- Email: [email protected]
The heterogeneity of circulating lung tumor cells from non-small cell lung cancer patients
S80
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
silencing of MCT1 or MCT4 was assessed in vitro. A lactate assay was used to investigate the effect on intra- and extracellular lactate under varying oxygen tensions. Effect on cell cycle progression was assessed using flow cytometry and toxicity of docetaxel was determined using MTT and SRB assays. Finally, a metabolome siRNA screen was carried out to determine if MCT4 expression was related to sensitivity to toxicity resulting from knockdown of a range of metabolic proteins. Results: Expression levels of MCT1 and MCT4 were confirmed by western blot and immunofluorescence. Silencing of MCT4 in DU145 and PC3 cell lines increased intracellular lactate under hypoxic conditions. Knockdown of MCT4 resulted in sensitization to docetaxel treatment under both normoxic and hypoxic conditions. Overexpression of MCT1 in PC3 cells caused cell cycle arrest in the G2/M phase in both normoxia and hypoxia. The metabolome screen indicated that, in wild type LNCaP cells, down-regulation of the protein product of the genes encoding 2,4-Dehydrocholesterol Reductase or Pyruvate Dehydrogenase Phosphatase Catalytic Subunit 2 was synthetically lethal. Overexpression of MCT4 resulted in protection against these lethal effects. Conclusions: Altering the expression of MCT1 and MCT4 in human prostate cancer cells had a variety of effects on cell function. Overexpression of MCT1 caused cell cycle arrest in PC3 cells which may impact on radiosensitivity as cells are most vulnerable to radiation treatment when in the G2/M phase. The identification of two proteins that cause toxicity when silenced in LNCaP wild-type cells, but not LNCaP cells showing overexpression of MCT4, is an interesting lead for further investigation. No conflict of interest. 385 The heterogeneity of circulating lung tumor cells from non-small cell lung cancer patients S.L. Kong1 , S.J. Tan2 , T.K.H. Lim3 , H.M. Poh1 , T.Z.X. Yeo2 , X. Liu1 , Y.W. Chua3 , A.A. Bhagat4 , W.T. Lim5 , A.M. Hillmer1 . 1 Genome Institute of Singapore- A*STAR, Cancer therapeutics and stratified oncology, Biopolis Street, Singapore, 2 Clearbridge Accelerator Pte Ltd, Science Park Drive, Singapore, 3 Singapore General Hospital, Department of Pathology, Outram Road, Singapore, 4 Clearbridge BioMedics Pte Ltd, Science Park Drive, Singapore, 5 National Cancer Center, Division of Medical Oncology, Hospital Drive, Singapore Background: The heterogeneity and plasticity of individual tumor subclones potentially drive development of resistance under selection pressure from anticancer therapy. Molecular analyses of single circulating tumor cells (CTCs) may allow us to understand the critical pathways that mediate the bloodborne dissemination of cancer, where bulk analyses of primary or metastatic tumors fail due to tumour heterogeneity. Material and Methods: In this study, we evaluated the heterogeneity of single CTCs isolated from non-small cell lung cancer (NSCLC) patients recruited from National Cancer Centre Singapore. A total of 7.5 ml EDTA blood was processed on the Clearbridge BioMedics ClearCell® FX system. Single CTCs were enumerated using a novel single cell isolation machine, DropCell that operates based on hydrodynamic focusing and negative antibody selection. Whole genome amplification was performed on the enumerated single cells followed by amplicon-based deep sequencing on a custom-designed lung cancer gene panel. Results: A total of 23 CD45-negative single cells were isolated from 7 NSCLC patients. As verified by the Sanger sequencing, we found similar EGFR driver mutation in the matched primary tumor and 5 single CTCs samples. A more comprehensive mutation profiling of the single CD45-negative cells and matched primary tumor is being processed and validated. Conclusions: This study demonstrates the possibility of single cell CTC isolation and targeted amplicon sequencing approach in characterizing the mutation profiles of NSCLC CTCs. This allows us to evaluate the degree of heterogeneity in CTCs and the matched primary tumor. No conflict of interest. 386 Induction of senescence in primary glioblastoma cells by serum and TGFb R. Kumar1 , A. Gont1 , T. Perkins2 , I. Lorimer1 . 1 Ottawa Hospital Research Institute, Cancer Therapeutics, Ottawa, Canada, 2 Ottawa Hospital Research Institute, Regenerative Medicine Program, Ottawa, Canada Introduction: Glioblastoma is the most common type of adult primary brain tumour and has a median survival after diagnosis of a little more than a year. Glioblastomas have a high frequency of mutations in the Telomerase reverse transcriptase (TERT) promoter and Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) locus that are expected to render them resistant to both replicative and oncogene-induced senescence. While traditional glioblastoma cell lines cultured in serum have demonstrated the ability to undergo premature senescence, the ability of primary glioblastoma cells to do so has not been studied in detail. Materials and Methods: Primary glioblastoma (PriGO) cells were harvested from human patients with glioblastoma and cultured in serum free media
under hypoxic conditions (5% oxygen). Senescence was determined primarily by the senescence-associated b galactosidase (SA-b-Gal) assay. Markers of senescence Promyelocytic Leukemia (PML) bodies and p21 were detected by immunofluorescence and immunoblotting respectively. Global gene expression profiling was determined using Affymetrix microarray human gene chips. Thrombospondin expression was determined by enzyme-linked immunosorbent assay (ELISA). Ras pathway inhibition was achieved with the MEK inhibitor U0126. Results and Discussion: Exposure of PriGO8A primary glioblastoma cells to media with 10% serum induced a senescence-like phenotype characterized by increased SA-B-Gal activity, PML bodies and p21 and morphological changes characteristic of senescence. This occurred in the absence of any increase in gH2AX foci or senescence-associated secretory phenotype (SASP). Microarray expression analysis showed that serum exposure increased the expression of genes associated with the Transforming growth factor beta (TGFb) pathway. Treatment of PriGO8A cells with TGFb was sufficient to induce senescence in these cells. The response of PriGO8A cells to serum was dependent on basal expression of the TGFb activator protein thrombospondin. Primary glioblastoma cells from three additional patients showed a variable ability to undergo senescence in response to serum. Cells that lacked basal thrombospondin expression did not undergo senescence in response to serum, but did in response to TGFb. Cells that had low level constitutive TGFb pathway activation driven by Ras pathway activation only underwent senescence in response to TGFb when Ras pathway activity was blocked. Conclusion: TGFb is a key mediator of premature senescence in primary glioblastoma cells, its activity dependent on the TGFb activator protein thrombospondin as well as Ras pathway activation. Primary glioblastoma cells therefore retain a functional senescence program which can potentially be exploited therapeutically. No conflict of interest. 387 Yeast as a model system to screen purine derivatives against human CDK1 and CDK2 kinases T. Mayi1 , M.E. Huang2 , L. Vernis2 , M. Legraverend3 , G. Faye2 . 1 Institut ˆ 110–112- Centre Universitaire- 91405 Orsay- France, INSERM Curie- Bat. ˆ 110–112- Centre UniversitaireU612, Orsay, France, 2 Institut Curie- Bat. ˆ 91405 Orsay- France, CNRS UMR3348, Orsay, France, 3 Institut Curie- Bat. 110–112- Centre Universitaire- 91405 Orsay- France, CNRS UMR176, Orsay, France Background: Cyclin-dependent kinases (Cdk) play crucial roles in cell cycle progression. Aberrant activation of Cdk1 has been observed in a number of primary tumors and Cdk2 is deregulated in various malignancies. The therapeutic value of targeting Cdk1 and Cdk2 has been explored in a number of experimental systems. Material and Methods: Taking advantage of the fact that deletion of the yeast CDC28 gene is functionally complemented by human CDK1 or CDK2, in vivo screen system have been set up to evaluate the inhibitory potency of purine derivatives against these two human Cdks. Three isogenic strains highly sensitive to small molecules and harboring genes CDK1, CDK2 or CDC28, have been constructed under the control of the CDC28 promoter. In a proof of principle assay, the inhibitory effect of 82 purine derivatives on the growth rate of these strains were determined. Results: Thirty-three of 82 purine derivatives were revealed to be able to inhibit the Cdk1- or Cdk2-harboring strains but not the Cdc28-harboring strain, suggesting a specific inhibitory effect on human Cdks. Conclusions: Our data demonstrate that the yeast-based assay is an efficient system to identify potential specific inhibitors that should be preferentially selected for further investigation in cancer cultured human cell lines. No conflict of interest. 388 The vacuolar ATPase subunit E controls apoptosis and is up-regulated in breast cancer S.N. Mohammed1 , M. Mourtada-Maarabouni1 . 1 Keele University, School of Life Sciences, Newcastle-Under-Lyme, United Kingdom Background: Vacuolar H+ATPase (V-ATPase) is a multi-subunit proton pump involved in the acidification of a wide variety of organelles. Dysfunction of the V-ATPase has been associated with several diseases, including cancer. Increased V-ATPase activity is reported to be critical for invasion of highly metastatic breast cancer cells and its expression at the plasma membrane correlates with the invasive characteristics of various malignant cells. Functional expression cloning identified V-ATPase subunit E (ATP6V1E) as a potential apoptosis regulatory gene. The aims of this study were to examine the effects of modulating ATP6V1E expression on the survival of breast cancer cells. In addition, we have examined the expression level of ATP6V1E in breast cancer samples. Materials and Methods: MCF7 and MDA-MB-231 cells were transfected either with a mammalian expression plasmid encoding ATP6V1E transcript, control cells received empty vector. Culture growth, cell viability, apoptosis,
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
silencing of MCT1 or MCT4 was assessed in vitro. A lactate assay was used to investigate the effect on intra- and extracellular lactate under varying oxygen tensions. Effect on cell cycle progression was assessed using flow cytometry and toxicity of docetaxel was determined using MTT and SRB assays. Finally, a metabolome siRNA screen was carried out to determine if MCT4 expression was related to sensitivity to toxicity resulting from knockdown of a range of metabolic proteins. Results: Expression levels of MCT1 and MCT4 were confirmed by western blot and immunofluorescence. Silencing of MCT4 in DU145 and PC3 cell lines increased intracellular lactate under hypoxic conditions. Knockdown of MCT4 resulted in sensitization to docetaxel treatment under both normoxic and hypoxic conditions. Overexpression of MCT1 in PC3 cells caused cell cycle arrest in the G2/M phase in both normoxia and hypoxia. The metabolome screen indicated that, in wild type LNCaP cells, down-regulation of the protein product of the genes encoding 2,4-Dehydrocholesterol Reductase or Pyruvate Dehydrogenase Phosphatase Catalytic Subunit 2 was synthetically lethal. Overexpression of MCT4 resulted in protection against these lethal effects. Conclusions: Altering the expression of MCT1 and MCT4 in human prostate cancer cells had a variety of effects on cell function. Overexpression of MCT1 caused cell cycle arrest in PC3 cells which may impact on radiosensitivity as cells are most vulnerable to radiation treatment when in the G2/M phase. The identification of two proteins that cause toxicity when silenced in LNCaP wild-type cells, but not LNCaP cells showing overexpression of MCT4, is an interesting lead for further investigation. No conflict of interest. 385 The heterogeneity of circulating lung tumor cells from non-small cell lung cancer patients S.L. Kong1 , S.J. Tan2 , T.K.H. Lim3 , H.M. Poh1 , T.Z.X. Yeo2 , X. Liu1 , Y.W. Chua3 , A.A. Bhagat4 , W.T. Lim5 , A.M. Hillmer1 . 1 Genome Institute of Singapore- A*STAR, Cancer therapeutics and stratified oncology, Biopolis Street, Singapore, 2 Clearbridge Accelerator Pte Ltd, Science Park Drive, Singapore, 3 Singapore General Hospital, Department of Pathology, Outram Road, Singapore, 4 Clearbridge BioMedics Pte Ltd, Science Park Drive, Singapore, 5 National Cancer Center, Division of Medical Oncology, Hospital Drive, Singapore Background: The heterogeneity and plasticity of individual tumor subclones potentially drive development of resistance under selection pressure from anticancer therapy. Molecular analyses of single circulating tumor cells (CTCs) may allow us to understand the critical pathways that mediate the bloodborne dissemination of cancer, where bulk analyses of primary or metastatic tumors fail due to tumour heterogeneity. Material and Methods: In this study, we evaluated the heterogeneity of single CTCs isolated from non-small cell lung cancer (NSCLC) patients recruited from National Cancer Centre Singapore. A total of 7.5 ml EDTA blood was processed on the Clearbridge BioMedics ClearCell® FX system. Single CTCs were enumerated using a novel single cell isolation machine, DropCell that operates based on hydrodynamic focusing and negative antibody selection. Whole genome amplification was performed on the enumerated single cells followed by amplicon-based deep sequencing on a custom-designed lung cancer gene panel. Results: A total of 23 CD45-negative single cells were isolated from 7 NSCLC patients. As verified by the Sanger sequencing, we found similar EGFR driver mutation in the matched primary tumor and 5 single CTCs samples. A more comprehensive mutation profiling of the single CD45-negative cells and matched primary tumor is being processed and validated. Conclusions: This study demonstrates the possibility of single cell CTC isolation and targeted amplicon sequencing approach in characterizing the mutation profiles of NSCLC CTCs. This allows us to evaluate the degree of heterogeneity in CTCs and the matched primary tumor. No conflict of interest. 386 Induction of senescence in primary glioblastoma cells by serum and TGFb R. Kumar1 , A. Gont1 , T. Perkins2 , I. Lorimer1 . 1 Ottawa Hospital Research Institute, Cancer Therapeutics, Ottawa, Canada, 2 Ottawa Hospital Research Institute, Regenerative Medicine Program, Ottawa, Canada Introduction: Glioblastoma is the most common type of adult primary brain tumour and has a median survival after diagnosis of a little more than a year. Glioblastomas have a high frequency of mutations in the Telomerase reverse transcriptase (TERT) promoter and Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) locus that are expected to render them resistant to both replicative and oncogene-induced senescence. While traditional glioblastoma cell lines cultured in serum have demonstrated the ability to undergo premature senescence, the ability of primary glioblastoma cells to do so has not been studied in detail. Materials and Methods: Primary glioblastoma (PriGO) cells were harvested from human patients with glioblastoma and cultured in serum free media
under hypoxic conditions (5% oxygen). Senescence was determined primarily by the senescence-associated b galactosidase (SA-b-Gal) assay. Markers of senescence Promyelocytic Leukemia (PML) bodies and p21 were detected by immunofluorescence and immunoblotting respectively. Global gene expression profiling was determined using Affymetrix microarray human gene chips. Thrombospondin expression was determined by enzyme-linked immunosorbent assay (ELISA). Ras pathway inhibition was achieved with the MEK inhibitor U0126. Results and Discussion: Exposure of PriGO8A primary glioblastoma cells to media with 10% serum induced a senescence-like phenotype characterized by increased SA-B-Gal activity, PML bodies and p21 and morphological changes characteristic of senescence. This occurred in the absence of any increase in gH2AX foci or senescence-associated secretory phenotype (SASP). Microarray expression analysis showed that serum exposure increased the expression of genes associated with the Transforming growth factor beta (TGFb) pathway. Treatment of PriGO8A cells with TGFb was sufficient to induce senescence in these cells. The response of PriGO8A cells to serum was dependent on basal expression of the TGFb activator protein thrombospondin. Primary glioblastoma cells from three additional patients showed a variable ability to undergo senescence in response to serum. Cells that lacked basal thrombospondin expression did not undergo senescence in response to serum, but did in response to TGFb. Cells that had low level constitutive TGFb pathway activation driven by Ras pathway activation only underwent senescence in response to TGFb when Ras pathway activity was blocked. Conclusion: TGFb is a key mediator of premature senescence in primary glioblastoma cells, its activity dependent on the TGFb activator protein thrombospondin as well as Ras pathway activation. Primary glioblastoma cells therefore retain a functional senescence program which can potentially be exploited therapeutically. No conflict of interest. 387 Yeast as a model system to screen purine derivatives against human CDK1 and CDK2 kinases T. Mayi1 , M.E. Huang2 , L. Vernis2 , M. Legraverend3 , G. Faye2 . 1 Institut ˆ 110–112- Centre Universitaire- 91405 Orsay- France, INSERM Curie- Bat. ˆ 110–112- Centre UniversitaireU612, Orsay, France, 2 Institut Curie- Bat. ˆ 91405 Orsay- France, CNRS UMR3348, Orsay, France, 3 Institut Curie- Bat. 110–112- Centre Universitaire- 91405 Orsay- France, CNRS UMR176, Orsay, France Background: Cyclin-dependent kinases (Cdk) play crucial roles in cell cycle progression. Aberrant activation of Cdk1 has been observed in a number of primary tumors and Cdk2 is deregulated in various malignancies. The therapeutic value of targeting Cdk1 and Cdk2 has been explored in a number of experimental systems. Material and Methods: Taking advantage of the fact that deletion of the yeast CDC28 gene is functionally complemented by human CDK1 or CDK2, in vivo screen system have been set up to evaluate the inhibitory potency of purine derivatives against these two human Cdks. Three isogenic strains highly sensitive to small molecules and harboring genes CDK1, CDK2 or CDC28, have been constructed under the control of the CDC28 promoter. In a proof of principle assay, the inhibitory effect of 82 purine derivatives on the growth rate of these strains were determined. Results: Thirty-three of 82 purine derivatives were revealed to be able to inhibit the Cdk1- or Cdk2-harboring strains but not the Cdc28-harboring strain, suggesting a specific inhibitory effect on human Cdks. Conclusions: Our data demonstrate that the yeast-based assay is an efficient system to identify potential specific inhibitors that should be preferentially selected for further investigation in cancer cultured human cell lines. No conflict of interest. 388 The vacuolar ATPase subunit E controls apoptosis and is up-regulated in breast cancer S.N. Mohammed1 , M. Mourtada-Maarabouni1 . 1 Keele University, School of Life Sciences, Newcastle-Under-Lyme, United Kingdom Background: Vacuolar H+ATPase (V-ATPase) is a multi-subunit proton pump involved in the acidification of a wide variety of organelles. Dysfunction of the V-ATPase has been associated with several diseases, including cancer. Increased V-ATPase activity is reported to be critical for invasion of highly metastatic breast cancer cells and its expression at the plasma membrane correlates with the invasive characteristics of various malignant cells. Functional expression cloning identified V-ATPase subunit E (ATP6V1E) as a potential apoptosis regulatory gene. The aims of this study were to examine the effects of modulating ATP6V1E expression on the survival of breast cancer cells. In addition, we have examined the expression level of ATP6V1E in breast cancer samples. Materials and Methods: MCF7 and MDA-MB-231 cells were transfected either with a mammalian expression plasmid encoding ATP6V1E transcript, control cells received empty vector. Culture growth, cell viability, apoptosis,