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. 2017 Aug 1;31(15):1573-1587.
doi: 10.1101/gad.300566.117. Epub 2017 Sep 8.

Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma

Ruba Halaoui  1   2 , Carlis Rejon  1 , Sudipa June Chatterjee  1   2 , Joseph Szymborski  1   2 , Sarkis Meterissian  3   4   5 , William J Muller  1   6 , Atilla Omeroglu  5   7 , Luke McCaffrey  1   2   3
Affiliations

Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma

Ruba Halaoui et al. Genes Dev. .

Abstract

Epithelial cancers (carcinoma) account for 80%-90% of all cancers. The development of carcinoma is associated with disrupted epithelial organization and solid ductal structures. The mechanisms underlying the morphological development of carcinoma are poorly understood, but it is thought that loss of cell polarity is an early event. Here we report the characterization of the development of human breast lesions leading to carcinoma. We identified a unique mechanism that generates solid ducts in carcinoma through progressive loss of polarity and collapse of the luminal architecture. This program initiates with asymmetric divisions of polarized cells that generate a stratified epithelium containing both polarized and depolarized cells. Stratified regions form cords that penetrate into the lumen, subdividing it into polarized secondary lumina. The secondary lumina then collapse with a concomitant decrease in RhoA and myosin II activity at the apical membrane and ultimately lose apical-basal polarity. By restoring RhoA activity in mice, ducts maintained lumen and cell polarity. Notably, disrupted tissue architecture through luminal collapse was reversible, and ducts with a lumen were re-established after oncogene suppression in vivo. This reveals a novel and common mechanism that contributes to carcinoma development by progressively disrupting cell and tissue organization.

Keywords: breast cancer; cancer progression; duct; epithelial; morphogenesis.

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Figures

Figure 1.
Figure 1.
Diversity of apical–basal polarity in DCIS. (A) Histological sections (panels i,iii) and immunofluorescence staining (panels ii,iv) of adjacent sections of DCIS showing examples of apical–basal polarity in DCIS. Arrowheads show examples of cells with polarized Par6. (B) Histogram of the distribution of DCIS with varying frequencies of cells with apical–basal polarity. n = 49. (C) Immunofluorescence staining of DCIS showing polarized apical Par6 (panels i,ii), basolateral Par6 (panels iv,v), and cytoplasmic Par6 (panels vii,viii). Panels iii, vi, and ix show immunofluorescence of serial sections stained for Ezrin and Dlg1. (D) Frequency of Par6 phenotypes observed in DCIS. (E) Immunofluorescence of Par6 and E-cadherin on individual patient samples containing DCIS and earlier stages. The arrow shows polarized Par6 in adjacent normal tissue. Bars: A,C, 50 µm; E, 20 µm.
Figure 2.
Figure 2.
Apical–basal polarity is progressively lost in preinvasive breast lesions. (A) Histological sections from biopsy material containing normal, FEA, ADH, and DCIS. (B) Fluorescence images of tissue sections immunostained for Par6 (red) and E-Cadherin (green) at the indicated stages, including DCIS adjacent to invasive carcinoma (DCIS adj. IDC) and IDC. Arrows show examples of cells lacking apical polarity. (C) Quantification of the percentage of cells that exhibit apical polarity at the indicated stages of progression. At least six image fields from five subjects were analyzed for each. Error bars indicate SEM. (D) Representative fluorescence images from DCIS showing collapsed lumen (white arrow) and apical patch (yellow arrowhead). (E) Quantification of lumen shape (circularity) between ADH and DCIS. Four subjects with both ADH and DCIS were used to measure 114 lumina for ADH and 153 lumina for DCIS. Black bars represent the mean. (***) P < 0.001. Bars: A, 100 µm; B, 50 µm; D, 20 µm.
Figure 3.
Figure 3.
Progressive polarity loss and luminal collapse during mammary tumor progression in mice. (A) Images of normal and tumor tissue from PyMT mice immunostained for ZO1 (red) and E-Cadherin (green). (B) Quantification of polarized and nonpolarized cells at the indicated stages during mammary tumor progression. (C) Schematic diagram of an experimental system for three-dimensional (3D) organotypic cultures. (D) Confocal images of organotypic cultures stained with phalloidin to mark the apical membrane following induction of PyMT. (E) Quantification of the indicated phenotypes at different time points following doxycycline-induced PyMT expression. Fifty structures from three mice were counted. (F) Images of tissue samples immunostained for α-Tubulin (red), phospho-Histone-H3 (green), and DNA (blue). (G) Quantification of the cell division angle relative to the plane of the lumen. n = 11 normal; n = 98 hyperplastic lesions. (H) Image series from time-lapse microscopy showing cell division in 3D organotypic cultures from MIC-PyMT cultures induced or not with doxycycline. Arrows show dividing cells. White dotted lines mark the apical surface, blue dotted lines mark the basal surface, and yellow dotted lines outline cells during division. (I) Quantification of the cell division angle relative to the plane of the lumen in organotypic cultures from MIC-PyMT mice. Cell divisions (n = 24) from 52 organotypic cultures were examined. Bars: A, 20 µm; D,F,H, 10 µm. Error bars represent SEM.
Figure 4.
Figure 4.
Luminal collapse is associated with disrupted apical integrity. (A) Image of mouse mammary glands with or without doxycycline-induced PyMT expression immunostained for phospho-myosin II (red) and E-Cadherin (green). Yellow arrowheads show open lumina, and white arrows show collapsed lumina. (B) Quantification of phospho-myosin II intensity at the apical membranes. Five tissue sections each were analyzed from three mice. (C) Images of mouse mammary glands with or without doxycycline-induced PyMT expression immunostained for Par6 (red) and active RhoA (RhoGTP; green). Yellow arrowheads show open lumina, and white arrows show collapsed lumina. (D) Image showing RhoA-GTP in open ducts in glands with or without doxycycline-induced PyMT. The bottom panel shows a duct that has not collapsed. (E) Quantification of active RhoA (RhoGTP) at the apical membrane. Three tissue sections each were analyzed from three mice. (F) Images of adjacent ADH and DCIS immunostained for Par6 (green) and RhoA-GTP (red). (G) Quantification of apical intensity of RhoA-GTP in ADH (n = 29 lumina) and DCIS (n = 52 lumina) from four human subjects. Bars: A,C, 20 µm; E, 50 µm.
Figure 5.
Figure 5.
Regulation of RhoA activity regulates lumen collapse. (A) Images of 3D organotypic cultures with or without doxycycline-induced PyMT expression in the presence of scrambled (shScr) or two different p190B-RhoGAP shRNAs and immunostained for Par6. GFP marks cells expressing shRNA. Asterisks indicate open lumina. (B) Quantification of lumen phenotypes in cysts. (C) Images of sections of mouse mammary glands with or without induction of PyMT and expression of p190B-RhoGAP shRNA immunostained for Par6 (green) and RhoA-GTP (red). (D) Quantification of ducts with open lumina. Two tissue sections were analyzed from each of four mice. (***) P < 0.001. Bars, 25 µm.
Figure 6.
Figure 6.
Disruption of tissue organization and cell polarity is reversible. (A) Scheme showing doxycycline treatment schedule for PyMT induction and deinduction. (B) Images of mammary tissue immunostained for Par6 and E-cadherin after doxycycline-mediated induction/deinduction for specified times. (C) Quantification of cells with apical–basal membrane polarity from induction/deinduction cycles. (D) Quantification of ducts with open lumina from induction/deinduction cycles. (E) Images of mammary tissue immunostained for Par6, E-cadherin, and cleaved Caspase 3 after doxycycline-mediated induction/deinduction for the indicated times. The white asterisk indicates fluorescence from debris in the lumen. (F) Quantification of the number of cleaved Caspase-3-positive cells in E-cadherin-positive ductal cells. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. Bars: 50 µm.
Figure 7.
Figure 7.
Model for loss of apical polarity and lumen filling in ER+ breast cancers. The model shows progressive loss of polarity (left) and early loss of polarity (right). See the Discussion for details.
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