Targeted Mass Spectrometry Assays for Specific Quantification of Urinary proPSA Isoforms.

Prostate cancer (PCa) is the second leading cause of male cancer-related deaths in the United States. The pre-mature forms of prostate-specific antigen (PSA), proPSA, were shown to be associated with PCa. However, there is a technical challenge in the development of antibody-based immunoassays for specific recognition of each individual proPSA isoform. Herein, we report the development of highly specific, antibody-free, targeted mass spectrometry assays for simultaneous quantification of [-2], [-4], [-5], and [-7] proPSA isoforms in voided urine. The newly developed proPSA assays capitalize on Lys-C digestion to generate surrogate peptides with appropriate length (9-16 amino acids) along with long-gradient liquid chromatography separation. The assay utility of these isoform markers was evaluated in a cohort of 30 well-established clinical urine samples for distinguishing PCa patients from healthy controls. Under the 95% confidence interval, the combination of [-2] and [-4] proPSA isoforms yields the area under curve (AUC) of 0.86, and the AUC value for the combined all four isoforms was calculated to be 0.85. We have further verified [-2]proPSA, the dominant isoform, in an independent cohort of 34 clinical urine samples. Validation of proPSA isoforms in large-scale cohorts is needed to demonstrate their potential clinical utility.

The opposing action of stromal cell proenkephalin and stem cell transcription factors in prostate cancer differentiation.

BACKGROUND: Loss of prostate cancer differentiation or de-differentiation leads to an untreatable disease. Patient survival would benefit if this can be prevented or reversed. Cancer de-differentiation transforms luminal-like (differentiated) adenocarcinoma into less luminal-like and more stem-like (undifferentiated) small cell carcinoma through a sequential activation of stem cell transcription factors (scTF) POU5F1, LIN28A, SOX2 and NANOG. Like stem cells, prostate small cell carcinoma express this quartet of scTF as well as a 10-fold lower level of ß2-microglobulin (B2M) than that of differentiated cell types. In organ development, prostate stromal mesenchyme cells mediate epithelial differentiation in part by secreted factors. METHODS: The identified prostate stromal-specific factor proenkephalin (PENK) was cloned, and transfected into scTF+B2Mlo stem-like small cell carcinoma LuCaP 145.1, reprogrammed luminal-like scTF-B2Mhi LNCaP, and luminal-like scTF-B2Mhi adenocarcinoma LuCaP 70CR. The expression of scTF, B2M and anterior gradient 2 (AGR2) was analyzed in the transfected cells. RESULTS: PENK caused down-regulation of scTF and up-regulation of B2M to indicate differentiation. When transfected into reprogrammed LNCaP, PENK reversed the reprogramming by down-regulation of scTF with attendant changes in cell appearance and colony morphology. When transfected into LuCaP 70CR, PENK up-regulated the expression of adenocarcinoma antigen AGR2, a marker associated with cancer cell differentiation. CONCLUSIONS: Prostate cancer cells appear to retain their responsiveness to stromal PENK signaling. PENK can induce differentiation to counter de-differentiation caused by scTF activation. The many mutations and aneuploidy characteristic of cancer cells appear not to hinder these two processes. Loss of prostate cancer differentiation is like reprogramming from luminal-like to stem-like.

Lineage relationship between prostate adenocarcinoma and small cell carcinoma.

BACKGROUND: Prostate cancer displays different morphologies which, in turn, affect patient outcome. This fact prompted questions about the lineage relationship between differentiated, more treatable prostate adenocarcinoma and poorly differentiated, less treatable non-adenocarcinoma including small cell carcinoma, and the molecular mechanism underlying prostate cancer differentiation. METHODS: Newly available non-adenocarcinoma/small cell carcinoma PDX LuCaP lines were analyzed for expression of stem cell transcription factors (scTF) LIN28A, NANOG, POU5F1, SOX2, which are responsible for reprogramming or de-differentiation. cDNA of these genes were cloned from small cell carcinoma LuCaP 145.1 into expression vectors to determine if they could function in reprogramming. RESULTS: Expression of scTF was detected in small cell carcinoma LuCaP 93, 145.1, 145.2, and non-adenocarcinoma LuCaP 173.1, 173.2A. Transfection of scTF from LuCaP 145.1 altered the gene expression of prostate non-small cell carcinoma cells, as well as fibroblasts. The resultant cells grew in stem-like colonies. Of note was a 10-fold lower expression of B2M in the transfected cells. Low B2M was also characteristic of LuCaP 145.1. Conversely, B2M was increased when stem cells were induced to differentiate. CONCLUSIONS: This work suggested a pathway in the emergence of non-adenocarcinoma/small cell carcinoma from adenocarcinoma through activation of scTF genes that produced cancer de-differentiation.

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.

High expression of AGR2 in lung cancer is predictive of poor survival.

BACKGROUND: Anterior gradient 2 (AGR2) is a protein disulfide isomerase-like protein widely expressed in many normal tissues as well as cancers. In our study, non-neoplastic bronchial epithelial cells as well as non-small cell lung cancer (NSCLC) cells express AGR2 protein. METHODS: AGR2 expression was analyzed on lung tissue microarrays. Tumor staining was correlated with clinical outcomes. RESULTS: On a lung cancer tissue microarray using immunohistochemistry, expression levels in cancer showed generally decreasing intensities in order from adenocarcinomas with mucinous components, other adenocarcinomas, squamous carcinomas, to large cell carcinomas. The study cohort was comprised of 400 cases. As a group, there was a slight trend of lower expression with increasing tumor grade. AGR2 expression level was a significant predictor of overall survival in younger patients only. Patients under 65 with lower levels showed a significantly better survival for both men and women. Patients over 65, in contrast, showed no such trend. CONCLUSIONS: Nearly all NSCLC tumors show AGR2 expression. Lung cancer expression of AGR2 has prognostic value for younger patients.

Reprogramming of prostate cancer cells--technical challenges.

Prostate cancer progression is characterized by tumor dedifferentiation. Cancer cells of less differentiated tumors have a gene expression/transcriptome more similar to that of stem cells. In dedifferentiation, cancer cells may follow a specific program of gene expression changes to a stem-like state. In order to treat cancer effectively, the stem-like cancer cells and cancer differentiation pathway need to be identified and studied. Due to the very low abundance of stem-like cancer cells, their isolation from fresh human tumors is technically challenging. Induced pluripotent stem cell technology can reprogram differentiated cells into stem-like, and this may be a tool to generate sufficient stem-like cancer cells.

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 research: tools, cell types, and molecular targets.

Multiple cancer cell types are found in prostate tumors. They are either luminal-like adenocarcinoma or less luminal-like and more stem-like non-adenocarcinoma and small cell carcinoma. These types are lineage related through differentiation. Loss of cancer differentiation from luminal-like to stem-like is mediated by the activation of stem cell transcription factors (scTF) such as LIN28A, NANOG, POU5F1 and SOX2. scTF expression leads to down-regulation of ß2-microglobulin (B2M). Thus, cancer cells can change from the scTF˜B2Mhi phenotype of differentiated to that of scTF˙B2Mlo of dedifferentiated in the disease course. In development, epithelial cell differentiation is induced by stromal signaling and cell contact. One of the stromal factors specific to prostate encodes proenkephalin (PENK). PENK can down-regulate scTF and up-regulate B2M in stem-like small cell carcinoma LuCaP 145.1 cells indicative of exit from the stem state and differentiation. In fact, prostate cancer cells can be made to undergo dedifferentiation or reprogramming by scTF transfection and then to differentiate by PENK transfection. Therapies need to be designed for treating the different cancer cell types. Extracellular anterior gradient 2 (eAGR2) is an adenocarcinoma antigen associated with cancer differentiation that can be targeted by antibodies to lyse tumor cells with immune system components. eAGR2 is specific to cancer as normal cells express only the intracellular form (iAGR2). For AGR2-negative stem-like cancer cells, factors like PENK that can target scTF could be effective in differentiation therapy.

Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion.

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM), for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ∼12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successfully quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies.