ABSTRACT
PURPOSE: LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells. EXPERIMENTAL DESIGN: Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions. RESULTS: Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm. CONCLUSIONS: Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Nuclear Localization Signals/metabolism , Prostatic Neoplasms/metabolism , Subcellular Fractions/metabolism , Ubiquitination/physiology , Amino Acid Sequence , Animals , Blotting, Western , Cell Adhesion Molecules/genetics , Cell Nucleus/genetics , Cells, Cultured , Cohort Studies , Cytoplasm/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunoenzyme Techniques , Immunoprecipitation , Male , Membrane Proteins , Mice , Molecular Sequence Data , NIH 3T3 Cells , Nuclear Localization Signals/genetics , Polymerase Chain Reaction , Prostatic Hyperplasia/genetics , Prostatic Hyperplasia/metabolism , Prostatic Neoplasms/genetics , RNA-Binding Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: Wnt signaling is thought to be important in prostate cancer, in part because proteins such as beta-catenin can also affect androgen receptor signaling. beta-Catenin forms a cell adhesion complex with E-cadherin raising the possibility that loss of expression or a change in beta-catenin distribution in the cell could also alter downstream signaling, decreased inter-cellular adhesion and the promotion of metastasis. A number of studies have reported the altered expression and/or localization of beta-catenin as a biomarker in prostate cancer. METHODS: Tissue microarrays comprised of BPH and low, moderate and high-grade prostate cancer (n=77) were assessed for beta-catenin expression and distribution using immunohistochemistry. Staining was also performed on a tissue microarray containing tissue from patients before and after hormone manipulation. The effects of fixation and different antibodies was assessed on fixed LNCaP cell pellets and small prostate tissue microarrays. RESULTS: We have observed increased beta-catenin expression in only high Gleason score (>7) prostate cancer. A nuclear re-distribution of beta-catenin has previously been reported. We noted nuclear beta-catenin in benign prostatic hyperplasia and a gradual loss in nuclear distribution with increasing Gleason grade. We found no evidence for an alteration in beta-catenin expression or re-distribution with hormone ablation. Altered fixation, antibodies and antibody concentration did affect the intensity and specificity of staining. CONCLUSIONS: A loss of nuclear beta-catenin is the most consistent feature in prostate cancer rather than absolute levels of expression. We also suggest that variation in immunohistochemical protocols may explain variations in the reported literature.
Subject(s)
Carcinoma/metabolism , Prostatic Neoplasms/metabolism , Signal Transduction/physiology , beta Catenin/metabolism , Animals , COS Cells , Carcinoma/secondary , Cattle , Cell Nucleus/metabolism , Chlorocebus aethiops , Hormones/metabolism , Humans , Immunohistochemistry , Kidney/cytology , Male , Mice , NIH 3T3 Cells , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/pathology , Protein Array AnalysisABSTRACT
BACKGROUND: Membrane proteins provide the interface between the cell and its environment and are responsible for cell adhesion, mobility, and intracellular signaling. Previous studies have focused on the LNCaP whole cell proteome and transcriptome but little is known about proteins at the prostate cell membrane and how they change in response to androgens. MATERIALS AND METHODS: Following treatment with R1881 or vehicle, membrane proteins of the prostate cancer LNCaP cell line were tagged with biotin using EZ-link sulfo-NHS-LC-biotin. Using the tag membrane proteins were purified and separated using two-dimensional gel electrophoresis and identified using mass spectrometry. E-cadherin and low density lipoprotein receptor (LDLR) were used as positive controls and also investigated following bicalutamide treatment. Membrane localization and androgen-regulation of proteins was confirmed using sub-cellular fractionation, Western blotting and microscopy. RESULTS: We have demonstrated efficient and specific protein biotinylation and purification of LNCaP plasma membrane proteins using Western analysis. E-cadherin and LDLR were regulated at the cell surface in response to R1881 and bicalutamide. Mass spectrometry identified several androgen-regulated membrane associated proteins including Prx-3 and GRP78 which are known to localize to other cellular compartments as well as the plasma membrane. We confirmed the localization of the identified proteins in LNCaP cells by co-localization with E-cadherin and immunohistochemistry of prostate tissue. CONCLUSION: Cell surface biotinylation is an effective technique for identifying membrane proteins in the LNCaP prostate cancer cell line. We have demonstrated the identification of androgen-regulated membrane proteins and their validation in tissue samples.
