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Akhism, highly influential in Islamic and Anatolian societies between 13th and 19th centuries is a professional, commercial, economic, cultural, social, moral and educational organization whose influence in this process exceeds the boundaries of time and space. It consists of highly moral and well-behaved people, each of its members interacting with each other, providing both material and spiritual guidance to its members, and aiming at the perfect human being. On the basis of its acceptance and strong influence for ages the role of its construction with a spirit and understanding inspired by Islamic sources is very great. It is very important scientifically in terms of dealing with Akhism in a way that sheds light on the present, to make sense of it by examining it, evaluate it, and to benefit from the results of a successful experience in the past. Considering that education is an activity of introducing ideal cultural elements to future generations, the introduction of institutions that affect the culture of society in history to today's people is also among the duties of education. In Akhism, educational values, both verbal and behavioral, have been inherited from the past to the present within the framework of the master-assistant master relationship. The aim of this study is to determine the prominent values in Akhism and to reveal the importance of advice value. In this context, the values in the Akhi organization and the value of “advice”, which stands out from these values, were investigated. At the same time, the role of the concept of advice in adding value to individuals and societies has been tried to be determined, although its value has weakened today. The study has been designed with a qualitative research design and the main Islamic sources have been examined in accordance with the document analysis method in the research. Based on the richness of meaning of the concept of advice, the aspect of advice that educates a person, guides, shapes life and makes it valuable and how it builds a lifestyle has been examined. In this study, according to the 5N+ 2K (What, why, where, how, when and who, to whom) technique, the use of advice and teaching methods in the Akhism have been tried to be determined. It is assessed that the results obtained from the richness of meaning of the advice, which has been determined to have an important place in theA khism, will shed light on today's educators and values education. Keywords: Religious Education, Akhism, Values, Advice, Advice in Akhism.

list of primer sequences, antibody details and primary fibroblasts used

Significant DDR genesets (from Leading-Edge analysis in GSEA) used to make the DDR_TGFB_signature geneset

macros for image quantifications

Myofibroblastic cancer-associated fibroblast (myoCAF)–rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species–producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti–PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance.Significance:ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

supplementary figures and legends

Non-redundant list of the leading-edge genes and ToppFun pathway analysis from the genesets in Suppl. File 4 used to make the DDR-TGFB_signature geneset

GSEA between different tumour stromal datasets from GEO and DDR & myoCAF genesets

Ṣaḥābīs, who passed the education of the last envoy of the Rahman, came to the fore with their special efforts in many areas, as well as conveying their Islamic heritage. Benefiting from the experience of the Ṣaḥābīs in the field of religious education can provide different perspectives for Religious Educators of all ages. Muadh b. Cebel has been one of the Ṣaḥābīs that stand out with their successful practices and spiritual guidance, especially in the field of religious education. In this study prepared with the documentation method, despite the short life of Muadh, the fact that he was among the distinguished teachers of the Ṣaḥābīs, the codes of being a successful and good educator, his educationalist characteristics, the methods he applied at the point of Religious Education, pioneering and exemplary practices, strategies were discussed in outline. After the examination, it has been determined that Muadh has carried out important works on Religious Education and has important examples for today's religious educators.Keywords: Religious Education, Ṣaḥābī, Teaching Method, Spiritual Education-Teaching Strategies., Rahmân’ın son elçisinin eğitiminden geçen sahâbîler, İslâm mirasını aktarmaları yanında pek çok alanda ortaya koydukları özel gayretlerle de öne çıkmışlardır. Sahâbîlerin din eğitim-öğretimi alanında ortaya koydukları tecrübeden faydalanmak, çağın her dönemindeki Din Eğitimcileri için farklı açılımlar kazandırabilir. Pek çok ilmi sahada adından söz ettirmesi yanında Muâz b. Cebel, özellikle din eğitim-öğretim alanında başarılı uygulamaları, manevi rehberliği ile öne çıkan sahabilerden biri olmuştur. Dokümantasyon yöntemi ile hazırlanan bu çalışmada Muâz b. Cebel’in, kısa ömrüne rağmen Ashab-ı Suffe’nin seçkin öğretmenleri arasına girmesi, başarılı ve iyi bir eğitimci olmasının kodları, eğitimci özellikleri, Din Eğitimi noktasında uyguladığı metotlar, öncü ve örnek uygulamalar, stratejiler ana hatlarıyla ele alınmıştır. Yapılan inceleme sonrası, Muâz b. Cebel’in, Din Eğitimi konusunda, önemli çalışmalara imza attığı, günümüz din eğitimcilerine önemli örneklikleri olduğu tespit edilmiştir.

Bilgi çağı olarak nitelendirilen yeni dönemde bilgiye ulaşım ve iletişim araçları da zenginleşmiş, dijital araçlar ve sosyal medya öne çıkmıştır. Bu değişimde; bireyler, yeni iletişim araçlarına hızla uyum sağlarken; kurumlar farklılaşmakta, her alanda değişim yaşayan kurumlar yanında, konunun önemini henüz kavrayamayanlar da bulunmaktadır. Bu çalışmanın amacı, ülkemizde özellikle yaygın din eğitim-öğretimi bağlamında önemli bir misyonu olan Diyanet İşleri Başkanlığı’nın yeni medya karşısındaki durumunu, merkez ve taşra teşkilatlarında (81 il müftülüğü), kamuoyuna din hizmeti götürme konusunda görevini ifa ederken, hangi sosyal medya mecralarını kullandığını nicel ve nitel kapsamıyla tespit etmektir. Özellikle 2019 Aralık ayında yaşanan Covid-19 pandemisi sonrası dünya genelindeki dijital dönüşüme paralel, ülkemizde de iletişim kanallarındaki değişimin Diyanet nezdinde nasıl gerçekleştiği ayrıca araştırmanın sorularındandır. Kurumun ve birimlerinin resmî web siteleri incelenerek hangi sosyal medya mecralarını kullandıkları tespit edilmiş, bu mecralarda öne çıkan birimler ve Facebook ve Twitter paylaşımları içerik analizine tabi tutulmuştur. Paylaşımların dini bilgilendirme, haber ve etkinlik ağırlıklı olduğu görülmüştür. Nicel veriler yanında kurumun online ve basılı yayınlarından sosyal medya konuları dokümantasyon yöntemi ile incelenmiştir. İl müftülüklerinin 54’ünün (%67’sinin) en az bir sosyal medya mecrasında yer aldığı 27’sinin henüz bu mecraları kullanmadığı tespit edilmiştir. Çalışmada elde edilen veriler ışığında, merkez teşkilatına yönelik ve yeni başlayacak, henüz başlangıç düzeyinde olan diğer birimlere yönelik sosyal medya stratejileri ışığında önerilerde bulunulmuştur.

Myofibroblastic cancer-associated fibroblast (myoCAF)–rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species–producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti–PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. Significance: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

Allah’ın varlığını kabul veya ret insanın hayata bakışını tümden etkileyen bir olgudur. Toplumlar huzur ve düzenlerinin devamı için sahip oldukları inancın kendilerinden sonraki nesil tarafından da sürdürülmesini istemektedirler. Allah’a iman eden bir ebeveynin ateist olmuş çocuklarıyla iletişimi ve sağlıklı diyalogu güçtür. Bu noktada son dönemde artışa geçtiği iddia edilen ateizm, deizm gibi tanrının varlığına yönelik inanç sapmaları dini ve kültürel değerlerine bağlı bir toplumun fertlerini kaygılandırmaktadır. Günümüz Türkiye’sinde gündeme gelen Allah’ın varlığına yönelik inanç problemlerini tespit ederek eğitimcilerin bu problemlere yaklaşımını teorik çerçevede ele almayı amaçlayan, dokümantasyon yöntemiyle hazırlanan çalışmada, alanyazındaki araştırmalar taranarak, mevcut durumun fotoğrafını çekip tespit edilen soru ve sorunlara çözüm önerileri ortaya konmaya çalışılmıştır. Son zamanlarda ortaya çıkan inanç problemlerinin birçok sebebi olsa da bunlar arasında ailenin, eğitimcilerin ve öğrenme ortamlarının rolü daha çok öne çıkmaktadır. Bu çalışmada özellikle problemin aile, eğitim ortamları ve eğitimcilerin yöntem ve üsluplarından kaynaklanan yönlerine dikkat çekilmesi amaçlandı. Her bir sorun alanına yönelik tespitler yapılarak bu sorunların çözümüne yönelik öneriler geliştirilmeye çalışılmıştır.

Bilgi çağı olarak nitelendirilen günümüzde, bilginin üretildiği, eğitim-öğretim faaliyetlerinin yapıldığı, öğrenimin gerçekleştiği okullardan, bu çağın gereklerine uygun, özgüveni yüksek bir öğrenci profili yetiştirmesi beklenmektedir. Bu konuda öğrenme iklimi önemlidir. İklim alan yazınında örgüt iklimi ve okul iklimi yerine kullanılan yeni bileşenlerden biri olan, öğrenme yetisini ilgilendiren her türlü faktörü içeren öğrenme iklimi; insan unsurları, öğrenmenin gerçekleştiği sanal ve gerçek mekân özellikleri başta olmak üzere pek çok boyutlarıyla ele alınmakta, öğrenme iklimini ölçmeye yönelik geliştirilen araçlarla ölçümler yapılmaktadır. Elde edilen veriler iyileştirme çalışmalarına kaynaklık etmektedir. Çalışmamızda okul öğrenme iklimi okula bağlılık, öğrenme ortamı, iletişim boyutlarıyla ele alınmıştır. Sosyal Bilişsel Kuramın temel kavramlarından özyeterliğin bir türü olarak, öğrencilerin akademik çalışma gerektiren konularda kendilerine olan öz güvenlerini ifade eden, bu kapsamda öğrenebilmek için bireyin etkili biliş stratejilerini kullanabilme, öğrenme çevrelerini ve öğrenme zamanlarını etkili bir şekilde yönetebilme ve kendi performansını etkili bir şekilde düzenleyebilmesi şeklinde de açıklanabilen akademik özyeterlik, araştırmacılarca farklı boyutlarla ele alınmakta, farklı ölçeklerle ölçümler yapılmaktadır. Çalışmamızda, akademik özyeterlik, bilişsel, sosyal ve teknik boyutlarda ele alınmıştır. İnsanı hayatın akışı içerisinde güçlü kılan asıl unsurlar kendine güven, eleştirel bakış, sosyal ilişkilerde başarı, herhangi bir sportif sanatsal ya da kültürel alana ilgi gibi temel beceriler, öğrencilerin okul ikliminden kazandıklarıyla mümkündür. Bu durumda, okulda iyi bir rüzgâr estirebilmenin, yaşanılası ve sevilesi bir hava oluşturabilmenin, özgün ve eleştirel bir zemine oturabilmenin ancak olumlu ve sürdürülebilir bir okul iklimiyle mümkün olduğunu söyleyebiliriz. Akademik özyeterliği olumlu besleyen okul iklimi, iklimin etkilediği insan unsurlarının başarısını da beraberinde getirecektir. Pozitif okul iklimi güçlü akademik özyeterlik akademik başarının da en büyük araçları haline gelecektir. Öğrenme iklimini ölçmek, okullardaki kişilerarası ilişkiler, rol modeller, beklentileri vb. boyutlarıyla hayatın kalitesini aynı zamanda öğrencilerin başarısını ve memnuniyetini anlamaya yardımcı olabilmektedir. Bu çalışmanın amacı, yüksek din öğrenim gören öğrencilerin öğrenme iklimi algıları ile akademik özyeterlik ve akademik başarıları arasındaki ilişkiyi incelemektir. Bu çerçevede dört temel araştırma sorusu cevaplanmaya çalışılmıştır: (i) Yüksek din öğrenimi öğrencilerinin öğrenme iklimi ve akademik özyeterlik algıları ne düzeydedir? (ii) Yüksek din öğrenimi öğrencilerinin öğrenme iklimi ve akademik özyeterlik algıları arasında anlamlı ilişki var mıdır? (iii) Yüksek din öğrenimi öğrencilerinin yaş, öğrenme iklimi ve akademik özyeterlik inançları akademik başarılarının anlamlı yordayıcısı mıdır? (iv) Yüksek din öğrenimi öğrencilerinin öğrenme iklimine dair olumsuz algılarına ilişkin görüşleri nelerdir? Karma yöntem ve yakınsak paralel araştırma deseni ile desenlenen araştırmada çeşitleme stratejisi kullanılmıştır. Araştırmada, Türkiye'de devlet üniversiteleri bünyesindeki muhtelif İlahiyat ve İslami İlimler Fakültelerinde yüksek din öğrenimi gören, altı fakülteden, nicel bölümde 1147 öğrenciye Owen ve Froman tarafından geliştirilen, Ekici tarafından Türkçe’ye uyarlaması yapılan Akademik Özyeterlik Ölçeği ve Terzi tarafından geliştirilen, Üniversite Öğrencilerine Yönelik Okul İklimi Ölçeği uygulanırken, nitel bölümde ise aynı fakültelerden 18 öğrenciye görüşme formu doldurtulmuştur. 2019 Mayıs ayında gerçekleştirilen araştırma sonrası elde edilen nicel veri setine normallik testi uygulanmış, verilerin analizinde; Bağımsız Gruplar t Testi, Mann Whitney U Testi, Levene testi, Tek Yönlü Varyans Analizi (ANOVA), Scheffe Testi, Kruskal Wallis H Testi, Pearson product-Moment Korelasyonu, Polyserial Korelasyonu ve Yol Analizi yapılmıştır. Nitel veriler betimsel analizle çözümlenmiştir. Araştırma sonuçlarına göre, yüksek din öğrenimi öğrencilerinin öğrenme iklimi algıları olumsuz çıkmıştır. Yüksek din öğrenimi öğrencilerinin genel olarak Akademik Özyeterlik algıları da olumsuz çıkmıştır. Okul İklimi Ölçeğinin genel ortalaması ile Akademik Özyeterlik Algısı Ölçeğinin genel ortalaması arasında korelasyon orta düzeydedir. Araştırma sonucuna göre elde edilen bulgular alanyazın eşliğinde tartışılmış ve yorumlar sonrası yüksek din öğretiminde pozitif bir öğrenme iklimini sağlayacağı, yüksek din öğretimi öğrencilerinin akademik özyeterliklerini güçlendireceği ve akademik başarılarını artıracağı düşünülen çeşitli çözüm önerileri sunulmuştur.

Epithelial-mesenchymal transition (EMT) is an embryonic program that is reactivated in cancer and regulates the invasion and metastasis of tumor cells. Zinc finger E-box binding homeobox 2 (ZEB2) induces EMT by upregulating matrix metalloproteinases (MMP), yet MMP genes lack ZEB2 binding motif in their promoters. Recently, expression of MMPs was associated to the activation of ETS1 transcription factor; however, a link between ZEB2 and ETS proto-oncogene 1, transcription factor (ETS1) remains to be elucidated. Hence, we investigated the transcriptional regulation of ETS1 by ZEB2 after our initial observation that ZEB2 and ETS1 are coexpressed in hepatocellular carcinoma cells (HCCs). Chromatin immunoprecipitation and luciferase reporter assays clearly showed that ZEB2 binds to E-box sequences on the promoter of ETS1. Elevated expression of ETS1 was found in DLD-ZEB2 and A431-ZEB2 inducible systems, and knockdown of ZEB2 caused an explicit downregulation of ETS1 in shZEB2-SNU398 and shZEB2-SK-HEP-1 cells. Repression of ETS1 expression in ZEB2-induced conditions substantially impaired the migration and invasive capacities of DLD1 cells. Mechanistically, knockdown of ETS1 in ZEB2-expressing cells resulted in the downregulation of established ZEB2 targets TWIST and MMP9. Correlation analyses in HCC lines, cancer complementary DNA arrays, and The Cancer Genome Atlas RNA-sequencing data set revealed that ZEB2 and ETS1 are coexpressed, and their expressions in human tumors show a highly significant positive correlation. Our results demonstrated that ZEB2 acts as an upstream regulator of ETS1 and, in turn, ETS1 maintains ZEB2-induced EMT. These findings add another level of complexity to the understanding of ZEB2 in the invasion and metastasis of cancer cells, and put ZEB2/ETS1 axis as a novel therapeutic target in human malignancies.

Covering: up to 2020 The indolocarbazoles, in particular indolo[2,3-a]pyrrolo[3,4-c]carbazole derivatives, are an important class of natural products that exhibit a wide range of biological activities. There has been a plethora of synthetic approaches to this family of natural products, leading to advances in chemical methodology, as well as affording access to molecular scaffolds central to protein kinase drug discovery programmes. In this review, we compile and summarise the synthetic approaches to the indolo[2,3-a]pyrrolo[3,4-c]carbazole derivatives, spanning the period from their isolation in 1980 up to 2020. The selected natural products include indolocarbazoles not functionalised at indolic nitrogen, pyranosylated indolocarbazoles, furanosylated indolocarbazoles and disaccharideindolocarbazoles.

Resistance to adjuvant chemotherapy is a major clinical problem in the treatment of colorectal cancer (CRC). The aim of this study was to elucidate the role of an epithelial to mesenchymal transition (EMT)‐inducing protein, ZEB2, in chemoresistance of CRC, and to uncover the underlying mechanism. We performed IHC for ZEB2 and association analyses with clinical outcomes on primary CRC and matched CRC liver metastases in compliance with observational biomarker study guidelines. ZEB2 expression in primary tumours was an independent prognostic marker of reduced overall survival and disease‐free survival in patients who received adjuvant FOLFOX chemotherapy. ZEB2 expression was retained in 96% of liver metastases. The ZEB2‐dependent EMT transcriptional programme activated nucleotide excision repair (NER) pathway largely via upregulation of the ERCC1 gene and other components in NER pathway, leading to enhanced viability of CRC cells upon oxaliplatin treatment. ERCC1‐overexpressing CRC cells did not respond to oxaliplatin in vivo, as assessed using a murine orthotopic model in a randomised and blinded preclinical study. Our findings show that ZEB2 is a biomarker of tumour response to chemotherapy and risk of recurrence in CRC patients. We propose that the ZEB2‐ERCC1 axis is a key determinant of chemoresistance in CRC., Here, we show that the expression of ZEB2 marks reduced overall and disease‐free survival in primary and secondary colorectal cancers (CRCs). ZEB2 overexpression promoted chemoresistance to oxaliplatin in vitro and in vivo by transcriptionally activating nucleotide excision repair genes ERCC1 and ERCC4. Overall ZEB2 is a promising biomarker predicting both therapy response and metastatic ability of CRCs.

Günlük hayata etkileriyle sosyal medya, kişiler arası iletişimin yanında kurumların da iletişimini etkilemiştir. Çok amaçlı iletişim stratejileri bağlamında, sosyal medya araçlarını da kullanmak zorunda kalan kurumlarda, adaptasyonun değişik hızda/anlayışlarda seyrettiği, günlük faaliyetlerini sosyal medyayla yürüten kurumların yanında bu durumun hâlâ gündeme gelmediği gözlenmiştir. Mevcut/potansiyel insan unsurlarıyla ve diğer kurumlarla çok yönlü etkileşimde, eğitim kurumları da bu mecralarda yerlerini almıştır. Çalışmada, Yüksek Din Öğretimi Kurumları’nın sosyal medyada hangi mecralarda, ne düzeyde yer aldıkları incelenmiştir. Kurumların web siteleri 10 Temmuz 2020’de incelenerek, sosyal medya mecraları ve varoldukları mecralardaki toplam takipçi sayısı tespit edilmiş ve mevcut 97 Yüksek Din Öğretimi Kurumu’ndan, Facebook, Instagram, Twitter ve YouTube’da aktif kullanımıyla ve en çok takipçi sayısıyla öne çıkanların sosyal medya stratejileri örnek olay olarak sunulmuş, tecrübeleri, hesapları yöneten uzman kişilerle görüşülerek derinlemesine ele alınmış ve anlaşılmaya çalışılmıştır. Betimsel tarama yöntemiyle nicel veriler, görüşme formu ile nitel veriler elde edilmiştir. Araştırma sonucu elde edilen bulgular bağlamında, İlahiyat/İslami İlimler Fakültelerinin muhtemel sosyal medya politikalarına yönelik stratejilere, geliştirici önerilere yer verilmiştir., Social media, with its effects on daily life, has affected the communication of institutions as well as interpersonal communication, and in institutions that have to use social media tools in the context of multi-purpose communication strategies, it is observed that the issue is still not on the agenda in some of them, besides conducting their daily activities with social media, where adaptation follows at different speeds / understandings. Educational institutions have also taken their place in these channels in multi-faceted interaction with existing / potential human elements and other institutions. In study, in which channels and at what level Higher Religious Education Institutions are involved in social media was examined. The websites of the institutions were examined on July 10, 2020, and the total number of followers in the social media channels and the channels they exist was determined and the social media strategies of the existing 97 Higher Religious Education Institutions, which stand out with their active use on Facebook, Instagram, Twitter and YouTube and with the highest number of followers, were presented as a case study, their experiences have been discussed in depth with the experts managing the accounts and they have been understood. Quantitative data were obtained by the descriptive screening method, and qualitative data were obtained by interview form. In the context of the findings obtained as a result of the research, strategies and developing suggestions for possible social media policies of the Faculty of Theology / Islamic Sciences are included.

Background Hepatocellular carcinoma (HCC) is common, but remains difficult to treat. Natural killer (NK) cells are cells of the innate immune system that have potent anti-cancer activity. Recent work has shown that stimulation with IL-12/15/18 leads to the generation of NK cells with enhanced functional and putative “memory” properties. We have investigated the activity of these NK cells against HCC cell lines in vitro and in a mouse model. Methods NK cells from healthy donors or individuals with HCC were activated with IL-12/15/18 in vitro and tested for cytotoxic activity against a panel of human HCC cell lines. IL-12/15/18 primed murine NK cells were then infused into a murine model of spontaneously arising HCC to test for anti-tumor activity. Results NK cells from patients and healthy controls had similar expression levels of activating and inhibitory NK cell receptors. However, proliferation of NK cells from HCC patients was weaker than healthy controls in response to IL-12/15/18 and IL-2 (p

Simple Summary AXL is a member of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases. In normal physiological conditions, AXL is involved in removing dead cells and their remains, and limiting the duration of immune responses. Both functions are utilized by cancers in the course of tumour progression. Cancer cells use the AXL pathway to detect toxic environments and to activate molecular mechanisms, thereby ensuring their survival or escape from the toxic zone. AXL is instrumental in controlling genetic programs of epithelial-mesenchymal and mesenchymal-epithelial transitions, enabling cancer cells to metastasize. Additionally, AXL signaling suppresses immune responses in tumour microenvironment and thereby helps cancer cells to evade immune surveillance. The broad role of AXL in tumour biology is the reason why its inhibition sensitizes tumours to a broad spectrum of anti-cancer drugs. In this review, we outline molecular mechanisms underlying AXL function in normal tissues, and discuss how these mechanisms are adopted by cancers to become metastatic and drug-resistant. Abstract The TAM proteins TYRO3, AXL, and MER are receptor tyrosine kinases implicated in the clearance of apoptotic debris and negative regulation of innate immune responses. AXL contributes to immunosuppression by terminating the Toll-like receptor signaling in dendritic cells, and suppressing natural killer cell activity. In recent years, AXL has been intensively studied in the context of cancer. Both molecules, the receptor, and its ligand GAS6, are commonly expressed in cancer cells, as well as stromal and infiltrating immune cells. In cancer cells, the activation of AXL signaling stimulates cell survival and increases migratory and invasive potential. In cells of the tumour microenvironment, AXL pathway potentiates immune evasion. AXL has been broadly implicated in the epithelial-mesenchymal plasticity of cancer cells, a key factor in drug resistance and metastasis. Several antibody-based and small molecule AXL inhibitors have been developed and used in preclinical studies. AXL inhibition in various mouse cancer models reduced metastatic spread and improved the survival of the animals. AXL inhibitors are currently being tested in several clinical trials as monotherapy or in combination with other drugs. Here, we give a brief overview of AXL structure and regulation and discuss the normal physiological functions of TAM receptors, focusing on AXL. We present a theory of how epithelial cancers exploit AXL signaling to resist cytotoxic insults, in order to disseminate and relapse.

In vivo studies in animal models are critical tools necessary to study the fundamental complexity of carcinogenesis. A constant strive to improve animal models in cancer exists, especially those investigating the use of chemotherapeutic effectiveness. In the present systematic review, colorectal cancer (CRC) is used as an example to highlight and critically evaluate the range of reporting strategies used when investigating chemotherapeutic agents in the preclinical setting. A systematic review examining the methodology and reporting of preclinical chemotherapeutic drug studies using CRC murine models was conducted. A total of 45 studies were included in this systematic review. The literature was found to be highly heterogeneous with various cell lines, animal strains, animal ages and chemotherapeutic compounds/regimens tested, proving difficult to compare outcomes between similar studies or indeed gain any significant insight into which chemotherapeutic regimen caused adverse events. From this analysis we propose a minimum core outcome dataset that could be regarded as a standardised way of reporting results from in vivo experimentation.

Background: Hepatocellular carcinoma (HCC) is a common and deadly cancer, however, very little improvement has been made towards its diagnosis and prognosis. The expression and functional contribution of the receptor tyrosine kinase ROR1 have not been investigated in HCC before. Hence, we investigated the expression of ROR1 in HCC cells and assessed its involvement in hepatocarcinogenesis. Methods: Recombinant bacterial ROR1 protein was used as an immunogen to generate ROR1 monoclonal antibodies. ROR1 transcript levels were detected by RT-qPCR and the protein expression of ROR1 in HCC was assessed by Western blotting by using homemade anti-ROR1 monoclonal antibodies. Apoptosis, cell cycle, trans-well migration, and drug efflux assays were performed in shRNA-ROR1 HCC cell clones to uncover the functional contribution of ROR1 to hepatocarcinogenesis. Results: New ROR1 antibodies specifically detected endogenous ROR1 protein in human and mouse HCC cell lines. ROR1-knockdown resulted in decreased proliferation and migration but enhanced resistance to apoptosis and anoikis. The observed chemotherapy-resistant phenotype of ROR1-knockdown cells was due to enhanced drug efflux and increased expression of multi-drug resistance genes. Conclusions: ROR1 is expressed in HCC and contributes to disease development by interfering with multiple pathways. Acquired ROR1 expression may have diagnostic and prognostic value in HCC.

Treatment of HER2+ breast cancer with trastuzumab is effective and combination anti-HER2 therapies have demonstrated benefit over monotherapy in the neoadjuvant and metastatic settings. This study investigated the therapeutic potential of targeting the BAG-1 protein co-chaperone in trastuzumab-responsive or -resistant cells. In the METABRIC dataset, BAG-1 mRNA was significantly elevated in HER2+ breast tumors and predicted overall survival in a multivariate analysis (HR = 0.81; p = 0.022). In a breast cell line panel, BAG-1 protein was increased in HER2+ cells and was required for optimal growth as shown by siRNA knockdown. Overexpression of BAG-1S in HER2+ SKBR3 cells blocked growth inhibition by trastuzumab, whereas overexpression of a mutant BAG-1S protein (BAG-1S H3AB), defective in binding HSC70, potentiated the effect of trastuzumab. Injection of a Tet-On SKBR3 clone, induced to overexpress myc-BAG-1S into the mammary fat pads of immunocompromised mice, resulted in 2-fold larger tumors compared to uninduced controls. Induction of myc-BAG-1S expression in two Tet-On SKBR3 clones attenuated growth inhibition by trastuzumab in vitro. Targeting endogenous BAG-1 by siRNA enhanced growth inhibition of SKBR3 and BT474 cells by trastuzumab, while BAG-1 protein-protein interaction inhibitor (Thio-S or Thio-2) plus trastuzumab combination treatment synergistically attenuated growth. In BT474 cells this reduced protein synthesis, caused G1/S cell cycle arrest and targeted the ERK and AKT signaling pathways. In a SKBR3 subpopulation with acquired resistance to trastuzumab BAG-1 targeting remained effective and either Thio-2 or BAG-1 siRNA reduced growth more compared to trastuzumab-responsive parental cells. In summary, targeting BAG-1 function in combination with anti-HER2 therapy might prove beneficial.

Background S100 proteins have been implicated in various aspects of cancer, including epithelial-mesenchymal transitions (EMT), invasion and metastasis, and also in inflammatory disorders. Here we examined the impact of individual members of this family on the invasion of pancreatic ductal adenocarcinoma (PDAC) cells, and their regulation by EMT and inflammation. Methods Invasion of PDAC cells was analysed in zebrafish embryo xenografts and in transwell invasion assays. Expression and regulation of S100 proteins was studied in vitro by immunoblotting, quantitative PCR and immunofluorescence, and in pancreatic lesions by immunohistochemistry. Results Whereas the expression of most S100 proteins is characteristic for epithelial PDAC cell lines, S100A4 and S100A6 are strongly expressed in mesenchymal cells and upregulated by ZEB1. S100A4/A6 and epithelial protein S100A14 respectively promote and represses cell invasion. IL-6/11-STAT3 pathway stimulates expression of most S100 proteins. ZEB1 synergises with IL-6/11-STAT3 to upregulate S100A4/A6, but nullifies the effect of inflammation on S100A14 expression. Conclusion EMT/ZEB1 and IL-6/11-STAT3 signalling act independently and congregate to establish the expression pattern of S100 proteins, which drives invasion. Although ZEB1 regulates expression of S100 family members, these effects are masked by IL-6/11-STAT3 signalling, and S100 proteins cannot be considered as bona fide EMT markers in PDAC.

Colorectal cancer (CRC) is a key public health concern and the second highest cause of cancer related death in Western society. A dynamic interaction exists between CRC cells and the surrounding tumor microenvironment, which can stimulate not only the development of CRC, but its progression and metastasis, as well as the development of resistance to therapy. In this chapter, we focus on the role of fibroblasts within the CRC tumor microenvironment and describe some of the key methods for their study, as well as the evaluation of dynamic interactions within this biological ecosystem.

Background and Aims. Hepatocellular carcinoma is an aggressive malignancy of the liver and is ranked as the sixth most common cancer worldwide. There is still room for novel markers to improve the diagnosis and monitoring of HCC. Our observations in cancer databases that PLXNC1 is upregulated in HCC led us to investigate the expression profile of Plexin C1 mRNA and protein in HCC cell lines and tissues. Methods. A recombinant protein encompassing part of the extracellular domain of Plexin C1 was used as an antigen for monoclonal antibody development. Transcript and protein levels of Plexin C1 in HCC cell lines were determined by RT-qPCR and Western blotting, respectively. In vivo evaluation of Plexin C1 expression in HCC tissues was accomplished by immunohistochemistry studies in tissue microarrays. Results. A monoclonal antibody, clone PE4, specific to Plexin C1, was generated. In silico and in vitro analyses revealed a Plexin C1-based clustering of well-differentiated HCC cell lines. Staining of HCC and nontumoral liver tissues with PE4 showed a membrane-localized overexpression of Plexin C1 in tumors (p=0.0118). In addition, this expression was correlated with the histological grades of HCC cases. Conclusions. Plexin C1 distinguishes HCC cells of epithelial characteristics from those with the mesenchymal phenotype. Compared to the nontumoral liver, HCC tissues significantly overexpress Plexin C1. The newly generated PE4 antibody can be evaluated in larger HCC cohorts and might be exploited for the examination of Plexin C1 expression pattern in other epithelial malignancies.

ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions. For ChIP-Seq we used an anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells exhibiting a high endogenous ZEB2 expression. The ChIP-Seq targets were validated using ChIP-qPCR, whereas ZEB2-dependent expression of target genes was assessed by RT-qPCR and Western blotting in shRNA-mediated ZEB2 silenced SNU398 cells and doxycycline-induced ZEB2 overexpressing colorectal carcinoma DLD1 cells. Changes in target gene expression were also assessed using primary human tumor cDNA arrays in conjunction with RT-qPCR. Additional differential expression and correlation analyses were performed using expO and Human Protein Atlas datasets. Over 500 ChIP-Seq positive genes were annotated, and intervals related to these genes were found to include the ZEB2 binding motif CACCTG according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed that the GALNT3 gene serves as a ZEB2 target with the highest, but inversely correlated, expression level. Remarkably, GALNT3 also exhibited the highest enrichment in the ChIP-qPCR validation assays. Through the analyses of primary tumor cDNA arrays and expO datasets a significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 expression were detected in most of the tumors. We also explored ZEB2 and GALNT3 protein expression using the Human Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression. Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.

Long non-coding RNAs (lncRNAs) are a diverse class of RNA transcripts which have limited protein coding potential. They perform a variety of cellular functions in health, but have also been implicated during malignant transformation. A further theme in recent years is the critical role of the tumour microenvironment and the dynamic interactions between cancer and stromal cells in promoting invasion and disease progression. Whereas the contribution of deregulated lncRNAs within cancer cells has received considerable attention, their significance within the tumour microenvironment is less well understood. The tumour microenvironment consists of cancer-associated stromal cells and structural extracellular components which interact with one another and with the transformed epithelium via complex extracellular signalling pathways. LncRNAs are directly and indirectly involved in tumour/stroma cross-talk and help stimulate a permissive tumour microenvironment which is more conducive for invasive tumour growth. Furthermore, lncRNAs play key roles in determining the phenotype of cancer associated stromal cells and contribute to angiogenesis and immune evasion pathways, extracellular-matrix (ECM) turnover and the response to hypoxic stress. Here we explore the multifaceted roles of lncRNAs within the tumour microenvironment and their putative pathophysiological effects.

The tumor microenvironment is a heterogeneous and dynamic network that exists between cancer and stroma, playing a critical role in cancer progression. Certain tumorigenic signals such as microRNAs are derived from the stroma and conveyed to cancer cells (and vice versa) in nanoparticles called exosomes. Their identification and characterization is an important step in better understanding cellular cross talk and its consequences. To this end we describe how to culture primary ex vivo derived fibroblasts from colorectal tissue, isolate their exosomes, extract exosomal RNA and perform microRNA profiling.

Fos-related antigen-1 (Fra-1) is a member of the Activator Protein-1 (AP-1) transcription factor superfamily that is overexpressed in a variety of cancers, including colon, breast, lung, bladder and brain. High Fra-1 levels are associated with enhanced cell proliferation, survival, migration and invasion. Despite its frequent overexpression, the molecular mechanisms that regulate the accumulation of Fra-1 proteins in tumour cells are not well understood. Here, we show that turnover of Fra-1, which does not require ubiquitylation, is cooperatively regulated by two distinct mechanisms—association with the 19S proteasomal subunit, TBP-1, and by a C-terminal degron, which acts independently of TBP-1, but is regulated by RAS–ERK (extracellular signal-regulated kinase) signalling. TBP-1 depletion stabilized Fra-1 and further increased its levels in tumour cells expressing RAS–ERK pathway oncogenes. These effects correlated with increased AP-1 transcriptional activity. We suggest that during Fra-1 degradation, association with TBP-1 provides a mechanism for ubiquitin-independent proteasomal recognition, while the C terminus of the protein regulates its subsequent proteolytic processing. This work was supported by grants from the National Health and Medical Research Council of Australia & SFI Deposited by bulk import

Importance: At present, patients with colorectal cancer (CRC) are risk stratified using TNM histologic features. More recently, an association between a mesenchymal phenotype and a high risk of disease recurrence and micrometastases has been recognized.Objective: To investigate the association of the epithelial to mesenchymal transition (EMT)-inducing transcription factor ZEB2 (zinc finger E box-binding homeobox 2), survival outcomes, and the efficacy of ZEB2 as a biomarker when added as refinement to TNM staging after curative intent surgery for CRC.Design, Setting, and Participants: ZEB2 expression was assessed using a previously validated scoring system as part of a prospective, observational, masked diagnostic study from January 1, 2008, to December 31, 2013. Data were prospectively collected and analyzed for association with oncologic outcomes from January 1, 2017, to December 31, 2018. An initial test cohort from an academic university medical center of 126 consecutive patients with CRC and, subsequently, an independent validation cohort of 210 patients were examined. ZEB2 positivity was scored by 2 independent, masked pathologists. External validity was tested using an open access gene expression portal. Nomograms were developed with or without ZEB2.Main Outcomes and Measures: Systemic and local recurrence of CRC.Results: The test cohort consisted of 126 consecutive patients (mean [SD] age, 72.7 [11.7] years; 61 [48.4%] male) and the validation cohort of 210 patients (mean [SD] age, 72.0 [10.6] years; 111 [52.9%] male). A total of 52 tumors (41.3%) in the test cohort and 104 (49.5%) in the validation cohort were scored nuclear ZEB2 positive. Survival analysis by the log-rank test found that ZEB2 expression was associated with a significant reduction in overall survival and disease-free survival in both cohorts. Cox proportional hazards regression analysis highlighted ZEB2 as an independent biomarker of shorter overall survival and disease-free survival. Analysis of node-negative disease (n = 222) identified ZEB2 as an independent biomarker of early recurrence and reduced survival. External validation confirmed these findings. Addition of ZEB2 expression to nomograms composed of conventional TNM risk factors improved the ability to identify patients at high risk of recurrence demonstrated by the improvement in concordance index in both test (0.73 to 0.77) and validation (0.82 to 0.87) cohorts.Conclusions and Relevance: The findings suggest that expression of ZEB2 is associated with poor oncologic outcome and distant recurrence. The study also found that the addition of ZEB2 to existing TNM classification improved the ability to stratify patients for risk of recurrence. The results of this study suggest that addition of ZEB2 expression status to the TNM staging system improves the ability to stratify patients at high risk of recurrence.

Zinc finger transcription factors of the Snail/Slug and ZEB-1/SIP1 families control epithelial-mesenchymal transitions in development in cancer. Here, we studied SIP1-regulated mesenchymal conversion of epidermoid A431 cells. We found that concomitant with inducing invasive phenotype, SIP1 inhibited expression of cyclin D1 and induced hypophosphorylation of the Rb tumor suppressor protein. Repression of cyclin D1 was caused by direct binding of SIP1 to three sequence elements in the cyclin D1 gene promoter. By expressing exogenous cyclin D1 in A431/SIP1 cells and using RNA interference, we demonstrated that the repression of cyclin D1 gene by SIP1 was necessary and sufficient for Rb hypophosphorylation and accumulation of cells in G1 phase. A431 cells expressing SIP1 along with exogenous cyclin D1 were highly invasive, indicating that SIP1-regulated invasion is independent of attenuation of G1/S progression. However, in another epithelial-mesenchymal transition model, gradual mesenchymal conversion of A431 cells induced by a dominant negative mutant of E-cadherin produced no effect on the cell cycle. We suggest that impaired G1/S phase progression is a general feature of cells that have undergone EMT induced by transcription factors of the Snail/Slug and ZEB-1/SIP1 families.

It is well established that the tumour microenvironment contributes to cancer progression. Stromal cells can be divided into mesenchymal, vascular and immune. Signalling molecules secreted by the tumour corrupts these cells to create ‘activated’ stroma. Equally, the extracellular matrix contributes to tumour development and invasion by forming a biologically active scaffold. In this review we describe the key structural, cellular and signalling components of the tumour microenvironment with a perspective on stromal soluble factors and microRNAs.

MicroRNAs (miRNAs) enable colonic epithelial cells to acquire malignant characteristics and metastatic capabilities. Recently, cancer relevant miRNAs deregulated during disease progression have also been identified in tumor-associated stroma. By combining laser-microdissection (LMD) with high-throughput screening and high-sensitivity quantitation techniques, miRNA expression in colorectal cancer (CRC) specimens and paired normal colonic tissue was independently characterized in stromal and epithelial tissue compartments. Notably, deregulation of the key oncogene miR-21 was identified exclusively as a stromal phenomenon and miR-106a, an epithelial phenomenon in the malignant state. MiRNAs identified in this study successfully distinguished CRC from normal tissue and metastatic from non-metastatic tumor specimens. Furthermore, in a separate cohort of 50 consecutive patients with CRC, stromal miR-21 and miR-556 and epithelial miR-106a expression predicted short disease free survival (DFS) and overall survival (OS) in stage II disease: miR-21 (DFS: HR = 2.68, p = 0.015; OS: HR = 2.47, p = 0.029); miR-556 (DFS: HR = 2.60, p = 0.018); miR-106a (DFS: HR = 2.91, p = 0.008; OS: HR = 2.25, p = 0.049); combined (All High vs. All Low. DFS: HR = 5.83, p = 0.002; OS: HR = 4.13, p = 0.007). These data support the notion that stromal as well as epithelial miRNAs play important roles during disease progression, and that mapping patterns of deregulated gene expression to the appropriate tumor strata may be a valuable aid to therapeutic decision making in CRC.

Cancer-associated fibroblasts are critical to tumor progression. There exists a dynamic crosstalk between cancer and stromal compartments, which maintains a permissive tumor microenvironment. Extracellular vesicles (EVs) play a significant role in this intercellular communication. Colorectal cancer (CRC) cells can be categorized according to epithelial-mesenchymal transition (EMT) status, and therefore metastatic capacity. We aimed to investigate the effect of EMT on EV-mediated cancer-fibroblast signaling. CRC cell lines (DLD-1, HCT116, SW620 and SW480) were characterized by western blotting to determine EMT status. EVs were isolated from conditioned media by serial centrifugation and validated by transmission electron microscopy, western blotting and nanoparticle tracking analysis. Fluorescently labeled EVs and cells were detected and evaluated by flow cytometry and fluorescence microscopy. Increasing concentrations of EVs from CRC cells were co-cultured with fibroblasts for 24h. Activation/inhibition of signaling pathways was examined by western blotting. EV microRNA (miRNA) profiles were obtained, validated by qPCR and submitted for target and pathway analysis. DLD-1, HCT116 and SW620 cells express E-cadherin and are considered epithelial, whereas SW480 lacks E-cadherin, expresses ZEB-1, and is considered mesenchymal. EVs were spherical, enriched in ALIX, TSG101, CD63 and had a mean diameter of 90nm. EVs from CRC cells were shown to transfer directly to primary ex vivo patient-derived fibroblasts and fibroblast cell lines. Transfer of EVs from epithelial CRC cells abrogated ERK activity in fibroblasts, even at the lowest concentration, and was associated with reduced fibroblast proliferation, whereas EVs from mesenchymal cells had no effect. MiRNA profiling of EVs from epithelial and mesenchymal CRC cells showed a 10-fold upregulation of miR-143-3p in epithelial compared to mesenchymal EVs. MiRNA target analysis and experimental validation show that miR-143-3p directly targets KRAS and HRAS, providing a potential miRNA-orchestrated mechanism of action for the downregulation of fibroblast ERK activity in the tumor microenvironment. Importantly, CRC cellular ERK activity is not reflected in fibroblasts treated with CRC EVs, suggesting that EVs do not directly transmit ERK protein or mRNA. However, miRNAs are the most stable EV cargo, and we show that epithelial but not mesenchymal CRC EVs contain upregulated miRNAs, which target critical components of the ERK pathway. Downregulation of ERK activity has been shown to induce fibroblast senescence, a phenotype linked to cancer progression. We hypothesize that differential regulation occurs because epithelial CRC cells are juxtaposed with fibroblasts in the tumor core, where senescent cancer associated fibroblasts are frequently observed, whereas mesenchymal CRC cells are at the invasive front or in the circulation. Citation Format: Rahul Bhome, Louise M. House, Tilman Sanchez-Elsner, Stephen M. Thirdborough, Emre Sayan, Alex H. Mirnezami. Metastatic and non-metastatic colorectal cancer cells differentially regulate fibroblast cell cycle via extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2982. doi:10.1158/1538-7445.AM2017-2982

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade. Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques. Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimensionally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions. Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision. By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.

Apoptosis or programmed cell death plays a pivotal role in embryonic development and maintenance of homeostasis. It is also involved in the etiology of pathophysiological conditions such as cancer, neurodegenerative, autoimmune, infectious, and heart diseases. Consequently, the study of apoptosis is now at center of both basic and clinical research applications. Therefore, sensitive and simple apoptosis detection techniques are required. Here we describe a monoclonal antibody–defined novel antigen, namely NAPO (negative in apoptosis), which is specifically lost during apoptosis. The anti-NAPO antibody recognizes two nuclear polypeptides of 60 and 70 kD. The antigen is maintained in quiescent and senescent cells, as well as in different phases of the cell cycle, including mitosis. Thus, immunodetection of NAPO antigen provides a specific, sensitive, and easy method for differential identification of apoptotic and nonapoptotic cells.

Epithelial–mesenchymal transition (EMT) is an embryonic trans-differentiation programme that is implicated in organ formation by facilitating the formation of highly motile cells with stem cell capabilities.1 Most tissues of cells derived from ectoderm or mesoderm retain certain elements of EMT and mesenchymal properties are their differentiation markers. On the other hand, cells of organs derived from endoderm only experience EMT and its reversal (mesenchymal-epithelial transition) at certain phases of development and become differentiated secretory epithelial cells. As de-differentiation is a common feature of cancer progression, acquisition of mesenchymal markers accompanied with the loss of epithelial features can be observed in most carcinomas and considered as the reason of cancer spread (metastasis). For the reasons that (1) more than 85% of all cancers are of epithelial origin and (2) epithelial (differentiated) and mesenchymal (undifferentiated) carcinoma cells are morphologically very different, EMT has been mostly studied in carcinoma setting. However, there is growing evidence suggesting that cancers of organs derived from mesoderm (e.g., hematopoietic malignancies and sarcomas) or ectoderm (e.g., glioblastomas and melanomas) also activate EMT programs to acquire an undifferentiated state and become resistant to conventional therapies.2 A good example of this is a recent article by Sanchez-Tillo et al. that investigated the role of EMT-inducing transcription factor ZEB1 in mantle cell lymphoma (MCL) progression.3 MCL is a B-cell malignancy and considered as the rarest form of non-Hodgkin lymphoma.4 Despite this statistical data, it is responsible for a significant portion of B-cell malignancy related mortality because it is an aggressive cancer with a continuous relapse pattern.5 In addition, most MCL patients show poor response to chemotherapy. The translocation of cyclin D1 to immunoglobulin heavy chain enhancer locus t(11;14)(q13;q32) is the genetic hallmark of MCLs; however Wnt, p53, Notch pathway aberrations and pro-survival gene overexpression (Mcl1 and Bcl2 overexpression) are also commonly observed. Sanchez-Tillo et al. investigated the expression of EMT-inducing transcription factor ZEB1 in a cohort of MCL patient samples.3 They found ZEB1 overexpression in half of the samples with a strong correlation with nuclear β-catenin presence (Wnt signalling active). In addition, the clinico-pathological analysis of patient data revealed ZEB1 positivity as a marker for shorter overall survival. This led them to investigate the mechanisms of ZEB1 upregulation and functional contribution of ZEB1 expression to the pathology of MCL. ZEB1 promoter has been previously reported (by the same group) to contain two TCF–LEF binding sites allowing β-catenin–TCF4 binding in colorectal cancer setting.6 They validated the presence of endogenous β-catenin on the respective DNA motifs of ZEB1 promoter by chromatin immunoprecipitation and also showed that ZEB1 mRNA levels are changing upon stimulation with Wnt-3a or knockdown of endogenous β-catenin using two different MCL cell lines. Following this observation, they also investigated if modulation of β-catenin signalling by chemical inhibitors such as salinomycin can alter ZEB1 expression. Salinomycin treatment, indeed, was able to decrease endogenous ZEB1 expression. These results mark the potential to control acquired ZEB1 expression by targeting Wnt pathway and inhibit its functional consequences in MCL. The cellular consequences of ZEB1 overexpression in MCL have not been investigated, however, carcinomas expressing ZEB1 were shown to have canonical hallmarks of EMT such as increased invasiveness and metastasis, resistance to chemotherapy and decreased proliferation.7 Sanchez-Tillo et al. systematically tested all these aspects in MCL by knocking down ZEB1 or β-catenin using MCL cell lines. They found that ZEB1 downregulation decreases baseline cell viability and proliferation. The gene expression of ZEB1-expressing cells indicated activation of survival and repression of cell death pathways as validated by increased levels of myc, cyclin D1, CENPF, Ki67, Bcl2, MCL1 and decreased p53, bax and PMAIP1 mRNAs. In addition, they also investigated whether ZEB1 expression confers resistance to chemotherapy. In line with their previous data regarding changes in baseline cell viability, ZEB1 downregulation increased sensitivity to commonly used chemotherapeutic drugs used in MCL treatment, such as Doxorubicin, Cytarabine and Gemcitabine but not Vincristine. The effect of ZEB1 knockdown on chemosensitization was also confirmed by pre-treatment of MCL cell lines with Salinomycin. EMT-induced chemoresistance has been described in different carcinoma models; however, the cellular mechanism of this phenomenon still remains to be clarified.8 The reasons for chemoresistance can be at least 3-fold. Cancer cells can alter drug influx/efflux to favour less chemotherapeutic agents in the cell (e.g., activate multidrug resistance proteins), they can activate efficient detoxification (e.g., by glutathione S-transferase) or enable efficient DNA repair to escape DNA damage-induced apoptosis. In this article, Sanchez-Tillo et al. investigated the regulation of drug influx/efflux pathways by ZEB1. Using two different MCL cell lines and validating by IHC in primary MCL samples, they showed that critical drug transporters are regulated by ZEB1 to favour survival of Wnt signalling active cancer cells. The authors also extended their in vitro studies to animal models. Subcutaneous injection of Granta-519 cells expressing or silenced ZEB1 showed significantly different responses; ZEB1 silenced cells showing delays in tumour growth and enhanced sensitivity to Doxorubicin treatment. Finally, they explored whether salinomycin can synergize with Doxorubicin for an enhanced response to chemotherapy. Using established MCL cell lines and primary cells from MCL patients, they showed that sublethal doses of Salinomycin and Doxorubicin can synergize and effectively kill MCL cells. These results support their in vivo data and provide evidence that β-catenin-activated ZEB1 expression contributes to tumour progression and chemoresistance (Figure 1). Figure 1 Role of EMT-inducing transcription factor ZEB1 in MCL. In carcinoma cells, activation of distinct signalling pathways such as TGFβ, Wnt and receptor tyrosine kinase (RTK) converges to ZEB1 for a fully de-differentiated and mesenchymal (EMT) phenotype. ... What is next? A couple of things remained to be done and questions answered to make use of the full potential of these exciting findings. First of all, Sanchez-Tillo et al. did not show the clinico-pathological variables of their patient cohort. In recent years, anti-B cell-based immunotherapeutic regimens, such as the anti-CD20 antibody rituximab, gained attention as an alternative in the treatment of B-cell malignancies. The provision of detailed patient data may allow stratification of MCL cases and potentially mark subgroups that can or cannot be treated with chemo and/or immunotherapies. In addition, similar correlative studies have to be performed using independent patient cohorts to externally validate the clinical importance of ZEB1 in MCL and allow clinical translation. Another point to be clarified is the functional link between ZEB1-induced alterations in drug influx/efflux and the actual presence of drugs in MCL cells. Genotoxic agents such as Doxorubicin can be imported through cell membrane by passive diffusion and effectively retained in the cell as DNA-bound form so they cannot be exported by drug transporters. This question can be easily answered due to the fluorescence nature of Doxorubicin using normal and transformed B cells, in the presence and absence of ZEB1. Finally, activation of EMT pathways in carcinoma results in generation of malignant cells with stem cell properties. A critical question that remains to be answered is whether ZEB1-expressing MCL cells are de-differentiated enough to be considered as B-cell precursors. As a follow-up of this work, it will be fascinating to explore if the tumour-promoting features of ZEB1 in MCL cells are also conserved in other malignancies, particularly in other mesoderm-derived tissues such as sarcomas or cancers such as hepatoblastomas that β-catenin activation is an early event in tumourigenesis.

As Eugene Garfield said, while it is easy to recognize a good paper, it could be more difficult to recognize a bad paper. In fact, the results could be weak, but the conclusion could still be right, even though not fully supported by the data shown. Preliminary reports could also fall in this category. After all, it was not a Cell but a BBRC paper – only a little BBRC of three impact factor – describing a novel experimental model in which to study ATP-dependent proteolysis.1 In a lysate from rabbit reticulocytes, where the proteolytic activity was not due to lysosomes (pH optimum of 7.8), they separated two fractions in a DEAE cellulose column, each one individually inactive, but after recombination of the two fractions ATP-dependent proteolysis was reconstituted. Immediately, Hershko and his young assistant, Ciechanover, went for a sabbatical to the Fox Chase Cancer Center in Philadelphia to work with Irwin Rose, and the three began unveiling the E1-E2-E3 ligase components of the Ubiquitin-Proteasome System (UPS),2 see for example some recent reviews.3, 4 For this work, Ciechanover, Hershko, and Rose were granted the 2004 Nobel Prize. Five clinical trials based on UPS inhibition were in progress in the same year:5 (i) Velcade-TM (dipeptide boronic acid, Bortezomid, PS-341, Millenium Pharmaceuticals Inc.) phase III, FDA approved for relapsed and refractory multiple myeloma and other solid cancers; it stabilizes cell-cycle and proapaoptotic proteins, inhibits antiapoptotic proteins, and affects tumor microenvironment; (ii) MLN519 (lactacystin derivative, PS-519, Millenium Pharmaceuticals Inc.) phase I for acute stroke and myocardial infections; it is a potent anti-inflammatory and neuroprotective compound; (iii) Epoxomicin-Eponemycin (Streptomyces epoxyketones) preclinical; it has cytotoxic effects in various tumor cells; (iv) NLVS (trileucine vinyl-sulfone) preclinical; it is an irreversible inhibitor of trypsin- and chemotrypsin-like proteasome activities; (v) Ritonavir (Peptidomimetic protease inhibitor; Abbott) phase II for AIDS and tumor patients; it is an HIV protease inhibitor, also inhibits chemotrypsin-like activity of proteasome. While the timeline of this story is outlined by Ciechanover himself,6 the present book describes the most advanced ongoing clinical trials on UPS inhibition. It is of particular interest that this book review comes out in the same issue containing manuscripts from scientists that have originated in this field.7, 8, 9, 10

MicroRNAs (miRNAs) are a class of small highly conserved RNAs that provide widespread expressional control through the translational repression of mRNA. MiRNAs have fundamental roles in the regulation of intracellular processes, and their importance during malignant transformation and metastasis is becoming increasingly well recognized. An important event in the metastatic cascade is epithelial to mesenchymal transition (EMT), a reversible phenotypic switch over, which endows malignant epithelial cells with the capacity to break free from one another and invade the surrounding stroma. Our understanding of EMT has been significantly improved by the characterization of miRNAs that influence the signalling pathways and downstream events that define EMT on a molecular level. Here, we detail the role of miRNAs in EMT, and in doing so demonstrate their importance in the early stages of the metastatic cascade; we discuss a significant body of data that suggest new opportunities for drug development, and we highlight critical knowledge gaps that remain to be addressed.

Despite recent advances, cancer remains a leading cause of death worldwide. In developed countries, the incidence of colorectal and breast cancer has been stable, but no improvement in prognosis has been observed if the patient presents with metastases at diagnosis. This fact highlights the importance of therapeutic approaches targeting cellular invasion and metastasis programmes as the next step in cancer treatment. During carcinoma progression a process called Epithelial-Mesenchymal Transition (EMT) results in enhanced invasion and motility which is directly linked with loss of epithelial polarity and epithelial junctions, migration permissive cytoskeleton alterations, and the acquisition of mesenchymal properties. The recent discovery of microRNAs (miRNAs) controlling key cellular pathways has opened a new era in understanding how EMT pathways are modulated. In this review, we classify EMT regulating proteins according to their cellular localization (membrane, cytoplasmic and nuclear), and summarize the current knowledge on how they are controlled by miRNAs and propose potential miRNAs for the transcripts that may control their expression.

Cataloged from PDF version of article. Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial-mesenchymal transition in development and tumor metastasis. Due to the lack of human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development. © 2010 Elsevier Inc.

Epithelial mesenchymal transitions (EMT), the generation of motile mesenchymal cells from epithelial sheets, are differentiation programs which take place at several critical steps of embryonic development and in metastatic cancer. Recent data have shown that the transcription factors which are master regulators of EMT also regulate cell cycle progression, apoptosis and senescence. In light of these new observations, the role of these factors in human cancer may be broader than previously anticipated. Here we review recent literature on non-EMT functions of EMT-controlling transcription factors. We will mainly focus on transcription factors belonging to the ZEB family, but some important results obtained by investigators studying other key EMT regulators, Snail and Twist are also discussed.