Androgen-regulated miR-32 targets BTG2 and is overexpressed in castration-resistant prostate cancer.

The androgen receptor (AR) signaling pathway is involved in the emergence of castration-resistant prostate cancer (CRPC). Here, we identified several androgen-regulated microRNAs (miRNAs) that may contribute to the development of CRPC. Seven miRNAs, miR-21, miR-32, miR-99a, miR-99b, miR-148a, miR-221 and miR-590-5p, were found to be differentially expressed in CRPC compared with benign prostate hyperplasia (BPH) according to microarray analyses. Significant growth advantage for LNCaP cells transfected with pre-miR-32 and pre-miR-148a was found. miR-32 was demonstrated to reduce apoptosis, whereas miR-148a enhanced proliferation. Androgen regulation of miR-32 and miR-148a was confirmed by androgen stimulation of the LNCaP cells followed by expression analyses. The AR-binding sites in proximity of these miRNAs were demonstrated with chromatin immunoprecipitation (ChIP). To identify target genes for the miRNAs, mRNA microarray analyses were performed with LNCaP cells transfected with pre-miR-32 and pre-miR-148a. Expression of BTG2 and PIK3IP1 was reduced in the cells transfected with pre-miR-32 and pre-miR-148a, respectively. Also, the protein expression was reduced according to western blot analysis. BTG2 and PIK3IP1 were confirmed to be targets by 3'UTR-luciferase assays. Finally, immunostainings showed a statistically significant (P<0.0001) reduction of BTG2 protein in CRPCs compared with untreated prostate cancer (PC). The lack of BTG2 staining was also associated (P<0.01) with a short progression-free time in patients who underwent prostatectomy. In conclusion, androgen-regulated miR-32 is overexpressed in CRPC, leading to reduced expression of BTG2. Thus, miR-32 is a potential marker for aggressive disease and is a putative drug target in PC.

Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer.

Androgen receptor (AR) is overexpressed in the majority of castration-resistant prostate cancers (CRPCs). Our goal was to study the effect of AR overexpression on the chromatin binding of the receptor and to identify AR target genes that may be important in the emergence of CRPC. We have established two sublines of LNCaP prostate cancer (PC) cell line, one overexpressing AR 2-3-fold and the other 4-5-fold compared with the control cells. We used chromatin immunoprecipitation (ChIP) and deep-sequencing (seq) to identify AR-binding sites (ARBSs). We found that the number of ARBSs and the AR-binding strength were positively associated with the level of AR when cells were stimulated with low concentrations of androgens. In cells overexpressing AR, the chromatin binding of the receptor took place in 100-fold lower concentration of the ligand than in control cells. We confirmed the association of AR level and chromatin binding in two PC xenografts, one containing AR gene amplification with high AR expression, and the other with low expression. By combining the ChIP-seq and expression profiling, we identified AR target genes that are upregulated in PC. Of them, the expression of ZWINT, SKP2 (S-phase kinase-associated protein 2 (p45)) and FEN1 (flap structure-specific endonuclease 1) was demonstrated to be increased in CRPC, while the expression of SNAI2 was decreased in both PC and CRPC. FEN1 protein expression was also associated with poor prognosis in prostatectomy-treated patients. Finally, the knock-down of FEN1 with small interfering RNA inhibited the growth of LNCaP cells. Our data demonstrate that the overexpression of AR sensitizes the receptor binding to chromatin, thus, explaining how AR signaling pathway is reactivated in CRPC cells.

[Quantification of microvascular obstruction in acute myocardial infarction using cardiac MRI]. / Kardiale MRT zur Quantifizierung der mikrovaskulären Obstruktion beim akuten Myokardinfarkt.

PURPOSE: Microvascular obstruction (MO) and the extent of infarction are important prognostic factors in acute myocardial infarction. Our study aimed to investigate the effect of the time interval between contrast administration and image acquisition on the quantification of microvascular obstruction and myocardial infarction. MATERIALS AND METHODS: 50 consecutive patients with acute myocardial infarction (40 male, mean age 58.1 +/- 11.7 years) treated by percutaneous coronary revascularization resulting in a grade 3 flow according to the thrombolysis in myocardial infarction flow classification were examined on a 1.5 T MR scanner within the first 5 days after infarction. 2, 5, 10, and 20 minutes after I.V. administration of 0.2 mmol/kg per kg body weight of Gadodiamid (Omniscan), GE Healthcare Buchler, Germany), a single shot IR-SSFP sequence (TR 2.4 ms, TE 1.08 ms, TI 180 - 280 ms, FA 50 degrees) covering the entire left ventricle was acquired. Areas of MO and myocardial infarction were measured for all times after contrast injection (p. i.). RESULTS: MO was detected in 32 of 50 patients two minutes p. i., while 23 patients showed evidence of MO (p = 0.002) 20 min. p. i. In all patients with MO, the extent of MO decreased over time (7.4 +/- 9.0 % of the LV myocardium 2 min. p. i. vs. 2.4 +/- 4.6 % 20 min. p. i. p < 0.0001). The area of myocardial infarction increased from 13.9 +/- 13.5 % 2 min. p. i. to 18.6 +/- 14.2 % 10 min. p. i. (p < 0.0001), and then remained unchanged (18.7 +/- 14.3 % at 20 min. p = 0.57). CONCLUSION: Our study shows that the time delay between contrast media injection and image acquisition has a significant impact on the delimitable extent of MO and infarct size.

Identification of ETS-like transcription factor 4 as a novel androgen receptor target in prostate cancer cells.

Transcriptional control by androgens via androgen receptor (AR) is strongly involved in prostate cancer development, but the critical target genes have remained elusive. We have characterized E twenty-six-like transcription factor 4 (ELK4) (also known as serum response factor accessory protein 1) as a novel AR target in human prostate cancer cells. In-silico screening identified three putative AR response elements (AREs) within -10 kb from the transcription start site of ELK4. Both ARE1 at -167/-153 and ARE2 at -481/-467 bound AR in vitro and mediated androgen induction as isolated elements in transcription assays in non-prostate cells. However, merely the ARE2 that cooperates with a proximal forkhead box A1-binding site was critical for the AR-dependent activation of ELK4 promoter in prostate cancer cells. Preferential loading of holo-AR onto the ARE2 and concomitant recruitment of RNA polymerase II onto the ELK4 promoter was confirmed in prostate cancer cells by chromatin immunoprecipitation. Database searches indicated that the expression of ELK4 is markedly increased in prostate cancers relative to normal prostates. Moreover, prostate cancer tissue immunostainings showed that nuclear ELK4 levels are significantly increased in androgen-refractory prostate cancers compared to untreated tumours. Reduction of the amount of ELK4 in LNCaP cells by RNAi retarded cell growth. In conclusion, ELK4 is a direct AR target in prostate cancer cells. Androgens may thus contribute to the growth of prostate cancer via influencing ELK4 levels.

Mutation analysis of aryl hydrocarbon receptor interacting protein (AIP) gene in colorectal, breast, and prostate cancers.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently identified in individuals with pituitary adenoma predisposition (PAP). These patients have prolactin (PRL) or growth hormone (GH) oversecreting pituitary adenomas, the latter exhibiting acromegaly or gigantism. Loss-of-heterozygosity (LOH) analysis revealed that AIP is lost in PAP tumours, suggesting that it acts as a tumour-suppressor gene. Aryl hydrocarbon receptor interacting protein is involved in several pathways, but it is best characterised as a cytoplasmic partner of the aryl hydrocarbon receptor (AHR). To examine the possible role of AIP in the genesis of common cancers, we performed somatic mutation screening in a series of 373 colorectal cancers (CRCs), 82 breast cancers, and 44 prostate tumour samples. A missense R16H (47G>A) change was identified in two CRC samples, as well as in the respective normal tissues, but was absent in 209 healthy controls. The remaining findings were silent, previously unreported, changes of the coding, non-coding, or untranslated regions of AIP. These results suggest that somatic AIP mutations are not common in CRC, breast, and prostate cancers.

[MR coronary angiography with MS-325, a blood pool contrast agent: comparison of an inversion recovery steady-state free precession with an inversion recovery fast low angle shot sequence in volunteers]. / MR-Koronarangiographie mit MS-325, einem Blood-Pool-Kontrastmittel: Vergleich einer Inversion Recovery Steady-State Free Precession und einer Inversion Recovery Fast Low Angle Shot Sequenz in Probanden.

PURPOSE: To compare the signal-to-noise and contrast-to-noise ratio as well as the image quality of 3D inversion recovery steady-state free precession (IR-SSFP) and 3D inversion recovery fast low angle shot (IR-FLASH) sequences for contrast-enhanced breath-hold MRCA. MATERIALS AND METHODS: 24 healthy volunteers (10 female, 14 male, mean age 29.8 +/- 6.1 years) were involved in this study. All examinations were performed on a 1.5 T MR scanner (Magnetom Sonata, Siemens, Germany) after injection of 0.05 mmol/kg body weight MS-325 (EPIX Pharmaceuticals, Cambridge, MA and Schering AG, Berlin, Germany). MRCA was performed using IR-SSFP (TR 3.8 ms, TE 1.6 ms, FA 65 degrees , 35 phase encoded steps, bandwidth 540 Hz/pixel, slice thickness 1.5 mm, in-plane resolution 1.2 x 0.9 - 1.4 x 1.0 mm) and IR-FLASH (TR 3.8 ms, TE 1.6 ms, FA 25 degrees , bandwidth 490 Hz/pixel, slice thickness 1.5 mm, in-plane resolution 1.2 x 0.9 - 1.4 x 1.0 mm) sequences. For all scans the inversion time was set to null the signal intensity of the myocardium. Signal-to-noise ratio (SNR) as well as contrast-to-noise ratio (CNR) measurements (blood versus myocardium) were performed. The image quality was assessed based on a 5-point scale ranging from 1 (excellent) to 5 (non-diagnostic) by two radiologists in consensus. RESULTS: The mean signal-to-noise ratio of blood (27.7 +/- 4.7 vs. 22.6 +/- 4.9, P < 0.0001) and the contrast-to-noise ratio (21.0 +/- 4.3 vs. 15.8 +/- 4.3, P < 0.0001) showed significantly higher values for IR-SSFP sequences. The mean image quality scores were significantly higher for SSFP (3.6 +/- 0.7) than FLASH (2.8 +/- 0.9) sequences (P < 0.05). CONCLUSION: IR-SSFP sequences show a considerable overall improvement in image quality compared to IR-FLASH sequences for MRCA after injection of a gadolinium-based blood pool contrast agent.

[Detection and characterization of left ventricular thrombi by MRI compared to transthoracic echocardiography]. / Detektion und Charakterisierung linksventrikulärer Thromben mittels MRT im Vergleich zur transthorakalen Echokardiographie.

PURPOSE: Transthoracic echocardiography is the routine diagnostic procedure in assessing patients with left ventricular thrombi, but is limited by the acoustic window and poor contrast between thrombus and adjacent myocardium. This study evaluates the role of cardiac MRI in the detection of left ventricular thrombi in patients with chronic myocardial infarction compared to standard transthoracic echocardiography. MATERIALS AND METHODS: In 82 patients (55 men and 27 women, age 36 to 79 years, median 59 +/- 11 years) who suffered a myocardial infarction more than 6 months earlier, transthoracic echocardiography and MRI were performed. The MRI protocol included steady state cine imaging (true FISP: TR 3.0 ms, TE 1.5 ms, FA 65 degrees ) in standard long and short axis orientation and contrast-enhanced imaging using a 3D IR-FLASH sequence with long inversion time (TR 4 ms, TE 1.43 ms, FA 10 degrees , TI 300 ms) early, and a 2D IR-FLASH sequence with optimized inversion time (TR 8 ms, TE 4.3 ms, FA 20 degrees , TI 180 - 280 ms) late after administration of gadolinium. RESULTS: Transthoracic echocardiography depicted 12 thrombi. Contrast-enhanced MRI confirmed these 12 thrombi and detected 23 additional thrombi. With the exception of 2 very small apical thrombi only visible on contrast-enhanced MR images, spherical thrombi were diagnosed by both techniques, whereas only contrast-enhanced MRI was able to visualize mural thrombi. Left ventricular thrombi were more frequently diagnosed in patients with moderate to severe impairment of the left ventricular systolic function, 32/42 (76 %), or in patients with left ventricular aneurysms, 21/24 (84 %). CONCLUSION: Contrast-enhanced MRI is mostly superior to transthoracic echocardiography in diagnosing mural left ventricular thrombi in patients who had suffered a myocardial infarction. Intracavitary thrombi are more frequently found in patients with impaired regional and global left ventricular function.

Optimization of contrast-enhanced peripheral MR angiography with mid-femoral venous compression (VENCO).

PURPOSE: To compare a standard protocol for contrast-enhanced three-dimensional magnetic resonance angiography (3D CE-MRA) of the lower extremities to a high spatial resolution protocol with venous compression (VENCO) at the mid-femoral level. MATERIAL AND METHODS: 12 patients with peripheral arterial occlusive disease (8-males; age range, 52 - 74 years; mean 67.1 years; 4 females; age range, 57 - 71 years; mean 62.1 years) were examined once with a standard MR angiography (MRA) protocol, and a second time with a high spatial resolution protocol in combination with mid-femoral venous compression (60 mm Hg) for the last two stations. All imaging was performed on a 1.5 T whole-body MR scanner (Magnetom Sonata, Siemens Medical Solutions, Erlangen, Germany) using a dedicated coil and paramagnetic contrast agent (gadodiamide, Omniscan, Amersham, Oslo, Norway). Signal-to-noise ratios (SNR) and contrast-to-noise ratios (CNR) were calculated and image quality as well as venous overlay were assessed on a five-point scale for both examinations. Statistical significance was established at p < 0.05. RESULTS: Mean SNR and CNR values of the two lower stations with VENCO were statistically significantly higher in comparison to the standard protocol (66 +/- 8 vs. 52 +/- 11 and 53 +/- 9 vs. 41 +/- 8, respectively; p < 0.01). The same was true for overall image quality with VENCO (4.0 +/- 0.2 vs. 3.4 +/- 0.8; p < 0.05) and presence of venous overlay (3.5 +/- 0.4 vs. 4.1 +/- 0.9; p < 0.05), respectively. CONCLUSION: VENCO 3D CE-MRA is simple to put into practice and advances the performance of multi-station MRA strategies for assessment of the peripheral arterial vasculature.