The clinical significance of circulating miR-21, miR-142, miR-143, and miR-146a in patients with prostate cancer

BackgroundProstate cancer (PCa) is the most common type of solid tissue cancer among men in western countries. In this study, we determined the levels of circulating miR-21, miR-142, miR-143, miR-146a, and RNU 44 levels as controls for early diagnosis of PCa.MethodsThe circulating miRNA levels in peripheral blood samples from 43 localized PCa patients, 12 metastatic PCa (MET) patients, and a control group of, 42 benign prostate hyperplasia (BPH) patients with a total of 97 volunteers were determined the by PCR method.ResultsNo differences in the DCT values were found among the groups. In PCa and PCaMet groups the expression of miR21 and miR142 were higher compared to the BHP group. No other differences were observed among the other groups. miR21 expression in the PCa group was 6.29 folds upregulated whereas in the PCaMet group 10.84 folds up-regulated. When the total expression of miR142 is evaluated, it showed a positive correlation with mir21 and mir 146 (both p<0.001). Also, the expression of miR146 shows a positive correlation with both miR21 and miR143 (both p<0.001). Expression of miRNAs was found to be an independent diagnostic factor in patients with Gleason score, PSA, and free PSA levels.ConclusionsOur study showed that co-expression of miR21, miR-142, miR-143, and miR-146a and the upregulation of miR-21 resulted in increased prostate carcinoma cell growth. In the PCaMet group, miR21 is the most upregulated of all miRNAs. These markers may provide a novel diagnostic tool to help diagnose PCa with aggressive behavior.     

Plasma-Derived Exosomal Survivin,a Plausible Biomarker for Early Detection of Prostate Cancer

Background

Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment.

Methods

Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively.

Results

Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six) or high (nine) Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls.

Conclusions

These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection.     

Two Different Serum MiRNA Signatures Correlate with the Clinical Outcome and Histological Subtype in Pleural Malignant Mesothelioma Patients

Pleural malignant mesothelioma (MPM) is a detrimental neoplasm affecting pleural sheets and determining a high rate of mortality. In this study, we have enrolled 14 consecutive patients (13 males and 1 female) with MPM (mean age: 70.3 ± 4.6 years). We have collected serum for the determination of a miRNA profiling using a low-density microarray real time PCR system in the serum of patients and comparing it with that one of 10 control counterparts affected by not-cancer-related pleural effusions. In the patients 5 miRNAs were up-regulated (miR101, miR25, miR26b, miR335 and miR433), 2 miRNA were downregulated (miR191, miR223) and two miRNAs were expressed exclusively in patients (miR29a and miR516). Based upon the changes in the expression of the above mentioned miRNAs we detected two distinctive miRNA signatures predicting histotype and survival in these patients: I) patients with more than 3/9 upregulated miRNAs or 3/9 upregulated miRNAs and miR516 not recordable or unchanged (signature A); II) patients with at least 3/9 downregulated or unchanged miRNAs and/or miR29a downregulated (signature B). Based upon these criteria, 5 patients were stratified in signature A and the remaining 9 in signature B. Patients with signature A had a significant shorter median survival than those with signature B (7 months vs. 17 months, 95% CI: 0.098–1.72, p = 0.0021), had a sarcomatoid or mixed histological MPM subtype and were diagnosed in stage II (3/5) and stage III (2/5). In conclusion, we suggest that miRNA signature A is predictive of sarcomatoid histotype and of worse prognosis in MPM.     

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

Background

Recent data show aberrant and altered expression of regulatory noncoding micro (mi) RNAs in prostate cancer (PCa). A large number of miRNAs are encoded in organized intronic clusters within many protein coding genes. While expression profiling studies of miRNAs are common place, little is known about the host gene and their resident miRNAs coordinated expression in PCa cells. Furthermore, whether expression of a subset of miRNAs is distinct in androgen-responsive and androgen-independent cells is not clear. Here we have examined the expression of mature miRNAs of miR 17–92, miR 106b-25 and miR 23b-24 clusters along with their host genes C13orf25, MCM7 and AMPO respectively in PCa cell lines.

Results

The expression profiling of miRNAs and host genes was performed in androgen-sensitive MDA PCa 2b and LNCaP as well as in androgen-refractory PC-3 and DU 145 cell culture models of PCa. No significant correlation between the miRNA expression and the intrinsic hormone-responsive property of PCa cells was observed. Androgen-sensitive MDA PCa 2b cells exhibited the highest level of expression of most miRNAs studied in this report. We found significant expression variations between host genes and their resident miRNAs. The expressions of C13orf25 and miR 17–92 cluster as well as MCM7 and miR 106b-25 cluster did not reveal statistically significant correlation, thus suggesting that host genes and resident miRNAs may be expressed independent of each other.

Conclusion

Our results suggest that miRNA expression profiles may not predict intrinsic hormone-sensitive environment of PCa cells. More importantly, our data indicate the possibility of additional novel mechanisms for intronic miRNA processing in PCa cells.     

DNA methylation changes correlate with Gleason score and tumor stage in prostate cancer

DNA methylation, a widely used epigenetic mark, has been associated with many tumors. However, few studies have addressed the role of cell-free plasma DNA methylation in discriminating aggressive prostate cancer (PCa) from indolent cases. We conducted a case series and a case-control study among histologically confirmed stage II/III cases and matched controls recruited at Columbia University Medical Center. The aim of this study was to investigate whether plasma DNA methylation levels are appropriate surrogate biomarker of PCa tumor tissue levels and whether these markers are associated with worse clinicopathological tumor characteristics, which correlate with poorer prognosis. Quantitative pyrosequencing was used to detect methylation levels of p16 (CDKN4A), APC, GSTP1, and LINE-1 in 24 pairs of prostate tumor and adjacent tissues, as well as 27 plasma samples of PCa patients and 24 of controls. DNA methylation levels were significantly higher in tumor tissue than in adjacent nontumor tissue for p16 (CDKN4A), GSTP1, and APC; GSTP1 had a higher average percentage methylation in tumor tissue (38.9%) compared with p16 (CDKN4A) (5.9%) and APC (14.5%). GSTP1, p16 (CDKN4A), and APC methylation in tumor tissue was statistically significantly higher for cases with Gleason score ≥7 compared with those with Gleason score <7 [49.0% vs. 21.9% (p=0.01), 6.6% vs. 4.5% (p=0.04), and 19.1% vs. 7.4% (p=0.02), respectively]. Plasma LINE-1 methylation levels were higher in those with higher Gleason (67.6%) than in those with Gleason's below 7 (64.6%, p=0.03). Significant plasma-tissue correlations were observed for GSTP1 and LINE-1 methylation. These data, although preliminary, suggest that aberrant methylation may be a useful marker to identify PCa patients with clinically aggressive disease.     

Circulating MicroRNAs as Easy-to-Measure Aging Biomarkers in Older Breast Cancer Patients: Correlation with Chronological Age but Not with Fitness/Frailty Status

Circulating microRNAs (miRNAs) hold great promise as easily accessible biomarkers for diverse (patho)physiological processes, including aging. We have compared miRNA expression profiles in cell-free blood from older versus young breast cancer patients, in order to identify “aging miRNAs” that can be used in the future to monitor the impact of chemotherapy on the patient’s biological age. First, we assessed 175 miRNAs that may possibly be present in serum/plasma in an exploratory screening in 10 young and 10 older patients. The top-15 ranking miRNAs showing differential expression between young and older subjects were further investigated in an independent cohort consisting of another 10 young and 20 older subjects. Plasma levels of miR-20a-3p, miR-30b-5p, miR106b, miR191 and miR-301a were confirmed to show significant age-related decreases (all p≤0.004). The remaining miRNAs included in the validation study (miR-21, miR-210, miR-320b, miR-378, miR-423-5p, let-7d, miR-140-5p, miR-200c, miR-374a, miR376a) all showed similar trends as observed in the exploratory screening but these differences did not reach statistical significance. Interestingly, the age-associated miRNAs did not show differential expression between fit/healthy and non-fit/frail subjects within the older breast cancer cohort of the validation study and thus merit further investigation as true aging markers that not merely reflect frailty.     

Genistein Up-Regulates Tumor Suppressor MicroRNA-574-3p in Prostate Cancer

Genistein has been shown to inhibit cancers both in vitro and in vivo, by altering the expression of several microRNAs (miRNAs). In this study, we focused on tumor suppressor miRNAs regulated by genistein and investigated their function in prostate cancer (PCa) and target pathways. Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion in vitro and in vivo. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3′ UTR of several target genes (such as RAC1, EGFR and EP300) that are components of ‘Pathways in cancer’. Quantitative real-time PCR and Western analysis showed that the mRNA and protein expression levels of the three target genes in PCa cells were markedly down-regulated with miR-574-3p. Loss-of-function studies demonstrated that the three target genes significantly affect cell proliferation, migration and invasion in PCa cell lines. Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa.     

Nuclear Ep-ICD Expression Is a Predictor of Poor Prognosis in “Low Risk” Prostate Adenocarcinomas

IntroductionMolecular markers for predicting prostate cancer (PCa) that would have poor prognosis are urgently needed for a more personalized treatment for patients. Regulated intramembrane proteolysis of Epithelial cell adhesion molecule results in shedding of the extracellular domain (EpEx) and release of its intracellular domain (Ep-ICD) which triggers oncogenic signaling and might correlate to tumor aggressiveness. This study aimed to explore the potential of Ep-ICD and EpEx to identify PCa that have poor prognosis.MethodsImmunohistochemical analysis of Ep-ICD and EpEx was carried out in normal prostate tissues (n = 100), benign prostate hyperplasia (BPH, n = 83), and prostate cancer (n = 249) using domain specific antibodies. The expression of Ep-ICD and EpEx was correlated with clinico- pathological parameters and disease free survival (DFS).ResultsReduced expression of nuclear Ep-ICD and membrane EpEx was observed in PCa in comparison with BPH and normal prostate tissues (p = 0.006, p < 0.001 respectively). For patients who had PCa with Gleason Score less than 7, preserved nuclear Ep-ICD emerged as the most significant marker in multivariate analysis for prolonged DFS, where these patients did not have recurrence during follow up of up to 12 years (p = 0.001).ConclusionReduced expression of nuclear Ep-ICD was associated with shorter disease free survival in patients with a Gleason Score less than 7 and may be useful in identifying patients likely to have aggressive tumors with poor prognosis. Furthermore, nuclear Ep-ICD can differentiate between normal and prostate cancer tissues for ambiguous cases.     

Hypertriglyceridemia Is a Potential Preoperative Predictor for Biochemical Recurrence after Radical Prostatectomy

Objectives

Many previous studies have suggested that the outcome of prostate cancer (PCa) may be closely related to abnormal lipid metabolism. Therefore, in this study, we evaluated the preoperative lipid profiles of patients with clinically localized prostate cancer (PCa) who underwent radical prostatectomy (RP), with particular emphasis on the relationship between these profiles and biochemical recurrence (BCR).

Patients and Methods

We evaluated 715 consecutive men with clinically localized PCa who underwent RP at our institution between January 2011 and December 2013. We defined hypertriglyceridemia as a fasting serum triglyceride (TG) level greater than 200 mg/dL. We used the Kaplan—Meier method to predict BCR-free survival and applied the log-rank test to determine the statistical significance between survival curves. Cox proportional hazard ratio (HR) models were used to identify the significant predictors of BCR according to clinicopathological variables.

Results

Of 663 patients who underwent RP for clinically localized PCa, 66 (10.0%) showed BCR during a median follow-up period of 21 months. Patients without BCR had higher levels of serum TG, and patients with hypertriglyceridemia were significantly more likely to achieve BCR-free survival in the Kaplan—Meier analysis (log-rank test, P = 0.009). In the multivariable analysis, the presence of hypertriglyceridemia (HR 0.22), pathologic Gleason score (≥8; HR 2.85), pathologic T stage (≥pT3; HR 3.44), and a positive surgical margin (HR, 2.39) were still significant BCR predictors.

Conclusions

We found that preoperative hypertriglyceridemia was associated with a lower risk of BCR after RP in patients with clinically localized PCa. Our results could help to clarify the currently conflicting evidence on the relationship between serum lipid profiles, particularly the presence of hypertriglyceridemia, and the risk of BCR in PC a patients after surgery.     

Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia

The histology‐based Gleason score (GS) of prostate cancer (PCa) tissue biopsy is the most accurate predictor of disease aggressiveness and an important measure to guide treatment strategies and patient management. The variability associated with PCa tumor sampling and the subjective determination of the GS are challenges that limit accurate diagnostication and prognostication. Thus, novel molecular signatures are needed to distinguish between indolent and aggressive forms of PCa for better patient management and outcomes. Herein, label‐free LC‐MS/MS proteomics is used to profile the proteome of 50 PCa tissues spanning five grade groups (n = 10 per group) relative to tissues from individuals with benign prostatic hyperplasia (BPH). Over 2000 proteins are identified albeit at different levels between and within the patient groups, revealing biological processes associated with specific grades. A panel of 11 prostate‐derived proteins including IGKV3D‐20, RNASET2, TACC2, ANXA7, LMOD1, PRCP, GYG1, NDUFV1, H1FX, APOBEC3C, and CTSZ display the potential to stratify patients from low and high PCa grade groups. Parallel reaction monitoring of the same sample cohort validate the differential expression of LMOD1, GYG1, IGKV3D‐20, and RNASET2. The four proteins associated with low and high PCa grades reported here warrant further exploration as candidate biomarkers for PCa aggressiveness.     

Identification of miRs-143 and -145 that is associated with bone metastasis of prostate cancer and involved in the regulation of EMT

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bone. MicroRNAs (miRNAs) play a crucial role in many tumor metastases. The importance of miRNAs in bone metastasis of PCa has not been elucidated to date. We investigated whether the expression of certain miRNAs was associated with bone metastasis of PCa. We examined the miRNA expression profiles of 6 primary and 7 bone metastatic PCa samples by miRNA microarray analysis. The expression of 5 miRNAs significantly decreased in bone metastasis compared with primary PCa, including miRs-508-5p, -145, -143, -33a and -100. We further examined other samples of 16 primary PCa and 13 bone metastases using real-time PCR analysis. The expressions of miRs-143 and -145 were verified to down-regulate significantly in metastasis samples. By investigating relationship of the levels of miRs-143 and -145 with clinicopathological features of PCa patients, we found down-regulations of miRs-143 and -145 were negatively correlated to bone metastasis, the Gleason score and level of free PSA in primary PCa. Over-expression miR-143 and -145 by retrovirus transfection reduced the ability of migration and invasion in vitro, and tumor development and bone invasion in vivo of PC-3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. Their upregulation also increased E-cadherin expression and reduced fibronectin expression of PC-3 cells which revealed a less invasive morphologic phenotype. These findings indicate that miRs-143 and -145 are associated with bone metastasis of PCa and suggest that they may play important roles in the bone metastasis and be involved in the regulation of EMT Both of them may also be clinically used as novel biomarkers in discriminating different stages of human PCa and predicting bone metastasis.     

Expression of Dicer and Its Related MiRNAs in the Progression of Prostate Cancer

Dicer is aberrantly expressed in several types of malignancies. Cleaved by Dicer, the small noncoding microRNAs (miRNAs) are considered potential tools for the diagnosis and prognosis of cancer. This study investigated the expression of miRNAs thought to target Dicer. Expression of 1,205 human miRNAs and miRNA*s were examined in four patients with prostate cancer (PCa) by miRNA array in which the threshold was set as two-fold. Seventy-three miRNAs and miRNA*s were significantly down-regulated while 10 were up-regulated in PCa tissues compared with matched histologically normal glands. Of these, miR-29b-1, miR-200a, miR-370, and miR-31, which were the most down/up-regulated and closely potentially target to the Dicer 3′ UTR, were investigated further. Tissues of primary tumors and matched normal prostate glands from 185 patients with PCa were collected for further investigation. Dicer mRNA levels were negatively correlated with miR-29b-1 (ρs = −0.177, p = 0.017), miR-200a (ρs = -0.489, p < 0.0001) and miR-31 (ρs = −0.314, p < 0.0001) expression. Compared with adjacent normal glands, PCa tissues showed significantly lower miR-200a and miR-31 expression levels. Furthermore, in metastatic PCa, the expression levels of miR-200a, miR-370, and miR-31 were dramatically higher than in localized PCa. Additionally, elevated expression levels of miR-200a and miR-31 appeared to be associated with castration-resistant PCa. These findings suggest possibilities that miR-200a and miR-31 target Dicer and are involved in the carcinogenesis, migration, and behavior of castration-resistant PCa, indicating that they could be potential biomarkers for monitoring PCa progression.     

miR‐15b‐5p facilitates the tumorigenicity by targeting RECK and predicts tumour recurrence in prostate cancer

MicroRNAs (miRNAs) have been reported to participate in many biological behaviours of multiple malignancies. Recent studies have shown that miR‐15b‐5p (miR‐15b) exhibits dual roles by accelerating or blocking tumour progression. However, the molecular mechanisms by which miR‐15b contributes to prostate cancer (PCa) are still elusive. Here, miR‐15b expression was found significantly up‐regulated in PCa in comparison with the normal samples and was positively correlated with age and Gleason score in patients with PCa. Notably, PCa patients with miR‐15b high expression displayed a higher recurrence rate than those with miR‐15b low expression (P = 0.0058). Knockdown of miR‐15b suppressed cell growth and invasiveness in 22RV1 and PC3 cells, while overexpression of miR‐15b reversed these effects. Then, we validated that RECK acted as a direct target of miR‐15b by dual‐luciferase assay and revealed the negative correlation of RECK with miR‐15b expression in PCa tissues. Ectopic expression of RECK reduced cell proliferation and invasive potential and partially abrogated the tumour‐promoting effects caused by miR‐15b overexpression. Additionally, miR‐15b knockdown inhibited tumour growth activity in a mouse PCa xenograft model. Taken together, our findings indicate that miR‐15b promotes the progression of PCa cells by targeting RECK and represents a potential marker for patients with PCa.     

Serum vascular endothelial growth factor C level in patients with prostate cancer and benign prostatic hyperplasia

OBJECTIVE: To compare serum vascular endothelial growth factor C (VEGF-C) levels in patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) and analyze VEGF-C levels in relation to clinicopathologic parameters. STUDY DESIGN: Fifty-eight patients with PCa and 61 patients with BPH were included in this study. Serum VEGF-C levels were assessed by enzyme-linked immunosorbent assay. RESULTS: The serum VEGF-C level for patients with PCa was 3,432.06 +/- 1,851.07 as opposed to 3,166.68 +/- 1,921.2 for patients with BPH. There was no statistically significant difference between the 2 groups (p = 0.4448). There was no correlation of VEGF-C to tumor stage, grading or the preoperative prostate-specific antigen values. CONCLUSION: We cannot recommend VEGF-C serum level as a marker for tumor growth in PCa.