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Hedgehog Pathway Misactivation Induces CDK6 to Drive Medulloblastoma
S56
International Journal of Radiation Oncology Biology Physics
Zagar: None. R.C. Chen: None. G.P. Gupta: None. E.L. Jones: None. M.A. Varia: None. B.S. Chera: None. S. Hong: Founder; Capio Biosciences. Research Funding; Durae. Founder and Patent Application; Capio Biosciences. Founder; Capio Biosciences. A.Z. Wang: Co-Founder; Capio Biosciences. Patent/License Fees/Copyright; PhoenixSongs. Stock and Ownership Interests; Gilead Sciences, Biogen Idec, Shire. Stock and Ownership Interests, Patent Pending; Capio Biosciences.
D. Vesprini: None. R.G. Bristow: None. P.C. Boutros: None. S.K. Liu: None.
124 The Biological Role and Clinical Significance of Long Noncoding RNA Urothelial Carcinoma Associated 1 (UCA1) in Prostate Cancer (PCa) A. Fotouhi Ghiam,1,2 S. Taeb,3 X. Huang,3 S. Jahangiri,3 J. Ray,3 C. Hoey,3 E. Fokas,4 D. Vesprini,1,5 R.G. Bristow,1,6 P.C. Boutros,7 and S.K. Liu1,2; 1 Department of Radiation Oncology, University of Toronto, Toronto, ON, Canada, 2Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada, 3Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, Canada, 4University of Oxford, Oxford, United Kingdom, 5Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada, 6Radiation Medicine Program, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada, 7Ontario Institute for Cancer Research, Toronto, ON, Canada Purpose/Objective(s): Urothelial carcinoma-associated 1 (UCA1) is a recently identified long non-coding RNA which plays an oncologic role in several cancers and enhances cellular proliferation, invasion, and tumor growth. It is upregulated in prostate tumors, but its involvement in therapy response has not been investigated. Materials/Methods: To simulate the clinical scenario of ionizing-radiation resistance (IRR), we created IRR PCa cell lines by treatment with mock irradiation (parental cells) or IR (conventional fractionation [CF]: 2 Gy daily X 59 for DU145-CF-IRR cells). We performed gene array profiling to discover dysregulated genes in DU145-CF-IRR cells and identified UCA1. We investigated the mechanism of UCA1 on aggression and response to radiation and chemotherapy by performing miRNA comparative profiling (NanoString platform), Proteome Profiler Array, proliferation assays, soft agar colony formation, clonogenic assays, invasion assay, g-H2AX assay, cell cycle profiling, MTS assay, and Western blotting. We also studied the clinical significance of UCA1 expression in two cohorts of PCa patients; CPC-GENE (n Z 210; patients with intermediate-risk PCa) and MSKCC (n Z 131; PCa patients treated with radical prostatectomy). Results: DU145-CF-IRR cells were radioresistant and acquired an aggressive phenotype. We found that UCA1 expression was significantly higher in DU145-CF-IRR compared to parental cells using gene array profiling and confirmatory qRT-PCR analysis (170-fold). Interestingly, UCA1 siRNA-knockdown reversed the aggressive phenotype and significantly increased sensitivity to IR and docetaxel. We demonstrated that UCA1 depletion inhibited growth, induced cell cycle arrest at the G2/M transition, and decreased activation of the pro-survival Akt pathway. Furthermore, we found that UCA1 overexpression was associated with a trend toward lower biochemical recurrence-free survival in CPC-GENE patients (HR Z 1.4, P Z 0.28) and its aberrant expression was significantly associated with decreased 5-year disease-free survival in publically available MSKCC database (84.5% vs 52%; HR Z 2.19, log-rank test P Z 0.005). Conclusion: We showed for the first time that UCA1 can influence cancer aggression, radiation, and chemotherapy response in PCa, which may occur through altered Akt signaling. Our results also suggest that UCA1 can have prognostic value in PCa. Future work will investigate UCA1 as a therapeutic target and prognostic biomarker for PCa. AF Ghiam is supported by a Canadian Association of Radiation Oncology (CARO) Fellowship Award. Author Disclosure: A. Fotouhi Ghiam: None. S. Taeb: None. X. Huang: None. S. Jahangiri: None. J. Ray: None. C. Hoey: None. E. Fokas: None.
125 Hedgehog Pathway Misactivation Induces CDK6 to Drive Medulloblastoma D. Raleigh,1 A.L. Krup,2 P.K. Choksi,2 and J.F. Reiter2; 1University of California, San Francisco, San Francisco, CA, 2University of California San Francisco, San Francisco, CA Purpose/Objective(s): Misregulation of the Hedgehog (Hh) pathway can cause cancers such as basal cell carcinoma (BCC), the most common cancer in the United States, and medulloblastoma, a common pediatric brain tumor. Gli2 is the principle effector of the Hh transcriptional program, but how Gli2 acts to drive cancer remains unknown. The objective of this study was to identify how misactivation of Gli2 in cancer causes cell proliferation. Materials/Methods: To study the Hh transcriptional program, we generated a novel mouse model of Hh-associated medulloblastoma with a knock-in of the Gli2 allele containing a GFP-tag that (i) is expressed under endogenous regulatory control; (ii) recapitulates Gli2 activity, interactions and localization; and (iii) allows us to efficiently isolate Gli2 and identify target genes. We misactivate the Hh pathway in this system using SmoM2c, an allele encoding an oncogenic form of the Hh pathway-activator Smoothened (Smo). Cre under the control of Math1 regulatory sequences conditionally expresses SmoM2 in medulloblastoma progenitor cells in the external granule layer. All Gli2-GFP; SmoM2c; and Math1-Cre mice develop neonatal cerebellar tumors. Protein and transcript levels were measured by immunoblot and qRT-PCR, respectively. All experiments were performed with at least 3 biologic replicates, repeated 3 times, and compared by t-tests. Data are displayed as mean SEM. Results: RNA sequencing of mouse Hh-associated medulloblastoma and comparison to human tumors demonstrates 98 33-fold enrichment of the cell cycle-activator Cdk6 in the setting of retinoblastoma (Rb) hyperphosphorylation and enhanced E2F transcription factor activity. To test if Cdk6 is a direct target of Gli2, NIH3T3 cells expressing Gli2-GFP (3T3GG) were treated with Smoothened agonist (SAG), the E2F inhibitor HLM006474 (HLM), or the translational inhibitor cycloheximide (CHX). Both HLM006474 and CHX block SAG-mediated induction of all cell cycle transcripts except Cdk6, suggesting that Cdk6 represents the point of cell cycle entry for the Hh transcriptional program. Gli2-GFP chromatin immunoprecipitation from mouse Hh-associated medulloblastoma and 3T3GG cells treated with SAG reveals Gli2 bound to the Cdk6 promoter in a manner that is reversed by the Smo inhibitor vismodegib. Consistently, a minimal Cdk6 promoter reporter was sufficient to respond to Hh signals in 3T3GG cells. To test if Cdk6 drives cell proliferation in Hh-associated medulloblastoma, P21 Gli2-GFP; SmoM2c; Math1-Cre mice were gavaged with the Cdk4/6 inhibitor palbociclib (150 mg/g; n Z 8) or vehicle control (n Z 9) every day for 2 weeks. Palbociclib reduced tumor weights by 40 1% (P < 0.05) and was associated with histopathologic changes indicative of tumor cell differentiation. Conclusion: Cdk6 is a direct target of Gli2 and drives proliferation of Hhassociated cancer. Outcomes with genetic inhibition of Cdk6 and palbociclib/vismodegib combination therapy in mouse Hh-associated medulloblastoma are under investigation. Author Disclosure: D. Raleigh: None. A.L. Krup: None. P.K. Choksi: None. J.F. Reiter: None.
126 Does Whole-Brain Radiation Therapy for Oligometastatic Brain Metastases Translate Into a Survival Benefit for Patients With a Limited Competing Risk From Extracranial Disease? A Secondary Analysis of EORTC 22952-26001 T.M. Churilla,1 E. Handorf,1 R. Soffietti,2 M. Kocher,3 A.A. Aizer,4 L. Collette,5 S. Collette,5 Y. Dong,1 B.M. Alexander,6 and S.E. Weiss1; 1 Fox Chase Cancer Center, Philadelphia, PA, 2University of Turin and City of Health and Science Hospital, Torino, Italy, 3Radiotherapy and Oncology
International Journal of Radiation Oncology Biology Physics
Zagar: None. R.C. Chen: None. G.P. Gupta: None. E.L. Jones: None. M.A. Varia: None. B.S. Chera: None. S. Hong: Founder; Capio Biosciences. Research Funding; Durae. Founder and Patent Application; Capio Biosciences. Founder; Capio Biosciences. A.Z. Wang: Co-Founder; Capio Biosciences. Patent/License Fees/Copyright; PhoenixSongs. Stock and Ownership Interests; Gilead Sciences, Biogen Idec, Shire. Stock and Ownership Interests, Patent Pending; Capio Biosciences.
D. Vesprini: None. R.G. Bristow: None. P.C. Boutros: None. S.K. Liu: None.
124 The Biological Role and Clinical Significance of Long Noncoding RNA Urothelial Carcinoma Associated 1 (UCA1) in Prostate Cancer (PCa) A. Fotouhi Ghiam,1,2 S. Taeb,3 X. Huang,3 S. Jahangiri,3 J. Ray,3 C. Hoey,3 E. Fokas,4 D. Vesprini,1,5 R.G. Bristow,1,6 P.C. Boutros,7 and S.K. Liu1,2; 1 Department of Radiation Oncology, University of Toronto, Toronto, ON, Canada, 2Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada, 3Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, Canada, 4University of Oxford, Oxford, United Kingdom, 5Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada, 6Radiation Medicine Program, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada, 7Ontario Institute for Cancer Research, Toronto, ON, Canada Purpose/Objective(s): Urothelial carcinoma-associated 1 (UCA1) is a recently identified long non-coding RNA which plays an oncologic role in several cancers and enhances cellular proliferation, invasion, and tumor growth. It is upregulated in prostate tumors, but its involvement in therapy response has not been investigated. Materials/Methods: To simulate the clinical scenario of ionizing-radiation resistance (IRR), we created IRR PCa cell lines by treatment with mock irradiation (parental cells) or IR (conventional fractionation [CF]: 2 Gy daily X 59 for DU145-CF-IRR cells). We performed gene array profiling to discover dysregulated genes in DU145-CF-IRR cells and identified UCA1. We investigated the mechanism of UCA1 on aggression and response to radiation and chemotherapy by performing miRNA comparative profiling (NanoString platform), Proteome Profiler Array, proliferation assays, soft agar colony formation, clonogenic assays, invasion assay, g-H2AX assay, cell cycle profiling, MTS assay, and Western blotting. We also studied the clinical significance of UCA1 expression in two cohorts of PCa patients; CPC-GENE (n Z 210; patients with intermediate-risk PCa) and MSKCC (n Z 131; PCa patients treated with radical prostatectomy). Results: DU145-CF-IRR cells were radioresistant and acquired an aggressive phenotype. We found that UCA1 expression was significantly higher in DU145-CF-IRR compared to parental cells using gene array profiling and confirmatory qRT-PCR analysis (170-fold). Interestingly, UCA1 siRNA-knockdown reversed the aggressive phenotype and significantly increased sensitivity to IR and docetaxel. We demonstrated that UCA1 depletion inhibited growth, induced cell cycle arrest at the G2/M transition, and decreased activation of the pro-survival Akt pathway. Furthermore, we found that UCA1 overexpression was associated with a trend toward lower biochemical recurrence-free survival in CPC-GENE patients (HR Z 1.4, P Z 0.28) and its aberrant expression was significantly associated with decreased 5-year disease-free survival in publically available MSKCC database (84.5% vs 52%; HR Z 2.19, log-rank test P Z 0.005). Conclusion: We showed for the first time that UCA1 can influence cancer aggression, radiation, and chemotherapy response in PCa, which may occur through altered Akt signaling. Our results also suggest that UCA1 can have prognostic value in PCa. Future work will investigate UCA1 as a therapeutic target and prognostic biomarker for PCa. AF Ghiam is supported by a Canadian Association of Radiation Oncology (CARO) Fellowship Award. Author Disclosure: A. Fotouhi Ghiam: None. S. Taeb: None. X. Huang: None. S. Jahangiri: None. J. Ray: None. C. Hoey: None. E. Fokas: None.
125 Hedgehog Pathway Misactivation Induces CDK6 to Drive Medulloblastoma D. Raleigh,1 A.L. Krup,2 P.K. Choksi,2 and J.F. Reiter2; 1University of California, San Francisco, San Francisco, CA, 2University of California San Francisco, San Francisco, CA Purpose/Objective(s): Misregulation of the Hedgehog (Hh) pathway can cause cancers such as basal cell carcinoma (BCC), the most common cancer in the United States, and medulloblastoma, a common pediatric brain tumor. Gli2 is the principle effector of the Hh transcriptional program, but how Gli2 acts to drive cancer remains unknown. The objective of this study was to identify how misactivation of Gli2 in cancer causes cell proliferation. Materials/Methods: To study the Hh transcriptional program, we generated a novel mouse model of Hh-associated medulloblastoma with a knock-in of the Gli2 allele containing a GFP-tag that (i) is expressed under endogenous regulatory control; (ii) recapitulates Gli2 activity, interactions and localization; and (iii) allows us to efficiently isolate Gli2 and identify target genes. We misactivate the Hh pathway in this system using SmoM2c, an allele encoding an oncogenic form of the Hh pathway-activator Smoothened (Smo). Cre under the control of Math1 regulatory sequences conditionally expresses SmoM2 in medulloblastoma progenitor cells in the external granule layer. All Gli2-GFP; SmoM2c; and Math1-Cre mice develop neonatal cerebellar tumors. Protein and transcript levels were measured by immunoblot and qRT-PCR, respectively. All experiments were performed with at least 3 biologic replicates, repeated 3 times, and compared by t-tests. Data are displayed as mean SEM. Results: RNA sequencing of mouse Hh-associated medulloblastoma and comparison to human tumors demonstrates 98 33-fold enrichment of the cell cycle-activator Cdk6 in the setting of retinoblastoma (Rb) hyperphosphorylation and enhanced E2F transcription factor activity. To test if Cdk6 is a direct target of Gli2, NIH3T3 cells expressing Gli2-GFP (3T3GG) were treated with Smoothened agonist (SAG), the E2F inhibitor HLM006474 (HLM), or the translational inhibitor cycloheximide (CHX). Both HLM006474 and CHX block SAG-mediated induction of all cell cycle transcripts except Cdk6, suggesting that Cdk6 represents the point of cell cycle entry for the Hh transcriptional program. Gli2-GFP chromatin immunoprecipitation from mouse Hh-associated medulloblastoma and 3T3GG cells treated with SAG reveals Gli2 bound to the Cdk6 promoter in a manner that is reversed by the Smo inhibitor vismodegib. Consistently, a minimal Cdk6 promoter reporter was sufficient to respond to Hh signals in 3T3GG cells. To test if Cdk6 drives cell proliferation in Hh-associated medulloblastoma, P21 Gli2-GFP; SmoM2c; Math1-Cre mice were gavaged with the Cdk4/6 inhibitor palbociclib (150 mg/g; n Z 8) or vehicle control (n Z 9) every day for 2 weeks. Palbociclib reduced tumor weights by 40 1% (P < 0.05) and was associated with histopathologic changes indicative of tumor cell differentiation. Conclusion: Cdk6 is a direct target of Gli2 and drives proliferation of Hhassociated cancer. Outcomes with genetic inhibition of Cdk6 and palbociclib/vismodegib combination therapy in mouse Hh-associated medulloblastoma are under investigation. Author Disclosure: D. Raleigh: None. A.L. Krup: None. P.K. Choksi: None. J.F. Reiter: None.
126 Does Whole-Brain Radiation Therapy for Oligometastatic Brain Metastases Translate Into a Survival Benefit for Patients With a Limited Competing Risk From Extracranial Disease? A Secondary Analysis of EORTC 22952-26001 T.M. Churilla,1 E. Handorf,1 R. Soffietti,2 M. Kocher,3 A.A. Aizer,4 L. Collette,5 S. Collette,5 Y. Dong,1 B.M. Alexander,6 and S.E. Weiss1; 1 Fox Chase Cancer Center, Philadelphia, PA, 2University of Turin and City of Health and Science Hospital, Torino, Italy, 3Radiotherapy and Oncology