The neoplastic tumour suppressors, Scribble, Dlg and Lgl, originally discovered in the vinegar fly Drosophila melanogaster, are currently being actively studied for their potential role in mammalian tumourigenesis. In Drosophila, these tumour suppressors function in a common genetic pathway to regulate apicobasal cell polarity and also play important roles in the control of cell proliferation, survival, differentiation and in cell migration/invasion. The precise mechanism by which Scribble, Dlg and Lgl function is not clear; however, they have been implicated in the regulation of signalling pathways, vesicle trafficking and in the Myosin II-actin cytoskeleton. We review the evidence for the involvement of Scribble, Dlg, and Lgl in cancer, and how the various functions ascribed to these tumour suppressors in Drosophila and mammalian systems may impact on the process of tumourigenesis.
We have shown previously that mutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (Ras ACT ) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with Ras ACT in tumorigenesis, we carried out a genome-wide screen for genes that when overexpressed throughout the developing Drosophila eye enhance Ras ACT -driven hyperplasia. Ras ACT -cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wildtype cells, only Rac1, an activated allele of Rho1 (Rho1 ACT ), RhoGEF2, and pbl cooperated with Ras ACT , resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or Rac1 with Ras ACT upregulated Jun kinase ( JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system, upregulation of JNK alone was not sufficient for cooperation with Ras ACT , while in the clonal setting, JNK upregulation was sufficient for Ras ACT -mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of Ras V12 -expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2 1 human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, our genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg and aPKC, with Ras ACT in the whole-tissue context. Collectively our analysis reveals the importance of the RhoGEF/ Rho-family/JNK pathway in cooperative tumorigenesis with Ras ACT .
Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression.
Epithelial to mesenchymal transition (EMT) and its reversion via mesenchymal to epithelial transition (MET), represent a stepwise cycle of epithelial plasticity that allows for normal tissue remodelling and diversification during development. In particular, epithelial-mesenchymal plasticity is central to many aspects of mammary development and has been proposed to be a key process in breast cancer progression. Such epithelial-mesenchymal plasticity requires complex cellular reprogramming to orchestrate a change in cell shape to an alternate morphology more conducive to migration. During this process, epithelial characteristics, including apical-basal polarity and specialised cell-cell junctions are lost and mesenchymal properties, such as a front-rear polarity associated with weak cell-cell contacts, increased motility, resistance to apoptosis and invasiveness are gained. The ability of epithelial cells to undergo transitions through cell polarity states is a central feature of epithelial-mesenchymal plasticity. These cell polarity states comprise a set of distinct asymmetric distributions of cellular constituents that are fashioned to allow specialized cellular functions, such as the regulated homeostasis of molecules across epithelial barriers, cell migration or cell diversification via asymmetric cell divisions. Each polarity state is engineered using a molecular toolbox that is highly conserved between organisms and cell types which can direct the initiation, establishment and continued maintenance of each asymmetry. Here we discuss how EMT pathways target cell polarity mediators, and how this EMT-dependent change in polarity states impact on the various stages of breast cancer. Emerging evidence places cell polarity at the interface of proliferation and morphology control and as such the changing dynamics within polarity networks play a critical role in normal mammary gland development and breast cancer progression.
Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease, and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by "conventional" therapies, memory T cell-mediated attack is a substantial challenge in islet transplantation, and this will extend to application of personalized approaches using stem cell-derived replacement b-cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement b-cells. Here, we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions, inactivates established memory CD8 + T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8 + memory T cell-mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell-mediated gene therapy effectively terminates antigenspecific memory T-cell responses, and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and, importantly, the clinical application of personalized b-cell replacement therapies using patient-derived stem cells.
BackgroundApplication of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a ‘niche’ so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained.MethodsBM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells.ResultsImmune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM.ConclusionsRapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0508-3) contains supplementary material, which is available to authorized users.
Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells.
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