Supplementary Materials and Figures 1-3 from p130Cas as a New Regulator of Mammary Epithelial Cell Proliferation, Survival, and HER2-Neu Oncogene–Dependent Breast Tumorigenesis

Introduction: The presence of positive correlations between the levels of oxalates in the bronchoalveolar lavage fluid and the severity of obstructive disorders was revealed. According to the authors, this indicates the potential role of poorly soluble oxalate fractions in the formation of bronchial obstruction and exfoliation of the bronchial epithelium. Next, Tao, J.L. et al. (Zhongguo Zhong Xi Yi Jie He Za Zhi 2017;37(3):319-325) found decrease of the content of oxalic acid in urine in children with bronchial asthma. Aims and objectives: To assess the daily urinary excretion of oxalates in children with allergic airway diseases (AAD). Methods: We examined 100 children aged 2 to 9 years, boys - 22% (22/100), girls - 78% (78/100) with symptoms of crystalluria. The children were divided into the main group (42 children) and the comparison group (58 people). The main group included children with established diagnoses of AAD, the comparison group included patients who did not have AAD. In addition to the general clinical examination, all patients underwent a biochemical study of daily urine. Results: It was found that the daily excretion of oxalates in patients with AAD is statistically significantly higher than in the comparison group, 26.5 [22.1; 32.6] mg / day and 23.3 [20.1; 27.6] mg / day, respectively, p = 0.017. 63 % of all patients have also high oxalate/creatinin ratio. Conclusions: The clinical and pathogenetic significance of higher oxalate excretion in children with AAD requires further study. Oxalates are end stage of the metabolism of the connective tissue. And connective tissue disorders can play role in pathogenesis of bronchial asthma.

Introduction: Hypertrophic and polyposis SM changes in patients with BA and AR negatively affect the course of these diseases. The features of systemic immune regulation in these patients remain unknown. Purpose of the study: To study the relationship of hypertrophic and polyposis SM changes with the content of serum immunoglobulins A, M, G and E. Methods: We examined 137 patients with atopic BA and AR at the age of 10.0 [7.0; 13.0] years, of which boys - 75.2% (103/137). Determination of the content of total IgE, as well as IgA, IgM, IgG in the blood serum was carried out by the enzyme-linked immunosorbent method. Results: In 19.7% (27/137) children, SM changes were revealed in the form of local hypertrophy of the medial surface of the turbinates, in 10.9% (15/137) polyposis SM changes. The content of serum total IgE was comparable in children with the absence and presence of synonasal hypertrophy (p = 0.65), as well as in serum Ig M (p = 0.74). At the same time, the serum IgA content in children with synonasal hypertrophy unexpectedly turned out to be statistically significantly higher (p = 0.024), and the serum IgG content had a clear tendency to increase (p = 0.053) compared with children who did not have hypertrophic SM changes. Conclusions: Children with hypertrophic and polyposis changes in SM showed a statistically significantly higher level of total serum IgA compared with children without SM hypertrophy. Apparently, BA and AR patients with hypertrophic SM changes have a unique systemic immunological profile that requires serious study.

Introduction: Th2-dependent allergic inflammation in patients with BA and AR can be associated with nasal mucosa remodeling, accompanied by hypertrophic and hyperplastic processes. TSLP is a key cytokine produced by epithelial cells and is actively involved in allergic inflammation. Purpose of the study: To study TSLP content in nasal secretion of children with atopic BA and AR. Methods: We examined 43 patients with atopic BA and AR at the age of 8,11 [7,0; 13,0] years, of which boys 67% (29/43), girls – 33% (14/43), nasal mucosa state was assessed using rhino-endoscopy and TSLP content in nasal secretion by the enzyme immunoassay by the test-sistem Human TSLP Quantikine ELISA Kit (R&D Systems, Inc.) Results: Hypertrophic or polypouschanges in the nasal mucosa and/or paranasal sinuses were detected in 30% (13/43) of patients. TSLP content in the nasal secretion in these patients was 908,5 [48,9; 2098,7] pg/mg, which is statistically significantly higher than in patients with BA and AR without sinonasal hypertrophy (132,3 [4,2; 325,9] pg/mg, p=0,036). AR exacerbation was accompanied by a statistically significant increase of TSLP content in the nasal secret compared with the remission period (p=0,002). Conclusion: Hypertrophic changes in the nasal mucosa are accompanied by a statistically significant increase of TSLP content in the nasal secret in children with atopic BA and AR, which allows to consider this biomarker as a potential non-invasive predictor of pathological airway remodeling in patients with chronic allergic airway inflammation.

Background: Currently, the relationship between bronchial obstruction and ECHO parameters in children with BA is not well understood. Aims and Objectives: To identify the relationship of ECHO and spirometric parameters in children with BA. Methods: 60 patients with uncontrolled BA were examined: 73.3% boys, 26.7% girls; mean age 9.48 ± 1.03 years. Children underwent spirometric examination, include forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), maximum expiratory flow at the point of 25%, 50% and 75% loop flow-volume (MEF 25%, 50%, 75%) and transthoracic echocardiography. Results: Patients were divided into 2 groups: 1- no ECHO abnormalites of the heart structures to the pulmonary circulation (EF HSPC: patent foramen ovale, tricuspid valve regurgitation) - 36.7%, 2- with the EF HSPC - 63.3% of children. We established a strong correlation between FVC and left ventricle end-diastolic diameter (LVED) - group 1 r=0.87, group 2 r=0.86, p = 0.0 and left ventricle end-systolic diameter (LVESD)- group 1 r=0.66, group 2 r=0.62, p <0.05); also FEV1 and LVED (1 group r=0.86, 2 group r=0.74, p = 0.0) and LVESD (1 group r=0.65, 2 group r=0.52, p <0.05). Analizez of the patency from large (MEF 75%) to small airways (MEF 25%) reveal a gradual weakening of the correlation relationships of the LVED and the spirometric parameters (from 0.65, p = 0.0 to 0, 41, p> 0.05). Conclusion: Significant correlation between of spirometric parameters and LVED and LVESD have been established in children with and without EF HSPC. This may have a negative effect on bronchial patency in children with a lack of BA control.

One of the common stages of technological processing of products is refining. In the refining process, socalled "ballast substances" are removed from the initial product. As a rule, vitamins, microelements, amino acids and other useful biologically active substances - antioxidants get into the ballast, and food is loaded with trans-fats, saturated fatty acids, etc. The purpose of the work was to assess the impact of nutrition features on some indicators of free radical status in EBC. The study involved young people (20 students) aged 22 to 35 years. During the experiment, the collection of EBC from patients took place several times: after a diet without refined foods and after a diet with a large number of refined foods. In the course of the work, the determination of the total concentration of nitrates/nitrites, as NO metabolites, and the level of iron in EBC was carried out. Antioxidant activity was assessed by catalase activity in EBC. As a result of this work, it was found that a diet with a large number of refined products leads to an increase in the body9s SRO processes (the concentration of nitrates / nitrites increases 3 times - from 2.91 ± 0.23 μM to 8.87 ± 0.32 μM ( P <0.05), and the concentration of iron is 2 times (from 1.29 ± 0.1 μM to 2.74 ± 0.16 μM (P<0.05)). Increased catalase activity in EBC with 0.161 ± 0.119 units. to 0,243 ± 0,019 units. (P <0.05) speaks of the activation and antioxidant system. Thus, the use of a large number of refined, processed products increases the production of free radicals, which is reflected in the performance of EBC. At the same time, an increase in the antioxidant indices of EBC occurs.

Integrin dimers α3/β1, α6/β1 and α6/β4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated by the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice, however myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins and underline an essential role of cell interactions with laminin for lactogenic differentiation.

Integrins, which bind laminin, a major component of the mammary basement membrane, are strongly expressed in basal stem cell-enriched populations, but their role in controlling mammary stem cell function remains unclear. We found that stem cell activity, as evaluated in transplantation and mammosphere assays, was reduced in mammary basal cells depleted of laminin receptors containing α3- and α6-integrin subunits. This was accompanied by low MDM2 levels, p53 stabilization, and diminished proliferative capacity. Importantly, disruption of p53 function restored the clonogenicity of α3/α6-integrin-depleted mammary basal stem cells, while inhibition of RHO or myosin II, leading to decreased p53 activity, rescued the mammosphere formation. These data suggest that α3/α6-integrin-mediated adhesion plays an essential role in controlling the proliferative potential of mammary basal stem/progenitor cells through myosin II-mediated regulation of p53 and indicate that laminins might be important components of the mammary stem cell niche.

The adult mammary epithelium is composed of basal and luminal cells. The luminal lineage comprises two major cell populations, positive and negative for estrogen and progesterone receptors (ER and PR, respectively), both containing clonogenic progenitor cells. Deregulated ER/PR− luminal progenitor cells are suspected to be at the origin of basal-type triple-negative (TNBC) breast cancers, a subtype frequently associated with loss of P53 function and MET signaling hyperactivation. Using mouse models, we recently reported that p53 restricts luminal progenitor cell amplification whereas paracrine Met activation stimulates their growth and favors a luminal-to-basal switch. Here, we analyzed how these two critical pathways interact to control luminal progenitor function. We have (i) established and analyzed the gene expression profile of luminal progenitors isolated by ICAM-1, a robust surface marker we previously identified; (ii) purified luminal progenitors from p53-deficient and p53-proficient mouse mammary epithelium to compare their functional and molecular characteristics; and (iii) analyzed their response to HGF, the major Met ligand, in three-dimensional cultures. We found that luminal progenitors, compared to non-clonogenic luminal cells, overexpress Trp53 and numerous p53 target genes. In vivo, loss of Trp53 induced the expansion of luminal progenitors, affecting expression of several important p53 target genes including those encoding negative regulators of cell cycle progression. Consistently, p53-deficient luminal progenitors displayed increased proliferative and self-renewal activities in culture. However, they did not exhibit perturbed expression of luminal-specific markers and major regulators, such as Hey1, Elf5, and Gata3. Moreover, although expressing Met at higher level than p53-proficient luminal progenitors, p53-deficient luminal progenitors failed to acquire basal-specific features when stimulated by HGF, showing that p53 promotes the plastic behavior of luminal progenitors downstream of Met activation. Our study reveals a crosstalk between Met- and p53-mediated signaling pathways in the regulation of luminal progenitor function. In particular, it shows that neither p53 loss alone nor p53 loss combined with Met signaling activation caused an early detectable cell fate alteration in luminal progenitors. Conceivably, additional events are required to confer basal-specific characteristics to luminal-derived TNBCs.

Stem cells (SCs) drive mammary development, giving rise postnatally to an epithelial bilayer composed of luminal and basal myoepithelial cells. Dysregulation of SCs is thought to be at the origin of certain breast cancers; however, the molecular identity of SCs and the factors regulating their function remain poorly defined. We identified the transmembrane protein podoplanin (Pdpn) as a specific marker of the basal compartment, including multipotent SCs, and found Pdpn localized at the basal-luminal interface. Embryonic deletion of Pdpn targeted to basal cells diminished basal and luminal SC activity and affected the expression of several Wnt/β-catenin signaling components in basal cells. Moreover, Pdpn loss attenuated mammary tumor formation in a mouse model of β-catenin-induced breast cancer, limiting tumor-initiating cell expansion and promoting molecular features associated with mesenchymal-to-epithelial cell transition. In line with the loss-of-function data, we demonstrated that mechanistically Pdpn enhances Wnt/β-catenin signaling in mammary basal cells. Overall, this study uncovers a role for Pdpn in mammary SC function and, importantly, identifies Pdpn as a new regulator of Wnt/β-catenin signaling, a key pathway in mammary development and tumorigenesis.

The aim was evaluated the total nitrate/nitrite (NO3-/ NO2-) concentration and level of malonic dialdehyde (MDA) in the exhaled breath condensate (EBC). It also evaluated the activity of antioxidant (AO) enzymes (SOD and catalase). The test group consisted of males, ages 37 to 69 years. All patients experienced an exacerbation of COPD II. The control group consisted of relatively healthy men. The study found that exacerbation of COPD II is associated with increased formation of free radicals and their derivatives (NO and MDA), which is reflected in an increase in the NO3-/ NO2- index by 2.16 times (P <0.05) and MDA by 3.83 times (P <0, 05) both in case of exacerbation of the disease and after treatment, these indicators are higher than those of healthy people (NO3-/ NO2- 2.16 times, MDA 3.16 times (P <0.05). The AO system (catalase enzymes and SOD) is activated in response on the increase in the number of free radicals in COPD. The activity of AO enzymes is maintained at a higher level, compared with healthy subjects, and after treatment (SOD at 1.05 times and catalase in 1.43 times (P <0.05). However, an increase in the activity of SOD occurs to an insufficient degree in comparison with the intensification of oxidative processes. The activity of SOD in EBC during exacerbation in COPD patients increases only 1.23 times in comparison with the control group of practically healthy people, while free radicals increase NO3-/ NO2- by 2.16 times and MDA 3.83 times. This indicates the lack of antioxidant protection in patients with chronic disease. Thus, exacerbation of COPD is accompanied by an intensification of both free radical and antioxidant processes, but to varying degrees

Bronchial asthma (BA) is associated with upper respiratory tract pathology. In recent years, attention has been focused on the multimorbidity of allergic diseases, but the spectrum of upper respiratory tract pathology in children with BA is not characterized enough. The study aim: to investigate the structure of the pathology of the upper respiratory tract in children with BA. We examined 358 children with BA, 9.91 [9.47; 10.35] years, of them boys - 67.9% (192/358) and 108 children comparable by age and sex, with complaints of nasal breathing, but not having BA. In addition to the standard all-clinical, allergological, functional examination, all patients underwent video endoscopic examination of the nasal cavity and nasopharynx. Results: All children with asthma were diagnosed with allergic rhinitis (AR) and / or allergic rhinosinusitis (ARS) with a predominance of persistent forms. The "isolated" course of AR/ARS occurred in 11.7% (42/358) of patients, the other children had nasal symptoms due to a combination of AR/ARS with other variants of the pathology of upper respiratory tract. Hypertrophy of the pharyngeal tonsil occurred in 61.2% (219/358) patients, hypertrophic rhinitis in 9.2% (33/358) of children with BA, anomalies of the architectonics of the nose were detected in 50.0% (179/358) patients. The combination of two nosological variants of nose pathology occurred in 47.8% (117/358) of patients with BA, 40.5% (145/358) of children with BA reported multimorbidity of the nasal pathology - a combination of three or more nosological units. Conclusion: Children with BA and nasal symptoms are characterized by combined and multimorbid forms of upper respiratory tract pathology.

The capacity of mammary myoepithelial cells to contract in response to suckling stimuli is essential for lactation. We describe here a protocol for studying the contractile activity of myoepithelial cells in vitro. This protocol includes the establishment of stable myoepithelial cell lines from mouse mammary glands and quantitative evaluation of the contraction and subsequent relaxation of cultured myoepithelial cells in response to oxytocin. It can be used for analyses of mouse mutants with gene deletions or overexpression altering myoepithelial cell function.

HGF/Met signaling has recently been associated with basal-type breast cancers, which are thought to originate from progenitor cells residing in the luminal compartment of the mammary epithelium. We found that ICAM-1 efficiently marks mammary luminal progenitors comprising hormone receptor-positive and receptor-negative cells, presumably ductal and alveolar progenitors. Both cell populations strongly express Met, while HGF is produced by stromal and basal myoepithelial cells. We show that persistent HGF treatment stimulates the clonogenic activity of ICAM1-positive luminal progenitors, controlling their survival and proliferation, and leads to the expression of basal cell characteristics, including stem cell potential. This is accompanied by the induction of Snai1 and Snai2, two major transcription factors triggering epithelial-mesenchymal transition, the repression of the luminal-regulatory genes Elf5 and Hey1, and claudin down-regulation. Our data strongly indicate that paracrine Met signaling can control the function of luminal progenitors and modulate their fate during mammary development and tumorigenesis.

In this issue, Tosoni et al. (2015. J. Cell Biol. hitp://dx.doi.org/10.1083/jcb.201505037) report that cell fate determinant and tumor suppressor Numb imposes asymmetric cell divisions in mammary stem cells by regulating p53. Numb thereby restricts mammary stem cell expansion and controls the proliferation and lineage-specific characteristics of their progeny.

The transition of ductal carcinoma in situ (DCIS) to invasive breast carcinoma requires tumor cells to cross the basement membrane (BM). However, mechanisms underlying BM transmigration are poorly understood. Here, we report that expression of membrane-type 1 (MT1)-matrix metalloproteinase (MMP), a key component of the BM invasion program, increases during breast cancer progression at the in situ to invasive breast carcinoma transition. In the intraductal xenograft model, MT1-MMP is required for BM transmigration of MCF10DCIS.com breast adenocarcinoma cells and is overexpressed in cell clusters overlying focal BM disruptions and at the invasive tumor front. Mirrored upregulation of p63 and MT1-MMP is observed at the edge of MCF10DCIS.com xenograft tumors and p63 is required for induction of MT1-MMP-dependent invasive program in response to microenvironmental signals. Immunohistochemistry and analysis of public database reveal that p63 and MT1-MMP are upregulated in human basal-like breast tumors suggesting that p63/MT1-MMP axis contributes to progression of basal-like breast cancers with elevated p63 and MT1-MMP levels.

The transplantation of mammary epithelial cells into the cleared fat pad allows their growth and differentiation in their normal physiological environment. This technique involves the grafting of tissue fragments or isolated cells into the mammary fat pads of prepubertal mice from which the endogenous epithelium has been surgically removed. Such transplantation assays are particularly useful for the analysis of morphogenetic potential and stem cell activity in normal mammary epithelium and breast tumors. We describe here the main steps in the transplantation of epithelial fragments and isolated cells from mouse mammary glands and the various approaches currently used to evaluate the regeneration and self-renewal properties of mammary stem cells.

The seventh annual meeting of the European Network of Breast Development and Cancer Laboratories, held in Weggis, Switzerland, in April 2015, was focused on techniques for the study of normal and cancer stem cells, cell fate decisions, cancer initiation and progression.

In this issue, Tosoni et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505037) report that cell fate determinant and tumor suppressor Numb imposes asymmetric cell divisions in mammary stem cells by regulating p53. Numb thereby restricts mammary stem cell expansion and controls the proliferation and lineage-specific characteristics of their progeny.