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The role of intracellular Ca2+ concentration for macromolecular drug carrier uptake into tumor cells in an acidotic tumor environment
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EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
data show that these mutations require a context of basal G protein-coupled receptor activity in order promote invasion. No conflict of interest. 759 Oncogenic reciprocal signalling C. Tape1 . 1 The Institute of Cancer Research, Cancer Biology, London, United Kingdom In a heterocellular cancer, tumor cell oncogenes drive aberrant signaling both within tumor cells (cell-autonomous signaling) and adjacent stromal cells (non-cell-autonomous signaling). As different cell-types process signals via distinct pathways, heterocellular systems (containing different cell-types) theoretically provide increased signal processing capacity over homocellular systems (containing a single cell-type). We hypothesized that the expanded signaling capacity provided by stromal heterocellularity allows oncogenes to establish a differential reciprocal signaling state in tumor cells. Here we show that oncogenic KRAS (KRAS-G12D) uniquely regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS-G12D signaling in Pancreatic Ductal Adenocarcinoma (PDA). Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS-G12D. Consequently, reciprocal KRAS-G12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS-G12D alone. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. No conflict of interest. 760 A single cell approach to understanding cell cycle entry in cancer A. Barr1 , F.S. Heldt2 , S. Cooper1 , B. Novak2 , C. Bakal1 . 1 Institute of Cancer Research, Division of Cancer Biology, London, United Kingdom, 2 University of Oxford, Department of Biochemistry, Oxford, United Kingdom Background: Signalling networks controlling the decision to proliferate or arrest are perturbed in cancer. Network perturbation leads to changes in signalling dynamics, however currently we do not understand how these dynamics are changed, or what affect these changes have on proliferation. In order to specifically inhibit cancer cell proliferation, we need to know which signalling pathways are driving cancer cells into S-phase. Much of our knowledge regarding the decision to arrest or proliferate comes from population-based studies, which mask the true signalling dynamics that determine these decisions. Therefore, we need to use single cell technologies to study signalling dynamics during cell cycle entry. Materials and Methods: To understand the regulation of cell fate decisions during the cell cycle, we use quantitative single cell imaging in tandem with mathematical modelling. We have tagged key regulators of the G1/S transition at their endogenous loci in cancer and non-cancer cell lines, in order to monitor protein expression and localisation over multiple cell cycles. In combination with automated image analysis, we can extract quantitative information describing protein dynamics at each cell cycle transition for hundreds of cells. The quantitative data is then used to parametrise mathematical models of cell cycle entry. Results: We have used these methods to understand the regulation of S-phase commitment in both cancer and non-cancer cells. In non-transformed cells, there is a high level of heterogeneity in S-phase entry timing. We find that this heterogeneity is partly dependent on a high degree of cell-to-cell variability in the expression of the cyclin-dependent kinase inhibitor, p21. Variability in p21 expression is driven by p53 and our data suggest that DNA replication stress can trigger expression of p21, through activation of the DNA damage response. p21 expression then maintains cells in G1 until the damage is repaired. In cancer cells lacking p53, and in non-transformed cells where p53 has been depleted, S-phase entry is much more homogeneous in timing. Conclusions: Non-genetic heterogeneity in cell cycle timing could be an important determinant of “fractional kill” in tumour cells. We are now investigating how heterogeneity in p21 expression, both within and between cancer cell lines, affects response to chemotherapy, and thus how we could drive cancer cells towards apoptosis. No conflict of interest. 761 The role of intracellular Ca2+ concentration for macromolecular drug carrier uptake into tumor cells in an acidotic tumor environment 1 , O. Thews2 . 1 Clinic of Nuclear Medicine, University HospitalD. Gundel ¨ Martin-Luther-University Halle-Wittenberg, Halle Saale, Germany, 2 Julius Bernstein Institute of Physiology, Medical Faculty- Martin-Luther-University Halle-Wittenberg, Halle Saale, Germany Background: In the last decade macromolecular drug carriers became of scientific and clinical interest. Due to the abnormal tumor vasculature, leading
to an enhanced permeation and retention (EPR) effect, macromolecules can be used for passive drug accumulation in solid tumors. After these molecules reached the tumor tissue, the nano-carriers have to be taken up into the cells via endocytosis and the cytotoxic drugs are cleaved intracellularly. Because of pronounced glycolytic activity and weak drainage of metabolic end products the tumor tissue often becomes acidotic. The aim of the study was to analyze whether this extracellular acidosis affects the intracellular Ca2+-signaling and by this the endocytotic pathway for taking up macromolecules. Methods: Cellular uptake was determined in AT-1 R-3327 prostate and Walker-256 mammary carcinoma cells of the rat. Intracellular [Ca2+] and pH were determined ratiometrically with fluorescence microscopy in superfused single cells. Uptake studies were performed with pH-stable Oregon Green 488 labeled 70 kDa dextran which was quantified by flow cytometry. Ca2+ dependent uptake studies were determined either with 1.2 mM CaCl2 containing or with almost Ca2+-free buffers. Typical endocytotic pathways were selectively inhibited by rottlerin, dynasore, chlorpromazine and nystatin. Results: Cellular uptake of dextran (micropinocytosis) varied with pH. In AT-1 cells the uptake increased with rising pH, but in Walker-256 cells a negative correlation was found. Also the inhibition of PKC d, an important regulator for micropinocytosis, with rottlerin led to similar effects. On the other hand, an inhibition of dynamin dependent endocytosis diminished uptake of the dextran in both cell lines. Measuring the intracellular [Ca2+] and pH during acidification of the extracellular space indicated that the intracellular [Ca2+] was rapidly decreasing in both cell lines by 20−40%. However, in AT-1 cells the Ca2+ level was spontaneously increasing within several minutes at low extracellular pH. Under low intracellular Ca2+ conditions, dextran uptake was independent from the extracellular pH in both cell lines. Conclusion: The present study demonstrates that the endocytotic uptake of macromolecules depends on the extracellular pH most probably due to a Ca2+-mediated mechanism. However, the consequence of low extracellular pH, which is a common feature of solid growing tumors, for the uptake of macromolecules and therefore for the cytotoxicity of these drugs seems to be cell line-specific. Acidosis was able to foster, but also to limit the endocytotic uptake of large molecules. Modulating the metabolic extracellular environment but also the relevant signaling pathways (e.g., PKC d) could be helpful to improve nano-carrier-based chemotherapy. No conflict of interest. 762 ADNP is a repressor of WNT signaling in colon cancer that can be therapeutically induced 2 , ¨ C. Blaj1 , A. Bringmann1 , M. Urbischek1 , S. Krebs2 , H. Blum2 , T. Frohlich G. Arnold2 , A. Jung1 , T. Kirchner1 , D. Horst1 . 1 Ludwig Maximilian Universtity, Pathologisches Institut, Munchen, ¨ Germany, 2 Ludwig Maximilian University, Gene Center, Munchen, ¨ Germany
Introduction: Overactivation of WNT signaling is a hallmark of human colon cancer. Considerable efforts have been made to develop therapeutic approaches that target WNT signaling but major hurdles in clinical applicability persist, since this pathway is required for various physiological processes including adult tissue and stem cell homeostasis. Here, we identified the transcription factor ADNP as a repressor of WNT signaling in colon cancer that can be induced pharmacologically by ketamine treatment. Material and Methods: We used transcriptomic and proteomic analyses to characterize ADNP expression, and silenced or overexpressed ADNP to determine its function in colon cancer cells. Furthermore, we manipulated ADNP by knockdown or ketamine induction in mouse xenografts of colon cancer cell lines and primary colon cancer cells to assess its function in vivo. Finally, we investigated on clinical relevance of ADNP expression in a cohort of 221 human colorectal cancer cases. Results: ADNP was overexpressed in colon cancer cells with high WNT activity, where it acted as a WNT repressor. Colon cancer cells with silenced ADNP showed enhanced migration, invasion and proliferation in vitro and accelerated tumor growth in xenografts in vivo. Treatment with sub-narcotic doses of ketamine, a known inducer of ADNP, significantly inhibited tumor growth and prolonged survival of tumor bearing animals. In human colon cancer patients, high ADNP expression emerged as a robust marker of good prognosis. Conclusion: This study indicates ADNP as a tumor suppressor and promising prognostic marker, and ketamine treatment with ADNP induction as a potential therapeutic approach that may improve current treatment protocols for colorectal cancer patients. No conflict of interest. 763 Thromboxane-mediated protein kinase C-related kinase (PRK) signalling: implications for thromboxane- and androgen-dependent neoplastic responses in prostate cancer A. O’Sullivan1 , E. Mulvaney1 , T. Kinsella1 . 1 University College Dublin, Conway Institute of Biomolecular & Biomedical Research, Dublin, Ireland Introduction: There is increasing evidence supporting the role of thromboxane (TX) A2 in neoplastic progression, not least due to findings that long-term
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
data show that these mutations require a context of basal G protein-coupled receptor activity in order promote invasion. No conflict of interest. 759 Oncogenic reciprocal signalling C. Tape1 . 1 The Institute of Cancer Research, Cancer Biology, London, United Kingdom In a heterocellular cancer, tumor cell oncogenes drive aberrant signaling both within tumor cells (cell-autonomous signaling) and adjacent stromal cells (non-cell-autonomous signaling). As different cell-types process signals via distinct pathways, heterocellular systems (containing different cell-types) theoretically provide increased signal processing capacity over homocellular systems (containing a single cell-type). We hypothesized that the expanded signaling capacity provided by stromal heterocellularity allows oncogenes to establish a differential reciprocal signaling state in tumor cells. Here we show that oncogenic KRAS (KRAS-G12D) uniquely regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS-G12D signaling in Pancreatic Ductal Adenocarcinoma (PDA). Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS-G12D. Consequently, reciprocal KRAS-G12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS-G12D alone. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. No conflict of interest. 760 A single cell approach to understanding cell cycle entry in cancer A. Barr1 , F.S. Heldt2 , S. Cooper1 , B. Novak2 , C. Bakal1 . 1 Institute of Cancer Research, Division of Cancer Biology, London, United Kingdom, 2 University of Oxford, Department of Biochemistry, Oxford, United Kingdom Background: Signalling networks controlling the decision to proliferate or arrest are perturbed in cancer. Network perturbation leads to changes in signalling dynamics, however currently we do not understand how these dynamics are changed, or what affect these changes have on proliferation. In order to specifically inhibit cancer cell proliferation, we need to know which signalling pathways are driving cancer cells into S-phase. Much of our knowledge regarding the decision to arrest or proliferate comes from population-based studies, which mask the true signalling dynamics that determine these decisions. Therefore, we need to use single cell technologies to study signalling dynamics during cell cycle entry. Materials and Methods: To understand the regulation of cell fate decisions during the cell cycle, we use quantitative single cell imaging in tandem with mathematical modelling. We have tagged key regulators of the G1/S transition at their endogenous loci in cancer and non-cancer cell lines, in order to monitor protein expression and localisation over multiple cell cycles. In combination with automated image analysis, we can extract quantitative information describing protein dynamics at each cell cycle transition for hundreds of cells. The quantitative data is then used to parametrise mathematical models of cell cycle entry. Results: We have used these methods to understand the regulation of S-phase commitment in both cancer and non-cancer cells. In non-transformed cells, there is a high level of heterogeneity in S-phase entry timing. We find that this heterogeneity is partly dependent on a high degree of cell-to-cell variability in the expression of the cyclin-dependent kinase inhibitor, p21. Variability in p21 expression is driven by p53 and our data suggest that DNA replication stress can trigger expression of p21, through activation of the DNA damage response. p21 expression then maintains cells in G1 until the damage is repaired. In cancer cells lacking p53, and in non-transformed cells where p53 has been depleted, S-phase entry is much more homogeneous in timing. Conclusions: Non-genetic heterogeneity in cell cycle timing could be an important determinant of “fractional kill” in tumour cells. We are now investigating how heterogeneity in p21 expression, both within and between cancer cell lines, affects response to chemotherapy, and thus how we could drive cancer cells towards apoptosis. No conflict of interest. 761 The role of intracellular Ca2+ concentration for macromolecular drug carrier uptake into tumor cells in an acidotic tumor environment 1 , O. Thews2 . 1 Clinic of Nuclear Medicine, University HospitalD. Gundel ¨ Martin-Luther-University Halle-Wittenberg, Halle Saale, Germany, 2 Julius Bernstein Institute of Physiology, Medical Faculty- Martin-Luther-University Halle-Wittenberg, Halle Saale, Germany Background: In the last decade macromolecular drug carriers became of scientific and clinical interest. Due to the abnormal tumor vasculature, leading
to an enhanced permeation and retention (EPR) effect, macromolecules can be used for passive drug accumulation in solid tumors. After these molecules reached the tumor tissue, the nano-carriers have to be taken up into the cells via endocytosis and the cytotoxic drugs are cleaved intracellularly. Because of pronounced glycolytic activity and weak drainage of metabolic end products the tumor tissue often becomes acidotic. The aim of the study was to analyze whether this extracellular acidosis affects the intracellular Ca2+-signaling and by this the endocytotic pathway for taking up macromolecules. Methods: Cellular uptake was determined in AT-1 R-3327 prostate and Walker-256 mammary carcinoma cells of the rat. Intracellular [Ca2+] and pH were determined ratiometrically with fluorescence microscopy in superfused single cells. Uptake studies were performed with pH-stable Oregon Green 488 labeled 70 kDa dextran which was quantified by flow cytometry. Ca2+ dependent uptake studies were determined either with 1.2 mM CaCl2 containing or with almost Ca2+-free buffers. Typical endocytotic pathways were selectively inhibited by rottlerin, dynasore, chlorpromazine and nystatin. Results: Cellular uptake of dextran (micropinocytosis) varied with pH. In AT-1 cells the uptake increased with rising pH, but in Walker-256 cells a negative correlation was found. Also the inhibition of PKC d, an important regulator for micropinocytosis, with rottlerin led to similar effects. On the other hand, an inhibition of dynamin dependent endocytosis diminished uptake of the dextran in both cell lines. Measuring the intracellular [Ca2+] and pH during acidification of the extracellular space indicated that the intracellular [Ca2+] was rapidly decreasing in both cell lines by 20−40%. However, in AT-1 cells the Ca2+ level was spontaneously increasing within several minutes at low extracellular pH. Under low intracellular Ca2+ conditions, dextran uptake was independent from the extracellular pH in both cell lines. Conclusion: The present study demonstrates that the endocytotic uptake of macromolecules depends on the extracellular pH most probably due to a Ca2+-mediated mechanism. However, the consequence of low extracellular pH, which is a common feature of solid growing tumors, for the uptake of macromolecules and therefore for the cytotoxicity of these drugs seems to be cell line-specific. Acidosis was able to foster, but also to limit the endocytotic uptake of large molecules. Modulating the metabolic extracellular environment but also the relevant signaling pathways (e.g., PKC d) could be helpful to improve nano-carrier-based chemotherapy. No conflict of interest. 762 ADNP is a repressor of WNT signaling in colon cancer that can be therapeutically induced 2 , ¨ C. Blaj1 , A. Bringmann1 , M. Urbischek1 , S. Krebs2 , H. Blum2 , T. Frohlich G. Arnold2 , A. Jung1 , T. Kirchner1 , D. Horst1 . 1 Ludwig Maximilian Universtity, Pathologisches Institut, Munchen, ¨ Germany, 2 Ludwig Maximilian University, Gene Center, Munchen, ¨ Germany
Introduction: Overactivation of WNT signaling is a hallmark of human colon cancer. Considerable efforts have been made to develop therapeutic approaches that target WNT signaling but major hurdles in clinical applicability persist, since this pathway is required for various physiological processes including adult tissue and stem cell homeostasis. Here, we identified the transcription factor ADNP as a repressor of WNT signaling in colon cancer that can be induced pharmacologically by ketamine treatment. Material and Methods: We used transcriptomic and proteomic analyses to characterize ADNP expression, and silenced or overexpressed ADNP to determine its function in colon cancer cells. Furthermore, we manipulated ADNP by knockdown or ketamine induction in mouse xenografts of colon cancer cell lines and primary colon cancer cells to assess its function in vivo. Finally, we investigated on clinical relevance of ADNP expression in a cohort of 221 human colorectal cancer cases. Results: ADNP was overexpressed in colon cancer cells with high WNT activity, where it acted as a WNT repressor. Colon cancer cells with silenced ADNP showed enhanced migration, invasion and proliferation in vitro and accelerated tumor growth in xenografts in vivo. Treatment with sub-narcotic doses of ketamine, a known inducer of ADNP, significantly inhibited tumor growth and prolonged survival of tumor bearing animals. In human colon cancer patients, high ADNP expression emerged as a robust marker of good prognosis. Conclusion: This study indicates ADNP as a tumor suppressor and promising prognostic marker, and ketamine treatment with ADNP induction as a potential therapeutic approach that may improve current treatment protocols for colorectal cancer patients. No conflict of interest. 763 Thromboxane-mediated protein kinase C-related kinase (PRK) signalling: implications for thromboxane- and androgen-dependent neoplastic responses in prostate cancer A. O’Sullivan1 , E. Mulvaney1 , T. Kinsella1 . 1 University College Dublin, Conway Institute of Biomolecular & Biomedical Research, Dublin, Ireland Introduction: There is increasing evidence supporting the role of thromboxane (TX) A2 in neoplastic progression, not least due to findings that long-term