- Email: [email protected]
O46. Reversal of resistance to standard therapy in oral squamous cell carcinoma cells by RNA interference
Oral AbstractsPoster ListOrals ListPan. Disc. & Symp. Abs.Keynote Abs.Keynote Bios.ProgramIAOOWelcomeCommittee Listings
72
Oral abstracts / Oral Oncology Supplement 3 (2009) 56–122
O46. Reversal of resistance to standard therapy in oral squamous cell carcinoma cells by RNA interference S.M. Gollin a,b,*, M. Sankunny a,b, R.E. Flores-Obando a, X. Huang a,b, R.A. Parikh a,b a University of Pittsburgh Graduate School of Public Health, United States b University of Pittsburgh Cancer Institute, United States
Tumor cell resistance to therapy leads to the death of cancer patients. We hypothesize that defects in the DNA damage response (DDR) result in loss of radiosensitivity and chemosensitivity in oral squamous cell carcinoma (OSCC). The DDR involves sensing DNA damage followed by transduction of the damage signal to a network of cellular pathways to repair DNA or kill irreparable cells. At the apex of the DDR are ATM and ATR, kinases that phosphorylate their substrates in response to DNA damage via intertwining signaling pathways that control cell cycle checkpoints, DNA repair, and apoptosis. Patients with mutant DDR genes are at increased risk of cancer, including OSCC. We are examining the role of DDR defects in chromosomal instability (CIN), one form of which, 11q13 amplification, is seen in 45% of OSCC, the highest frequency of any cancer, and is an indicator of poor prognosis. Loss of distal chromosome 11q (including the key DDR gene, ATM) precedes the breakage-fusionbridge (BFB) cycles that lead to 11q13 amplification. Our preliminary studies of CIN in OSCC cell lines reveal DDR defects; 10% of cells with distal 11q loss survive 10 Gy of ionizing radiation, regardless of the presence of 11q13 amplification. Thus, we have broken with standard thinking to test the novel idea that haploinsufficiency for critical DDR genes on distal 11q (a key facet of our biomarker signature) is a critical component of the gene amplification mechanism, associated with poor prognosis, CIN, and LORS. We can reverse this radioresistance in biomarker signature-positive OSCC, lung, and ovarian cancer cells using RNA interference. This suggests that our biomarker signature may serve as a patient selection test for a small molecule inhibitor under development, which may reverse resistance to standard therapies in cancer patients and thus, save lives. doi:10.1016/j.oos.2009.06.131
O47. The epithelial-mesenchymal transition inducer, Snail, inhibits terminal differentiation and promotes inflammation and invasion in squamous cell carcinomas J.G. Lyons a,b,*, N. Roue a,b, V. Patel c, J.S. Gutkind c, G.M. Halliday b a
Sydney Head & Neck Cancer Institute, Australia University of Sydney, Australia c National Institute of Dental and Craniofacial Research, United States b
Introduction: The Snail family of transcription factors causes epithelial cells to undergo an epithelial-mesenchymal transition and is frequently over-expressed in carcinomas. Snail2 is more highly expressed in head and neck squamous cell carcinomas (HNSCCs) than in normal oral epithelium, and in recurrent than in non-recurrent HNSCCs. The aim of this study was to identify the cellular and molecular mechanisms by which Snails contribute to the pathogenesis of HNSCCs. Methods: We used in vitro squame formation assays to study differentiation. Microarrays, qRTPCR and transfections were used to identify Snail target genes and analyse their promoters. Results: We have identified a regulatory cascade required for upregulation of terminal differentiation genes and determined the stages at which Snail acts directly as a repressor of promoter activity or indirectly by repressing intermediary factors. The expression of
Snail1 or Snail2 inhibited keratinocyte differentiation, the stage of desquamation being particularly strongly affected. Snail reduces the expression of genes critical for terminal differentiation, including ELF3 and matriptase-1. Snail also strongly up-regulates the expression of the genes for the cytokines/chemokines IL6, IL8 and CXCL1, and the prostaglandin synthetic enzyme COX2, coinciding with an enhanced ability to attract monocytes and invade extracellular matrix. Nuclear factor kappa B (NFkB) activity is a pre-requisite for this pro-inflammatory effect of Snail; however, Snail does not increase NFkB activity. Discussion: The data indicate that Snail can contribute to HNSCC progression at early stages, by down-regulating factors required for the terminal differentiation of keratinocytes, and at later stages, by creating a pro-inflammatory, invasive environment in the tumour. The up-regulation of pro-inflammatory and down-regulation of terminal differentiation Snail-target genes correlates with malignancy and local recurrence in HNSCC patients, suggesting that Snail is an important contributor to their poor prognosis. doi:10.1016/j.oos.2009.06.132
O48. Tumour stem cells in head and neck squamous cell carcinoma (HNSCC) S.J. Goldie a,c,*, F.M. Watt a,c, R. Price b a
Cambridge Research Institute, United Kingdom Department of Plastic Surgery, Addenbrooke’s Hospital, United Kingdom c Department of Genetics, University of Cambridge, United Kingdom b
Despite advances in surgery and adjuvant therapies for head and neck squamous cell carcinoma (HNSCC), many patients develop recurrent disease following radical treatment. The tumour stem cell hypothesis proposes that a population of malignant cells may evade current therapies and the immune system and allow a cancer to regrow. By targeting the tumour stem cell population, clinicians may be more successful at controlling the disease and cause less harm to the patient’s normal tissues. Having obtained full ethical approval, patients undergoing resection of an SCC from the oral cavity were asked to donate a sample of their tissue. Cell cultures derived from these tumours were then infected using lentivirus to add a luciferase marker. Using flow cytometry the SCC lines were enriched based on their expression of potential stem cell markers. The enriched populations were cultured in vitro and their growth potential tested with colony forming assays, then xenografted in plastic chambers grafted onto the backs of severely immunocompromised NOD/SCID gamma (NSG) mice to show in vivo changes in tumorigenicity and epithelial mesenchymal transition (EMT) potential. Patterns of growth in vitro and in vivo were compared with the histological grade of the tumour of origin. Behaviour of the cell lines in vitro and the tumorigenicity and metastasis in vivo differ according to the cell line of origin. This is further altered by enriching for stem cell markers. Using this model allows us to study how alterations in gene expression can affect the growth and metastasis of human head and neck SCCs. This model could also be used to test tumour responses to therapies targeted at the stem cell population. Overexpression or knock down of stem cell markers may affect tumour growth. doi:10.1016/j.oos.2009.06.133
72
Oral abstracts / Oral Oncology Supplement 3 (2009) 56–122
O46. Reversal of resistance to standard therapy in oral squamous cell carcinoma cells by RNA interference S.M. Gollin a,b,*, M. Sankunny a,b, R.E. Flores-Obando a, X. Huang a,b, R.A. Parikh a,b a University of Pittsburgh Graduate School of Public Health, United States b University of Pittsburgh Cancer Institute, United States
Tumor cell resistance to therapy leads to the death of cancer patients. We hypothesize that defects in the DNA damage response (DDR) result in loss of radiosensitivity and chemosensitivity in oral squamous cell carcinoma (OSCC). The DDR involves sensing DNA damage followed by transduction of the damage signal to a network of cellular pathways to repair DNA or kill irreparable cells. At the apex of the DDR are ATM and ATR, kinases that phosphorylate their substrates in response to DNA damage via intertwining signaling pathways that control cell cycle checkpoints, DNA repair, and apoptosis. Patients with mutant DDR genes are at increased risk of cancer, including OSCC. We are examining the role of DDR defects in chromosomal instability (CIN), one form of which, 11q13 amplification, is seen in 45% of OSCC, the highest frequency of any cancer, and is an indicator of poor prognosis. Loss of distal chromosome 11q (including the key DDR gene, ATM) precedes the breakage-fusionbridge (BFB) cycles that lead to 11q13 amplification. Our preliminary studies of CIN in OSCC cell lines reveal DDR defects; 10% of cells with distal 11q loss survive 10 Gy of ionizing radiation, regardless of the presence of 11q13 amplification. Thus, we have broken with standard thinking to test the novel idea that haploinsufficiency for critical DDR genes on distal 11q (a key facet of our biomarker signature) is a critical component of the gene amplification mechanism, associated with poor prognosis, CIN, and LORS. We can reverse this radioresistance in biomarker signature-positive OSCC, lung, and ovarian cancer cells using RNA interference. This suggests that our biomarker signature may serve as a patient selection test for a small molecule inhibitor under development, which may reverse resistance to standard therapies in cancer patients and thus, save lives. doi:10.1016/j.oos.2009.06.131
O47. The epithelial-mesenchymal transition inducer, Snail, inhibits terminal differentiation and promotes inflammation and invasion in squamous cell carcinomas J.G. Lyons a,b,*, N. Roue a,b, V. Patel c, J.S. Gutkind c, G.M. Halliday b a
Sydney Head & Neck Cancer Institute, Australia University of Sydney, Australia c National Institute of Dental and Craniofacial Research, United States b
Introduction: The Snail family of transcription factors causes epithelial cells to undergo an epithelial-mesenchymal transition and is frequently over-expressed in carcinomas. Snail2 is more highly expressed in head and neck squamous cell carcinomas (HNSCCs) than in normal oral epithelium, and in recurrent than in non-recurrent HNSCCs. The aim of this study was to identify the cellular and molecular mechanisms by which Snails contribute to the pathogenesis of HNSCCs. Methods: We used in vitro squame formation assays to study differentiation. Microarrays, qRTPCR and transfections were used to identify Snail target genes and analyse their promoters. Results: We have identified a regulatory cascade required for upregulation of terminal differentiation genes and determined the stages at which Snail acts directly as a repressor of promoter activity or indirectly by repressing intermediary factors. The expression of
Snail1 or Snail2 inhibited keratinocyte differentiation, the stage of desquamation being particularly strongly affected. Snail reduces the expression of genes critical for terminal differentiation, including ELF3 and matriptase-1. Snail also strongly up-regulates the expression of the genes for the cytokines/chemokines IL6, IL8 and CXCL1, and the prostaglandin synthetic enzyme COX2, coinciding with an enhanced ability to attract monocytes and invade extracellular matrix. Nuclear factor kappa B (NFkB) activity is a pre-requisite for this pro-inflammatory effect of Snail; however, Snail does not increase NFkB activity. Discussion: The data indicate that Snail can contribute to HNSCC progression at early stages, by down-regulating factors required for the terminal differentiation of keratinocytes, and at later stages, by creating a pro-inflammatory, invasive environment in the tumour. The up-regulation of pro-inflammatory and down-regulation of terminal differentiation Snail-target genes correlates with malignancy and local recurrence in HNSCC patients, suggesting that Snail is an important contributor to their poor prognosis. doi:10.1016/j.oos.2009.06.132
O48. Tumour stem cells in head and neck squamous cell carcinoma (HNSCC) S.J. Goldie a,c,*, F.M. Watt a,c, R. Price b a
Cambridge Research Institute, United Kingdom Department of Plastic Surgery, Addenbrooke’s Hospital, United Kingdom c Department of Genetics, University of Cambridge, United Kingdom b
Despite advances in surgery and adjuvant therapies for head and neck squamous cell carcinoma (HNSCC), many patients develop recurrent disease following radical treatment. The tumour stem cell hypothesis proposes that a population of malignant cells may evade current therapies and the immune system and allow a cancer to regrow. By targeting the tumour stem cell population, clinicians may be more successful at controlling the disease and cause less harm to the patient’s normal tissues. Having obtained full ethical approval, patients undergoing resection of an SCC from the oral cavity were asked to donate a sample of their tissue. Cell cultures derived from these tumours were then infected using lentivirus to add a luciferase marker. Using flow cytometry the SCC lines were enriched based on their expression of potential stem cell markers. The enriched populations were cultured in vitro and their growth potential tested with colony forming assays, then xenografted in plastic chambers grafted onto the backs of severely immunocompromised NOD/SCID gamma (NSG) mice to show in vivo changes in tumorigenicity and epithelial mesenchymal transition (EMT) potential. Patterns of growth in vitro and in vivo were compared with the histological grade of the tumour of origin. Behaviour of the cell lines in vitro and the tumorigenicity and metastasis in vivo differ according to the cell line of origin. This is further altered by enriching for stem cell markers. Using this model allows us to study how alterations in gene expression can affect the growth and metastasis of human head and neck SCCs. This model could also be used to test tumour responses to therapies targeted at the stem cell population. Overexpression or knock down of stem cell markers may affect tumour growth. doi:10.1016/j.oos.2009.06.133