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2019 Medical Technologies Congress, TIPTEKNO 2019; Palm Wings Ephesus HotelIzmir; Turkey; 3 October 2019 through 5 October 2019, Phase contrast optical microscopy is a preferred imaging technique for live-cell, temporal analysis. Segmentation of cells from time series data acquired with this technique is a labor-intensive and time-consuming task that cell biology researchers need solution for. In this study traditional image processing and deep learning based approaches for automated cell segmentation from phase contrast optical microscopy time series are presented, and their performances are evaluated against manually annotated datasets. © 2019 IEEE., Faz kontrast optik mikroskopi hücrelerin canlı ortamlarında zamana bağlı incelenmesi için tercih edilen görüntüleme yöntemidir. Bu yöntem ile elde edilen zaman serisi görüntülerinde hücrelerin bölütlenmesi işi hücre biyolojisi araştırmacılarının çözümüne ihtiyaç duyduğu emek yoğun ve zaman alan bir iştir. Bu çalışmada faz kontrast optik mikroskopi zaman serilerinde hücrelerin otomatik bölütlenmesi için geleneksel görüntü işleme ve derin öğrenme temelli yöntemler önerilmiş ve başarımları elle işaretlenmiş veri kümelerinde nicel olarak ölçülmüştür.

Thesis (Master)--Izmir Institute of Technology, Molecular Biology and Genetics, Izmir, 2019, Includes bibliographical references (leaves: 45-51), Text in English; Abstract: Turkish and English, Breast cancer, the most commonly diagnosed cancer type and the leading cause of cancer-associated deaths, is the major health issue among women worldwide. In many cancer types, the expression of the semaphorins and their receptors such as plexins and neuropilins are dysregulated. SEMA6D is a member of class-6 family transmembrane semaphorin proteins and acts through Plexin-A1 receptor. It was previously shown that overexpression of SEMA6D in breast cancer cell line MCF-7 leads to a reduction in proliferation and an increase in migration. On the other hand, in the MDA-MB-231 breast cancer cell line, overexpression of SEMA6D had no significant effect on proliferation but enhanced migration. In this study, we aimed to analyze the effects of SEMA6D overexpression in normal breast cell line MCF10A and investigate the invasive behavior and transformation capacity of SEMA6D overexpressing breast cancer cell lines. We demonstrated that overexpression of SEMA6D leads to elevated proliferation, viability and migration in MCF10A cells, whereas it did not trigger their anchorage-independent growth. On the other hand, MDA-MB-231 and MCF7 cells stably expressing SEMA6D showed reduced colony formation in the soft-agar assay. Furthermore, the invasiveness of MDA-MB-231 cells was elevated with SEMA6D overexpression, whereas SEMA6D overexpression did not stimulate the invasiveness of MCF-7 cells through matrigel microenvironment, whereas slightly trigger invasion through bone microenvironment. In conclusion, SEMA6D overexpression has cell-specific effects on breast cancer. The exact role of SEMA6D in breast cancer development remains undefined and must be further investigated., Meme kanseri, kadınlarda en sık teşhis edilen kanser türü olmakla birlikte dünya çapında kansere bağlı ölümlerin ikinci önde gelen nedenidir. Bir çok insanda bulunan kanser çeşitlerinde semaphorinler ve receptörleri olan plexin ve neuropilinler anormal bir şekilde ekspres edilmektedir. SEMA6D, grup 6 transmembran semaphorin proteinlerinin bir üyesidir ve sinyalini hedef yerlere reseptörü olan Plexin-A1 ile iletir. Önceki çalışmalarımızda, SEMA6D aşırı ekspresyonunun MCF-7 meme kanseri hücrelerinin proliferasyonunda azalmaya sebep olduğu ancak hücre göçünde artışa sebep olduğu gösterilmiştir. MDA-MB-231 meme kanseri hücresinde ise, SEMA6D fazla ekspresyonu proliferasyonu anlamlı olarak etkilemezken hücre göçünü arttırdığı gözlemlenmiştir. Bu çalışmada, SEMA6D aşırı ekspresyonunun MCF10A normal meme epitel hücrelerindeki etkisinin araştırılması ve meme kanseri hücrelerindeki invazif davranışları ve transformasyon kapasitesi üzerine etkisinin incelenmesi hedeflenmiştir. MCF10A hücrelerinde SEMA6D fazla ekspresyonunun proliferasyonda ve hücre göçünde artışa sebep olduğu gösterilmiştir, ancak yumuşak agarda herhangi bir koloni oluşumu gözlenmemiştir. Meme kanseri hücreleri olan MDA-MB-231 ve MCF-7 da SEMA6D fazla-ifadesi yumuşak agarda koloni oluşumunu azaltmıştır. SEMA6D fazla-ifade edildiğinde MDA-MB-231 hücrelerinin invazif karakterini arttırmıştır ancak invazif olmayan MCF-7 hücre hattında matrigel mikro çevresine doğru invasyonu tetiklememiştir fakat kemik mikro ortamına doğru invazyon görülmüştür. Sonuç olarak SEMA6D kanser ilerlemesinde hücre spesifik olarak önemli bir role sahiptir. SEMA6D' nin meme kanseri gelişimindeki net rolü halen belirsizliğini korumaktadır ve daha detaylı olarak incelenmelidir.

Medical Technologies Congress (TIPTEKNO); OCT 03-05, 2019; Izmir, Turkey, The quantitative and qualitative ascertainment of cell culture is integral to the robust determination of the cell structure analysis. Microscopy cell analysis and the epithet structures of cells in cell cultures are momentous in the fields of the biological research process. In this paper, we addressed the problem of phase-contrast microscopy under cell segmentation application. In our proposed method, we utilized the state-of-the-art deep learning models trained on our proposed dataset. Due to the low number of annotated images, we propose a multi-resolution network which is based on the U-Net architecture. Moreover, we applied multi-combination augmentation to our dataset which has increased the performance of segmentation accuracy significantly. Experimental results suggest that the proposed model provides superior performance in comparison to traditional state-of-the-art segmentation algorithms.

Meme kanseri, kadınlarda en sık teşhis edilen kanser türü olmakla birlikte dünya çapında kansere bağlı ölümlerin ikinci önde gelen nedenidir. Bir çok insanda bulunan kanser çeşitlerinde semaphorinler ve receptörleri olan plexin ve neuropilinler anormal bir şekilde ekspres edilmektedir. SEMA6D, grup 6 transmembran semaphorin proteinlerinin bir üyesidir ve sinyalini hedef yerlere reseptörü olan Plexin-A1 ile iletir. Önceki çalışmalarımızda, SEMA6D aşırı ekspresyonunun MCF-7 meme kanseri hücrelerinin proliferasyonunda azalmaya sebep olduğu ancak hücre göçünde artışa sebep olduğu gösterilmiştir. MDA-MB-231 meme kanseri hücresinde ise, SEMA6D fazla ekspresyonu proliferasyonu anlamlı olarak etkilemezken hücre göçünü arttırdığı gözlemlenmiştir. Bu çalışmada, SEMA6D aşırı ekspresyonunun MCF10A normal meme epitel hücrelerindeki etkisinin araştırılması ve meme kanseri hücrelerindeki invazif davranışları ve transformasyon kapasitesi üzerine etkisinin incelenmesi hedeflenmiştir. MCF10A hücrelerinde SEMA6D fazla ekspresyonunun proliferasyonda ve hücre göçünde artışa sebep olduğu gösterilmiştir, ancak yumuşak agarda herhangi bir koloni oluşumu gözlenmemiştir. Meme kanseri hücreleri olan MDA-MB-231 ve MCF-7 da SEMA6D fazla-ifadesi yumuşak agarda koloni oluşumunu azaltmıştır. SEMA6D fazla-ifade edildiğinde MDA-MB-231 hücrelerinin invazif karakterini arttırmıştır ancak invazif olmayan MCF-7 hücre hattında matrigel mikro çevresine doğru invasyonu tetiklememiştir fakat kemik mikro ortamına doğru invazyon görülmüştür.Sonuç olarak SEMA6D kanser ilerlemesinde hücre spesifik olarak önemli bir role sahiptir. SEMA6D' nin meme kanseri gelişimindeki net rolü halen belirsizliğini korumaktadır ve daha detaylı olarak incelenmelidir. Breast cancer, the most commonly diagnosed cancer type and the leading cause of cancer-associated deaths, is the major health issue among women worldwide. In many cancer types, the expression of the semaphorins and their receptors such as plexins and neuropilins are dysregulated. SEMA6D is a member of class-6 family transmembrane semaphorin proteins and acts through Plexin-A1 receptor. It was previously shown that overexpression of SEMA6D in breast cancer cell line MCF-7 leads to a reduction in proliferation and an increase in migration. On the other hand, in the MDA-MB-231 breast cancer cell line, overexpression of SEMA6D had no significant effect on proliferation but enhanced migration. In this study, we aimed to analyze the effects of SEMA6D overexpression in normal breast cell line MCF10A and investigate the invasive behavior and transformation capacity of SEMA6D overexpressing breast cancer cell lines. We demonstrated that overexpression of SEMA6D leads to elevated proliferation, viability and migration in MCF10A cells, whereas it did not trigger their anchorage-independent growth. On the other hand, MDA-MB-231 and MCF7 cells stably expressing SEMA6D showed reduced colony formation in the soft-agar assay. Furthermore, the invasiveness of MDA-MB-231 cells was elevated with SEMA6D overexpression, whereas SEMA6D overexpression did not stimulate the invasiveness of MCF-7 cells through matrigel microenvironment, whereas slightly trigger invasion through bone microenvironment.In conclusion, SEMA6D overexpression has cell-specific effects on breast cancer. The exact role of SEMA6D in breast cancer development remains undefined and must be further investigated. 61

Gravity is an important biomechanical signal effecting the morphology and function of organisms. Reduction of gravitational forces, as experienced during spaceflight, cause alterations in the biological systems. Magnetic levitation technique is one of the most recent ground-based technology to mimic weightlessness environment. In addition to providing a platform to investigate biological effects of the weightlessness, this platform presents a novel opportunity to biofabricate 3-dimensional (3D) structures in a scaffold-and nozzle-free fashion. In this study, various controllable self-assembled 3D living structures were fabricated via magnetic levitation technique. This strategy may offer an easy and cost-effective opportunity for a wide range of space biotechnology researches., TUBITAK (215S86)

Magnetic levitation though negative magnetophoresis is a novel technology to simulate weightlessness and has recently found applications in material and biological sciences. Yet little is known about the ability of the magnetic levitation system to facilitate biofabrication of in situ three dimensional (3D) cellular structures. Here, we optimized a magnetic levitation though negative magnetophoresis protocol appropriate for long term levitated cell culture and developed an in situ 3D cellular assembly model with controlled cluster size and cellular pattern under simulated weightlessness. The developed strategy outlines a potential basis for the study of weightlessness on 3D living structures and with the opportunity for real-time imaging that is not possible with current ground-based simulated weightlessness techniques. The low-cost technique presented here may offer a wide range of biomedical applications in several research fields, including mechanobiology, drug discovery and developmental biology., Scientific and Technological Research Council of Turkey (215S862)

Notch sinyal yolağı gelişim ve tümör oluşumunda yer alan ana yolaklardan biridir. Notch aktivasyonu meme kanserinde migrasyon, invazyon ve mezenkimal geçiş (EMT) ile ilişkilidir. Fakat Notch aracılığıyla kanser gelişiminin mekanizması tam olarak anlaşılamamıştır. CYR61 salgılanan bir proteindir ve fazla ifadelenmesinin meme tümörleşmesi ile ilgili olduğu bilinmektedir. Microarray analizi CYR61 geninin Notch aktivasyonuyla farklı ifadelendiği açığa çıkarmıştır. Bu tez çalışmasında, CYR61'in EMT, migrasyon ve invazyon sırasında Notch alt aracısı olduğu hipotez edilmiştir.Notch yolağının EMT, migrasyon ve invazyon sırasında CYR61 e ihtiyacı olup olmadığını anlamak için iki tamamlayıcı yaklaşım takip edildi: (i) MCF10A epitelyum meme hücre hattında Notch varlığında CYR61 ifadelenmesi shRNA kullanılarak susturuldu, (ii) MDA-MB-231 meme kanseri hücre hattında Notch eksikliğinde CYR61 fazla ifadelendi. Sonrasında, EMT belirteçlerinin ifadelenmesi mRNA ve protein seviyesinde sırasıyla RT-qPCR ve immün-blotlama yöntemleriyle analiz edilmiştir. Hücrelerin migrasyon ve invazyon yetenekleri sırasıyla yara iyileşme analizi ve lab-on-a-chip sistemi ile araştırıldı. Burada, CYR61'in MCF10A hücre hattında Notch tarafından indüklenen EMT, migrasyon ve invazyonda önemli olduğu ve Notch aktivitesi olmadığında CYR61 in fazla ifadelenmesinin Notch tarafından indüklenen invazyonu MDA-MB-231 kanser hücrelerinde kısmi olarak kurtardığı gösterilmiştir. Bu bulgular CYR61 in Notch yolağının alt mekanizmasında rol alabileceğini ve Notch tarafından kontrol edilebileceğini önermektedir. CYR61'in Notch tarafından indüklenen EMT ve kanser gelişimdeki önemi düşünüldüğünde, CYR61'in hedef alınması, meme kanserini erken aşamalarda tedavi etmek için yeni tedavi stratejileri geliştirmede ümit vaat etmektedir. Notch signaling is one of the major pathways involved in development and tumorigenesis. Activated Notch is correlated with increased migration, invasion and epithelial-to-mesenchymal-transition (EMT) in breast cancer. However, mechanism of Notch-mediated cancer progression is poorly understood. CYR61 is a secreted protein and its upregulation is also related to increased breast tumorigenesis and EMT. Microarray analyses revealed that CYR61 was differentially expressed in response to Notch activation in breast epithelial cells. We hypothesized that CYR61 is a downstream mediator of Notch during EMT, migration and invasion.To test whether Notch requires CYR61 during EMT, migration and invasion, two complementary approaches were followed: (i) CYR61 expression was silenced by using shRNA in MCF10A epithelial breast cell line in the presence of Notch activation, (ii) CYR61 was over-expressed in MDA-MB-231 cancer breast cell line in the absence of Notch activity. Then, expression of EMT markers was analyzed in mRNA and protein levels via RT-qPCR and immuno-blotting, respectively. Migration and invasion ability of cells were investigated by wound healing assay and a lab-on-a-chip-system, respectively. Here, it was shown that CYR61 inhibition decreased Notch-induced EMT, migration and invasion of MCF10A and CYR61 overexpression in the absence of Notch activity partially rescued Notch-mediated invasion in MDA-MB-231 cell lines. Our findings suggest that CYR61 may act in downstream of Notch and is regulated by Notch. When we consider importance of CYR61 in Notch-induced EMT and cancer progression, targeting CYR61 may hold promise to develop novel strategies for treatment of breast cancer in early stages. 50

Meme kanseri dünyada yaygın görülen bir kanser çeşidi olmakla birlikte meme kanseriyle alakalı ölümler metastazdan kaynaklanmaktadır. Metastaz yapabilmek için kanser hücreleri göç etme ve invazyon özellikleri kazanırlar ve hücreler bu süreçte epitel-mezenkimal geçiş gösterebilir. SEMA6D, semaforin ailesine bağlı transmembran bir proteindir. SEMA6D embriyonik kıkırdak hücrelerinin göç etmesinde görev alır. Son zamanlarda osteosarkoma'da onkogen olarak değerlendirilmektedir. Ayrıca onkogen fonksiyonları, mide kanseri ve mezotelyoma'da da keşfedilmiştir. Biyoinformatik analize göre, SEMA6D'nin gen ifadesi üçlü negatif meme kanseri olan hastaların yaşamlarıyla ilişkilendirilmiştir fakat SEMA6D'nin meme kanseri üzerindeki görevleri ve fonksiyonları ile ilgili yayınlanmış bir çalışma bulunmamaktadır. Bu sebeple, bu tez çalışmasında SEMA6D'nin meme kanseri hücrelerinde çoğalma, epitel-mezenkimal geçiş ve göç etme özelliklerini anlamak amaç edinilmiştir. Bu çalışmada, SEMA6D'nin aşırı ekspresyonu, invazyon yeteneği olmayan meme kanseri hücre hattı MCF7'da çoğalmayı azaltmış fakat göç etmeyi arttırmıştır. SEMA6D'nin metastatik özelliği ayrıca epitel-mezenkimal geçiş ile de desteklenmiştir. Diğer yandan, SEMA6D'nin aşırı ekspresyonu metastatik ve agresif olan MDA-MB-231 hücre hattında çoğalmayı etkilememiş fakat göç etme yeteneğini azalttığı gözlemlenmiştir. Sonuç olarak, SEMA6D, MCF7 hücre hattında çoğalmayı, epitel-mezenkimal geçiş aracılığıyla göç etme yeteneğini arttırırken, metastatik olan MDA-MB-231 hücre hattında önemli bir değişiklik yapmamıştır. Bu yüzden, SEMA6D'nin görevlerini değerlendirirken farklı hücre hatlarında farklı etkilere yol açabileceğini göz önünde bulundurmak ve görevlerini daha detaylı anlamak için daha fazla çalışma yapmak gerekmektedir. Breast cancer is one of the most common cancer types around world and the second leading cause of cancer related deaths among women. Not the primary tumor but distant metastases are mainly the reason of deaths. For metastasis, the cells may go through epithelial-mesenchymal transition (EMT), and acquire migration and invasion abilities. SEMA6D is a transmembrane protein that belongs to a large semaphorin family. SEMA6D is involved in the migration of embryonic cardiac cells. Recently it was validated as an oncogene in osteosarcoma. Also, its oncogenic roles were investigated in gastric cancer and mesothelioma. According to in silico analysis of the Cancer Genome Atlas (TCGA), high SEMA6D expression level is associated with better survival of triple negative breast cancer patients. However, there is not any published study which investigates roles of SEMA6D in breast cancer yet, other than bioinformatic analysis. Therefore, we aimed to understand role of SEMA6D in proliferation, EMT and migration of breast cancer cells. We observed that overexpression of SEMA6D reduces proliferation but enhances migration in non-invasive breast cancer cell line MCF7. Thereby, SEMA6D may increase metastatic ability of MCF7 cells. Its metastatic ability was also supported by changes in EMT markers. On the other hand, proliferation of metastatic breast cancer cell line MDAMB231 was not significantly changed by overexpression of SEMA6D and migration ability was slightly reduced but mesenchymal markers tended to increase in SEMA6D overexpressing MDAMB231 cells. As a conclusion, SEMA6D tends to enhance proliferation, migration through EMT in MCF7 cell line whereas overexpression of SEMA6D did not demonstrate significant effect on metastatic MDA-MB-231 cell line. Therefore, we should separately evaluate role of SEMA6D in different breast cancer cell lines and further studies are required to understand role of SEMA6D in breast cancer. 45

Bisphenol A [BPA, 2,2,-bis (hydroxyphenyl) propane] is one of the highest-volume chemicals produced worldwide. It is detected in body fluids of more than 90% of the human population. Originally synthesized as an estrogenic compound, it is currently utilized to manufacture food and beverage containers resulting in uptake with food and drinks. There is concern that exposure to low doses of BPA, defined as less than or equal to 5 mg/kg body weight /d, may have developmental effects on various hormone-responsive organs including the mammary gland. Here, we asked whether perinatal exposure to a range of low doses of BPA is sufficient to alter mammary gland hormone response later on in life, with a possible impact on breast cancer risk. To mimic human exposure, we added BPA to the drinking water of C57/Bl6 breeding pairs. Analysis of the mammary glands of their daughters at puberty showed that estrogen-dependent transcriptional events were perturbed and the number of terminal end buds, estrogen-induced proliferative structures, was altered in a dose-dependent fashion. Importantly, adult females showed an increase in mammary epithelial cell numbers comparable to that seen in females exposed to diethylbestrol, a compound exposure to which was previously linked to increased breast cancer risk. Molecularly, the mRNAs encoding Wnt-4 and receptor activator of nuclear factor kB ligand, two key mediators of hormone function implicated in control of mammary stem cell proliferation and carcinogenesis, showed increased induction by progesterone in the mammary tissue of exposed mice. Thus, perinatal exposure to environmentally relevant doses of BPA alters long-term hormone response that may increase the propensity to develop breast cancer. © 2011 by The Endocrine Society., Swiss Science Foundation (NRP50); Swiss Federal Public Health Office

Notch yolağı'nın meme kanserindeki rolü ilk olarak MMTV (Mouse mammary tumor virus) virüsünün Notch4 lokusuna (Int3 lokus) entegre olup yolağın sürekli olarak aktif olmasına ve buna bağlı olarak meme tümörü oluşumuna yol açmasıyla ortaya çıkmıştır. Meme kanseri hasta örneklerinde aktif Notch1 ifadesinin fazla olduğu ve Notch1'in sürekli aktivasyonunun neoplazm oluşumuna yol açtığı gözlenmiştir. Meme kanserinde, Notch1 almaçının aşırı ekspresyonunun epitelyal-mezenkimal geçişi (EMG), Snai1 üzerinden tetiklediği gösterilmiştir. Fakat Notch yolağının EMG, migrasyon ve invazyonu gerçekleştirmek için hangi alt mekanizmaları kullandığı henüz tam olarak anlaşılmamıştır. Bu çalışmada, Notch'a bağlı EMG, migrasyon ve invazyona yol açan yeni Notch hedef genlerinin tanımlanması amaçlanmıştır. Mikrodizi analizlerinden faydalanılarak, Notch ile ilgisi bilinmeyen fakat, metastazla ilgili çalışmalarda yer alan yedi gen aday olarak belirlenmiştir. Normal meme hücrelerinde, Notch aktivasyonuna bağlı olarak aday genlerin ifadelerinin değiştiği gösterilmiştir. Bu genler, SEMA3C, SEMA6D, CXCL14, CXCR7, CCL20, CYR61 ve HMGA dır. MCF10A normal meme epitel hücre hattında, Notch1 aktivasyonu SEMA6D ve CYR61 mRNA ve protein ifadelerinde anlamlı artışa sebep olurken, HMGA2 ve SEMA3C genlerinin ifadesinde anlamlı azalışa sebep olmuştur. MDA-MB-231 meme kanseri hücre hattında, Notch yolağının inhibasyonu CYR61, CCL20 ve HMGA2 mRNA ifadelerinde ve sadece CYR61 protein ifadesinde anlmalı azalışa sebep olurken, diğer aday genlerinin ifadesinde çok az değişme sebep olmuştur. Bu geneler arasından SEMA6D daha ileri ki araştırmalar için seçilmiştir çünkü literatürde SEMA6D'nin meme kanserindeki rolünü gösteren bir çalışma yoktur. SEMA6D'nin Notch inhibasyonunun EMG belirteç molekülleri üzerindeki negatif etkisini kısmen kurtardığı görülmüştür ve Notch yolağının, EMG ve migrasyonu tetiklemek için SEMA6D yi aracı olarak kullanıyor olabileceği gösterilmiştir. SEMA6D'nin Notch sinyalinden bağımsız etkisi araştırılmıştır ve SEMA6D'nin MDA-MB-231 meme kanseri hücrelerinin yaşama kapasitesini anlamlı artırdığı ve migrasyonu tetiklediği gözlemlenmiştir. Notch signaling has first been described in murine mammary gland by the proviral integration of mouse mammary tumor virus (MMTV) into the Notch4 locus (Int3 locus) which resulted expression of constitutively active form of Notch4 and transformation of mammary epithelial cells. Notch1 is highly expressed in breast cancer and constitutively active form of Notch1 induces neoplasm. In breast cancer, overexpression of active Notch1 receptor (NICD) promotes epithelial-mesenchymal transition (EMT) via Snail induction which demonstrates the role of Notch signaling in induction of metastasis through EMT. However, the downstream mediators of Notch in EMT, migration and invasion processes are still elusive. In this study, we hypothesized that Notch signaling induces EMT and migration via regulating one or more of the seven candidate genes that are SEMA6D, SEMA3C, CXCR7, CXCL14, CCL20, HMGA2 and CYR61 which were shown to be differentially regulated by Notch signaling in breast cells in microarray data. The candidate genes are involved in EMT and migration in different cell types and tissues. We showed that Notch1 activation in normal breast epithelial cell line MCF10A significantly increased both mRNA and protein expressions of SEMA6D and CYR61 while it significantly reduced SEMA3C and HMGA2 mRNA levels. Notch inhibition led to significant reduction in mRNA expression of CYR61, CCL20 and HMGA2 and protein expression of CYR61 only, while the rest of candidate genes were affected slightly in breast cancer cell line, MDA-MB-231. We chose SEMA6D for further investigation because there is no data indicating the role of SEMA6D in breast cancer in the literature. SEMA6D could be mediator of Notch signaling to induce EMT because it partially rescues negative effect of Notch inhibition on EMT markers. Notch independent effect of SEMA6D suggested that SEMA6D may be involved in inhibiting EMT whereas, it induced migration and cell viability in MDA-MB-231 cell line. Further analysis is required to reveal the role of SEMA6D in EMT and migration. 67

Breast cancer, a heterogeneous disease comprised of tumors with different histological characteristics and clinical outcomes, is the leading cause of cancer deaths in women. Heterogeneous nature of the breast cancer demands delicate approaches to diagnose and follow the most appropriate strategy for clinical management. Based on microarray analysis of mRNA expression, four main molecular subtypes were identified: (a) luminal A, (b) luminal B, (c) basal-like, and (d) ERBB2(+). Even though molecular subtypes provided novel insights into our understanding of breast cancer heterogeneity, there is still room for improvement for better diagnostic, prognostic and therapeutic approaches. In this sense, epigenetics, specifically DNA methylation and histone modifications, have emerged as prominent candidates since several epigenetic factors were observed to be recurrently mutated in cancers including breast cancer. Recent advances in the field implicated that DNA methylation profiles and histone modifications are strongly associated with breast tumor subtypes and patient prognosis. Therefore, understanding contributions of epigenetics to breast cancer biology will lead to better diagnostic and prognostic strategies and will enable development of novel therapeutic approaches. © Springer International Publishing Switzerland 2016.

Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf-κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR+ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal-myoepithelial crosstalk with Wnt4 acting independent of PR perinatally. Synopsis This paper ascribes a new role for Wnt4 in pre-pubertal mammary gland development while revealing luminal cells to respond to Wnt activation. During regeneration, Wnt4 interacts with progesterone receptor signaling, correcting previous notions on RANKL signaling in this context. Wnt4 is an essential control factor for mammary epithelial stem cell function. RANKL is not required for mammary gland regeneration potential. Wnt4 activates canonical Wnt signaling in the basal/myoepithelial compartment. Progesterone receptor signaling is required for mammary epithelial Wnt4 expression already during puberty. This paper ascribes a new role for Wnt4 in pre-pubertal mammary gland development while revealing luminal cells to respond to Wnt activation. During regeneration, Wnt4 interacts with progesterone receptor signaling, correcting previous notions on RANKL signaling in this context., Swiss National Center of Competence in Research (NCCR) in Molecular Oncology (SNF3100A0-112090); Marie Heim-Vogtlin fellowship from Swiss National Science Foundation (MHV - PMPDP3_129024); Academy of Finland (206038--121647--251314); Sigrid Juselius Foundation; Novonordisk Fonden; European Union (LSHG-CT-2004-005085-HEALTH-F5-2008-223007 STAR-T REK EURenOmics)

Estrogens and progesterones are major drivers of breast development but also promote carcinogenesis in this organ. Yet, their respective roles and the mechanisms underlying their action in the human breast are unclear. Receptor activator of nuclear factor kB ligand (RANKL) has been identified as a pivotal paracrine mediator of progesterone function in mouse mammary gland development and mammary carcinogenesis. Whether the factor has the same role in humans is of clinical interest because an inhibitor for RANKL, denosumab, is already used for the treatment of bone disease and might benefit breast cancer patients. We show that progesterone receptor (PR) signaling failed to induce RANKL in PR + breast cancer cell lines and in dissociated, cultured breast epithelial cells. In clinical specimens from healthy donors and intact breast tissue microstructures, hormone response was maintained and RANKL expression was under progesterone control, which increased RNA stability. RANKL was sufficient to trigger cell proliferation and was required for progesterone-induced proliferation. The findings were validated in vivo where RANKL protein expression in the breast epithelium correlated with serum progesterone levels and the protein was expressed in a subset of luminal cells that express PR. Thus, important hormonal control mechanisms are conserved across species, making RANKL a potential target in breast cancer treatment and prevention. Copyright 2013 by the American Association for the Advancement of Science; all rights reserved., National Center of Competence in Research Molecular Oncology, Oncosuisse (SNF-3100A0112090/SNF-31003-138065); Marie-Curie incoming fellowship; European Union (115188)

The breast epithelium has two major compartments, luminal and basal cells, that are established and maintained by poorly understood mechanisms. The p53 homolog, p63, is required for the formation of mammary buds, but its function in the breast after birth is unknown. We show that in primary human breast epithelial cells, maintenance of basal cell characteristics depends on continued expression of the p63 isoform, ΔNp63, which is expressed in the basal compartment. Forced expression of ΔNp63 in purified luminal cells confers a basal phenotype. Notch signaling downmodulates ΔNp63 expression and mimics ΔNp63 depletion, whereas forced expression of ΔNp63 partially counteracts the effects of Notch. Consistent with Notch activation specifying luminal cell fate in the mammary gland, Notch signaling activity is specifically detected in mice at sites of pubertal ductal morphogenesis where luminal cell fate is determined. Basal cells in which Notch signaling is active show decreased p63 expression. Both constitutive expression of ΔNp63 and ablation of Notch signaling are incompatible with luminal cell fate. Thus, the balance between basal and luminal cell compartments of the breast is regulated by antagonistic functions of ΔNp63 and Notch. © 2010 Macmillan Publishers Limited All rights reserved., NCCR Molecular Oncology, Oncosuisse, SNF-3100A0–112090

IRF6, a member of Interferon Regulatory Factors (IRF) family, is involved in orofacial and epidermal development. In breast cancer cell lines ectopic expression of IRF6 reduces cell numbers suggesting a role as negative regulator of cell cycle. IRF6 is a direct target of canonical Notch signaling in keratinocyte differentiation. Notch is involved in luminal cell fate determination and stem cell regulation in the normal breast and is implicated as an oncogene in breast cancer. Notch activation is sufficient to induce proliferation and transformation in non-tumorigenic breast epithelial cell line, MCF10A. ΔNp63, which is downregulated by Notch activation in the breast, regulates IRF6 expression in keratinocytes. In this report, we investigate Notch-IRF6 and ΔNp63-IRF6 interactions in MCF10A and MDA MB 231 cells. We observed that in these cells, IRF6 expression is partially regulated by canonical Notch signaling and ΔNp63 downregulation. Furthermore, we demonstrate that IRF6 abrogation impairs Notch-induced proliferation and transformation in MCF10A cells. Thus, we confirm the previous findings by showing a tissue independent regulation of IRF6 by Notch signaling, and extend them by proposing a context dependent role for IRF6, which acts as a positive regulator of proliferation and transformation in MCF10A cells downstream of Notch signaling., Scientific and Technological Research Council of Turkey (110T895); Izmir Institute of Technology