Iduronate-2-Sulfatase-Regulated Dermatan Sulfate Levels Potentiate the Invasion of Breast Cancer Epithelia through Collagen Matrix

Abstract

Cancer epithelia show elevation in levels of sulfated proteoglycans including dermatan sulfates (DS). The effect of increased DS on cancer cell behavior is still unclear. We hypothesized that decreased expression of the enzyme Iduronate-2-sulfatase (IDS) can lead to increased DS levels, which would enhance the invasion of cancer cells. Breast cancer sections shows depleted IDS levels in tumor epithelia, when compared with adjacent untransformed breast tissues. IDS signals showed a progressive decrease in the non-transformed HMLE, transformed but non-invasive MCF-7 and transformed and invasive MDA-MB-231 cells, respectively, when cultured on Type 1 collagen scaffolds. DS levels measured by ELISA increased in an inverse-association with IDS levels. Knockdown of IDS in MCF-7 epithelia also increased the levels of DS. MCF-7 cells with depleted IDS expression, when imaged using two photon-excited fluorescence and second harmonic generation microscopy, exhibited a mesenchymal morphology with multiple cytoplasmic projections compared with epithelioid control cells, interacted with their surrounding matrix, and showed increased invasion through Type 1 collagen matrices. Both these traits were phenocopied when control MCF-7 cells were cultivated on Type 1 collagen gels polymerized in the presence of DS. In monolayer cultures, DS had no effect on MCF-7 migration. In the context of our demonstration that DS enhances the elastic modulus of Type 1 collagen gels, we propose that a decrease of IDS expression leads to accumulation within cancer epithelia of DS: the latter remodels the collagen around cancer cells leading to changes in cell shape and invasiveness through fibrillar matrix milieu.

Keywords: breast cancer; dermatan sulfate; iduronate-2-sulfatase.

Figure 1

Sulfated proteoglycans are elevated in breast cancer epithelia in vivo and in culture. (A) Breast cancer (top) and patient-matched adjacent non-transformed (bottom) breast tissue sections were stained with Alcian blue dye (blue), which detects sulfated proteoglycans (sPGs) at pH 1. Scale bar = 50 µm. (B) 3D profile plots representing sPGs levels in tissue sections of breast cancer (top) and adjacent normal breast (bottom) with bright colored peaks showing higher staining for Alcian blue in breast cancer tissues (blue-low; white-high). (C) Scatter plot showing pixel intensities of Alcian blue staining in breast cancer and adjacent normal breast tissue sections (n = 5). (D) Confocal micrographs of Alcian blue autofluorescence (green signal) in stained immortalized breast epithelial cells HMLE (left), non-invasive malignant MCF-7 (middle) and the triple negative invasive MDA-MB-231 cells cultured on top of 1 mg/mL Type 1 collagen gels. Scale bar = 20 µm. (E) Scatter plot showing pixel intensities of Alcian blue autofluorescence in stained HMLE, MCF-7 and MDA-MB-231 cells cultured on top of 1 mg/mL Type 1 collagen gels (lines in graphs represents mean ± SE of three independent experiments). Significance was measured using one-way ANOVA (* p < 0.05) and student’s t test (** p < 0.01).

Figure 2

Levels of iduronate-2 sulfatase (IDS) are decreased in cancer epithelia in vivo and in culture. (A) Scatter plot of dermatan sulfate (DS) levels normalized to total protein measured using ELISA in HMLE, MCF-7 and MDA-MB-231 cells cultured in 3D Type 1 collagen scaffolds. (B) Scatter plot of IDS mRNA levels in HMLE, MCF-7 and MDA-MB-231 cells cultured in 3D Type 1 collagen scaffolds, as determined by real time PCR with 18S rRNA as internal control. (C) (left) Epifluorescence micrographs of matched normal breast sections (top) and breast cancer tissues (bottom) stained for DNA (using DAPI; blue), and IDS (using antibody; red), scale = 200 μm. (middle) 3D profile plots of IDS levels in normal (top) and cancer (bottom) sections with bright colored peaks showing higher staining for IDS in nontransformed cells. (right) Bar graph showing a statistically significant decrease in IDS levels in breast cancer tissues compared with adjacent breast epithelial cells (50 cells, 5 fields, 2 sample sets). (D) Confocal micrographs of HMLE, MCF-7 and MDA-MB-231 cells cultured on top of Type 1 collagen scaffolds and stained for DNA (with DAPI; blue), F-actin (with phalloidin; pink), acidic compartment (with antibody against LAMP2; green), and for IDS (antibody; red), scale = 20 µm. Significance was measured using one-way ANOVA and student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Decrease in IDS levels increases DS levels and alters the shape of MCF-7 cells. (A) Graphical representation showing a downregulation of IDS mRNA levels in MCF-7 cells upon lentiviral transduction of 2 shRNA clones, compared with scrambled control shRNA transduction, using qRT-PCR. 18S rRNA was used as an internal control. (B) Confocal micrographs of MCF-7 cells with scrambled- and IDS-specific shRNA transduction, stained for DNA (using DAPI, blue), F-actin (using phalloidin, pink), acidic compartment (using anti-LAMP-2 antibody, green) and IDS (using anti-IDS antibody, red). Depletion of IDS is accompanied with change in shape of MCF-7 cells from polyhedral to a spindle-like morphology, scale = 10 µm. (C) Confocal micrographs of MCF-7 cells with scrambled- and IDS-specific shRNA transduction, stained for DNA (using DAPI, blue), and sulfated proteoglycans (using Alcian Blue, green), scale = 20 µm. (D) Scatter plot representation of the pixel intensities of autofluorescent signals from Alcian Blue staining from 3C. (E) Scatter plot representation depicting dermatan sulfate (DS) levels in control and IDS knockdown MCF-7 cells when cultured in 3D Type 1 collagen scaffolds, analyzed using ELISA. Levels are represented as scatter plots (mean ± SE of three independent experiments). Significance was measured using one-way ANOVA (* p < 0.05).

Figure 4

Low levels of IDS increase invasion of MCF-7 and correlate with poorer prognosis of breast cancer patients. (A) Two photon micrographs of MCF-7 control cells (left) and with IDS knockdown (right) showing phalloidin staining of actin cytoskeleton (top) and second harmonic generation signals (bottom), scale = 10 µm. (B) Epifluorescence micrographs of the invasion of MCF-7 cells stained for DNA (using DAPI), lentivirally transduced with scrambled shRNA and 2 shRNA clones against IDS through transwells coated with Type 1 collagen. DAPI was used as indicator of invaded cells. Graphical representation of the number of invaded cells shown in 4B. (C,D) Kaplan–Meier plots of risk-free survival and overall survival, respectively, reveal a significant correlation between higher IDS expression and better survival (lines in graphs represent mean ± SE of 2–3 independent experiments). Significance was measured using one-way ANOVA (* p < 0.05).

Figure 5

Increase in DS levels phenocopies IDS depletion and increases MCF-7 invasion. (A) Epifluorescence micrographs showing lack of migration of MCF-7 within scratches made in monolayers (left, control; right, upon treatment with 50 µg/mL Dermatan Sulfate (DS)). The cells were visualized by staining DNA using DAPI. (B) Epifluorescence micrographs showing invasion of MCF-7 through Type 1 collagen-coated transwells (top, control; bottom, transwells coated with Type 1 collagen scaffold polymerized in the presence of 50 µg/mL DS). The cells were visualized by using DNA stain DAPI. (C) Scatter plot showing the number of invaded cells in 5B. (D) Two-photon micrographs of MCF-7 cells cultured in 3D Type 1 collagen scaffold (left) and in 3D Type 1 collagen scaffolds polymerized in the presence of 50 µg/mL DS (right) showing F-actin staining (using phalloidin, red) (lines in graphs represent mean ± SE of three independent experiments). Significance was measured using Student’s t test (* p < 0.05).