Pathology-Driven Comprehensive Proteomic Profiling of the Prostate Cancer Tumor Microenvironment

Prostate cancer is the second most common cancer in men worldwide. Gleason grading is an important predictor of prostate cancer outcomes and is influential in determining patient treatment options. Clinical decisions based on a Gleason score of 7 are difficult as the prognosis for individuals diagnosed with Gleason 4+3 cancer is much worse than for those diagnosed with Gleason 3+4 cancer. Laser capture microdissection (LCM) is a highly precise method to isolate specific cell populations or discrete microregions from tissues. This report undertook a detailed molecular characterization of the tumor microenvironment in prostate cancer to define the proteome in the epithelial and stromal regions from tumor foci of Gleason grades 3 and 4. Tissue regions of interest were isolated from several Gleason 3+3 and Gleason 4+4 tumors using telepathology to leverage specialized pathology expertise to support LCM. Over 2,000 proteins were identified following liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of all regions of interest. Statistical analysis revealed significant differences in protein expression (>100 proteins) between Gleason 3 and Gleason 4 regions—in both stromal and epithelial compartments. A subset of these proteins has had prior strong association with prostate cancer, thereby providing evidence for the authenticity of the approach. Finally, validation of these proteins by immunohistochemistry has been obtained using an independent cohort of prostate cancer tumor specimens. Implications: This unbiased strategy provides a strong foundation for the development of biomarker protein panels with significant diagnostic and prognostic potential. Mol Cancer Res; 15(3); 281–93. ©2017 AACR.

[1]  Lydie Lane,et al.  Metrics for the Human Proteome Project 2016: Progress on Identifying and Characterizing the Human Proteome, Including Post-Translational Modifications. , 2016, Journal of proteome research.

[2]  Jeffrey R. Whiteaker,et al.  Commercially available antibodies can be applied in quantitative multiplexed peptide immunoaffinity enrichment targeted mass spectrometry assays , 2016, Proteomics.

[3]  Jouhyun Jeon,et al.  Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer , 2016, Nature Communications.

[4]  Yongheng Chen,et al.  Proteomic analysis of stromal proteins in different stages of colorectal cancer establishes Tenascin-C as a stromal biomarker for colorectal cancer metastasis , 2016, Oncotarget.

[5]  J. Foekens,et al.  The advantage of laser‐capture microdissection over whole tissue analysis in proteomic profiling studies , 2016, Proteomics.

[6]  Alexander L. Vahrmeijer,et al.  Selecting Tumor-Specific Molecular Targets in Pancreatic Adenocarcinoma: Paving the Way for Image-Guided Pancreatic Surgery , 2016, Molecular Imaging and Biology.

[7]  A. Laurinavičienė,et al.  Comprehensive Immunohistochemistry: Digital, Analytical and Integrated , 2016, Pathobiology.

[8]  S. Carr,et al.  Targeted MS Assay Predicting Tamoxifen Resistance in Estrogen-Receptor-Positive Breast Cancer Tissues and Sera. , 2016, Journal of proteome research.

[9]  L. Egevad,et al.  A Contemporary Prostate Cancer Grading System: A Validated Alternative to the Gleason Score. , 2016, European urology.

[10]  M. Mason,et al.  Prostate stromal cell proteomics analysis discriminates normal from tumour reactive stromal phenotypes , 2016, Oncotarget.

[11]  Jingxia Li,et al.  p85α promotes nucleolin transcription and subsequently enhances EGFR mRNA stability and EGF-induced malignant cellular transformation , 2016, Oncotarget.

[12]  K. Guru,et al.  Clinical significance of prospectively assigned Gleason tertiary pattern 4 in contemporary Gleason score 3+3=6 prostate cancer , 2016, The Prostate.

[13]  Joe Y. Chang,et al.  Prognostic significance of nuclear or cytoplasmic nucleolin expression in human non-small cell lung cancer and its relationship with DNA-PKcs , 2016, Tumor Biology.

[14]  M. Loda,et al.  Profiling the tumor microenvironment proteome in prostate cancer using laser capture microdissection coupled to LC⿿MS⿿A technical report , 2015, EuPA open proteomics.

[15]  S. Ramsey,et al.  Cost-Effectiveness of a Biopsy-Based 8-Protein Prostate Cancer Prognostic Assay to Optimize Treatment Decision Making in Gleason 3 + 3 and 3 + 4 Early Stage Prostate Cancer. , 2015, The oncologist.

[16]  Ana C. Gregório,et al.  Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination. , 2015, Biomaterials.

[17]  A. Giordano,et al.  Nucleolin antagonist triggers autophagic cell death in human glioblastoma primary cells and decreased in vivo tumor growth in orthotopic brain tumor model , 2015, Oncotarget.

[18]  P. Kantoff,et al.  The role of miRNAs in prostate cancer. , 2015, European urology.

[19]  Sarah J. Kurley,et al.  The spliceosome is a therapeutic vulnerability in MYC-driven cancer , 2015, Nature.

[20]  Francesca Salipur,et al.  Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation , 2015, Molecular oncology.

[21]  Y. Guan,et al.  Computational Inferences of the Functions of Alternative/Noncanonical Splice Isoforms Specific to HER2+/ER-/PR- Breast Cancers, a Chromosome 17 C-HPP Study. , 2015, Journal of proteome research.

[22]  L. Rassenti,et al.  Targeting the spliceosome in chronic lymphocytic leukemia with the macrolides FD-895 and pladienolide-B , 2015, Haematologica.

[23]  P. Bouvet,et al.  The roles of nucleolin subcellular localization in cancer. , 2015, Biochimie.

[24]  M. Loda,et al.  Development and Clinical Validation of an In Situ Biopsy-Based Multimarker Assay for Risk Stratification in Prostate Cancer , 2015, Clinical Cancer Research.

[25]  S. Carr,et al.  Antibody-based capture of target peptides in multiple reaction monitoring experiments. , 2015, Methods in molecular biology.

[26]  M. Wicha,et al.  Breast cancer stem cells: current advances and clinical implications. , 2015, Methods in molecular biology.

[27]  M. M. Vivanco Mammary Stem Cells , 2015, Methods in Molecular Biology.

[28]  Matthias Mann,et al.  The Q Exactive HF, a Benchtop Mass Spectrometer with a Pre-filter, High-performance Quadrupole and an Ultra-high-field Orbitrap Analyzer* , 2014, Molecular & Cellular Proteomics.

[29]  Kyungeun Kim,et al.  AZGP-1 Immunohistochemical Marker in Prostate Cancer: Potential Predictive Marker of Biochemical Recurrence in Post Radical Prostatectomy Specimens , 2014, Applied immunohistochemistry & molecular morphology : AIMM.

[30]  Zhaohai Wang,et al.  Increased level of nucleolin confers to aggressive tumor progression and poor prognosis in patients with hepatocellular carcinoma after hepatectomy , 2014, Diagnostic Pathology.

[31]  P. Febbo,et al.  A 17-gene assay to predict prostate cancer aggressiveness in the context of Gleason grade heterogeneity, tumor multifocality, and biopsy undersampling. , 2014, European urology.

[32]  Maolin Yan,et al.  Nucleolin identified by comparative mass‑spectra analysis is a potential marker for invasive progression of hepatocellular carcinoma. , 2014, Molecular medicine reports.

[33]  M. Loda,et al.  Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error , 2014, British Journal of Cancer.

[34]  David L Rimm,et al.  Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality , 2014, Proteome Science.

[35]  S. Pennington,et al.  Why have so few proteomic biomarkers “survived” validation? (Sample size and independent validation considerations) , 2014, Proteomics.

[36]  G. Klein Evolutionary aspects of cancer resistance. , 2014, Seminars in cancer biology.

[37]  William S. Hancock,et al.  Distinct splice variants and pathway enrichment in the cell-line models of aggressive human breast cancer subtypes. , 2014, Journal of proteome research.

[38]  Amos Bairoch,et al.  Metrics for the Human Proteome Project 2013-2014 and strategies for finding missing proteins. , 2014, Journal of proteome research.

[39]  Malte Buchholz,et al.  MALDI mass spectrometric imaging based identification of clinically relevant signals in prostate cancer using large‐scale tissue microarrays , 2013, International journal of cancer.

[40]  H. Tsuda,et al.  Laser microdissection and two-dimensional difference gel electrophoresis reveal proteomic intra-tumor heterogeneity in colorectal cancer. , 2013, Journal of proteomics.

[41]  A. Vlahou,et al.  Zinc α2‐glycoprotein as a potential novel urine biomarker for the early diagnosis of prostate cancer , 2012, BJU international.

[42]  Albert J R Heck,et al.  High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies , 2012, Nature Methods.

[43]  L. Kavoussi,et al.  Clinical evaluation of a novel bipolar radiofrequency ablation system for renal masses , 2012, BJU international.

[44]  R. Weil,et al.  Targeted tissue proteomic analysis of human astrocytomas. , 2012, Journal of proteome research.

[45]  A. Bergh,et al.  The Stroma—A Key Regulator in Prostate Function and Malignancy , 2012, Cancers.

[46]  D. Piwnica-Worms,et al.  Knocking down nucleolin expression in gliomas inhibits tumor growth and induces cell cycle arrest , 2012, Journal of Neuro-Oncology.

[47]  B. Engels,et al.  Targeting stroma to treat cancers. , 2012, Seminars in cancer biology.

[48]  D R Mani,et al.  Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma* , 2011, Molecular & Cellular Proteomics.

[49]  Ying Liang,et al.  Identification of PRDX4 and P4HA2 as metastasis-associated proteins in oral cavity squamous cell carcinoma by comparative tissue proteomics of microdissected specimens using iTRAQ technology. , 2011, Journal of proteome research.

[50]  Richard D Smith,et al.  Recommendations for Mass Spectrometry Data Quality Metrics for Open Access Data (Corollary to the Amsterdam Principles)* , 2011, Molecular & Cellular Proteomics.

[51]  M. Mann,et al.  Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer* , 2011, Molecular & Cellular Proteomics.

[52]  Michel Salzet,et al.  Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer , 2011, Analytical and bioanalytical chemistry.

[53]  K. Iczkowski,et al.  Current Perspectives on Gleason Grading of Prostate Cancer , 2011, Current urology reports.

[54]  V. Karantza,et al.  Keratins in health and cancer: more than mere epithelial cell markers , 2011, Oncogene.

[55]  Zhuchu Chen,et al.  Proteomic analysis of the stroma-related proteins in nasopharyngeal carcinoma and normal nasopharyngeal epithelial tissues , 2010, Medical oncology.

[56]  Peng Wang,et al.  Development of mass spectrometry-based shotgun method for proteome analysis of 500 to 5000 cancer cells. , 2010, Analytical chemistry.

[57]  R. Shah,et al.  Current perspectives on the Gleason grading of prostate cancer. , 2009, Archives of pathology & laboratory medicine.

[58]  Lisa H Cazares,et al.  Imaging Mass Spectrometry of a Specific Fragment of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Kinase 2 Discriminates Cancer from Uninvolved Prostate Tissue , 2009, Clinical Cancer Research.

[59]  M. Mann,et al.  In-gel digestion for mass spectrometric characterization of proteins and proteomes , 2006, Nature Protocols.

[60]  E. Petricoin,et al.  Laser Capture Microdissection , 1996, Science.

[61]  Beth Katcher,et al.  Development of an integrated prostate cancer research information system. , 2006, Clinical genitourinary cancer.

[62]  P. Humphrey,et al.  Gleason grading and prognostic factors in carcinoma of the prostate , 2004, Modern Pathology.

[63]  B. Hammock,et al.  The soluble epoxide hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate phosphatase activity , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[64]  M. Mann,et al.  Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. , 2003, Analytical chemistry.

[65]  Complexity staff Research article: merging bottom-up and top-down approaches to study prostate cancer biology , 2002 .

[66]  W. Demark-Wahnefried,et al.  Zinc α-2-Glycoprotein Is Expressed by Malignant Prostatic Epithelium and May Serve as a Potential Serum Marker for Prostate Cancer , 2001 .

[67]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.