Basal Keratin Research Articles

A CD146 FACS Protocol Enriches for Luminal Keratin 14/19 Double Positive Human Breast Progenitors

Human breast cancer is believed to arise in luminal progenitors within the normal breast. A subset of these are double positive (DP) for basal and luminal keratins and localizes to a putative stem cell zone within ducts. We here present a new protocol based on a combination of CD146 with CD117 and CD326 which provides an up to thirty fold enrichment of the DP cells. We show by expression profiling, colony formation, and morphogenesis that CD146high/CD117high/CD326high DP cells belong to a luminal progenitor compartment. While these DP cells are located quite uniformly in ducts, with age a variant type of DP (vDP) cells, which is mainly CD146-negative, accumulates in lobules. Intriguingly, in specimens with BRCA1 mutations known to predispose for cancer, higher frequencies of lobular vDP cells are observed. We propose that vDP cells are strong candidates for tracing the cellular origin of breast cancer.

Overexpression and alternative splicing of NF-YA in breast cancer

NF-Y is a CCAAT-binding trimeric transcription factor, whose regulome, interactome and oncogenic potential point to direct involvement in cellular transformation. Yet little is known about the levels of NF-Y subunits in tumors. We focused on breast carcinomas, and analyzed RNA-Seq datasets of TCGA and 54 BRCA cell lines at gene and isoforms level. We partitioned all tumors in the four major subclasses. NF-YA, but not histone-fold subunits NF-YB/NF-YC, is globally overexpressed, correlating with the proliferative Ki67 marker and a common set of 840 genes, with cell-cycle, metabolism GO terms. Their promoters are enriched in NF-Y, GC-rich and E2F sites. Surprisingly, there is an isoform switch, with the “short” isoform -NF-YAs- becoming predominant in tumors. E2F genes are also overexpressed in BRCA, but no switch in isoforms is observed. In Basal-like Claudinlow cell lines and tumors, expression of NF-YAl -long- isoform is high, together with 11 typical EMT markers and low levels of basal Keratins. Analysis of Progression-Free-Intervals indicates that tumors with unbalance of NF-YA isoforms ratios have worst clinical outcomes. The data suggest that NF-YA overexpression increases CCAAT-dependent, pro-growth genes in BRCA. NF-YAs is associated with a proliferative signature, but high levels of NF-YAl signal loss of epithelial features, EMT and acquisition of a more aggressive behavior in a subset of Claudinlow Basal-like tumors.

Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4.

The epidermis, the outer layer of the skin, is formed by stratified keratinocyte layers. The self-renewal of the epidermis is provided by sustained proliferation and differentiation of the keratinocyte stem cells localized to the basal layer of the epidermis. Receptor-interacting protein kinase 4 (RIPK4) is an important regulator of keratinocyte differentiation, mutations of which are associated with congenital ectodermal malformations. In an attempt to identify the molecular basis of RIPK4’s function, we applied yeast two-hybrid screen (Y2H) and found basal layer-specific keratin filament component keratin 14 (KRT14) as a novel RIPK4-interacting partner. During keratinocyte differentiation, layer-specific keratin composition is tightly regulated. Likewise, the basal layer specific KRT14/keratin 5 (KRT5) heterodimers are replaced by keratin 1 (KRT1)/keratin 10 (KRT10) in suprabasal layers. The regulation of keratin turnover is under the control of signaling associated with posttranslational modifications in which phosphorylation plays a major role. In this study, we verified the KRT14-RIPK4 interaction, which was identified with Y2H, in mammalian cells and showed that the interaction was direct by using proteins expressed in bacteria. According to our results, the N-terminal kinase domain of RIPK4 is responsible for KRT14-RIPK4 interaction; however, the RIPK4 kinase activity is dispensable for the interaction. In accordance with their interaction, RIPK4 and KRT14 colocalize within the cells, particularly at keratin filaments associated with perinuclear ring-like structures. Moreover, RIPK4 did not show any effect on KRT14/KRT5 heterodimer formation. Our results suggest that RIPK4 may regulate the keratin turnover required for keratinocyte differentiation through interacting with KRT14.

Krt5+ urothelial cells are developmental and tissue repair progenitors in the kidney.

Congenital urinary tract obstruction (UTO) is the leading cause of chronic kidney disease in children; however, current management strategies do not safeguard against progression to end-stage renal disease, highlighting the need for interventions to limit or reverse obstructive nephropathy. Experimental UTO triggers renal urothelial remodeling that culminates in the redistribution of basal keratin 5-positive (Krt5+) renal urothelial cells (RUCs) and the generation of uroplakin-positive (Upk)+ RUCs that synthesize a protective apical urothelial plaque. The cellular source of Upk+ RUCs is currently unknown, limiting the development of strategies to promote renal urothelial remodeling as a therapeutic approach. In the present study, we traced the origins of adult Upk+ RUCs during normal development and in response to UTO. Fate mapping analysis demonstrated that adult Upk+ RUCs derive from embryonic and neonatal Krt5+ RUCs, whereas Krt5+ RUCs lose this progenitor capacity and become lineage restricted by postnatal day 14. However, in response to UTO, postnatal day 14-labeled adult Krt5+ RUCs break their lineage restriction and robustly differentiate into Upk+ RUCs. Thus, Krt5+ RUCs drive renal urothelial formation during normal ontogeny and after UTO by differentiating into Upk+ RUCs in a temporally restricted manner.

Abstract 4666: p21CIP1 promotes mammary cancer initiating cells via activation of Wnt/TCF1/CyclinD1 signaling

Abstract Cancer stem cells (CSCs) generate and sustain tumors due to tumor initiating potential, resulting in recurrence or metastasis. We showed previously that knockout of the cell cycle inhibitor, p21CIP1, in the PyMT mammary tumor model, inhibits metastasis; however the exact mechanism remained unknown. Here, we show a pivotal role for p21 in potentiating a cancer stem cell-like phenotype. p21 knockout in PyMT mammary tumor cells suppressed tumorsphere formation, ALDH1 expression, and tumor initiating potential, that were in turn rescued by p21 overexpression. Consistent with these effects, p21 loss caused mesenchymal to epithelial transition that was accompanied by downregulation of Slug, Sox9, basal keratins and upregulation of luminal keratins and Claudin-3. Furthermore, p21 knockout suppressed Wnt/β-catenin signaling which was due to TCF1 downregulation. In turn, TCF1 knockdown in PyMT cells, attenuated Wnt signaling and tumorsphere formation. Interestingly, p21 rescue in p21 knockout cells, caused dramatic upregulation of Cyclin D1, consistent with known inhibition of Cyclin D1 nuclear export by p21. These data suggest that p21 promotes a cancer stem like phenotype via Wnt-TCF1 signaling along with direct upregulation of Cyclin D1 by p21. Citation Format: Rachel B. Hazan. p21CIP1 promotes mammary cancer initiating cells via activation of Wnt/TCF1/CyclinD1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4666.

The Anticipated Effect of Both Zamzam Water and Epidermal Growth Factor (EGF) Separately on Tongue Tissues and Lingual Salivary Glands of Diabetic Albino Rats.

 Purpose: The aim of this study was to evaluate the anticipated effect of both Zamzam water and Epidermal growth factor (EGF) separately on tongue tissues and associated lingual salivary glands of diabetic albino rats,( Histological and Ultrastructure evaluation) Materials and Methods Eighteen male albino rats were selected and divided into four groups. Group I (control group) received no drugs, while rats of group II (diabetic group) received a single dose of alloxan intraperitoneal injection. Moreover, rats of group III diabetic and received zamzam water for 3 months and group IV diabetic and received EGF. All rats were sacrificed; tongue was dissected and processed for hematoxylin &eosin and transmission electron microscopy evalution. Results The diabetic group showed histological & ultrastructural changes in the tongue epithelium and underlying lamina propria, a condition which almost restored to normal in zamzam treated group and relatively restored in EGF group. Concolusion Zamzam water provides safer alternative nutritional strategy in restoring the integrity of the damaged epithelium of the tongue tissues after degenerative effect of DM. EGF cause relatively improvement of the damaged epithelium especially in basal cell layer, keratin layer and blood vessels in lamina propria of tongue tissue.

Loss of Limb-Bud-and-Heart (LBH) attenuates mammary hyperplasia and tumor development in MMTV-Wnt1 transgenic mice

WNT/β-catenin signaling plays pivotal roles in mammary development and tumorigenesis; and aberrant activation of this pathway is frequently observed in human breast cancer, correlating with poor outcome. However, the mechanisms underlying WNT-driven mammary tumorigenesis remain incompletely understood. Here, we used mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice, which develop aggressive mammary adenocarcinomas, to examine whether Limb-Bud-and-Heart (LBH) - a WNT/β-catenin target transcription co-factor overexpressed in human triple-negative breast cancers with WNT pathway hyperactivation, contributes to WNT-induced tumorigenesis. We found LBH is specifically overexpressed in basal epithelial tumor cells of MMTV-Wnt1 mammary tumors reminiscent of its basal cell-restricted expression in the normal postnatal mammary gland. To determine the role of LBH in mammary tumorigenesis, we crossed MMTV-Wnt1 mice with basal epithelial-specific Keratin 14/K14-Cre;LbhloxP knockout mice. Mammary glands from virgin LBH-deficient MMTV-Wnt1 mice exhibited reduced hyperplasia, cell proliferation and increased apoptosis. Importantly, LBH inactivation in mammary epithelium significantly delayed tumor onset in MMTV-Wnt1 transgenic mice, with a median tumor-free survival of 32.5 weeks compared to 22.5 weeks in control LBH wild type MMTV-Wnt1 mice (p < 0.05). This data provides the first evidence that LBH plays an essential role in WNT-induced mammary tumorigenesis by promoting hyperplastic growth and tumor formation.

Abstract A05: Deep immunofluorescence imaging of solvent-cleared mouse mammary glands

Abstract We seek to trace the progression of epithelial lesions originating from Lgr5+ cells in mouse mammary glands using the 3DISCO protocol for solvent-cleared tissue. Lineage tracing is increasingly being used to probe different cell types that exist within the mammary gland. Lgr5 is a stem cell marker and it is expressed in several different tissues including colon, stomach, and breast. It has been reported that Lgr5+ cells are necessary for mammary gland organogenesis. However, the cells that initiate tumor formation have remained intangible. Lgr5+ cells reside within the basal cell population of mammary ducts. Lgr5 is a downstream target of Wnt and therefore Lgr5+ cells may have a role in breast cancer progression. Since heterogeneity of tumors is shaped by several factors, including the cell of origin, specific oncogenic events, and interactions between tumor cells and cells of the microenvironment, it is important to obtain 3-dimensional information of tumor development. We used the 3DISCO protocol for solvent-clearing mammary fat pads for deep immunofluorescence staining of transgenic mice harboring a EGFP reporter for Lgr5+ cells (mouse: Lgr5-EGFP-Ires-CreERT2). We performed deep immunofluorescence staining with DAPI and an antibody against the basal cell marker keratin 5 (K5). Mammary fat pads were successfully solvent-cleared and prepared for deep immunofluorescence staining. Epithelial mammary ducts were easily recognized within the fat tissue using DAPI nuclear staining and an antibody against K5. Three-dimensional confocal microscope imaging protocols for whole mammary glands, including epithelial ducts, were established. We optimized the method using tile scans covering the entire fat pad, and multiple z-stacks of selected areas of Lgr5+ cells expressing EGFP were also detected and colocalized with K5+ cells. The whole-organ tissue clearing, assessed in the mouse mammary gland, is suitable to obtain 3-dimensional information of various cell populations using specific molecular markers. This technique enables high-resolution, 3D imaging and phenotyping of tissue and might significantly enhance our understanding of mammary gland tumorigenesis. We will use the established protocol to localize the appearance of early lesions in mouse mammary glands and determine the role of Lgr5+ cells in mammary tumor development. Citation Format: Anna Polec, Jens Henrik Norum, Andreas Brech, Therese Sorlie. Deep immunofluorescence imaging of solvent-cleared mouse mammary glands [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A05.

The role of keratins in the digestive system: lessons from transgenic mouse models.

Keratins are the largest subfamily of intermediate filament proteins. They are either type I acidic or type II basic keratins. Keratins form obligate heteropolymer in epithelial cells and their expression patterns are tissue-specific. Studies have shown that keratin mutations are the cause of many diseases in humans or predispose humans to acquiring them. Using mouse models to study keratin-associated human diseases is critical, because they allow researchers to get a better understanding of these diseases and their progressions, and so many such studies have been conducted. Acknowledging the importance, researches with genetically modified mice expressing human disease-associated keratin mutants have been widely done. Numerous studies using keratin knockout mice, keratin-overexpressed mice, or transgenic mice expressing keratin mutants have been conducted. This review summarizes the mouse models that have been used to study type I and type II keratin expression in the digestive organs, namely, the liver, pancreas, and colon.

UVB exposure of a humanized skin model reveals unexpected dynamic of keratinocyte proliferation and Wnt inhibitor balancing.

We developed human dermo-epidermal skin substitutes that are presently applied in phase I and II clinical trials. Here, we used these very same skin equivalents containing melanocytes, named MelSkin, as an experimental skin model. We investigated the effects of ultraviolet B (UVB) irradiation on the skin grafts transplanted on immune-compromised rats. The irradiation induces a strong wound healing response going along with massive proliferation of basal keratinocytes, basically quiescent under nonirradiated, homeostatic conditions. As a consequence of UVB irradiation, the initially clearly defined basal keratinocyte (mono)layer expands into about 3 layers of keratinocytes, all of which still express the basal keratinocyte marker keratin 15. In contrast, epidermal melanocytes remain quiescent under these circumstances. Moreover, the Wnt inhibitors Dickkopf 3 and Wif1 are downregulated upon UVB irradiation in basal keratinocytes, whereas melanocytes continue to express Wnt inhibitors. These findings suggest that there is (a) a suprabasal population, proliferating in the homeostatic state, hence maintaining the integrity of the epidermis, and (b) a basal, usually quiescent keratinocyte population that is induced to massively proliferate upon irradiation. Importantly, the finding that MelSkin responds in a physiological fashion to UVB is of paramount importance in light of the planned clinical application.

Threonine 150 Phosphorylation of Keratin 5 Is Linked to Epidermolysis Bullosa Simplex and Regulates Filament Assembly and Cell Viability

A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII-/-) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.

135 Comparative genomics of skin barrier proteins reveals environment-dependent dispensability of the suprabasal epidermal keratins K1, K2, and K10

The epidermis has been shaped by evolutionary adaptations to the environment. In humans, the most abundant proteins of the suprabasal epidermis are keratins K1, K2 and K10 which together form the cytoskeleton at the skin barrier. Mutations in K1 and K10 cause epidermolytic hyperkeratosis. Here, we performed comparative genomics to determine whether mutations in keratin genes were tolerated under different environmental constraints. Genome sequences of a phenotypically diverse set of mammalian species were screened by bioinformatic methods. The basal epidermal keratins K5 and K14 as well as the hyperproliferation-associated keratins K6 and K17 are highly conserved among mammals. By contrast, the late differentiation-associated keratin K2 has been lost in at least 4 phylogenetic lineages, indicating dispensability of K2 for skin barrier formation. K1 and K10 have been conserved in terrestrial mammals, but both have been inactivated in fully aquatic mammals of the phylogenetic clade comprising dolphins and whales. The results of this study identify specific components of the epidermal cytoskeleton that are associated with the skin barrier function in a terrestrial environment.

Ectopic expression of the transcription factor MafB in basal keratinocytes induces hyperproliferation and perturbs epidermal homeostasis.

Mammalian epidermis is composed of four morphologically and functionally distinct layers of keratinocytes. The innermost basal layer consists of proliferating self-renewing keratinocytes, which also undergo asymmetric cell division to differentiate into postmitotic suprabasal cells throughout life. Control of the balance between growth and differentiation of basal cells is important for epidermal homeostasis to prevent skin disorders including malignancies; however, the underlying mechanism remains to be elucidated. Recently, MafB was identified as one of the transcription factors that regulate epidermal keratinocyte differentiation. MafB is expressed in postmitotic differentiating keratinocytes, and epidermal differentiation is partially impaired in MafB-deficient mice. To further establish the roles of MafB in the epidermis in vivo, we generated mice transgenic for MafB under the control of the basal cell-specific keratin (Krt) 14 promoter. In the epidermis of transgenic mice at embryonic day 18.5, the number of proliferating Krt14-positive basal-like cells was increased, and the granular and cornified layers were thickened. Furthermore, these MafB transgenic mice developed papillomas spontaneously with age. Therefore, MafB promotes differentiation in postmitotic keratinocytes and simultaneously has potential to promote growth when ectopically expressed in undifferentiated basal keratinocytes.

Multicolor immunofluorescence reveals that p63- and/or K5-positive progenitor cells contribute to normal breast epithelium and usual ductal hyperplasia but not to low-grade intraepithelial neoplasia of the breast.

We contend that knowledge about the cellular composition of normal breast epithelium is a prerequisite for understanding proliferative breast disease. Against this background, we used multicolor immunofluorescence to study normal breast epithelium and two types of intraepithelial proliferative breast lesion for expression of the p63, basal keratin K5, glandular keratin K8/18, SMA, ER-alpha, and Ki67. We studied eight normal breast epithelium samples, 12 cases of usual ductal hyperplasia, and 33 cases of low-grade intraepithelial neoplasia (9 flat epithelial atypia, 14 low-grade ductal carcinoma in situ and 10 cases of lobular neoplasia). Usual ductal hyperplasia showed striking similarity to normal luminal breast epithelium including p63+ and/or K5+ luminal progenitor cells and the full spectrum of luminal progeny cells. In normal breast epithelium and usual ductal hyperplasia, expression of ER-alpha was associated with lack of expression of the proliferation antigen Ki67. In contrast, we found in both types of low-grade intraepithelial neoplasia robust expression of keratin K8/18 and a positive association between ER-alpha and Ki67 expression. However, these lesions were consistently negative for p63 and/or K5. Our observational study supports the view that usual ductal hyperplasia and low-grade intraepithelial neoplasia are different entities rather than part of a spectrum of the same disease. We propose a new operational model of cell differentiation that may serve to better understand correlations between normal breast epithelium and proliferative breast diseases. From our data we conclude that p63+ and/or K5+ progenitor cells contribute to maintenance of normal epithelium and usual ductal hyperplasia, but not to low-grade intraepithelial neoplasia of the breast.

Retinoic acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium.

Retinoic acid (RA), the active metabolite of vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified. Here, we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and nonneuronal mesenchyme. By culturing epithelium explants in isolation from other tissues, we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling. RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs. Developmental Dynamics 246:135-147, 2017. © 2016 Wiley Periodicals, Inc.

Developmental expression of Toll‑like receptors in the guinea pig lung.

The guinea pig is a useful model for investigating infectious and non-infectious lung diseases due to the sensitivity of its respiratory system and susceptibility to infectious agents. Toll-like receptors (TLRs) are important components of the innate immune response and are critical for lung immune function. In the present study, the differentiation of epithelial cells in the guinea pig lung was examined during gestation by studying anatomic morphology and the major epithelial cell types using cell type-specific markers. The developmental expression of all 9 TLRs and the TLR signaling adaptors myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor associated factor 6 (TRAF-6) were investigated by reverse transcription-quantitative polymerase chain reaction and western blotting analysis. The formation of lung lobes in guinea pigs was observed at 45 days of gestation (dGA), along with the expression of the basal cell marker keratin 14 and the alveolar type II cell marker pro-surfactant protein. However, the cube cell marker secretoglobin family1A member 1 and ciliated cell marker b-tubulin IV were only detected in the lungs from 52 dGA onward. The expression levels of all TLRs, MyD88 and TRAF-6 were determined in lung tissues harvested from embryos, newborn, postnatal and adult animals. The expression levels of all TLR signaling components displayed similar dynamic expression patterns with gestation age and postnatal maturation time, except for TLR-4 and TLR-7. mRNA expression levels of TLR components were significantly increased in the lungs at 45 and 52 dGA, compared with later developmental stages. These results suggest that TLR expression in the guinea pig lung is developmentally regulated, enhancing the understanding of lung biology in guinea pig models.

Proteomic Analysis of Primary Human Airway Epithelial Cells Exposed to the Respiratory Toxicant Diacetyl.

Occupational exposures to the diketone flavoring agent, diacetyl, have been associated with bronchiolitis obliterans, a rare condition of airway fibrosis. Model studies in rodents have suggested that the airway epithelium is a major site of diacetyl toxicity, but the effects of diacetyl exposure upon the human airway epithelium are poorly characterized. Here we performed quantitative LC-MS/MS-based proteomics to study the effects of repeated diacetyl vapor exposures on 3D organotypic cultures of human primary tracheobronchial epithelial cells. Using a label-free approach, we quantified approximately 3400 proteins and 5700 phosphopeptides in cell lysates across four independent donors. Altered expression of proteins and phosphopeptides were suggestive of loss of cilia and increased squamous differentiation in diacetyl-exposed cells. These phenomena were confirmed by immunofluorescence staining of culture cross sections. Hyperphosphorylation and cross-linking of basal cell keratins were also observed in diacetyl-treated cells, and we used parallel reaction monitoring to confidently localize and quantify previously uncharacterized sites of phosphorylation in keratin 6. Collectively, these data identify numerous molecular changes in the epithelium that may be important to the pathogenesis of flavoring-induced bronchiolitis obliterans. More generally, this study highlights the utility of quantitative proteomics for the study of in vitro models of airway injury and disease.

CXCL11 Expression by Keratinocytes Occurs Transiently Between Reaching Confluence and Cellular Compaction.

Objective: To investigate whether differentiation or cellular confluence is responsible for CXCL11 expression patterns in re-epithelialization. Approach:In vitro model systems of re-epithelialization using the HaCaT keratinocyte cell line were utilized in monitoring expression of differentiation markers, including desmoplakin and various cytokeratins while evaluating for an association with chemokine CXCL11 expression. Results: CXCL11 expression was elevated in sparse culture with peak expression near the time of confluence. This somewhat followed the accumulation of desmoplakin in detergent-insoluble pool of proteins. However, in postconfluent, despite continued accumulation of desmoplakin within cells, CXCL11 expression decreased to baseline levels. This biphasic pattern was also seen in low calcium culture, an environment that inhibits keratinocyte differentiation and accumulation of desmosomal proteins. Highest CXCL11-expressing areas best correlated with newly confluent areas within culture expressing basal keratin 14, but also activated keratin 6. Innovation: Achievement of a threshold cellular density induces cell signaling cascade through CXCR3 that, in addition to other undiscovered pathways, can progress cutaneous wounds from the proliferative into the remodeling phases of cutaneous wound healing. Conclusion: These results suggest that the achievement of confluence with increased cellular density by migrating keratinocytes at the wound edge triggers expression of CXCL11. Since CXCR3 stimulation in endothelial cells results in apoptosis and causes neovascular pruning, whereas stimulation of CXCR3 in fibroblasts results decreased motility and cellular contraction, we speculate that CXCL11 expression by epidermal cells upon achieving cellular confluence could be the source of CXCR3 stimulation in the dermis ushering a transition from proliferative to remodeling phases of wound healing.

The ErbB2ΔEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment.

Amplification and over expression of erbB2/neu proto-oncogene is observed in 20–30% human breast cancer and is inversely correlated with the survival of the patient. Despite this, somatic activating mutations within erbB2 in human breast cancers are rare. However, we have previously reported that a splice isoform of erbB2, containing an in-frame deletion of exon 16 (herein referred to as ErbB2ΔEx16), results in oncogenic activation of erbB2 due to constitutive dimerization of the ErbB2 receptor. Here, we demonstrate that the ErbB2ΔEx16 is a major oncogenic driver in breast cancer that constitutively signals from the cell surface. We further show that inducible expression of the ErbB2Ex16 variant in mammary gland of transgenic mice results in the rapid development of metastatic multifocal mammary tumors. Genetic and biochemical characterization of the ErbB2ΔEx16 derived mammary tumors exhibit several unique features that distinguish it from the conventional ErbB2 models expressing the erbB2 proto-oncogene in mammary epithelium. Unlike the wild-type ErbB2 derived tumors that express luminal keratins, ErbB2ΔEx16 derived tumors exhibit high degree of intra-tumoral heterogeneity co-expressing both basal and luminal keratins. Consistent with these distinct pathological features, the ErbB2ΔEx16 tumors exhibited distinct signaling and gene expression profiles that correlated with activation of number of key transcription factors implicated in breast cancer metastasis and cancer stem cell renewal.

Analysis of immunohistochemical characteristics of lymphoid stroma and epithelium of the salivary gland Warthin tumor

The aim of our study was to determine using immunohistochemical method profi and relationship between lymphocytes in lymphoid rich stroma of the Warhins tumors, as well as, the relationship between lymphocytes and tumors epithelial cells. Our retrospective-prospective study we conducted in the Department of Clinical Pathology KBC Zemun on 25 samples of tissue from the parotid gland with WT tumor surgically removed at the Department of ENT KBC Zemun in the period from 2000 to 2016 year. Sections of tumor tissue, fixed in 10% formalin were stained with hematoxylin-eosin standard method, and then immunohistochemical method to: antibody B lymphocytes (CD20) antibody for T lymphocytes (CD3), keratin antibody that marks the basal and columnar epithelial cells (CK7 ) and keratin antibody that marks only the basal epithelial cells (CK5 / 6). We have found that in relation to the gender distribution of all 25 patients, there were more male patients: 16 (64%) men and 9 (36%) women. Immunohistochemical analysis with keratin antibody CK7 showed that luminal columnar cells intensely positive (+++) more than 75% in 23 cases (92%), whereas in 2 cases were intensely positive (+++) in percentages ranging from 25 to 75%. In contrast to the luminal, basal cells were always showed little immunoreactivity (+) antibody to CK7 and in 20 cases in the group of <25% of the cells respectively in the cases of 5% in the group 25-75. Evaluation of the presence, intensity, type and distribution of cells with each other and in relation to the tumor epithelial component would contribute to establishing unclear histogenesis lymphoid stroma at the tumor.