- Email: [email protected]
Selenium Potentiates the Antitumor Activity of Radiation Therapy in Human A549 Lung Cancer Xenografts
Proceedings of the 49th Annual ASTRO Meeting effects on cell autonomous functions such as proliferation, apoptosis and DNA damage repair and cell non-autonomous functions such as angiogenesis, the sum of which can drive the process of oncogenesis. The murine counterpart to ‘‘oncogene-addicted’’ human BACs are Ras-induced ‘‘oncogene-addicted’’ non-small cell lung cancers (NSCLCs). Corroborating this supposition are human mutational studies that suggest Ras and EGFR mutations are mutually exclusive in NSCLC. Materials/Methods: We constructed mouse preclinical lung cancer model systems to address these issues. Using a conditional lung specific gene expression system we analyzed transgenic mouse models of Myc, Ras or Myc/Ras induced lung adenocarcinoma. Results: These transgenic mice develop primary lung adenocarcinomas with variable latency (26–51 weeks) and clinical presentation that is dependent on their genotype. Upon oncogene-inactivation in these murine models, we have found that Ras, but not Myc induced lung adenocarcinomas regress in a matter of weeks completely. We measured quantitatively the clinical behavior of murine lung tumors in situ after oncogene-inactivation by use of serial micro-computed tomography (microCT) imaging. By modeling the regression curves of each transgenic system, we were able to express the difference mathematically between the tumor regression of Ras- and Myc-induced tumors. The modeling also allowed us to distinguish that double mutant, Ras/Myc-induced tumors, were likely composed of two dominant populations with behavior similar to single Ras- and Myc-induced tumors. We then used our data array as a training set for a predictive support vector machine algorithm which is highly accurate at predicting the regression of murine tumors based on only three serial weekly microCT scans at the initiation of oncogene-inactivation. Conclusions: Our work incorporates highly penetrant spontaneously arising murine lung tumor models, non-invasive serial tumor imaging and predictive mathematically modeling. This system serves as a more clinically relevant bridge for studies transitioning from the bench to the clinic and can also be used to probe basic questions of oncogene-addiction. Furthermore, these preclinical studies suggest a novel method for predicting tumor responses in human BACs. Author Disclosure: P.T. Tran, None; J. Lin, None; P. Bendapudi, None; S. Koh, None; K. Komatsubara, None; G. Horng, None; J. Chen, None; C. Shachaf, None; D. Paik, None; D.W. Felsher, None.
2717
Exploiting Breast Cancer Cells Stress Response to Ionizing Radiation to Improve the Effectiveness of Immunotherapy
S. Demaria, B. Wang, M. Badura, S. Matsumura, N. Kawashima, T. Cameron, M. Dustin, R. J. Schneider, S. C. Formenti New York University School of Medicine, New York, NY Purpose/Objective: Vaccination can be successful in boosting pre-existing or inducing de novo immune responses to the tumor. However, activated anti-tumor T cells do not efficiently home and perform effector functions within tumors. Chemokines and their receptors regulate trafficking of immune cells to secondary lymphoid organs, and to inflamed peripheral tissues. Little is known about the chemokines that regulate homing of effector T cells to tumors. Cancer cells produce several chemokines, mostly to recruit leukocytes that promote tolerance and immune escape, and aid tumor growth by enhanced angiogenesis. Ionizing radiation therapy (RT), in addition to its cytocidal effects, alters the phenotype of the surviving neoplastic and stromal cells. We and others have shown in preclinical syngeneic models of cancer that local RT used in combination with different immunotherapy approaches promotes immune-mediated tumor destruction. These data suggest that radiation-induced changes in the tumor microenvironment may facilitate tumor rejection. We have tested the hypothesis that RT can alter the phenotype and the pattern of chemokine secretion by tumor cells resulting in improved T cell recruitment and function within irradiated tumors. Materials/Methods: Expression of MHC class I, Fas/CD95, and ICAM-1 was tested by immunostaining with specific antibodies and flow cytometry analysis. CXCL16 expression was analyzed by real time RT-PCR and immunofluorescence. Soluble CXCL16 was measured by ELISA in supernatants of 4T1 cells cultured for 4 hours in the presence or absence of MPase activators/inhibitors. Expression of CXCL16 in growing tumors in vivo was analyzed by immunohistochemistry. Chemotaxis assays were performed using a transwell system. Results: RT induced a marked dose-dependent up-regulation of surface MHC class I, Fas and ICAM-1 molecules by poorly immunogenic, highly aggressive and metastatic BALB/c-derived 4T1 breast cancer cells. Surprisingly, 4T1 cells expressed the proinflammatory chemokine CXCL16 that plays an important role in recruiting activated CD8+ T cells to sites of inflammation. CXCL16 was rapidly shed in soluble form from 4T1 cells by metalloprotease (MPase)-mediated cleavage. Although the levels of CXCL16 released by untreated 4T1 cells were too low to promote a significant chemotaxis of CXCR6+ activated CD8+ T cells, release of CXCL16 was markedly increased following irradiation of 4T1 cells, resulting in strong increase in migration of activated T cells towards 4T1 cells. Antibody blocking experiments and specific knockdown of the CXCR6 receptor on T cells, and of CXCL16 in 4T1 cells confirmed that increased migration was largely driven by CXCL16. Conclusions: These data provide the first evidence that RT can alter the secretion of pro-inflammatory chemotactic factors by cancer cells. This effect, together with increased expression of surface molecules that improve recognition of cancer cells by anti-tumor T cells strongly support the use of RT in combination with immunotherapy. Author Disclosure: S. Demaria, None; B. Wang, None; M. Badura, None; S. Matsumura, None; N. Kawashima, None; T. Cameron, None; M. Dustin, None; R.J. Schneider, None; S.C. Formenti, None.
2718
Selenium Potentiates the Antitumor Activity of Radiation Therapy in Human A549 Lung Cancer Xenografts
G. Y. Yang, F. A. Durrani, S. Cao, Y. Park, G. M. Thompson, J. L. Martin, W. Jaggernauth, Y. M. Rustum Roswell Park Cancer Institute, Buffalo, NY Purpose/Objective(s): We have previously demonstrated synergistic antitumor activity between selenium containing compounds, Se-(methyl)-Selenocysteine (MSC) and Seleno-L-Methionine (SLM), with a wide range of anticancer drugs and tumor types. The observed therapeutic synergy was highly selenium dose and schedule dependent. Studies were extended to determine whether therapeutic synergy also exists between selenium and radiation (RT) in nude mice bearing A549 human non-small cell lung cancer xenografts.
S597
I. J. Radiation Oncology d Biology d Physics
S598
Volume 69, Number 3, Supplement, 2007
Materials/Methods: Tumor tissues were transplanted into the shoulder of nude mice. Treatments were started 7 days after transplantation when tumors reached 200 mg. Animals were randomized in different groups: 1) Untreated control; 2) SLM or MSC alone; 3) RT alone; 4) RT+SLM; 5) RT+MSC. Tumor bed was radiated for 5 consecutive daily fractions of 8 Gy (40 Gy total) using 150 kV x-rays. Selenium was administered orally at 0.01 or 0.2 mg/mouse/day for 19 days with the first dose administered seven days prior to radiation treatment. Tumors were measured with the aid of a Vernier caliper daily during the RT and 3-4 times/week after treatment for the first month and twice a week thereafter. Tumor response was expressed as partial response (PR, $50% reduction in initial tumor size), and complete response (CR) when tumor was undetectable by palpation and surviving without tumor for more than 3 months when the animal was sacrificed. Toxicity includes body weights and physical changes which were also evaluated at the same time with tumor measurements. Results: There were no changes in body weight and no toxicity apparent from addition of the selenium to the maximally tolerated dose of radiation. Results indicate that while SLM or MSC alone exhibited no antitumor activity, 10% CR were achieved with RT alone, in combination with either SLM(0.01 mg/d), SLM(0.2 mg/d) or MSC, CR were increased to 20%, 46% and 60%, respectively. In addition, the total response rate (RR, CR+PR) with RT alone and in combination with selenium was increased from 30% to 100% (Table). Both SLM and MSC significantly increased the therapeutic synergy in this tumor model when combined with RT. Conclusions: Our findings indicate that the combination of radiation and selenium results in significant synergistic effects in nude mice bearing A549 human lung cancer xenografts. The observed therapeutic synergy between selenium and radiation was highly selective with no additional toxicity observed. These encouraging data could provide the rationale for clinical validation of this new and novel concept.
Treatment
Animal #
PR (%)
CR (%)
RR (%)
Untreated Control MSC or SLM(0.2 mg/d 19) RT RT + SLM(0.01 mg/d 19) RT + SLM(0.2 mg/d 19) RT + MSC(0.2 mg/d 19)
20 20 20 10 15 10
0 0 20 80 54 40
0 0 10 20 46 60
0 0 30 100 100 100
Author Disclosure: G.Y. Yang, None; F.A. Durrani, None; S. Cao, None; Y. Park, None; G.M. Thompson, None; J.L. Martin, None; W. Jaggernauth, None; Y.M. Rustum, None.
2719
Identification of a Small Moleculue That Stimulates Human RAD51 Protein
K. Jayathilaka, S. Sheridan, T. Bold, D. K. Bishop, P. P. Connell University of Chicago Hospitals, Chicago, IL Purpose/Objective(s): DNA repair via homologous recombination (HR) confers cellular resistance to ionizing radiation and certain DNA damaging agents. RAD51 protein momomers assemble into helical filaments at sites of double-stranded DNA breaks; this is a critical event required for the initiation of HR. A central hypothesis of this project is that pharmacologic manipulation of RAD51 may be used to modulate cellular resistance. This represents the final report based on funding from an ASTRO Junior Faculty Research Training Award. Materials/Methods: A microplate-based assay was developed for high throughput measurement of RAD51 binding to DNA. The assay makes use of the increase in fluorescence polarization (FP) that occurs upon filament formation on a single stranded DNA oligonucleotide (dT 45mer). This method was modified to allow use of 384-well plates and a fluorescent plate reader. Results: A 10,000 compound library of small molecules was screened leading to the identification of three compounds that increased FP by at least 50%. Further study of the most active of these three compounds confirmed that it enhances human RAD51 binding. Ultrastructural analysis of filaments formed on single strand DNA (1000 mer) showed that the compound can increase both the efficiency of filament initiation and average filament length. Salt titrations provided evidence that the compound can enhance filament stability. D-loop assays, using a supercoiled double-stranded plasmid and a homologous 32P-labeled ssDNA oligonucleotide, demonstrated concentration-dependent stimulation of D-loop formation by the compound with a maximum stimulation of about 5-fold. This stimulation of D-loops occurred in various buffer conditions (Mg2+ or Ca2+) and nucleotide triphosphates (ATP or AMP-PNP) indicating that the mechanism of action was distinct from that conferred by known RAD51-stimulatory conditions. No D-loop activity was observed in the absence of nucleotide cofactor indicating that the compound does not substitute for this requirement. Conclusions: Further experiments are underway to determine whether the compound can stimulate HR repair in cells. If in vivo activities are ultimately observed, this small molecule could potentially be used to protect normal tissues against the toxic effects of oncologic therapies or accidental exposures to radiation and mutagenic agents. Author Disclosure: K. Jayathilaka, None; S. Sheridan, None; T. Bold, None; D.K. Bishop, None; P.P. Connell, This represents the final report based on funding from an ASTRO Junior Faculty Research Training Award, B. Research Grant.
2720
Exonuclease Activity is Essential for Mre11 in Controlling Microhomology Mediated End Joining of DNA Double-Strand Breaks
D. S. Yu1, J. Zhuang1, E. Nam1, H. Willers2, F. Xia1 1
Vanderbilt University Medical Center, Nashville, TN, 2Massachusetts General Hospital, Boston, MA
Purpose/Objective(s): Mre11, in a complex with Rad50 and NBS1, is critical in DNA damage response. Patients with germline mutations in Mre11 share similar clinical presentations as Ataxia-Telangiectasia (AT) patients. Loss of Mre11 results in increased radiosensitivity and chromosomal instability. Previously we have shown that Mre11 may play a direct role in DNA double-strand
2717
Exploiting Breast Cancer Cells Stress Response to Ionizing Radiation to Improve the Effectiveness of Immunotherapy
S. Demaria, B. Wang, M. Badura, S. Matsumura, N. Kawashima, T. Cameron, M. Dustin, R. J. Schneider, S. C. Formenti New York University School of Medicine, New York, NY Purpose/Objective: Vaccination can be successful in boosting pre-existing or inducing de novo immune responses to the tumor. However, activated anti-tumor T cells do not efficiently home and perform effector functions within tumors. Chemokines and their receptors regulate trafficking of immune cells to secondary lymphoid organs, and to inflamed peripheral tissues. Little is known about the chemokines that regulate homing of effector T cells to tumors. Cancer cells produce several chemokines, mostly to recruit leukocytes that promote tolerance and immune escape, and aid tumor growth by enhanced angiogenesis. Ionizing radiation therapy (RT), in addition to its cytocidal effects, alters the phenotype of the surviving neoplastic and stromal cells. We and others have shown in preclinical syngeneic models of cancer that local RT used in combination with different immunotherapy approaches promotes immune-mediated tumor destruction. These data suggest that radiation-induced changes in the tumor microenvironment may facilitate tumor rejection. We have tested the hypothesis that RT can alter the phenotype and the pattern of chemokine secretion by tumor cells resulting in improved T cell recruitment and function within irradiated tumors. Materials/Methods: Expression of MHC class I, Fas/CD95, and ICAM-1 was tested by immunostaining with specific antibodies and flow cytometry analysis. CXCL16 expression was analyzed by real time RT-PCR and immunofluorescence. Soluble CXCL16 was measured by ELISA in supernatants of 4T1 cells cultured for 4 hours in the presence or absence of MPase activators/inhibitors. Expression of CXCL16 in growing tumors in vivo was analyzed by immunohistochemistry. Chemotaxis assays were performed using a transwell system. Results: RT induced a marked dose-dependent up-regulation of surface MHC class I, Fas and ICAM-1 molecules by poorly immunogenic, highly aggressive and metastatic BALB/c-derived 4T1 breast cancer cells. Surprisingly, 4T1 cells expressed the proinflammatory chemokine CXCL16 that plays an important role in recruiting activated CD8+ T cells to sites of inflammation. CXCL16 was rapidly shed in soluble form from 4T1 cells by metalloprotease (MPase)-mediated cleavage. Although the levels of CXCL16 released by untreated 4T1 cells were too low to promote a significant chemotaxis of CXCR6+ activated CD8+ T cells, release of CXCL16 was markedly increased following irradiation of 4T1 cells, resulting in strong increase in migration of activated T cells towards 4T1 cells. Antibody blocking experiments and specific knockdown of the CXCR6 receptor on T cells, and of CXCL16 in 4T1 cells confirmed that increased migration was largely driven by CXCL16. Conclusions: These data provide the first evidence that RT can alter the secretion of pro-inflammatory chemotactic factors by cancer cells. This effect, together with increased expression of surface molecules that improve recognition of cancer cells by anti-tumor T cells strongly support the use of RT in combination with immunotherapy. Author Disclosure: S. Demaria, None; B. Wang, None; M. Badura, None; S. Matsumura, None; N. Kawashima, None; T. Cameron, None; M. Dustin, None; R.J. Schneider, None; S.C. Formenti, None.
2718
Selenium Potentiates the Antitumor Activity of Radiation Therapy in Human A549 Lung Cancer Xenografts
G. Y. Yang, F. A. Durrani, S. Cao, Y. Park, G. M. Thompson, J. L. Martin, W. Jaggernauth, Y. M. Rustum Roswell Park Cancer Institute, Buffalo, NY Purpose/Objective(s): We have previously demonstrated synergistic antitumor activity between selenium containing compounds, Se-(methyl)-Selenocysteine (MSC) and Seleno-L-Methionine (SLM), with a wide range of anticancer drugs and tumor types. The observed therapeutic synergy was highly selenium dose and schedule dependent. Studies were extended to determine whether therapeutic synergy also exists between selenium and radiation (RT) in nude mice bearing A549 human non-small cell lung cancer xenografts.
S597
I. J. Radiation Oncology d Biology d Physics
S598
Volume 69, Number 3, Supplement, 2007
Materials/Methods: Tumor tissues were transplanted into the shoulder of nude mice. Treatments were started 7 days after transplantation when tumors reached 200 mg. Animals were randomized in different groups: 1) Untreated control; 2) SLM or MSC alone; 3) RT alone; 4) RT+SLM; 5) RT+MSC. Tumor bed was radiated for 5 consecutive daily fractions of 8 Gy (40 Gy total) using 150 kV x-rays. Selenium was administered orally at 0.01 or 0.2 mg/mouse/day for 19 days with the first dose administered seven days prior to radiation treatment. Tumors were measured with the aid of a Vernier caliper daily during the RT and 3-4 times/week after treatment for the first month and twice a week thereafter. Tumor response was expressed as partial response (PR, $50% reduction in initial tumor size), and complete response (CR) when tumor was undetectable by palpation and surviving without tumor for more than 3 months when the animal was sacrificed. Toxicity includes body weights and physical changes which were also evaluated at the same time with tumor measurements. Results: There were no changes in body weight and no toxicity apparent from addition of the selenium to the maximally tolerated dose of radiation. Results indicate that while SLM or MSC alone exhibited no antitumor activity, 10% CR were achieved with RT alone, in combination with either SLM(0.01 mg/d), SLM(0.2 mg/d) or MSC, CR were increased to 20%, 46% and 60%, respectively. In addition, the total response rate (RR, CR+PR) with RT alone and in combination with selenium was increased from 30% to 100% (Table). Both SLM and MSC significantly increased the therapeutic synergy in this tumor model when combined with RT. Conclusions: Our findings indicate that the combination of radiation and selenium results in significant synergistic effects in nude mice bearing A549 human lung cancer xenografts. The observed therapeutic synergy between selenium and radiation was highly selective with no additional toxicity observed. These encouraging data could provide the rationale for clinical validation of this new and novel concept.
Treatment
Animal #
PR (%)
CR (%)
RR (%)
Untreated Control MSC or SLM(0.2 mg/d 19) RT RT + SLM(0.01 mg/d 19) RT + SLM(0.2 mg/d 19) RT + MSC(0.2 mg/d 19)
20 20 20 10 15 10
0 0 20 80 54 40
0 0 10 20 46 60
0 0 30 100 100 100
Author Disclosure: G.Y. Yang, None; F.A. Durrani, None; S. Cao, None; Y. Park, None; G.M. Thompson, None; J.L. Martin, None; W. Jaggernauth, None; Y.M. Rustum, None.
2719
Identification of a Small Moleculue That Stimulates Human RAD51 Protein
K. Jayathilaka, S. Sheridan, T. Bold, D. K. Bishop, P. P. Connell University of Chicago Hospitals, Chicago, IL Purpose/Objective(s): DNA repair via homologous recombination (HR) confers cellular resistance to ionizing radiation and certain DNA damaging agents. RAD51 protein momomers assemble into helical filaments at sites of double-stranded DNA breaks; this is a critical event required for the initiation of HR. A central hypothesis of this project is that pharmacologic manipulation of RAD51 may be used to modulate cellular resistance. This represents the final report based on funding from an ASTRO Junior Faculty Research Training Award. Materials/Methods: A microplate-based assay was developed for high throughput measurement of RAD51 binding to DNA. The assay makes use of the increase in fluorescence polarization (FP) that occurs upon filament formation on a single stranded DNA oligonucleotide (dT 45mer). This method was modified to allow use of 384-well plates and a fluorescent plate reader. Results: A 10,000 compound library of small molecules was screened leading to the identification of three compounds that increased FP by at least 50%. Further study of the most active of these three compounds confirmed that it enhances human RAD51 binding. Ultrastructural analysis of filaments formed on single strand DNA (1000 mer) showed that the compound can increase both the efficiency of filament initiation and average filament length. Salt titrations provided evidence that the compound can enhance filament stability. D-loop assays, using a supercoiled double-stranded plasmid and a homologous 32P-labeled ssDNA oligonucleotide, demonstrated concentration-dependent stimulation of D-loop formation by the compound with a maximum stimulation of about 5-fold. This stimulation of D-loops occurred in various buffer conditions (Mg2+ or Ca2+) and nucleotide triphosphates (ATP or AMP-PNP) indicating that the mechanism of action was distinct from that conferred by known RAD51-stimulatory conditions. No D-loop activity was observed in the absence of nucleotide cofactor indicating that the compound does not substitute for this requirement. Conclusions: Further experiments are underway to determine whether the compound can stimulate HR repair in cells. If in vivo activities are ultimately observed, this small molecule could potentially be used to protect normal tissues against the toxic effects of oncologic therapies or accidental exposures to radiation and mutagenic agents. Author Disclosure: K. Jayathilaka, None; S. Sheridan, None; T. Bold, None; D.K. Bishop, None; P.P. Connell, This represents the final report based on funding from an ASTRO Junior Faculty Research Training Award, B. Research Grant.
2720
Exonuclease Activity is Essential for Mre11 in Controlling Microhomology Mediated End Joining of DNA Double-Strand Breaks
D. S. Yu1, J. Zhuang1, E. Nam1, H. Willers2, F. Xia1 1
Vanderbilt University Medical Center, Nashville, TN, 2Massachusetts General Hospital, Boston, MA
Purpose/Objective(s): Mre11, in a complex with Rad50 and NBS1, is critical in DNA damage response. Patients with germline mutations in Mre11 share similar clinical presentations as Ataxia-Telangiectasia (AT) patients. Loss of Mre11 results in increased radiosensitivity and chromosomal instability. Previously we have shown that Mre11 may play a direct role in DNA double-strand