Bois tropicaux : [2002]

Reference data DNA/RNA analysis and individual pipeline steps DNA/RNA (DOCX 22 kb

Hypoxic Tumor Kinase Signaling Mediated by STAT5A in Development of Castration-Resistant Prostate Cancer

<div><p>In this study, we hypothesized that androgen-deprivation therapy (ADT) in prostate cancer, although initially efficient, induces changes in the tumor kinome, which subsequently promote development of castration-resistant (CR) disease. Recognizing the correlation between tumor hypoxia and poor prognosis in prostate cancer, we further hypothesized that such changes might be influenced by hypoxia. Microarrays with 144 kinase peptide substrates were applied to analyze CWR22 prostate carcinoma xenograft samples from ADT-naïve, androgen-deprived (AD), long-term AD (ADL), and CR disease stages. The impact of hypoxia was assessed by matching the xenograft kinase activity profiles with those acquired from hypoxic and normoxic prostate carcinoma cell cultures, whereas the clinical relevance was evaluated by analyzing prostatectomy tumor samples from patients with locally advanced disease, either in ADT-naïve or early CR disease stages. By using this novel peptide substrate microarray method we revealed high kinase activity mediated by signal transducer and activator of transcription 5A (STAT5A) in CR prostate cancer. Additionally, we uncovered high STAT5A kinase activity already in regressing ADL xenografts, before renewed CR growth was evidenced. Finally, since increased STAT5A kinase activity also was detected after exposing prostate carcinoma cells to hypoxia, we propose long-term ADT to induce tumor hypoxia and stimulate STAT5A kinase activity, subsequently leading to renewed CR tumor growth. Hence, the study detected STAT5A as a candidate to be further investigated for its potential as marker of advanced prostate cancer and as possible therapeutic target protein.</p></div

Hypoxia-induced increase in STAT5A substrate phosphorylation by prostate carcinoma cells.

<p>(A) 22Rv1 cells were exposed to moderate (1% O₂) and severe (<0.02% O₂) hypoxia for 24 hours in a hypoxia chamber. Protein lysates from three biological replicates from each of the three experimental groups (normoxia, moderate hypoxia, severe hypoxia) were analyzed to generate the kinase activity profiles. Kinase activity profiling of hypoxic and normoxic cell lysates detected increased STAT5A substrate phosphorylation with increasing hypoxia (blue bars), whereas the mean phosphorylation level for all 22 kinase peptide substrates with significant changes was low (grey bars). *The increase from normoxia to severe hypoxia was significant (p = 0.020). The difference in mean phosphorylation level at moderate versus severe hypoxia was not significant. (B) The proportion of decreased versus increased kinase activity in hypoxic versus normoxic 22Rv1 cell lysates. Listed are the gene names of the affected kinase peptide substrates, with start and end positions for the peptide sequence within the protein in brackets. (C) Western blot of anti-phospho-STAT5A/B and anti-STAT5A expression in normoxic (−) and severely hypoxic (<0.02% O₂, 24 h) (+) cell lysates from PC3, DU145 and 22Rv1 cells. Anti-γ-tubulin served as loading control. (D) Western blot of anti-HIF-1α, anti-phospho-STAT5A/B and anti-STAT5A expression of lysates from PC3, DU145 and 22Rv1 cells with (+) or without (−) 4 hours of exposure to 100 µM of the hypoxia-mimetic agent cobalt chloride (CoCl₂), a chemical inducer of HIF-1α. Anti-γ-tubulin served as loading control.</p

Kinase activity in xenografts following androgen-deprivation therapy (ADT).

<p>(A) Decreased (blue; negative log₂ ratio) and increased (red; positive log₂ ratio) phosphorylation levels of kinase peptide substrates by androgen-deprived (AD), long-term AD (ADL), and castration-resistant (CR) prostate carcinoma xenografts compared to ADT-naïve tumors (mean ratio and s.e.m. per substrate). Protein lysates from three biological replicates from each of the four experimental groups (ADT-naïve, AD, ADL, CR) were analyzed to generate the xenograft kinase activity profiles. Listed are the substrates’ corresponding gene names, with start and end positions for the peptide sequence within the protein in brackets. (B) Volcano plots (−log₁₀ p-values versus ratios) visualizing kinase peptide substrates with most significant and/or highest log₂ ratios between treated and ADT-naïve tumors’ phosphorylation levels. Color labeling: yellow: p<0.01 and ratio >1.0; green: p<0.01 and ratio = 0.5–1.0; dark pink: p = 0.01–0.05 and ratio >1.0; orange: p = 0.01–0.05 and ratio = 0.5–1.0; blue: p = 0.01–0.05 and ratio <0.5; light pink: p>0.05.</p

Additional file 1: Table S1. of Favorable outcomes in locally advanced and node positive prostate cancer patients treated with combined pelvic IMRT and androgen deprivation therapy

Univariate Cox proportional hazards analysis for associations between covariates and survival endpoints. (DOC 48 kb

Kinase activity in androgen-deprivation therapy (ADT)-naïve xenografts versus ADT-naïve patient tumors.

<p>An ADT-naïve kinase activity signature was defined as the 100 kinase peptide substrates with highest phosphorylation levels. For prostate carcinoma xenografts, the signature was represented by the mean of all ADT-naïve substrate phosphorylation levels. For prostate cancer patients, a top 100 signature was produced by calculating the mean of the three ADT-naïve (case 1, 2, and 3; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063723#pone-0063723-t001" target="_blank">Table 1</a>) tumor phosphorylation levels. (A) Seventy-four of the 100 substrates were shared by the xenograft and the patients’ tumor ADT-naïve signatures. (B) Phosphorylated kinase peptide substrates shared by ADT-naïve prostate carcinoma xenografts (n = 3) and ADT-naïve tumors from prostate cancer patients (mean of case 1, 2, and 3). Listed are the substrates’ corresponding gene names, with start and end positions for the peptide sequence within the protein in brackets. (C) The exclusively phosphorylated kinase peptide substrates (n = 26) in the xenograft signature (left) and in the patient tumor signature (right).</p

Experimental design.

<p>(A) Xenograft diameters versus time in the transition from androgen-deprivation therapy (ADT)-naïve, via androgen-deprived (AD) and long-term AD (ADL), to castration-resistant (CR) disease, after ADT by castration. The time to develop CR disease in patients is ∼2 to 5 years. In our prostate carcinoma xenograft model, the corresponding time was 5.4±0.4 months. The four circles represent the four experimental groups in this study. Blood samples for prostate-specific antigen (PSA) measurements were withdrawn from all of the mice before tumors were excised and snap-frozen for kinase activity profiling. (B) Serum PSA levels reflected the clinical pattern of disease progression. PSA was decreased in AD and ADL tumors, before being restored in CR tumors. Significant differences (p<0.05) compared to ADT-naïve tumors are indicated (*). Each bar represents mean and s.e.m of 3–6 tumors.</p

Kinase peptide substrates with significantly affected (reduced or increased) phosphorylation levels by samples from androgen-deprived (AD), long-term AD (ADL), or castration-resistant (CR) prostate carcinoma xenografts, versus androgen-deprivation therapy (ADT)-naïve xenografts.

a<p>Kinase peptide substrate names on the Tyrosine Kinase PamChip® microarray.</p>b<p>Start and end positions for the kinase peptide sequence within the protein.</p>c<p>Site(s) of tyrosine phosphorylation within the protein.</p>d<p>Corresponding gene names and ^ecommon names, retrieved from UniProt or HUGO Gene Nomenclature Committee (HGNC).</p>f<p>Kinase peptide substrates are grouped according to the main cellular process they are involved in.</p>g<p>FLT1 and KDR are also known as VEGFR1 and VEGFR2.</p

STAT5A substrate phosphorylation and HIF-1α expression in prostatectomy tumor samples.

<p>(A) For the androgen-deprivation therapy (ADT)-naïve prostate cancer patients, the log₂ ratios between the substrate phosphorylation levels generated by the prostatectomy tumor sample versus the respective contralateral lobe normal tissue sample were calculated. For the patient that received long-term ADT before radical prostatectomy, the log₂ ratio between the substrate phosphorylation levels from the tumor sample versus the mean substrate phosphorylation levels from the ADT-naïve patients’ normal tissue samples were calculated (since this patient’s normal tissue also was exposed to ADT). (B) Western immunoblot analysis of the prostatectomy tumor samples’ lysates with an antibody against hypoxia-inducible factor-1α (HIF-1α), the main protein known to be activated and stabilized under hypoxic conditions, revealing increased HIF-1α expression in the lysate from the early CR tumor (case 4). No HIF-1α expression was seen in lysates from ADT-naïve tumors.</p

Patient characteristics.

<p>Abbreviations: pTN, histopathologic tumor (T) and node (N) staging; PSA, prostate-specific antigen; ADT, androgen-deprivation therapy.</p