Serum Of Prostate Cancer Patients Research Articles

Non-enzymatic electrochemical detection of sarcosine in serum of prostate cancer patients by CoNiWBO/rGO nanocomposite

Selective and sensitive sarcosine detection is crucial due to its recent endorsement as a prostate cancer (PCa) biomarker in clinical diagnosis. The reduced graphene oxide-cobalt nickel tungsten boron oxides (CoNiWBO/rGO) nanocomposite is developed as a non-enzymatic electrochemical sensor for sarcosine detection in PCa patients’ serum. CoNiWBO/rGO is synthesized by the chemical reduction method via a one-pot reduction method followed by calcination at 500 °C under a nitrogen environment for 2 h and characterized by UV-Vis, XRD, TGA, and SEM. CoNiWBO/rGO is then deposited on a glassy carbon electrode, and sarcosine sensing parameters are optimized, including concentration and pH. This non-enzymatic sensor is employed to directly determine sarcosine in serum samples. Differential pulse voltammetry (DPV) and linear sweep voltammetry (LSV) are employed to monitor the electrochemical behavior where sarcosine binding leads to oxidation. Chronoamperometric studies show the stability of the developed sensor. The results demonstrate a wide linear range from 0.1 to 50 µM and low limits of detection, i.e., 0.04 µM and 0.07 µM using DPV and LSV respectivel. Moreover, the calculated recovery of sarcosine in human serum of prostate cancer patients is 78–96%. The developed electrochemical sensor for sarcosine detection can have potential applications in clinical diagnosis.

Diagnostic Value of GSTP1, RASSF1, AND RASSF2 Methylation in Serum of Prostate Cancer Patients.

Considering the inadequacy of PSA measurement in the diagnosis of prostate cancer, it is aimed to establish a potential liquid biopsy diagnostic panel. 39 patients who underwent TRUS-biopsy and 15 healthy volunteers were included. Approximately 15 ml of venous blood samples taken from healthy volunteers and patients before biopsy were separated as plasma. Hypermethylation status of GSTP1 and RASSF1:RASSF2 genes was revealed in cfDNA materials collected from plasma samples. Correlation of this epigenetic change detected in PCa, BPH and healthy volunteer groups with pathology results was examined. Pathology reports of 39 patients included were 13 PCa, 3 ASAP, 3 HGPIN, and 20 BPH. In total, 3 of the patients with PCa had positive GSTP1, 4 had RASSF1 and 9 had positive RASSF2 methylation. It was seen that RASSF2 had the highest sensitivity (69%), specificity (39%) and NPV (80%), while RASSF1 had the highest PPV (30%). When the binary combinations of genes were examined it was observed that the GSTP1:RASSF1 combination had the highest sensitivity (46%), specificity (76%) and NPV (82%). When the methylation of all three genes was examined, it was observed that the sensitivity was quite low (8%), but the specificity (83%) increased significantly. Although we observed that the GSTP1 and RASSF1 methylation positivity rates that we examined in our study were higher in patients without prostate cancer, we found that the RASSF2 methylation rate was higher in patients with prostate cancer. randomized controlled studies are needed.

Abstract 2146: Race-related differential immunoreactivity of enolase autoantibodies in patients with prostate cancer

Abstract INTRODUCTION AND OBJECTIVE: Enolase-1 (ENO1) and -2 (ENO2) are implicated in tumor metabolic and aggressive functions. Like prostate specific membrane antigen (PSMA), they localize to the tumor cell surface and are potential theranostics targets. ENO autoantibodies were reported by our group in prostate cancer (PCa) patients but their immunoreactivity to ENO1/2 isoforms was not defined. Differential ENO immunoreactivity in docetaxel-resistant (DR) PCa cells between African American (AA) and European American (EA) PCa patients was also reported. Our objective was to investigate the role of ENO isoforms in the race-related differential ENO immunoreactivity of PCa patients. METHODS: PCa patient sera (41 AA, 68 EA) were obtained from Loma Linda University Biospecimen Laboratory. Western blotting (WB) analysis, using patient sera and monoclonal antibodies to ENO1 and ENO2, was done against lysates from docetaxel-sensitive (DS) and DR PCa cell lines and against recombinant (rec) ENO1 and ENO2 to assess ENO isoform expression and detect ENO autoantibodies. Scratch-wound assay was performed to evaluate AA and EA anti-ENO driven inhibition of DR PCa cell migration. Student’s T-test and Chi-squared analyses were performed with p<0.05 deemed significant. RESULTS: ENO1 expression was robust in all assessed DS and DR cell lines. ENO2 was detected in DS cells but was significantly reduced in DR cells. PSMA expression was lost in cells expressing ENO1 and neuroendocrine markers. Anti-ENO EA sera reacted in WB with the expected 50 kD band in both DS and DR PCa cells, but anti-ENO AA sera reacted preferentially with this band in DS cells. Strong immunoreactivity was noted against recENO1 in AA and EA sera (7% vs 28%, p<0.01) and against recENO2 in AA and EA sera (24% vs 35%, p=0.23). EA, but not AA, ENO autoantibodies significantly inhibited DR cell migration, and their pre-absorption with recENO1 diminished this inhibitory effect. CONCLUSIONS: Autoantibodies to ENO1 and ENO2 were identified in EA and AA PCa patient sera. Weaker ENO1 immunoreactivity was seen in sera from AA PCa patients, corresponding with weaker inhibition of DR PCa cell migration compared to EA patient sera. Unlike PSMA and ENO2, ENO1 expression was robust in neuroendocrine-like DR PCa cell lines. Future studies will correlate the presence of ENO autoantibodies with clinicopathologic parameters in a larger patient cohort. Defining mechanisms underlying the race-related differential ENO immunoreactivity may identify novel biological determinants of PCa health disparities and uncover the theranostic potential of tumor surface ENO1. Citation Format: Kai Wen Cheng, Carlos J. Diaz Osterman, Krystal Santiago, Michael Reeves, Diano Lozano, Pedro Ochoa, Alfonso Duran, Saied Mirshahidi, Brian R. Hu, Frankis Almaguel, Carlos A. Casiano. Race-related differential immunoreactivity of enolase autoantibodies in patients with prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2146.

The value of a panel of circulating microRNAs in screening prostate cancer.

Prostate cancer (PCa) remains a worldwide public health problem that poses a serious threat to the health of men worldwide. Many studies have found that microRNA (miRNA) in serum has the potential to be a biomarker for cancer screening. Our study was conducted to investigate the value of serum miRNAs in PCa screening. We selected 12 miRNAs from past studies for its association with PCa. We checked the expression levels of these miRNAs in the serum of 112 PCa patients and 112 healthy controls in a two-stage experiment. We plotted the receiver operating characteristic curve of miRNAs in the validation stage and constructed a four-miRNA panel with the highest diagnostic value using stepwise logistic regression. We also predicted the target genes with these four miRNAs through online databases and performed Gene Ontology functional annotation and pathway analysis. The results showed that six miRNAs (miR-429, miR-10a-5p, miR-183-5p, miR-181a-5p, miR-1231, miR-129-5p) were abnormally expressed in the serum of PCa patients. We used four of these miRNAs including miR-1231, miR-10a-5p, miR-429 and miR-129-5p to construct a combination of miRNAs with high specificity and sensitivity in screening PCa (area under the curve =0.878). Bioinformatics analysis showed that the genes targeted by these miRNAs can be linked to the development of PCa. Our study detected and identified a set of miRNAs that serves as screening marker for PCa, which may assist in early diagnosis and treatment of PCa.

Rationally Designed LaWO₄ Nanoflakes for the Simultaneous Electrochemical Detection of Valine and Leucine in Serum of Prostate Cancer Patients

Rationally Designed LaWO₄ Nanoflakes for the Simultaneous Electrochemical Detection of Valine and Leucine in Serum of Prostate Cancer Patients

Abstract 2168: Identifying a surrogate biomarker for neuropilin-2 axis in serum of prostate cancer patients

Abstract The purpose of this study is to investigate the correlation of neuropilin-2 (NRP2) and its ligand, vascular endothelial growth factor-C (VEGF-C), with treatment outcomes and resistance in prostate cancer patients. 166 prostate cancer patient serum samples were obtained from the Integrated Cancer Repository for Cancer Research at the University of Nebraska Medical Center. 11 patients were excluded for having a non-prostate genitourinary cancer or more than one concurrent cancer. Patients were categorized as untreated, primary treatment, remission, and secondary treatment based on when their serum sample was collected. Primary treatment indicated current treatment with first-line medical therapy, remission indicated those without evidence of disease after prostatectomy or first-line medical therapy, and secondary therapy indicated second-line treatment currently being given due to treatment-resistant disease. Other variables collected include age, race, ethnicity, prostate-specific antigen (PSA) measured at the time of diagnosis, PSA measured at the time of serum sample collection, Gleason score, and clinical stage using the American Joint Committee on Cancer (AJCC) tumor, node, metastasis (TNM) system. Finally, VEGF-C levels in patient serum were measured using a commercially available ELISA assay (R&D System, Minneapolis, MN). The final data was analyzed using a linear regression model to compare VEGF-C levels between treatment groups, as well as VEGF-C level correlation with each of the other variables collected. Results: All patients were male with an age range of 47 to 85 years, with a mean age of 65.3 years. Most were white (n=135) and non-Hispanic (n=148). VEGF-C levels ranged from 341 pg/ml to 15,983 pg/ml, with a mean of 3,787 pg/ml. Of the 155 patients, 8 were untreated, 26 were in primary therapy, 66 were in remission, and 52 were in secondary therapy. Results showed no statistically significant correlation between VEGF-C levels and PSA at diagnosis, PSA at serum collection, Gleason score, or clinical stage. The most interesting difference between treatment groups was a higher VEGF-C level in remission (mean 63.48 pg/ml) versus secondary therapy (mean 55.31 pg/ml), although this was not statistically significant (p=0.089). Conclusions: Our results indicated that plasma VEGF-C alone is not a prognostic indicator for prostate cancer. Since our studies using cell culture and animal models of prostate cancer have indicated the role of VEGF-C and the NRP2 axis in promoting metastasis and therapy resistance in prostate cancer, it would be useful to determine both the VEGF-C and NRP2 levels in the plasma of the same patients and study whether their level together would predict the outcome of medical therapy. Our ongoing experiments to determine plasma NRP2 levels in this patient cohort should be able to test this hypothesis. Citation Format: Sarah A. Mullen, Dipanwita Das, Samikshan Dutta, Robin R. High, Lynette M. Smith, Benjamin A. Teply, Kaustubh Datta. Identifying a surrogate biomarker for neuropilin-2 axis in serum of prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2168.

A novel fluorescence aptasensor based on PCN-223 as an efficient quencher for sensitive determination of prostate-specific antigen.

A novel fluorescence aptasensor based on PCN-223 as an efficient quencher was developed to sensitively detect prostate-specific antigen (PSA). The 5-carboxytetramethylrhodamine (TAMRA)-labeled PSA aptamer was adsorbed on PCN-223 by π-π stacking and hydrogen-bonding interactions, which contributed to fluorescence quenching because of the photoinduced electron transfer from TAMRA to PCN-223. In addition, the amount ofquenched fluorescence of the PSA-binding aptamer complex-PCN-223 was lower than that of TAMRA aptamer-PCN-223 without PSA (at excitation/emission peaks of 545/582nm), which can be explained by the factthat the PSA-binding aptamer complexes contributed to the separation of the aptamer from PCN-223. ∆F value of fluorescence intensities for TAMRA aptamer-PCN-223 with and without PSA showed a good linear relationship with PSA concentration over a range of 0.1 to 24ngmL-1, with a detection limit of 0.05ngmL-1. Compared with three metal-organic frameworks (MOFs) of UiO-66-NH2, ZIF-67, and Ni3(HITP)2 as quenchers, PCN-223 as a Zr-MOF exhibited the highest ∆F value for PSA detection. The advantage of PCN-223 could be attributed to its carboxyl, benzene, and porphyrin groups, the large specific surface area and good biocompatibility. This proposed aptasensor can be successfully used to detect PSA in sera of prostate cancer patients. The PSA detection results of this aptasensor were consistent with those which were obtained from hospital by Archtecti2000sr automatic chemiluminescence immunoanalyzer. The proposed aptasensor has potential clinical detection application.

Recent updates on analysis of alteration of Zn level in cancer patients

Zn micronutrient is involved in many physiological processes that include enzyme activity, genomic stability, DNA repairing, apoptosis, immunity, neurological function, response to oxidative stress, and cell signaling. The deficiency of trace element Zn may be responsible for the mutation of DNA, tumor genesis, and carcinoma cell production. The Zn level has been found to decrease in cancer patients as compared to the healthy controls. Various cancer such as breast, ovarian, prostate, lung, Ewing, endometrial, brain, and bladder cancer have been correlated with Zinc deficiency. The polymorphism may be correlated with the ZINC deficiency in several cancer patients. The polymorphism has been observed in MT2A, Matrix metalloproteinase (MMP)-1, MMP-2, MMP-7, and MMP-13 genes significantly change in the Zn levels in the serum of prostate cancer patients. The genotypes GSTM1 and GSTT1 significantly change the concentration of zinc concentration in lung cancer patients. The presence of polymorphism rs1805502 in the GRIN2B gene within the brain is associated with a notably reduced concentration of zinc in the serum. The results of these studies associated with deficiency of zinc in the body may cause DNA damage, mutation in DNA, and tumor growth. This review article provides a detailed description of the deficiency of the Zn element in cancer patients and polymorphisms in genes encoding zinc-dependent proteins associated with different cancers.

IL-8 Produced Via Bidirectional Communication Between Prostate Cancer and M2 Macrophages as a Potential Diagnostic and Prognostic Biomarker.

Owing to the mortality associated with metastatic prostate cancer and the shortcomings of the current parameters in predicting the disease prognosis, we require the identification of viable biomarkers, which would help in the diagnosis and prognosis of the disease. We aimed to determine whether the interleukin-8 level in the tumor microenvironment could serve as a potential clinical diagnostic marker and prognostic factor for prostate cancer. The migration assay of prostate cancer cells was performed in an in vitro co-culture model. Cell lines PC3 and DU145 were divided into two groups and co-cultured with M0 and M2 macrophages, respectively. We used reverse transcription-quantitative polymerase chain reaction to detect M2 macrophage marker expression levels. Immunohistochemistry analyses of tissue microarrays were performed to analyze the correlation between the increased expression of interleukin-8 and the prognosis of prostate cancer. A retrospective analysis based on 142 residual serum specimens was performed to analyze the level of interleukin-8. We observed that M2 macrophages promoted the migration of prostate cancer cells and significantly increased the concentrations of interleukin-8 in the co-culture supernatants. We observed increased expression of CD163 and interleukin-8 in prostate cancer tissues. Furthermore, the levels of interleukin-8 in the serum of prostate cancer patients were higher than those in healthy controls. Untreated patients had higher levels of interleukin-8, which could be a predictor of a higher metastasis rate. These results suggest that interleukin-8 produced via bidirectional communication between prostate cancer cells and M2 macrophages is a putative biomarker for prostate cancer diagnosis and treatment.

Association between interleuleukin-1β polymorphism (rs16944) and biomarkers levels in Iraqi patients with prostate cancer.

Prostate cancer (PCa) is the second-leading cause of mortality in men and the most commonly diagnosed non-cutaneous male malignancy. Host genetic factors, and inflammation-induced cytokines, play a key role in prostate oncogenesis. Single Nucleotide Polymorphisms (SNP) in cytokine genes were suggested to increase the susceptibility for PCa development and progression. This study aimed to investigate the association between the SNP (rs16944) in the interleukin-1 β (IL-1β) gene and the serum levels of Prostate Specific Antigen (PSA) Prolactine (PRL), testosterone, and IL-1β in Iraqi PCa patients versus healthy controls. Taqman Real Time-PCR, was performed to investigate the IL-1β (rs16944) polymorphism in 100 Iraqi PCa patients and 50 age-matched healthy controls in a case-control study. Serum levels of PSA, PRL, and testosterone were determined by ELISA and FIA, and associated with the IL-1β serum level as well as with the SNP (rs 16944). The association between the clinico-pathological parameters and the genotype distribution of PCa patients was also studied. There level of IL-1β was significant increased in the serum of PCa patients compared to controls (P = 8.19 10-7). Serum levels for other biomarkers such as PSA, PRL and testosterone were also significantly elevated in patients compared to controls (P < 0.0001). No differences were seen for genotype and allele distribution between PCa patients and controls. Nevertheless, in the group of controls, we found that 36% carried the GG genotype against only 26% in the patients group.This suggests that this could be a protective genotype (OR 0.62, P = 0.254). In addition, we found that the GA genotype is slightly more frequent in patients as compared to controls (OR 1.22, P = 0.605). Interestingly, serum levels of IL-1β, PSA, PRL and testosterone were significantly higher in PCa patients carrying the GA genotype, and the GA and AA genotypes are strongly associated with the aggressive behavior of the disease such as advanced TNM, and high Gleason score. Our data suggest that both serum IL-1β level and IL-1β SNP (rs16944) may be considered as candidate biomarkers for PCa. Moreover, the GA, and AA genotypes carriers along with high sera levels of IL-1β, PSA and PRL, have an increased risk for PCa with aggressive behavior in Iraqi men.

DIFFERENTIAL EXPRESSION PATTERN OF AIP, UCKL1, AND PKN1 GENES IN PROSTATE CANCER PATIENTS.

The evolution of research on the therapy of prostate cancer (PC) depends on a study of molecules that are involved in the progression of this disease. Nevertheless, there is a need for additional biomarkers that would help to refine the molecular profile of PC and propose the personalized therapeutic approach. To study differential expression patterns of the AIP, UCKL1, and PKN1genes in blood sera and tumor tissue of patients with PC with different Gleason scores. The total extracellular RNA was isolated from blood sera of 44PC patients and 4healthy donors. cDNAs were synthesized and quantitative polymerase chain reaction (qPCR) was performed. Immunohistochemical study of the UCKL, AIP and PKN1proteins was performed on deparaffinized sections of tumors. The study was supplemented by a bioinformatic analysis of the publicly available databases. The UCKL1, AIP, PKN1genes were overexpressed at the mRNA level in blood sera of PC patients, compared to healthy donors. Extracellular mRNA levels of AIP and UCKL-1were 100-1000-fold increased in all PC samples compared to the healthy donors but without significant inequality between the groups of PC cases differing by the Gleason score. The highest levels were detected in the samples from PC patients with the Gleason score > 9. The PKN1expression was higher in PC patients compared with healthy donors but without significant difference between the groups. From the three chosen genes, AIP and UCKL1showed similar pattern of expression assessed either by extracellular mRNA levels in patient sera or the protein in PC tissues. AIP was up to 1000-fold increased in all PC samples, compared to the healthy donors, with the highest levels in PC cases with Gleason score > 9. Expression levels of the AIP and UCKL1genes in the PC patient sera may be used as an additional criterion for prognosis of tumor progression.

Hepatitis A Virus Cellular Receptor 1 (HAVcr-1) Initiates Prostate Cancer Progression in Human Cells via Hepatocyte Growth Factor (HGF)-Induced Changes in Junctional Integrity.

Background: HAVcR-1 has been linked to cancer aetiology and may regulate junctional complexes, with its role in prostate cancer still unexplored. This study aims to investigate the expression of HAVcR-1 in prostate cancer samples and the exploration of the cellular/molecular impact of HAVcR-1. Methods: Levels of HAVcR-1 ectodomain in the serum of prostate cancer patients were compared to healthy controls, and assessed as the total protein and gene expression of HAVcR-1 and tissues sections. The manipulation of HAVcR-1 levels within prostate cancer cell lines determined changes in cell behaviour using in vitro cell models and barrier function assays. Protein/phosphoprotein levels were assessed using Western blotting. Results: Levels of HAVcR-1 ectodomain from serum were decreased in patients with prostate cancer. Ectodomain levels correlated with the Gleason score. Histologically, the total protein/gene expression of HAVcR-1 was overexpressed in prostate cancer. The overexpression of HAVcR-1 in prostate cancer cell lines resulted in key changes in cell behaviour and the phosphorylation of β-catenin with a concurrent decrease in membranous E-cadherin, increased nuclear β-catenin and increased cyclin D1 protein expression, which were associated with HGF-promoted changes in the barrier function. Conclusions: HAVcR-1 expression and ectodomain release coincides with the presence of prostate cancer; thus, indicating HAVcR-1 as a potential biomarker to aid in diagnostics, and implicating HAVcR-1 in the dysregulation of junctional complexes.

Уровень протеолитических фрагментов IgG как дополнительный прогностический биомаркер рака предстательной железы

Introduction. Prostate cancer (PC) leads the world structure of male cancer mortality. Understanding the prognosis for PC patients is vitally important and requires diagnostic enhancements such as developing biomarker panels. The studyaimed to estimate sensitivity, specificity, and prognostic significance of the level of immunoglobulin G (IgG) proteolysis products in the blood serum depending on PC Gleason score and detect how the levels of IgG proteolytic fragments were associated with the diagnostic marker expression. Materials and methods. The study included 90 PC patients. We used the Gleason score to evaluate tumor grade. The level of the IgG proteolysis products and total prostate-specific antigen (PSA) in the serum was detected with ELISA. We performed an immunohistochemical assay with Ki-67, plasminogen-binding protein enolase-1 (ENO-1), and prostate-specific membrane antigen (PSMA). Results. ROC analysis demonstrated 100% specificity of the IgG proteolysis products test regardless of Gleason score, while the sensitivity reached 72.2%, 57.5%, 52.6%, and 46.2% for ISUP stages 1, 2, 3, and 4, respectively. An intergroup comparison showed significant differences between patient groups according to Gleason score, preoperative PSA and IgG proteolysis product serum levels and Ki-67 index. We detected a positive correlation between the level of IgG proteolysis products in the PC patients’ serum and ENO-1 expression and a negative correlation with the Gleason score and PSMA expression. Conclusion. Preoperative detection of high levels of proteolytic IgG fragments in the serum of PC patients even with low Gleason score and PSMA expression may help predict a more aggressive disease course and choose the management strategy. Keywords: prostate cancer, plasminogen activation system, proteolytic fragments of immunoglobulin G, oncomarkers

An improved amperometric sarcosine biosensor based on graphene nanoribbon/chitosan nanocomposite for detection of prostate cancer

We describe herein an improved amperometric biosensor for detection of sarcosine, a potential biomarker for prostate cancer. The biosensor is based on covalent immobilization of sarcosine oxidase (SOx) onto nanocomposite of chitosan (CHIT) and graphene nanoribbons (GNRs) electrodeposited onto Au electrode. The GNRs were studied by transmission electron microscopy and UV spectroscopy. The working electrode (SOx/CHIT/GNRs/AuE) exhibited maximum current at a potential of 0.1V against Ag/AgCl, generated from electrochemical oxidation of H2O2, from sarcosine by immobilized SOx. The biosensor showed optimum response i.e. current (mA) within 2s at pH 7.3 and 35 ​°C. There was a linearity between current (mA) and sarcosine concentration in a wider range 0.001–100 ​μM with a minimum detection limit of 0.001 ​μM and a high sensitivity of 277.5 ​μA/μM/cm. Analytical recoveries of added sarcosine in sera were 97.35%, within and between-batch coefficients of variation were 1.08% and 1.40% respectively. A good correlation (R2 ​= ​0.99) was obtained between sera sarcosine values, as measured by the standard immuno kit method and present biosensor. The biosensor measured sarcosine levels in sera of prostate cancer patients, which was significantly higher than in apparently healthy persons. The enzyme electrode lost 20% of its initial activity during 180days, when stored dry at 4°C.

Exosomal lncAY927529 enhances prostate cancer cell proliferation and invasion through regulating bone microenvironment

ABSTRACT Exosomes mediate the interaction between cancer cells and their microenvironment, and play a key role in tumor development. Although exosomes can package lncRNAs to mediate extracellular communication, the role of exosomal lncRNA AY927529 in prostate cancer (PCa) remains unclear. Exosomes were extracted from normal human prostatic epithelial cell lines (BPH-1 and RWPE-1) and PCa cell lines (VCaP and LNCaP, DU145, PC3) by ultrahigh speed centrifugation. Results of Western blot indicated that Alix, HSC70 and TSGl01 protein levels were upregulated in exosomes derived from PCa cells. LncAY927529 level was upregulated in PCa cells and exosomes derived from PCa patient serum and human PCa cells. CCK-8, Transwell and Flow cytometry assays demonstrated that bone marrow stromal cell line (ST2) conditioned medium (ST2-CM), treated with exosomes derived from PCa cells with high lncAY927529 level, promoted proliferation and invasion of PC3 and DU145 cells, and inhibited cell apoptosis. RT-qPCR assay indicated that lncAY927529 level was downregulated in PC3 and DU145 cells, exosomes derived from PCa cells (PCa-Exo) and ST2-CM treated with PCa-Exo with low expression of lncAY927529, and overexpression of lncAY927529 had the opposite results. In addition, Western blot assay showed that the autophagy related protein LC3II level was increased in ST2 cells treated with exosomes derived from DU145 cells with high expression of lncAY927529, and LC3I protein level was decreased. CXCL14 acted as a RNA-binding protein of lncAY927529, and exosome-mediated lncAY927529 positively regulated CXCL14 levels in ST2 cells. In general, exosome-mediated lncAY927529 could promote PCa cell proliferation and invasion by regulating bone microenvironment, suggesting that exosomal lncAY927529 may be a potential molecular diagnostic marker of PCa.

Circulating miRNAs as Potential Biomarkers in Prostate Cancer Patients Undergoing Radiotherapy

IntroductionDisease recurrence is a major concern in patients with localized prostate cancer (PCa) following treatment with radiotherapy (RT), and few studies have evaluated the clinical relevance of microRNAs (miRNAs) prior and post-RT.PurposeWe aimed to investigate the significance of miRNAs in the outcomes of prostate cancer patients undergoing radiotherapy and to identify the related pathways through bioinformatics analysis.Materials and MethodsThe expression levels of miR-21, miR-106b, miR-141 and miR-375 involved in the response to radiotherapy were assessed by RT-qPCR in the serum of PCa patients (n=56) prior- and post-RT.ResultsLow expression levels of miR-106b prior-RT were associated with extracapsular extension and seminal vesicles invasion by the tumor (p=0.031 and 0.044, respectively). In the high-risk subgroup (n=47), post-RT expression levels of miR-21 were higher in patients with biochemical relapse (BR) compared to non-relapse (p=0.043). Also, in the salvage treatment subgroup (post-operative BR; n=20), post-RT expression levels of miR-21 and miR-106b were higher in patients with BR compared to non-relapse (p=0.043 and p=0.032, respectively). In the whole group of patients, high expression levels of miR-21 prior-RT and of miR-106b post-RT were associated with significantly shorter overall survival (OS; p=0.049 and p=0.050, respectively). No associations were observed among miR-141 and miR-375 expression levels with clinicopathological features or treatment outcome. Bioinformatics analysis revealed significant enrichment in DNA damage response pathways.ConclusionCirculating miRNAs prior or post-RT may hold prognostic implications in patients with PCa.

Increased Sensitivity of Detection of RASSF1A and GSTP1 DNA Fragments in Serum of Prostate Cancer Patients: Optimisation of Diagnostics Using OBBPA-ddPCR.

Simple SummaryProstate carcinoma (PCa) incidence rates have continued to increase over the last 3 decades worldwide and PCa is the most common cancer in men in Germany. Classical tumour markers exhibit unsatisfactory sensitivity and specificity, leading to unnecessary prostate biopsies and associated clinical complications. New, minimally invasive biomarkers with high sensitivity and specificity are required for an early diagnosis and a better prognosis. In the present study, we evaluated the performance of a previously developed analytical method to detect trace amounts of aberrant methylated tumour DNA. The optimised method which was developed in this study evaluates DNA methylation changes of RASSF1A and GSTP1 in the blood of PCa patients at different stages compared to benign prostatic hyperplasia patients and healthy individuals. RASSF1A methylation analysis was found to be beneficial as a complementary biomarker for PCa diagnosis in combination with classical PCa markers. Both RASSF1A and GSTP1 exhibited pathological DNA methylation levels in all metastatic PCa patients, which may improve early detection of PCa metastases and therapeutic outcomes.Identification of aberrant DNA methylation is a promising tool in prostate cancer (PCa) diagnosis and treatment. In this study, we evaluated a two-step method named optimised bias-based preamplification followed by digital PCR (OBBPA-dPCR). The method was used to identify promoter hypermethylation of 2 tumour suppressor genes RASSF1A and GSTP1 in the circulating cell-free DNA (cfDNA) from serum samples of PCa patients (n = 75), benign prostatic hyperplasia (BPH, n = 58), and healthy individuals (controls, n = 155). The PCa cohort was further subdivided into subgroups comprising (I) patients with Gleason Scores (GS) ≤ 7 (n = 55), (II) GS ≥ 8 (n = 10), and (III) patients with metastatic PCa diagnosis (n = 10). We found that RASSF1A methylation levels were significantly increased in all 3 PCa subgroups compared to the controls and BPH cohorts (p < 0.01 for all comparisons). Fractional abundances of methylated GSTP1 DNA fragments were significantly increased in subgroup III of metastatic PCa patients (p < 0.001). RASSF1A methylation analysis was found to be beneficial as a complementary biomarker where further diagnostic validation is most crucial. In combination with free PSA, RASSF1A methylation status helps to identify PCa patients with GS ≥ 8 and grey-zone total PSA values between 2–10 ng/mL. In our study, PCR biases between 80–90% were sufficient to detect minute amounts of tumour DNA with high signal-to-noise ratios as well as high analytical sensitivity and specificity. Both RASSF1A and GSTP1 exhibited strongly increased DNA methylation levels in all metastatic PCa patients. Our data indicates a superior sensitivity of epigenetic biomarker analyses in early detection of PCa metastases that should also help to improve PCa therapy.

Exosomal circRNA HIPK3 knockdown inhibited cell proliferation and metastasis in prostate cancer by regulating miR-212/BMI-1 pathway

Prostate cancer (PCa) is the second frequent malignancy among men in the world. Exosomal circular RNAs (circRNAs) have been reported to function in PCa progression. The current study aimed to investigate the role of exosomal circRNA homeodomain-interacting protein kinase 3 (circHIPK3) in PCa development. Exosomes were extracted from serum and cells utilizing commercial kit, and identified by transmission electron microscopy (TEM), Western blot assay and nanoparticle tracking analyzer. Relative expression of circHIPK3, microRNA (miR)-212 and B-cell specific MMLV insertion site-1 (BMI-1) was examined by quantitative realtime PCR or Western blot assay. Receiver Operating Characteristic (ROC) analysis was conducted to assess the diagnostic potential of exosomal miR-212. Cell viability, and metastasis including migration and invasion, were detected by Methyl thiazolyl tetrazolium (MTT) assay and Transwell assay, respectively. Cell apoptosis was monitored using flow cytometry. The interaction between miR-212 and circHIPK3 or BMI-1 was validated by dual-luciferase reporter assay. Xenograft tumor assay was employed to explore the role of exosomal circHIPK3 in vivo. Exosomal circHIPK3 was increased in serum of PCa patients, and could discriminate PCa patients from normal volunteers. Depletion of exosomal circHIPK3 or overexpression of exosomal miR-212 reduced viability, migration and invasion, but promoted cell apoptosis in PCa cells, which was attenuated by miR-212 inhibition or BMI-1, respectively. MiR-212 targeted BMI-1, and downregulated BMI-1 expression. Exosomal circHIPK3 knockdown also suppressed tumor growth in vivo. Exosomal circHIPK3 knockdown inhibited PCa progression by regulating miR-212/BMI-1 axis, at least in part, offering a new insight into the molecular mechanism of PCa.

Diagnostic and prognostic potential of the proteomic profiling of serum-derived extracellular vesicles in prostate cancer

Extracellular vesicles (EVs) and their cargo represent an intriguing source of cancer biomarkers for developing robust and sensitive molecular tests by liquid biopsy. Prostate cancer (PCa) is still one of the most frequent and deadly tumor in men and analysis of EVs from biological fluids of PCa patients has proven the feasibility and the unprecedented potential of such an approach. Here, we exploited an antibody-based proteomic technology, i.e. the Reverse-Phase Protein microArrays (RPPA), to measure key antigens and activated signaling in EVs isolated from sera of PCa patients. Notably, we found tumor-specific protein profiles associated with clinical settings as well as candidate markers for EV-based tumor diagnosis. Among others, PD-L1, ERG, Integrin-β5, Survivin, TGF-β, phosphorylated-TSC2 as well as partners of the MAP-kinase and mTOR pathways emerged as differentially expressed endpoints in tumor-derived EVs. In addition, the retrospective analysis of EVs from a 15-year follow-up cohort generated a protein signature with prognostic significance. Our results confirm that serum-derived EV cargo may be exploited to improve the current diagnostic procedures while providing potential prognostic and predictive information. The approach proposed here has been already applied to tumor entities other than PCa, thus proving its value in translational medicine and paving the way to innovative, clinically meaningful tools.

Proteomic analysis of extracellular vesicles identified PI3K pathway as a potential therapeutic target for cabazitaxel-resistant prostate cancer.

Cabazitaxel (CBZ) is now widely used for prostate cancer (PC) patients resistant to docetaxel (DOC), however, most patients eventually acquire resistance. It will, therefore, be of great benefit to discover novel therapeutic target for the resistance. We aimed to identify candidate therapeutic targets for CBZ-resistance by proteomic analysis of extracellular vesicles (EVs) isolated from serum of DOC-resistant PC patients who later developed CBZ-resistance as well as those harvested from culture medium of DOC- and CBZ-resistant PC cell lines. Using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) conjugated to magnetic beads, EVs were purified from serum of PC patients with DOC-resistance that was collected before and after acquiring CBZ-resistance and conditioned medium of DOC-resistant (22Rv1DR) and CBZ-resistant (22Rv1CR) PC cell lines. Protein analysis of EVs was performed by nanoLC-MS/MS, followed by a comparative analysis of protein expression and network analysis. The cytotoxic effect of a phosphatidylinositol-3-kinase (PI3K) inhibitor, ZSTK474, was evaluated by WST-1 assay. The expression and phosphorylation of PI3K and PTEN were examined by western blot analysis. Among differentially regulated proteins, 77 and 61 proteins were significantly increased in EVs from CBZ-resistant PC cell line and patients, respectively. A comparison between the two datasets revealed that six proteins, fructose-bisphosphate aldolase, cytosolic nonspecific dipeptidase, CD63, CD151, myosin light chain 9, and peroxiredoxin-6 were elevated in EVs from both cell line and patients. Network analysis of the increased EV proteins identified pathways associated with CBZ-resistance including PI3K signaling pathway. ZSTK474 significantly inhibited growth of 22Rv1CR cells and improved their sensitivity to CBZ. In 22Rv1CR cells, PI3K was activated and PTEN that inhibits PI3K was deactivated. Proteomic analysis of serum EVs was successfully accomplished by using Tim-4 as a tool to isolate highly purified EVs. Our results suggest that the combination use of CBZ and PI3K inhibitor could be a promising treatment option for CBZ-resistant PC patients.