Ezh2 promotes mammary tumor initiation through epigenetic regulation of the Wnt and mTORC1 signaling pathways.

The regulation of gene expression through histone posttranslational modifications plays a crucial role in breast cancer progression. However, the molecular mechanisms underlying the contribution of histone modification to tumor initiation remain unclear. To gain a deeper understanding of the role of the histone modifier Enhancer of Zeste homology 2 (Ezh2) in the early stages of mammary tumor progression, we employed an inducible mammary organoid system bearing conditional Ezh2 alleles that faithfully recapitulates key events of luminal B breast cancer initiation. We showed that the loss of Ezh2 severely impairs oncogene-induced organoid growth, with Ezh2-deficient organoids maintaining a polarized epithelial phenotype. Transcriptomic profiling showed that Ezh2-deficient mammary epithelial cells up-regulated the expression of negative regulators of Wnt signaling and down-regulated genes involved in mTORC1 (mechanistic target of rapamycin complex 1) signaling. We identified Sfrp1, a Wnt signaling suppressor, as an Ezh2 target gene that is derepressed and expressed in Ezh2-deficient epithelium. Furthermore, an analysis of breast cancer data revealed that Sfrp1 expression was associated with favorable clinical outcomes in luminal B breast cancer patients. Finally, we confirmed that targeting Ezh2 impairs mTORC1 activity through an indirect mechanism that up-regulates the expression of the tumor suppressor Pten. These findings indicate that Ezh2 integrates the mTORC1 and Wnt signaling pathways during early mammary tumor progression, arguing that inhibiting Ezh2 or therapeutically targeting Ezh2-dependent programs could be beneficial for the treatment of early-stage luminal B breast cancer.

3D chromatin remodeling potentiates transcriptional programs driving cell invasion.

The contribution of deregulated chromatin architecture, including topologically associated domains (TADs), to cancer progression remains ambiguous. CCCTC-binding factor (CTCF) is a central regulator of higher-order chromatin structure that undergoes copy number loss in over half of all breast cancers, but the impact of this defect on epigenetic programming and chromatin architecture remains unclear. We find that under physiological conditions, CTCF organizes subTADs to limit the expression of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K) and cell adhesion networks. Loss of a single CTCF allele potentiates cell invasion through compromised chromatin insulation and a reorganization of chromatin architecture and histone programming that facilitates de novo promoter-enhancer contacts. However, this change in the higher-order chromatin landscape leads to a vulnerability to inhibitors of mTOR. These data support a model whereby subTAD reorganization drives both modification of histones at de novo enhancer-promoter contacts and transcriptional up-regulation of oncogenic transcriptional networks.

Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma.

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.

Girdin is a component of the lateral polarity protein network restricting cell dissemination.

Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. Here we show that Drosophila Girdin and its human ortholog (GIRDIN) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, we observed cell dissemination from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, we discovered a core mechanism contributing to epithelial cell polarization from flies to humans. Our data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination.

A long isoform of GIV/Girdin contains a PDZ-binding module that regulates localization and G-protein binding.

PDZ domains are one of the most abundant protein domains in eukaryotes and are frequently found on junction-localized scaffold proteins. Various signaling molecules bind to PDZ proteins via PDZ-binding motifs (PBM) and fine-tune cellular signaling. However, how such interaction affects protein function is difficult to predict and must be solved empirically. Here we describe a long isoform of the guanine nucleotide exchange factor GIV/Girdin (CCDC88A) that we named GIV-L, which is conserved throughout evolution, from invertebrates to vertebrates, and contains a PBM. Unlike GIV, which lacks PBM and is cytosolic, GIV-L localizes onto cell junctions and has a PDZ interactome (as shown through annotating Human Cell Map and BioID-proximity labeling studies), which impacts GIV-L's ability to bind and activate trimeric G-protein, Gαi, through its guanine-nucleotide exchange modulator (GEM) module. This GEM module is found exclusively in vertebrates. We propose that the two functional modules in GIV may have evolved sequentially: the ability to bind PDZ proteins via the PBM evolved earlier in invertebrates, whereas G-protein binding and activation may have evolved later only among vertebrates. Phenotypic studies in Caco-2 cells revealed that GIV and GIV-L may have antagonistic effects on cell growth, proliferation (cell cycle), and survival. Immunohistochemical analysis in human colon tissues showed that GIV expression increases with a concomitant decrease in GIV-L during cancer initiation. Taken together, these findings reveal how regulation in GIV/CCDC88A transcript helps to achieve protein modularity, which allows the protein to play opposing roles either as a tumor suppressor (GIV-L) or as an oncogene (GIV).

Genetic alterations of epithelial polarity genes are associated with loss of polarity in invasive breast cancer.

Breast cancer remains a leading cause of cancer-related death for women. The stepwise development of breast cancer through preinvasive to invasive disease is associated with progressive disruption of cellular and tissue organization. Apical-basal polarity is thought to be a barrier to breast cancer development, but the extent and potential mechanisms that contribute to disrupted polarity are incompletely understood. To investigate the cell polarity status of invasive breast cancers, we performed multiplex imaging of polarity markers on tissue cores from 432 patients from a spectrum of grades, stages and molecular subtypes. Apical-basal cell polarity was lost in 100% of cells in all cases studied, indicating that loss of epithelial polarity may be a universal feature of invasive breast cancer. We then analyzed genomic events from the TCGA dataset for an 18-gene set of core polarity genes. Coamplification of polarity genes with established breast oncogenes was found, which is consistent with functional cooperation within signaling amplicons. Gene-expression levels of several polarity genes were significantly associated with survival, and protein localization of Par6 correlated with higher grade, nodal metastasis and molecular subtype. Finally, multiple hotspot mutations in protein-protein interaction domains critical for cell polarity were identified. Our data indicate that genomic events likely contribute to pervasive disruption of epithelial polarity observed in invasive breast cancer.

The Par3/aPKC interaction is essential for end bud remodeling and progenitor differentiation during mammary gland morphogenesis.

Mammalian polarity proteins have been studied predominantly in cell culture systems, and little is known about their functions in vivo. To address this issue, we used a shRNA lentiviral system to manipulate gene expression in mouse mammary stem/progenitor cells. Transplantation of Par3-depleted stem/progenitor cells into the mammary fat pad severely disrupted mammary development, and glands were characterized by ductal hyperplasia, luminal filling, and highly disorganized end bud structures that were unable to remodel into normal ductal structures. Unexpectedly, Par3-depleted mammary glands also had an expanded progenitor population. We identified a novel function for the atypical protein kinase C (aPKC)-binding domain of Par3 in restricting Par3 and aPKC to the apical region in mammary epithelia in vivo, and found that mammary morphogenesis is dependent on the ability of Par3 to directly bind aPKC. These results reveal a new function for Par3 in the regulation of progenitor differentiation and epithelial morphogenesis in vivo and demonstrate for the first time an essential requirement for the Par3-aPKC interaction.

Cell Polarity Proteins in Breast Cancer Progression.

Breast cancer, one of the leading causes of cancer related death in women worldwide, is a heterogeneous disease with diverse subtypes that have different properties and prognoses. The developing mammary gland is a highly proliferative and invasive tissue, and some of the developmental programs may be aberrantly activated to promote breast cancer progression. In the breast, luminal epithelial cells exhibit apical-basal polarity, and the failure to maintain this organizational structure, due to disruption of polarity complexes, is implicated in promoting hyperplasia and tumors. Therefore, understanding the mechanisms underlying loss of polarity will contribute to our knowledge of the early stages leading to the pathogenesis of the disease. In this review, we will discuss recent findings that support the idea that loss of apical-basal cell polarity is a crucial step in the acquisition of the malignant phenotype. Oncogene induced loss of tissue organization shares a conserved cellular mechanism with developmental process, we will further describe the role of the individual polarity complexes, the Par, Crumbs, and Scribble, to couple cell division orientation and cell growth. We will examine symmetric or asymmetric cell divisions in mammary stem cell and their contribution to the development of breast cancer subtypes and cancer stem cells. Finally, we will highlight some of the recent advances in our understanding of the molecular mechanisms by which changes in epithelial polarity programs promote invasion and metastasis through single cell and collective cell modes. J. Cell. Biochem. 117: 2215-2223, 2016. © 2016 Wiley Periodicals, Inc.

LGN loss randomizes spindle orientation and accelerates tumorigenesis in PTEN-deficient epidermis.

Loss of cell polarity and disruption of tissue organization are key features of tumorigenesis that are intrinsically linked to spindle orientation. Epithelial tumors are often characterized by spindle orientation defects, but how these defects impact tumor formation driven by common oncogenic mutations is not fully understood. Here, we examine the role of spindle orientation in adult epidermis by deleting a key spindle regulator, LGN, in normal tissue and in a PTEN-deficient mouse model. We report that LGN deficiency in PTEN mutant epidermis leads to a threefold increase in the likelihood of developing tumors on the snout, and an over 10-fold increase in tumor burden. In this tissue, loss of LGN alone increases perpendicular and oblique divisions of epidermal basal cells, at the expense of a planar orientation of division. PTEN loss alone does not significantly affect spindle orientation in these cells, but the combined loss of PTEN and LGN fully randomizes basal spindle orientation. A subset of LGN- and PTEN-deficient animals have increased amounts of proliferative spinous cells, which may be associated with tumorigenesis. These results indicate that loss of LGN impacts spindle orientation and accelerates epidermal tumorigenesis in a PTEN-deficient mouse model.

Polarity in breast development and cancer.

Mammary gland development and breast cancer progression are associated with extensive remodeling of epithelial tissue architecture. Apical-basal polarity is a key feature of epithelial cells that coordinates key elements of epithelial morphogenesis including cell organization, proliferation, survival, and migration. In this review we discuss advances in our understanding of how apical-basal polarity programs are used in breast development and cancer. We describe cell lines, organoids, and in vivo models commonly used for studying apical-basal polarity in breast development and disease and discuss advantages and limitations of each. We also provide examples of how core polarity proteins regulate branching morphogenesis and lactation during development. We describe alterations to core polarity genes in breast cancer and their associations with patient outcomes. The impact of up- or down-regulation of key polarity proteins in breast cancer initiation, growth, invasion, metastasis, and therapeutic resistance are discussed. We also introduce studies demonstrating that polarity programs are involved in regulating the stroma, either through epithelial-stroma crosstalk, or through signaling of polarity proteins in non-epithelial cell types. Overall, a key concept is that the function of individual polarity proteins is highly contextual, depending on developmental or cancer stage and cancer subtype.

Ultrasoft edge-labelled hydrogel sensors reveal internal tissue stress patterns in invasive engineered tumors.

Measuring internal mechanical stresses within 3D tissues can provide important insights into drivers of morphogenesis and disease progression. Cell-sized hydrogel microspheres have recently emerged as a powerful technique to probe tissue mechanobiology, as they can be sufficiently soft as to deform within remodelling tissues, and optically imaged to measure internal stresses. However, measuring stresses at resolutions of ∼10 Pa requires ultrasoft, low-polymer content hydrogel formulations that are challenging to label with sufficiently fluorescent materials to support repeated measurements, particularly in optically dense tissues over 100 µm thick, as required in cancer tumor models. Here, we leverage thermodynamic partitioning of hydrogel components to create "edge-labelled" ultrasoft hydrogel microdroplets, in a single polymerization step. Bright and stable fluorescent nanoparticles preferentially polymerize at the hydrogel droplet interface, and can be used to repeatedly track sensor surfaces over long-term experiments, even when embedded deep in light-scattering tissues. We utilize these edge-labelled microspherical stress gauges (eMSGs) in inducible breast cancer tumor models of invasion, and demonstrate distinctive internal stress patterns that arise from cell-matrix interactions at different stages of breast cancer progression. Our studies demonstrate a long-term macroscale compaction of the tumor during matrix encapsulation, but only a short-term increase in local stress as non-invasive tumors rapidly make small internal reorganizations that reduce the mechanical stress to baseline levels. In contrast, once invasion programs are initiated, internal stress throughout the tumor is negligible. These findings suggest that internal tumor stresses may initially prime the cells to invade, but are lost once invasion occurs. Together, this work demonstrates that mapping internal mechanical stress in tumors may have utility in advancing cancer prognostic strategies, and that eMSGs can have broad utility in understanding dynamic mechanical processes of disease and development.

Studying Cell Polarity Dynamics During Cancer Initiation Using Inducible 3D Organotypic Cultures.

Epithelial tissues are highly organized structures that are structured at both the cellular and tissue levels. Individual cells are characterized by an apical membrane facing a central lumen, and a basolateral membrane that contacts adjacent cells and the basement membrane. The maintenance of apical-basal polarity is crucial for maintaining epithelial homeostasis and is considered a barrier to carcinogenesis. Apical-basal cell polarity is compromised in many epithelial cancers, such as breast, lung, and prostate, and has been associated with disease progression. Three-dimensional (3D) organotypic cultures recapitulate the 3D tissue architecture and mechanical properties found in vivo. This chapter describes methods to establish 3D organoids from human cell lines or mouse primary cells with inducible oncogene expression in polarized epithelial structures to investigate mechanisms of tumor initiation, luminal filling, and growth. The method is versatile, and simple modifications can be made to study diverse cell/tissue types and oncogenes.

Deep Interpretable Classification and Weakly-Supervised Segmentation of Histology Images via Max-Min Uncertainty.

Weakly-supervised learning (WSL) has recently triggered substantial interest as it mitigates the lack of pixel-wise annotations. Given global image labels, WSL methods yield pixel-level predictions (segmentations), which enable to interpret class predictions. Despite their recent success, mostly with natural images, such methods can face important challenges when the foreground and background regions have similar visual cues, yielding high false-positive rates in segmentations, as is the case in challenging histology images. WSL training is commonly driven by standard classification losses, which implicitly maximize model confidence, and locate the discriminative regions linked to classification decisions. Therefore, they lack mechanisms for modeling explicitly non-discriminative regions and reducing false-positive rates. We propose novel regularization terms, which enable the model to seek both non-discriminative and discriminative regions, while discouraging unbalanced segmentations. We introduce high uncertainty as a criterion to localize non-discriminative regions that do not affect classifier decision, and describe it with original Kullback-Leibler (KL) divergence losses evaluating the deviation of posterior predictions from the uniform distribution. Our KL terms encourage high uncertainty of the model when the latter inputs the latent non-discriminative regions. Our loss integrates: (i) a cross-entropy seeking a foreground, where model confidence about class prediction is high; (ii) a KL regularizer seeking a background, where model uncertainty is high; and (iii) log-barrier terms discouraging unbalanced segmentations. Comprehensive experiments and ablation studies over the public GlaS colon cancer data and a Camelyon16 patch-based benchmark for breast cancer show substantial improvements over state-of-the-art WSL methods, and confirm the effect of our new regularizers (our code is publicly available at https://github.com/sbelharbi/deep-wsl-histo-min-max-uncertainty).

Revisiting tissue tensegrity: Biomaterial-based approaches to measure forces across length scales.

Cell-generated forces play a foundational role in tissue dynamics and homeostasis and are critically important in several biological processes, including cell migration, wound healing, morphogenesis, and cancer metastasis. Quantifying such forces in vivo is technically challenging and requires novel strategies that capture mechanical information across molecular, cellular, and tissue length scales, while allowing these studies to be performed in physiologically realistic biological models. Advanced biomaterials can be designed to non-destructively measure these stresses in vitro, and here, we review mechanical characterizations and force-sensing biomaterial-based technologies to provide insight into the mechanical nature of tissue processes. We specifically and uniquely focus on the use of these techniques to identify characteristics of cell and tissue "tensegrity:" the hierarchical and modular interplay between tension and compression that provide biological tissues with remarkable mechanical properties and behaviors. Based on these observed patterns, we highlight and discuss the emerging role of tensegrity at multiple length scales in tissue dynamics from homeostasis, to morphogenesis, to pathological dysfunction.

CD13 orients the apical-basal polarity axis necessary for lumen formation.

Polarized epithelial cells can organize into complex structures with a characteristic central lumen. Lumen formation requires that cells coordinately orient their polarity axis so that the basolateral domain is on the outside and apical domain inside epithelial structures. Here we show that the transmembrane aminopeptidase, CD13, is a key determinant of epithelial polarity orientation. CD13 localizes to the apical membrane and associates with an apical complex with Par6. CD13-deficient cells display inverted polarity in which apical proteins are retained on the outer cell periphery and fail to accumulate at an intercellular apical initiation site. Here we show that CD13 is required to couple apical protein cargo to Rab11-endosomes and for capture of endosomes at the apical initiation site. This role in polarity utilizes the short intracellular domain but is independent of CD13 peptidase activity.

A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation.

Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRASG12V exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRASG12V-expressing cells. This represents an applicable approach to explore protein-protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells.

Architectural control of metabolic plasticity in epithelial cancer cells.

Metabolic plasticity enables cancer cells to switch between glycolysis and oxidative phosphorylation to adapt to changing conditions during cancer progression, whereas metabolic dependencies limit plasticity. To understand a role for the architectural environment in these processes we examined metabolic dependencies of cancer cells cultured in flat (2D) and organotypic (3D) environments. Here we show that cancer cells in flat cultures exist in a high energy state (oxidative phosphorylation), are glycolytic, and depend on glucose and glutamine for growth. In contrast, cells in organotypic culture exhibit lower energy and glycolysis, with extensive metabolic plasticity to maintain growth during glucose or amino acid deprivation. Expression of KRASG12V in organotypic cells drives glucose dependence, however cells retain metabolic plasticity to glutamine deprivation. Finally, our data reveal that mechanical properties control metabolic plasticity, which correlates with canonical Wnt signaling. In summary, our work highlights that the architectural and mechanical properties influence cells to permit or restrict metabolic plasticity.

Mapping cellular-scale internal mechanics in 3D tissues with thermally responsive hydrogel probes.

Local tissue mechanics play a critical role in cell function, but measuring these properties at cellular length scales in living 3D tissues can present considerable challenges. Here we present thermoresponsive, smart material microgels that can be dispersed or injected into tissues and optically assayed to measure residual tissue elasticity after creep over several weeks. We first develop and characterize the sensors, and demonstrate that internal mechanical profiles of live multicellular spheroids can be mapped at high resolutions to reveal broad ranges of rigidity within the tissues, which vary with subtle differences in spheroid aggregation method. We then show that small sites of unexpectedly high rigidity develop in invasive breast cancer spheroids, and in an in vivo mouse model of breast cancer progression. These focal sites of increased intratumoral rigidity suggest new possibilities for how early mechanical cues that drive cancer cells towards invasion might arise within the evolving tumor microenvironment.

CCDC88B is required for mobility and inflammatory functions of dendritic cells.

The Coiled Coil Domain Containing Protein 88B (CCDC88B) gene is associated with susceptibility to several inflammatory diseases in humans and its inactivation in mice protects against acute neuroinflammation and models of intestinal colitis. We report that mice lacking functional CCDC88B (Ccdc88bMut ) are defective in several dendritic cells (DCs)-dependent inflammatory and immune reactions in vivo. In these mice, an inflammatory stimulus (LPS) fails to induce the recruitment of DCs into the draining lymph nodes (LNs). In addition, OVA-pulsed Ccdc88bMut DCs injected in the footpad do not induce recruitment and activation of antigen-specific CD4+ and CD8+ T cells in their draining LN. Experiments in vitro indicate that this defect is independent of the ability of mutant DCs to capture and present peptide antigen to T cells. Rather, kinetic analyses in vivo of wild-type and Ccdc88bMut DCs indicate a reduced migration capacity in the absence of the CCDC88B protein expression. Moreover, using time-lapse light microscopy imaging, we show that Ccdc88bMut DCs have an intrinsic motility defect. Furthermore, in vivo studies reveal that these reduced migratory properties lead to dampened contact hypersensitivity reactions in Ccdc88b mutant mice. These findings establish a critical role of CCDC88B in regulating movement and migration of DCs. Thus, regulatory variants impacting Ccdc88b expression in myeloid cells may cause variable degrees of DC-dependent inflammatory response in situ, providing a rationale for the genetic association of CCDC88B with several inflammatory and autoimmune diseases in humans.

RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote.

Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been recently implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. Here we show that "regulator of G protein signaling-14" (RGS14) is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. Our results indicate that RGS14 is one of the earliest essential product of the mammalian embryonic genome yet described and has a general role in mitosis.