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MP52-10 RNA SEQUENCING OF CIRCULATING TUMOR CELLS FROM MEN WITH CASTRATION-RESISTANT PROSTATE CANCER
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Monday, May 19, 2014
However, resistance will inevitably develop via a multitude of possible mechanisms which include insulin-like growth factor (IGF) signaling. Targeting insulin-like growth factor-binding protein (IGFBP) -2 and IGFBP-5 using a novel bispecific antisense oligodeoxynucleotide (ASO), designated OGX-225, presents as one strategy to treat castration and enzalutamide-resistant prostate cancer. METHODS: Effects of OGX-225 on IGFBP-2 and IGFBP-5 expression levels, cell growth, and apoptosis were evaluated in human castrate sensitive (LNCaP), castrate resistant (PC-3, 22RV1 and V16D), and enzalutamide-resistant (MR42D and MR49F) prostate cancer cell lines in vitro. The effect of OGX-225 on LNCaP and PC-3 xenograft growth was also evaluated. RESULTS: Whole transcriptome analysis demonstrated that mRNA for IGFBP-2 is overexpressed (4 fold) in castrate resistant prostate cancer cell lines; and that the expression of IGFBP-5 increases over 300 fold in enzalutamide-resistant MR42D prostate cancer cells compared to LNCaP parental cells. OGX-225 induced potent dosedependent, sequence-specific knockdown of IGFBP-2 in LNCaP, 22Rv1, MR42D, and MR49F, and IGFBP-5 expression in PC3 cells. This treatment decreased cell viability by over 50% in all cell lines through induction of a strong apoptotic response. Compared to control ASO, OGX-225 significantly suppressed castrate-resistant progression of LNCaP xenografts as measured by delayed tumor growth and serum prostate-specific antigen levels. OGX-225 also inhibited growth of androgen-independent PC-3 xenografts. Pharmacodynamic activity of OGX-225 in vivo was demonstrated through significant down-regulation of IGFBP-2 and IGFBP-5 mRNA levels in the LNCaP and PC3 xenografts, respectively. CONCLUSIONS: This study reports the first preclinical proof-ofprinciple data that a novel bispecific inhibitor of IGFBP-2 and IGFBP-5, OGX-225, delays prostate cancer progression and displays specific anti-cancer activity in enzalutamide-resistant prostate cancer cell lines. Source of Funding: None
MP52-09 NOVEL LIPID-REGULATORY FUNCTION FOR THROMBOSPONDIN-1 IN PROSTATE CANCER Susan Wcislak, Rockford, IL; Ayesha Chawla, Richmond, VA; Beth A. Plunkett, Charles B. Brendler, Evanston, IL; Jennifer A. Doll*, Milwaukee, WI INTRODUCTION AND OBJECTIVES: We have previously shown that expression of the angiogenesis inhibitor, thrombospondin-1 (TSP-1), is decreased in prostate cancer (PCa) tissues and that TSP-1 knockout (TKO) mice develop prostatic hyperplasia. The objective of this study was to determine if TSP-1 regulates lipid metabolism in the prostate. There is a positive correlation between hypertriglyceridemia and PCa risk, and a diet high in fat promotes PCa progression; however, the molecular mechanisms are unclear. Here, we hypothesized that challenging TKO mice with a higher fat diet would alter lipid metabolism and promote PCa progression. METHODS: In vitro, PC-3 and LNCaP PCa cells were treated with TSP-1 (1-20 nM). Lipolytic levels were measured by the free glycerol assay. TKO and wildtype (WT) mice (n¼6/group; 2 mo.) were fed a modestly higher fat diet (mHFD; 32% kcal from fat vs 12% kcal in control diet) for 8 weeks. A mHFD was used because of existing lipid defects in the TKO mice. The prostates were assessed by H&E and IHC for adipose triglyceride lipase (ATGL; a lipid droplet marker). To identify specific lipid targets, serum metabolite analyses were performed by Metabolon with samples processed for GC/MS and LC/MS/MS platforms as described (Gall et al., 2010). RESULTS: With the mHFD, the hyperplasia present in TKO prostates progressed to cancer. On the control diet, ATGL staining in TKO prostates was increased compared to WT mice, and was further increased on the mHFD. Expected changes in serum lipid levels with mHFD were observed in WT mice (e.g. increased total lipid levels, FA metabolism markers, propionylcarnitine and butyrylcarnitine, and tricarboxylic acid cycle intermediates). Surprisingly, TKO mice
e583
on the control diet had increased levels of propionlycarnitine and butyrylcarnitione compared to WT, but on the mHFD, these markers were decreased, rather than increased. Moreover, in vitro, TSP-1 treatment stimulated lipolysis in PCa cells. CONCLUSIONS: Together these data provide compelling evidence that TSP-1 has a significant impact on organismal lipid metabolism and prostate cancer cell lipid metabolism. These observations also suggest that loss of TSP-1 in tumors not only removes an antiangiogenic barrier, but also alters lipid metabolism which may contribute to PCa progression. Future studies will examine whether the in vivo effects of the mHFD on the TKO prostate are via systemic effects and/or local effects within the prostate. Source of Funding: This study was funded in parts by a Pilot grant and RCDA award from NorthShore University HealthSystem, the Northwestern University Prostate SPORE P50-CA090386, and philanthropic support secured through the Division of Urology and the John and Carol Walter Center for Urological Health, NorthShore University HealthSystem.
MP52-10 RNA SEQUENCING OF CIRCULATING TUMOR CELLS FROM MEN WITH CASTRATION-RESISTANT PROSTATE CANCER Mary Nakazawa*, Changxue Lu, Yan Chen, Emmanuel Antonarakis, Jun Luo, Baltimore, MD INTRODUCTION AND OBJECTIVES: The genomic profile of circulating tumor cells isolated from prostate cancer patients is poorly defined. One of the main challenges is the rarity of the target cells. To address this challenge, the present study focuses on men with high disease burden and thus likely to have high number of circulating tumor cells compatible with genomic analysis. The goal is to define the genomic profile of circulating tumor cells and facilitate development of noninvasive methods to personalize treatment. METHODS: Patients with metastatic prostate cancer progressing on existing therapies are enrolled into the study under an IRB approved protocol. Circulating tumor cells were isolated through magnetic beads coated with prostate epithelial and tumor-cell specific antibodies. Five milliliter of blood of fresh blood was used to make a single preparation of RNA. Following quality assessment and initial evaluation, qualified RNA specimens were subjected to 100 bp paired-end RNAseq. Sequencing data was analyzed using RSEM and SpliceMap. RESULTS: The androgen receptor signature is the dominant signature in circulating tumor cells. Canonical androgen receptor regulated genes are highly expressed in all CTC specimens when compared with RNA extracted from leucocytes. In particular, mutations and splice variants of the androgen receptor were discovered and ascertained using this non-invasive approach. Longitudinal RNA-seq data were generated from a small subset of patients before and after treatment with enzalutamide, in which androgen receptor splice variants were frequently detected. Androgen receptor mutations are generally rare events. When detected, androgen receptor mutations may coexist with splice variants, suggesting that these putative mechanisms of castration resistance that are not mutually excluseive. CONCLUSIONS: RNA-seq is a powerful technology compatible with analysis of circulating prostate tumor cells. The initial analysis in men with high disease burden will help to drive the development of noninvasive methods of genomic profiling integral to personalized medicine. Source of Funding: Prostate Cancer Foundation
MP52-11 COMPLEMENT THERAPY - A NOVEL AXIS FOR PROSTATE CANCER Hidekazu Yamamoto*, Antonella Fara, Martin Kolev, Ashish Chandra, Prokar Dasgupta, Claudia Kemper, London, United Kingdom INTRODUCTION AND OBJECTIVES: Most tumours are characterized by a ‘tolerogenic milieu’ that suppresses infiltrating CD8+ and
Vol. 191, No. 4S, Supplement, Monday, May 19, 2014
However, resistance will inevitably develop via a multitude of possible mechanisms which include insulin-like growth factor (IGF) signaling. Targeting insulin-like growth factor-binding protein (IGFBP) -2 and IGFBP-5 using a novel bispecific antisense oligodeoxynucleotide (ASO), designated OGX-225, presents as one strategy to treat castration and enzalutamide-resistant prostate cancer. METHODS: Effects of OGX-225 on IGFBP-2 and IGFBP-5 expression levels, cell growth, and apoptosis were evaluated in human castrate sensitive (LNCaP), castrate resistant (PC-3, 22RV1 and V16D), and enzalutamide-resistant (MR42D and MR49F) prostate cancer cell lines in vitro. The effect of OGX-225 on LNCaP and PC-3 xenograft growth was also evaluated. RESULTS: Whole transcriptome analysis demonstrated that mRNA for IGFBP-2 is overexpressed (4 fold) in castrate resistant prostate cancer cell lines; and that the expression of IGFBP-5 increases over 300 fold in enzalutamide-resistant MR42D prostate cancer cells compared to LNCaP parental cells. OGX-225 induced potent dosedependent, sequence-specific knockdown of IGFBP-2 in LNCaP, 22Rv1, MR42D, and MR49F, and IGFBP-5 expression in PC3 cells. This treatment decreased cell viability by over 50% in all cell lines through induction of a strong apoptotic response. Compared to control ASO, OGX-225 significantly suppressed castrate-resistant progression of LNCaP xenografts as measured by delayed tumor growth and serum prostate-specific antigen levels. OGX-225 also inhibited growth of androgen-independent PC-3 xenografts. Pharmacodynamic activity of OGX-225 in vivo was demonstrated through significant down-regulation of IGFBP-2 and IGFBP-5 mRNA levels in the LNCaP and PC3 xenografts, respectively. CONCLUSIONS: This study reports the first preclinical proof-ofprinciple data that a novel bispecific inhibitor of IGFBP-2 and IGFBP-5, OGX-225, delays prostate cancer progression and displays specific anti-cancer activity in enzalutamide-resistant prostate cancer cell lines. Source of Funding: None
MP52-09 NOVEL LIPID-REGULATORY FUNCTION FOR THROMBOSPONDIN-1 IN PROSTATE CANCER Susan Wcislak, Rockford, IL; Ayesha Chawla, Richmond, VA; Beth A. Plunkett, Charles B. Brendler, Evanston, IL; Jennifer A. Doll*, Milwaukee, WI INTRODUCTION AND OBJECTIVES: We have previously shown that expression of the angiogenesis inhibitor, thrombospondin-1 (TSP-1), is decreased in prostate cancer (PCa) tissues and that TSP-1 knockout (TKO) mice develop prostatic hyperplasia. The objective of this study was to determine if TSP-1 regulates lipid metabolism in the prostate. There is a positive correlation between hypertriglyceridemia and PCa risk, and a diet high in fat promotes PCa progression; however, the molecular mechanisms are unclear. Here, we hypothesized that challenging TKO mice with a higher fat diet would alter lipid metabolism and promote PCa progression. METHODS: In vitro, PC-3 and LNCaP PCa cells were treated with TSP-1 (1-20 nM). Lipolytic levels were measured by the free glycerol assay. TKO and wildtype (WT) mice (n¼6/group; 2 mo.) were fed a modestly higher fat diet (mHFD; 32% kcal from fat vs 12% kcal in control diet) for 8 weeks. A mHFD was used because of existing lipid defects in the TKO mice. The prostates were assessed by H&E and IHC for adipose triglyceride lipase (ATGL; a lipid droplet marker). To identify specific lipid targets, serum metabolite analyses were performed by Metabolon with samples processed for GC/MS and LC/MS/MS platforms as described (Gall et al., 2010). RESULTS: With the mHFD, the hyperplasia present in TKO prostates progressed to cancer. On the control diet, ATGL staining in TKO prostates was increased compared to WT mice, and was further increased on the mHFD. Expected changes in serum lipid levels with mHFD were observed in WT mice (e.g. increased total lipid levels, FA metabolism markers, propionylcarnitine and butyrylcarnitine, and tricarboxylic acid cycle intermediates). Surprisingly, TKO mice
e583
on the control diet had increased levels of propionlycarnitine and butyrylcarnitione compared to WT, but on the mHFD, these markers were decreased, rather than increased. Moreover, in vitro, TSP-1 treatment stimulated lipolysis in PCa cells. CONCLUSIONS: Together these data provide compelling evidence that TSP-1 has a significant impact on organismal lipid metabolism and prostate cancer cell lipid metabolism. These observations also suggest that loss of TSP-1 in tumors not only removes an antiangiogenic barrier, but also alters lipid metabolism which may contribute to PCa progression. Future studies will examine whether the in vivo effects of the mHFD on the TKO prostate are via systemic effects and/or local effects within the prostate. Source of Funding: This study was funded in parts by a Pilot grant and RCDA award from NorthShore University HealthSystem, the Northwestern University Prostate SPORE P50-CA090386, and philanthropic support secured through the Division of Urology and the John and Carol Walter Center for Urological Health, NorthShore University HealthSystem.
MP52-10 RNA SEQUENCING OF CIRCULATING TUMOR CELLS FROM MEN WITH CASTRATION-RESISTANT PROSTATE CANCER Mary Nakazawa*, Changxue Lu, Yan Chen, Emmanuel Antonarakis, Jun Luo, Baltimore, MD INTRODUCTION AND OBJECTIVES: The genomic profile of circulating tumor cells isolated from prostate cancer patients is poorly defined. One of the main challenges is the rarity of the target cells. To address this challenge, the present study focuses on men with high disease burden and thus likely to have high number of circulating tumor cells compatible with genomic analysis. The goal is to define the genomic profile of circulating tumor cells and facilitate development of noninvasive methods to personalize treatment. METHODS: Patients with metastatic prostate cancer progressing on existing therapies are enrolled into the study under an IRB approved protocol. Circulating tumor cells were isolated through magnetic beads coated with prostate epithelial and tumor-cell specific antibodies. Five milliliter of blood of fresh blood was used to make a single preparation of RNA. Following quality assessment and initial evaluation, qualified RNA specimens were subjected to 100 bp paired-end RNAseq. Sequencing data was analyzed using RSEM and SpliceMap. RESULTS: The androgen receptor signature is the dominant signature in circulating tumor cells. Canonical androgen receptor regulated genes are highly expressed in all CTC specimens when compared with RNA extracted from leucocytes. In particular, mutations and splice variants of the androgen receptor were discovered and ascertained using this non-invasive approach. Longitudinal RNA-seq data were generated from a small subset of patients before and after treatment with enzalutamide, in which androgen receptor splice variants were frequently detected. Androgen receptor mutations are generally rare events. When detected, androgen receptor mutations may coexist with splice variants, suggesting that these putative mechanisms of castration resistance that are not mutually excluseive. CONCLUSIONS: RNA-seq is a powerful technology compatible with analysis of circulating prostate tumor cells. The initial analysis in men with high disease burden will help to drive the development of noninvasive methods of genomic profiling integral to personalized medicine. Source of Funding: Prostate Cancer Foundation
MP52-11 COMPLEMENT THERAPY - A NOVEL AXIS FOR PROSTATE CANCER Hidekazu Yamamoto*, Antonella Fara, Martin Kolev, Ashish Chandra, Prokar Dasgupta, Claudia Kemper, London, United Kingdom INTRODUCTION AND OBJECTIVES: Most tumours are characterized by a ‘tolerogenic milieu’ that suppresses infiltrating CD8+ and