Carrie S. Shemanko

Mutations in the plakophilin 1 gene result in ectodermal dysplasia/skin fragility syndrome

Members of the armadillo protein gene family, which includes plakoglobin and β-catenin, have important functions in cytoskeleton/cell membrane interactions1,2. These proteins may act as linker molecules at adherens junctions and desmosomes at the plasma membrane3; in addition, they may have pivotal roles in signal transduction pathways and significant effects on cell behaviour during development4–7. Here, we describe the first human mutations in one of these dual function proteins, plakophilin 1 (band-6 protein; refs 8–10). The affected individual has a complete absence of immunostaining for plakophilin 1 in the skin and is a compound heterozygote for autosomal-recessively inherited premature termination codons of translation on both alleles of the plakophilin 1 gene (PKP1). Clinically, there are features of both cutaneous fragility and congenital ectodermal dysplasia affecting skin, hair and nails. There is no evidence of significant abnormalities in other epithelia or tissues. Desmosomes in the skin are small and poorly formed with widening of keratinocyte intercellular spaces and perturbed desmosome/keratin intermediate filament interactions. The molecular findings and clinical observations in this patient attest to the dual importance of plakophilin 1 in both cutaneous cell–cell adhesion and epidermal morphogenesis.

Comparative Proteomic Analysis of Proliferating and Functionally Differentiated Mammary Epithelial Cells

Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.

Heat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation

IntroductionThe prolactin-Janus-kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) pathway is essential for the development and functional differentiation of the mammary gland. The pathway also has important roles in mammary tumourigenesis. Prolactin regulated target genes are not yet well defined in tumour cells, and we undertook, to the best of our knowledge, the first large genetic screen of breast cancer cells treated with or without exogenous prolactin. We hypothesise that the identification of these genes should yield insights into the mechanisms by which prolactin participates in cancer formation or progression, and possibly how it regulates normal mammary gland development.MethodsWe used subtractive hybridisation to identify a number of prolactin-regulated genes in the human mammary carcinoma cell line SKBR3. Northern blotting analysis and luciferase assays identified the gene encoding heat shock protein 90-alpha (HSP90A) as a prolactin-JAK2-STAT5 target gene, whose function was characterised using apoptosis assays.ResultsWe identified a number of new prolactin-regulated genes in breast cancer cells. Focusing on HSP90A, we determined that prolactin increased HSP90A mRNA in cancerous human breast SKBR3 cells and that STAT5B preferentially activated the HSP90A promoter in reporter gene assays. Both prolactin and its downstream protein effector, HSP90α, promote survival, as shown by apoptosis assays and by the addition of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in both untransformed HC11 mammary epithelial cells and SKBR3 breast cancer cells. The constitutive expression of HSP90A, however, sensitised differentiated HC11 cells to starvation-induced wild-type p53-independent apoptosis. Interestingly, in SKBR3 breast cancer cells, HSP90α promoted survival in the presence of serum but appeared to have little effect during starvation.ConclusionsIn addition to identifying new prolactin-regulated genes in breast cancer cells, we found that prolactin-JAK2-STAT5 induces expression of the HSP90A gene, which encodes the master chaperone of cancer. This identifies one mechanism by which prolactin contributes to breast cancer. Increased expression of HSP90A in breast cancer is correlated with increased cell survival and poor prognosis and HSP90α inhibitors are being tested in clinical trials as a breast cancer treatment. Our results also indicate that HSP90α promotes survival depending on the cellular conditions and state of cellular transformation.

A pathway map of prolactin signaling.

Prolactin (PRL) is a pleiotropic polypeptide hormone secreted primarily by the lactotrophic cells of anterior pituitary gland in vertebrates (Freeman et al. 2000). This hormone family includes placental lactogen (PL) and growth hormone (GH) (Corbacho et al. 2002). Prolactin plays a major role in lactation and reproduction and has been shown to have a multitude of effects relating to growth, development, metabolism, immunoregulation and protection (Ben-Jonathan et al. 2006). The prolactin signaling pathway is initiated by the binding of prolactin with the prolactin receptor (PRLR), a member of class I cytokine receptor superfamily (Binart et al. 2000), which is expressed in a variety of tissues. The PRLR comprises of an extracellular ligand binding domain, a transmembrane domain and an intracellular domain. The PRLR lacks intrinsic kinase activity and transduces signal through kinases that interact with its cytoplasmic tail. Three constitutively active variants of the receptor have been reported in humans (Goffin et al. 2010). Though the signaling reactions downstream of the longest isoform of prolactin receptor have been well established, little is known about prolactin signaling initiated by six other isoforms (Bouilly et al. 2011). Studies also indicate that binding affinity of the human prolactin receptor to nonhuman prolactin is lower than human prolactin (Utama et al. 2009). The prolactin receptor also binds to hPL and hGH leading to the activation of downstream pathways. However, we have not considered these reactions in the current study. This study reports only those reactions, which occur upon stimulation of prolactin receptor with prolactin, based on the criteria described previously (Nanjappa et al. 2011). Availability of signaling pathway information is useful to the biomedical research community, especially for systems biology approaches. Considering this, we have developed ‘NetPath’ as a resource of ligand-receptor specific signal transduction pathways (Kandasamy et al. 2010). As a part of this, we have carried out manual annotation of available information from the published literature for ligand-receptor signaling pathways (Raju et al. 2011a; Nanjappa et al. 2011; Telikicherla et al. 2011; Goel et al. 2012). Similarly, in this study, we enriched publicly available information pertaining to prolactin-prolactin receptor dependent signaling pathways and also generated a graphic map depicting the prolactin signaling pathway.

Characterization of mammary epithelial cell line HC11 using the NIA 15k gene array reveals potential regulators of the undifferentiated and differentiated phenotypes

Differentiation of undifferentiated mammary epithelial stem and/or progenitor cells results in the production of luminal-ductal and myoepithelial cells in the young animal and upon pregnancy, the production of luminal alveolar cells. A few key regulators of differentiation have been identified, though it is not known yet how these proteins function together to achieve their well-orchestrated products. In an effort to identify regulators of early differentiation, we screened the NIA 15k gene array of 15,247 developmentally expressed genes using mouse mammary epithelial HC11 cells as a model of differentiation. We have confirmed a number of genes preferentially expressed in the undifferentiated cells (Lgals1, Ran, Jam-A and Bmpr1a) and in those induced to undergo differentiation (Id1, Nfkbiz, Trib1, Rps21, Ier3). Using antibodies to the proteins encoded by Lgals1, and Jam-A, we confirmed that their proteins levels were higher in the undifferentiated cells. Although the amounts of bone morphogenetic protein receptor-1A (BMPR1A) protein were present at all stages, we found the activity of its downstream signal transduction pathway, as measured by the presence of phosphorylated-SMAD1, -SMAD5, and -SMAD8, is elevated in undifferentiated cells and decreases in fully differentiated cells. This evidence supports that the BMPR1A pathway functions primarily in undifferentiated mammary epithelial cells. We have identified a number of genes, of known and unknown function, that are candidates for the maintenance of the undifferentiated phenotype and for early regulators of mammary alveolar cell differentiation.

A sparing procedure to clear the mouse mammary fat pad of epithelial components for transplantation analysis

Summary Transplantation of epithelial cells into cleared fat pads is a widely used technique in the study of mammary gland biology. It was first described in 1959 and has remained a valuable technique, most recently in conjunction with the analysis of mammary anlagen from knockout mice with an embryonic lethal phenotype or reproductive defect, and for mammary epithelial stem-cell assays or analysis of precancerous cells. Mammary glands, unlike most other organs, mainly develop postnatally. When the small amount of endogenous epithelium present in the fat pad of a prepubertal mouse is removed, this clearance leaves a natural microenvironment that can be repopulated with exogenously supplied epithelial cells. Cells with the appropriate developmental potential (stem cells or progenitor cells) can regenerate the epithelial portion of the mammary gland after puberty and pregnancy. The conventional clearance of the fat pad is an involved surgical procedure. We have improved the technique and minimized surgery and recovery time, while maintaining an efficient removal of endogenous epithelium from the mammary fat pad.

The Breast Cancer to Bone (B2B) Metastases Research Program: a multi-disciplinary investigation of bone metastases from breast cancer

BackgroundBone is the most common site of breast cancer distant metastasis, affecting 50–70 % of patients who develop metastatic disease. Despite decades of informative research, the effective prevention, prediction and treatment of these lesions remains elusive. The Breast Cancer to Bone (B2B) Metastases Research Program consists of a prospective cohort of incident breast cancer patients and four sub-projects that are investigating priority areas in breast cancer bone metastases. These include the impact of lifestyle factors and inflammation on risk of bone metastases, the gene expression features of the primary tumour, the potential role for metabolomics in early detection of bone metastatic disease and the signalling pathways that drive the metastatic lesions in the bone.Methods/DesignThe B2B Research Program is enrolling a prospective cohort of 600 newly diagnosed, incident, stage I-IIIc breast cancer survivors in Alberta, Canada over a five year period. At baseline, pre-treatment/surgery blood samples are collected and detailed epidemiologic data is collected by in-person interview and self-administered questionnaires. Additional self-administered questionnaires and blood samples are completed at specified follow-up intervals (24, 48 and 72 months). Vital status is obtained prior to each follow-up through record linkages with the Alberta Cancer Registry. Recurrences are identified through medical chart abstractions. Each of the four projects applies specific methods and analyses to assess the impact of serum vitamin D and cytokine concentrations, tumour transcript and protein expression, serum metabolomic profiles and in vitro cell signalling on breast cancer bone metastases.DiscussionThe B2B Research Program will address key issues in breast cancer bone metastases including the association between lifestyle factors (particularly a comprehensive assessment of vitamin D status) inflammation and bone metastases, the significance or primary tumour gene expression in tissue tropism, the potential of metabolomic profiles for risk assessment and early detection and the signalling pathways controlling the metastatic tumour microenvironment. There is substantial synergy between the four projects and it is hoped that this integrated program of research will advance our understanding of key aspects of bone metastases from breast cancer to improve the prevention, prediction, detection, and treatment of these lesions.

The Role of Prolactin in Bone Metastasis and Breast Cancer Cell-Mediated Osteoclast Differentiation.

BACKGROUND Metastasis to the bone is a deleterious aspect of breast cancer and is a preferred site that results in bone loss. Hormones such as prolactin (PRL) have not yet been studied for their role in modulating the secondary tumor bone microenvironment. METHODS We used quantitative immunohistochemistry with 134 samples of human primary breast cancer and 17 matched primary breast cancers and bone metastases. A Cox proportional hazards regression model was fitted to evaluate the associations between high prolactin receptor (PRLR) expression and time to bone metastasis, adjusting for estrogen receptor status, lymph node status, and chemotherapy status. We assessed osteoclast differentiation, osteoclast size, and measured pit formation in dentine slices. Statistical tests were two-sided. RESULTS High PRLR expression in the primary breast tumor was associated with a shorter time to metastasis that includes bone (PRLRAQUA Max-per 100 unit hazard ratio = 1.04, 95% confidence interval = 1.00 to 1.07, P = .03). We observed the PRLR in rare samples of bone metastases and matched primary breast cancer. PRL treatment of breast cancer cells induced osteoclast differentiation and bone lysis via secreted factors and was abrogated by a PRLR antagonist (delta1-9-G129R-hPRL). We demonstrated that sonic hedgehog is a PRL-regulated cytokine in breast cancer cells and part of the mechanism that induces osteoclast differentiation. CONCLUSIONS Our evidence indicates that PRL-PRLR can escalate the impact of breast cancer on bone metastasis and that the presence of the PRLR in the tumor microenvironment of breast cancer bone metastasis has the potential to modulate the microenvironment to induce lytic osteoclast formation.

Prolactin receptor in breast cancer: marker for metastatic risk

Prolactin and prolactin receptor signaling and function are complex in nature and intricate in function. Basic, pre-clinical and translational research has opened up our eyes to the understanding that prolactin and prolactin receptor signaling function differently within different cellular contexts and microenvironmental conditions. Its multiple roles in normal physiology are subverted in cancer initiation and progression, and gradually we are teasing out the intricacies of function and therapeutic value. Recently, we observed that prolactin has a role in accelerating the time to bone metastasis in breast cancer patients and identified the mechanism by which prolactin stimulated breast cancer cell-mediated lytic osteoclast formation. The possibility that the prolactin receptor is a marker for metastasis, and specifically bone metastasis, is one that may have to be put into the context of the different variants of prolactin, different prolactin receptor isoforms and intricate signaling pathways that are regulated by the microenvironment. The more complete the picture, the better one can test biomarker identity and design clinical trials to test therapeutic intervention. This review will cover the recent advances and highlight the complexity of prolactin receptor biology.

Mammary epithelial stem and progenitor cells and the prolactin pathway.

Prolactin is a pleiotropic peptide hormone and cytokine that is secreted from the pituitary gland and locally within various tissues of the body for autocrine and paracrine signal transduction. It controls proliferation and differentiation in a number of body tissues and increasing evidence indicates that it controls these functions in undifferentiated stem and progenitor cells of adult tissues, such as mesenchymal stem cells, hematopoietic progenitors, neural stem cells, oligodendrocyte precursor cells and possibly in mammary gland stem/progenitor cells. These roles in these undifferentiated cell types also implicate prolactin in the stem cell theory of cancer, supporting its known roles in cancer formation and progression.