The authors have declared that no competing interests exist.
Conceived and designed the experiments: IQ AH AP SH LK PV. Performed the experiments: IQ AH AP EP MR. Analyzed the data: IQ AH CA AP SH KO EP EK YS RS LK PV. Contributed reagents/materials/analysis tools: EK. Wrote the paper: IQ AH CA AP SH KO EP EK LK PV.
The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP−/−) with C57BL/6J background. The PAP−/− mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and
The association between prostate cancer and serum prostatic acid phosphatase (PAP;
PAP is a histidine acid phosphatase
The prostate gland is fundamentally a secretory organ, and it is known that the secretion of specialized exosomes (prostasomes) is essential for the maintenance of the spermatozoa
PAP exerts its phosphatase activity
SNARE proteins comprise a large family found in yeast and mammalian cells, with the primary function to mediate docking and fusion of vesicles with the cell membranes
The mouse prostate consists of three different lobes: anterior (AP), dorsolateral (DLP) and ventral prostate (VP); and it does not show spontaneous development of neoplasia
To understand the physiological function of PAP, we studied the prostate of our PAP-deficient mouse model (PAP−/−)
The animal protocols were approved by the Animal Experimentation Committee of the University of Oulu and ELLA – The National Animal Experiment Board of Finland. The project license numbers are 044/11 and STH705A/ESLH-2009–08353/Ym-2.
Mice deficient in PAP were generated by replacing exon 3 (
DLP samples from age-matched PAP−/− and PAP+/+ mice were fixed in a mixture of 1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer for TEM. The samples were post-fixed in 1% osmium tetroxide, dehydrated in acetone, embedded in Epon Embed 812 (Electron Microscopy Sciences) and analyzed at the Biocenter Oulu EM core facility using Philips 100 CM Transmission Electron Microscope with CCD camera.
To screen for interacting partners of human TMPAP, yeast two-hybrid screening was performed using the Matchmaker Gal4 two-hybrid System 3 (Clontech) in accordance with the manufacturer's instructions. The bait construct consisted of the coding region of human TMPAP (GeneBank accession BC007460, nucleotides 51–1304, except the starting methionine was changed to valine) cloned in frame into NcoI/SmaI sites of pGBKT7 using PCR generated linkers. A human thymus cDNA library cloned in pACT2 (Clontech) was used as the prey. The bait and prey plasmids were co-transformed into
The FRET variant acceptor photobleaching was used. In this technique, the efficiency of energy transfer between two molecules (and consequently the interaction between them) is measured by comparing the fluorescence of the donor molecule before and after the selective photobleaching of the acceptor moleclule
Cultured cells were mounted 24 hours after transfection and epifluorescent images were acquired using an Olympus CellR imaging system with 60× oil immersion NA 1.45 objective. Images were collected with a CCD camera (Orca, Hamamatsu). The system was equipped with automated filter wheels for excitation filters and emission beam-splitter/emission-filter cubes for epifluorescence imaging. GFP fluorescence was excited at 450 nm and collected at 510/40 nm. DsRed fluorescence was excited at 575 nm and collected at 640/50 nm. Acceptor fluorescence was bleached for 5 minutes with maximal burner power.
Images were quantified and processed using Olympus Biosystems AnalySIS software, ImageJ (freely available at
The detailed methodology can be found in the Supplementary Methodology in
Gene expression files containing microarray raw-data can be accessed from ArrayExpress repository database (accession number E-MTAB-1191).
PAP-deficiency led to slow development of prostate neoplasia in DLP and AP. The progressive changes in mouse DLP were observed in all the PAP−/−mice examined (
The panels show an overview of the 12-old mice prostate dissected lobes. The DLP, AP and VP lobes were dissected from WT and PAP−/− mouse. The monolayer epithelium (white arrows) is seen in all the lobes of the WT mouse, whereas in the PAP−/− mouse an increased amount of cells is present in the lumen of the DLP lobe (black arrows). The AP and VP of PAP−/− mouse show no significant changes. Scale bars: 100 µm.
A, age related histological changes in PAP−/− mouse DLP. Epithelial hyperplasia was present in DLP of 3 month-old PAP−/− mice. The lumen was filled with dysplastic epithelial cells, and mPIN structures were observed in 6 month-old animals. Bulging of epithelial cells into the stroma (black arrows) through loosen fibromuscular sheath and prostate adenocarcinoma were present in 12 month-old mice. Scale bars: 100 µm. (
All the PAP−/−mice analyzed at the age of 12 months had developed prostate adenocarcinoma (
A, smooth muscle actin (SMA) immunohistochemistry in 12 month-old mice. Monolayer epithelium (mL) and open lumen in PAP+/+ DLP. White arrows show the broken fibromuscular sheath (SM, smooth muscle) and bulging of epithelial cells to the stroma. Prostate adenocarcinoma (black arrows) is present in AP and DLP, showing a multilayer epithelium (ML) and inflammatory cells (black arrowhead) spreading in neighboring areas. Scale bars: 100 µm. (
The breakdown of the fibromuscular sheath and invasion of the epithelial cells into stroma were also detected with smooth muscle β-actin (SMA) staining (
Important changes observed by transmission electron microscopy (TEM) included irregularities and invaginations of the basement membrane into the epithelium (
A, electronmicroscopy images show the presence of electron-dense (white arrow) and electron-lucent (black arrow) microvesicles (∼30 to 80 nm) in the lumen of the acini, and MVE containing microvesicles in the apical part of the cell. Scale bar: 1,000 nm. B, numerous microvesicles are present in the apical region of PAP−/− DLP and secreted into the lumen, decreased amount of microvilli is observed (black arrowheads) (scale bar: 1,000 nm). C, microvesicles are secreted into basolateral intercellular space of PAP−/− DLP (scale bar: 2000 nm). D, lamellar body-like structures (*) are inside the epithelial cell (scale bar: 500 nm) and E, released into the lumen (*). Scale bar: 200 nm. F, TMPAP and snapin are also present in exosomes. Immunoblots of exosomes isolated from TMPAP/LNCaP cell culture medium. Flotillin and CD13 were used as exosomal and prostasomal marker respectively.
Due to the gradually increased number of cells in the prostate acini, we determined the status of proliferation and apoptosis in the tissue. As a result, the proliferation was significantly increased in the three- (P-value = 4.3×10−3,
A, bar plot showing the ratio (as percentage) between proliferative cell counts and total amount of cells. (**, P value <0.01; ***, P value <0.001). Error bars indicate S.E.M. values. B, bar plot showing the ratio (as percentage) between apoptotic cell counts and total amount of cells. Error bars indicate S.E.M. values.
To determine the effects of PAP deficiency at gene expression level, we performed microarray experiments of mouse prostatic tissue. The Gene Ontology analyses of differentially expressed genes showed, among the most significant groups, those genes associated with regulated secretory pathway, neurotransmitter secretion (
GO ID | Term | Genes | |
5576 | <0.0001 | extracellular region | |
4293 | <0.0001 | tissue kallikrein activity | |
42044 | <0.0001 | fluid transport | |
32501 | 0.0001 | multicellular organismal process | |
7267 | 0.0001 | cell-cell signaling | |
19226 | 0.0001 | transmission of nerve impulse | |
3001 | 0.0001 | generation of a signal involved in cell-cell signaling | |
45055 | 0.0001 | regulated secretory pathway | |
30672 | 0.0001 | synaptic vesicle membrane | |
15026 | 0.0001 | coreceptor activity | |
5372 | 0.0001 | water transporter activity | |
15250 | 0.0001 | water channel activity | |
15722 | 0.0001 | canalicular bile acid transport | |
5179 | 0.0002 | hormone activity | |
7269 | 0.0002 | neurotransmitter secretion | |
1772 | 0.0002 | immunological synapse | |
6833 | 0.0003 | water transport | |
5232 | 0.0006 | serotonin-activated cation-selective channel activity | |
5615 | 0.0007 | extracellular space | |
48812 | 0.0007 | neurite morphogenesis | |
48667 | 0.0007 | neuron morphogenesis during differentiation | |
7409 | 0.0008 | axonogenesis |
The disturbed exocytosis observed in the ultrastructural studies of PAP−/− prostates and the differential expression of genes related to vesicle fusion, such as
To validate the yeast two-hybrid result, double-immunofluorescence staining of PAP and snapin in TMPAP/LNCaP cells showed co-localization of these two proteins in vesicular structures and cell membrane (
A, co-localization (yellow) of TMPAP (green) with snapin (red) was observed in the vesicles and lamellipodia of the TMPAP/LNCaP cells. Arrows mark the co-localization points in the upper panel (scale bar: 20 µm). Lower panel (scale bar: 3 µm) showing the lamellipodia region, amplification of the area marked with a box in the upper panel (left). B, intensification of donor (TMPAP-GFP) fluorescence in LNCaP cells was observed after acceptor (snapin-DsRed) photobleaching which confirms FRET between two molecules (Scale bar: 2 µm).
Hence our previous results have showed the co-localization of PAP and flotillin, which is a protein also used as exosomal marker
Prostate cancer is a disease of complex etiology, in which genetic and epigenetic mechanisms are involved. PAP was the first prostate cancer marker, and its usefulness was based in the assessment of its serum activity levels. We have previously shown that in addition to the secretory PAP, a transmembrane isoform is widely expressed in different mouse organs such as prostate, salivary glands, thymus, lung, kidney and brain, amongst others. TMPAP is also present in androgen sensitive prostate cancer cells (LNCaP), but absent in androgen insensitive prostate cancer cells (PC3)
In all the PAP−/− mice, progressive changes were observed in the prostatic tissue leading to the development of prostate adenocarcinoma at the age of 12 months. Despite the presence of prostate cancer, we have not detected any metastatic lesions. Histologically, the mouse DLP has been considered to be the analogous area to the peripheral zone of the human prostate, where the majorities of adenocarcinomas reside
In humans, it has been observed that PTEN (phosphatase and tensin homolog deleted on chromosome 10) is downregulated in prostate cancer tissue specimens
The ultrastructural studies of PAP−/− mouse prostates showed a high amount of nanovesicles, compatible in size with exosomes. According to this finding, other authors have reported a significant increment of exosomes in plasma of prostate cancer patients compared to healthy donors or benign hyperplasia
Our results in addition to our previous knowledge of the presence of PAP in the endosomal-lysosomal pathway
In
TMPAP synthesized in the endoplasmic reticulum is transported in vesicles to the plasma membrane through the trans-Golgi network (TGN). After the vesicle docking and fusion events leading to release of vesicle content, TMPAP inserted in plasma membrane exerts its phosphatase function over AMP. The resulting product adenosine (Ado) activates the adenosine receptors, which are GPCRs, A1 or A3with Gαi (inhibitory G-protein β-subunit) specificity leading to the inhibition of adenylate cyclase (AC) activity, and A2 adenosine receptors with Gαs (stimulatory G-protein α-subunit) producing the stimulation of AC activity. Activated AC produces cAMP, which activates PKA responsible for the phosphorylation of snapin. The turnover is completed by clathrin-mediated endocytosis of SNARE components and TMPAP for recycling and degradation in lysosomes vía the endosomal-lysosomal pathway. From early endosomes, the cargo can be sorted to late endosomes or to MVE, which can follow the route leading to exosome release. Additional dephosphorylation events by TMPAP can occur while trafficking between different compartments. From late endosomes, TMPAP can go to lysosomes or back to TGN via the retrograde pathway. ATP: adenosine triphosphate, ADP: adenosine diphosphate, AMP: adenosine monophosphate, Ado: adenosine, TGN: trans-Golgi network, P: phosphate group, AP-2: adaptor protein complex 2, ADORA: adenosine receptor A (types A1, A2 and A3), AC: adenylate cyclase, Gαs, Gαi, Gβ, Gγ: G-protein subunits, VDCC: Voltage-gated calcium channel. Synaptobrevin, syntaxin and SNAP25 are SNARE proteins. PI (4,5) P2: phosphatidylinositol 4,5-bisphosphate.
According to this model, the lack of TMPAP would lead to the observed dysregulation of vesicular traffic, exocytosisand release of exosomes in PAP−/− mouse prostate. This could establish a significant starting point for uncontrolled cell proliferation and the development of prostate adenocarcinoma. Interestingly, in DRG of PAP−/− mice the levels of PI (4,5) P2 are increased when compared to wild-type mice
In summary, this PAP−/− mouse model shows that TMPAP is required for the normal function of prostate in mice, and its deficiency leads to prostate adenocarcinoma. This suggests that TMPAP acts as a regulator of endo-/exocytosis mechanism.
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We acknowledge CSC (The Finnish IT Center for Science) for the allocation of computational resources. We acknowledge Biomedicum Imaging Unit (BIU) at Biomedicum Helsinki for providing access to the equipment and technical support, and the Biocenter Oulu EM Core Facility, University of Oulu, for processing of TEM samples. We thank Dr. Maija Wolf at the Institute for Molecular Medicine Finland, FIMM, for the CGH analyses. We thank Prof. T. Kitamura at the Institute of Medical Science, The University of Tokyo, for providing retroviral plasmids.