Multiple Epithelial Tissues Research Articles

An engineered Trop2/CD3 bispecific antibody for treatment of gastric, pancreatic, and colorectal cancers.

e14521 Background: Given its over-expression and poor prognosis in a variety of solid tumors, trophoblast cell surface antigen 2 (Trop2) is regarded as a potential therapeutic target. Trop2-directed antibody-drug conjugates have been developed for treatment of solid tumors; however, their clinical benefits can be limited by the associated undesirable effects which is likely attributed to the on-target off-tumor effect arising from the low basal Trop2 expression on the surface of multiple normal epithelial tissues. The present study aims to develop a humanized Trop2/CD3 bispecific T-cell engager antibody that can promote immunological synapse formation with tumor cells overexpressing Trop2, redirecting T cell-mediated killing specifically to cancers with reduced risk of on-target off-tumor effects. Methods: A Trop2/CD3 bispecific antibody was generated by linking a humanized anti-Trop2 monoclonal antibody with a CD3-binding scFv. To tune the affinity of the bispecific antibody for tumor target, in silico paratope prediction was firstly done using ProABC-2 (Ambrosetti F et al., 2020), following by alanine substitution of the identified paratope residues individually. A total of 6 CDR variants were generated with their binding to Trop2 examined using biolayer interferometry. Selected candidates were further characterized for their binding to Trop2-positive cancer cells and activation of T cells upon target engagement. Redirection of T cell-mediated killing on cancer cells expressing high Trop2 level was also studied in vitro. Results: Substitution of a complementarity-determining residue (CDR) on the heavy chain of anti-Trop2 antibody with an alanine significantly reduced binding affinity to Trop2. This CDR variant was selected for further functional characterization. Apart from weaker binding to Trop2-positive tumor cells, the variant also demonstrated a slower cytokine induction, a less acute T-cell activation, and a cytotoxic effect which was more restricted to Trop2-high cancer cells. Conclusions: Our new Trop2/CD3 bispecific antibody demonstrates potent anti-tumor activity, while at the same time shows a reduced risk of on-target off-tumor toxicity. The drug candidate warrants further investigations including toxicity study in monkeys. [Table: see text]

Suprabasin-derived polypeptides: SBSN(50-63) induces inflammatory response via TLR4-mediated mast cell activation.

Psoriasis is a chronic, relapsing, immune-mediated, and papulosquamous skin disorder. Excessive mast cell activation, in psoriatic lesions, contributes to inflammation. Various endogenous peptides can participate in the pathogenesis of inflammatory diseases by activating mast cells. Suprabasin (SBSN) is expressed in multiple epithelial tissues and it regulates the normal epidermal barrier function. We have recently shown that suprabasin-derived polypeptides, SBSN(50-63), are significantly increased in psoriatic lesions, through differential peptide analysis. This study was conducted to clarify whether SBSN(50-63) plays a pivotal role in activating mast cells and mediating proinflammatory cytokines and chemokines production in psoriasis. The increased expression of SBSN in psoriatic lesions was confirmed by bioinformatics analysis, PCR and ELISA. Wild-type mice injected subcutaneously with SBSN(50-63) exhibited infiltration of inflammatory cells and the release of cytokines in vivo. SBSN(50-63) stimulated mouse primary mast cells (MPMC) and the laboratory of allergic disease 2 (LAD2) human mast cells to produce more inflammatory mediators than the control group, which were measured ex vivo and in vitro. Toll-like receptor 4 was identified as the receptor of SBSN on mast cells by molecular docking analysis, molecular dynamics simulation, and siRNA transfection. Collectively, SBSN(50-63) could activate mast cells through TLR4, which may increase the inflammatory response in psoriasis.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac

The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

3021 - LGR5 Mediates Positive B-Cell Selection and is Critical for Survival of Normal and Transformed B Cells

Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5), through binding to its ligand R-spondin, increases strength and duration of Wnt signaling in multiple epithelial tissues, however a role of Lgr5 in hematopoietic cells was not explored. Successful completion of immunoglobulin V(D)J gene recombination and initial encounter of antigen in germinal centers represent key events in the life of developing B cells that promote survival and positive selection. Here, we found that both events result in strong upregulation of Lgr5 surface expression in B cell precursors in the bone marrow and germinal center B cells.

Abstract 4515: Lgr5 mediates positive B-cell selection and is critical for initiation and survival of B-cell malignancies

Abstract Introduction: In B-cell leukemia and lymphoma, leukemia initiating cells occur at a high frequency (Rehe 2013), are phenotypically diverse (Aoki 2015) and can arise from any stage of B-cell development (Le Viseur 2008). Unlike stem cells, where self-renewal is regulated by a developmental hierarchy, in the B-cell lineage, positive selection events, i.e. induced by antigen-receptor (BCR) signaling dictates their ability to self-renew. Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) is a Wnt target gene and through binding to its ligand R-spondin, Lgr5 modulates Wnt signaling strength. Lgr5 is widely used as stem cell marker in multiple epithelial tissues, however the role of Lgr5 in hematopoietic cells was not explored. Results: Upon successful completion of immunoglobulin V(D)J gene recombination and first encounter of antigen represent key events in the life of a B-cell that promote survival and positive selection. Here, we found that both events result in upregulation of Lgr5 expression in B cell precursors in the bone marrow and germinal center B cells. Likewise, engagement of BCR signaling on B-cell lymphomas and oncogenic BCR-signaling mimics in leukemia strongly increased LGR5 expression, which was sensitive to inhibition of SYK and BTK kinases in the BCR pathway.In patients with B-cell leukemia, higher than median mRNA levels of LGR5 at the time of diagnosis were associated with poor clinical outcome and higher likelihood of drug-resistance and relapse. Inducible ablation of Lgr5 during earliest stages of B-cell development resulted in a >100-fold reduction of absolute B-cell numbers. Studies in epithelial cells suggest a role of Lgr5 as potentiator of WNT-signaling. However, deletion of Lgr5 in B cells caused cell death in parallel with massive accumulation of nuclear β-catenin and increased expression of β -catenin target genes. Deletion of Lgr5 abolished colony forming capacity and reduced the ability of leukemia cells to initiate fatal disease in transplant recipients. Likewise, inducible activation of a gain-of-function mutant of β-catenin resulted in rapid cell death in normal and malignant B cells. Conclusion: Lgr5-expression and positive B-cell selection is induced by BCR-engagement by antigen or oncogenic mimics of BCR signaling in B-cell malignancies (e.g. transforming oncogenes that engage the BCR pathway). Unlike in epithelial cells, LGR5 expression in B cells restricts the levels of nuclear β-catenin and enables B-cell survival and transformation through negative regulation of Wnt-signaling. Targeting Lgr5 using a novel Lgr5-ADC seems promising to deplete B-cell leukemia- and lymphoma-initiating cells. Citation Format: Kadriye Nehir Cosgun, Anna Hecht, Xin Yang, Maurizio Mangolini,, Ali Aghajanirefah, Gang Xiao, Teresa Sadras, Zhengshan Chen, Lars Klemm, Huimin Geng, Chao Hong, Qingxiao Song, Deye Zeng, Hassan Jumaa, Defu Zeng, Hans Clevers, Markus Muschen. Lgr5 mediates positive B-cell selection and is critical for initiation and survival of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4515.

LMO2 attenuates tumor growth by targeting the Wnt signaling pathway in breast and colorectal cancer.

The proto-oncogene LIM-domain only 2 (lmo2) was traditionally considered to be a pivotal transcriptional regulator in hematopoiesis and leukemia. Recently, the cytosolic localization of LMO2 was revealed in multiple epithelial tissues and a variety of solid tumors. However, the function of LMO2 in these epithelia and solid tumors remains largely unclear. The Wnt signaling pathway is a crucial determinant of development, and abnormalities in several key segments of this pathway contribute to oncogenesis. The current study demonstrated that LMO2 participates in the regulation of canonical Wnt signaling in the cytoplasm by binding to Dishevelled-1/2 (DVL-1/2) proteins. These interactions occurred at the PDZ domain of Dishevelled, and LMO2 subsequently attenuated the activation of the key factor β-catenin in the canonical Wnt signaling pathway. Meanwhile, significantly decreased expression of LMO2 was detected in breast and colorectal cancers, and the downregulation of LMO2 in these cells increased cell proliferation and reduced apoptosis. Taken together, the data in this study revealed a novel crosstalk between LMO2 and the Wnt signaling pathway during tumorigenesis and suggested that LMO2 might be a tumor suppressor in certain solid tumors, in contrast to its traditional oncogenic role in the hematopoietic system.

Sodium channel Nax is a regulator in epithelial sodium homeostasis.

The mechanisms by which the epidermis responds to disturbances in barrier function and restores homeostasis are unknown. With a perturbation of the epidermal barrier, water is lost, resulting in an increase in extracellular sodium concentration. We demonstrate that the sodium channel Nax functions as a sodium sensor. With increased extracellular sodium, Nax up-regulates prostasin, which results in activation of the sodium channel ENaC, resulting in increased sodium flux and increased downstream mRNA synthesis of inflammatory mediators. Nax is present in multiple epithelial tissues, and up-regulation of its downstream genes is found in hypertrophic scars. In animal models, blocking Nax expression results in improvement in scarring and atopic dermatitis-like symptoms, both of which are pathological conditions characterized by perturbations in barrier function. These findings support an important role for Nax in maintaining epithelial homeostasis.

Immunohistochemistry on decalcified rat nasal cavity: trials and successes

Immunohistochemical (IHC) staining for Casein Kinase 2 alpha (CK2a), cyclin-dependent kinase inhibitor 1B (p27Kip1), and heat shock protein 90 (HSP90) was performed on four levels of decalcified rat nasal cavity from four male and three female controls following nose-only exposure to ambient air for 5 days. Successful staining with negligible background artifact was obtained on multiple epithelial and neuroepithelial soft tissues, without antigen retrieval. Independent optimization was needed for each of the three antibodies. A major challenge in this project was maintaining tissue adherence to the slides during tissue incubations. This report documents immunolocalization of CK2a, p27Kip1, and HSP90 in respiratory and olfactory tissue of the rat nose.

Abstract 1577: Subtype dependent carcinoma initiation by Rb tumor suppressor inactivation in multiple epithelial tissues.

Abstract Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by mesenchymal cells. Of major importance is to understand the relationship among cell types, initiation event, and ultimate cancer outcome. Mouse models facilitate the prospective examination of early oncogenic events, which is not feasible in humans, permitting an analysis of epithelial subtype susceptibility. Since most solid tumors harbor aberrations in the pRb network, we assessed the susceptibility of epithelial subtypes expressing cytokeratin (K) 18 or K19 to the disruption of Rb tumor suppression (Rb-TS) in vivo. Tumorigenesis could be initiated in either K18- or K19-expressing cells. However, the susceptibility of epithelial tissues was subtype-dependent. K19 cells were more prone to hyperplasia and neoplasia than K18 targeted cells. Given that a single genetic event was engineered, a small percentage of animals developed carcinoma/adenocarcinoma, providing the opportunity to define progression events via engineering or spontaneous evolution. To extend our analysis to a specific tissue, we employed a prostate-specific Cre line to target either K18- or K19-expressing prostate epithelial cells. Differential responses observed in the frequency and extent of premalignant hyperplastic lesions strongly supported the notion of cell type-dependent susceptibility to Rb-TS inactivation. Our data suggest that the tumorigenic consequences of a single initiation event are dictated by cellular subtype distinctiveness, underscoring the importance of the tumor cell of origin. Citation Format: Yurong Song, Chunyu Yang, Wenqi Pan, Alisan Fathalizadeh, Lucy Lu, Debra Gilbert, T. Norene O'Sullivan, Diana C. Haines, Philip L. Martin, Terry Van Dyke. Subtype dependent carcinoma initiation by Rb tumor suppressor inactivation in multiple epithelial tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1577. doi:10.1158/1538-7445.AM2013-1577

The matriptase-prostasin proteolytic cascade in epithelial development and pathology

The type II transmembrane serine protease matriptase has an essential role in the integrity and function of multiple epithelial tissues. In the epidermis, matriptase activates the glycosylphosphatidylinositol (GPI) anchored membrane serine protease prostasin to initiate a proteolytic cascade that is required for the development of the stratum corneum barrier function. Accordingly, mice deficient for matriptase phenocopy mice deficient for epidermal prostasin and present with impaired corneocyte differentiation, imparied lipid matrix formation, loss of profilaggrin processing and loss of tight junction formation and function. Together, these defects lead to a compromised epidermal barrier and result in fatal dehydration during the neonatal period. Proteolytic activity of the matriptase-prostasin cascade is regulated in the epidermis via inhibition by the Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Importantly, targeted post-natal ablation of matriptase in mice perturbs the function of multiple adult tissues, indicating an ongoing requirement for matriptase proteolysis in the maintenance of diverse types of epithelia. Impaired matriptase proteolytic activity has been linked to human Autosomal Recessive Icthyosis with Hypotrichosis (ARIH), whereas aberrant matriptase activity has been implicated in Netherton's Syndrome. This review will summarize information pertaining to the role of matriptase in epithelial biology and will discuss recent advancements in our understanding of how matriptase activity is regulated and the down-stream effectors of matriptase proteolysis.

Correction: Dietary Energy Balance Modulates Signaling through the Akt/Mammalian Target of Rapamycin Pathways in Multiple Epithelial Tissues

Correction: Dietary Energy Balance Modulates Signaling through the Akt/Mammalian Target of Rapamycin Pathways in Multiple Epithelial Tissues

Mammary epithelial cell transformation is associated with deregulation of the vitamin D pathway

The vitamin D endocrine system mediates anti-proliferative and pro-differentiating signaling in multiple epithelial tissues, including mammary gland and breast tumors. The vitamin D metabolite 1alpha,25(OH)2D3 mediates growth inhibitory signaling via activation of the vitamin D receptor (VDR), a ligand dependent transcription factor. 1alpha,25(OH)(2)D3 is synthesized from 25(OH)D3 (the major circulating form of the vitamin) by the mitochondrial enzyme CYP27b1 in renal and other tissues. Human mammary epithelial (HME) cells express VDR and CYP27b1 and undergo growth inhibition when exposed to physiological concentrations of 25(OH)D3, suggesting that autocrine or paracrine vitamin D signaling contributes to maintenance of differentiation and quiescence in the mammary epithelium. In the current studies we tested the hypothesis that cancer cells would exhibit reduced sensitivity to vitamin D mediated negative growth regulation. We used a series of progressively transformed HME cell lines expressing known oncogenic manipulations to study the effects of transformation per se on the vitamin D pathway. We report that mRNA and protein levels of VDR and CYP27b1 were reduced greater than 70% upon stable introduction of known oncogenes (SV40 T antigens and H-rasV12) into HME cells. Oncogenic transformation was also associated with reduced 1alpha,25(OH)2D3 synthesis, and cellular sensitivity to growth inhibition by 1alpha,25(OH)2D3 and 25(OH)D3 was decreased approximately 100-fold in transformed cells. These studies provide evidence that disruption of the vitamin D signaling pathway occurs early in the cancer development process.

Simplified purification procedure of laminin-332 and laminin-511 from human cell lines

Laminins are glycoproteins expressed in the basement membrane of multiple epithelial tissues. Previously described purification procedures for the human laminin variants laminin-5 (LN-332) and laminin-10 (LN-511) use tissue as starting material and have multiple steps. We demonstrate a two-step laminin immunoaffinity purification method to produce consistent quantities of intact and biologically active LN-332 and LN-511 from human keratinocyte (HaCaT) and human lung carcinoma (A549) cell lines, respectively. The purification of LN-332 and LN-551 was demonstrated by PAGE analysis, silver staining and Western blot analysis. The purification procedure includes instruction on removing a cell adhesion contaminant known as galectin-3 binding protein from purified LN-511. The biological activity of purified laminin was tested in a standard cell adhesion assay and compared to commercially available LN-111. This rapid and reproducible purification method will contribute to understanding the role of LN-332 and LN-511 in cell behavior, signaling, and gene expression.

Facial clefting in Tp63 deficient mice results from altered Bmp4, Fgf8 and Shh signaling

During embryogenesis, the transcription factor Tp63 is expressed in the basal cells of multiple epithelial tissues. In humans, mutations in TP63 have been identified in five distinct human developmental disorders that are characterized by limb abnormalities, ectodermal dysplasia, and facial anomalies. To dissect the molecular pathogenesis of the bilateral cleft lip and cleft palate that results from mutation of Tp63, we analysed Tp63 mutant mice. At E10.5, Tp63-deficient mice exhibited abnormal morphogenesis of the medial nasal, lateral nasal and maxillary processes. Analysis of key signaling molecules revealed that these defects result from increased Bmp4 signaling in the epithelia of the facial processes. Acting antagonistically on Fgf8 and Shh, this aberrant signaling led to a reduction in mesenchymal cell proliferation and increased cell death in specific regions of the facial processes. In addition, a proliferative defect in the mesenchyme of the maxillary processes at E11.5 resulted in absence of the anterior region of the palatal shelves and, subsequently, cleft palate. Our results are consistent with a role for Tp63 in the regulation of Bmp signaling controlling the growth, modelling and fusion events underlying facial development and shed new light on the complex abnormality of facial clefting.

Dietary Energy Balance Modulates Signaling through the Akt/Mammalian Target of Rapamycin Pathways in Multiple Epithelial Tissues

The prevalence of obesity, an established risk factor for several types of cancer, has increased steadily over the past several decades in the United States. New targets and strategies for offsetting the effect of obesity on cancer risk are urgently needed. In the present study, we examined the effect of dietary energy balance manipulation on steady-state signaling in multiple epithelial tissues, with a focus on the Akt and mammalian target of rapamycin (mTOR) pathways. For these experiments, male FVB/N and C57BL/6 and female ICR mice were maintained on a control (10 kcal% fat) diet, a diet-induced obesity (DIO; 60 kcal% fat) regimen, or a 30% calorie restriction (CR) regimen for 15 to 17 weeks. Relative to the control group, the DIO regimen increased, whereas CR decreased, circulating insulin-like growth factor-I (IGF-I) as has previously been reported. Western blot analyses showed that the DIO regimen enhanced, whereas CR inhibited, activation of Akt and mTOR, regardless of epithelial tissue or genetic background. In contrast, activation of AMP-activated protein kinase was modulated by dietary energy balance manipulation in the liver but not in the epidermis or dorsolateral prostate. Western blot analyses of epidermal extracts taken from ICR mice also revealed reduced activation of both the IGF-I receptor and epidermal growth factor receptor in CR mice, compared with control mice or mice maintained on the DIO regimen. Taken together, these novel findings suggest that dietary energy balance modulates signaling through cell-surface receptors (i.e., IGF-I receptor and epidermal growth factor receptor), affecting activation of multiple downstream pathways including Akt and mTOR, thus providing important dietary and pharmacologic targets for disrupting the obesity-cancer link.

Cutaneous Cancer Stem Cells: β-Catenin Strikes Again

Cancer stem cells (CSCs) are a subpopulation of tumor cells that retain properties of tissue-specific stem cells, including the ability to self-renew. In a recent article in Nature, Malanchi et al. (2008) identified a population of CD34(+) cells in epidermal tumors that require beta-catenin signaling to maintain a CSC phenotype.

Regressing amphibian tail as a model for the cadherin/β-catenin complex disruption and glycosylation alteration during epithelial apoptosis

Epidermis is one of the many tissues that are resorbed during metamorphosis in the regressing tail of amphibian tadpoles. Apoptotic mechanisms play an important role in this process. In this study, loss of intercellular contacts and alterations in plasma membrane glycosylation were observed during apoptosis. The cadherin/beta-catenin complex represents one of the major adhesive systems in multiple epithelial tissues. Here, we analysed the fate of cadherin/beta-catenin complex and alterations of plasma membrane glycoconjugate compositions in apoptotic epithelial cells. Our results showed that the cadherin molecules were cleaved into extracellular and beta-catenin associated cytosolic domains by an intracellular mechanism. However, the extracellular domains were probably removed completely by matrix metalloproteinases. Lectin histochemistry studies suggested that mannose and alpha(2-->6) linked (but not alpha(2-->3) linked) sialic acids were major sugar motifs in plasma membranes of apoptotic tadpole epithelial cells. Although previous studies indicated reduced levels of sialic acid residues during apoptosis, elevated Sambucus nigra agglutinin (SNA) reactivity might be due to the degradation of high molecular weight glycoproteins (probably including cadherin) that masked the SNA-binding residues of the plasma membrane prior to apoptosis.