Benign, persistent, and invasive: mechanistic and translational approaches to middle‑ear cholesteatoma.

Cholesteatoma is a destructive squamous epithelial lesion of the temporal bone which gradually expands and leads to serious complications by destruction of nearby bony structures. Erosion caused by bone resorption of the ossicular chain and bony labyrinth may result in hearing loss, vestibular dysfunction, facial paralysis, labyrinthine fistulae and intracranial complications. The exact underlying cellular and molecular mechanism of bone resorption in acquired cholesteatoma still remains unexplained. Pubmed database and China National Knowledge Infrastructure were screened for articles focusing on bone resorption in acquired cholesteatoma. Osteoclast activation, pressure necrosis, acid lysis, enzyme mediation, inflammatory mediators and several newly discovered biomolecules are outlined as main theories behind bone resorption in acquired cholesteatoma, aiming to facilitate the development of potential therapeutic targets for preventing intracranial and extracranial complications caused by bone resorption in acquired middle ear cholesteatoma.

Leukocytes Induce Epithelial to Mesenchymal Transition after Unilateral Ureteral Obstruction in Neonatal Mice

Cancer Metastasis Is Accelerated through Immunosuppression during Snail-Induced EMT of Cancer Cells

From the earliest stages of embryonic development, cells of epithelial and mesenchymal origin contribute to the structure and function of developing organs. However, these phenotypes are not always permanent, and instead, under the appropriate conditions, epithelial and mesenchymal cells convert between these two phenotypes. These processes, termed Epithelial-Mesenchymal Transition (EMT), or the reverse Mesenchymal-Epithelial Transition (MET), are required for complex body patterning and morphogenesis. In addition, epithelial plasticity and the acquisition of invasive properties without the full commitment to a mesenchymal phenotype are critical in development, particularly during branching morphogenesis in the mammary gland. Recent work in cancer has identified an analogous plasticity of cellular phenotypes whereby epithelial cancer cells acquire mesenchymal features that permit escape from the primary tumor. Because local invasion is thought to be a necessary first step in metastatic dissemination, EMT and epithelial plasticity are hypothesized to contribute to tumor progression. Similarities between developmental and oncogenic EMT have led to the identification of common contributing pathways, suggesting that the reactivation of developmental pathways in breast and other cancers contributes to tumor progression. For example, developmental EMT regulators including Snail/Slug, Twist, Six1, and Cripto, along with developmental signaling pathways including TGF-β and Wnt/β-catenin, are misexpressed in breast cancer and correlate with poor clinical outcomes. This review focuses on the parallels between epithelial plasticity/EMT in the mammary gland and other organs during development, and on a selection of developmental EMT regulators that are misexpressed specifically during breast cancer.

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.

Aim: To assess radiological findings in Chronic otitis media (COM), its involvement in middle ear and adjacent structure and to compare with similar published data. The ability of the radiological investigations to detect the various pathological and anatomical variations were evaluated and compared with intraoperative findings. COM is a long standing inflammation of the middle ear cleft without reference to etiology or pathogenesis. Due to the strategic location of the tympanomastoid compartment, separated from the middle and posterior cranial fossa by the thinnest of bony partitions, otitis media has the potential for intracranial extension. Hence, it becomes very important to know the location and extent of the disease before proceeding to surgical treatment. Radiological examination of the temporal bone helps us to achieve this objective. The present work has been undertaken to study the role of radiological imaging of the temporal bone as a diagnostic modality in COM and its use in determining the lines of management as in the type of surgical intervention required. Materials and Methods: This is a prospective study in which total of 50 cases with COM were studied. Results: HRCT is reliable for all the parameters like scutum erosion, ossicular erosion, mastoid pneumatisation, low lying dura, anterior lying sigmoid, Korner's septum, cholesteatoma extension in the middle ear and mastoid, and presence of complications such as mastoiditis and mastoid abscess, mastoid cortex dehiscence, sigmoid sinus plate erosion, facial canal dehiscence, tegmen mastoideum erosion and labyrinthine fistula and intracranial complications with a P < 0.05 but not reliable for tegmen tympani erosion and posterior fossa dural plate erosion. Conclusion: HRCT is highly reliable and findings are in par with intraoperative findings in this study.

Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and trans-differentiation to mesenchymal-like cells with invasive behavior.

Background: The androgen receptor (AR) is a key gene involved in prostate cancer (PC) biology, including disease development, response to initial hormonal therapies, and subsequent resistance to hormonal therapies. AR variants lacking the ligand-binding domain, such as AR-V7, have been observed and enriched in metastatic castration resistant prostate cancer. These variants contribute to resistance mechanisms against agents that target hormonal regulation of the AR, including anti-androgens, such as enzalutamide. In addition to AR variants, epithelial plasticity, including an epithelial-to-mesenchymal transition (EMT), has been implicated in PC metastatic dissemination during castration-resistance. In response to androgen deprivation therapy, PC xenografts have been found to display an acute and reversible up-regulation of both EMT phenotypic markers and AR variants. Methods: To explore the mechanism by which an EMT program may regulate AR variant expression and metastatic disease, we used pre- and post- EMT cells to compare AR expression, migration, invasion and enzalutamide resistance. To induce EMT, we stably transfected LNCaP95 PC cells with a plasmid encoding inducible Snail, a master regulator of EMT. In addition, LNCAP95 cells were serially passaged under increasing enzalutamide conditions and we assessed biomarkers of EMT and invasion/growth in resistant cells. Results: We found that Snail activation was sufficient to induce an EMT and increased expression of both AR-FL and AR-V7 as compared to pre-EMT cells. Nuclear localization of AR, migration and invasion were also increased in post-EMT cells. Importantly, cells were more resistant to enzalutamide treatment. Likewise, in a second model of acquired enzalutamide resistance, we found that Snail, AR-FL and AR-V7 were upregulated in enzalutamide resistant vs. sensitive cell lines. Conclusion: Our study suggests that Snail activation promotes both an aggressive and therapy resistant tumor cell through the induction of EP and both AR-FL and AR-V7 isoforms. Citation Format: Kathryn E. Ware, Daneen Schaeffer, Tian Zhang, Mariano A. Garcia-Blanco, Andrew J. Armstrong. AR-V7 regulation during epithelial plasticity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1847. doi:10.1158/1538-7445.AM2015-1847

Epithelia are physical barriers that constitute a functional interface between distinct body compartments and the outside. Under healthy condition, cells that composed the epithelial sheets are tightly bound to neighboring cells and to underlying basement membranes by adherens junctions, tight junctions, desmosomes and hemi-desmosomes (Farquhar, M. G. & Palade, G. E., 1963). However, epithelial cells empower high degree of plasticity and under certain circumstances such as developmental processes, fibrogenesis or tumor progression, they loss their static phenotype and acquire migratory and invasive behavior (Grunert, S., et al., 2003). Epithelial plasticity could be limited to relocalization of junctional proteins or to a more drastic epithelial to mesenchymal transition (EMT) which is characterized by disruption of intercellular contacts, loss of epithelium-specific proteins, switch to a mesenchymal gene expression pattern, and gain of invasive properties (Thiery, J. P., 2002). It is to note that EMT is different than collective cell movement which occurs when two or more cells that retain their genetic and phenotypic feature move together across a twodimensional (layer of extracellular matrix) or through a three-dimensional interstitial tissue (Ilina, O. & Friedl, P., 2009).

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating invivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.

Epithelial plasticity, the ability of epithelial cells to change phenotypes, is a fascinating phenomenon that has been extensively studied for decades. Most commonly epithelial plasticity refers to the conversion between epithelial and mesenchymal phenotype, known as epithelialto-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). Both EMT and MET are common features in embryonic development, tissue responses to damage, e.g., inflammation, repair, and cancer. Under the Research Topic "Epithelial plasticity and complexity in development, disease and regeneration" a collection of original research articles and research report have been gathered addressing the fascinating and complex states of epithelial plasticity, their distinctions and functions, in human and other model systems. Epithelial to mesenchymal transition (EMT) has been classically defined as a developmental program that is instrumental in early embryo patterning for many organs, characterized by epithelial cells losing cell-to-cell adhesion, epithelial tight junctions, and desmosomes. Evolutionally, EMT processes enabled organisms to acquire more-complex structures by creating mesenchymal cells of mesoderm from primitive ectoderm. EMT is a fundamental process in physiologic repair and pathologic fibrosis of tissues and organs. More recently it has been recognized that EMT also plays a critical role in facilitating the creation of a pro-tumoral microenvironment, promoting tumorigenesis and metastasis. Reorganization of intercellular junctions, particularly tight junctions is a key event of the EMT process during tumor progression. In this issue Neyrinck-Leglantier et al. published their research work investigating how the tight junction protein Zonula occludens-1 (ZO-1) is involved in modulating tumor microenvironment. Using both in vitro and in vivo models they demonstrated that relocation of membrane-associated ZO-1 towards the cyto-nuclear compartments can modulate the secretion of pro-inflammatory chemokines hence

BackgroundThe majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as epithelial to mesenchymal transition (EMT), cells change their genetic and trancriptomic program leading to phenotypic and functional alterations. The challenge of understanding this dynamic process resides in unraveling regulatory networks involving master transcription factors (e.g. SNAI1/2, ZEB1/2 and TWIST1) and microRNAs. Here we investigated microRNAs regulated by SNAI1 and their potential role in the regulatory networks underlying epithelial plasticity.ResultsBy a large-scale analysis on epithelial plasticity, we highlighted miR-203 and its molecular link with SNAI1 and the miR-200 family, key regulators of epithelial homeostasis. During SNAI1-induced EMT in MCF7 breast cancer cells, miR-203 and miR-200 family members were repressed in a timely correlated manner. Importantly, miR-203 repressed endogenous SNAI1, forming a double negative miR203/SNAI1 feedback loop. We integrated this novel miR203/SNAI1 with the known miR200/ZEB feedback loops to construct an a priori EMT core network. Dynamic simulations revealed stable epithelial and mesenchymal states, and underscored the crucial role of the miR203/SNAI1 feedback loop in state transitions underlying epithelial plasticity.ConclusionBy combining computational biology and experimental approaches, we propose a novel EMT core network integrating two fundamental negative feedback loops, miR203/SNAI1 and miR200/ZEB. Altogether our analysis implies that this novel EMT core network could function as a switch controlling epithelial cell plasticity during differentiation and cancer progression.

BackgroundIdiopathic pulmonary fibrosis is a common interstitial lung disease; it is a chronic, progressive, and fatal lung disease of unknown etiology. Over the last two decades, knowledge about the underlying mechanisms of pulmonary fibrosis has improved markedly and facilitated the identification of potential targets for novel therapies. However, despite the large number of antifibrotic drugs being described in experimental pre-clinical studies, the translation of these findings into clinical practices has not been accomplished yet. NADH:quinone oxidoreductase 1 (NQO1) is a homodimeric enzyme that catalyzes the oxidation of NADH to NAD+ by various quinones and thereby elevates the intracellular NAD+ levels. In this study, we examined the effect of increase in cellular NAD+ levels on bleomycin-induced lung fibrosis in mice.MethodsC57BL/6 mice were treated with intratracheal instillation of bleomycin. The mice were orally administered with β-lapachone from 3 days before exposure to bleomycin to 1-3 weeks after exposure to bleomycin. Bronchoalveolar lavage fluid (BALF) was collected for analyzing the infiltration of immune cells. In vitro, A549 cells were treated with transforming growth factor β1 (TGF-β1) and β-lapachone to analyze the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT).Resultsβ-Lapachone strongly attenuated bleomycin-induced lung inflammation and fibrosis, characterized by histological staining, infiltrated immune cells in BALF, inflammatory cytokines, fibrotic score, and TGF-β1, α-smooth muscle actin accumulation. In addition, β-lapachone showed a protective role in TGF-β1–induced ECM expression and EMT in A549 cells.ConclusionOur results suggest that β-lapachone can protect against bleomycin-induced lung inflammation and fibrosis in mice and TGF-β1–induced EMT in vitro, by elevating the NAD+/NADH ratio through NQO1 activation.

Background: Chronic suppurative otitis media (CSOM) poses a significant burden in low- and middle-income countries, with India showing a prevalence of 7.8%. Cholesteatoma, an aggressive subtype of CSOM, can lead to destructive complications such as ossicular damage, facial nerve involvement, and intracranial extension if left untreated. High-resolution computed tomography (HRCT) of the temporal bone is widely used for preoperative evaluation; however, its utility hinges on its diagnostic accuracy in reliably identifying cholesteatoma and related complications. Objective: To evaluate the sensitivity, specificity, and overall diagnostic accuracy of HRCT in detecting cholesteatoma and its complications in patients with unsafe CSOM, using intraoperative findings as the gold standard. Methods: A prospective, observational study was conducted at CMH Rawalpindi from March 2024 to December 2024, involving 50 patients clinically diagnosed with unsafe CSOM and suspected cholesteatoma. HRCT temporal bone imaging was performed prior to surgery, assessing soft tissue density, ossicular erosion, scutum erosion, tegmen tympani erosion, facial canal dehiscence, and labyrinthine fistula. All patients subsequently underwent surgical intervention, and intraoperative findings were meticulously recorded. Diagnostic performance was assessed by comparing radiological and surgical data to calculate sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy. Results: HRCT showed a sensitivity of 95.8%, specificity of 90%, PPV of 96%, NPV of 89.5%, and overall diagnostic accuracy of 94% for detecting cholesteatoma. Ossicular erosion was identified in 41 patients (82%), scutum erosion in 38 (76%), tegmen tympani erosion in 9 (18%), facial canal dehiscence in 6 (12%), and labyrinthine fistula in 3 (6%). Cholesteatoma was surgically confirmed in 48 of 50 patients, resulting in a concordance rate of 96%. Conclusion: HRCT demonstrates high sensitivity and diagnostic accuracy in evaluating cholesteatoma in unsafe CSOM, with excellent agreement to surgical findings. It remains an indispensable tool for preoperative assessment and surgical planning in cholesteatoma management.

Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis.