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Isolation and characterization of castration-resistant prostate cancer LNCaP95 clones.

ABSTRACT: The androgen receptor (AR) is a validated therapeutic target for prostate cancer and has been a focus for drug development for more than six decades. Currently approved therapies that inhibit AR signaling, such as enzalutamide, rely solely on targeting the AR ligand-binding domain and, therefore, have limited efficacy on prostate cancer cells that express truncated, constitutively active AR splice variants (AR-Vs). The LNCaP95 cell line is a human prostate cancer cell line that expresses both functional full-length AR and AR-V7. LNCaP95 is a heterogeneous cell population that is resistant to enzalutamide, with its proliferation dependent on transcriptionally active AR-V7. The purpose of this study was to identify a LNCaP95 clone that would be useful for evaluating therapies for their effectiveness against enzalutamide-resistant prostate cancer cells. Seven clones from the LNCaP95 cell line were isolated and characterized using morphology, in vitro growth rate, and response to ralaniten (AR N-terminal domain inhibitor) and enzalutamide (antiandrogen). In vivo growth of the clones as subcutaneous xenografts was evaluated in castrated immunodeficient mice. All of the clones maintained the expression of full-length AR and AR-V7. Cell proliferation of the clones was insensitive to androgen and enzalutamide but importantly was inhibited by ralaniten, which is consistent with AR-Vs driving the proliferation of parental LNCaP95 cells. In castrated immunodeficient animals, the growth of subcutaneous xenografts of the D3 clone was the most reproducible compared to the parental cell line and other clones. These data support that the enzalutamide-resistant LNCaP95-D3 subline may be suitable as a xenograft tumor model for preclinical drug development with improved reproducibility.

SUBMITTER: Leung JK

PROVIDER: S-EPMC8693726 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Enzalutamide is a novel antiandrogen with proven efficacy in metastatic castration-resistant prostate cancer (mCRPC).To evaluate enzalutamide's effects on cancer and on androgens in blood and bone marrow, and associate these with clinical observations.In this prospective phase 2 study, 60 patients with bone mCRPC received enzalutamide 160mg orally daily and had transilial bone marrow biopsies before treatment and at 8 wk of treatment.Androgen signaling components (androgen receptor [AR], AR splice variant 7 (ARV7), v-ets avian erythroblastosis virus E26 oncogene homolog [ERG], cytochrome P450, family 17, subfamily A, polypeptide 1 [CYP17]) and molecules implicated in mCRPC progression (phospho-Met, phospho-Src, glucocorticoid receptor, Ki67) were assessed by immunohistochemistry; testosterone, cortisol, and androstenedione concentrations were assessed by liquid chromatography-tandem mass spectrometry; AR copy number was assessed by real-time polymerase chain reaction. Descriptive statistics were applied.Median time to treatment discontinuation was 22 wk (95% confidence interval, 19.9-29.6). Twenty-two (37%) patients exhibited primary resistance to enzalutamide, discontinuing treatment within 4 mo. Maximal prostate-specific antigen (PSA) decline ? 50% and ? 90% occurred in 27 (45%) and 13 (22%) patients, respectively. Following 8 wk of treatment, bone marrow and circulating testosterone levels increased. Pretreatment tumor nuclear AR overexpression (> 75%) and CYP17 (> 10%) expression were associated with benefit (p = 0.018). AR subcellular localization shift from the nucleus was confirmed in eight paired samples (with PSA decline) of 23 evaluable paired samples. Presence of an ARV7 variant was associated with primary resistance to enzalutamide (p = 0.018). Limited patient numbers warrant further validation.The observed subcellular shift of AR from the nucleus and increased testosterone concentration provide the first evidence in humans that enzalutamide suppresses AR signaling while inducing an adaptive feedback. Persistent androgen signaling in mCRPC was predictive of benefit and ARV7 was associated with primary resistance.We report a first bone biopsy study in metastatic prostate cancer in humans that searched for predictors of outcome of enzalutamide therapy. Benefit is linked to a pretreatment androgen-signaling signature.ClinicalTrials.gov identifier NCT01091103.

Project description:The number of circulating tumor cells (CTCs) in metastatic prostate cancer patients provides prognostic and predictive information. However, it is the molecular characterization of CTCs that offers insight into the biology of these tumor cells in the context of personalized treatment.We developed a novel approach to isolate CTCs away from hematopoietic cells with high purity, enabling genomic analysis of these cells. The isolation protocol involves immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS). To evaluate the feasibility of isolation of CTCs by IE/FACS and downstream genomic profiling, we conducted a pilot study in patients with metastatic castration resistant prostate cancer (CRPC). Twenty (20) sequential CRPC patients were assayed using CellSearch™. Twelve (12) patients positive for CTCs were subjected to immunomagnetic enrichment and fluorescence activated cell sorting (IE/FACS) to isolate CTCs. Genomic DNA of CTCs was subjected to whole genome amplification (WGA) followed by gene copy number analysis via array comparative genomic hybridization (aCGH).CTCs from nine (9) patients successfully profiled were observed to have multiple copy number aberrations including those previously reported in primary prostate tumors such as gains in 8q and losses in 8p. High-level copy number gains at the androgen receptor (AR) locus were observed in 7 (78%) cases. Comparison of genomic profiles between CTCs and archival primary tumors from the same patients revealed common lineage. However, high-level copy number gains in the AR locus were observed in CTCs, but not in the matched archival primary tumors.We developed a new approach to isolate prostate CTCs without significant leukocyte admixture, and to subject them to genome-wide copy number analysis. Our assay may be utilized to explore genomic events involved in cancer progression, e.g. development of castration resistance and to monitor therapeutic efficacy of targeted therapies in clinical trials in a relatively non-invasive manner.

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Isolation and characterization of castration-resistant prostate cancer LNCaP95 clones.

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