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114 Hypoxia-associated protein expression in prostate cancer
$36
March 12 - 15
P r o f f e r e d Papers
113 ~I-INTEGRIN OR INTEGRIN-LINKED KINASE OVEREXPRESSION PROMOTES RADIATION-INDUCED A P O P T O S I S I N HUMAN HL60 LEUKEMIA CELLS
F. Hess, D. Estrugo, A. Fischer, N. Cordes OncoRay - Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, 01307 Dresden, Germany; Bundeswehr Institute of Radiobiology, Neuherbergstrasse 11, 80937 Munich, Germany Objective: Integrin-mediated cell adhesion to extracellular matrix (ECM) proteins reduces apoptosis following radiationinduced genotoxic injury. This mechanism effectively hampers therapeutic eradication of malignant cells at the primary tumor site and at peripheral sites after metastasization. To evaluate the importance of this phenomenon in suspension cells as well as the role of ~l-integrin and integrin-linked kinase (ILK), HL60 human acute promyelocytic leukemia cells were examined on fibronectin (FN) or in suspension. Material and methods: Cells were stably transfected with 131-integrin or ILK wild-type or hyperactive overexpression vectors. Suspension (polystyrene, BSA) or FN adhesion cultures were exposed to inhibitory anti- ~l-integrin antibodies or stimulatory RGD peptides and irradiated (0 - 10 Gy). Apoptosis, mitochondrial transmembrane potential (MTP) and caspase activation were analyzed by DAPI staining, flow cytometry and Western blotting technique (caspase-3, -9, -8, poly(ADP-ribose) polymerase (PARP); pulldown assays). Results: Adhesion to FN significantly (P<0.01) reduced radiation-induced apoptosis in HL60 cells (2 Gy: N15%) relative to suspension (N35%) at 48 h. Intriguingly, overexpression of J31-integrin or ILK dramatically enhanced apoptosis (90-100%) in 2-Gy irradiated adhesion and suspension cells. Adhesion to FN suppressed MTP breakdown, caspase-9 and PARP cleavage but permitted strong activation of caspase-8 and -3 in irradiated pl-integrin transfectants. In contrast, suspension conditions allowed both caspase-8 and 9-mediated activation of caspase-3. ILK overexpression resulted in combined activation of caspase-3 through caspase8 and -9 in irradiated FN adhesion cultures. Irradiation of ILK suspension cultures lacked caspase-8 activation but showed serial cleavage of caspase-9, -3 and PARP. Pulldown assays demonstrated that ~l-integrin recruits caspase-8 under both suspension and FN adhesion condition for proapoptotic signaling in DNA-damaged HL60 cells. In contrast, ILK directly interacts with caspase-8 exceptionally in irradiated FN adhesion cultures. Conclusions: These findings confirm our view of ILK as strong anti-survival regulator in genotoxically injured cells. New insights on cell adhesion-mediated radiation resistance comprise that (i) ~l-integrins are potent promoters of radiation-induced apoptosis, (ii) 131-integrin and ILK show overlapping signaling for enhancement of apoptosis and (iii) ~l-integrins and ILK recruit death receptor pathway-related initiator caspase-8 after irradiation. Our data further demonstrate that both the ligated and the unligated integrin states provide information to the cell about its microenvironment. This critically determines the repertoire of proteins available to execute apoptosis after genotoxic injury. Further, these data might provide the basis for developing new targeted chemo- and radiotherapeutic treatment options.
114 HYPOXIA-ASSOCIATED PROSTATE CANCER.
PROTEIN
EXPRESSION
G.D. Wilson, A.D. Powell, M. Che, J.D. Forman Department of Radiation Oncology, Karmanos Institute, Wayne State University, Detroit, USA
IN
Cancer
Objective: The aim of this Study was to investigate the expression of hypoxia inducible factor-lalpha (HIF-lalpha), glucose transporter protein-1 (GLUT-l) and carbonic anhydrase-IX (CA-IX) in a series of prostatectomy specimens, tissue microarrays and needle biopsies from prostate cancer patients. Oxygen electrode studies have suggested that prostate cancer has significant hypoxia which may be a confounding factor for the current trend of hypofractionation based on the low alpba/beta ratio. None of these markers have so far been studied in the context of radiotherapy in
prostate cancer. Material and methods: A series of 39 prostatectomy specimens, a tissue microarray (TMA) containing 79 individual tumors and 41 needle biopsies were stained for HIF-lalpha, GLUT-1 and CA-IX using standard immunohistochemistry and assessed semi-quantitatively using a scoring system based on the level of positivity. Results: HIF-lalpha and GLUT-1 showed the expected staining .pattern of nuclear and cytoplasmic for HIF-lalpha and cytoplasmic staining for GLUT-1. Interestingly, CA-IX failed to show any staining using the standard staining technique without microwave pretreatment. However, using microwave unmasking, revealed a strong and specific nuclear localization of CA-IX in prostate cancer cells. 30%, 48% and 36% of the prostatectomies stained positively for HIF-lalpha, GLUT-1 and CA-IX respectively. In the TMA, 79%, 70% and 34% of tumors expressed the proteins whilst in biopsy material the figures were 60%, 45% and 55% respectively. The biopsy material was not significantly different to the prostatectomies for either HIF-laplha or CA-IX. However, there was a significant difference in the distribution of GLUT-1 scores (p=0.04). The biopsy material was significantly different to the TMA for both HIF-laplha (p=0.03) and GLUT-1 (p<0.01) but not CAIX. The TMA data was also different to the prostatectomy data for HIFlaplha (p<0.01) but not GLUT-1 or CAIX. In the biopsy material, HIF-lalpha showed a significant correlation with GLUT-1 (r=0.498, p=0.02) but not with CA-IX; GLUT-1 and CA-IX also did not correlate. In the prostatectomies, the correlation between HIF-lalpha and GLUT-1 reached near significance (r=0.365, p=0.0558) whilst the relationship with CA-IX was significant (r=0.341, p=0.0361). However, like the biopsies there was no correlation between GLUT-1 and CA-IX. In the TMA samples, HIF-lalpha showed a highly significant correlation with GLUT-1 (r=0.408, p=0.0015) whilst neither of these markers correlated with CA-IX. The data were pooled from all the clinical specimens to study the relationship between hypoxia-regulated protein expression and clinicopathological features. HIF-lalpha expression decreased significantly (p=0.0021, n=136) with increasing tumor grade. Neither GLUT-1 nor CA-IX showed an association with tumor grade. When the total Gleason score was reduced to above and below 7, GLUT-1 showed a significant (p=0.0153, n=123) reduction expression in the lower Gleason score category (45% +ve) compared to the higher Gleason score tumors (68% +ve). A similar effect was seen with CA-IX where the incidence of higher Gleason scores was 79% in the CA-IX positive tumors (p=0.0242, n=123); HIF-laplha showed no association with Gleason score. PSA levels did not correlate with any of the hypoxia-regulated proteins. Conclusions: This study has demonstrated that hypoxia-regulated protein expression is prevalent in prostate cancer confirming previous oxygen electrode studies. The unusual expression pattern of CA-IX warrants further investigation. Although correlations existed between some of the markers, it was evident hat differences existed both in prevalence and association with clinicopathological feutres. The feasibility of investigating hypoxia-related protein expression in biopsy material will facilitate future studies involving retrospective and prospective analysis of neutron and photon-treated patients at Wayne State University to establish whether surrogate markers of hyopxia can discriminate patients who gain benefit from different forms of radiotherapy. 115 CONFORMAL R A D I O B I O L O G Y - C L I N I C A L G A I N S FROM 'SAFE' H Y P O F R A C T I O N A T I O N
A. Nahum, A. J. Johansson, Clatterbridge Milano, Italy;
Kacperek, M. Cattaneo, R. Calandrino, C. Fiorino, B. Glimelius Centre for Oncology, UK; Istituto San Raffaele University of Uppsala, Sweden
Objective: The aim of this study is to demonstrate how conformal therapy including tomotherapy can yield clinical gains by reducing the fraction number as an alternative to escalating the prescription dose at fixed fraction size, especially for OARs exhibiting 'parallel' behaviour. Material and methods: The tools that advanced computer technology has placed at the disposal of radiotherapy can only be used to full clinical advantage if guided by radiobiological principles. The 'small
March 12 - 15
P r o f f e r e d Papers
113 ~I-INTEGRIN OR INTEGRIN-LINKED KINASE OVEREXPRESSION PROMOTES RADIATION-INDUCED A P O P T O S I S I N HUMAN HL60 LEUKEMIA CELLS
F. Hess, D. Estrugo, A. Fischer, N. Cordes OncoRay - Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, 01307 Dresden, Germany; Bundeswehr Institute of Radiobiology, Neuherbergstrasse 11, 80937 Munich, Germany Objective: Integrin-mediated cell adhesion to extracellular matrix (ECM) proteins reduces apoptosis following radiationinduced genotoxic injury. This mechanism effectively hampers therapeutic eradication of malignant cells at the primary tumor site and at peripheral sites after metastasization. To evaluate the importance of this phenomenon in suspension cells as well as the role of ~l-integrin and integrin-linked kinase (ILK), HL60 human acute promyelocytic leukemia cells were examined on fibronectin (FN) or in suspension. Material and methods: Cells were stably transfected with 131-integrin or ILK wild-type or hyperactive overexpression vectors. Suspension (polystyrene, BSA) or FN adhesion cultures were exposed to inhibitory anti- ~l-integrin antibodies or stimulatory RGD peptides and irradiated (0 - 10 Gy). Apoptosis, mitochondrial transmembrane potential (MTP) and caspase activation were analyzed by DAPI staining, flow cytometry and Western blotting technique (caspase-3, -9, -8, poly(ADP-ribose) polymerase (PARP); pulldown assays). Results: Adhesion to FN significantly (P<0.01) reduced radiation-induced apoptosis in HL60 cells (2 Gy: N15%) relative to suspension (N35%) at 48 h. Intriguingly, overexpression of J31-integrin or ILK dramatically enhanced apoptosis (90-100%) in 2-Gy irradiated adhesion and suspension cells. Adhesion to FN suppressed MTP breakdown, caspase-9 and PARP cleavage but permitted strong activation of caspase-8 and -3 in irradiated pl-integrin transfectants. In contrast, suspension conditions allowed both caspase-8 and 9-mediated activation of caspase-3. ILK overexpression resulted in combined activation of caspase-3 through caspase8 and -9 in irradiated FN adhesion cultures. Irradiation of ILK suspension cultures lacked caspase-8 activation but showed serial cleavage of caspase-9, -3 and PARP. Pulldown assays demonstrated that ~l-integrin recruits caspase-8 under both suspension and FN adhesion condition for proapoptotic signaling in DNA-damaged HL60 cells. In contrast, ILK directly interacts with caspase-8 exceptionally in irradiated FN adhesion cultures. Conclusions: These findings confirm our view of ILK as strong anti-survival regulator in genotoxically injured cells. New insights on cell adhesion-mediated radiation resistance comprise that (i) ~l-integrins are potent promoters of radiation-induced apoptosis, (ii) 131-integrin and ILK show overlapping signaling for enhancement of apoptosis and (iii) ~l-integrins and ILK recruit death receptor pathway-related initiator caspase-8 after irradiation. Our data further demonstrate that both the ligated and the unligated integrin states provide information to the cell about its microenvironment. This critically determines the repertoire of proteins available to execute apoptosis after genotoxic injury. Further, these data might provide the basis for developing new targeted chemo- and radiotherapeutic treatment options.
114 HYPOXIA-ASSOCIATED PROSTATE CANCER.
PROTEIN
EXPRESSION
G.D. Wilson, A.D. Powell, M. Che, J.D. Forman Department of Radiation Oncology, Karmanos Institute, Wayne State University, Detroit, USA
IN
Cancer
Objective: The aim of this Study was to investigate the expression of hypoxia inducible factor-lalpha (HIF-lalpha), glucose transporter protein-1 (GLUT-l) and carbonic anhydrase-IX (CA-IX) in a series of prostatectomy specimens, tissue microarrays and needle biopsies from prostate cancer patients. Oxygen electrode studies have suggested that prostate cancer has significant hypoxia which may be a confounding factor for the current trend of hypofractionation based on the low alpba/beta ratio. None of these markers have so far been studied in the context of radiotherapy in
prostate cancer. Material and methods: A series of 39 prostatectomy specimens, a tissue microarray (TMA) containing 79 individual tumors and 41 needle biopsies were stained for HIF-lalpha, GLUT-1 and CA-IX using standard immunohistochemistry and assessed semi-quantitatively using a scoring system based on the level of positivity. Results: HIF-lalpha and GLUT-1 showed the expected staining .pattern of nuclear and cytoplasmic for HIF-lalpha and cytoplasmic staining for GLUT-1. Interestingly, CA-IX failed to show any staining using the standard staining technique without microwave pretreatment. However, using microwave unmasking, revealed a strong and specific nuclear localization of CA-IX in prostate cancer cells. 30%, 48% and 36% of the prostatectomies stained positively for HIF-lalpha, GLUT-1 and CA-IX respectively. In the TMA, 79%, 70% and 34% of tumors expressed the proteins whilst in biopsy material the figures were 60%, 45% and 55% respectively. The biopsy material was not significantly different to the prostatectomies for either HIF-laplha or CA-IX. However, there was a significant difference in the distribution of GLUT-1 scores (p=0.04). The biopsy material was significantly different to the TMA for both HIF-laplha (p=0.03) and GLUT-1 (p<0.01) but not CAIX. The TMA data was also different to the prostatectomy data for HIFlaplha (p<0.01) but not GLUT-1 or CAIX. In the biopsy material, HIF-lalpha showed a significant correlation with GLUT-1 (r=0.498, p=0.02) but not with CA-IX; GLUT-1 and CA-IX also did not correlate. In the prostatectomies, the correlation between HIF-lalpha and GLUT-1 reached near significance (r=0.365, p=0.0558) whilst the relationship with CA-IX was significant (r=0.341, p=0.0361). However, like the biopsies there was no correlation between GLUT-1 and CA-IX. In the TMA samples, HIF-lalpha showed a highly significant correlation with GLUT-1 (r=0.408, p=0.0015) whilst neither of these markers correlated with CA-IX. The data were pooled from all the clinical specimens to study the relationship between hypoxia-regulated protein expression and clinicopathological features. HIF-lalpha expression decreased significantly (p=0.0021, n=136) with increasing tumor grade. Neither GLUT-1 nor CA-IX showed an association with tumor grade. When the total Gleason score was reduced to above and below 7, GLUT-1 showed a significant (p=0.0153, n=123) reduction expression in the lower Gleason score category (45% +ve) compared to the higher Gleason score tumors (68% +ve). A similar effect was seen with CA-IX where the incidence of higher Gleason scores was 79% in the CA-IX positive tumors (p=0.0242, n=123); HIF-laplha showed no association with Gleason score. PSA levels did not correlate with any of the hypoxia-regulated proteins. Conclusions: This study has demonstrated that hypoxia-regulated protein expression is prevalent in prostate cancer confirming previous oxygen electrode studies. The unusual expression pattern of CA-IX warrants further investigation. Although correlations existed between some of the markers, it was evident hat differences existed both in prevalence and association with clinicopathological feutres. The feasibility of investigating hypoxia-related protein expression in biopsy material will facilitate future studies involving retrospective and prospective analysis of neutron and photon-treated patients at Wayne State University to establish whether surrogate markers of hyopxia can discriminate patients who gain benefit from different forms of radiotherapy. 115 CONFORMAL R A D I O B I O L O G Y - C L I N I C A L G A I N S FROM 'SAFE' H Y P O F R A C T I O N A T I O N
A. Nahum, A. J. Johansson, Clatterbridge Milano, Italy;
Kacperek, M. Cattaneo, R. Calandrino, C. Fiorino, B. Glimelius Centre for Oncology, UK; Istituto San Raffaele University of Uppsala, Sweden
Objective: The aim of this study is to demonstrate how conformal therapy including tomotherapy can yield clinical gains by reducing the fraction number as an alternative to escalating the prescription dose at fixed fraction size, especially for OARs exhibiting 'parallel' behaviour. Material and methods: The tools that advanced computer technology has placed at the disposal of radiotherapy can only be used to full clinical advantage if guided by radiobiological principles. The 'small