A new insight on serum microRNA expression as novel biomarkers in breast cancer patients

Breast cancer (BC) is one of the widespread lethal diseases affecting a large number of women worldwide. As such, employing and identifying significant markers for detecting BC in different stages can assist in better diagnosis and management of the disease. Several diverse markers have been introduced for diagnosis, but their limitations, including low specificity and sensitivity, reduce their application. microRNAs (miRNAs), as short noncoding RNAs, have been shown to significantly influence gene expression in different disease pathologies, especially BC. Clearly, among different samples used for detecting miRNA expressions, circulating miRNAs present as promising and useful biomarkers. Among different body fluid samples, serum serves as one of the most reliable samples, thanks to its high stability under various severe conditions and some unique features. Extensive research has suggested that BC-related miRNAs can remain stable in the serum. The objective of this review is to describe different samples used for detecting miRNAs in BC subjects with emphasis on serum miRNAs. So, this study highlights serum miRNAs with the potential of acting as biomarkers for different stages of BC. We reviewed the possible correlation between potential miRNAs and the risk of early breast cancer, metastatic breast cancer, response to chemotherapy, and relapse.     

Identification of novel serum peptides biomarkers for female breast cancer patients in Western China

This study aimed to identify novel serum peptides biomarkers for female breast cancer (BC) patients. We analyzed the serum proteomic profiling of 247 serum samples from 96 BC patients, 48 additional paired pre‐ and postoperative BC patients, 39 fibroadenoma patients as benign disease controls, and 64 healthy controls, using magnetic‐bead‐based separation followed by MALDI‐TOF MS. ClinProTools software identified 78 m/z peaks that differed among all analyzed groups, ten peaks were significantly different (P < 0.0001), with Peaks 1–6 upregulated and Peaks 7–10 downregulated in BC. Moreover, three peaks of ten (Peak 1, m/z: 2660.11; Peak 2, m/z: 1061.09; Peak 10, m/z: 1041.25) showed a tendency to return to healthy control values after surgery. And these three peptide biomarkers were identified as FGA605‐629, ITIH4 347–356, and APOA2 43–52. Methods used in this study could generate serum peptidome profiles of BC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.     

Bladder cancer determination via two urinary metabolites: a biomarker pattern approach

The purpose of this study was to use metabonomic profiling to identify a potential specific biomarker pattern in urine as a noninvasive bladder cancer (BC) detection strategy. A liquid chromatography-mass spectrometry based method, which utilized both reversed phase liquid chromatography and hydrophilic interaction chromatography separations, was performed, followed by multivariate data analysis to discriminate the global urine profiles of 27 BC patients and 32 healthy controls. Data from both columns were combined, and this combination proved to be effective and reliable for partial least squares-discriminant analysis. Following a critical selection criterion, several metabolites showing significant differences in expression levels were detected. Receiver operating characteristic analysis was used for the evaluation of potential biomarkers. Carnitine C9:1 and component I, were combined as a biomarker pattern, with a sensitivity and specificity up to 92.6% and 96.9%, respectively, for all patients and 90.5% and 96.9%, respectively for low-grade BC patients. Metabolic pathways of component I and carnitine C9:1 are discussed. These results indicate that metabonomics is a practicable tool for BC diagnosis given its high efficacy and economization. The combined biomarker pattern showed better performance than single metabolite in discriminating bladder cancer patients, especially low-grade BC patients, from healthy controls.     

Holistic metabonomic profiling of urine affords potential early diagnosis for bladder and kidney cancers

The clinical exploration of urinary metabonomic analysis on discriminating between the top-two-incidence urological cancers, bladder and kidney cancers (BC and KC), is still virgin land. In this study, we discovered and evaluated the clinical utility of holistic metabonomic profiling and current single biomarker methods, and ultimately suggested a potential screening test for BC and KC. Urine metabonomic profiling for 19 BC patients, 25 KC patients, and 24 healthy controls was carried out using an LC–MS based platform, which utilized both reversed phase chromatography and hydrophilic interaction chromatography. The holistic method that refers to orthogonal partial least-squares-discriminant analysis based on all qualified profile data, successfully classified BC, KC and healthy control groups, showing 100 % sensitivity and specificity. Taurine, hippuric acid, phenylacetylglutamine and carnitine species contributed most to the BC and KC discrimination. The predictive power of each above metabolite is evaluated using receiver operator characteristic technique. Hippuric acid was found 10-fold decrease in concentration relative healthy controls, and performed the best as a biomarker for BC diagnosis, with its sensitivity and specificity of 78.9 and 86.5 %, respectively. Carnitine C10:3 was found 1.5-fold decrease, and reached 84.0 % of sensitivity and 60.5 % of specificity for KC diagnosis. In view of both sensitivity and specificity, the holistic method is more accurate for detecting BC and KC, than current single metabonomic biomarker methods, and it could be advocated as a prescreen to other forms of more invasive or uncomfortable screening. Moreover, this approach also demonstrates attractive performance in diagnosis of early stage (ES) BC and KC patients.     

Discovery of urine biomarkers for bladder cancer via global metabolomics

Bladder cancer (BC) is latent in its early stage and lethal in its late stage. Therefore, early diagnosis and intervention are essential for successful BC treatment. Considering the limitations of current diagnostic tools, noninvasive biomarkers that are both highly sensitive and specific are needed to improve the overall survival and quality of life of patients. With the advent of systems biology, “-omics” technologies have been developed over the past few decades. As a promising member, global metabolomics has increasingly been found to have clear potential for biomarker discovery. However, urinary metabolomics studies related to BC have lagged behind those of other urinary cancers, and major findings have not been systematically reported. The objective of this review is to comprehensively list the currently identified potential urinary metabolite biomarkers for BC.     

Circulating microRNAs,miR‐92a,miR‐100 and miR‐143, as non‐invasive biomarkers for bladder cancer diagnosis

The application of microRNAs (miRNAs) as potential biomarkers and therapy targets has been widely investigated in many kinds of cancers. Recent advantages of serum miRNAs open a new realm of possibilities for non‐invasive diagnosis and prognosis of bladder cancer (BC). The aim of our study was to identify plasma miR‐92a, miR‐100 and miR‐143 expression signatures in patients with BC to introduce new markers for establishing BC diagnosis and prognosis. Blood samples were collected from 70 BC patients and 62 controls. An expression of three target miRNAs (miR‐92a, miR‐100 and miR‐143) was measured using quantitative real‐time PCR method. Results were correlated with clinicopathological data and analysed. Plasma levels of miR‐92a, miR‐100 and miR‐143 were significantly lower in BC patients than in control group. Receiver operator characteristic analysis revealed that the sensitivity and specificity values of miR‐92a were 97·1% and 76·7%, respectively, with a cut‐off value of 0·573. The sensitivity and specificity values of miR‐100 were 90% and 66·7%, respectively, with a cut‐off value of 0·644. The sensitivity and specificity values of miR‐143 were 78·6% and 93·3%, respectively, with a cut‐off value of 0·164. This study explores the existence of specific plasma miRNAs as early diagnostic biomarkers for BC in Egyptian patients; and these findings suggest that plasma miR‐92a, miR‐100 and miR‐143 could be promising novel circulating biomarkers in clinical detection of BC. Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluation of serum exosomal LncRNA‐based biomarker panel for diagnosis and recurrence prediction of bladder cancer

Exosomes are small membrane vesicles released by many cells. These vesicles can mediate cellular communications by transmitting active molecules including long non‐coding RNAs (lncRNAs). In this study, our aim was to identify a panel of lncRNAs in serum exosomes for the diagnosis and recurrence prediction of bladder cancer (BC). The expressions of 11 candidate lncRNAs in exosome were investigated in training set (n = 200) and an independent validation set (n = 320) via quantitative real‐time PCR. A three‐lncRNA panel (PCAT‐1, UBC1 and SNHG16) was finally identified by multivariate logistic regression model to provide high diagnostic accuracy for BC with an area under the receiver‐operating characteristic curve (AUC) of 0.857 and 0.826 in training set and validation set, respectively, which was significantly higher than that of urine cytology. The corresponding AUCs of this panel for patients with Ta, T1 and T2‐T4 were 0.760, 0.827 and 0.878, respectively. In addition, Kaplan‐Meier analysis showed that non‐muscle‐invasive BC (NMIBC) patients with high UBC1 expression had significantly lower recurrence‐free survival (P = 0.01). Multivariate Cox analysis demonstrated that UBC1 was independently associated with tumour recurrence of NMIBC (P = 0.018). Our study suggested that lncRNAs in serum exosomes may serve as considerable diagnostic and prognostic biomarkers of BC.     

The identification of new biomarkers for bladder cancer: A study based on TCGA and GEO datasets

Bladder cancer (BC) is one of the most common neoplastic diseases worldwide. With the highest recurrence rate among all cancers, treatment of BC only improved a little in the last 30 years. Available biomarkers are not sensitive enough for the diagnosis of BC, whereas the standard diagnostic method, cystoscopy, is an invasive test and expensive. Hence, seeking new biomarkers of BC is urgent and challenging. With that order, we screened the overlapped differentially expressed genes (DEGs) of GSE13507 and TCGA BLCA datasets. Subsequent protein–protein interactions network analysis recognized the hub genes among these DEGs. Further functional analysis including Gene Ontology and KEGG pathway analysis and gene set enrichment analysis were processed to investigate the role of these genes and potential underlying mechanisms in BC. Kaplan–Meier analysis and Cox hazard ratio analysis were carried out to clarify the diagnostic and prognostic role of these genes. In conclusion, our present study demonstrated that ACTA2, CDC20, MYH11, TGFB3, TPM1, VIM, and DCN are all potential diagnostic biomarkers for BC. And may also be potential treatment targets for clinical implication in the future.     

乳腺癌早期筛查和诊断生物标志物研究进展

2020年全球乳腺癌(breast cancer,BC)新发病例达226万例,占全部肿瘤新发病例的11.7%,是全世界发病率最高的癌症。早期发现、早期诊断和早期治疗是降低乳腺癌死亡率及改善预后的关键。尽管乳房X光筛查被广泛用作乳腺癌筛查的工具,但其假阳性、辐射性和过度诊断仍是亟待解决的问题。因此,亟需开发易于获取且稳定可靠的生物标志物,用于乳腺癌无创筛查和诊断。近年来多项研究显示来自乳腺癌患者血液中的循环肿瘤细胞DNA(circulating tumor cell DNA,ctDNA)、癌胚抗原(carcinoembryonic antigen,CEA)、糖类抗原15-3(carbohydrate antigen 15-3,CA15-3)、细胞外囊泡(extracellular vesicles,EV)、循环miRNA和BRCA基因突变等生物标志物,以及来自人体尿液、呼出气体(volatile organic compounds,VOCs)和乳头吸出液(nipple aspirate fluid,NAF)中的磷脂、miRNA、苯乙酮和十六烷等多种生物标志物与乳腺癌早期筛查和诊断密切相关。本文综述了上述生物标志物在乳腺癌早期筛查和诊断中的应用。     

Profilin 1 is a potential biomarker for bladder cancer aggressiveness

Of the most important clinical needs for bladder cancer (BC) management is the identification of biomarkers for disease aggressiveness. Urine is a "gold mine" for biomarker discovery, nevertheless, with multiple proteins being in low amounts, urine proteomics becomes challenging. In the present study we applied a fractionation strategy of urinary proteins based on the use of immobilized metal affinity chromatography for the discovery of biomarkers for aggressive BC. Urine samples from patients with non invasive (two pools) and invasive (two pools) BC were subjected to immobilized metal affinity chromatography fractionation and eluted proteins analyzed by 1D-SDS-PAGE, band excision and liquid chromatography tandem MS. Among the identified proteins, multiple corresponded to proteins with affinity for metals and/or reported to be phosphorylated and included proteins with demonstrated association with BC such as MMP9, fibrinogen forms, and clusterin. In agreement to the immobilized metal affinity chromatography results, aminopeptidase N, profilin 1, and myeloblastin were further found to be differentially expressed in urine from patients with invasive compared with non invasive BC and benign controls, by Western blot or Elisa analysis, nevertheless exhibiting high interindividual variability. By tissue microarray analysis, profilin 1 was found to have a marked decrease of expression in the epithelial cells of the invasive (T2+) versus high risk non invasive (T1G3) tumors with occasional expression in stroma; importantly, this pattern strongly correlated with poor prognosis and increased mortality. The functional relevance of profilin 1 was investigated in the T24 BC cells where blockage of the protein by the use of antibodies resulted in decreased cell motility with concomitant decrease in actin polymerization. Collectively, our study involves the application of a fractionation method of urinary proteins and as one main result of this analysis reveals the association of profilin 1 with BC paving the way for its further investigation in BC stratification.     

Differentially Expressed Extracellular Vesicle-Contained microRNAs before and after Transurethral Resection of Bladder Tumors

Bladder cancer (BC) is currently diagnosed and monitored by cystoscopy, a costly and invasive procedure. Potential biomarkers in urine, blood, and, more recently, extracellular vesicles (EVs), have been explored as non-invasive alternatives for diagnosis and surveillance of BC. EVs are nanovesicles secreted by most cell types containing diverse molecular cargo, including different types of small RNAs, such as microRNA (miRNA). In this study, we performed next-generation sequencing of EV-contained miRNA isolated from urine and serum of 41 patients with non-muscle invasive BC (27 stage Ta, 14 stage T1) and 15 non-cancer patients (NCP) with benign cystoscopy findings. MiRNA sequencing was also performed on serum supernatant samples for T1 patients. To identify potential BC-specific biomarkers, expression levels of miRNA in presurgery samples were compared to those at postsurgery check-ups, and to NCPs. Results showed that two miRNAs, urinary EV-contained miR-451a and miR-486-5p, were significantly upregulated in presurgery samples from T1 patients compared to postsurgery check-up samples. This was confirmed in a replica EV/RNA isolation and sequencing run of 10 T1 patients from the primary run; however, analyses revealed no differential expression of miRNAs in serum EVs, serum supernatant, or when comparing BC patients to NCPs. This is the first study to investigate EV-containing miRNA sequencing in pre- and postsurgery BC patient samples and our findings suggest that urinary EV-contained miR-451a and miR-486-5p may be potential biomarkers for recurrence-free survival of BC patients with stage T1 disease.     

Matrix metalloproteases and TIMPs as prognostic biomarkers in breast cancer patients treated with radiotherapy: A pilot study

Breast cancer (BC) is the most common tumour in women and one of the most important causes of cancer death worldwide. Radiation therapy (RT) is widely used for BC treatment. Some proteins have been identified as prognostic factors for BC (Ki67, p53, E‐cadherin, HER2). In the last years, it has been shown that variations in the expression of MMPs and TIMPs may contribute to the development of BC. The aim of this pilot work was to study the effects of RT on different MMPs (‐1, ‐2, ‐3, ‐7, ‐8, ‐9, ‐10, ‐12 and ‐13) and TIMPs (‐1 to ‐4), as well as their relationship with other variables related to patient characteristics and tumour biology. A group of 20 BC patients treated with RT were recruited. MMP and TIMP serum levels were analysed by immunoassay before, during and after RT. Our pilot study showed a slight increase in the levels of most MMP and TIMP with RT. However, RT produced a significantly decrease in TIMP‐1 and TIMP‐3 levels. Significant correlations were found between MMP‐3 and TIMP‐4 levels, and some of the variables studied related to patient characteristics and tumour biology. Moreover, MMP‐9 and TIMP‐3 levels could be predictive of RT toxicity. For this reason, MMP‐3, MMP‐9, TIMP‐3 and TIMP‐4 could be used as potential prognostic and predictive biomarkers for BC patients treated with RT.     

Lipidomic analysis of phospholipids from human mammary epithelial and breast cancer cell lines

Alterations of phospholipid (PL) profiles have been associated to disease and specific lipids may be involved in the onset and evolution of cancer; yet, analysis of PL profiles using mass spectrometry (MS) in breast cancer cells is a novel approach. Previously, we reported a lipidomic analysis of PLs from mouse mammary epithelial and breast cancer cells using off‐line thin layer chromatography (TLC)‐MS, where several changes in PL profile were found to be associated with the degree of malignancy of cells. In the present study, lipidomic analysis has been extended to human mammary epithelial cells and breast cancer cell lines (MCF10A, T47‐D, and MDA‐MB‐231), using TLC‐MS, validated by hydrophilic interaction liquid chromatography‐MS. Differences in phosphatidylethanolamine (PE) content relative to total amount of PLs was highest in non‐malignant cells while phosphatidic acid was present with highest relative abundance in metastatic cells. In addition, the following differences in PL molecular species associated to cancer phenotype, metastatic potential, and cell morphology were found: higher levels of alkylacyl PCs and phosphatidylinositol (PI; 22:5/18:0) were detected in migratory cells, epithelial cells had less unsaturated fatty acyl chains and shorter aliphatic tails in PE and sphingomyelin classes, while PI (18:0/18:1) was lowest in non‐malignant cells compared to cancer cells. To date, information about PL changes in cancer progression is scarce, therefore results presented in this work will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for cancer therapy. J. Cell. Physiol. 228: 457–468, 2013. © 2012 Wiley Periodicals, Inc.     

MicroRNA expression profile in serum reveals novel diagnostic biomarkers for endometrial cancer

Purpose: Circulating microRNAs (miRNAs) prove to be promising diagnostic biomarkers for various cancers, including endometrial cancer (EC). The present study aims to identify serum microRNAs that can serve as potential biomarkers for EC diagnosis.Patients and methods: A total of 92 EC and 102 normal control (NC) serum samples were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) in this four-phase experiment. The logistic regression method was used to construct a diagnostic model based on the differentially expressed miRNAs in serum. The receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value. To further validate the diagnostic capacity of the identified signature, the 6-miRNA marker was compared with previously reported biomarkers and verified in three public datasets. In addition, the expression characteristics of the identified miRNAs were further explored in tissue and serum exosomes samples.Results: Six miRNAs (miR-143-3p, miR-195-5p, miR-20b-5p, miR-204-5p, miR-423-3p, and miR-484) were significantly overexpressed in the serum of EC compared with NCs. Areas under the ROC of the 6-miRNA signatures were 0.748, 0.833, and 0.967 for the training, testing, and the external validation phases, respectively. The identified signature has a very stable diagnostic performance in the large cohorts of three public datasets. Compared with previously identified miRNA biomarkers, the 6-miRNA signature in the present study has superior performance in diagnosing EC. Moreover, the expression of miR-143-3p and miR-195-5p in tissues and the expression of miR-20b-5p in serum exosomes were consistent with those in serum.Conclusions: We established a 6-miRNA signature in serum and they could function as potential non-invasive biomarker for EC diagnosis.     

Clinical relevance of glycerophospholipid,sphingomyelin and glutathione metabolism in the pathogenesis of pharyngolaryngeal cancer in smokers: the Korean Cancer Prevention Study-II

Introduction

Although smoking is a major risk factor for pharyngolaryngeal cancer, most smokers do not develop pharyngolaryngeal cancer.

Objectives

In the prospective Korean Cancer Prevention Study-II (KCPS-II), we investigated the application of metabolomics to differentiate smokers with incident pharyngolaryngeal cancer (pharyngolaryngeal cancer group) from smokers who remained free from cancer (controls) during a mean follow-up period of 7 years and aimed to discover valuable early biomarkers of pharyngolaryngeal cancer.

Methods

We used baseline serum samples from 30 smoking men with incident pharyngolaryngeal cancer and 59 age-matched cancer-free smoking men. Metabolic alterations associated with the incidence of pharyngolaryngeal cancer were investigated by performing metabolomics on baseline serum samples using ultra-performance liquid chromatography-linear-trap quadrupole-Orbitrap mass spectrometry.

Results

Compared to the control group, the pharyngolaryngeal cancer group showed significantly higher oxidized LDL levels. Seventeen metabolites were differentially abundant between the two groups. At baseline, compared to controls, smokers with incident pharyngolaryngeal cancer during follow-up showed significantly higher levels of pyroglutamic acid (glutathione metabolism) but lower levels of lysophosphatidylcholines (lysoPCs) C14:0, C15:0, C16:0, C17:0, C18:0, and C20:5; glycerophosphocholine; PC C36:5; lysoPEs C16:0, C20:1, and C22:0 (glycerophospholipid metabolism); SM (d18:0/16:1); and SM (d18:1/18:1) (sphingomyelin metabolism). Furthermore, smokers with incident pharyngolaryngeal cancer showed significantly higher levels of oleamide and lower levels of tryptophan and linoleyl carnitine at baseline than cancer-free smokers.

Conclusion

This prospective study showed the clinical relevance of dysregulated metabolism of glutathione, glycerophospholipids and sphingolipids to the pathogenesis of pharyngolaryngeal cancer among smokers. These data suggest that the dysregulation of these metabolic processes may be a key mechanism underlying pharyngolaryngeal cancer progression and development.

     

Biomarkers for detection and prognosis of breast cancer identified by a functional hypermethylome screen

Breast cancer (BC) is a disease with diverse tumor heterogeneity, which challenges conventional approaches to develop biomarkers for early detection and prognosis. To identify effective biomarkers, we performed a genome-wide screen for functional methylation changes in BC, i.e., genes silenced by promoter hypermethylation, using a functionally proven gene expression approach. A subset of candidate hypermethylated genes were validated in primary BCs and tested as markers for detection and prognosis prediction of BC. We identified 33 cancer specific methylated genes and, among these, two categories of genes: (1) highly frequent methylated genes that detect early stages of BC. Within that category, we have identified the combination of NDRG2 and HOXD1 as the most sensitive (94%) and specific (90%) gene combination for detection of BC; (2) genes that show stage dependent methylation frequency pattern, which are candidates to help delineate BC prognostic signatures. For this category, we found that methylation of CDO1, CKM, CRIP1, KL and TAC1 correlated with clinical prognostic variables and was a significant prognosticator for poor overall survival in BC patients. CKM [Hazard ratio (HR) = 2.68] and TAC1 (HR = 7.73) were the strongest single markers and the combination of both (TAC1 and CKM) was associated with poor overall survival independent of age and stage in our training (HR = 1.92) and validation cohort (HR = 2.87). Our study demonstrates an efficient method to utilize functional methylation changes in BC for the development of effective biomarkers for detection and prognosis prediction of BC.     

Potential plasma lipid biomarkers in early-stage breast cancer

Objective

To find new biomarkers for early diagnosis of breast cancer.

Results

847 lipid species were identified from 78 plasma samples (37 breast cancer samples and 41 healthy controls) by ultra HPLC coupled with quadrupole time-of-flight tandem mass spectrometry. These include 321 glycerophospholipids (GPs), 265 glycerolipids (GLs), 91 sphingolipids (SPs), 77 fatty acyls (FAs), 68 sterol lipids (STs), 18 prenol lipids (PRs), 6 polyketides (PKs), and 1 saccharolipid (SL). Separation was observed from an orthogonal signal correction Partial Least Square Discrimination Analysis model. Based on this analysis, six differentiating lipids were identified: PC (20:2/20:5), PC (22:0/24:1), TG (12:0/14:1), and DG (18:1/18:2) had high levels, whereas PE (15:0/19:1) and N-palmitoyl proline had low levels in the breast cancer samples compared with the healthy controls. Furthermore, significant differences in metabolites were found among some clinical characteristics.

Conclusions

Our results reveal that six specific lipids could serve as potential biomarkers for early diagnosis of breast cancer.

     

Melatonin and human cancer

A number of studies performed in vitro and on experimental animals supported the view that pineal gland inhibits neoplastic growth. Data in humans are scanty and controversial. In the present study we measured serum melatonin (MT), prolactin (PRL) and growth hormone (GH) concentrations, at 08.00 and 24.00, in 132 cancer patients and in 58 healthy control subjects. The patients were stratified according to histology and stage of disease as follows: 30 stage I–II and 45 stage III–IV breast cancer (BC); 39 stage III–IV lung cancer; 18 advanced gastrointestinal (GI) cancer. We also measured MT levels, at the same time-points, in 20 women with primary BC before and after radical mastectomy. Finally, we evaluated the circadian rhythm of serum MT in 18 patients with advanced cancer. On the whole, the patients with advanced tumors showed serum MT levels significantly higher than controls, without any correlation with PRL and GH values. When looking at stage III–IV vs stage I–II BC patients, significantly higher MT levels have been found in the former group. The surgical removal of the primary BC was not associated with any changes in MT values at both time points considered. A highly significant rhythm of serum MT was recorded in advanced cancer patients and the rhythmic parameters were substantially superimposable on those of the control subjects.     

Proteomic analysis of conditioned media from the PC3, LNCaP, and 22Rv1 prostate cancer cell lines: discovery and validation of candidate prostate cancer biomarkers

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen (PSA) test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from three human prostate cancer cell lines of differing origin [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)] to identify secreted proteins that could serve as novel prostate cancer biomarkers. Each cell line was cultured in triplicate, followed by a bottom-up analysis of the peptides by two-dimensional chromatography and tandem mass spectrometry. Approximately, 12% (329) of the proteins identified were classified as extracellular and 18% (504) as membrane-bound among which were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. On the basis of this, four novel candidates, follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2, were validated in the serum of patients with and without prostate cancer. The proteins presented in this study represent a comprehensive sampling of the secreted and shed proteins expressed by prostate cancer cells, which may be useful as diagnostic, prognostic or predictive serological markers for prostate cancer.