Developmental signaling pathways regulating mammary stem cells and contributing to the etiology of triple-negative breast cancer.

Cancer has been considered as temporal and spatial aberrations of normal development in tissues. Similarities between mammary embryonic development and cell transformation suggest that the underlying processes required for mammary gland development are also those perturbed during various stages of mammary tumorigenesis and breast cancer (BC) development. The master regulators of embryonic development Cripto-1, Notch/CSL, and Wnt/ß-catenin play key roles in modulating mammary gland morphogenesis and cell fate specification in the embryo through fetal mammary stem cells (fMaSC) and in the adult organism particularly within the adult mammary stem cells (aMaSC), which determine mammary progenitor cell lineages that generate the basal/myoepithelial and luminal compartments of the adult mammary gland. Together with recognized transcription factors and embryonic stem cell markers, these embryonic regulatory molecules can be inappropriately augmented during tumorigenesis to support the tumor-initiating cell (TIC)/cancer stem cell (CSC) compartment, and the effects of their deregulation may contribute for the etiology of BC, in particular the most aggressive subtype of BC, triple-negative breast cancer (TNBC). This in depth review will present evidence of the involvement of Cripto-1, Notch/CSL, and Wnt/ß-catenin in the normal mammary gland morphogenesis and tumorigenesis, from fMaSC/aMaSC regulation to TIC generation and maintenance in TNBC. Specific therapies for treating TNBC by targeting these embryonic pathways in TICs will be further discussed, providing new opportunities to destroy not only the bulk tumor, but also TICs that initiate and promote the metastatic spread and recurrence of this aggressive subtype of BC.

Cripto-1 ablation disrupts alveolar development in the mouse mammary gland through a progesterone receptor-mediated pathway.

Cripto-1, a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic family, is critical for early embryonic development. Together with its ligand Nodal, Cripto-1 has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Several studies have clearly shown that Cripto-1 is involved in regulating branching morphogenesis and epithelial-mesenchymal transition of the mammary gland both in vitro and in vivo and together with the cofactor GRP78 is critical for the maintenance of mammary stem cells ex vivo. Our previous studies showed that mammary-specific overexpression of human Cripto-1 exhibited dramatic morphological alterations in nulliparous mice mammary glands. The present study shows a novel mechanism for Cripto-1 regulation of mammary gland development through direct effects on progesterone receptor expression and pathways regulated by progesterone in the mammary gland. We demonstrate a strict temporal regulation of mouse Cripto-1 (mCripto-1) expression that occurs during mammary gland development and a stage-specific function of mCripto-1 signaling during mammary gland development. Our data suggest that Cripto-1, like the progesterone receptor, is not required for the initial ductal growth but is essential for subsequent side branching and alveologenesis during the initial stages of pregnancy. Dissection of the mechanism by which this occurs indicates that mCripto-1 activates receptor activator NF-κB/receptor activator NF-κB ligand, and NF-κB signaling pathways.

The multifaceted role of the embryonic gene Cripto-1 in cancer, stem cells and epithelial-mesenchymal transition.

Cripto-1 (CR-1)/Teratocarcinoma-derived growth factor1 (TDGF-1) is a cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein that can function either in cis (autocrine) or in trans (paracrine). The cell membrane cis form is found in lipid rafts and endosomes while the trans acting form lacking the GPI anchor is soluble. As a member of the epidermal growth factor (EGF)/Cripto-1-FRL-1-Cryptic (CFC) family, CR-1 functions as an obligatory co-receptor for the transforming growth factor-ß (TGF-ß) family members, Nodal and growth and differentiation factors 1 and 3 (GDF1/3) by activating Alk4/Alk7 signaling pathways that involve Smads 2, 3 and 4. In addition, CR-1 can activate non-Smad-dependent signaling elements such as PI3K, Akt and MAPK. Both of these pathways depend upon the 78kDa glucose regulated protein (GRP78). Finally, CR-1 can facilitate signaling through the canonical Wnt/ß-catenin and Notch/Cbf-1 pathways by functioning as a chaperone protein for LRP5/6 and Notch, respectively. CR-1 is essential for early embryonic development and maintains embryonic stem cell pluripotentiality. CR-1 performs an essential role in the etiology and progression of several types of human tumors where it is expressed in a population of cancer stem cells (CSCs) and facilitates epithelial-mesenchymal transition (EMT). In this context, CR-1 can significantly enhance tumor cell migration, invasion and angiogenesis. Collectively, these facts suggest that CR-1 may be an attractive target in the diagnosis, prognosis and therapy of several types of human cancer.

Cripto-1: an extracellular protein - connecting the sequestered biological dots.

Cripto-1 (CR-1) is a multifunctional embryonic protein that is re-expressed during inflammation, wound repair, and malignant transformation. CR-1 can function either as a tethered co-receptor or shed as a free ligand underpinning its flexible role in cell physiology. CR-1 has been shown to mediate cell growth, migration, invasion, and induce epithelial to mesenchymal transition (EMT). The main signaling pathways mediating CR-1 effects include Nodal-dependent (Smad2/3) and Nodal-independent (Src/p44/42/Akt) signaling transduction pathways. In addition, there are several naturally occurring binding partner proteins (BPPs) for CR-1 that can either agonize or antagonize its bioactivity. We will review the collective role of CR-1 as an extracellular protein, discuss caveats to consider in developing a quantitation assay, define possible mechanistic avenues applicable for drug discovery, and report on our experimental approaches to overcome these problematic issues.

Trp53 regulates Notch 4 signaling through Mdm2.

Notch receptors and their ligands have crucial roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch 4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal-controlling mechanism is mediated by physical interactions between Mdm2 and Notch 4 and suggest the existence of a trimeric complex between Trp53, Notch 4 and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4-induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 has a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland.

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells.

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/ß-catenin pathway, or short hairpin RNA targeting ß-catenin expression demonstrated that the invasive activity was not mediated by ß-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the ß-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.

Expression of Notch receptors, ligands, and target genes during development of the mouse mammary gland.

Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study, we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context.

Dual neurotoxic and neuroprotective role of metabotropic glutamate receptor 1 in conditions of trophic deprivation - possible role as a dependence receptor.

Group I metabotropic glutamate receptors have been often implicated in various models of neuronal toxicity, however, the role played by the individual receptors and their putative mechanisms of action contributing to neurotoxicity or neuroprotection remain unclear. Here, using primary cultures of rat cerebellar granule cells and mouse cortical neurons, we show that conditions of trophic deprivation increased mGlu1 expression which correlated with the developing cell death. The inhibition of mGlu1 expression by specific siRNA attenuated toxicity, while adenovirus-mediated overexpression of mGlu1 resulted in increased cell death, indicating a causal relationship between the level of receptor expression and neuronal survival. In pharmacological experiments selective mGlu1 antagonists failed to protect from mGlu1-induced cell death, instead, neuronal survival was promoted by glutamate acting at mGlu1 receptors. Such properties are characteristics of a novel heterogeneous family of dependence receptors which control neuronal apoptosis. Our findings indicate that increased expression of mGlu1 in neurons creates a state of cellular dependence on the presence of its endogenous agonist glutamate. We propose a new role and a new mechanism for mGlu1 action. This receptor may play a crucial role in determining the fate of individual neurons during the development of the nervous system.

Cripto-1 as a novel therapeutic target for triple negative breast cancer.

Triple-negative breast cancer (TNBC) presents the poorest prognosis among the breast cancer subtypes and no current standard therapy. Here, we performed an in-depth molecular analysis of a mouse model that establishes spontaneous lung metastasis from JygMC(A) cells. These primary tumors resembled the triple-negative breast cancer (TNBC) both phenotypically and molecularly. Morphologically, primary tumors presented both epithelial and spindle-like cells but displayed only adenocarcinoma-like features in lung parenchyma. The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively. Cripto-1, an embryonic stem cell marker, was present in spindle-like areas and its promoter showed activity in primary tumors. Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis. Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.

Recombinant R-spondin2 and Wnt3a up- and down-regulate novel target genes in C57MG mouse mammary epithelial cells.

R-spondins (Rspos) comprise a family of four secreted proteins that have important roles in cell proliferation, cell fate determination and organogenesis. Rspos typically exert their effects by potentiating the Wnt/ß-catenin signaling pathway. To systematically investigate the impact of Rspo/Wnt on gene expression, we performed a microarray analysis using C57MG mouse mammary epithelial cells treated with recombinant Rspo2 and/or Wnt3a. We observed the up- and down-regulation of several previously unidentified target genes, including ones that encode proteins involved in immune responses, effectors of other growth factor signaling pathways and transcription factors. Dozens of these changes were validated by quantitative real time RT-PCR. Time course experiments showed that Rspo2 typically had little or no effect on Wnt-dependent gene expression at 3 or 6 h, but enhanced expression at 24 h, consistent with biochemical data indicating that Rspo2 acts primarily to sustain rather than acutely increase Wnt pathway activation. Up-regulation of gene expression was inhibited by pre-treatment with Dickkopf1, a Wnt/ß-catenin pathway antagonist, and by siRNA knockdown of ß-catenin expression. While Dickkopf1 blocked Rspo2/Wnt3a-dependent down-regulation, a number of down-regulated genes were not affected by ß-catenin knockdown, suggesting that in these instances down-regulation was mediated by a ß-catenin-independent mechanism.

Heat shock enhances CMV-IE promoter-driven metabotropic glutamate receptor expression and toxicity in transfected cells.

In CHO-K1 cells, heat shock strongly activated reporter-gene expression driven by the cytomegalovirus immediate-early (CMV-IE) promoter from adenoviral and plasmid vectors. Heat shock treatment (2h at 42.5 °C) significantly enhanced the promoter DNA-binding activity in nuclear extracts. In CHO cells expressing mGluR1a and mGluR5a receptors under the control of the CMV promoter, heat shock increased receptor protein expression, mRNA levels and receptor function estimated by measurement of PI hydrolysis, intracellular Ca²+ and cAMP. Hyperthermia increased average amplitudes of Ca²+ responses, the number of responding cells, and revealed the toxic properties of mGluR1a receptor. Heat shock also effectively increased the expression of EGFP. Hence, heat shock effects on mGluR expression and function in CHO cells may be attributed to the activation of the CMV promoter. Moreover, this effect was not limited to CHO cells as heat shock also increased EGFP expression in PC-12 and HEK293 cells. Heat shock treatment may be a useful tool to study the function of proteins expressed in heterologous systems under control of the CMV promoter. It may be especially valuable for increasing protein expression in transient transfections, for enhancing receptor expression in drug screening applications and to control the expression of proteins endowed with toxic properties. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function.

R-spondins (Rspos) potentiate Wnt/beta-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein,demonstrating for the first time specific amino acid residues in Rspos that are critical for beta-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a beta-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.