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Background & Aims: The class I‐ phosphatidylinositol‐3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform‐specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation. Approach & Results: Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT‐qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ‐overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8‐based‐PI3Kδ overexpression. These PI3Kδ‐mediated cell morphogenesis and stroma remodelling were dependent on TGFβ/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression. Conclusions: Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFβ/src/Notch‐dependent mechanisms and open new directions for the classification and treatment of CCA patients. [ABSTRACT FROM AUTHOR]

The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T‐cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)‐cell fate in T cells. In addition, T cells upregulate the NK cell‐associated receptors and transcription factors, lyse NK‐cell targets, and are reprogrammed into NK‐like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1‐mediated elevated OXPHOS enhances cellular acetyl‐CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T‐cell fate directly by repressing NK cell‐associated transcription and indirectly through a metabolic‐epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs. Synopsis: While BCL11b is known to bind the NuRD complex and be required for T cell development, the function of these factors in mature T cells has so far been unclear. This work demonstrates how the NuRD complex and BCL11B together maintain T‐cells from reprogramming into NK cells. T cells are reprogrammed into NK‐like cells upon deletion of MTA2, MBD2, or CHD4.BCL11B associates with the NuRD complex to repress OPA1 expression in T cells.OPA1‐mediated mitochondrial fusion facilitates ITNK reprogramming and cytotoxicity by increasing OXPHOS.Acetyl‐CoA elevated by OXPHOS promotes NK‐cell‐associated genes expression and antitumor effects in ITNKs by regulating H3K27 acetylation. [ABSTRACT FROM AUTHOR]

Frizzled family receptor 7 (FZD7), a Wnt signaling receptor, is associated with the maintenance of stem cell properties and cancer progression. FZD7 has emerged as a potential therapeutic target because it is capable of transducing both canonical and noncanonical Wnt signals. In this study, we investigated the regulatory pathway downstream of FZD7 and its functional roles. We found that FZD7 expression was crucial to the maintenance of the mesenchymal phenotype, anoikis resistance, and spheroid and tumor formation in ovarian cancer (OC). We identified TWIST1 as the crucial downstream effector of the FZD7 pathway. TWIST1, a basic helix loop helix transcription factor, is known to associate with mesenchymal and cancer stem cell phenotypes. Manipulating TWIST1 expression mimicked the functional consequences observed in the FZD7 model, and overexpression of TWIST1 partially rescued the functional phenotypes abolished by FZD7 knockdown. We further proved that FZD7 regulated TWIST1 expression through epigenetic modifications of H3K4me3 and H3K27ac at the TWIST1 proximal promoter. We also identified that the FZD7‐TWIST1 axis regulates the expression of BCL2, a gene that controls apoptosis. Identification of this FZD7‐TWIST1‐BCL2 pathway reaffirms the mechanism of anoikis resistance in OC. We subsequently showed that the FZD7‐TWIST1 axis can be targeted by using a small molecule inhibitor of porcupine, an enzyme essential for secretion and functional activation of Wnts. In conclusion, our results identified that the FZD7‐TWIST1 axis is important for tumorigenesis and anoikis resistance, and therapeutic inhibition results in cell death in OCs. Frizzled family receptor 7 (FZD7) expression maintains the mesenchymal phenotype and anchorage independence via epigenetic regulation of TWIST1. Knockdown of FZD7 results in inhibition of TWIST1 with consequent downregulation of epithelial to mesenchymal transition and BCL2 leading to anoikis and loss of tumorigenesis. Our results demonstrate that the FZD7‐TWIST1 axis is important for tumorigenesis and anoikis in ovarian cancer. [ABSTRACT FROM AUTHOR]

Tumor biopsy is the gold standard for the assessment of clinical biomarkers for treatment. However, tumors change dynamically in response to therapy, and there remains a need for a more representative biomarker that can be assayed over the course of treatment. Circulating tumor cells (CTCs) may provide clinically important and comprehensive tumoral information that is predictive of treatment response and outcome. Blood samples were processed for CTCs from 56 patients using the ClearCell FX system. Captured cells were phenotyped for CTC clusters and markers for immunotherapy (PD‐L1) CTC chromosomal architecture (ALK, EGFR). CTCs were isolated in 11/23 (47.8%) of head and neck cancer (HNC) patients and 17/33 (51.5%) of non‐small‐cell lung cancer (NSCLC) patients. CTCs were determined to be PD‐L1‐positive in 6/11 (54.4%) HNC and 11/17 (64.7%) NSCLC cases, respectively. 3D chromosomal DNA FISH for ALK and EGFR molecular targets showed better resolution than in 2D when imaging CTCs. HNC CTC‐positive patients had shorter progression‐free survival (PFS) (hazard ratio[HR]: 4.946; 95% confidence internal[CI]:1.571‐15.57; P = 0.0063), and PD‐L1‐positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011‐26.33; P = 0.0485). In the advanced stage NSCLC patient cohort, PFS was not found to be associated with CTCs prior to therapy ([HR]:2.246; 95% [CI]:0.9565‐5.273; P = 0.0632), nor the presence of PD‐L1 expression ([HR]:1.646; 95% [CI]:0.5128‐5.283; P = 0.4023). This study demonstrated that CTCs are predictive of poorer outcomes in HNC and provides distinct and separate utility for CTCs in HNC and NSCLC, which may be more representative of the disease burden and overall survival than the parameters used to measure them. 3D DNA FISH of circulating tumor cells demonstrates a higher resolution for genomic aberrations compared to 2D imaging modalities. [ABSTRACT FROM AUTHOR]

Abstract: In ovarian cancer, the prometastatic RTK AXL promotes motility, invasion and poor prognosis. Here, we show that reduced survival caused by AXL overexpression can be mitigated by the expression of the GPI‐anchored tumour suppressor OPCML. Further, we demonstrate that AXL directly interacts with OPCML, preferentially so when AXL is activated by its ligand Gas6. As a consequence, AXL accumulates in cholesterol‐rich lipid domains, where OPCML resides. Here, phospho‐AXL is brought in proximity to the lipid domain‐restricted phosphatase PTPRG, which de‐phosphorylates the RTK/ligand complex. This prevents AXL‐mediated transactivation of other RTKs (cMET and EGFR), thereby inhibiting sustained phospho‐ERK signalling, induction of the EMT transcription factor Slug, cell migration and invasion. From a translational perspective, we show that OPCML enhances the effect of the phase II AXL inhibitor R428 in vitro and in vivo. We therefore identify a novel mechanism by which two spatially restricted tumour suppressors, OPCML and PTPRG, coordinate to repress AXL‐dependent oncogenic signalling. [ABSTRACT FROM AUTHOR]

Novel immunotherapy approaches have provided durable remission in a significant number of cancer patients with cancers previously considered rapidly lethal. Nonetheless, the high degree of nonresponders, and in some cases the emergence of resistance in patients who do initially respond, represents a significant challenge in the field of cancer immunotherapy. These issues prompt much more extensive studies to better understand how cancer cells escape immune surveillance and resist immune attacks. Here, we review the current knowledge of how cellular heterogeneity and plasticity could be involved in shaping the tumor microenvironment (TME) and in controlling antitumor immunity. Indeed, recent findings have led to increased interest in the mechanisms by which cancer cells undergoing epithelial-mesenchymal transition ( EMT), or oscillating within the EMT spectrum, might contribute to immune escape through multiple routes. This includes shaping of the TME and decreased susceptibility to immune effector cells. Although much remains to be learned on the mechanisms at play, cancer cell clones with mesenchymal features emerging from the TME seem to be primed to face immune attacks by specialized killer cells of the immune system, the natural killer cells, and the cytotoxic T lymphocytes. Recent studies investigating patient tumors have suggested EMT as a candidate predictive marker to be explored for immunotherapy outcome. Promising data also exist on the potential utility of targeting these cancer cell populations to at least partly overcome such resistance. Research is now underway which may lead to considerable progress in optimization of treatments. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) represents the most common type of kidney cancer with high mortality in its advanced stages. Our study aim was to explore the correlation between tumor epithelial-to-mesenchymal transition (EMT) and patient survival. Renal biopsies of tumorous and adjacent nontumorous tissue were taken with a 16 g needle from our patients (n = 26) undergoing partial or radical nephrectomy due to ccRCC. RNA sequencing libraries were generated using Illumina TruSeq® Access library preparation protocol and TruSeq Small RNA library preparation kit. Next generation sequencing (NGS) was performed on Illumina HiSeq2500. Comparative analysis of matched sample pairs was done using the Bioconductor Limma/voom R-package. Liquid chromatography-tandem mass spectrometry and immunohistochemistry were applied to measure and visualize protein abundance. We detected an increased generic EMT transcript score in ccRCC. Gene expression analysis showed augmented abundance of AXL and MMP14, as well as downregulated expression of KL (klotho). Moreover, microRNA analyses demonstrated a positive expression correlation of miR-34a and its targets MMP14 and AXL. Survival analysis based on a subset of genes from our list EMTrelated genes in a publicly available dataset showed that the EMT genes correlated with ccRCC patient survival. Several of these genes also play a known role in fibrosis. Accordingly, recently published classifiers of solid organ fibrosis correctly identified EMT-affected tumor samples and were correlated with patient survival. EMT in ccRCC linked to fibrosis is associated with worse survival and may represent a target for novel therapeutic interventions. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition ( EMT) is a reversible and dynamic process hypothesized to be co-opted by carcinoma during invasion and metastasis. Yet, there is still no quantitative measure to assess the interplay between EMT and cancer progression. Here, we derived a method for universal EMT scoring from cancer-specific transcriptomic EMT signatures of ovarian, breast, bladder, lung, colorectal and gastric cancers. We show that EMT scoring exhibits good correlation with previously published, cancer-specific EMT signatures. This universal and quantitative EMT scoring was used to establish an EMT spectrum across various cancers, with good correlation noted between cell lines and tumours. We show correlations between EMT and poorer disease-free survival in ovarian and colorectal, but not breast, carcinomas, despite previous notions. Importantly, we found distinct responses between epithelial- and mesenchymal-like ovarian cancers to therapeutic regimes administered with or without paclitaxel in vivo and demonstrated that mesenchymal-like tumours do not always show resistance to chemotherapy. EMT scoring is thus a promising, versatile tool for the objective and systematic investigation of EMT roles and dynamics in cancer progression, treatment response and survival. [ABSTRACT FROM AUTHOR]

High-grade serous ovarian carcinoma (HG-SOC) is a heterogeneous, poorly classified, lethal disease that frequently exhibits altered expressions of microRNAs. Let-7 family members are often reported as tumor suppressors; nonetheless, clinicopathological functions and prognostic values of individual let-7 family members have not been addressed in HG-SOC. In our work, we performed an integrative study to investigate the potential roles, clinicopathological functions and prognostic values of let-7 miRNA family in HG-SOC. Using microarray and clinical data of 1,170 HG-SOC patients, we developed novel survival prediction and system biology methods to analyze prognostic values and functional associations of let-7 miRNAs with global transcriptome and clinicopathological factors. We demonstrated that individual let-7 members exhibit diverse evolutionary history and distinct regulatory characteristics. Statistical tests and network analysis suggest that let-7b could act as a global synergistic interactor and master regulator controlling hundreds of protein-coding genes. The elevated expression of let-7b is associated with poor survival rates, which suggests an unfavorable role of let-7b in treatment response for HG-SOC patients. A novel let-7b-defined 36-gene prognostic survival signature outperforms many clinicopathological parameters, and stratifies HG-SOC patients into three high-confidence, reproducible, clinical subclasses: low-, intermediate- and high-risk, with 5-year overall survival rates of 56-71%, 12-29% and 0-10%, respectively. Furthermore, the high-risk and low-risk subclasses exhibit strong mesenchymal and proliferative tumor phenotypes concordant with resistance and sensitivity to primary chemotherapy. Our results have led to identification of promising prognostic markers of HG-SOC, which could provide a rationale for genetic-based stratification of patients and optimization of treatment regimes. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer (EOC) is hallmarked by a high degree of heterogeneity. To address this heterogeneity, a classification scheme was developed based on gene expression patterns of 1538 tumours. Five, biologically distinct subgroups - Epi-A, Epi-B, Mes, Stem-A and Stem-B - exhibited significantly distinct clinicopathological characteristics, deregulated pathways and patient prognoses, and were validated using independent datasets. To identify subtype-specific molecular targets, ovarian cancer cell lines representing these molecular subtypes were screened against a genome-wide shRNA library. Focusing on the poor-prognosis Stem-A subtype, we found that two genes involved in tubulin processing, TUBGCP4 and NAT10, were essential for cell growth, an observation supported by a pathway analysis that also predicted involvement of microtubule-related processes. Furthermore, we observed that Stem-A cell lines were indeed more sensitive to inhibitors of tubulin polymerization, vincristine and vinorelbine, than the other subtypes. This subtyping offers new insights into the development of novel diagnostic and personalized treatment for EOC patients. [ABSTRACT FROM AUTHOR]

wnt11r is a recently identified member of the Wnt family of genes, which has been proposed to be the true Xenopus homologue to the mammalian wnt11 gene. In this study we have examined the role of wnt11r on neural crest development. Expression analysis of wnt11r and comparison with the neural crest marker snail2 and the noncanonical Wnt, wnt11, shows wnt11r is expressed at the medial or neural plate side of the neural crest while wnt11 is expressed at the lateral or epidermal side. Injection of wnt11r morpholino leads to strong inhibition of neural crest migration with no effect on neural crest induction or maintenance. This effect can be rescued by co-injection of Wnt11r but not by Wnt11 mRNA, demonstrating the specificity of the loss of function treatment. Finally, neural crest graft experiments show that wnt11r is required in a non-cell-autonomous manner to control neural crest migration. Developmental Dynamics 237:3404-3409, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

In this study, we compared qualitative and quantitative changes in the lateral mobility of phospholipid molecules in the plasma membrane of intact cells under various conditions of specific interaction of integrins in the cell membrane with two extracellular matrix (ECM) components viz. fibronectin (FN) and laminin (LN). We found a strong and specific correlation between the lower lateral mobility of phosphatidylcholine (PC) and higher lateral mobility of phosphatidylethanolamine (PE) when cells were expressing high levels of α5β1 integrin and thus were adherent and motile on FN. The interaction between PC and FN in α5 integrin expressing cells was aided by the strong affinity of α5 integrin to the FN matrix. Cholesterol was involved in regulating the lateral mobility of PC to a great extent and of PE to a lesser extent without affecting the overall microviscosity of the plasma membrane or the distribution of caveolin-marked domains. The distribution and mobility of PC and PE molecules in the lamellipodial regions differed from that in the rest of the membrane and also in the more motile and in the less motile cells. We propose that these differences in distribution of PC and PE in different regions of cell membrane and their respective lateral mobility are observed due to the specific interaction of PC molecules with FN molecules in the ECM. Our results outline a new role of integrin–matrix interactions in the regulation of membrane phospholipid behavior. J. Cell. Physiol. 215: 550–561, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: P-cadherin expression is restricted to the basal layer of stratified epithelia including that of the mammary gland. Although evidence for an important role of P-cadherin in mammary morphogenesis and tumorigenesis is increasing, the mechanisms that regulate its expression are poorly understood. We show that in basal mammary epithelial cells, β-catenin is associated with the P-cadherin promoter and activates its expression independently of LEF/TCF in a cell-type specific manner. Down-regulation of endogenous β-catenin levels by RNA interference technique inhibited P-cadherin promoter activity. In vivo, in skin and mammary gland of mutant mice, activation of β-catenin signalling correlates with up-regulation of P-cadherin expression. These data suggest that β-catenin-dependent modulation of P-cadherin expression can contribute to the establishment of the basal phenotype. [Copyright &y& Elsevier]

E‐cadherin is the predominant adhesion molecule of epithelia. The interaction between extracellular segments of E‐cadherin in the membrane of opposing cells is homophilic and calcium dependent. Whereas it is widely accepted that the specificity of the adhesive interaction is localized to the N‐terminal domain, the kinetics of the recognition process are unknown. We report the first quantitative data describing the dissociation kinetics of individual E‐cadherin interactions. Aggregation assays indicate that the two outermost domains of E‐cadherin (E/EC1–2) retain biological activity when chemically immobilized on glass beads. Cadherin fragment trans‐interaction was analysed using a flow chamber technique. Transient tethers had first‐order kinetics, suggesting a unimolecular interaction. The unstressed lifetime of individual E‐cadherin interactions was as brief as 2 s. A fast off rate and the low tensile strength of the E‐cadherin bond may be necessary to support the high selectivity and plasticity of epithelial cell interactions. [ABSTRACT FROM AUTHOR]

Adhesion to extracellular matrix (ECM) induces intracellular signals that modulate cell proliferation, survival and differentiation. To study signalling events triggered by cell-ECM interactions in vivo we used transgenic mice exhibiting reduced mammary epithelial cell proliferation and increased apoptosis rates during the growth phase in pregnancy and lactation due to expression of a β1-integrin dominant-negative mutant in the mammary gland epithelium. Here we show that ERK and JNK MAPKs were markedly less activated in lactating transgenic glands thereby accounting for the growth defects. The FAK pathway was not affected suggesting a mechanism of activation additional to the ECM signal. On the contrary, the significant decrease of Shc phosphorylation, Grb2 recruitment and the reduced phosphorylation level of Akt Thr308 and Akt substrates FKHR and Bad detected in transgenic glands show that activation of the Shc and the Akt pathways require intact cell-ECM interactions. These results provide an insight into the mechanisms of growth control by integrin-mediated adhesion that operate in vivo. [ABSTRACT FROM AUTHOR]

The kinetics of degradation of calf thymus DNA by the DNAase from Helix aspersa has been investigated in its middle and terminal phase by determining both hyperchromic shift and average chain length of the digest. The results obtained are practically identical with those previously reported for acid DNAase from hog spleen. In contrast, the distribution of the isostichs in snail DNAase digests is significantly different from those found for spleen acid DNAase digests of similar sizes. The 3′phosphate and 5′-hydroxy terminal nucleotides were found not to vary in their compositions in the average size range 34 to 10 and 70 to 12, respectively. In the 3′-phosphate terminal position, deoxyadenosine is by far predominant (78%) and deoxycytidine almost absent (less than 1%); deoxyguanosinc and thymidine form 6% and 16%, respectively, of the terminals. In the 5′-hydroxy terminal position deoxyguanosine is predominant (45%), followed by deoxycytidine (31%), thymidine (14%) and deoxyadenosine (10%); in the 5′-hydroxy penultimate position thymidine forms 38% of the nucleotides, deoxyguanosine 24%, deoxy-adenosine 21% and deoxycytidine 17%. [ABSTRACT FROM AUTHOR]

The kinetics of the middle and terminal phases of calf thymus DNA degradation by spleen acid DNAase has been investigated in detail with the main purpose of providing a background for a study of the specificity of the enzyme and its use in investigations of nucleotide sequences in DNA. During the middle phase the ultraviolet absorption, the acid solubility and the reciprocal average degree of polymerization of the hydrolysate, &Pmacr;n-1 increase linearly with digestion time, while the elution profile of the DEAE-cellulose-urea chromatogram is progressively shifted to the left. Empirical relationships between &Pmacr;n-1 and hyperchromic shift have been established. Extrapolation of &Pmacr;n-1 to zero digestion time indicates that bond splitting proceeds at a faster, yet decreasing rate during the early part of digestion. The beginning of the terminal phase is characterized by a sudden slow down of the reaction rate; this phenomenon has been shown to be essentially due to the melting of double-stranded DNA fragments; the single-stranded fragments so originated arc a poor substrate for the enzyme, in spite of the fact that they still contain a large number of sequences which can be spit. During the terminal phase, the ultraviolet absorption, the acid solubility and &Pmacr;n-1 increase at a progressively slower rate until an end-point is practically reached. Investigations on the liberation of mono- and dinucleotides, the base composition and termini of isostichs and the effects of digestion temperature and of dephosphorylation on the isostich patterns are also reported. [ABSTRACT FROM AUTHOR]

The kinetics of liberation of 3′-phosphate terminal and of 5′-hydroxy terminal and penultimate nucleotides (termini) by spleen acid DNAase was investigated using as substrates calf thymus DNA, three bacterial DNAs of different base composition, and poly-[d(A-T] · d(A-T)]. The relative amounts of the nucleotides deoxyguanosine, deoxyadenosine, thymidine and deoxycytidine in the termini released by the enzyme from all DNAs were found to be constant in the terminal phase of digestion. In contrast, some variations were seen during the middle phase, particularly in the amount of 3′-phosphate terminal deoxyguanosine, which decreased with digestion time, specially in the calf thymus DNA digest. This phenomenon appears to he due to an initial accumulation of breaks in some nucleotide sequences which are preferentially split at the beginning of digestion, possibly by a diplotomic mechanism, with predominant release of 3′-phosphate deoxyguanosine. In contrast, no change in the composition of 3′-phosphate terminals released from poly[d(A-T) · d(A-T)] took place in the 40–15 average size range; deoxyadenosine and thymidine formed 80% and 20%, respectively, of these terminals. The composition of the 3′-phosphate terminal nucleotides was very little or not at all affected by changes in the ionic environment of the incubation mixture, whereas it was significantly changed in degradations of native DNA digested at neutrality and low ionic strength, or of heat-denatured DNA. Linear relationships were found to hold, at all degradation stages investigated here, between the relative amounts of termini liberated from different bacterial DNAs and their (dG + dC) contents. In contrast, the termini obtained from calf thymus DNA were released in relative amounts which did not fit the relationships established for bacterial DNAs. [ABSTRACT FROM AUTHOR]

The expression of a transgene coding for a chimeric molecule, containing the cytoplasmic and transmembrane domains of the β1-integrin chain and the extracellular domain of the T-cell differentiation antigen CD4, was targeted to the mouse mammary gland by the mouse mammary tumor virus (MMTV) promoter. The chimera does not interact with the extracellular ligands; however, its expression in cultured cells was shown to interfere with focal adhesion kinase (FAK) phosphorylation following ligation of endogenous β1-integrin. Therefore, expression of the transgenic protein on the cell surface should uncouple adhesion from intracellular events associated with the β1-cytoplasmic domain and thus perturb β1-integrin functions. Although most of the transgenic females were able to lactate, their mammary glands had a phenotype clearly distinct from that of wild-type mice. At mid-pregnancy and the beginning of lactation, transgenic glands were underdeveloped and the epithelial cell proliferation rates were decreased, while the apoptosis levels were higher than in wild-type glands. In lactation, the amounts of the whey acidic protein (WAP) and β-casein gene transcripts were diminished, and the basement membrane component, laminin and the β4-integrin chain accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our observations prove that in vivo, β1-integrins are involved in control of proliferation, apoptosis, differentiation and maintenance of baso­apical polarity of mammary epithelial cells, and therefore are essential for normal mammary gland development and function. [ABSTRACT FROM AUTHOR]

We have demonstrated previously that Src controls the epidermal growth factor (EGF)-induced dispersion of NBT-II carcinoma epithelial cells. Here we show that while only Src and Yes were expressed and activated by EGF, microinjected kinase-inactive mutants of Src (SrcK-) and Fyn (FynK-) were able to exert a dominant negative effect on the scattering response. Both SH2 and SH3 domains of FynK- were required for inhibition of cell scattering. Expression of dominant-negative N17Ras also abrogated EGF-induced dispersion, showing that Ras is another regulator of cell dispersion. Expression of SrcK- did not alter EGF-evoked Shc tyrosine phosphorylation, Shc-Grb2 complex formation and MAPK activation, three elements of the Ras pathway. Furthermore, the expression of Jun-Fos and Slug rescued the block induced by N17Ras but not by SrcK-, showing that Src kinases and Ras operate in separate pathways. In addition, actinomycin D inhibition of RNA synthesis repressed the ability of the activated mutant L61Ras but not that of F527Src to induce epithelial cell scattering. Since tyrosine phosphorylation of cytoskeleton-associated proteins pp125FAK and cortactin were abolished in EGF-stimulated SrcK- cells, we concluded that, in contrast to Ras, Src kinases may control epithelial cell dispersion in the absence of gene expression and by directly regulating the organization of the cortical cytoskeleton. [ABSTRACT FROM AUTHOR]