ABSTRACT
The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level. The overexpression of ASNS was validated by analyzing other public CRPC data sets. ASNS protein expression, as detected by reverse-phase protein lysate array, was tightly correlated with gene copy number. In addition, ASNS protein expression, as determined by IHC analysis, was associated with progression to a therapy-resistant disease state in TMAs that included 77 castration-resistant and 40 untreated prostate cancer patient samples. Knockdown of ASNS by small-interfering RNAs in asparagine-deprived media led to growth inhibition in both androgen-responsive (ie, LNCaP) and castration-resistant (ie, C4-2B) prostate cancer cell lines and in cells isolated from a CRPC xenograft (ie, MDA PCa 180-30). Together, our results suggest that ASNS is up-regulated in cases of CRPC and that depletion of asparagine using ASNS inhibitors will be a novel strategy for targeting CRPC cells.
Subject(s)
Aspartate-Ammonia Ligase/genetics , Prostatic Neoplasms/enzymology , Animals , Aspartate-Ammonia Ligase/metabolism , DNA/genetics , DNA Copy Number Variations/genetics , Gene Expression Profiling , Gene Knockdown Techniques , Genome-Wide Association Study , Male , Mice , Mice, Inbred C57BL , Mice, SCID , Neoplasms, Hormone-Dependent/enzymology , Neoplasms, Hormone-Dependent/genetics , Orchiectomy , Prostatic Neoplasms/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
PTEN haploinsufficiency is common in hormone-sensitive prostate cancer, though the incidence of genomic deletion and its downstream effects have not been elucidated in clinical samples of hormone refractory prostate cancer (HRPC). Progression to androgen independence is pivotal in prostate cancer and mediated largely by the androgen receptor (AR). Since this process is distinct from metastatic progression, we examined alterations of the PTEN gene in locally advanced recurrent, non-metastatic human HRPC tissues. Retrospective analyses of PTEN deletion status were correlated with activated downstream phospho-Akt (p-Akt) pathway proteins and with the androgen receptor. The prevalence of PTEN genomic deletions in transurethral resection samples of 59 HRPC patients with known clinical outcome was assessed by four-colour FISH analyses. FISH was performed using six BAC clones spanning both flanking PTEN genomic regions and the PTEN gene locus, and a chromosome 10 centromeric probe. PTEN copy number was also evaluated in a subset of cases using single nucleotide polymorphism (SNP) arrays. In addition, the samples were immunostained with antibodies against p-Akt, p-mTOR, p-70S6, and AR. The PTEN gene was deleted in 77% of cases, with 25% showing homozygous deletions, 18% homozygous and hemizygous deletions, and 34% hemizygous deletions only. In a subset of the study group, SNP array analysis confirmed the FISH findings. PTEN genomic deletion was significantly correlated to the expression of downstream p-Akt (p < 0.0001), AR (p = 0.025), and to cancer-specific mortality (p = 0.039). PTEN deletion is common in HRPC, with bi-allelic loss correlating to disease-specific mortality and associated with Akt and AR deregulation.
Subject(s)
PTEN Phosphohydrolase/genetics , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Androgen/metabolism , Signal Transduction/genetics , Aged , Aged, 80 and over , Androgen Antagonists/therapeutic use , Chromosomes, Human, Pair 10 , Gene Deletion , Genome , Genotype , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , PTEN Phosphohydrolase/analysis , Phenotype , Polymorphism, Single Nucleotide , Prostatic Neoplasms/drug therapy , Statistics, Nonparametric , Treatment FailureABSTRACT
OBJECTIVE: To further evaluate the association between the cytoplasmic or nuclear localization of ErbB3 with biochemical recurrence (BCR) in patients with prostate cancer and positive surgical margins, as there is a greater risk of BCR for such patients after radical prostatectomy (RP). PATIENTS AND METHODS: We recently noted that ErbB3, which is normally associated with the plasma membrane, can translocate to the nucleus, an event which appears to be associated with disease progression. We evaluated ErbB3 expression and localization using immunohistochemistry on tissue samples from 55 patients with positive surgical margins after RP; 30 of these 55 (55%) had BCR after 3 years of follow-up. The relationship between ErbB3 nuclear localization and BCR (prostate-specific antigen, PSA, >0.3 ng/mL) after RP was analysed by Kaplan-Meier survival analysis and Cox regression models. RESULTS: The BCR-free survival probability at 3 years was 0.65 and 0.35 for positive and negative nuclear ErbB3, respectively (Kaplan-Meier, P = 0.029). Patients negative for nuclear ErbB3 had a 2.47-fold increase in BCR frequency in a univariate Cox model (P = 0.008) and it remained an independent prognostic marker when combined with clinical prognostic variables in a multivariate model (P = 0.023). CONCLUSION: Low nuclear localization of ErbB3 is a predictor of BCR in patients with prostate cancer and positive surgical margins after RP.
