Multiple immunofluorescence assay identifies upregulation of Active β-catenin in prostate cancer
To apply a systems pathology-based approach to the quantification of nuclear Active β-catenin and human leukocyte antigen class I, and assess the biomarker involvement in a cohort of prostate tumor patients.
The systems pathology approach applied allows a precise quantification of the marker expression in the different cell compartments as well as the determination of the areas that coexpress two markers. Our data shows that the accumulation of β-catenin in the nuclear compartment is significantly decreased in the adjacent normal areas when compared to tumor of the same patients (p < 0.001). In conclusion, the application of this novel multiple immunofluorescence assay demonstrates that the upregulation of Active β-catenin is a relatively common feature of prostate tumor development, and further supports the activation of the Wnt/β-catenin pathway in prostate cancer progression.
KeywordsActive β-catenin HLA class1 Wnt/β-catenin pathway Prostate cancer Systems pathology
castrate resistant prostate cancer
heat-induced epitope retrieval
The origin of the data is a summary of single observations derived from a more comprehensive study encompassing seven protein biomarkers that were assembled in two multiplex assays and applied to a cohort of prostate cancer patients (n = 505). Quantitative and cellular attributes representing biomarkers from both multiplex assays containing Active β-catenin (ABC), HLA-1 and CK18, among others, were correlated with clinical variables including biochemical recurrence (BCR). One of the objectives of the comprehensive work (manuscript in preparation) was to describe correlations between clinicopathological data and results from the quantification and colocalization of individual marker expressions. When we started analyzing the data we observed that the expression of Active β-catenin was higher in tumor areas than in adjacent normal tissue. In this research note we describe for the first time how a systems biology-based methodology yields a precise quantification of the nuclear Active β-catenin (effector fraction) and thus unequivocally demonstrates the implication of this marker in prostate tumor formation.
Prostate cancer is the most common epithelial malignancy after skin cancer in men from western countries . Systems pathology is a multidisciplinary approach that integrates proteomic, molecular and imaging data with the patients’ clinical history using machine learning . By applying multiplex immunofluorescence and image analysis, we have been able to construct quantitative features for interrogating several biomarkers at the microscopic cellular level. This allows for investigations of co-localization and expression of cellular proteins, specifically during activation of specific signaling pathways. Of paramount importance, the colocalization of a given marker with DAPI can be applied in the quantification of the marker that is present within the nucleus .
HLA class I expression on the cell surface of cancer cells is critical for an appropriate anti-tumor immune response by presenting antigenic peptides to cytotoxic T lymphocytes . One of the most frequent escape mechanisms that tumor cells develop to avoid immune system recognition is the loss of HLA-1 expression, which correlates with properties associated with ‘stemness’ resulting in treatment resistance and a poor prognosis .
The Wnt/β-catenin signaling pathway is an evolutionarily conserved developmental pathway involved in several physiological processes . Aberrant activation of the Wnt/β-catenin pathway is implicated in many tumor types. β-catenin, a key protein in the Wnt pathway, can trigger the transcription of important regulators of cell cycle progression, cell proliferation and cell stemness upon activation and nuclear translocation . β-catenin expression has been shown to be associated with more malignant, high Gleason score tumors. Both β-catenin and wnt-1 expressions are located in the advancing edge of the tumor . The correlation between higher malignancy related to castrate resistant prostate cancer (CRPC) and β-catenin suggests Wnt/β-catenin signaling pathway upregulation in CRPC . Here we demonstrate the association between nuclear localization of Active β-catenin and prostate tumor progression.
Materials and methods
Patient selection and TMA production
Prostate cancer samples were obtained from 505 patients (age range 40–75, median 63 years) with localized and locally advanced disease that underwent a radical prostatectomy (Hospital de la Vall d’Hebron, Hospital Clínic de Barcelona and Hospital 12 de Octubre). The Gleason breakdown of the tumors was as follows: 81 tumors Gleason < 6, 130 tumors Gleason 6, 293 tumors Gleason > 6. Whole tissue sections from each patient were first stained and three tumor cores and a normal core previously selected were included in tissue microarrays (TMAs). Blank 3.5 µM thick consecutive sections of the TMA were used for IF analysis.
Markers included in the multiplex reaction
HLA class 1
Nuclear Active β-catenin assessment
The antibody for Active β-catenin (ABC) is able to detect nuclear, cytoplasmic and membranous ABC. The values for nuclear β-catenin, accounting for the effector fraction of the transcription factor, were deduced from the co-localization between DAPI and ABC in cells that were expressing CK18 (see Additional file 1: Figure S1 for details on subcellular localization of ABC). ABC can be membranous, cytoplasmic and nuclear in cells with activation of the wnt/β-catenin pathway. To avoid any potential overestimation of nuclear ABC due to some background issue related to the cytoplasmic ABC expression a correction value was added. The correction factor, the co-localization value between CK18 and DAPI, is based on the fact that CK18 is only expressed both in membrane and in cytoplasm and should not be found in co-localization with DAPI. Thus, the correction factor should be zero in samples that are not affected by nuclear ABC overestimation.
A Wilcoxon signed-rank test was applied to elucidate differences in the marker expression measures between tumor and adjacent normal areas.
Results and discussion
Marker expression data
HLA Class 1 in tumor formation
Wilcoxon signed-rank test for paired samples (n = 383)
Median [Q1; Q3]
Tumor expression Median [Q1; Q3]
Nuclear Active β-catenin
2.05 [0.00; 7.12]
4.96 [0.46; 10.9]
Total Active β-catenin
0.28 [0.13; 0.58]
0.49 [0.20; 0.89]
HLA class I
0.29 [0.10; 0.70]
0.28 [0.16; 0.50]
Active β-catenin in tumor formation
Nuclear Active β-catenin expression was found associated with prostate tumorigenesis according to the Wilcoxon signed-rank test calculation (p < 0.001). This correlation was obtained comparing the median values of normalized nuclear ABC in the tumor cores with the values of the same normalized ABC fraction in normal cores (n = 383 tumor-normal paired samples). The same level of significance (p < 0.001) was obtained when total ABC expression was assessed and compared in tumor cores vs. normal cores (Table 2).
In the current study, we present nuclear accumulation of Active β-catenin in prostate malignancy development. This finding is in agreement with the published literature [7, 8, 12, 13, 14]. β-catenin, a dual effector protein that was first described in the regulation of intracellular adhesion, is also a key nuclear signaling protein in the activation of the Wnt/β-catenin pathway. Downstream targets of β-catenin including c-Myc, Cyclin D1 and CD44, among others, are proliferation agents that are involved in oncogenesis . β-catenin signaling may play important roles in prostate cancer progression  and in the acquisition of tumor malignant phenotypes and the capacity for invasion through the induction of AR activity .
Interestingly, the correlation between tumor progression and nuclear Active β-catenin is also found with total ABC (nuclear, cytoplasmic and membranous). However, when average expression values are compared, the difference between tumor cores and normal cores is bigger for nuclear Active β-catenin than for total Active β-catenin. Our data further supports a role for the Wnt/β-catenin signaling pathway in prostate cancer formation and as a potential therapeutic target. Furthermore, applications which interrogate biomarkers at the intact tissue-cellular level will continue to advance our understanding of basic tumor biology.
To summarize, our multiplex-based systems pathology approach is a novel tool for the precise quantification of CK18 epithelial nuclear Active β-catenin through colocalization of DAPI and ABC. The additional evaluation of HLA provides insight into the biology underpinning prostate cancer progression. Thus, the use of the multiplex approach is essential for a more comprehensive analysis of various markers, including DAPI, HLA class I, Active β-catenin and CK18. Our results show a trend for HLA Class I downregulation, and strongly support the implication of Active β-catenin, both nuclear and total, in prostate cancer development.
The study needs an expanded cohort of patients and a more robust assessment of HLA and nuclear ABC to further characterize such discrete cell populations.
Project design and supervision: PP, NE, MJD, and VGR. Data collection: NE, LM, MTAO, MO, AA, ARA, JALM, IT, and JM. Pathological analysis at the hospitals: AA, JLRP, IT, and JM. Pathological analysis in Atryshealth: MT. Statistical analysis: IS. Methodology Setup: PP, JGG, and MJD. Writing—original draft: PP. Writing review and editing: PP, NE, MT, AAP, MJD, LM, MTAO, MO, IT, and VGR. All authors read and approved the final manuscript.
We would like to thank the technical support received from the technician staff of the involved centers and the accurate language edition of Mr. Anthony Brady.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due to the personal data they contain, but are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The study was approved by the ethics committee of the hospitals, and all enrolled patients provided signed informed consent. Hospital de la Vall d’Hebron: Clinical Research Ethics Committee (CEIC), PR_AG_313-2012. Hospital Clínic de Barcelona: Clinical Research Ethics Committee (CEIC), 2012-8040. Hospital 12 de Octubre: Clinical Research Ethics Committee (CEIC), 13/165.
Funding support has been received from The Spanish Ministery of Economy and Competitivity and the European Regional Development Funds (ERDF/FEDER). Grant # IPT-2012-1311-300000, cofunded by the ERDF and the Ministery of Economy and Competitivity in the frame of the Programa Nacional de cooperación Público-Privada, Subprograma INNPACTO, within the línea Instrumental de Articulación e Internacionalización del Sistema, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011.
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- 3.Donovan MJ, Hamann S, Clayton M, Khan FM, Sapir M, Bayer-Zubek V, Fernandez G, Mesa-Tejada R, Teverovskiy M, Reuter VE, Scardino PT, Cordon-Cardo C. Systems pathology approach for the prediction of prostate cancer progression after radical prostatectomy. J Clin Oncol. 2008;26(24):3923–9.CrossRefGoogle Scholar
- 4.Garrido C, Algarra I, Maleno I, Stefanski J, Collado A, Garrido F, Garcia-Lora AM. Alterations of HLA class I expression in human melanoma xenografts in immunodeficient mice occur frequently and are associated with higher tumorigenicity. Cancer Immunol Immunother. 2010;59(1):13–26.CrossRefGoogle Scholar
- 11.Carretero FJ, Del Campo AB, Flores-Martín JF, Mendez R, García-Lopez C, Cozar JM, Adams V, Ward S, Cabrera T, Ruiz-Cabello F, Garrido F, Aptsiauri N. Frequent HLA class I alterations in human prostate cancer: molecular mechanisms and clinical relevance. Cancer Immunol Immunother. 2016;65(1):47–59. https://doi.org/10.1007/s00262-015-1774-5.CrossRefPubMedGoogle Scholar
- 15.Mohammed MK, Shao C, Wang J, W Q, Wang X, Collier Z, Tang S, Liu H, Zhang F, Huang J, Guo D, Minpeng L, Liu F, Liu J, Ma C, Shi L, Athiviraham A, He TC, Lee Michael J. Wnt/β-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance. Genes Dis. 2016;3(1):11–40.CrossRefGoogle Scholar
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