Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell-cell junctions, luminal iniltration and basal invasion induced by AGE-modiied basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180 Ex2-6/ Ex2-6 mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These indings indicate that AGE-dependent modiication of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors.
Epithelial cell-cell contacts maintain normal glandular tissue homeostasis, and their breakage can trigger epithelialto-mesenchymal transition (EMT), a fundamental step in the development of metastatic cancer. Despite the ability of C-type lectin domains (CTLD) to modulate cell-cell adhesion, it is not known if they modulate epithelial adhesion in EMT and tumor progression. Here, the multi-CTLD mannose receptor, Endo180 (MRC2/uPARAP), was shown using the Kaplan-Meier analysis to be predictive of survival outcome in men with early prostate cancer. A proteomic screen of novel interaction partners with the fourth CTLD (CTLD4) in Endo180 revealed that its complex with CD147 is indispensable for the stability of three-dimensional acini formed by nontransformed prostate epithelial cells (PEC). Mechanistic study using knockdown of Endo180 or CD147, and treatment with an Endo180 mAb targeting CTLD4 (clone 39.10), or a dominant-negative GST-CTLD4 chimeric protein, induced scattering of PECs associated with internalization of Endo180 into endosomes, loss of E-cadherin (CDH1/ECAD), and unzipping of cell-cell junctions. These findings are the first to demonstrate that a CTLD acts as a suppressor and regulatory switch for EMT; thus, positing that stabilization of Endo180-CD147 complex is a viable therapeutic strategy to improve rates of prostate cancer survival.Implications: This study identifies the interaction between CTLD4 in Endo180 and CD147 as an EMT suppressor and indicates that stabilization of this molecular complex improves prostate cancer survival rates.
Background:Endo180 (CD280; MRC2; uPARAP)-dependent collagen remodelling is dysregulated in primary tumours and bone metastasis. Here, we confirm the release and diagnostic accuracy of soluble Endo180 for diagnosing metastasis in breast cancer (BCa).Methods:Endo180 was quantified in BCa cell conditioned medium and plasma from BCa patients stratified according to disease status and bisphosphonate treatment (n=88). All P-values are from two-sided tests.Results:Endo180 is released by ectodomain shedding from the surface of MCF-7 and MDA-MB-231 BCa cell lines. Plasma Endo180 was significantly higher in recurrent/metastatic (1.71±0.87; n=59) vs early/localised (0.92±0.37; n=29) BCa (P<0.0001). True/false-positive rates for metastasis classification were: 85%/50% for the reference standard, CA 15-3 antigen (28 U ml−1); ⩽97%/⩾36% for Endo180; and ⩽97%/⩾32% for CA 15-3 antigen+Endo180. Bisphosphonate treatment was associated with reduced Endo180 levels in BCa patients with bone metastasis (P=0.011; n=42). True/false-positive rates in bisphosphonate-naive patients (n=57) were: 68%/45% for CA 15-3 antigen; ⩽95%/⩾20% for Endo180; and ⩽92%/⩾21% for CA 15-3 antigen+Endo180.Conclusion:Endo180 is a potential marker modulated by bisphosphonates in metastatic BCa.
The diverse composition and structure of extracellular matrix (ECM) interfaces encountered by tumor cells at secondary tissue sites can influence metastatic progression. Extensive in vitro and in vivo data has confirmed that metastasizing tumor cells can adopt different migratory modes in response to their microenvironment. Here we present a model that uses human stromal cell-derived matrices to demonstrate that plasticity in tumor cell movement is controlled by the tumor-associated collagen receptor Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) and the crosslinking of collagen fibers by stromal-derived lysyl oxidase (LOX). Human osteoblast-derived and fibroblast-derived ECM supported a rounded ‘amoeboid-like’ mode of cell migration and enhanced Endo180 expression in three prostate cancer cell lines (PC3, VCaP, DU145). Genetic silencing of Endo180 reverted PC3 cells from their rounded mode of migration towards a bipolar ‘mesenchymal-like’ mode of migration and blocked their translocation on human fibroblast-derived and osteoblast-derived matrices. The concomitant decrease in PC3 cell migration and increase in Endo180 expression induced by stromal LOX inhibition indicates that the Endo180-dependent rounded mode of prostate cancer cell migration requires ECM crosslinking. In conclusion, this study introduces a realistic in vitro model for the study of metastatic prostate cancer cell plasticity and pinpoints the cooperation between tumor-associated Endo180 and the stiff microenvironment imposed by stromal-derived LOX as a potential target for limiting metastatic progression in prostate cancer.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-015-9765-7) contains supplementary material, which is available to authorized users.
Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.
<p>Supplementary Figures S1-S5, Tables S1-S3. Figure S1. Silencing Endo180 disrupts normal and benign PEC acini, but not PTC spheroids. Figure S2. None of the treatment used in culture affected cell cycle and/or cell viability. Figure S3. Epitope mapping of the Mouse monoclonal anti-Endo180 39.10. Figure S4. Targeted blockade of Endo180 does not alter spheroid formation by PTCs. Figure S5. Highly glycosylated CD147 can be precipitated from normal PEC lysates using CTLD4 as bait. Supplementary Table S1. Antibodies and reagents used for Immunoblotting, Immunofluorescence and Immunohistochemistry. Supplementary Table S2. NCLPC1/4 prostate cancer tissue microarray patient characteristics, N = 157. Supplementary Table S3. NCLPC1/4 prostate cancer tissue microarray - patient deaths [censored] after three, five, seven and ten years.</p>
<div>Abstract<p>Epithelial cell–cell contacts maintain normal glandular tissue homeostasis, and their breakage can trigger epithelial-to-mesenchymal transition (EMT), a fundamental step in the development of metastatic cancer. Despite the ability of C-type lectin domains (CTLD) to modulate cell–cell adhesion, it is not known if they modulate epithelial adhesion in EMT and tumor progression. Here, the multi-CTLD mannose receptor, Endo180 (MRC2/uPARAP), was shown using the Kaplan–Meier analysis to be predictive of survival outcome in men with early prostate cancer. A proteomic screen of novel interaction partners with the fourth CTLD (CTLD4) in Endo180 revealed that its complex with CD147 is indispensable for the stability of three-dimensional acini formed by nontransformed prostate epithelial cells (PEC). Mechanistic study using knockdown of Endo180 or CD147, and treatment with an Endo180 mAb targeting CTLD4 (clone 39.10), or a dominant-negative GST-CTLD4 chimeric protein, induced scattering of PECs associated with internalization of Endo180 into endosomes, loss of E-cadherin (CDH1/ECAD), and unzipping of cell–cell junctions. These findings are the first to demonstrate that a CTLD acts as a suppressor and regulatory switch for EMT; thus, positing that stabilization of Endo180–CD147 complex is a viable therapeutic strategy to improve rates of prostate cancer survival.</p><p><b>Implications:</b> This study identifies the interaction between CTLD4 in Endo180 and CD147 as an EMT suppressor and indicates that stabilization of this molecular complex improves prostate cancer survival rates.</p><p><b>Visual Overview:</b> <a href="http://mcr.aacrjournals.org/content/13/3/538/F1.large.jpg" target="_blank">http://mcr.aacrjournals.org/content/13/3/538/F1.large.jpg</a></p><p><i>Mol Cancer Res; 13(3); 538–47. ©2014 AACR</i>.</p></div>
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