Multiplexed targeted mass spectrometry assays for prostate cancer-associated urinary proteins.

Biomarkers for effective early diagnosis and prognosis of prostate cancer are still lacking. Multiplexed assays for cancer-associated proteins could be useful for identifying biomarkers for cancer detection and stratification. Herein, we report the development of sensitive targeted mass spectrometry assays for simultaneous quantification of 10 prostate cancer-associated proteins in urine. The diagnostic utility of these markers was evaluated with an initial cohort of 20 clinical urine samples. Individual marker concentration was normalized against the measured urinary prostate-specific antigen level as a reference of prostate-specific secretion. The areas under the receiver-operating characteristic curves for the 10 proteins ranged from 0.75 for CXL14 to 0.87 for CEAM5. Furthermore, MMP9 level was found to be significantly higher in patients with high Gleason scores, suggesting a potential of MMP9 as a marker for risk level assessment. Taken together, our work illustrated the feasibility of accurate multiplexed measurements of low-abundance cancer-associated proteins in urine and provided a viable path forward for preclinical verification of candidate biomarkers for prostate cancer.

Cancer-secreted AGR2 induces programmed cell death in normal cells.

Anterior Gradient 2 (AGR2) is a protein expressed in many solid tumor types including prostate, pancreatic, breast and lung. AGR2 functions as a protein disulfide isomerase in the endoplasmic reticulum. However, AGR2 is secreted by cancer cells that overexpress this molecule. Secretion of AGR2 was also found in salamander limb regeneration. Due to its ubiquity, tumor secretion of AGR2 must serve an important role in cancer, yet its molecular function is largely unknown. This study examined the effect of cancer-secreted AGR2 on normal cells. Prostate stromal cells were cultured, and tissue digestion media containing AGR2 prepared from prostate primary cancer 10-076 CP and adenocarcinoma LuCaP 70CR xenograft were added. The control were tissue digestion media containing no AGR2 prepared from benign prostate 10-076 NP and small cell carcinoma LuCaP 145.1 xenograft. In the presence of tumor-secreted AGR2, the stromal cells were found to undergo programmed cell death (PCD) characterized by formation of cellular blebs, cell shrinkage, and DNA fragmentation as seen when the stromal cells were UV irradiated or treated by a pro-apoptotic drug. PCD could be prevented with the addition of the monoclonal AGR2-neutralizing antibody P3A5. DNA microarray analysis of LuCaP 70CR media-treated vs. LuCaP 145.1 media-treated cells showed downregulation of the gene SAT1 as a major change in cells exposed to AGR2. RT-PCR analysis confirmed the array result. SAT1 encodes spermidine/spermine N1-acetyltransferase, which maintains intracellular polyamine levels. Abnormal polyamine metabolism as a result of altered SAT1 activity has an adverse effect on cells through the induction of PCD.

Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

Anterior gradient 2 (AGR2) is a cancer-associated secreted protein found predominantly in adenocarcinomas. Given its ubiquity in solid tumors, cancer-secreted AGR2 could be a useful biomarker in urine or blood for early detection. However, normal organs express and might also secrete AGR2, which would impact its utility as a cancer biomarker. Uniform AGR2 expression is found in the normal bladder urothelium. Little AGR2 is secreted by the urothelial cells as no measurable amounts could be detected in urine. The urinary proteomes of healthy people contain no listing for AGR2. Likewise, the blood proteomes of healthy people also contain no significant peptide counts for AGR2 suggesting little urothelial secretion into capillaries of the lamina propria. Expression of AGR2 is lost in urothelial carcinoma, with only 25% of primary tumors observed to retain AGR2 expression in a cohort of lymph node-positive cases. AGR2 is secreted by the urothelial carcinoma cells as urinary AGR2 was measured in the voided urine of 25% of the cases analyzed in a cohort of cancer vs. non-cancer patients. The fraction of AGR2-positive urine samples was consistent with the fraction of urothelial carcinoma that stained positive for AGR2. Since cancer cells secrete AGR2 while normal cells do not, its measurement in body fluids could be used to indicate tumor presence. Furthermore, AGR2 has also been found on the cell surface of cancer cells. Taken together, secretion and cell surface localization of AGR2 are characteristic of cancer, while expression of AGR2 by itself is not.

Conversion of Prostate Adenocarcinoma to Small Cell Carcinoma-Like by Reprogramming.

The lineage relationship between prostate adenocarcinoma and small cell carcinoma was studied by using the LuCaP family of xenografts established from primary neoplasm to metastasis. Expression of four stem cell transcription factor (TF) genes, LIN28A, NANOG, POU5F1, SOX2, were analyzed in the LuCaP lines. These genes, when force expressed in differentiated cells, can reprogram the recipients into stem-like induced pluripotent stem (iPS) cells. Most LuCaP lines expressed POU5F1, while LuCaP 145.1, representative of small cell carcinoma, expressed all four. Through transcriptome database query, many small cell carcinoma genes were also found in stem cells. To test the hypothesis that prostate cancer progression from "differentiated" adenocarcinoma to "undifferentiated" small cell carcinoma could involve re-expression of stem cell genes, the four TF genes were transduced via lentiviral vectors into five adenocarcinoma LuCaP lines-70CR, 73CR, 86.2, 92, 105CR-as done in iPS cell reprogramming. The resultant cells from these five transductions displayed a morphology of small size and dark appearing unlike the parentals. Transcriptome analysis of LuCaP 70CR* ("*" to denote transfected progeny) revealed a unique gene expression close to that of LuCaP 145.1. In a prostate principal components analysis space based on cell-type transcriptomes, the different LuCaP transcriptome datapoints were aligned to suggest a possible ordered sequence of expression changes from the differentiated luminal-like adenocarcinoma cell types to the less differentiated, more stem-like small cell carcinoma types, and LuCaP 70CR*. Prostate cancer progression can thus be molecularly characterized by loss of differentiation with re-expression of stem cell genes. J. Cell. Physiol. 231: 2040-2047, 2016. © 2016 Wiley Periodicals, Inc.

Processing of voided urine for prostate cancer RNA biomarker analysis.

BACKGROUND: Voided urine samples have been shown to contain cells released from prostate tumors. Could good quality RNA from cells in urine be obtained from every donor for multimarker analysis? In addition, could urine donation be as simple as possible, a practical consideration for a lab test, without involving a prostate massage (as indicated for PCA3 testing), which precludes frequent collection; needing it done at a specific time of day (e.g., first or second urine); and requiring prompt processing of samples in clinics with limited molecular biology capability? METHODS: Collected urine samples were pelleted, and the RNA isolated was processed for cDNA synthesis and in vitro transcription to generate amplified sense aRNA. The resultant aRNA was rigorously analyzed for possible introduced changes. DMSO was used as a cell preservative for frozen storage of urine samples. RESULTS: Good quality aRNA was obtained for over 100 samples collected at two different institutions. The process of RNA amplification removed co-isolated DNA in some samples, which did not affect RNA amplification. Amplification did not amplify genes that were absent and produce other expression alterations. The sense aRNA could be used to generate urinary transcriptomes specific to individual patients. No chaotropic agents for RNA preservation were added to the urine samples so that the supernatant could be used for analysis of secreted protein biomarkers. The time of donation was not important since patients were seen during the entire day. DMSO was an effective cell preservative for freezing urine. CONCLUSIONS: Urinary RNA can be readily isolated and amplified for prostate cancer biomarker analysis. Individual patients had unique set of transcripts derived from their tumor.

A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum.

Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase liquid chromatography (LC) separations to fractionate and select target fractions for follow-on LC-selected reaction monitoring (LC-SRM) analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and limit of quantification (LOQ) values of ∼130 pg/mL in serum and ∼10 pg per 100 µg of total protein mass in urine, respectively. A good correlation (R(2) = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and enzyme-linked immunosorbent assay (ELISA). On the basis of an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between noncancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.

Prostate cancer cell phenotypes based on AGR2 and CD10 expression.

The combination of expression patterns of AGR2 (anterior gradient 2) and CD10 by prostate cancer provided four phenotypes that correlated with clinical outcome. Based on immunophenotyping, CD10(low)AGR2(high), CD10(high)AGR2(high), CD10(low)AGR2(low), and CD10(high)AGR2(low) were distinguished. AGR2(+) tumors were associated with longer recurrence-free survival and CD10(+) tumors with shorter recurrence-free survival. In high-stage cases, the CD10(low)AGR2(high) phenotype was associated with a ninefold higher recurrence-free survival than the CD10(high)AGR2(low) phenotype. The CD10(high)AGR2(high) and CD10(low)AGR2(low) phenotypes were intermediate. The CD10(high)AGR2(low) phenotype was most frequent in high-grade primary tumors. Conversely, bone and other soft tissue metastases, and derivative xenografts, expressed more AGR2 and less CD10. AGR2 protein was readily detected in tumor metastases. The CD10(high)AGR2(low) phenotype in primary tumors is predictive of poor outcome; however, the CD10(low)AGR2(high) phenotype is more common in metastases. It appears that AGR2 has a protective function in primary tumors but may have a role in the distal spread of tumor cells.

A multiplex assay to measure RNA transcripts of prostate cancer in urine.

The serum prostate-specific antigen (PSA) test has a high false positive rate. As a single marker, PSA provides limited diagnostic information. A multi-marker test capable of detecting not only tumors but also the potentially lethal ones provides an unmet clinical need. Using the nanoString nCounter gene expression system, a 20-gene multiplex test was developed based on digital gene counting of RNA transcripts in urine as a means to detect prostate cancer. In this test, voided urine is centrifuged to pellet cells and the purified RNA is amplified for hybridization to preselected probesets. Amplification of test cell line RNA appeared not to introduce significant bias, and the counts matched well with gene abundance levels as measured by DNA microarrays. For data analysis, the individual counts were compared to that of ß2 microglobulin, a housekeeping gene. Urine samples of 5 pre-operative cases and 2 non-cancer were analyzed. Pathology information was then retrieved. Signals for a majority of the genes were low for non-cancer and low Gleason scores, and 6/6 known prostate cancer markers were positive in the cases. One case of Gleason 4+5 showed, in contrast, strong signals for all cancer-associated markers, including CD24. One non-cancer also showed signals for all 6 cancer markers, and this man might harbor an undiagnosed cancer. This multiplex test assaying a natural waste product can potentially be used for screening, early cancer detection and patient stratification. Diagnostic information is gained from the RNA signatures that are associated with cell types of prostate tumors.

Development of an ELISA to detect the secreted prostate cancer biomarker AGR2 in voided urine.

BACKGROUND: Comparative transcriptomics between sorted cells identified AGR2 as one of the highest up-regulated genes in cancer. Overexpression in primary tumors was verified by tissue microarray analysis. AGR2 encodes a 19-kDa secreted protein that might be found in urine. METHODS: Monoclonal antibodies were generated against AGR2. One antibody pair, P1G4 (IgG1) to capture and P3A5 (IgG2a) to detect, showed good performance characteristics in a sandwich ELISA. This assay could detect AGR2 at sub ng/ml quantities. RESULTS: AGR2 was detected in tissue digestion media of tumor specimens and culture media of AGR2-secreting prostate cancer cell lines. Additional testings involved frozen section immunohistochemistry, immunoprecipitation, and Western blot analysis. Voided urine samples were collected from pre-operative cancer patients, and urinary protein was desalted and concentrated by filtration. The amount of AGR2 detected was scored as pg/100 µg total protein, and then converted to pg/ml urine. The developed ELISA detected AGR2 protein, ranging from 3.6 to 181 pg/ml, in an initial cohort of samples. AGR2 was not detected in the urine of non-cancer and a bladder cancer patient. CONCLUSIONS: For prostate cancer, an AGR2 urine test could be used for diagnosis. The data, although derived from a small number of samples assayed, showed that developing such a test for clinical application is viable because AGR2 is specific to cancer cells, and apparently secreted into urine.

Arterial and venous vessels are required for modulating developmental relocalization and laterality of the interrenal tissue in zebrafish.

During zebrafish embryogenesis, the endothelium signals to emergent bilateral interrenal primordia to converge toward the midline, yet the merged interrenal tissue has been found to be situated lateral to the midline. We show in this study that bilateral interrenal tissue clusters fused at the central midline, before relocating laterally to be juxtaposed between the dorsal aorta and the posterior cardinal vein. In ets1b morphants where the midtrunk vasculature failed to assemble, various degrees of interrenal fusion defects were displayed, and the interrenal laterality was lost. As either arterial or venous endothelium was specifically reduced, the interrenal tissue was defective in its relocalization and laterality, yet remained closely associated with the malformed vasculature. Our results showed evidence to support that assembly of the axial artery and vein, and its resulting vascular topology at the midtrunk, is required for patterning relocalization and laterality of the interrenal tissue after the initial medial fusion.

Transcriptional activation of zebrafish cyp11a1 promoter is dependent on the nuclear receptor Ff1b.

The cytochrome P450scc (cholesterol side-chain cleavage enzyme) encoded by CYP11A1 catalyzes the first step in steroidogenesis by converting cholesterol to pregnenolone, and thus, controls the synthesis rate of steroid hormones. In mammals, steroidogenic factor 1 (SF1) has been implicated in the cAMP-mediated transcriptional activation of CYP11A1 promoter. In zebrafish, Ff1b has been established as the homolog of SF1. To assess the dependency of cyp11a1 expression on Ff1b, the putative promoter of zebrafish cyp11a1, spanning 1.7 kb, was isolated and bioinformatic analysis revealed two conserved FF1 response elements (FREs) that potentially bind Ff1b. Transfection studies in cell lines of different lineages confirmed that this promoter fragment contained the necessary regulatory elements required for its basal transcription. Truncation and mutagenesis studies performed in Y1 adrenocortical cells revealed that only the proximal FRE was essential for transcriptional activation. Electrophoretic mobility shift assay, however, indicated that Ff1b bound to both FREs, while their in vivo occupancy was confirmed using a chromatin immunoprecipitation assay. Lastly, the cyp11a1 promoter was able to direct EGFP expression specifically to the interrenal gland and genital ridge when transiently expressed in microinjected zebrafish embryos, and the promoter activity is potentiated by ff1b overexpression as measured from luciferase reporter activity in zebrafish embryos.

Cloning, genomic organization, and expression analysis of zebrafish nuclear receptor coactivator, TIF2.

Thyroid hormone receptors (TRs) are involved in numerous diverse biological processes such as growth and differentiation, thermogenesis, neurulation, homeostasis, and metamorphosis. In zebrafish, TRbeta1 has been implicated to be involved in the obligatory embryonic-to-larval transitory phase. In order to understand if nuclear receptor coactivators could modulate the transcriptional activities of TRs during this transitory phase, the transcriptionary intermediary factor 2 (TIF2), a member of the p160 coactivator, was isolated from zebrafish. The zebrafish tif2 cDNA encodes a polypeptide of 1,505 amino acids. The tif2 gene is made up of 23 exons with the AUG and stop codon located in Exon IV and XXIII, respectively. The overall genomic organization of human and zebrafish tif2 genes are very similar. Four tif2 isoforms were identified by RT-PCR. The N-terminus mRNA variants are generated as a result of multiple initiation start sites located upstream of the noncoding Exon I and Exon II. The C-terminus isoforms, E20a and E20b, resulted from the alternative splicing of Exon XX. Although E20a and E20b isoforms were ubiquitously expressed, they were very highly expressed in reproductive tissues. The availability of TIF2 cDNA will allow the analysis of its functional roles in mediating the actions of TRs in various aspects of zebrafish developmental biology.