AGE ‐modified basement membrane cooperates with Endo180 to promote epithelial cell invasiveness and decrease prostate cancer survival

Abstract

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.

Survival Outcome and EMT Suppression Mediated by a Lectin Domain Interaction of Endo180 and CD147

“…Endo180 and CD147 have both been correlated with low levels of E-cadherin in tumor tissue, and both are under the transcriptional regulation of the core EMT trigger TGFb (7,8,19,20). In an aligned study, we have demonstrated that the stiff microenvironment associated with advanced glycation endproduct accumulation and collagen crosslinking of the basement membrane in the prostate gland is sufficient to activate an EMTlike phenotype in PEC acini by forcing the relocalization of Endo180 into endosomes and initiating an Endo180-dependent contractile signaling cascade that promotes cell invasion (21). Indeed, ECM stiffness has been identified as a regulator that can release matricellular TGFb in a number of pathologies (22).…”

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

“…5. Monitor acinar morphology in growing cultures using brightfield microscopy 13 . Note: After 3 days in culture individual cells will form a cluster of >3 cells and after 1 week prostate gland acini with a diameter of ~50 µm will be observed.…”

“…These acini also have the characteristics of highly organized PECs with apical-to-basal polarity and a visible luminal space 13,20 .…”

“…Analyse 25 µl of each BM sample on a 12% v/v polyacrylamide gel 10,11 . Following SDS-PAGE, carry out silver staining of the polyacrylamide gel 12 to visualize the electrophoretic pattern of cyanogen bromide-matrix peptides 13 .…”

“…Immunofluorescent Microscopy Analysis 1. Perform immunofluorescent staining of GLA treated and control rBM gels with anti-AGE/pentosidine, anti-collagen IV and anti-laminin antibodies followed by confocal microscopy to visualize accumulated AGEs and collagen IV/laminin fibre structural rearrangements in the crosslinked rBM gels 13 . Note: Always use a sufficient volume to cover the entire rBM gel during incubations and washes without touching the rBM surface with the pipette tip.…”