Bylgja Hilmarsdottir

Functional Role of the microRNA-200 Family in Breast Morphogenesis and Neoplasia

Branching epithelial morphogenesis is closely linked to epithelial-to-mesenchymal transition (EMT), a process important in normal development and cancer progression. The miR-200 family regulates epithelial morphogenesis and EMT through a negative feedback loop with the ZEB1 and ZEB2 transcription factors. miR-200 inhibits expression of ZEB1/2 mRNA, which in turn can down-regulate the miR-200 family that further results in down-regulation of E-cadherin and induction of a mesenchymal phenotype. Recent studies show that the expression of miR-200 genes is high during late pregnancy and lactation, thereby indicating that these miRs are important for breast epithelial morphogenesis and differentiation. miR-200 genes have been studied intensively in relation to breast cancer progression and metastasis, where it has been shown that miR-200 members are down-regulated in basal-like breast cancer where the EMT phenotype is prominent. There is growing evidence that the miR-200 family is up-regulated in distal breast metastasis indicating that these miRs are important for colonization of metastatic breast cancer cells through induction of mesenchymal to epithelial transition. The dual role of miR-200 in primary and metastatic breast cancer is of interest for future therapeutic interventions, making it important to understand its role and interacting partners in more detail.

Tyrosine kinase mutations in gastrointestinal stromal tumors in a nation-wide study in Iceland.

Tryggvason G, Hilmarsdottir B, Gunnarsson GH, Jónsson JJ, Jónasson JG, Magnússon MK. Tyrosine kinase mutations in gastrointestinal stromal tumors in a nation‐wide study in Iceland. APMIS 2010; 118: 648–56.

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal breast epithelium, what effects they have on cellular differentiation and how they participate in neoplastic transformation. D492 is a breast epithelial cell line with stem cell properties that can undergo epithelial to mesenchyme transition (EMT), generate luminal- and myoepithelial cells and form complex branching structures in three-dimensional (3D) culture. Here, we show that overexpression of HER2 in D492 (D492HER2) resulted in EMT, loss of contact growth inhibition and increased oncogenic potential in vivo. HER2 overexpression, furthermore, inhibited endogenous EGFR expression. Re-introducing EGFR in D492HER2 (D492HER2/EGFR) partially reversed the mesenchymal state of the cells, as an epithelial phenotype reappeared both in 3D cultures and in vivo. The D492HER2/EGFR xenografts grow slower than the D492HER2 tumors, while overexpression of EGFR alone (D492EGFR) was not oncogenic in vivo. Consistent with the EGFR-mediated epithelial phenotype, overexpression of EGFR drove the cells toward a myoepithelial phenotype in 3D culture. The effect of two clinically approved anti-HER2 and EGFR therapies, trastuzumab and cetuximab, was tested alone and in combination on D492HER2 xenografts. While trastuzumab had a growth inhibitory effect compared with untreated control, the effect of cetuximab was limited. When administered in combination, the growth inhibitory effect of trastuzumab was less pronounced. Collectively, our data indicate that in HER2-overexpressing D492 cells, EGFR can behave as a tumor suppressor, by pushing the cells towards epithelial differentiation.

Expression and functional role of sprouty-2 in breast morphogenesis.

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.

Metabolic re-wiring of isogenic breast epithelial cell lines following epithelial to mesenchymal transition

Epithelial to mesenchymal transition (EMT) has implications in tumor progression and metastasis. Metabolic alterations have been described in cancer development but studies focused on the metabolic re-wiring that takes place during EMT are still limited. We performed metabolomics profiling of a breast epithelial cell line and its EMT derived mesenchymal phenotype to create genome-scale metabolic models descriptive of both cell lines. Glycolysis and OXPHOS were higher in the epithelial phenotype while amino acid anaplerosis and fatty acid oxidation fueled the mesenchymal phenotype. Through comparative bioinformatics analysis, PPAR-γ1, PPAR- γ2 and AP-1 were found to be the most influential transcription factors associated with metabolic re-wiring. In silico gene essentiality analysis predicts that the LAT1 neutral amino acid transporter is essential for mesenchymal cell survival. Our results define metabolic traits that distinguish an EMT derived mesenchymal cell line from its epithelial progenitor and may have implications in cancer progression and metastasis. Furthermore, the tools presented here can aid in identifying critical metabolic nodes that may serve as therapeutic targets aiming to prevent EMT and inhibit metastatic dissemination.

Clinical phenotype in heterozygote and biallelic Bernard‐Soulier syndrome—A case control study

Bernard‐Soulier syndrome (BSS) is a rare severe autosomal recessive bleeding disorder. To date heterozygous carriers of BSS mutations have not been shown to have bleeding symptoms. We assessed bleeding using a semi‐quantitative questionnaire, platelet parameters, PFA‐100 closure times, ristocetin response, GP Ib/IX expression and VWF antigen in 14 BSS patients, 30 heterozygote carriers for related mutations and 29 controls. Eight mutations in GP1BA, GP1BB or GP9 were identified including four previously unknown pathogenic mutations. Subjects with BSS reported markedly more mucocutaneous bleeding than controls. Increased bleeding was also observed in heterozygotes. Compared to controls, patients with BSS had lower optical platelet counts (P < 0.001), CD61‐platelet counts (P < 0.001) and higher mean platelet volume (17.7 vs. 7.8 fL, P < 0.001) and ristocetin response and closure times were unmeasurable. Heterozygotes had higher MPV (9.7 fL, P < 0.001) and lower platelet counts (P < 0.001) than controls but response to ristocetin and closure times were normal. The VWF was elevated in both BSS and in heterozygotes (P = 0.005). We conclude that heterozygotes for BSS mutations have lower platelet counts than controls and show a bleeding phenotype albeit much milder than in BSS. Both patients with BSS and heterozygote carriers of pathogenic mutations have raised VWF. Am. J. Hematol. 90:149–155, 2015.

Context-Dependent Function of Myoepithelial Cells in Breast Morphogenesis and Neoplasia

Myoepithelial cells (MEPs) are specialized cells derived from epithelial progenitor cells, yet they also express the contractile machinery of smooth muscle cells. MEPs are prominent in glandular tissues where their function is to help expel secretions generated by the glandular epithelial cells. In the breast, MEPs are part of the bi-layered breast epithelium that line ducts and alveoli positioned perpendicular to the luminal epithelial cells (LEPs), separated from the surrounding stroma by the basement membrane. Researchers have recognized MEPs as important regulators of structural and functional behavior of LEPs, namely having role in polarization of LEPs, and regulating milk production. Furthermore, they have also been proposed to act as tumor suppressors as their presence inhibits invasion of cancer cells into the surrounding stroma. There is, however, accumulating evidence that MEPs in normal breast, carcinoma in situ and in invasive breast cancer differ significantly in terms of marker expression and this may truly interfere with their ability to behave as tumor suppressors. The term myoepithelial cell is often used synonymously with basal cell. While all MEPs, due to their position, can be referred to as basal cells, some basal cells do not fulfill the criteria of being MEPs. Synonymous use of these terms may hold true under normal conditions but careful interpretation of these terms should be used in breast cancer. In recent years, partial myoepithelial differentiation and epithelial to mesenchymal transition (EMT) have been shown to be associated with, and in some cases, necessary for cancer invasion and metastasis. In this review, we will discuss the context-dependent role of MEPs in breast morphogenesis, tumor suppression, and also the appearance of basal or partial myoepithelial differentiation in aggressive forms of breast cancer.

Abstract 5251: Branching morphogenesis in the mammary gland is regulated by sprouty-2

Branching morphogenesis is a conserved mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including the epidermal growth factor receptor (EGFR), and their intracellular regulators, the sprouty protein family is believed to be a critical regulator of branching morphogenesis. In this study, we show that, Sprouty-2 (Spry-2) is predominantly expressed in the luminal epithelial cells both in ducts and lobuli in the human breast gland. We have also analyzed the expression of Spry-2 and EGFR pathway in virgin, lactating and pregnant mouse mammary gland. Spry-2 is expressed at branching epithelial buds during pregnancy with increased expression during lactation. The expression of phosphorylated EGFR (Y1068) shows a similar expression pattern as Spry-2. Using D492 a breast epithelial cell line with stem cell properties, that generate branching structures in 3D laminin rich gel we show that Spry-2 expression increases during the formation of branching. Immunostaining locates expression of Spry-2 and active EGFR at the tip of lobular-like, branching ends. Interestingly, knocking down (KD) spry-2 expression using shRNA resulted in increased migration. Spry-2 knockdown also gives larger and more complex branching structures indicating loss of negative feedback control of branching morphogenesis. In co-culture with endothelial cells, D492 spry-2 KD cells generate predominantly spindle like colonies reminiscent of epithelial to mesenchymal tansition. In conclusion, these data indicate that Spry-2 is an important regulator of branching morphogenesis and dysregulation of sprouty expression can lead to aberrant development related to breast cancer, such as epithelial to mesenchymal transition in the mammary gland. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5251. doi:1538-7445.AM2012-5251