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Polo-like Kinase 4 (Plk4) enhances cancer cell motility via RhoGTPase activation
Vol. 215, No. 3S, September 2012
Abstracts S129
INTRODUCTION: Venous thromboembolism (VTE) rates vary by tumor type, and pancreatic cancer is associated with the highest risk. Virchow’s triad describes three factors contributing to Vte: hypercoagulability, stasis, and endothelial injury. Rotational thromboelastography (ROTEM) is a new point of care test that allows rapid quantification of coagulation status, for which there is limited information in cancer. We used ROTEM to compare preoperative clot kinetics/strength in patients undergoing surgery for malignant disease. METHODS: After informed consent, preoperative blood samples were analyzed with ROTEM. Data are mean⫾SD or median (IQR). ANOVA and chi-squared assessed significance at p⬍0.05. RESULTS: Seventy-two patients aged 65⫾15 yrs and 63% male were enrolled. The most common cancers involved the esophagus (n⫽19, 26%), pancreas (n⫽21, 29%), liver (n⫽13, 18%), stomach (n⫽7, 10%), and bile ducts (n⫽6, 8%). Preoperative hypercoagulability was detected in 31% (n⫽22), and these patients were more likely to have lymphovascular invasion (88% vs. 50%, p⫽0.011), perineural invasion (77% vs. 36%, p⫽0.007), and stage III/IV disease (80% vs. 61%, p⫽0.039). More patients with pancreatic tumors (5/21, 24%) were hypercoagulable than with liver (1/13, 8%) or esophageal (0/19), p⫽0.05. When only resectable malignancies were considered, clot formation was more rapid (low CFT, high alpha) with enhanced maximum clot strength (high MCF) in pancreatic vs. esophageal or liver cancers. CONCLUSIONS: Preoperative hypercoagulability can be identified with ROTEM and is associated with lymphovascular/perineural invasion and advanced-staged disease in cancer. Compared to other tumor types, pancreatic adenocarcinomas have the greatest risk for hypercoagulability, which might explain increased VTE rates in this population. Pancreatic (nⴝ18)
Esophageal (nⴝ17)
Liver (nⴝ8)
Intrinsic Pathway 59 ⫾ 15*† 77 ⫾ 19† 8 0⫾ 14* 64 ⫾ 6ⴱⴱ 60 ⫾ 4* 58 ⫾ 4* 78 ⫾ 3ⴱⴱ 76 ⫾ 3* 75 ⫾ 2* Extrinsic Pathway CFT, sec 69 ⫾ 20 81 ⫾ 21 90 ⫾ 21 MCF, mm 68 ⫾ 6* 64 ⫾ 4 62 ⫾ 5* Alpha, degrees 77 ⫾ 4 75 ⫾ 4 73 ⫾ 6 Final Common Pathway (Fibrin) MCF, mm 21 (7)ⴱⴱ 18 (6)* 16 (9)* CFT, sec MCF, mm Alpha, degrees
pⴝ
0.003 0.005 0.007 0.031 0.050 0.076 0.017
Post-hoc Bonferoni test. *p⬍0.05; † p⬍0.01; Data are mean⫾SD or median (IQR).
RON overexpression drives macrophage differentiation toward an alternatively activated (M2) phenotype in a mouse model of spontaneous pancreatic carcinogenesis Michele L Babicky, MD, Megan M Harper, BSc(Hons), Evangeline Mose, BSc(Hons), Dawn Jaquish, BSc(Hons), Randy French, PhD, Andrew M Lowy, MD, FACS University of California, San Diego, San Diego, CA
INTRODUCTION: The RON receptor is frequently overexpressed in pancreatic cancer. We developed a mouse model of spontaneous pancreatic carcinogenesis driven by RON overexpression and oncogenic KRAS (RCK). RCK mice demonstrate accelerated PanIN progression to invasive cancer marked by desmoplasia that is highly characteristic of human disease. RON signaling in macrophages induces differentiation towards the alternatively activated (M2) phenotype, which promotes tumorigenesis. We hypothesized that pancreatic RON overexpression in RCK mice may promote changes in the tumor microenvironment, specifically by driving the differentiation of infiltrating macrophages towards the M2 phenotype. METHODS: Pancreatic tissues from RCK and CK controls (3-12 months) were analyzed via immunofluorescent (IMF) staining to quantify myeloid infiltration. Single cell suspensions were generated from pancreatic and splenic tissues. CD11b⫹F4/80⫹ myeloid cell populations, as well as CD11c⫹ M1 and MRC1⫹ M2 subpopulations, were analyzed via FACS. RESULTS: RCK pancreata demonstrated increased CD11b⫹F4/ 80⫹ myeloid infiltration at 6-12 months. In RCK, pancreatic myeloid cells were skewed toward the M2 phenotype compared with CK controls (MRC1⫹ 39% vs 10% p⬍0.05, respectively). The expression profile of myeloid cells from the spleens was unchanged in CK vs. RCK (4-15% CD11c⫹, ⬍1% MRC1⫹). There was no change in the myeloid derived suppressor cell (MDSC) populations (CD11b⫹Gr1⫹) in the spleen or pancreas of these animals. CONCLUSIONS: Pancreatic RON overexpression shifts infiltrating myeloid differentiation toward the M2 phenotype in this murine model. This data reveals a direct connection between aberrant RON signaling and the tumor permissive inflammatory infiltrate present within the pancreatic cancer microenvironment.
Polo-like Kinase 4 (Plk4) enhances cancer cell motility via RhoGTPase activation Francis Si Wai Zih, MD, MS, Carla O Rosario, PhD, Olga Brashavitskaya, BSc(Hons), Karineh Kazazian, MD, BSc(Hons), Carol J Swallow, MD, PhD, FACS, FRCS Mount Sinai Hospital, Toronto, ON INTRODUCTION: Plk4 is one of a small suite of death-from-cancer signature genes that predict treatment failure in metastatic cancer. mRNA arrays showed altered motility gene expression in Plk4ⴞ vs. Plk4⫹/⫹ murine embryonic fibroblasts (MEFs), with functional validation in wound healing and spreading assays. Plk4 is essential for cytokinesis, a function mediated through activation of GTPase RhoA. Based on these findings, we hypothesized that Plk4 increases cancer cell motility in a RhoGTPase-dependent manner. METHODS: Invasion assays were performed using Boyden chamber and Real-time Cell Analyzer (RTCA) System. Plk4 siRNA treated cells were compared to buffer and scrambled siRNA controls. DLD-1 colorectal cancer cells were stably transfected to express mCherry-Plk4 in response to tetracycline. Activated RhoA and RhoGEF Ect2 were detected in spreading cells by indirect immunofluorescence.
S130
Abstracts
RESULTS: Invasion was significantly reduced in Plk4⫾compared to Plk4⫹/⫹MEFs (p⬍0.05). Depletion of Plk4 using siRNA impaired invasion by MEFs and HeLa cervical cancer cells. Tetinduced expression of Plk4 enhanced invasion in a dosedependent manner in DLD-1 clones. Transfection of HeLa cells with Flag-Plk4 resulted in markedly elongated protrusions. Activated RhoA and Ect2 were identified in protrusions of motile cells, with a temporal pattern that coincided with localization of active Plk4. Levels of activated RhoA and Rac1 were reduced in cells with haploid levels of Plk4. CONCLUSIONS: Plk4 enhances invasion in cancer cells, with evidence of RhoGTPase dependence. Intelligent targeting of Plk4 as a cancer therapy will require this better understanding of how it regulates invasion, and thereby cancer progression.
Inhibition of dendritic cells fatty acid synthesis enhance their immune-stimulatory potential Adeel Rehman, MD, Keith C Hemmert, BA, Justin R Henning, MD, Constantinos P Zambirinis, MD, Shahzad Rahim, MD, Mohsin Jamal, MD, Rocky M Barilla, BSc(Hons), Nina Fallon, BA, Christopher S Graffeo, BA, George Miller, MD, FACS New York University School of Medicine, New York City, NY INTRODUCTION: Dendritic cells (DC) are professional antigen presenting cells (APC) that hold significant promise for the development of cancer vaccines. Endoplasmic reticular (ER)-stress can enhance the immunogenicity of APC. Since blockade of fatty acid synthesis increases ER-stress, we postulated that inhibition of fatty acid synthesis in DC would enhance their immune-stimulatory properties. METHODS: Human moDC were generated from PMBC by culture in GM-CSF/IL-4. Murine DC were generated from bone marrow using GM-CSF. TOFA--an acetyl-CoA carboxylase inhibitor--was used to block fatty acid synthesis. RESULTS: Blockade of fatty acid synthesis decreased DC development from precursors and mitigated DC proliferation in mice and humans. The mechanism was related to downregulation of Cyclin B-1—a pro-proliferative gene—and upregulation of Caspase 3 and PARP—apoptosis-related genes. Blockade of fatty acid synthesis enhanced DC activation and capacity for stimulation of immune effecter cells. TOFA-DC expressed elevated activated MAP-K, NF-kB, and PI3K/Akt intermediates and produced higher cytokines (Table). Peptide-pulsed TOFA-DC induced more vigorous proliferation of antigen-restricted CD4⫹ and CD8⫹ T cells, induced T cell IFN-g and TNF-a production, and enhanced CTL in vivo (Table). TOFA-DC induced higher NK expression of CD25 and production of IFN-g (Table). Accordingly, we found increased capacity for Notch signaling in TOFA-DC. TOFA-treated DC exhibited high ER-stress including increased expression of GRP-78, eIF2-a, p-eIF2-a and XBP-1, and blockade of ER-stress by a chaperone protein mitigated their enhanced immune-stimulation.
J Am Coll Surg
DC cytokines
DC IL-6 (pg/mL) DC MCP1 (pg/mL) T cell CD4 Proliferation stimulation CPM (⫻103) CD4 TNF-␣ (pg/ mL) CD8 Proliferation CPM (⫻103) CD8 TNF-␣ (pg/ mL) IFN-␥ CTL (pg/mL) NK stimulation IFN-␥ (pg/mL) CD25 (pg/mL)
p Value
DC
TOFA-DC
30 597 38
79 2315 90
⬍0.01 ⬍0.001 ⬍0.001
441
715
⬍0.0001
76
200
⬍0.01
10
38
⬍0.01
338 100 65
512 378 146
⬍0.01 ⬍0.0001 ⬍0.0001
CONCLUSIONS: Blockade of fatty acid synthesis markedly increases DC immunogenicity and capacity to induce CTL by augmenting ER stress. Our findings have significant implications for the design of immunotherapy vaccines. Ascorbate-induced autophagy: Mode of cell death or mechanism for survival? Jessemae L Welsh, MD, Juan Du, PhD, Zita Sibenaller, PhD, Joseph J Cullen, MD, FACS University of Iowa, Iowa City, IA INTRODUCTION: Pharmacological ascorbate induces autophagy in pancreatic cancer (Du et al. 2010), which is characterized by accumulation of autophagosomes and can be detected by the processing of LC3 to the lipidated form referred to as LC3-II. LC3-II is also upregulated in pancreatic cancer cells after chemoradiation (Mukubou et al. 2010). The aim of our study was to determine the role of autophagy in ascorbate-induced cytotoxicity. METHODS: Pancreatic cancer cells that stably overexpress LC3 protein linked to GFP were generated (MiaPaca-LC3-GFP). In addition, we used prostate cancer cells (PC-3E) which express low basal levels of autophagy; an isogeneic cell line derived from PC-3E with high basal autophagy after induction of epithelial to mesenchymal transition (TEM418VC); and the same cell line with short hairpin knockdown of autophagy protein 5 (ATG5), which is necessary for autophagosome formation, (TEM418shATG5) with the knockdown leading to diminished basal autophagy. Cells were treated with 5 mM ascorbate and Western blots and clonogenic survival were determined. RESULTS: Overexpression of LC3 induced resistance to ascorbateinduced cytotoxicity and demonstrated a higher LC3-II/LC3-I ratio at 6 and 24 hours after ascorbate treatment. Ascorbate-induced cytotoxicity was similar in cells that have low and high basal levels of autophagy. However, cancer cells with ATG5 knockdown showed increased sensitivity to ascorbate compared to the parental cell line. CONCLUSIONS: These data suggest autophagy is an important mechanism for cell survival after ascorbate treatment. Modulation of the autophagy pathway may be a mechanism to enhance ascorbate-
Abstracts S129
INTRODUCTION: Venous thromboembolism (VTE) rates vary by tumor type, and pancreatic cancer is associated with the highest risk. Virchow’s triad describes three factors contributing to Vte: hypercoagulability, stasis, and endothelial injury. Rotational thromboelastography (ROTEM) is a new point of care test that allows rapid quantification of coagulation status, for which there is limited information in cancer. We used ROTEM to compare preoperative clot kinetics/strength in patients undergoing surgery for malignant disease. METHODS: After informed consent, preoperative blood samples were analyzed with ROTEM. Data are mean⫾SD or median (IQR). ANOVA and chi-squared assessed significance at p⬍0.05. RESULTS: Seventy-two patients aged 65⫾15 yrs and 63% male were enrolled. The most common cancers involved the esophagus (n⫽19, 26%), pancreas (n⫽21, 29%), liver (n⫽13, 18%), stomach (n⫽7, 10%), and bile ducts (n⫽6, 8%). Preoperative hypercoagulability was detected in 31% (n⫽22), and these patients were more likely to have lymphovascular invasion (88% vs. 50%, p⫽0.011), perineural invasion (77% vs. 36%, p⫽0.007), and stage III/IV disease (80% vs. 61%, p⫽0.039). More patients with pancreatic tumors (5/21, 24%) were hypercoagulable than with liver (1/13, 8%) or esophageal (0/19), p⫽0.05. When only resectable malignancies were considered, clot formation was more rapid (low CFT, high alpha) with enhanced maximum clot strength (high MCF) in pancreatic vs. esophageal or liver cancers. CONCLUSIONS: Preoperative hypercoagulability can be identified with ROTEM and is associated with lymphovascular/perineural invasion and advanced-staged disease in cancer. Compared to other tumor types, pancreatic adenocarcinomas have the greatest risk for hypercoagulability, which might explain increased VTE rates in this population. Pancreatic (nⴝ18)
Esophageal (nⴝ17)
Liver (nⴝ8)
Intrinsic Pathway 59 ⫾ 15*† 77 ⫾ 19† 8 0⫾ 14* 64 ⫾ 6ⴱⴱ 60 ⫾ 4* 58 ⫾ 4* 78 ⫾ 3ⴱⴱ 76 ⫾ 3* 75 ⫾ 2* Extrinsic Pathway CFT, sec 69 ⫾ 20 81 ⫾ 21 90 ⫾ 21 MCF, mm 68 ⫾ 6* 64 ⫾ 4 62 ⫾ 5* Alpha, degrees 77 ⫾ 4 75 ⫾ 4 73 ⫾ 6 Final Common Pathway (Fibrin) MCF, mm 21 (7)ⴱⴱ 18 (6)* 16 (9)* CFT, sec MCF, mm Alpha, degrees
pⴝ
0.003 0.005 0.007 0.031 0.050 0.076 0.017
Post-hoc Bonferoni test. *p⬍0.05; † p⬍0.01; Data are mean⫾SD or median (IQR).
RON overexpression drives macrophage differentiation toward an alternatively activated (M2) phenotype in a mouse model of spontaneous pancreatic carcinogenesis Michele L Babicky, MD, Megan M Harper, BSc(Hons), Evangeline Mose, BSc(Hons), Dawn Jaquish, BSc(Hons), Randy French, PhD, Andrew M Lowy, MD, FACS University of California, San Diego, San Diego, CA
INTRODUCTION: The RON receptor is frequently overexpressed in pancreatic cancer. We developed a mouse model of spontaneous pancreatic carcinogenesis driven by RON overexpression and oncogenic KRAS (RCK). RCK mice demonstrate accelerated PanIN progression to invasive cancer marked by desmoplasia that is highly characteristic of human disease. RON signaling in macrophages induces differentiation towards the alternatively activated (M2) phenotype, which promotes tumorigenesis. We hypothesized that pancreatic RON overexpression in RCK mice may promote changes in the tumor microenvironment, specifically by driving the differentiation of infiltrating macrophages towards the M2 phenotype. METHODS: Pancreatic tissues from RCK and CK controls (3-12 months) were analyzed via immunofluorescent (IMF) staining to quantify myeloid infiltration. Single cell suspensions were generated from pancreatic and splenic tissues. CD11b⫹F4/80⫹ myeloid cell populations, as well as CD11c⫹ M1 and MRC1⫹ M2 subpopulations, were analyzed via FACS. RESULTS: RCK pancreata demonstrated increased CD11b⫹F4/ 80⫹ myeloid infiltration at 6-12 months. In RCK, pancreatic myeloid cells were skewed toward the M2 phenotype compared with CK controls (MRC1⫹ 39% vs 10% p⬍0.05, respectively). The expression profile of myeloid cells from the spleens was unchanged in CK vs. RCK (4-15% CD11c⫹, ⬍1% MRC1⫹). There was no change in the myeloid derived suppressor cell (MDSC) populations (CD11b⫹Gr1⫹) in the spleen or pancreas of these animals. CONCLUSIONS: Pancreatic RON overexpression shifts infiltrating myeloid differentiation toward the M2 phenotype in this murine model. This data reveals a direct connection between aberrant RON signaling and the tumor permissive inflammatory infiltrate present within the pancreatic cancer microenvironment.
Polo-like Kinase 4 (Plk4) enhances cancer cell motility via RhoGTPase activation Francis Si Wai Zih, MD, MS, Carla O Rosario, PhD, Olga Brashavitskaya, BSc(Hons), Karineh Kazazian, MD, BSc(Hons), Carol J Swallow, MD, PhD, FACS, FRCS Mount Sinai Hospital, Toronto, ON INTRODUCTION: Plk4 is one of a small suite of death-from-cancer signature genes that predict treatment failure in metastatic cancer. mRNA arrays showed altered motility gene expression in Plk4ⴞ vs. Plk4⫹/⫹ murine embryonic fibroblasts (MEFs), with functional validation in wound healing and spreading assays. Plk4 is essential for cytokinesis, a function mediated through activation of GTPase RhoA. Based on these findings, we hypothesized that Plk4 increases cancer cell motility in a RhoGTPase-dependent manner. METHODS: Invasion assays were performed using Boyden chamber and Real-time Cell Analyzer (RTCA) System. Plk4 siRNA treated cells were compared to buffer and scrambled siRNA controls. DLD-1 colorectal cancer cells were stably transfected to express mCherry-Plk4 in response to tetracycline. Activated RhoA and RhoGEF Ect2 were detected in spreading cells by indirect immunofluorescence.
S130
Abstracts
RESULTS: Invasion was significantly reduced in Plk4⫾compared to Plk4⫹/⫹MEFs (p⬍0.05). Depletion of Plk4 using siRNA impaired invasion by MEFs and HeLa cervical cancer cells. Tetinduced expression of Plk4 enhanced invasion in a dosedependent manner in DLD-1 clones. Transfection of HeLa cells with Flag-Plk4 resulted in markedly elongated protrusions. Activated RhoA and Ect2 were identified in protrusions of motile cells, with a temporal pattern that coincided with localization of active Plk4. Levels of activated RhoA and Rac1 were reduced in cells with haploid levels of Plk4. CONCLUSIONS: Plk4 enhances invasion in cancer cells, with evidence of RhoGTPase dependence. Intelligent targeting of Plk4 as a cancer therapy will require this better understanding of how it regulates invasion, and thereby cancer progression.
Inhibition of dendritic cells fatty acid synthesis enhance their immune-stimulatory potential Adeel Rehman, MD, Keith C Hemmert, BA, Justin R Henning, MD, Constantinos P Zambirinis, MD, Shahzad Rahim, MD, Mohsin Jamal, MD, Rocky M Barilla, BSc(Hons), Nina Fallon, BA, Christopher S Graffeo, BA, George Miller, MD, FACS New York University School of Medicine, New York City, NY INTRODUCTION: Dendritic cells (DC) are professional antigen presenting cells (APC) that hold significant promise for the development of cancer vaccines. Endoplasmic reticular (ER)-stress can enhance the immunogenicity of APC. Since blockade of fatty acid synthesis increases ER-stress, we postulated that inhibition of fatty acid synthesis in DC would enhance their immune-stimulatory properties. METHODS: Human moDC were generated from PMBC by culture in GM-CSF/IL-4. Murine DC were generated from bone marrow using GM-CSF. TOFA--an acetyl-CoA carboxylase inhibitor--was used to block fatty acid synthesis. RESULTS: Blockade of fatty acid synthesis decreased DC development from precursors and mitigated DC proliferation in mice and humans. The mechanism was related to downregulation of Cyclin B-1—a pro-proliferative gene—and upregulation of Caspase 3 and PARP—apoptosis-related genes. Blockade of fatty acid synthesis enhanced DC activation and capacity for stimulation of immune effecter cells. TOFA-DC expressed elevated activated MAP-K, NF-kB, and PI3K/Akt intermediates and produced higher cytokines (Table). Peptide-pulsed TOFA-DC induced more vigorous proliferation of antigen-restricted CD4⫹ and CD8⫹ T cells, induced T cell IFN-g and TNF-a production, and enhanced CTL in vivo (Table). TOFA-DC induced higher NK expression of CD25 and production of IFN-g (Table). Accordingly, we found increased capacity for Notch signaling in TOFA-DC. TOFA-treated DC exhibited high ER-stress including increased expression of GRP-78, eIF2-a, p-eIF2-a and XBP-1, and blockade of ER-stress by a chaperone protein mitigated their enhanced immune-stimulation.
J Am Coll Surg
DC cytokines
DC IL-6 (pg/mL) DC MCP1 (pg/mL) T cell CD4 Proliferation stimulation CPM (⫻103) CD4 TNF-␣ (pg/ mL) CD8 Proliferation CPM (⫻103) CD8 TNF-␣ (pg/ mL) IFN-␥ CTL (pg/mL) NK stimulation IFN-␥ (pg/mL) CD25 (pg/mL)
p Value
DC
TOFA-DC
30 597 38
79 2315 90
⬍0.01 ⬍0.001 ⬍0.001
441
715
⬍0.0001
76
200
⬍0.01
10
38
⬍0.01
338 100 65
512 378 146
⬍0.01 ⬍0.0001 ⬍0.0001
CONCLUSIONS: Blockade of fatty acid synthesis markedly increases DC immunogenicity and capacity to induce CTL by augmenting ER stress. Our findings have significant implications for the design of immunotherapy vaccines. Ascorbate-induced autophagy: Mode of cell death or mechanism for survival? Jessemae L Welsh, MD, Juan Du, PhD, Zita Sibenaller, PhD, Joseph J Cullen, MD, FACS University of Iowa, Iowa City, IA INTRODUCTION: Pharmacological ascorbate induces autophagy in pancreatic cancer (Du et al. 2010), which is characterized by accumulation of autophagosomes and can be detected by the processing of LC3 to the lipidated form referred to as LC3-II. LC3-II is also upregulated in pancreatic cancer cells after chemoradiation (Mukubou et al. 2010). The aim of our study was to determine the role of autophagy in ascorbate-induced cytotoxicity. METHODS: Pancreatic cancer cells that stably overexpress LC3 protein linked to GFP were generated (MiaPaca-LC3-GFP). In addition, we used prostate cancer cells (PC-3E) which express low basal levels of autophagy; an isogeneic cell line derived from PC-3E with high basal autophagy after induction of epithelial to mesenchymal transition (TEM418VC); and the same cell line with short hairpin knockdown of autophagy protein 5 (ATG5), which is necessary for autophagosome formation, (TEM418shATG5) with the knockdown leading to diminished basal autophagy. Cells were treated with 5 mM ascorbate and Western blots and clonogenic survival were determined. RESULTS: Overexpression of LC3 induced resistance to ascorbateinduced cytotoxicity and demonstrated a higher LC3-II/LC3-I ratio at 6 and 24 hours after ascorbate treatment. Ascorbate-induced cytotoxicity was similar in cells that have low and high basal levels of autophagy. However, cancer cells with ATG5 knockdown showed increased sensitivity to ascorbate compared to the parental cell line. CONCLUSIONS: These data suggest autophagy is an important mechanism for cell survival after ascorbate treatment. Modulation of the autophagy pathway may be a mechanism to enhance ascorbate-