination, tumour cell extravasation, colonization of distant organs and the role of the tumour microenvironment in modulating therapeutic response. In this project, an in vivo shRNA screen was peformed with the goal of identifying novel modulators of breast cancer metastasis. Materials and Methods: An in vivo shRNA screen using the Cancer 1000 mouse shRNA retroviral library was conducted. 4T1-Luc cells, derived from a spontaneously arisen mammary tumour in a female BALB/cfC3H mouse, were infected with the Cancer 1000 library in subpools and each subpool injected intravenously into syngeneic recipient mice. 21 days later the lungs of the mice were collected at necroscopy. gDNA was extracted from the combined plasmid pool, combined infected cells, and lung samples. DNA samples were PCR amplified and the shRNAs were subjected to next generation sequencing. Results and Discussion: The effect of each shRNA on metastasis is assessed using paired statistics. shRNAs with a Z score −2 considered as significantly enriched and shRNAs with a Z score <−2 considered as significantly depleted. In the first instance, we investigated enriched shRNAs and identified the sialyltransferase ST6GalNAc2 as a novel metastasis suppressor (Murugaesu et al., 2014. Cancer Discovery). Currently we are investigating the depletion of shRNAs targeting metastasis enhancers. Among the shRNAs that showed significant depletion are shRNAs targeting the known metastasis enhancer Met and a number of anti-apoptotic genes. Novel putative metastasis enhancers are currently being validated in in vivo functional analysis. Potential novel therapies targeting identified metastatic modulators will be explored and the clinical relevance of obtained results determined. Conclusion: An in vivo shRNA screen combined with next generation sequencing is proving to be an effective tool in identifying novel breast cancer metastasis suppressors and enhancers. No conflict of interest. 370 Lymphotoxin signalling alters the vasculature to increase tumour cell metastasis N. Simonavicius1 , B. Seubert2 , L. Borsig3 , D. Wohlleber4 , J. Browning5 , 2 , M. Heikenwalder1 . 1 Helmholtz Zentrum Munchen, ¨ Institute A. Kruger ¨ ¨ Munchen, of Virology, Muenchen, Germany, 2 Technische Universitat ¨ 1Institute for Experimental Oncology and Therapy Research/Institute of Molecular Immunology, Muenchen, Germany, 3 University of Zurich, Institute of Physiology, Zurich, Switzerland, 4 University Hospital Bonn, Institutes of Molecular Medicine and Experimental Immunology, Bonn, Germany, 5 Boston University School of Medicine, Department of Microbiology and Section of Rheumatology, Boston, USA The importance of an inflammatory microenvironment in affecting carcinogenesis and metastasis is becoming increasingly evident. One of the key steps of tumour cell extravasation to secondary sites is the transmigration through the blood vessel, which consists of a barrier of endothelial cells and closely associated pericytes. The impact of an inflammatory microenvironment to the vascular wall and the consequences for metastasis remain elusive. Here, we investigate the role of the pro-inflammatory cytokine lymphotoxin (LT) − either provided systemically or produced by the tumour cells − in altering the vasculature to facilitate tumour cell extravasation. In an in vivo experimental metastasis assay we observed that the T-lymphoma cells express the LT ligands to enable their own extravasation through the liver vasculature as both the knockdown of LT as well as a blocking antibody decreased the number of T-lymphoma metastases in the liver. Congruently, preconditioning of the liver with an agonistic lymphotoxin b receptor (LTbR) antibody, mimicking a pro-carcinogenic environment, leads to an increased number of T-lymphoma cells. The effects are neither specific to lymphoma cells nor the liver as experimental metastasis assays using melanoma or colon carcinoma cells also showed increased lung metastases after LTbR agonisation. Importantly, the stimulation of the LTbR pathway led to vascular disintegration as monitored by Evans Blue leakage. In vivo the liver vasculature responds to LTbR agonisation via induction of target genes, such as matrix metalloproteinases, that may be responsible for the increase in vascular permeability and metastases. Interestingly, neither macrophages nor the LTbR on hepatocytes mediate the increased vascular permeability after LTbR agonisation. In conclusion, we elucidate a mechanism how the presence of proinflammatory LTs either administered systemically or via expression by the tumour cells themselves changes the vasculature to enhance tumour cell metastasis. This not only provides insight into the link of inflammation and metastasis but may also provide novel therapeutic opportunities. No conflict of interest.