Fig 1.
Loss of apicobasal polarity occurs in low-grade endometrial cancer.
Human endometrial tissue (a, a’, d, d’, d”), normal; (b, b’, e, e’, e”) grade 1 endometrioid endometrial carcinoma, G1 EEC; and (c, c’, f, f’, f”) grade 2 endometrioid endometrial carcinoma, G2 EEC stained with antibodies for the apical proteins (a-c) Ezrin or (d-f) Par3, E-cadherin and DAPI. Scale bar, 20 μm. Asterisks indicate glandular lumen and arrows show apical localizing protein (a’) Ezrin or (d’) Par3. Scale bar, 20 μm. (g and h) Quantification of (a-c) Ezrin or (d-f) Par3 apical localization in 10 lumens of each sample (n = 3 normal, n = 2 G1 EEC, n = 2 G2 EEC) showing loss of apical protein localization in low-grade EEC. Error bars represent SEM.
Fig 2.
E-cadherin localization in endometrial cancer with disrupted polarity.
(a, a’) Normal endometrium, (b, b’) G1 EEC and (c, c’) G2 EEC stained with an antibody against E-cadherin and DAPI. (a’-c’) Images of (a-c) E-cadherin staining showing localization to the apical junctions and lateral border in normal endometrium, G1 EEC, and G2 EEC. Asterisks indicate glandular lumen. (d) G3 EEC stained with antibodies against E-cadherin (green) with DAPI or (d’) with E-cadherin staining only showing loss of localization to the apical junctions and lateral borders. (e) Ratio of the apical localization of Par3 to the basolateral localization of E-cadherin. Localization of Par3 or E-cadherin was determined from 10 lumens of each sample (n = 3 normal, n = 4 EEC). Error bars represent SEM. * <0.05. Scale bar, 20 μm.
Fig 3.
Depletion of apical polarity proteins cause a decrease in differentiation markers in epithelial 3D cell culture.
(a-c) Immunofluorescence staining on (a) Scramble-shRNA (Scr-shRNA), (b) Par3-shRNA (Par3-kd), and (c) Ezrin-shRNA (Ezrin-kd) 3D cysts for primary cilia by acetylated tubulin (ac. tub) and actin by Phalloidin. Scale bar, 20 μm (d, e) Quantification of the (d) number of lumens (n = 2) and (e) cilia (n = 1) present within all 3D cysts compared to scramble control cells. Par3-kd, Ezrin-kd, and Scr-shRNA had at least 49 cysts, 13 cysts, or 70 cysts examined per independent experiment. Abnormal cilia include cysts without cilia present or cilia that appears abnormal. Error bars represent SEM. (f) Transepithelial resistance demonstrates loss of functional TJ in Par3-kd and Ezrin-kd cells compared to Scr-shRNA cells calculated by Ohms per cm2. (g-i) Immunofluorescence staining for ZO-1 shows altered TJ protein localization in Par3-kd and Ezrin-kd cells indicative of decrease in differentiation by loss of epithelial cell junctions. E-cadherin is also stained to label junctional complexes. Scale bar, 20 μm (g’-i’) ZO-1 only staining to show the aggregation of ZO-1 at tricellular junctions in white. (j-l) Line plots of (g-i) showing intensity of E-cadherin (green line) and ZO-1 (red line) on the yellow line in the respective image, Scr-shRNA (j), Par3-kd (k), and Ezrin-kd (l). Note the overlap in E-cadherin and ZO-1 intensities in Scr-shRNA compared to distinct peaks of ZO-1 intensity in Par3-kd and Ezrin-kd indicative of mislocalized ZO-1. (m-o) Increased BrdU incorporation observed in Par3-kd and Ezrin-kd cell lines compared to the Scr-shRNA cells. (m’-o’) BrdU only staining of (m-o). Scale bar, 20 μm.
Fig 4.
Notch signaling decreases in Par3 depleted epithelial cells.
(a-g) qRT-PCR analysis of Notch1 (a), Notch2 (b), JAG1 (c), JAG2 (d), p21 (e), HEYL (f), or HEY1 (g) expression in Scr-shRNA cells and Par3-kd cells. Error bars signify SEM. * <0.05, ** <0.001, ***<0.0001.
Fig 5.
Notch downstream signaling and receptor localization is disrupted in low-grade endometrial cancer.
(a-f) qRT-PCR of Notch receptors, ligands, and downstream targets in normal and in low-grade endometrial cancer (G1 & G2 EEC). The Notch receptor (a) Notch1 and (b) Notch2 show no change in expression while (c) Notch4 is decreased. Notch ligand (d) Jag1 and downstream targets (e) HEYL and (f) HES1 are down regulated in low-grade endometrial cancer. (a) Notch1 and (b) Notch2 data was analyzed using ΔCT with the Tata box binding protein (DBP) as the reference gene with 7 samples for Normal, G1 EEC, and G2 EEC. (c-f) Notch4, Jag1, HES1, and HEYL were analyzed by calculating the number of molecules of the gene of interest compared to 18SrRNA (%18SrRNA). Tukey box plots were used with SEM where + is the mean value and • are outliers. Normal (n = 10), G1 EEC (n = 9), and G2 EEC (n = 22). (g-l) Immunofluorescence of Notch receptors showing localization of (g-i, g’-i’) Notch1 and (j-l, j’-l’) Notch2 in (g, j) normal endometrium, (h, k) G1 EEC, and (i, l) G2 EEC. E-cadherin marks the basolateral cell:cell contacts (g-l) (*) signifies the lumen. Scale bar, 20 μm. (g’-l’) Images of (g-l) with Notch1 or Notch2 respectively showing localization to the lumen (*). Arrows denote lateral localization of Notch1 or Notch2.
Fig 6.
Expressing Par3 in endometrial cancer cell lines blocks proliferation and promotes differentiation.
(a) Western blot analysis of Par3 in control transfection (control) and Myc-Par3 overexpression Hec-1-A cells. (b,c) Parental Hec-1-A or Hec-1-A with exogenous Par3 stained with DAPI and Notch1. (d-g, d’-g’) Staining of parental (d, f) and Par3 overexpressing (e, g) Hec-1-A cells. Cells were stained for Myc-Par3, ZO-1, and DAPI (d,e) or Par3, BrdU, and DAPI (f, g). Scale bar, 20 μM. (d’, e’) ZO-1 only staining (d”-d””, e”-e””) Z-plane showing ZO-1, Myc and/or DAPI staining. (f’, g’) Images of (f,g) with BrdU staining only. (h) Quantification of disorganized ZO-1 in the parental (n = 3) and Par3 overexpression Hec-1-A cells (n = 3) for at least three regions of interest (ROI) per experiment. Error bars represent SEM *<0.05. (i) Quantification of BrdU incorporation in the parental (n = 3) and Par3 overexpression Hec-1-A (n = 3) cells for at least three ROI per experiment. Error bars represent SEM *0.05. (j) Schematic of proposed model for how apicobasal polarity controls differentiation of endometrial epithelial cells by regulating Notch receptor localization and, Notch downstream targets that modulate proliferation and differentiation.
Fig 7.
Inhibiting Notch signaling rescues Par3 mediated changes in migration and proliferation.
(a) Quantification of cell migration for parental Hec-1-A cells, Par3 overexpression Hec-1-A cells, and Hec-1-A cells treated the gamma secretase inhibitor (DAPT) to block Notch signaling. (b) Quantification of BrdU incorporation in the parental, Par3 overexpression, and DAPT treated Hec-1-A cells. (c) qRT-PCR analysis of the Notch target HES-1 in parental, Par3 overexpression and DAPT treated Hec-1-A cells. (d-g) Photos showing specific times during the migration assay performed to examine rate of migration for Hec-1-A parental cells (d-d”), Hec-1-A with Par3 expression (e-e”), Hec-1-A parental cells treated with DAPT (f-f”), and Hec-1-A Par3 expressing cells treated with DAPT (g-g”). Immunofluorescence analysis of BrdU incorporation in parental Hec-1-A cells (h, i), Hec-1-A overexpressing Par3 cells (h’, i’), parental cells treated with DAPT (h”, i”) or Par3 expressing cells treated with DAPT (h’”, i’”). Top panels (h-h’”) show BrdU (green) with DAPI (blue) staining and panels (i-i’”) show BrdU staining alone. Scale bar, 20 μM.