Parathyroid Hormone-Related Protein Specifies the Mammary Mesenchyme and Regulates Embryonic Mammary Development

Parathyroid Hormone related Protein (PTHrP) is a critical regulator of mammary gland morphogenesis in the mouse embryo. Loss of PTHrP, or its receptor, PTHR1, results in arrested mammary buds at day 15 of embryonic development (E15). In contrast, overexpression of PTHrP converts the ventral epidermis into hairless nipple skin. PTHrP signaling appears to be critical for mammary mesenchyme specification, which in turn maintains mammary epithelial identity, directs bud outgrowth, disrupts the male mammary rudiment and specifies the formation of the nipple. In the embryonic mammary bud, PTHrP exerts its effects on morphogenesis, in part, through epithelial-stromal crosstalk mediated by Wnt and BMP signaling. Recently, PTHLH has been identified as a strong candidate for a novel breast cancer susceptibility locus, although PTHrP’s role in breast cancer has not been clearly defined. The effects of PTHrP on the growth of the embryonic mammary rudiment and its invasion into the dermis may, in turn, have connections to the role of PTHrP in breast cancer.     

ErbB4 modulates tubular cell polarity and lumen diameter during kidney development

ErbB4 receptor tyrosine kinase contributes to the development of the heart, the central nervous system, and the lactating mammary gland, but whether it has a role in the development of the kidney epithelium is unknown. Here, we found that expression of Erbb4 isoforms JM-a CYT-1 and JM-a CYT-2 was first detectable around embryonic day 13 in the mouse, mainly in the collecting ducts and both the proximal and distal tubules. In vitro, overexpression of a relevant ErbB4 isoform promoted proliferation and disturbed polarization of kidney epithelial cells when cultured as three-dimensional structures. We examined ErbB4 function in developing kidney tubules in vivo with Pax8-Cre-mediated conditional overexpression of Rosa26 locus-targeted ERBB4 and with conditional Erbb4 knock-out mice. The Pax8-Cre-driven ERBB4 overexpression enhanced proliferation in the collecting ducts, reduced the size of epithelial duct lumens, and promoted formation of cortical tubular cysts. These defects were associated with changes in the subcellular distribution of markers of epithelial cell polarity. Similarly, the Pax8-Cre-mediated Erbb4 knock-out mice manifested dysfunctional kidneys with larger duct lumens and epithelial cell mispolarization. Taken together, these data suggest that ErbB4 signaling modulates proliferation and polarization, cellular functions critical for the development of epithelial ducts in the kidney.     

The Role of NRG3 in Mammary Development

The Neuregulin gene family encodes EGF-containing ligands which mediate their effects by binding to the ERBB receptor tyrosine kinases, a signalling network with important roles in both mammary gland development and breast cancer. Neuregulin3 (NRG3), a ligand for ERBB4, promotes early mammary morphogenesis and acts during specification of the mammary placode, an aggregate of epithelial cells that forms during mid-embryogenesis. Recent studies have shown that NRG3 can alter the cell fate of other epidermal progenitor populations when NRG3 is mis-expressed throughout the basal layer of the developing epidermis with the K14 promoter. Here evidence for a key function for NRG3 in promoting early mammary morphogenesis and the implication for the role of NRG3 in breast cancer and establishment of the mammary lineage are discussed.     

Wnt-Signalling in the Embryonic Mammary Gland

The first member of the Wnt-family ligands was identified 30 years ago as a factor in mouse mammary tumours whose expression was deregulated due to the promoter activity emanating from the proximal integration of the Mouse Mammary Tumour Virus genome (Nusse and Varmus, Embo J 31:2670–84, 2012). The Wnt-ligands invoke a number of molecular-genetic signalling cascades fundamental to the patterning of developing tissues and organs during embryogenesis as well as during postnatal development. The Wnt-signalling cascade that controls the activities of β-catenin and the T-cell Factor (Tcf)/Lympoid enhancer factor (Lef1) plays a fundamental role in control of all stages of embryonic mammary gland development. We provide here a brief overview of the known aspects of Wnt-signalling activities in the embryonic mammary gland and its interactions with other signalling cascades in this developing tissue.     

ErbB/EGF Signaling and EMT in Mammary Development and Breast Cancer

Activation of the ErbB family of receptor tyrosine kinases via cognate Epidermal Growth Factor (EGF)-like peptide ligands constitutes a major group of related signaling pathways that control proliferation, survival, angiogenesis and metastasis of breast cancer. In this respect, clinical trials with various ErbB receptor blocking antibodies and specific tyrosine kinase inhibitors have proven to be partially efficacious in the treatment of this heterogeneous disease. Induction of an embryonic program of epithelial-to-mesenchymal transition (EMT) in breast cancer, whereupon epithelial tumor cells convert to a more mesenchymal-like phenotype, facilitates the migration, intravasation, and extravasation of tumor cells during metastasis. Breast cancers which exhibit properties of EMT are highly aggressive and resistant to therapy. Activation of ErbB signaling can regulate EMT-associated invasion and migration in normal and malignant mammary epithelial cells, as well as modulating discrete stages of mammary gland development. The purpose of this review is to summarize current information regarding the role of ErbB signaling in aspects of EMT that influence epithelial cell plasticity during mammary gland development and tumorigenesis. How this information may contribute to the improvement of therapeutic approaches in breast cancer will also be addressed.     

A Novel Effect of β-Adrenergic Receptor on Mammary Branching Morphogenesis and its Possible Implications in Breast Cancer

Understanding the mechanisms that govern normal mammary gland development is crucial to the comprehension of breast cancer etiology. β-adrenergic receptors (β-AR) are targets of endogenous catecholamines such as epinephrine that have gained importance in the context of cancer biology. Differences in β₂-AR expression levels may be responsible for the effects of epinephrine on tumor vs non-tumorigenic breast cell lines, the latter expressing higher levels of β₂-AR. To study regulation of the breast cell phenotype by β₂-AR, we over-expressed β₂-AR in MCF-7 breast cancer cells and knocked-down the receptor in non-tumorigenic MCF-10A breast cells. In MCF-10A cells having knocked-down β₂-AR, epinephrine increased cell proliferation and migration, similar to the response by tumor cells. In contrast, in MCF-7 cells overexpressing the β₂-AR, epinephrine decreased cell proliferation and migration and increased adhesion, mimicking the response of the non-tumorigenic MCF-10A cells, thus underscoring that β₂-AR expression level is a key player in cell behavior. β-adrenergic stimulation with isoproterenol induced differentiation of breast cells growing in 3-dimension cell culture, and also the branching of murine mammary epithelium in vivo. Branching induced by isoproterenol was abolished in fulvestrant or tamoxifen-treated mice, demonstrating that the effect of β-adrenergic stimulation on branching is dependent on the estrogen receptor (ER). An ER-independent effect of isoproterenol on lumen architecture was nonetheless found. Isoproterenol significantly increased the expression of ERα, Ephrine-B1 and fibroblast growth factors in the mammary glands of mice, and in MCF-10A cells. In a poorly differentiated murine ductal carcinoma, isoproterenol also decreased tumor growth and induced tumor differentiation. This study highlights that catecholamines, through β-AR activation, seem to be involved in mammary gland development, inducing mature duct formation. Additionally, this differentiating effect could be resourceful in a breast tumor context.     

TrkC Is Essential for Nephron Function and Trans-Activates Igf1R Signaling

BackgroundInjury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling pathways contributing to glomerular disease. Neurotrophic tyrosine kinase receptor 3 (TrkC) is expressed in podocytes and the protein transmits signals to the podocyte actin cytoskeleton.MethodsNephron-specific TrkC knockout (TrkC-KO) and nephron-specific TrkC-overexpressing (TrkC-OE) mice were generated to dissect the role of TrkC in nephron development and maintenance.ResultsBoth TrkC-KO and TrkC-OE mice exhibited enlarged glomeruli, mesangial proliferation, basement membrane thickening, albuminuria, podocyte loss, and aspects of FSGS during aging. Igf1 receptor (Igf1R)–associated gene expression was dysregulated in TrkC-KO mouse glomeruli. Phosphoproteins associated with insulin, erb-b2 receptor tyrosine kinase (Erbb), and Toll-like receptor signaling were enriched in lysates of podocytes treated with the TrkC ligand neurotrophin-3 (Nt-3). Activation of TrkC by Nt-3 resulted in phosphorylation of the Igf1R on activating tyrosine residues in podocytes. Igf1R phosphorylation was increased in TrkC-OE mouse kidneys while it was decreased in TrkC-KO kidneys. Furthermore, TrkC expression was elevated in glomerular tissue of patients with diabetic kidney disease compared with control glomerular tissue.ConclusionsOur results show that TrkC is essential for maintaining glomerular integrity. Furthermore, TrkC modulates Igf-related signaling in podocytes.     

The Epidermal Growth Factor Receptor in Breast Cancer

The epidermal growth factor receptor (EGFR)³ is a 170 kD transmembrane tyrosine kinase activated by several ligands. It is required for normal mammary development and lactation and is aberrantly expressed in approximately 40% of breast carcinomas, particularly those with a poor prognostic phenotype. Since EGF receptor levels are elevated in a high proportion of many tumor types its potential as a therapy target is being investigated using the EGF receptor to target toxins, as well as drugs that interfere with signaling and anti-receptor antibodies. These approaches are likely to be most effective when used in the adjuvant situation in combination with chemotherapy.     

Parathyroid Hormone-Related Protein: A Developmental Regulatory Molecule Necessary for Mammary Gland Development

Parathyroid hormone-related protein (PTHrP) wasoriginally identified as the tumor factor responsiblefor a clinical syndrome known as humoral hypercalcemiaof malignancy. It is now appreciated that PTHrP3 is a developmental regulatory moleculeexpressed during the formation of a wide variety oforgans. Recently, our laboratory has demonstrated thatPTHrP is necessary for mammary gland development. Ourstudies have suggested that this molecule participatesin the regulation of epithelial-mesenchymal interactionsduring embryonic mammary development and perhaps alsoduring adolescent ductal morphogenesis. In addition, it has been suggested that PTHrP plays acritical role in the establishment of bone metastases inbreast cancer. In this article, we will discuss thecurrent knowledge of the mechanisms underlying PTHrPs actions during normal mammary development andin breast cancer.     

Next Stop, the Twilight Zone: Hedgehog Network Regulation of Mammary Gland Development

The hedgehog signal transduction network is a critical mediator of cell-cell communication during embryonic development. Evidence also suggests that properly regulated hedgehog network function is required in some adult organs for stem cell maintenance or renewal. Mutation, or misexpression, of network genes is implicated in the development of several different types of cancer, particularly that of skin, brain, lung, and pancreas. Recent studies in the mouse mammary gland have demonstrated roles for hedgehog network genes at virtually every phase of mammary gland development where it regulates such diverse processes as embryonic mammary gland induction, establishment of ductal histoarchitecture, and functional differentiation in lactation. Further, studies suggest a role for misregulated network function in the progression of breast cancer.     

The Cell of Origin of <Emphasis Type="Italic">BRCA1</Emphasis> Mutation-associated Breast Cancer: A Cautionary Tale of Gene Expression Profiling

Breast tumours are highly heterogeneous with several distinct sub-types recognised according to their histological and molecular features. The biological basis for this heterogeneity is largely unknown, although there are some distinct phenotype–genotype correlations. These include BRCA1 mutation-associated breast cancers, which are typically high grade invasive ductal carcinomas of no special type (IDC-NSTs) with pushing margins that do not express estrogen receptor (ER), progesterone receptor (PR) or the HER2 receptor tyrosine kinase (‘triple negative’). Gene expression analysis of these tumours has grouped them with so called ‘basal-like’ breast cancers and this, together with evidence that knock-down of BRCA1 in vitro blocked luminal differentiation, led to speculation that these tumours arose from the normal basal stem cells within the mammary gland. Recently, however, human breast tissue from BRCA1 mutation carriers was shown to contain an expanded population of luminal progenitor cells which have increased in vitro clonogenic ability. In the mouse, targeted deletion of Brca1 in luminal ER negative progenitors resulted in the formation of mammary tumours which phenocopied human BRCA1 breast tumour pathology, while the deletion of Brca1 in basal stem cells resulted in the formation of tumours which neither resembled human BRCA1 tumours or sporadic basal-like breast tumours. Importantly, however, both sets of mouse tumours were classified as ‘basal-like’ by methods used for human tumour classification based on gene expression profiles. This demonstrates that, as it stands, expression profiling is poor at distinguishing tumour histological subtypes and is also a poor guide to the cell of tumour origin. These human and rodent studies support an origin of BRCA1-mutation associated breast cancer (and indeed of the majority of sporadic basal-like breast cancers) in a luminal ER negative mammary epithelial progenitor. This is a key finding, as identification of the cells of origin in breast cancer subtypes makes possible the identification of key processes associated with initiation, progression and maintenance of each tumour subtype, the development of novel targeted therapies and, potentially, of new preventative approaches in high risk groups.     

Human breast cancer cell lines as models of growth regulation and disease progression

The routine isolation and culture of human breast cancer cells from patients samples has been a goal of breast cancer cell biologists for over 30 years. Despite extensive work in this area and the development of many human breast cancer cell lines, the proportion of patient samples that give rise to immortalized breast cancer cell lines is still disappointingly low. The majority of human breast cancer cell lines that have been established were isolated many years ago and have been grown continuously under poorly defined culture conditions. These cell lines have been useful for studies of the estrogen receptor biology in human breast cancer cells, in identifying growth factors synthesized by breast cancer cells, and for the characterization of genetic alterations in oncogenes and tumor suppressor genes present in these cells. More recently, tissue culture methods have improved, resulting in the ability to culture routinely normal human mammary epithelial cells of specific lineages and this has resulted in the development of new human breast cancer cell lines. The ability to isolate and culture normal and neoplastic human mammary epithelial cells under similar culture conditions has improved these models dramatically and has resulted in the identification of altered cellular phenotypes of human breast cancer cells.     

Novel chimeric parapoxvirus CF189 as an oncolytic immunotherapy in triple-negative breast cancer

Background

Triple-negative breast cancer is an aggressive subtype of breast cancer with high recurrence rate and poor prognosis. Here we describe a novel, genetically engineered parapoxvirus that efficiently kills triple-negative breast cancer.

Prolactin as an Autocrine/Paracrine Factor in Breast Tissue

The neuroendocrine hormone prolactin (PRL)³ stimulates breast growth and differentiation during puberty, pregnancy, and lactation. Despite extensive and convincing data indicating that PRL significantly contributes to the pathogenesis and progression of rodent mammary carcinoma, parallel observations for human breast cancer have not been concordant. In particular, the therapeutic alteration of somatolactogenic hormone levels has not consistently altered the course of human breast cancer. Recent data, however, suggest that extra-pituitary tissues are capable of elaborating PRL; indeed, the observation of sustained serum levels of PRL in post-hypophysectomy patients supports this hypothesis. Proof of an autocrine/paracrine loop for PRL within normal and malignant human breast tissues requires that the following three criteria be met: (1) PRL must be synthesized and secreted within mammary tissues; (2) the receptor for PRL (PRLR) must be present within these tissues; and, (3) proliferative responses to autocrine/paracrine PRL must be demonstrated. These criteria have now been fulfilled in several laboratories. With the demonstration of a PRL autocrine/paracrine loop in mammary glands, the basis for the ineffective treatment of human breast cancer by prior endocrine-based anti-somatolactogenic therapies is evident. These findings provide the precedent for novel therapeutic strategies aimed at interrupting the stimulation of breast cancer growth by PRL at both endocrine and autocrine/paracrine levels.     

Hedgehog Signaling in Mammary Gland Development and Breast Cancer

The Hedgehog pathway is critical for many developmental processes, including the formation of several epidermal appendages. In the mammary gland strict regulation of the Hedgehog pathway is required for normal development. Alterations in Hedgehog signaling result in defects in both the embryonic and postnatal mammary gland. Activation of Hedgehog signaling either by mutation or misexpression of pathway members can lead to the development and/or progression of cancers in multiple organs. This review addresses the current understanding and controversies of Hedgehog signaling in mammary gland development and its potential role in promoting breast carcinogenesis and cancer progression.