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Survival Signaling Through Nuclear Epidermal Growth Factor Receptor and STAT3 Mediates Photodynamic Therapy Resistance in Ovarian and Lung Cancer Cells
I. J. Radiation Oncology d Biology d Physics
S98
Volume 69, Number 3, Supplement, 2007
Author Disclosure: C.C. Park, None; H. Zhang, None; S. Kim, None; R. Lee, None; M.J. Bissell, None.
175
SF1126, a Novel Integrin-Targeting Phosphatidylinositol-3 Kinase Inhibitor, Has Radiosensitizing and Anti-Tumor Effects in Glioma Model Systems
H. G. Shu1, H. Gao1, C. Chang1, D. L. Durden2 1 Emory University, Atlanta, GA, 2Children’s Healthcare of Atlanta, Atlanta, GA Purpose/Objective(s): Outcomes for patients with glioblastoma multiforme (GBM) are poor despite multimodality therapy. Phosphatidylinositol-3 kinase (PI-3K) is often activated in these tumors and may be a good target for inhibition. However, general PI-3K inhibitors have poor toxicity profiles. SF1126 is a modified pan PI-3K inhibitor that contains an RGDS peptide side chain moiety that permits targeting to integrins (aVb3/a5b1) improving therapeutic index. Here, we examine the effect of this novel agent on in vitro and in vivo glioma models. Materials/Methods: Glioma cell lines used in this study include 1) SF767 [high endogenous epidermal growth factor receptor (EGFR)/mutant PTEN], 2) LN229 (low EGFR/wild-type PTEN) engineered to express constitutively-active mutant EGFRvIII (LNvIII) with the corresponding vector only control (LNpuro) and 3) U251MG (low EGFR/mutant PTEN) also engineered to express EGFRvIII (U251vIII) with corresponding vector control (U251tk). Cells were treated with the general PI-3K inhibitor LY294002, SF1126 (Semafore) and temozolomide (Schering Plough), as indicated. Clonogenic survival assays were performed by standard techniques with drug treatment starting 2 hours prior to irradiation and maintained for 48 hours in total. Enhancement ratios (value without drug/value with drug) for SF2 (Surviving Fraction w/2 Gy) and ID50 (Inhibitory radiation Dose required to suppress colonies by 50%) were estimated based on linear-quadratic modeling. Subcutaneous tumors were established on the flanks of nude mice by injection of 5 106 tumor cells with SF1126 administered 3/week with subcutaneous injections. Results: Significant PI-3K inhibition with LY294002 and SF1126, as measured by loss of AKT phosphorylation, was attained with 15 mM or less of each agent. Clonogenic survival after radiation exposure was then determined with or without LY294002 or SF1126 in SF767, LNpuro and LNvIII. All showed significant enhancement of radiosensitivity after PI-3K inhibition with SF1126 displaying at least as potent, if not greater, radiosensitizing effect as LY294002. Enhancement ratios for SF2 and ID50 with 40 mM SF1126 were 1.5–1.9 and 1.7–2.0, respectively. These results compare favorably with those obtained using 5 mM temozolomide (enhancement ratios for SF2 and ID50 of 1.2–1.5 and 1.3–1.6, respectively). SF1126 concentrations in the 40 mM range are achievable based on intratumoral measurements of drug in mice after subcutaneous administration at 50 mg/kg. Besides its radiosensitizing activity, SF1126 had direct anti-tumor effects in glioma tumor models. While both the LN229 and U251 control and EGFRvIII-expressing isogenic cell line pairs formed tumors in nude mice, EGFRvIII-expressing lines formed larger tumors with greater microvascular density than their corresponding controls. Treatment of tumor-bearing mice with SF1126 resulted in significant inhibition of tumor growth with the effect being particularly pronounced in the EGFRvIII-expressing tumors. Conclusions: SF1126 is a novel pan PI-3K inhibitor that displays ‘‘homing’’ to tumors. In malignant gliomas, it may not only act as a radiosensitizer but also has direct anti-tumor activity. This agent warrants further evaluation in GBMs and may be a useful adjunct to standard therapy for these aggressive brain tumors. Author Disclosure: H.G. Shu, None; H. Gao, None; C. Chang, None; D.L. Durden, Funding from Semafore Pharmaceutical, B. Research Grant; Semafore stock options, E. Ownership Interest.
176
Survival Signaling Through Nuclear Epidermal Growth Factor Receptor and STAT3 Mediates Photodynamic Therapy Resistance in Ovarian and Lung Cancer Cells
K. A. Cengel, M. Chaudhury, S. Hagan, S. M. Hahn University of Pennsylvania Medical Ctr., Philadelphia, PA Purpose/Objective(s): Patients with serosal (pleural or peritoneal) spread of malignancy have few definitive treatment options and consequently have a very poor prognosis. We have previously shown that photodynamic therapy (PDT), a modality that combines photosensitizer, light and oxygen to mediate cancer cell cytotoxicity, can be an effective treatment for these patients. However, in these studies, the therapeutic index of PDT for serosal malignancies was relatively narrow. Recently studies showed that Cetuximab, an inhibitor of epidermal growth factor receptor (EGFR) signaling, synergistically enhanced the therapeutic efficacy of benzoporphyrin derivative monoacid (BPD)-mediated PDT in a mouse model of ovarian carcinomatosis. To investigate the mechanism and begin to develop molecular predictors of response for this novel cancer therapy, we have examined the impact of BPD-PDT on direct cell cytotoxicity and EGFR signaling in ovarian cancer (OvCa) and non-small cell lung cancer (NSCLC) cells. Materials/Methods: Cells were treated with 100 ng/ml BPD for 3 h, washed and exposed to 690 nm diode laser light. PDT cytotoxicity was measured using clonogenic cell survival assays. For signaling studies, a PDT dose was used to give a 10–20% surviving cell fraction to study PDT initiated signaling at levels of cell killing when clinical tumor recurrence following therapy is likely.
Proceedings of the 49th Annual ASTRO Meeting Results: Cetuximab pre-treatment of OVCAR-5 OvCa and H460 NSCLC cells resulted in dose-dependent inhibition of EGFR tyrosine phosphorylation and enhancement of PDT cytotoxicity. The EGFR-specificity of this effect was demonstrated by the ability of siRNA-mediated knockdown of EGFR expression to recapitulate this cytotoxicity enhancement. To further investigate interactions between EGFR and PDT, whole cell lysates were analyzed by immunoprecipitation of EGFR followed by probing with an anti-phosphotyrosine (anti-pY, not sequence-specific) antibody. These studies demonstrate that EGFR tyrosine phosphorylation increases over a 2 hr time course in response to BPD-PDT. Western blots of total protein lysates with antibodies specific for EGFR pY-1068 and pY-1173 residues suggest that this PDT-mediated increase in tyrosine phosphorylation occurs on residue 1068 (associated with activation of PI 3-kinase/AKT), but not 1173 (associated with activation of MAP kinase). Since nuclear relocalization of activated EGFR has been found to result in activation of STAT3-mediated anti-apoptotic signals, we next examined nucleocytoplasmic redistribution of EGFR and STAT3 following BPD-PDT. In these studies, we found that BPD-PDT results in a 4–5 fold induction of nuclear EGFR (pY1068) and STAT3 levels 6 hours following PDT. Conclusions: These results show that PDT activates EGFR signaling and promotes nuclear redistribution of EGFR and STAT3. Moreover, by interfering with these signals, EGFR inhibitors such as Cetuximab increase the efficacy of BPD-PDT in killing OvCa and NSCLC cells. Taken together, this study identifies a novel mechanism for cancer cell PDT resistance that involves EGFR, STAT3 and possibly PI3K/Akt and represents a critical first step in defining molecular predictors of treatment response with this novel combination therapy. Author Disclosure: K.A. Cengel, None; M. Chaudhury, None; S. Hagan, None; S.M. Hahn, None.
177
Caspase-3/7 Deficiency Promotes ER Stress-induced Autophagy via PERK-eIF2a Under Ionizing Radiation
K. Kim Vanderbilt University Medical Center North, Nashville, TN Purpose/Objective(s): Autophagy, a non-apoptotic cell death mode, has recently been associated with radiation in presence of deficient intrinsic apoptotic molecules, Bak/Bax. In this study, we examined whether caspase inactivation is necessary to autophagic cell death through PERK-eIF2a signaling in response of radiation-induced ER stress. Materials/Methods: Clonogenic assay was used to determine radiosensitivity of WT, Caspase3/7 / , Perk / and IRE1ab / . Protein levels of LC3 and Caspase-3 and Caspase-7 were determined by western blotting. Autophagy was determined with the punctate GFP-LC3 fluorescence, which observed under a confocal fluorescence microscope. Apoptosis was measured using the Annexin V-fluorescien isothiocyante apoptosis detection kit I (Pharmingen) with flow cytometry. Results: Either absence or knockdown of caspase 3/7 results in radiosensitization, primarily by induction of autophagy (DER = 1.65, p \ 0.004). These apoptosis defective cells revealed amplified ER stress through PERK-eIF2a signaling, suggesting that autophagy cell death occurs in response to radiation-induced ER stress (Figure 1). On the contrary, knockdown or absence of PERK inhibits autophagy and confers radiation resistance. In addition, knockdown of essential autophagy molecules, Beclin-1 and ATG-5, causes reversal of autophagy death in caspase 3/7 double knockout cells. Finally, irradiation of two human cancer models that were made caspase-deficient also resulted in radiosensitization, mainly by induction of autophagy. Either absence or knockdown of caspase 3/7 results in radiosensitization, primarily by induction of autophagy (DER = 1.65, p \ 0.004). These apoptosis defective cells revealed amplified ER stress through PERK-eIF2a signaling, suggesting that autophagy cell death occurs in response to radiation-induced ER stress (Figure 1). On the contrary, knockdown or absence of PERK inhibits autophagy and confers radiation resistance. In addition, knockdown of essential autophagy molecules, Beclin-1 and ATG-5, causes reversal of autophagy death in caspase 3/7 double knockout cells. Finally, irradiation of two human cancer models that were made caspase-deficient also resulted in radiosensitization, mainly by induction of autophagy. Conclusions: These results suggest that autophagy mediated by ER stress becomes an effective death pathway in apoptosis-defective cancer cells treated with ionized radiation. Therefore, autophagy may be utilized as novel targets for enhancing cancer therapy.
Author Disclosure: K. Kim, Vanderbilt, A. Employment.
S99
S98
Volume 69, Number 3, Supplement, 2007
Author Disclosure: C.C. Park, None; H. Zhang, None; S. Kim, None; R. Lee, None; M.J. Bissell, None.
175
SF1126, a Novel Integrin-Targeting Phosphatidylinositol-3 Kinase Inhibitor, Has Radiosensitizing and Anti-Tumor Effects in Glioma Model Systems
H. G. Shu1, H. Gao1, C. Chang1, D. L. Durden2 1 Emory University, Atlanta, GA, 2Children’s Healthcare of Atlanta, Atlanta, GA Purpose/Objective(s): Outcomes for patients with glioblastoma multiforme (GBM) are poor despite multimodality therapy. Phosphatidylinositol-3 kinase (PI-3K) is often activated in these tumors and may be a good target for inhibition. However, general PI-3K inhibitors have poor toxicity profiles. SF1126 is a modified pan PI-3K inhibitor that contains an RGDS peptide side chain moiety that permits targeting to integrins (aVb3/a5b1) improving therapeutic index. Here, we examine the effect of this novel agent on in vitro and in vivo glioma models. Materials/Methods: Glioma cell lines used in this study include 1) SF767 [high endogenous epidermal growth factor receptor (EGFR)/mutant PTEN], 2) LN229 (low EGFR/wild-type PTEN) engineered to express constitutively-active mutant EGFRvIII (LNvIII) with the corresponding vector only control (LNpuro) and 3) U251MG (low EGFR/mutant PTEN) also engineered to express EGFRvIII (U251vIII) with corresponding vector control (U251tk). Cells were treated with the general PI-3K inhibitor LY294002, SF1126 (Semafore) and temozolomide (Schering Plough), as indicated. Clonogenic survival assays were performed by standard techniques with drug treatment starting 2 hours prior to irradiation and maintained for 48 hours in total. Enhancement ratios (value without drug/value with drug) for SF2 (Surviving Fraction w/2 Gy) and ID50 (Inhibitory radiation Dose required to suppress colonies by 50%) were estimated based on linear-quadratic modeling. Subcutaneous tumors were established on the flanks of nude mice by injection of 5 106 tumor cells with SF1126 administered 3/week with subcutaneous injections. Results: Significant PI-3K inhibition with LY294002 and SF1126, as measured by loss of AKT phosphorylation, was attained with 15 mM or less of each agent. Clonogenic survival after radiation exposure was then determined with or without LY294002 or SF1126 in SF767, LNpuro and LNvIII. All showed significant enhancement of radiosensitivity after PI-3K inhibition with SF1126 displaying at least as potent, if not greater, radiosensitizing effect as LY294002. Enhancement ratios for SF2 and ID50 with 40 mM SF1126 were 1.5–1.9 and 1.7–2.0, respectively. These results compare favorably with those obtained using 5 mM temozolomide (enhancement ratios for SF2 and ID50 of 1.2–1.5 and 1.3–1.6, respectively). SF1126 concentrations in the 40 mM range are achievable based on intratumoral measurements of drug in mice after subcutaneous administration at 50 mg/kg. Besides its radiosensitizing activity, SF1126 had direct anti-tumor effects in glioma tumor models. While both the LN229 and U251 control and EGFRvIII-expressing isogenic cell line pairs formed tumors in nude mice, EGFRvIII-expressing lines formed larger tumors with greater microvascular density than their corresponding controls. Treatment of tumor-bearing mice with SF1126 resulted in significant inhibition of tumor growth with the effect being particularly pronounced in the EGFRvIII-expressing tumors. Conclusions: SF1126 is a novel pan PI-3K inhibitor that displays ‘‘homing’’ to tumors. In malignant gliomas, it may not only act as a radiosensitizer but also has direct anti-tumor activity. This agent warrants further evaluation in GBMs and may be a useful adjunct to standard therapy for these aggressive brain tumors. Author Disclosure: H.G. Shu, None; H. Gao, None; C. Chang, None; D.L. Durden, Funding from Semafore Pharmaceutical, B. Research Grant; Semafore stock options, E. Ownership Interest.
176
Survival Signaling Through Nuclear Epidermal Growth Factor Receptor and STAT3 Mediates Photodynamic Therapy Resistance in Ovarian and Lung Cancer Cells
K. A. Cengel, M. Chaudhury, S. Hagan, S. M. Hahn University of Pennsylvania Medical Ctr., Philadelphia, PA Purpose/Objective(s): Patients with serosal (pleural or peritoneal) spread of malignancy have few definitive treatment options and consequently have a very poor prognosis. We have previously shown that photodynamic therapy (PDT), a modality that combines photosensitizer, light and oxygen to mediate cancer cell cytotoxicity, can be an effective treatment for these patients. However, in these studies, the therapeutic index of PDT for serosal malignancies was relatively narrow. Recently studies showed that Cetuximab, an inhibitor of epidermal growth factor receptor (EGFR) signaling, synergistically enhanced the therapeutic efficacy of benzoporphyrin derivative monoacid (BPD)-mediated PDT in a mouse model of ovarian carcinomatosis. To investigate the mechanism and begin to develop molecular predictors of response for this novel cancer therapy, we have examined the impact of BPD-PDT on direct cell cytotoxicity and EGFR signaling in ovarian cancer (OvCa) and non-small cell lung cancer (NSCLC) cells. Materials/Methods: Cells were treated with 100 ng/ml BPD for 3 h, washed and exposed to 690 nm diode laser light. PDT cytotoxicity was measured using clonogenic cell survival assays. For signaling studies, a PDT dose was used to give a 10–20% surviving cell fraction to study PDT initiated signaling at levels of cell killing when clinical tumor recurrence following therapy is likely.
Proceedings of the 49th Annual ASTRO Meeting Results: Cetuximab pre-treatment of OVCAR-5 OvCa and H460 NSCLC cells resulted in dose-dependent inhibition of EGFR tyrosine phosphorylation and enhancement of PDT cytotoxicity. The EGFR-specificity of this effect was demonstrated by the ability of siRNA-mediated knockdown of EGFR expression to recapitulate this cytotoxicity enhancement. To further investigate interactions between EGFR and PDT, whole cell lysates were analyzed by immunoprecipitation of EGFR followed by probing with an anti-phosphotyrosine (anti-pY, not sequence-specific) antibody. These studies demonstrate that EGFR tyrosine phosphorylation increases over a 2 hr time course in response to BPD-PDT. Western blots of total protein lysates with antibodies specific for EGFR pY-1068 and pY-1173 residues suggest that this PDT-mediated increase in tyrosine phosphorylation occurs on residue 1068 (associated with activation of PI 3-kinase/AKT), but not 1173 (associated with activation of MAP kinase). Since nuclear relocalization of activated EGFR has been found to result in activation of STAT3-mediated anti-apoptotic signals, we next examined nucleocytoplasmic redistribution of EGFR and STAT3 following BPD-PDT. In these studies, we found that BPD-PDT results in a 4–5 fold induction of nuclear EGFR (pY1068) and STAT3 levels 6 hours following PDT. Conclusions: These results show that PDT activates EGFR signaling and promotes nuclear redistribution of EGFR and STAT3. Moreover, by interfering with these signals, EGFR inhibitors such as Cetuximab increase the efficacy of BPD-PDT in killing OvCa and NSCLC cells. Taken together, this study identifies a novel mechanism for cancer cell PDT resistance that involves EGFR, STAT3 and possibly PI3K/Akt and represents a critical first step in defining molecular predictors of treatment response with this novel combination therapy. Author Disclosure: K.A. Cengel, None; M. Chaudhury, None; S. Hagan, None; S.M. Hahn, None.
177
Caspase-3/7 Deficiency Promotes ER Stress-induced Autophagy via PERK-eIF2a Under Ionizing Radiation
K. Kim Vanderbilt University Medical Center North, Nashville, TN Purpose/Objective(s): Autophagy, a non-apoptotic cell death mode, has recently been associated with radiation in presence of deficient intrinsic apoptotic molecules, Bak/Bax. In this study, we examined whether caspase inactivation is necessary to autophagic cell death through PERK-eIF2a signaling in response of radiation-induced ER stress. Materials/Methods: Clonogenic assay was used to determine radiosensitivity of WT, Caspase3/7 / , Perk / and IRE1ab / . Protein levels of LC3 and Caspase-3 and Caspase-7 were determined by western blotting. Autophagy was determined with the punctate GFP-LC3 fluorescence, which observed under a confocal fluorescence microscope. Apoptosis was measured using the Annexin V-fluorescien isothiocyante apoptosis detection kit I (Pharmingen) with flow cytometry. Results: Either absence or knockdown of caspase 3/7 results in radiosensitization, primarily by induction of autophagy (DER = 1.65, p \ 0.004). These apoptosis defective cells revealed amplified ER stress through PERK-eIF2a signaling, suggesting that autophagy cell death occurs in response to radiation-induced ER stress (Figure 1). On the contrary, knockdown or absence of PERK inhibits autophagy and confers radiation resistance. In addition, knockdown of essential autophagy molecules, Beclin-1 and ATG-5, causes reversal of autophagy death in caspase 3/7 double knockout cells. Finally, irradiation of two human cancer models that were made caspase-deficient also resulted in radiosensitization, mainly by induction of autophagy. Either absence or knockdown of caspase 3/7 results in radiosensitization, primarily by induction of autophagy (DER = 1.65, p \ 0.004). These apoptosis defective cells revealed amplified ER stress through PERK-eIF2a signaling, suggesting that autophagy cell death occurs in response to radiation-induced ER stress (Figure 1). On the contrary, knockdown or absence of PERK inhibits autophagy and confers radiation resistance. In addition, knockdown of essential autophagy molecules, Beclin-1 and ATG-5, causes reversal of autophagy death in caspase 3/7 double knockout cells. Finally, irradiation of two human cancer models that were made caspase-deficient also resulted in radiosensitization, mainly by induction of autophagy. Conclusions: These results suggest that autophagy mediated by ER stress becomes an effective death pathway in apoptosis-defective cancer cells treated with ionized radiation. Therefore, autophagy may be utilized as novel targets for enhancing cancer therapy.
Author Disclosure: K. Kim, Vanderbilt, A. Employment.
S99