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Polo-like kinase 4 (Plk4) promotes cancer cell invasion
S128
Surgical Forum Abstracts
INTRODUCTION: We have previously shown that the immune system contributes to the antitumor effects of imatinib in GIST. T cell exhaustion is an important barrier to antitumor immunity. PD-1 (programmed cell death-1) and TIM-3 (T cell immunoglobulin mucin 3) are markers of exhausted T cells and clinical agents are now available that target both receptors. METHODS: We characterized the activation and exhaustion status of T cells in human and murine GISTs using flow cytometry. In human GISTs, intratumoral T cells were compared with matched peripheral blood lymphocytes, and exhaustion markers were correlated with treatment response to imatinib. In GIST mice, we analyzed lymphocytes from the tumor, draining lymph node, and spleen. RESULTS: In untreated human GISTs (n¼11), intratumoral CD4 T cells had higher TIM-3 and PD-1 expression than peripheral blood (p<0.05). In human GISTs that had responded to imatinib (n¼9), CD4 T cells in the tumor had increased PD-1 expression compared with peripheral blood (p<0.05). In murine GISTs, TIM-3 staining on CD8 T cells was low in the tumor, draining lymph node, and spleen. However, PD-1 was upregulated 20-fold on intratumoral CD8 T cells compared to nodal and splenic CD8 T cells (11.8% vs 0.5% and 0.3%, p<0.005). CONCLUSIONS: Intratumoral T cells in both murine and human GISTs show signs of exhaustion by upregulated expression of PD-1 and TIM-3. T cell-targeted immunotherapy may therefore improve the effects of imatinib in GIST. Extracellular superoxide dismutase reduces pancreatic cancer invasion and metastasis Brianne O’Leary, PhD, Jennifer E Hrabe, MD, Michael D Henry, PhD, Frederick E Domann, PhD, James J Mezhir, MD, FACS University of Iowa Hospitals and Clinics, Iowa City, IA INTRODUCTION: Pancreatic cancer cells are known to produce excessive reactive oxygen species (ROS), particularly superoxide radical, which degrades the extracellular matrix (ECM) to facilitate invasion and metastasis. Extracellular superoxide dismutase (EcSOD) is a superoxide scavenger that protects the ECM from superoxide degradation, and we have previously shown that its expression is lost in pancreatic cancer specimens. Our objective is to evaluate the potential for EcSOD overexpression as a novel therapy for pancreatic cancer. METHODS: EcSOD was constitutively overexpressed in a primary pancreatic cancer cell line (BxPC3). Superoxide radical production was quantified using electron paramagnetic resonance (EPR), ECM integrity with immunoreactivity to intact heparan sulfate, tumor cell growth in vitro, and Matrigel invasion (all in triplicate). Cells were then administered via IP injection and peritoneal metastasis quantified using bioluminescence imaging. RESULTS: Constitutive EcSOD expression resulted in an 80% reduction in superoxide production compared to controls by
J Am Coll Surg
EPR spectra (p<0.001). EcSOD overexpressing cell lines had an decrease in ECM degradation, shorter doubling time (34.4h versus 24.0h, p¼0.02) and a greater than 40% decrease in invasive capacity on Matrigel (p¼0.008). Following IP injection, EcSOD overexpressing cells had a significant decrease in peritoneal metastasis evidenced by lower photon emission at 28d post-injection (6.45x107 vs 6.72x108 photons/cell/second, p¼0.003). CONCLUSIONS: Overexpression of EcSOD resulted in reduced superoxide radical production which correlated with reduced pancreatic cancer cell ECM degradation, growth, invasion, and peritoneal metastasis. The manipulation of the redox balance with EcSOD may provide new insights into tumor biology and act as a potential novel therapy for pancreatic cancer.
Polo-like kinase 4 (Plk4) promotes cancer cell invasion Karineh Kazazian, MD, Roland Xu, Francis Zih, MD, MS, Carla Rosario, PhD, Carol J Swallow, MD, PhD, FRCSC, FACS University of Toronto, Toronto, Ontario, Canada INTRODUCTION: High expression of Plk4 has been identified as a molecular predictor of aggressive behavior and resistance to therapy in breast, pancreas and colorectal cancers. We hypothesize that Plk4 facilitates cancer progression by increasing cancer cell invasion; here we test this by manipulating Plk4 levels in cancer cells. METHODS: HeLa cells were transfected with Flag-Plk4 or Plk4 siRNA. Cell phenotype was assessed at 48h using indirect immunofluorescence. Transfected cells were plated onto polycarbonate filters allowing for pseudopodial protrusion through filter pores; Isolated protrusion fraction protein levels were assessed using the BCA assay. Real-time wound-healing migration assays were performed on confluent cells transfected with Plk4 or Luciferase siRNA; Images were obtained every 4h. Cell invasion through Matrigel was assessed using a transwell invasion assay employing the RTCA system. RESULTS: An arborized spreading phenotype with increased number and length of filopodia was noted in Flag-Plk4 transfected cells, while Plk4 knockdown caused a rounded morphology compared with Luciferase control (p<0.05). Cell protrusions were isolated, and the purity of this fraction was confirmed by Western blot, probing for Hsp-70. Protrusion protein levels were decreased in Plk4 siRNA treated cells (p<0.05), and wound healing was delayed at 4h, 8h and 12h with Plk4 knockdown (p<0.04) compared to control. Plk4 siRNA treated HeLa cells also showed decreased invasion through Matrigel in the RTCA system. CONCLUSIONS: Plk4 promotes HeLa cancer cell invasion by enhancing protrusion formation and directional migration. This is one mechanism through which Plk4 can facilitate metastatic spread, and represents an attractive and novel target for kinase inhibitor therapy.
Surgical Forum Abstracts
INTRODUCTION: We have previously shown that the immune system contributes to the antitumor effects of imatinib in GIST. T cell exhaustion is an important barrier to antitumor immunity. PD-1 (programmed cell death-1) and TIM-3 (T cell immunoglobulin mucin 3) are markers of exhausted T cells and clinical agents are now available that target both receptors. METHODS: We characterized the activation and exhaustion status of T cells in human and murine GISTs using flow cytometry. In human GISTs, intratumoral T cells were compared with matched peripheral blood lymphocytes, and exhaustion markers were correlated with treatment response to imatinib. In GIST mice, we analyzed lymphocytes from the tumor, draining lymph node, and spleen. RESULTS: In untreated human GISTs (n¼11), intratumoral CD4 T cells had higher TIM-3 and PD-1 expression than peripheral blood (p<0.05). In human GISTs that had responded to imatinib (n¼9), CD4 T cells in the tumor had increased PD-1 expression compared with peripheral blood (p<0.05). In murine GISTs, TIM-3 staining on CD8 T cells was low in the tumor, draining lymph node, and spleen. However, PD-1 was upregulated 20-fold on intratumoral CD8 T cells compared to nodal and splenic CD8 T cells (11.8% vs 0.5% and 0.3%, p<0.005). CONCLUSIONS: Intratumoral T cells in both murine and human GISTs show signs of exhaustion by upregulated expression of PD-1 and TIM-3. T cell-targeted immunotherapy may therefore improve the effects of imatinib in GIST. Extracellular superoxide dismutase reduces pancreatic cancer invasion and metastasis Brianne O’Leary, PhD, Jennifer E Hrabe, MD, Michael D Henry, PhD, Frederick E Domann, PhD, James J Mezhir, MD, FACS University of Iowa Hospitals and Clinics, Iowa City, IA INTRODUCTION: Pancreatic cancer cells are known to produce excessive reactive oxygen species (ROS), particularly superoxide radical, which degrades the extracellular matrix (ECM) to facilitate invasion and metastasis. Extracellular superoxide dismutase (EcSOD) is a superoxide scavenger that protects the ECM from superoxide degradation, and we have previously shown that its expression is lost in pancreatic cancer specimens. Our objective is to evaluate the potential for EcSOD overexpression as a novel therapy for pancreatic cancer. METHODS: EcSOD was constitutively overexpressed in a primary pancreatic cancer cell line (BxPC3). Superoxide radical production was quantified using electron paramagnetic resonance (EPR), ECM integrity with immunoreactivity to intact heparan sulfate, tumor cell growth in vitro, and Matrigel invasion (all in triplicate). Cells were then administered via IP injection and peritoneal metastasis quantified using bioluminescence imaging. RESULTS: Constitutive EcSOD expression resulted in an 80% reduction in superoxide production compared to controls by
J Am Coll Surg
EPR spectra (p<0.001). EcSOD overexpressing cell lines had an decrease in ECM degradation, shorter doubling time (34.4h versus 24.0h, p¼0.02) and a greater than 40% decrease in invasive capacity on Matrigel (p¼0.008). Following IP injection, EcSOD overexpressing cells had a significant decrease in peritoneal metastasis evidenced by lower photon emission at 28d post-injection (6.45x107 vs 6.72x108 photons/cell/second, p¼0.003). CONCLUSIONS: Overexpression of EcSOD resulted in reduced superoxide radical production which correlated with reduced pancreatic cancer cell ECM degradation, growth, invasion, and peritoneal metastasis. The manipulation of the redox balance with EcSOD may provide new insights into tumor biology and act as a potential novel therapy for pancreatic cancer.
Polo-like kinase 4 (Plk4) promotes cancer cell invasion Karineh Kazazian, MD, Roland Xu, Francis Zih, MD, MS, Carla Rosario, PhD, Carol J Swallow, MD, PhD, FRCSC, FACS University of Toronto, Toronto, Ontario, Canada INTRODUCTION: High expression of Plk4 has been identified as a molecular predictor of aggressive behavior and resistance to therapy in breast, pancreas and colorectal cancers. We hypothesize that Plk4 facilitates cancer progression by increasing cancer cell invasion; here we test this by manipulating Plk4 levels in cancer cells. METHODS: HeLa cells were transfected with Flag-Plk4 or Plk4 siRNA. Cell phenotype was assessed at 48h using indirect immunofluorescence. Transfected cells were plated onto polycarbonate filters allowing for pseudopodial protrusion through filter pores; Isolated protrusion fraction protein levels were assessed using the BCA assay. Real-time wound-healing migration assays were performed on confluent cells transfected with Plk4 or Luciferase siRNA; Images were obtained every 4h. Cell invasion through Matrigel was assessed using a transwell invasion assay employing the RTCA system. RESULTS: An arborized spreading phenotype with increased number and length of filopodia was noted in Flag-Plk4 transfected cells, while Plk4 knockdown caused a rounded morphology compared with Luciferase control (p<0.05). Cell protrusions were isolated, and the purity of this fraction was confirmed by Western blot, probing for Hsp-70. Protrusion protein levels were decreased in Plk4 siRNA treated cells (p<0.05), and wound healing was delayed at 4h, 8h and 12h with Plk4 knockdown (p<0.04) compared to control. Plk4 siRNA treated HeLa cells also showed decreased invasion through Matrigel in the RTCA system. CONCLUSIONS: Plk4 promotes HeLa cancer cell invasion by enhancing protrusion formation and directional migration. This is one mechanism through which Plk4 can facilitate metastatic spread, and represents an attractive and novel target for kinase inhibitor therapy.