Cellular Plasticity and Heterotypic Interactions during Breast Morphogenesis and Cancer Initiation

Publisher Copyright: © 2022 by the authors.The human breast gland is a unique organ as most of its development occurs postnatally between menarche and menopause, a period ranging from 30 to 40 years. During this period, the monthly menstruation cycle drives the mammary gland through phases of cell proliferation, differentiation, and apoptosis, facilitated via a closely choreographed interaction between the epithelial cells and the surrounding stroma preparing the gland for pregnancy. If pregnancy occurs, maximal differentiation is reached to prepare for lactation. After lactation, the mammary gland involutes to a pre-pregnant state. These cycles of proliferation, differentiation, and involution necessitate the presence of epithelial stem cells that give rise to progenitor cells which differentiate further into the luminal and myoepithelial lineages that constitute the epithelial compartment and are responsible for the branching structure of the gland. Maintaining homeostasis and the stem cell niche depends strongly on signaling between the stem and progenitor cells and the surrounding stroma. Breast cancer is a slowly progressing disease whose initiation can take decades to progress into an invasive form. Accumulating evidence indicates that stem cells and/or progenitor cells at different stages, rather than terminally differentiated cells are the main cells of origin for most breast cancer subgroups. Stem cells and cancer cells share several similarities such as increased survival and cellular plasticity which is reflected in their ability to switch fate by receiving intrinsic and extrinsic signals. In this review, we discuss the concept of cellular plasticity in normal breast morphogenesis and cancer, and how the stromal environment plays a vital role in cancer initiation and progression.Peer reviewe

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.Peer reviewe

Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum

Publisher's version (útgefin grein)Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.This research was supported by the Icelandic Technology Development Fund with Grant No. 163922-0611, Landsvirkjun Energy Research Fund and NYUAD faculty research funds (AD060).Peer Reviewe

Melflufen, a peptide‐conjugated alkylator, is an efficient anti‐neo‐plastic drug in breast cancer cell lines

Publisher's version (útgefin grein)Melphalan flufenamide (hereinafter referred to as “melflufen”) is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins ƴH2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.We thank Inovotion for their work and support in the CAM assays.Peer Reviewe

Samspil æðaþels og eðlilegs og illkynja þekjuvefjar úr brjóstkirtli í þrívíðri frumurækt

Brjóstkirtillinn samanstendur af tveimur megingerðum þekjuvefsfruma, kirtilþekju- og vöðvaþekjufrumum. Saman mynda þessar frumugerðir hina greinóttu formgerð brjóstkirtilsins. Kirtilvefurinn er umlukinn æðaríkum stoðvef sem inniheldur margar mismunandi frumugerðir, þ.m.t. bandvefsfrumur og æðaþelsfrumur. Þroskun og sérhæfing kirtilsins er mjög háð samskiptum hans við millifrumuefni brjóstsins og frumur stoðvefjarins. Mest áhersla hefur verið lögð á rannsóknir á bandvefsfrumum í þessu tilliti, en minni athygli beint að æðaþelsfrumum, sem voru lengi taldar gegna því hlutverki einu að miðla súrefni og næringu um líkamann. Á síðustu árum hefur verið sýnt fram á að nýmyndun æða í krabbameinsæxlum spili stórt hlutverk í framþróun æxlisvaxtar og hefur það verið tengt slæmum horfum. Nýlegar rannsóknir hafa sýnt fram á mikilvægt hlutverk æðaþels í þroskun og sérhæfingu ýmissa líffæra, til dæmis í heila, lifur og beinmerg sem og í framþróun krabbameins. Nýleg þekking bendir einnig til mikilvægra áhrifa æðaþels á þroskun eðlilegs og illkynja brjóstvefjar. Markmið verkefnisins er að kanna áhrif brjóstaæðaþels á eðlilegar og illkynja brjóstaþekjufrumulínur og nota til þess þrívíð ræktunarlíkön sem þróuð voru á rannsóknastofunni, sem og að endurbæta þessi líkön til frekari rannsókna á samskiptum æðaþels og þekjufruma. Flestar frumulínur eru af kirtilþekjuuppruna en skortur er á frumulínum sem endurspegla svipgerð vöðvaþekjunnar. Búin var til ný frumulína af vöðvaþekju uppruna úr vef fengnum úr brjóstaminnkunaraðgerð. Frumulínan hefur svipgerð samskonar þeirri sem sjá má í vöðvaþekjufrumum. Í samrækt með æðaþeli mátti sjá mikla aukningu á stærð frumuþyrpinga, sem og á fjölda frumuþyrpinga miðað við viðmið án æðaþels, bæði hjá eðlilegum og illkynja þekjufrumum. Til að kanna betur þessi áhrif var þróað nýtt samræktunarlíkan sem nota má til að greina áhrif leysanlegra þátta á þekjufrumur. Niðurstöður benda til að áhrifum æðaþelsfrumanna sé miðlað af leysanlegum þáttum sem dreifast um gelið, en ekki af beinni snertingu á milli frumugerðanna. Áframhaldandi rannsóknir á samskiptum æðaþels og þekjufruma og greining á þáttum sem eiga þar hlut munu varpa mikilvægu ljósi á hlutverk æðaþels í þroskun brjóstkirtilsins og í framþróun krabbameins

Modeling the role of the EGFR receptor family in the normal and malignant breast gland

Brjóstkirtillinn er gerður úr greinóttum kirtilgöngum með upphaf í geirvörtu en greinist svo inn á við og endar í kirtilberjum, sem eru seytunarhluti kirtilsins. Kirtilgangarnir eru umluktir æðaþelsríkum bandvef. Stofnfrumur í brjóstkirtlinum gera kirtlinum kleift að ganga í gegnum endurtekna hringrás frumufjölgunar, sérhæfingar og frumudauða tengt meðgöngu og mjólkurframleiðslu. Stofnfrumur í brjóstkirtli hafa á undanförnum árum verið tengdar myndun brjóstakrabbameina. Brjóstakrabbamein má flokka í marga undirhópa, byggt á tjáningu kennipróteina. Sýnt hefur verið fram á að EGF viðtakafjölskyldan gegnir mikilvægu hlutverki í bæði eðlilegum brjóstkirtli, en einnig í krabbameinsmyndun. Lokastig krabbameins¬framvindu er myndun meinvarpa, þar sem frumur losna frá æxlinu og mynda meinvörp í öðrum líffærum. Hlutverk æðaþelsins í þessu ferli hefur yfirleitt verið álitið einskorðað við flutning næringarefna og súrefnis til æxlisins, en nokkrar rannsóknir hafa sýnt fram á beint hlutverk æðaþels í þroskun, meðal annars með því að ýta undir bandvefsummyndun þekjufruma (epithelial to mesenchymal transition, EMT). Markmið þessa doktorsverkefnis er að þróa þrívítt ræktunarlíkan til að kanna samspil æðaþels og þekjuvefjar í brjóstkirtli. Ennfremur er markmiðið að nota þetta ræktunarlíkan til að kanna hlutverk EGFR og ErbB2 í þroskun og sérhæfingu eðlilegra og illkynja brjóstaþekjufruma. Í fyrstu greininni sem lögð er fram með ritgerðinni sýnum við hvernig æðaþelsfrumur sem einangraðar voru úr heilbrigðum brjóstvef geta hvatað vöxt heilbrigðra óummyndaðra brjóstaþekjufruma. Samræktanir á æðaþeli og þekjufrumufrumulínum sýndi fram á að æðaþel virkar sem vaxtarhvetjandi þáttur í umhverfinu. Í grein 2 könnum við áhrif þess að fjarlægja EGF úr ræktunaræti frumulínunnar A163 – þessi breyting leiðir til mikillar mögnunar á EGFR geninu í A163, sem breytir svipgerð hennar í þrívíðu umhverfi. Þessi breyting varð til þess að við skoðuðum hlutverk EGFR boðleiðarinnar í myndun greinóttrar formgerðar. Við notuðum stofnfrumulínuna D492 í grein 3 og slógum niður Sprouty-2, sem er neikvæður afturkasthindri á Týrósín kínasa boðleiðum. Við sáum að með því að slá niður Sprouty fengum við fram mikla aukningu í myndun greinóttra frumuþyrpinga. Ennfremur sáum við að í samrækt með æðaþeli kom fram mikil aukning á myndun bandvefslíkra frumuþyrpinga, þar sem frumur höfðu undirgengist EMT. Í fjórðu greininni könnuðum við áhrif þess að yfirtjá EGFR og ErbB2 í D492. Við sáum að ErbB2 yfirtjáning leiddi til taps á bæði EGFR og þekjuvefspróteinum og aukningar á bandvefspróteinum, eða EMT. Þessari svipgerð gátum við snúið að hluta við með því að yfirtjá EGFR með ErbB2. Þegar þessar frumur voru græddar í mýs mynduðu ErbB2 yfirtjáandi frumur stór bandvefslík æxli, en æxli með bæði EGFR og ErbB2 voru mun minni, og sýndu þekjuvefssvipgerð. Niðurstaða verkefnisins er að æðaþel getur haft mikil áhrif á svipgerð þekjuvefsfruma í þrívíðri rækt, ennfremur að EGF viðtakafjölskyldan, sérstaklega EGFR gegnir mjög mismunandi hlutverkum í frumum, og getur jafnvel þjónað hlutverki krabbameinsbælipróteins, en hingað til hefur EGFR verið álitinn hvetja krabbameinsmyndun. Líkanið sem við höfum þróað má nota til áframhaldandi rannsókna á samspili þessara viðtaka í klínísku samhengiThe mammary gland consists of epithelial ducts originating from the nipple and branches inwards, terminating in acini, the functional, milk producing unit of the breast. Stem cells within the mammary gland enable repeated cycles of proliferation, differentiation and involution during pregnancy and lactation, and also to a lesser degree during each menstrual cycle. Breast cancer can be stratified into several subgroups, based on marker expression. Formation of distant metastases represents the final progression of breast cancer. The role of endothelium during development and cancer progression has been considered that of oxygen and nutrient transport, in addition to facilitating cancer cell metastasis, while some research has shown the importance of endothelial derived signals during normal development. The aim of this thesis was to design a culture system that could be utilized for modeling endothelial-epithelial interaction. Additionally, the aim was to use this culture system to analyze the role of EGFR and ErbB2 signaling pathways in the epithelial stem cell line D492, which can form branching colonies when cultured in a three-dimensional environment. The first paper submitted with this thesis demonstrates how normal primary endothelial cells facilitate increased proliferation of primary epithelial cells. This was further supported by co-culturing endothelial cells with established cell lines. Paper two shows how removal of EGF from the culture media results in strong upregulation of EGFR in the breast cell lines A163 through amplification of the EGFR gene. This change results in a stronger basal-like phenotype, loss of polarity and abnormal three-dimensional behavior. These data emphasized the importance of EGFR signaling during morphogenesis. We used the D492 cell line in paper three to analyze the role of sprouty-2, a modulator of receptor tyrosine kinase signaling. We observed that knockdown of Sprouty-2 resulted in greatly enhanced branching morphogenesis. Additionally, we observed an increased tendency for D492 cells to undergo epithelial to mesenchymal transition (EMT) in co-culture with endothelial cells. In the fourth paper we overexpressed ErbB2 and EGFR in D492 cells. We saw that ErbB2 overexpression resulted in downregulation of EGFR, in addition to loss of epithelial cadherin and keratin expression, and gain of mesenchymal markers and N-cadherin. This phenotype could be partially reversed by overexpressing EGFR along with ErbB2. ErbB2 overexpression also enhanced tumor formation in mice, where large tumors with a mesenchymal phenotype were observed. Dual expression of EGFR and ErbB2 reduced tumor growth, and tumors exhibited a stronger epithelial phenotype. The result of this thesis is that endothelial cells can greatly influence proliferation and morphology of epithelial cells in three-dimensional culture. Additionally, the EGF receptor plays a highly varied role in epithelial cells, and could have different prognostic values in breast cancer. Traditionally EGFR has been described as an oncogene, facilitating tumor formation, but in the context of ErbB2 expression, EGFR could serve as a tumor suppressor, by maintaining epithelial marker expression, thereby reducing invasiveness. These data indicate that EGFR and ErbB2 must be valued together when tumors are evaluated for treatment options