The Retinoblastoma Tumor Suppressor Modulates DNA Repair and Radioresponsiveness

Published OnlineFirst August 27, 2014; DOI: 10.1158/1078-0432.CCR-14-0326

Clinical Cancer Cancer Therapy: Preclinical Research

Chellappagounder Thangavel1, Ettickan Boopathi2, Steve Ciment1, Yi Liu1, Raymond O' Neill3, Ankur Sharma4, Steve B. McMahon4,5, Hestia Mellert4,5,6,7, Sankar Addya5,8, Adam Ertel5,8, Ruth Birbe3, Paolo Fortina5,8, Adam P. Dicker1,5, Karen E. Knudsen1,4,5,9, and Robert B. Den1,4,5

Abstract Purpose: Perturbations in the retinoblastoma pathway are over-represented in advanced prostate cancer; retinoblastoma loss promotes bypass of first-line hormone therapy. Conversely, preliminary studies suggested that retinoblastoma-deficient tumors may become sensitized to a subset of DNA-damaging agents. Here, the molecular and in vivo consequence of retinoblastoma status was analyzed in models of clinical relevance. Experimental Design: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of retinoblastoma using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts. Results: Loss of retinoblastoma enhanced the radioresponsiveness of both hormone-sensitive and castrate-resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. Retinoblastoma loss led to alteration in DNA damage repair and activation of the NF-kB pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of retinoblastoma-deficient tumors exhibited diminished tumor mass, lower PSA kinetics, and decreased tumor growth after treatment with ionizing radiation (P < 0.05). Conclusions: Loss of retinoblastoma confers increased radiosensitivity in prostate cancer. This hypersensitization was mediated by alterations in apoptotic signaling. Combined, these not only provide insight into the molecular consequence of retinoblastoma loss, but also credential retinoblastoma status as a putative biomarker for predicting response to radiotherapy. Clin Cancer Res; 20(21); 5468–82. 2014 AACR.

Introduction 1Department of Radiation Oncology, Thomas Jefferson University, Phila- delphia, Pennsylvania. 2Department of Surgery, Division of Urology, Uni- The retinoblastoma protein (RB1) is a tumor suppressor versity of Pennsylvania, Glenolden, Pennsylvania. 3Department of Pathol- protein and is functionally inactivated in several major ogy, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania. 4Department of Cancer Biology, Thomas Jefferson Univer- cancers (1). Retinoblastoma belongs to the pocket protein sity, Philadelphia, Pennsylvania. 5Kimmel Cancer Center, Thomas Jeffer- family (pRb, p107, and p130), whose members have a 6 son University, Philadelphia, Pennsylvania. Biomedical Graduate Studies, pocket for the functional binding of other proteins and University of Pennsylvania, Philadelphia, Pennsylvania. 7Department of Molecular, Cellular and Developmental Biology, University of Colorado at while present throughout the cell cycle, its function is Boulder, Boulder, Colorado. 8Cancer Genomics, Thomas Jefferson Uni- regulated in a cell-cycle–dependent manner (2). In quies- versity, Philadelphia, Pennsylvania. 9Department of Urology, Thomas Jef- ferson University, Philadelphia, Pennsylvania. cent cells, retinoblastoma is hypophosphorylated and forms a repressive transcriptional complex on E2F-regulated Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). gene promoters to inhibit cell cycle. However, in response to mitogenic signals, retinoblastoma phosphorylation E. Boopathi and S. Ciment contributed equally to this article. disrupts the retinoblastoma–E2F interactions facilitating K.E. Knudsen and R.B. Den share senior authorship of this article. G1–S cell-cycle progression. Corresponding Authors: Karen E. Knudsen, Department of Cancer Biol- Retinoblastoma is a key regulator of multiple cellular ogy, Urology, and Radiation Oncology, Kimmel Cancer Center, Sidney functions including cell proliferation, apoptosis, differen- Kimmel Medical College, Thomas Jefferson University, 233 South 10th St, BLSB 1008, Philadelphia, PA 19107. Phone: 215-503-8574; Fax: 215-923- tiation, genome integrity, quiescence, senescence, and DNA 4498; E-mail: [email protected]; and Robert B. Den, E-mail: repair (3–6). Retinoblastoma function is altered in several [email protected] tumor types through distinct mechanisms, which are often doi: 10.1158/1078-0432.CCR-14-0326 tissue speciﬁc. Within prostate cancer, RB1 loss is deregu- 2014 American Association for Cancer Research. lated in approximately 5% of primary tumors, and up to

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addition, the results are recapitulated using human xeno- Translational Relevance grafts. Together, these in vitro and in vivo data reveal a new Loss-of-function of the retinoblastoma tumor sup- paradigm for the role of retinoblastoma in regulating cell pressor protein in prostate cancer yields a castrate-resis- survival in prostate cancer after treatment with radiotherapy tant phenotype. Given the survival benefit of radiother- and reveal the potential to personalize therapy in prostate apy in the management of men with advanced prostate cancer patients based on retinoblastoma status. cancer, one outstanding issue is the impact of retinoblastoma function on sensitization to DNA-damaging Materials and Methods agents. In this study, we show that retinoblastoma loss Cell culture promotes sensitization to genotoxic stress through LNCaP and C4-2 cells were maintained in improved mechanisms distinct from cell-cycle checkpoint control minimum essential medium supplemented with 5% FBS and identify retinoblastoma as a potent effector of the (heat-inactivated FBS). LAPC4 cells were maintained in response to radiotherapy. The findings of this study Iscove modified Dulbecco medium supplemented with suggest that future radiation trials should interrogate 10% DFBS. 22Rv1 cells were maintained in RPMI supple- retinoblastoma status as a potential biomarker of ther- mented with 10% FBS (Atlanta Biologicals). For steroid- apy response. depleted conditions, cells were plated in appropriate phe- nol red–free media supplemented with 5% to 10% CDT (GE Healthcare Life Sciences, Hyclone Laboratories).

30% to 40% in metastatic or castration-resistant prostate Immunofluoresence analysis cancer (CRPC) samples (7). Loss of retinoblastoma func- Immunofluorescence staining was performed as previ- tion, most commonly via allelic loss, facilitates develop- ously described (10). Immunolocalization of gH2AX, ment of resistance to hormone ablative therapies (8). 53BP1, cleaved caspase-3, and NFkBp50 was carried out Retinoblastoma status has also been shown to alter by using a confocal microscopy (Nikon, Core Facility at response to genotoxic insults (9). Despite challenge with Thomas Jefferson University, Philadelphia, PA). various chemotherapeutics, retinoblastoma-deficient MEFs failed to halt cell-cycle progression resulting in incorrect Cell growth assay DNA repair and cell death. However, within the context of Retinoblastoma-proficient and -deficient LNCaP, LAPC4, prostate cancer cells, retinoblastoma loss failed to alter cell C4-2, and 22Rv1 cells were seeded at equal densities (1 growth despite challenge with an HDAC inhibitor and, 105), exposed to IR (PanTakOrthovoltage X-ray irradiator, surprisingly, lead to resistance with cisplatin exposure calibrated daily using a Victoreen dosimeter), and harvested in vitro (10). Moreover, treatment with antimicrotubule at indicated time points. At the time of harvest, cell number agents and a topoisomerase inhibitor yielded increased was determined using Trypan blue exclusion dye by using a sensitivity in the retinoblastoma-depleted cells suggesting hemocytometer. Cells were seeded at the above densities that cellular response to therapeutic intervention in prostate and transfected and infected with PLK3 cDNA (Addgene) or cancer cells is agent specific. Radiotherapy is a well-estab- adenovirus harboring IkBa DN (SA mutation; Vector lished treatment modality for localized and locally Biolabs). advanced prostate cancer. However, the role of radiotherapy has expanded with the introduction of radium-223 (11), RNA isolation and microarray analysis which has yielded an improvement in survival in men with Actively growing retinoblastoma-proficient and retino- metastatic castrate-resistant prostate cancer. Despite the blastoma-deficient LNCaP cells were exposed to IR (10 Gy) high frequency of retinoblastoma inactivation, few studies and the cells were harvested 24 hours after IR administration have addressed the impact of this event on cellular response (three independent biologic replicates). Total RNA was to ionizing radiation (IR). extracted using TRIzol reagent (Invitrogen, Life Technolo- Herein, we delineated the impact of retinoblastoma gies). Microarray was carried out as described (13). A 1.5-fold function on response to IR using a panel of human isogenic differentially expressed gene list was generated. The differ- prostate cancer lines with stable knockdown of retinoblas- entially expressed gene list was loaded into Ingenuity Path- toma. In this study, we show for the first time that loss of way Analysis (IPA) 8.0 software (http://www.ingenuity.com) retinoblastoma function results in increased radiosensitiza- to perform biologic network and functional analyses and tion of human prostate cancer cells, using both short-term genes were highlighted. Microarray data was deposited in growth as well as clonogenic survival assays. Furthermore, NCBI/GEO website and the GEO accession number the increased sensitivity is mediated through alterations in GSE58711. P < 0.05 was considered as statistically significant. both apoptotic as well as DNA damage and repair pathways. Microarray validation was conducted using quantitative real- Furthermore, the study identified a key mechanism of time (qRT)-PCR and quantified using a DDCt method. NF-kB–mediated cellular apoptosis through polo-like kinase 3 (PLK3) modulation. PLK3 is a cytokine inducible Flow cytometry analysis kinase and has been shown to function as potent inducer of Cell proliferation was assessed by bromodeoxyuridine apoptosis via NF-kB binding to the PLK3 promoter (12). In (BrdUrd) incorporation using flow cytometry as previously

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described (14). Flow cytometry analysis was performed (GE Life Technologies). Retrovirus encoding shRb plasmid Healthcare) and the data were analyzed by using FlowJo (MSCV-LMP Rb88; targeted sequence: 5-GAAAGGCATGT- version 8.8 software. GAACTTA-3) or control plasmid (MSCV donor) were used to create retinoblastoma knockdown or control LAPC-4 Clonogenic assay stable clones. After selection with puromycin for 6 to 7 Clonogenic survival assays were carried out as previously days, stable clones were isolated and characterized. Puro- described (15). Only colonies of 50 or more cells were mycin-selected clones were subjected to retinoblastoma counted. Three replicates per dose were studied. Survival mRNA (qRT-PCR) and retinoblastoma protein analysis curves were generated. The surviving fraction value was (retinoblastoma immunoblotting) and selected retinoblas- corrected for cellular multiplicity to provide single-cell toma-deficient clones were further utilized for the study. survival. Comet assay Western blotting analysis Alkaline comet assay was performed as described in ref. For protein analysis, cells were harvested by trypsiniza- (16) using the Trevigen comet assay kit (Trevigen Inc). tion and cell lysis was performed. The membranes were Slides were visualized using epifluorescence microscopy. immunoblotted for pRB (BD Biosciences), actin, LaminB, The images were analyzed using CometScoring software p21, NFkB p50, NFkB p65, IkBa cleaved caspase-3 (Santa (TriTek Corp). Cruz Biotechnology Inc and Abcam), PLK3 (Sigma- Aldrich), DNA ligase IV (Abcam), CDC25A, CDK2, and Transcription factor ELISA p53 (Santa Cruz Biotechnology Inc) by standard techniques Transcription factor ELISA (TF ELISA) were performed as and visualized using enhanced Western Lightening Chemi- previously described (17). The protein–DNA complex was luminescence (Perkin-Elmer Life Sciences). detected by chromogenic substrate. Absorbance of the samples was measured at 450 nm using microplate reader Chromatin immunoprecipitation assay (Bio-Tek I Spectrophotometer instruments). Following IR, as described above, LNCaP and LAPC4 cells were cross-linked with formaldehyde and processed for Xenografts and IR treatments chromatin immunoprecipitation (ChIP) analysis as previ- Xenografts were generated as described previously (8). ously described (8). Equal concentrations of chromatin Retinoblastoma-proficient and -deficient LNCaP cells (4 from all treatment groups were precleared with protein 106) were individually mixed (1:1) with Matrigel in a Agarose beads in the presence of BSA to reduce nonspecific 200 mL volume (BD Biosciences) and the cells were background. After removal of beads by centrifugation, 2 mg implanted subcutaneously into the flanks of NCR/nu/nu of NFkBp50 or NFkBp65 antibodies (Santa Cruz Biotech- (athymic) male mice. Once the tumors reached 150 mm3 nology) were added and kept at 4C for overnight on a volume, retinoblastoma-proficient and retinoblastoma- rotary platform. The immunoprecipitated DNA was puri- deficient tumors were exposed to IR (10 Gy, PanTa- fied using PCR purification kit (Qiagen) and resuspended in kOrthovoltage X-ray irradiator). Tumor volumes were 50 mL of sterile water. The purified DNAs and input DNAs measured weekly with calipers, serum prostate-specific were analyzed by semiquantitative PCR using PLK3 pro- antigen (PSA) levels were determined, and PSA doubling moter targeting by using a forward 50-GCC CGT GTC TAG times were calculated. In shRB xenografts, maintenance of CAT TTG AG-30 and a reverse primer 50-CCA TCA CAC CCG retinoblastoma knockdown in vivo was verified by qRT- GCT AAT TT-30 sequences, as described in refs. (8, 12). Input PCR analysis of the human RB1 transcript as described in DNA served as a positive control, whereas rabbit IgG and a ref. (8, 18). Retinoblastoma transcript levels as measured non-E2F/retinoblastoma target albumin promoter served as by qRT-PCR ranged from 10% to 90%. Maintenance of negative controls. PCR products were resolved on agarose retinoblastoma silencing was defined as having 30% or gel and the images were captured using Bio-Rad HemiDoc less of RB1 transcript level when compared with wild-type Imager (Bio-Rad Laboratories Inc), the band intensities xenograft tumors. Thirty percent of tumors failed to were measured using ImageJ, and the results were presented maintain loss of retinoblastoma transcript and were not graphically using GraphPad prism version 6 (GraphPad included in the analysis given the uncertainty of the Software, Inc.). The quantitative PCR results were quanti- timing of retinoblastoma function. Animal studies were tated using the DDCt method. conducted in accordance with the principles and proce- dures outlined by the NIH guidelines and the Institution- Retinoblastoma knockdown al Animal Care and Use Committee of Thomas Jefferson Retinoblastoma knockdown was carried out as described University (Philadelphia, PA). in ref. (8). Control and knockdown LNCaP, LAPC4, 22RV1, and C4-2 cells were generated by transfection with PLK3 ectopic expression and knockdown either shRNA plasmid directed against retinoblastoma PLK3 cDNA transfection and knockdown was carried as (MSCV-Rb3C; targeted sequence: 50-CGCATACTCCGGT- described in the manufacturer’s protocol. Briefly, 10 mgof TAGGACTGTTATGAA-30) or a control plasmid (MSCV PLK3 cDNA (Addgene) or control vector (PCDNA3) or donor) using Lipofectin Transfection Reagent (Invitrogen, PLK3 shRNA (Applied Biological Materials, Inc.) or shRNA

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vector alone were transiently transfected in LNCaP shCon or tasis samples. Pearson correlation coefficient was calculated LNCaP shRB or LAPC4 shCon or LAPC4 shRB cells with within normal prostate and prostate cancer groups. RB1 Lipofectamine 2000 (Invitrogen, Life Technologies). The transcript levels were used to infer prostate cancer sample PLK3 overexpression or PLK3 knockdown was confirmed by retinoblastoma status, where retinoblastoma-deficient sam- PLK3 immunoblotting. The cells expressing PLK3 or PLK3- ples were defined below the median RB1 expression and deficient cells were used for generating growth curve with retinoblastoma-proficient samples were defined above the radiation or no radiation. Cells were also analyzed for median. A two-tailed t test for populations of unequal cleaved caspase-3 immunoblotting. variance was used to evaluate the significance of differential PLK3 expression in retinoblastoma-proficient versus nor- Ectopic expression of dominant-negative IkBaSA mal and retinoblastoma-deficient versus retinoblastoma- shRB cells (LNCaP and LAPC4) were infected with ade- proficient groups. novirus harboring dominant-negative IkBaSA (Vector Bio- labs) and exposed to IR 10 Gy, processed for cell growth Statistical analysis assay and cleaved caspase-3 apoptotic marker immunoflu- Statistical analyses were performed using GraphPad orescence, and Western blotting analysis. Prism (version 6.0) software (GraphPadPrism Software, Inc.). All the data were analyzed for statistical significance Retinoblastoma immunohistochemical analyses using Student t test/one-way ANOVA. For all experiments, Retinoblastoma immunohistochemistry was performed P < 0.05 was considered statistically significant. as described in ref. (8, 19). Eleven patients were identified in our institution that had biopsy-proven local recurrence Results following primary radiotherapy (5 patients received exter- Retinoblastoma status dictates the cellular response to nal beam radiotherapy and received brachytherapy). Biopsy radiation in both early- and late-stage cancers specimens or whole mount glands were used for immuno- The retinoblastoma tumor suppressor protein (pRB) histochemical analyses. Five micrometer sections were pathway is a key regulator of cell cycle in coordination deparaffinized, antigen retrieval was performed in 10 with E2Fs. Retinoblastoma inactivation occurs during mmol/L EDTA (pH 9) for 10 minutes in a pressure cooker, prostate cancer progression and has been correlated with and slides were incubated with 3% H2O2 for 10 minutes and poor outcome (8). In human samples, retinoblastoma then blocked with avidin/biotin blocking solution (Vector loss-of-function occurs commonly via loss of heterozygos- Biolabs) for 30 minutes and incubated in a 5% chicken/ ity (20). To delineate the role of retinoblastoma in mod- goat/horse serum solution for 2 hours. Sections were incu- ulating the response to radiotherapy, clinically relevant bated with anti-retinoblastoma antibody overnight at 4C hormone-sensitive and castrate-resistant human isogenic [(4H1) Mouse mAb #9309, Cell Signaling Technology, Inc.; prostate cancer cell lines were utilized that expressed either concentration 1:200]. Negative control slides were incubat- shRB or control as previously described (8). Retinoblas- ed with mouse anti-MOPC21 (generated from a hybridoma toma knockdown was verified using protein analysis across obtained from ATCC) at the same concentration as the multiple prostate cancer cell lines (Fig. 1A and B, left). primary antibody. Slides were then incubated with horse First, in vitro clonogenic assays were performed. In the anti-mouse biotinylated secondary antibody (1:150, Vector presence of androgen, retinoblastoma-deficient LNCaP Biolabs) for 30 minutes, developed using Vectastain ABC and LAPC4 cells showed increased radiosensitivity across (Vector Biolabs) and stable DAB (Invitrogen, Life Technol- all radiation doses studied, as compared with the isogenic ogies) counterstained with hematoxylin, dehydrated, and retinoblastoma-proficient pairs. Statistical significance was mounted with Cytoseal XYL (Richard Allan Scientific, observed after both 8 and 10 Gy exposure, indicating Thermo Fisher Scientific). The stain was interpreted as the conservation of this result across multiple ranges of DNA percentage of tumor cells with nuclear staining and inten- damage (Fig. 1C). Short-term growth assays using 10 Gy of sity of staining as 3þ (strong staining similar to positive IR confirmed these findings, as control LNCaP and LAPC4 control), 2þ (moderate staining), 1þ (weak staining), and 0 demonstrated greater viable cell numbers than their iso- (no staining). Photomicrographs were taken by using bright genic pairs (Fig. 1A) in both hormone-enriched and hor- field microscope (20) and the scoring was performed by a mone-free media. Given that retinoblastoma knockdown clinical pathologist (an experienced staff pathologist; had been demonstrated to result in a growth advantage in a Thomas Jefferson University Hospital, Philadelphia, PA). hormone-free environment (10), short-term growth assays were performed with isogenic pairs (shRB and shControl) Analysis of PLK3 and RB1 correlation in prostate derived from castrate-resistant prostate cancer–deficient cancer C4-2 and 22Rv1 cell lines (Fig. 1B) in both hormone- A normalized mRNA expression dataset of prostate ade- competent and hormone-free conditions. Together, these nocarcinoma (7) was downloaded from the cBioPortal results suggest that retinoblastoma loss sensitizes prostate for cancer genomics (http://www.cbioportal.org/public- cancer to IR, irrespective of hormone microenvironment. portal/) and was used to evaluate coexpression of RB1 and Given the prominent role of cell cycle in the manifestation PLK3 transcript levels. This dataset includes mRNA profiles of DNA damage, the impact of cell-cycle checkpoints was for 29 normal prostate, 131 primary tumor, and 19 metas- investigated.

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A LNCaP (IR) C 1 shCon shRB shCon LNCaP 0.25 shRB 0.1 0.20 * * shRB shCon 0.01 0.15 * * RB * * * * * 0.10 * 0.001 * * Actin * (LNCaP) 0.05

Surviving fraction 0.0001

Lane 1 2 Relative cell number (normalized to 0 Gy) 0.00 Hormone Hormone 0.00001 proficient deficient 86420 10 IR dose (Gy) LAPC4 LAPC4 (IR)

1.0 shCon shRB 1 shCon shCon shRB 0.8 shRB * * 0.1 RB 0.6 * * * 0.4 * 0.01 Actin * * 0.2 (LAPC4) 0.001

Lane 1 2 Relative cell number (normalized to 0 Gy) 0.0 Surviving fraction Hormone Hormone 0.0001 proficient deficient 86420 10 IR dose (Gy)

B C4-2 (IR) C4-2 0.15 shCon shRB shCon shRB 0.10 RB * * * * 0.05 Actin Relative cell number (normalized to 0 Gy) Lane 1 2 0.00 Hormone Hormone proficient deficient

22Rv1 (IR)

22Rv1 0.15 shCon shRB 0.10 RB * * * * Actin 0.05 Lane 1 2 (normalized to 0 Gy) Relative cell number 0.00 Hormone Hormone proficient deficient

Figure 1. Retinoblastoma (RB) status dictates the cellular response to radiation in both hormone-sensitive and castrate-resistant cancers. Actively growing LNCaP, LAPC4, C4-2, and 22RV1 cells were exposed to radiotherapy and processed for further analysis. A, retinoblastoma Western blotting analysis in hormone-responsive LNCaP and LAPC4 cells and actin loading control (top left). Cell number analysis of hormone-responsive LNCaP and LAPC4 cells in hormone-enriched and hormone-free media in response to radiotherapy (10 Gy; top right). B, retinoblastoma Western blotting analysis in castrate-resistant C4-2 and 22Rv1 cells and actin loading control (bottom left). Cell number analysis of C4-2 and 22Rv1 cells in hormone-enriched and hormone-free media in response to radiotherapy (10 Gy; bottom right). C, clonogenic assay in LNCaP and LAPC4 cells. Each data point is a mean SD from three or more independent experiments. , P < 0.05 were considered as statistically signiﬁcant.

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A IR (10 Gy) B shCon shRB # Biological function P

1 Cancer 1.74E−04-2.14E-02 2 Cell-to-cell signaling 1.27E−04-2.14E-02 − 3 Cell- death and survival 5E-04 2.14E-02 − 4 Cellular movement 3.24E 04-2.14E-02 − 5 Cell morphology 4.36E 03-2.14E-02 6 DNA replication, recombination, and 2.95E−05-2.14E-02 repair 7 Cell cycle 1.84E−03-2.14E-02 8 Cellular growth and proliferation 2.61E−03-2.98E-02 9 Posttranslational modifications 2.14E−02-2.14E-02 10 Cellular function and maintenance 1.35E−03-2.14E-02

C SOCS3 TICAM1 CYP3A5 BIK 4 * * * * * * * * IGFR1 2

0 shCon+IR (10 Gy) shRB+IR (10 Gy) −2 Relative mRNA expression * *

Color range

−2.5 0 2.5

Figure 2. Retinoblastoma (RB) status alters the transcriptional response to DNA damage. Actively growing LNCaP and LAPC4 cells were exposed to radiotherapy and processed for further analysis. A, microarray analysis (in silico) generated heatmap of 1,131 differentially regulated genes in retinoblastoma- proficient and deficient LNCaP cells 24 hours post-IR (10 Gy). B, IPA software generated deregulated functional pathways in retinoblastoma-proficient and deficient LNCaP cells in response to radiotherapy. C, qRT-PCR validation of functionally important genes from the microarray data. Each data point is a mean SD from three or more independent experiments. , P < 0.05 were considered as statistically significant.

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10.0 shCon shRB A LNCaP + IR (10 Gy) * * 7.5 shCon shRB D LNCaP 5.0 VIM γ IGFR1 -H2AX 53BP-1 DAPI Merge FN1 2.5 DNAPTP3 * * SRC LOC100 Relative γ -H2AX foci 0.0

VIM shCon AKAP13 0 4 8 24 AKAP13 AKAP13

IR (0 Gy) Post-IR (h) SOCS3 LZTS2 shRB 10.0 KCD13 shCon shRB RLN2 TREX1 PIK3R1 7.5 ORC5 REV3L shCon BIK 5.0 BMX

TBRG1 IR (10 Gy) PDGFA shRB 2.5 POLB SDC2

MLH3 Relative 53BP1 foci ING5 0.0 PMS1 0 4 8 24 JAK2 ETV6 Post-IR (h) PMS2 TYMP POLK ATRIP shCon shRB POLK 15 SOCS3 * * SYCP3 PRKRA MSH5 LAPC4 10 MSH5 E GCG Color range γ-H2AX 53BP-1 DAPI Merge 5 −0.6 0 0.6 LNCaP shCon + IR LNCaP shRB + IR Relative γ -H2AX foci (10 Gy) (10 Gy) shCon 0 B 0 4 8 24 IR (0 Gy) Post-IR (h)

5 shRB 15 shCon shRB 4 * *

shCon 10 3

IR (10 Gy) 5 2 shRB Relative 53BP1 foci Relative mRNA 1 0 0 4 8 24 0 Post-IR (h) POLB POLK REV3L SYCP3 F Post-20 Gy IR (min) Post-20 Gy IR (min) No IR 0 15 30 No IR 0 15 30

C shCon shCon LAPC4 LNCaP LAPC4 LNCaP shRB shRB 100 shCon shRB 40 shCon shRB ** LNCaP LAPC4 shCon shRB shRB shCon 80 30 * * 60 * * DNA ligase IV 20 * * 40 * * * * 10 LaminB 20 Median tail DNA (%) Median tail DNA (%) 0 1 2 3 4 Lane 0 No IR 0 15 30 No IR 0 15 30 IR 10 Gy Post-20 Gy IR (min) Post-20 Gy IR (min)

Figure 3. Retinoblastoma (RB) loss alters DNA damage and repair capacity in response to radiation. Actively growing LNCaP and LAPC4 cells were exposed to radiotherapy and processed for further analysis. A, in silico analysis generated heatmap of DNA damage and repair pathway genes from retinoblastoma- proﬁcient and deﬁcient LNCaP cells after 24 hours post-IR (10 Gy). (Continued on the following page.)

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The impact of retinoblastoma on the radiation ficient and -deficient LNCaP cells were exposed to IR and response is independent of p53 followed by mRNA microarray analysis. After background DNA damage induces cell-intrinsic checkpoints, including correction and normalization, a gene list consisting of 1,131 p21, p53, and retinoblastoma (21). Further prior reports differentially (at least 1.5-fold change in expression) regu- demonstrated that p53 and retinoblastoma can exert par- lated genes was observed (Fig. 2A). Using biologic network tially overlapping roles in the setting of inactivation of either and functional analysis, gene ontology demonstrated that tumor suppressor (22). Thus, complementary upregulation 11 important functional pathways were significantly altered of p21 or p53 may occur in the setting of retinoblastoma (Fig. 2B) including cell death and survival as well as DNA knockdown after exposure to IR. In prostate cancer models, replication, recombination, and repair, as a result of reti- upregulation and stabilization of p53 has been correlated noblastoma knockdown in the setting of DNA damage. An with the induction of apoptosis (23). However, no difference extended qRT-PCR validation was performed (Fig. 2C), in either protein induction or stabilization was observed for confirming the findings of the microarray. Together, these either p21 or p53 after exposure to DNA damage (Supple- data suggest that cell survival may be regulated through mentary Fig. S1A and S1B) in either LNCaP or LAPC4 alterations of DNA damage/repair and increased apoptosis regardless of retinoblastoma status. These findings indicate in retinoblastoma-deficient cells. that cell proliferation and cell-cycle control may be altered in retinoblastoma knockdown cells given the lack of compen- Retinoblastoma loss alters DNA damage repair sation for other prominent checkpoint regulators. pathway Microarray transcript analysis demonstrated that DNA Retinoblastoma status does not impinge on the damage and repair pathway genes were elevated in reti- alterations of cell cycle after radiation noblastoma-deficient LNCaP cells (Fig. 3A). IR results in Given the prominent role of retinoblastoma as a G1 cell- multiple DNA aberrations including base excision as well cycle checkpoint through the regulation of E2F family tran- as double-strand DNA breaks (24), and microarray val- scription factors, it was hypothesized that retinoblastoma idation reveals that multiple base excision repair genes knockdown would alter cell-cycle regulation after radiation. including POLB, POLK,andREV3L (Fig. 3B) as well as the Furthermore, in multiple cell lines with disrupted retino- DNA damage-induced meiotic recombination gene blastoma function, it has been consistently shown that SYCP3 (25) were upregulated and alternatively DNA retinoblastoma deficiency allows cells to efficiently bypass ligase IV was downregulated (Fig. 3C) in shRB cells in the cell-cycle inhibitory response to DNA-damaging chemo- the setting of radiation-induced damage (5). DNA ligase therapeutic agents such as cisplatin (9). In both retinoblas- IV is an ATP-dependent DNA ligase that joins double- toma-proficient and -deficient prostate cancer cells, exposure strand breaks during the nonhomologous endjoining to IR resulted in a G1 cell-cycle arrest. However, it was pathway of double-strand break repair (26). These find- surprising that no differential response in cell-cycle progres- ingssuggestthatretinoblastomamayimpacttheDNA sion as measured by BrdUrd incorporation was noted after repair machinery. Given that SYCP3, gH2AX, and 53BP1 exposure to IR regardless of retinoblastoma status (Supple- are involved in DNA repair via similar mechanisms, we mentary Fig. S1C–S1F). No differences were observed in assessed the ability to repair DNA double-strand breaks CDC25A expression regardless of retinoblastoma status with (27). gH2AX and 53BP1 foci were significantly elevated or without exposure to IR (Supplementary Fig. SS1G). Upre- in shRB LNCaP cells as compared with control cells, gulation of CDK2 expression occurred in retinoblastoma- indicating that radiation-induced DNA damage depends deficient cells, but was not altered by exposure to IR (Sup- on the retinoblastoma status of the cells (Fig. 3D, left with plementary Fig. SS1G). Given that alterations in cell survival quantification on right panel). The results were recapit- do not appear to be accounted through alterations in cell- ulated in LAPC4 (Fig. 3E); shRB cells showed markedly cycle proliferation or cell-cycle checkpoint response, other diminished capacity to repair double-strand breaks up to mechanisms were explored, which could putatively impinge 24 hours after treatment. Comet assay recapitulated these on cell survival after genotoxic insult. findings demonstrating that retinoblastoma deficiency radiosensitizes prostate cancer cells as shown with longer Retinoblastoma status alters the transcriptional tail moments indicating higher amounts of DNA breaks response to DNA damage (Fig.3F).Takentogether,markersofDNAdamagesup- Given that cell survival is a complex process and regulated port the hypothesis that retinoblastoma loss alters DNA by multiple underlying mechanisms, retinoblastoma-pro- repair mechanisms. Thus, these data reveal for the first

(Continued.) B, microarray validation of POLB, POLK, REV3L, and SYCP3 transcripts in LNCaP cells exposed to IR. C, immunoblot analysis of DNA ligase IV in LNCaP and LAPC4 (shCon and shRB) cells in response to 10 Gy IR. D, confocal microscopic images of DNA damage-induced gH2AX and 53BP1 foci (left) and a graphic representation of gH2AX and 53BP1 foci (right) in retinoblastoma-proficient and deficient LNCaP cells post-IR. E, confocal microscopic images of DNA damage-induced gH2AX and 53BP1 foci (left) and a graphic representation of gH2AX and 53BP1 foci (right) in retinoblastoma-proficient and deficient LAPC4 cells post-IR (10 Gy). F, photomicrographs of alkaline comet assay in LNCaP shCon, shRB LAPC4 shCon, and shRB exposed to 20 Gy radiation (top) and graphic representation of alkaline comet assay (bottom). For each data point there is a mean SD from three or more independent experiments. , P < 0.05 were considered as statistically significant. Scale bar "" ¼ 20 mm.

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A LNCaP LAPC4 D shRB shRB shCon shCon shRB shRB NF-κB p50 LNCaP shCon+Con Ad shCon+IKBA DN NF-κB p65 shRB+CON Ad

) 50 shRB+IKBA DN 4 Nuclear extract 40 Cont Ad Cont Ad LaminB Ad IKB-α DN Ad IKB-α DN 30 α κ IKB- NF- B p50 20 * *

10 * NF-κB p50 NF-κB p65

Cell number (1×10 Cell number 0 0 24 48 72 κ GAPDH NF- B 65 Cytoplasmic extract 1 2 1 2 Lane Post 10 Gy IR (h) GAPDH B IR 10 Gy Cytoplasmic extract NF-κB p50 DAPI Merge LAPC4 NF-κB p50 shCon+Con Ad )

4 50 shCon+IKBA DN shRB+CON Ad NF-κB p65 40 shRB+IKBA DN 30 LaminB

shCon 20 Nuclear extract 10 * 1 2 1 2 Lane * * 0 Cell number (1×10 Cell number 0 24 48 72 LNCaP LAPC4 IR 10 Gy

shRB Post 10 Gy IR (h) LNCaP + IR (10 Gy)

NF-κB p50 DAPI Merge E No IR

shCon IR 10 Gy LNCaP LAPC4 LNCaP LAPC4

shConshRB shConshRB shConshRB shConshRB Cleaved Cleaved shRB LAPC4 + IR (10 Gy) Caspase-3 Caspase-3 LaminB LaminB C 1.0 * * * * * * * * * * 1 2 1 2 Lane 0.8 shCon + IR (10 Gy) 1 2 1 2 Lane shRB + IR (10 Gy) 0.6 Total cell extract LNCaP Total cell extract 0.4 * * * * OD 450 nm 0.2

0.0 1X 5X 20X 50X 1X 5X 20X 50X F Cleaved CASP3 DAPI Merge Wild-type NF-κB p50 Mutant NF-κB p50 oligo concentration oligo concentration 1.0 * * * * * * * * 0.8 shCon + IR (10 Gy) * * shRB + IR (10 Gy) shRB+Con Ad shRB+Con 0.6 LAPC4

0.4 DN * * α OD 450 nm 0.2 * * LNCaP+ IR (10 Gy) 0.0 1X 5X 20X 50X 1X 5X 20X 50X Wild-type NF-κB p50 Mutant NF-κB p50 shRB+IKB- oligo concentration oligo concentration

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time that retinoblastoma promotes DNA double-strand promoter (Fig. 5A). These increased bindings correlated break repair independent of the ability of retinoblastoma with increased expression of the PLK3 transcript in the to regulate cell-cycle progression. setting of retinoblastoma knockdown (Fig. 5B). Further- more, inhibition of NF-kB nuclear translocation resulted Retinoblastoma deficiency leads to activation of in diminished PLK3 expression (Fig. 5C). An in silico NF-kB pathway and cellular apoptosis in response analysis of human prostate tumor samples demonstrated to IR a statistically significant inverse correlation of PLK3 and Retinoblastoma has been shown to function as an anti- retinoblastoma (Fig. 5C) suggesting that retinoblastoma apoptotic factor (28) and microarray analysis demonstrated loss may prime towards a proapoptotic pathway. Over- induction of proapoptotic transcripts in the setting of expression of PLK3 in the setting of retinoblastoma defi- retinoblastoma depletion (Supplementary Fig. S2). Many ciency inhibited growth (Fig. 5D, left and Supplementary of the altered gene transcripts are proapoptotic genes reg- Fig. S4A, left) and induced apoptosis (Fig. 5D, right and ulated via NF-kB including DR4, TRAIL, RIP1 Fas, FasL, Supplementary Fig. S4A, right). Furthermore, knockdown MYC, NOTCH2, and PLK3 (29). The behavior of the tran- of PLK3 in the retinoblastoma-deficient setting reversed scription factor NF-kB as a promoter or antagonist of this phenotype through decreased levels of apoptosis Fig. 5E apoptosis depends on the apoptotic stimulus. Higher total and Supplementary Fig. S4B). Thus, retinoblastoma loss levels as well as increased nuclear translocation of NF-kB results in activation of Plk3, a NF-kB–regulated gene that p50 and p65 were observed in retinoblastoma-deficient induces apoptosis and radiosensitivity. cells in response to radiation (Fig. 4A). Further immunolocalization and TF ELISA analysis reveal that nuclear and Retinoblastoma depletion results in marked in vivo DNA-bound NF-kB p50 was elevated in retinoblastoma- radiosensitization deficient LNCaP and LAPC4 cells (Fig. 4B and C). To mimic To further mimic the clinical setting, the role of retino- NF-kB knockdown, we exogenously expressed a dominant- blastoma in response to IR was explored in vivo retinoblas- negative IkBa (SA), which retains NF-kB in the cytoplasm, toma-proficient and retinoblastoma-deficient LNCaP cells thus preventing NF-kB–mediated downstream signaling. were implanted into nude mice. As previously reported This modulation decreased nuclear translocation (Fig. 4D, (10), no significant growth advantage was noted (Fig. 6A, Western blots) and decreased the radiosensitivity of shRB left) between shCon and shRB LNCaP cells. After reaching LNCaP and LAPC4 cells (Fig. 4D, growth curves). TF ELISA 100 to 150 mm3, irradiation of the isogenic pairs unmasked analysis reveals that nuclear and DNA-bound NF-kB p50 a radiosensitivity advantage specific to retinoblastoma-defi- was diminished in retinoblastoma-deficient LNCaP and cient tumors (Fig. 6A, right). These data suggested that LAPC4 cells with the introduction of the dominant-negative retinoblastoma depletion confers a clear alteration in IkBa (Supplementary Fig. S3A and S3B). Elevated levels of response to IR, as monitored by tumor growth kinetics; NF-kB p50 and p65 protein have been demonstrated to consonantly, there was a significant decrease in tumor mass activate the apoptotic pathway (29). Knockdown of retino- among shRB compared with shCon1 tumors at the time of blastoma resulted in increased apoptosis in the setting of IR sacrifice (Fig. 6B). In addition, serum PSA (also known as (Fig. 4E) and inhibition of NF-kB via IkBa DN diminished KLK3) was monitored. PSA is used clinically as a marker of cleaved caspase-3 levels (Fig. 4F). Thus, retinoblastoma loss prostate cancer detection, burden, and progression (30), results in upregulation and increased nuclear translocation and is not expressed in mice; thus, serum PSA was moni- of NF-kB with subsequent induction of apoptosis in the tored as a measure of tumor growth. Serum PSA levels were setting of IR. significantly low in animals carrying the shRB xenografts (Fig. 6C). In addition, to determine the clinical impact of Modulation of PLK3 alters cellular apoptosis through radiation sensitivity and retinoblastoma status, we retro- an NF-kB–dependent manner spectively identified all cases of biopsy proven local recur- NF-kB proapoptotic signaling has been demonstrated rence following radiotherapy over the past 10 years. Eleven to be mediated via direct binding to the polo-kinase 3 patients were identified (5 cases treated with external beam promoter (12). This activation of PLK3 resulted in induc- therapy and 6 treated with brachytherapy). All samples tion of apoptosis. ChIP assay and qRT-PCR revealed stained positively for retinoblastoma (Supplementary Fig. higher levels of NF-kB p50 and p65 bound to the PLK3 S5) further suggesting the diminished radiosensitivity of

Figure 4. Retinoblastoma (RB) deficiency leads to activation of NF-kB pathway and cellular apoptosis in response to IR. A, immunoblotting analysis nuclear and cytoplasmic NFkB p50, NFkB p65, LaminB, GAPDH (left), and apoptotic marker cleaved caspase-3 (A, right). B, immunolocalization of nuclear NFkB p50 in retinoblastoma-proficient and retinoblastoma-deficient LNCaP cells 24 hours after radiotherapy (10 Gy). C, graphic representation of NFkB p50 binding to consensus sequence by TF ELISA in retinoblastoma-proficient and retinoblastoma-deficient LNCaP cells (10 Gy) in the presence of wild-type oligonucleotides and mutant oligonucleotides 24 hours after radiotherapy (10 Gy). D, growth curve and immunoblotting analysis of cytoplasmic and nuclear NFkBp50, NFkBp65, LaminB, GAPDH, and IkBa in LNCaP and LAPC4 shCon and shRB cells expressing IkBa-DN. E, immunoblotting analysis of cleaved caspase-3 in LNCaP or LAPC4 shCon and shRB cells in the absence (left) and presence (right) of IR. F, immunolocalization of cleaved caspase-3 in LNCaP shRB cells with and without concomitant expression of IkBa-DN in response to radiotherapy. Each data point is a mean SD from three or more independent experiments. , P < 0.05 were considered as statistically significant over shControl. Scale bar "" ¼ 20 mm.

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A shCon+IR (10 Gy) shRB+IR (10 Gy) shCon+IR (10 Gy) shRB+IR (10 Gy) 4.0 * * 4.0 * * * * * * 3.5 Input IgG α-NFkBp50 Input IgG α-NFkBp65 3.5 Input IgG α-NFkBp50 Input IgG α-NFkBp65 3.0 3.0 shRB shRB shCon shCon shRB shCon shCon shCon shCon shCon shCon shCon shRB shRB shRB shRB shRB shRB shRB shCon shCon shCon shRB shRB 2.5 PLK3 PLK3 PLK3 PLK3 promoter promoter 2.5 promoter promoter 2.0 Albumin Albumin Albumin Albumin promoter promoter 2.0 promoter promoter 1.5 1.5 1.0 1.0 (fold over control) 0.5 (fold over control) 0.5

Relative NFkB p50/p65 binding 0.0

Rabbit IgG NF-κBp50 Rabbit IgG NF-κBp65 Relative NFkB p50/p65 binding 0.0 Rabbit IgG NF-κBp50 Rabbit IgG NF-κBp65 LNCaP LAPC4

B LNCaP D shCon + IR (10Gy) shRB shRB shRB +IR (10Gy)

shCon vector shRB vector

3 100 shCon PLK3 shRB PLK3 ) 4 shRB+PLK3 cDNA shRB+Vector shCon+PLK3 cDNA Cont AdAd IKB-α DNCont Ad shCon+Vector Ad IKB-α DN 80 2 PLK3 60 PLK3 1 Relative mRNA LaminB 40 Cleaved Total cell extract LNCaP 1 2 1 2 Lane Caspase-3 0 20 LNCaP PLK3 NOTCH2 * * LaminB LNCaP LAPC4 (1×10 Cell number 0 * * 0 24 48 72 (h) 1 2 3 4 Lane

C Prostate tumor E

RB1 LNCaP − PLK3 R = 0.45 p = 1.03e−08

LNCaP 1.5 PLK3

shRB+shPLK3 shcon1 * 150 shRNA PLK3 shRB+shCon 4 1 PLK3 100 0.5 Cleaved 50 0 caspase-3 * * * * PLK3 expression −0.5 1×10 Cell number 0 LaminB 0 24 48 72

−1 Post IR (h) 1 2 Lane PNA PCA PCA RB+ RB− 10 Gy

Figure 5. Modulation of PLK3 alters cellular apoptosis through NF-kB–dependent manner. A, ChIP assay showing recruitment of NFkB p50 on the PLK3 promoter. B, microarray data validation of PLK3 and NOTCH2 (qRT-PCR; left) and immunobloting analysis of PLK3 in shRB expressing IkBa-DN. C, in silico analysis of RB1 and PLK3 transcripts from human prostate tumor samples. Samples are ordered from low (blue) to high (red) RB1 and PLK3 expression (top). Boxplots show differential expression of PLK3 in normal prostate and tumor, grouped by retinoblastoma (RB) status (bottom; , P ¼ 1.46E5and statistically significant). D, cell growth and immunoblot analysis of PLK3, cleaved caspase-3, and laminB in LNCaP shCon and shRB cells ectopically overexpressing PLK3. E, cell growth and immunoblot analysis of PLK3, cleaved caspase-3, and laminB in PLK3-deficient LNCaP shRB cells. Each data point is a mean SD from three or more independent experiments. , P < 0.05 were considered as statistically significant over shControl.

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A 200 LNCaP shCon 15 shCon shRB )

3 LNCaP shRB LNCaP 150 * * 10 * * 100 5

50 Tumor volume (mm Relative tumor volume 0 0 1 2 3 4 5 21 23 25 27 29 31 33 35 Post-IR (weeks) (Days after post xenograft)

Figure 6. Retinoblastoma (RB) B 4 C deficiency results in marked in vivo LNCaP shCon shRB 15 * * radiosensitization. A, tumor 3 ** volume analysis in LNCaP shCon LNCaP and LNCaP shRB xenografts with 2 10 no radiation (left) and after radiation (right). B, tumor mass (grams) 1 analysis 1 to 5 weeks post-IR Tumor mass * * 5 (5 Gy) in LNCaP shCon and LNCaP 0 PSA Relative shCon shRB shRB xenografts. C, measurement 0 of serum PSA levels 1 to 5 weeks 1 2 3 4 5 post-IR. D, working model Post-IR (weeks) (schematics) shows the NF-kB– mediated apoptosis through PLK3 in retinoblastoma-deficient D Ionizing radiation radiosensitized prostate cancer model. Each data point is a mean SD from 5 or more mice. , P < 0.05 were considered as AR+ statistically significant.

RB loss or inactivation

Upregulation Nucleus Nuclear Transcription translocation p50 p50 p50 p65 p50 Caspase-3 p50

p65 activation

p65 Hetero dimer PLK3 promoter p65 p65 NF k B p50/p65 formation Apoptosis

tumors with intact retinoblastoma. The working model of effective therapeutic strategies and sequencing thera- illustrates that NF-kB pathway drives the radiosensitized pies. The present study identifies for the first time the cells to cellular apoptosis through PLK3 (Fig. 6D). Collec- impact of retinoblastoma status on radiation sensitivity. tively, these results indicate that active retinoblastoma pro- Key findings show that (i) regardless of hormonal envi- motes resistance to radiation, using both in vitro and in vivo ronment, retinoblastoma loss sensitizes both hormone- models of disease progression. sensitive and CRPC to IR both in vitro and in vivo; (ii) retinoblastoma loss alters multiple functionally important pathways critical in the response to radiotherapy Discussion including DNA damage and repair as well as apoptosis; Given the frequency and importance of retinoblastoma (iii) modulation of NF-kB or PLK3 alters the cellular inactivation in prostate cancer, understanding the apoptosis and strongly suggests that retinoblastoma loss response to radiotherapy is crucial for the development correlates with an increased nuclear translocation of NF-

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kB resulting in cellular apoptosis mediated via PLK3 in Notably, retinoblastoma loss is associated with altered response to radiotherapy. AR activity and is causative for the transition to CRPC (8). The concept that retinoblastoma inactivation alters radio- Recent studies from our laboratory and others demonstrat- sensitivity via upregulation of apoptotic pathways as ed that AR is a mediator of double-strand DNA break repair opposed to alterations in cell cycle was unexpected. DNA and can alter cell survival in response to DNA damage (42). damage elicits arrest at both G1 and G2 phases of the cell Data herein demonstrate that retinoblastoma is dominant cycle (31). G1 arrest occurs due to activation of the p53/p21 to these effects and that as mediated by deregulation of regulatory pathway and is dependent on functional retino- apoptotic signaling events, confers a robust radiosensitiza- blastoma (32). Retinoblastoma is a known regulator of cell tion phenotype. proliferation, and functional retinoblastoma is required to Retinoblastoma status may be a viable biomarker from induce a cell-cycle arrest in G1 after DNA damage (9). G2 which to base therapeutic decisions (43). In tumors that arrest generally relies on Chk1-dependent inactivation of retain retinoblastoma function, next-generation cyclin- the cyclin B1/Cdc2 kinase with maintenance of arrest after dependent kinase (CDK) inhibitors may provide a robust genotoxic stress being further regulated by p21, p53, and approach to engage retinoblastoma tumor suppressor retinoblastoma (33). IR induces a G2 arrest in mouse activity and halt cellular proliferation. Both preclinical embryo fibroblasts (MEF) with intact retinoblastoma, while (44) and early phase I trials demonstrate early tolerability MEF with knockout of retinoblastoma continue to prolif- and efficacy (44). For retinoblastoma-deficient cells, use erate and eventually undergo cell death (33). In the current of DNA-damaging agents is intriguing as this study con- study, there were no differential alterations in cell cycle fers with previous reports that retinoblastoma loss confers noted between control and retinoblastoma knockdown hypersensitization to genotoxic stress (10, 45). Clinical prostate cancer cells after exposure to IR. However, retino- observations from breast (46), bladder (47), and head blastoma can inhibit cellular proliferation through distinct and neck cancer (48) support the hypothesis that retino- mechanisms: alterations in cell cycle and induction of cell blastoma-deficient cells have a compromised response to death (34). DNA damage. Within the context of clinical prostate Retinoblastoma inhibits apoptosis in both normal tis- cancer, retinoblastoma function has been incompletely sue as well as tumor models. IR induces apoptosis in defined due to the dual function of retinoblastoma loss SAOS-2 cells, which lack functional retinoblastoma and increasing radiosensitivity while driving castrate-resistant this phenotype is reversed by stable transfection of reti- growth (8). Low p16 expression, which is hypothesized to noblastoma (35). Furthermore, E2F1, which is inactive compromise retinoblastoma function, was associated when present in complex with retinoblastoma, is capable with an increased risk of development of distant metas- of inducing apoptosis (36). Prior studies have demonstrat- tases in RTOG 9202, yet investigation of locally advanced ed that E2F1 and E2F3 are upregulated in the setting of disease indicates that loss of retinoblastoma and loss of retinoblastoma knockdown in prostate cancer (8, 36). p16 are not redundant (49, 50). Given that retinoblas- Furthermore, etoposides sensitize retinoblastoma-defi- toma loss-of-function is noted in the context of metastatic cient prostate cancer (8) and this is mediated via E2F1- castrate-resistant prostate cancer (7), retinoblastoma may mediated sensitivity to apoptotic stimuli. In this study, we serve as a marker of response in the context of palliative demonstrated that proapoptotic pathways were upregu- radiation or radium-223 (11). lated after exposure to IR in the setting of retinoblastoma In summary, the findings herein present a paradigm for loss. Retinoblastoma inactivation primes cells for apopto- retinoblastoma function in protecting prostate cancer sis via induction of procaspases (37). Caspase activation against IR. Retinoblastoma loss confers radiosensitivity via leads to apoptosis and increases the radiosensitivity of increased apoptosis. Given the resurgent role of radiation in prostate cancer (38). the management of men with advanced castrate-resistant In the current study, cellular apoptosis was mediated prostate cancer, a context in which retinoblastoma loss is via increased nuclear translocalization of NF-kBand common, retinoblastoma status may be a biomarker to induction of PLK3 and cleaved caspase-3. PLK3 is a therapeutic response. NF-kB–regulated gene that induces apoptosis in both p53-dependent and independent signaling pathways Disclosure of Potential Conflicts of Interest (12). The function of NF-kB as either a proapoptotic or S.B. McMahon is a consultant/advisory board member for CellCentric antiapoptotic signal is context dependent (39). In the Ltd. No potential conflicts of interest were disclosed by the other authors. context of LNCaP cells, increased levels of NF-kB activity led to increased apoptosis mediated via caspase activation Authors' Contributions (40). Docetaxel treatment also increases NF-kB activity in Conception and design: C. Thangavel, S.B. McMahon, K.E. Knudsen, a dose-dependent manner leading to decreased cell sur- R.B. Den vival in RWPE-2 prostate cells (41). Docetaxel and pac- Development of methodology: C. Thangavel, E. Boopathi, S. Ciment, A. Sharma, A.P. Dicker, R.B. Den litaxel demonstrate enhanced sensitization to cell death Acquisition of data (provided animals, acquired and managed patients, in the context of retinoblastoma loss (10). Our study provided facilities, etc.): C. Thangavel, S. Ciment, Y. Liu, R. O’Neill, H. Mellert, R. Birbe, P. Fortina, A.P. Dicker, R.B. Den further clarifies that NF-kB–mediated apoptosis is regu- Analysis and interpretation of data (e.g., statistical analysis, biosta- lated via PLK3 expression. tistics, computational analysis): C. Thangavel, E. Boopathi, S. Ciment,

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Y. Liu, S. Addya, A. Ertel, R. Birbe, P. Fortina, A.P. Dicker, K.E. Knudsen, R.B. Grant Support Den The study was supported by a Young Investigator Award from the Prostate Writing, review, and/or revision of the manuscript: C. Thangavel, Y. Liu, Cancer Foundation (to R.B. Den), a Physician Research Training Award from R. Birbe, A.P. Dicker, K.E. Knudsen, R.B. Den the Department of Defense Grant (PC101841; to R.B. Den), a PA Cure Award Administrative, technical, or material support (i.e., reporting or orga- (to K.E. Knudsen), NIH grants (R01 CA176401; to K.E. Knudsen), and a nizing data, constructing databases): C. Thangavel, Y. Liu, R. Birbe, A.P. Movember-PCF Challenge Award (to K.E. Knudsen). Dicker, R.B. Den The costs of publication of this article were defrayed in part by the Study supervision: K.E. Knudsen, R.B. Den payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Acknowledgments this fact. The authors thank members of the K. Knudsen laboratory for input and commentary. The authors speciﬁcally thank M. Augello, J. Dean, Received February 11, 2014; revised July 17, 2014; accepted August 8, M. Schiewer, and R. DeLeuw for insightful discussion. 2014; published OnlineFirst August 27, 2014.

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5482 Clin Cancer Res; 20(21) November 1, 2014 Clinical Cancer Research

The Retinoblastoma Tumor Suppressor Modulates DNA Repair and Radioresponsiveness

Chellappagounder Thangavel, Ettickan Boopathi, Steve Ciment, et al.

Clin Cancer Res 2014;20:5468-5482. Published OnlineFirst August 27, 2014.

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