Jianhe Peng

Initial Development and Validation of a Novel Extraction Method for Quantitative Mining of the Formalin-Fixed, Paraffin-Embedded Tissue Proteome for Biomarker Investigations

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography−mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250−300 proteins per 500 ng of tissue with 1D LC−MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10−15). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.

Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function.

Parkin is an ubiquitin‐protein ligase (E3), mutations of which cause juvenile onset – autosomal recessive Parkinsons disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2‐DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (±2‐fold change; p<0.05) using 2‐DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2‐DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.

A systematic analysis of the effects of increasing degrees of serum immunodepletion in terms of depth of coverage and other key aspects in top-down and bottom-up proteomic analyses

Immunodepletion of clinical fluids to overcome the dominance by a few very abundant proteins has been explored but studies are few, commonly examining only limited aspects with one analytical platform. We have systematically compared immunodepletion of 6, 14, or 20 proteins using serum from renal transplant patients, analysing reproducibility, depth of coverage, efficiency, and specificity using 2‐D DIGE (‘top‐down’) and LC‐MS/MS (‘bottom‐up’). A progressive increase in protein number (≥2 unique peptides) was found from 159 in unfractionated serum to 301 following 20 protein depletion using a relatively high‐throughput 1‐D‐LC‐MS/MS approach, including known biomarkers and moderate–lower abundance proteins such as NGAL and cytokine/growth factor receptors. On the contrary, readout by 2‐D DIGE demonstrated good reproducibility of immunodepletion, but additional proteins seen tended to be isoforms of existing proteins. Depletion of 14 or 20 proteins followed by LC‐MS/MS showed excellent reproducibility of proteins detected and a significant overlap between columns. Using label‐free analysis, greater run‐to‐run variability was seen with the Prot20 column compared with the MARS14 column (median %CVs of 30.9 versus 18.2%, respectively) and a corresponding wider precision profile for the Prot20. These results illustrate the potential of immunodepletion followed by 1‐D nano‐LC‐LTQ Orbitrap Velos analysis in a moderate through‐put biomarker discovery process.

Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel‐Lindau transfectant cell line model

The von Hippel‐Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL‐defective RCC cell line UMRC2 by transfection with vector control or VHL (−/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin‐affinity chromatography and analysis by GeLC‐MS/MS, VHL‐associated changes in plasma membrane proteins were analysed. Comparative analysis of ‐/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the α3 and β1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL‐defective cells. Western blotting confirmed these changes and also revealed VHL‐dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient‐matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL‐dependent proteins that may be important in tumorigenesis.

Differential effects of wild-type and A53T mutant isoform of alpha-synuclein on the mitochondrial proteome of differentiated SH-SY5Y cells.

Increased levels of wild-type (WT) alpha-synuclein (alpha-syn) and mutant A53T alpha-syn are associated with Parkinsons disease (PD), a disease linked to abnormal mitochondrial function. This study compared mitochondria prepared from differentiated SH-SY5Y cells overexpressing WT or A53T alpha-syn with control cells, using 2-D difference in-gel electrophoresis. Statistical analysis was carried out primarily using ANOVA (p < 0.01; Host:WT:A53T) and subsequently using independent t tests (host vs WT, host vs A53T). Of the protein spots found to be differentially expressed (n = 71; p < 0.01, >1.8/<-1.8 fold change), 63 proteins were identified by LC-MS/MS, with the majority (77%) significantly altered in WT samples only. Twenty-three proteins known to be integral components of the mitochondria were abnormally expressed including those with roles in ATP synthesis, oxidoreduction, motor activity, carbohydrate metabolism, protein transcription, and protein folding. Thirteen forms of cytoskeletal proteins were also found to be overexpressed in the mitochondrial preparations from WT alpha-syn cells, suggesting an increased interaction of mitochondria with the cytoskeletal network. Altered levels of four mitochondrial proteins (HSPA9 (mortalin), NDUFS1, DLAT, ATP5A1) were confirmed using Western blot analysis. Furthermore, a significant reduction in OXPHOS 1 activity was observed in the WT alpha-syn cells, suggesting that there are functional consequences of the observed altered protein expression changes in the mitochondria.

Shotgun proteomics of human bile in hilar cholangiocarcinoma

The need to find biomarkers for hepatobiliary diseases including cholangiocarcinoma (CCA) has led to an interest in using bile as a proximal fluid in biomarker discovery experiments, although there are inherent challenges both in its acquisition and analysis. The study described here greatly extends previous studies that have started to characterise the bile proteome. Bile from four patients with hilar CCA was depleted of albumin and immunoglobulin G and analysed by GeLC‐MS/MS. The number of proteins identified per bile sample was between 378 and 741. Overall, the products of 813 unique genes were identified, considerably extending current knowledge of the malignant bile proteome. Of these, 268 were present in at least 3 out of 4 patients. This data set represents the largest catalogue of bile proteins to date and together with other studies in the literature constitutes an important prelude to the potential promise of expression proteomics and subsequent validation studies in CCA biomarker discovery.

CAPG and GIPC1: Breast Cancer Biomarkers for Bone Metastasis Development and Treatment

Background: Bone is the predominant site of metastasis from breast cancer, and recent trials have demonstrated that adjuvant bisphosphonate therapy can reduce bone metastasis development and improve survival. There is an unmet need for prognostic and predictive biomarkers so that therapy can be appropriately targeted. Methods: Potential biomarkers for bone metastasis were identified using proteomic comparison of bone-metastatic, lung-metastatic, and nonmetastatic variants of human breast cancer MDA-MB-231 cells. Clinical validation was performed using immunohistochemical staining of tumor tissue microarrays from patients in a large randomized trial of adjuvant zoledronic acid (zoledronate) (AZURE-ISRCTN79831382). We used Cox proportional hazards regression, the Kaplan-Meier estimate of the survival function, and the log-rank test to investigate associations between protein expression, clinical variables, and time to distant recurrence events. All statistical tests were two-sided. Results: Two novel biomarker candidates, macrophage-capping protein (CAPG) and PDZ domain–containing protein GIPC1 (GIPC1), were identified for clinical validation. Cox regression analysis of AZURE training and validation sets showed that control patients (no zoledronate) were more likely to develop first distant recurrence in bone (hazard ratio [HR] = 4.5, 95% confidence interval [CI] = 2.1 to 9.8, P < .001) and die (HR for overall survival = 1.8, 95% CI = 1.01 to 3.24, P = .045) if both proteins were highly expressed in the primary tumor. In patients with high expression of both proteins, zoledronate had a substantial effect, leading to 10-fold hazard ratio reduction (compared with control) for first distant recurrence in bone (P = .008). Conclusions: The composite biomarker, CAPG and GIPC1 in primary breast tumors, predicted disease outcomes and benefit from zoledronate and may facilitate patient selection for adjuvant bisphosphonate treatment.

Changes in the urinary proteome post-operatively in renal cancer patients – a reflection of tumour or kidney removal?

During the initial phases of a study focussed on discovering new urinary biomarkers for renal cell carcinoma, a number of challenges and limitations were identified, which we subsequently investigated. The purpose of this report is to provide insight into experimental design for such investigations and potential confounding factors that can impact on such studies. Sixty urine samples from 20 patients with clear cell renal cell carcinoma and ten live renal transplant donor patients, pre‐ and post‐nephrectomy, were profiled using SELDI‐TOF‐MS incorporating stringent quality control and in‐house data processing/analysis. There were 65 significantly differentially expressed peaks (five solitary peaks and four peak clusters that increased post nephrectomy and four peak clusters that decreased). Peak 3934 Da m/z and peaks within 11731–11961 Da m/z, which increased post nephrectomy were identified as the 36 amino acid isoform of β‐defensin‐1 and β2‐microglobulin, respectively. However, changes in these two protein forms were also seen in healthy donors following nephrectomy implying a relationship with kidney removal per se rather than tumour removal. This study indicates the difficulties in identifying SELDI peaks for subsequent validation and illustrates the need for appropriate controls in biomarker studies to determine whether changes are indirect consequences of treatment.

VHL-dependent regulation of a β-dystroglycan glycoform and glycogene expression in renal cancer

Identification of novel biomarkers and targets in renal cell carcinoma (RCC) remains a priority and one cellular compartment that is a rich potential source of such molecules is the plasma membrane. A shotgun proteomic analysis of cell surface proteins enriched by cell surface biotinylation and avidin affinity chromatography was explored using the UMRC2- renal cancer cell line, which lacks von Hippel-Lindau (VHL) tumour suppressor gene function, to determine whether proteins of interest could be detected. Of the 814 proteins identified ∼22% were plasma membrane or membrane-associated, including several with known associations with cancer. This included β-dystroglycan, the transmembrane subunit of the DAG1 gene product. VHL-dependent changes in the form of β-dystroglycan were detected in UMRC2−/+VHL transfectants. Deglycosylation experiments showed that this was due to differential sialylation. Analysis of normal kidney cortex and conventional RCC tissues showed that a similar change also occurred in vivo. Investigation of the expression of genes involved in glycosylation in UMRC2−/+VHL cells using a focussed microarray highlighted a number of enzymes involved in sialylation; upregulation of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) was validated in UMRC2− cells compared with their +VHL counterparts and also found in conventional RCC tissue. These results implicate VHL in the regulation of glycosylation and raise interesting questions regarding the extent and importance of such changes in RCC.

Abstract P2-11-07: Proteins predictive of bone metastasis development in breast cancer patients

Background Bone is the commonest site of metastasis in breast cancer and bone metastasis is associated with skeletal complications and reduced quality of life. Adjuvant use of zoledronic acid (ZA) has been explored to prevent or reduce development of bone metastases. In the large international AZURE trial (N = 3360), early stage (II/III) breast cancer patients were randomised to standard therapy (control arm) or to standard therapy + ZA. There is an unmet need for biomarkers to identify early stage patients at high risk of developing bone metastasis so that therapy can be appropriately targeted. We report a study using proteomics and primary tumour tissue microarrays (TMAs) from patients in the AZURE trial to address this need. Methods Bone- and lung-homed variants of the MDA-MB-231 cell line were compared to the parental (non-bone homing) cell type using proteomics (difference gel electrophoresis and mass spectrometry) to identify differentially regulated proteins for clinical validation using TMAs from the AZURE trial. Following characterisation on breast cancer TMAs of different grade, protein expression of candidate biomarkers on AZURE TMAs was assessed semi-quantitatively (low, medium, or high) based on immunohistochemical staining intensity. Statistical analysis investigated associations between protein expression, clinical variables (e.g. ER/PR/HER2 status) and time to local and distant recurrence events (updated to 59 months follow-up). Results Over 140 proteins were differentially expressed and two were chosen for validation based on fold change, biological relevance and antibody availability: Macrophage-capping protein (CAPG) and PDZ domain-containing protein GIPC1. Cox proportional hazards regression analysis of 378 AZURE breast tumour samples showed that patients who did not receive ZA were 4.5-fold more likely to develop bone-only metastasis (p = 0.006) if both proteins were highly expressed in the primary tumour (adjusted for systemic therapy plan, ER status, lymph node involvement, Table 1). This effect was not seen in patients who received ZA. Kaplan-Meier analysis indicated that the effect was not linked to menopausal status. Discussion We have identified two proteins expressed in primary breast tumours of patients which are significantly associated with subsequent development of bone-only metastases and appear to predict for benefit from ZA. Biologically, the two proteins are reported to be involved in cellular structures and signalling, and are implicated in cancers, but their association with breast cancer bone metastasis appears to be novel. Ongoing analysis will extend validation in a further AZURE TMA sample set. These proteins have potential as biomarkers to predict development of bone metastasis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-07.