Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
Permanent URI for this collectionhttps://hdl.handle.net/11147/9
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Review Citation - WoS: 14Citation - Scopus: 14Recent Advances in Lab-On Systems for Breast Cancer Metastasis Research(Royal Society of Chemistry, 2023) Fıratlıgil Yıldırır, Burcu; Yalçın Özuysal, Özden; NonappaBreast cancer is the leading cause of cancer-related deaths in women. Multiple molecular subtypes, heterogeneity, and their ability to metastasize from the primary site to distant organs make breast cancer challenging to diagnose, treat, and obtain the desired therapeutic outcome. As the clinical importance of metastasis is dramatically increasing, there is a need to develop sustainable in vitro preclinical platforms to investigate complex cellular processes. Traditional in vitro and in vivo models cannot mimic the highly complex and multistep process of metastasis. Rapid progress in micro- and nanofabrication has contributed to soft lithography or three-dimensional printing-based lab-on-a-chip (LOC) systems. LOC platforms, which mimic in vivo conditions, offer a more profound understanding of cellular events and allow novel preclinical models for personalized treatments. Their low cost, scalability, and efficiency have resulted in on-demand design platforms for cell, tissue, and organ-on-a-chip platforms. Such models can overcome the limitations of two- and three-dimensional cell culture models and the ethical challenges involved in animal models. This review provides an overview of breast cancer subtypes, various steps and factors involved in metastases, existing preclinical models, and representative examples of LOC systems used to study and understand breast cancer metastasis and diagnosis and as a platform to evaluate advanced nanomedicine for breast cancer metastasis.Article Citation - WoS: 7Citation - Scopus: 7Connexin 32 Overexpression Increases Proliferation, Reduces Gap Junctional Intercellular Communication, Motility and Epithelial-To Transition in Hs578t Breast Cancer Cells(Springer, 2022) Uğur, Deniz; Güngül, Taha Buğra; Yücel, Simge; Özçivici, Engin; Yalçın Özuysal, Özden; Meşe Özçivici, GülistanConnexins (Cx) are primary components of gap junctions that selectively allow molecules to be exchanged between adjacent cells, regulating multiple cellular functions. Along with their channel forming functions, connexins play a variety of roles in different stages of tumorigenesis and their roles in tumor initiation and progression is isoform- and tissue-specific. While Cx26 and Cx43 were downregulated during breast tumorigenesis, Cx32 was accumulated in the cytoplasm of the cells in lymph node metastasis of breast cancers and Cx32 was further upregulated in metastasis. Cx32's effect on cell proliferation, gap junctional communication, hemichannel activity, cellular motility and epithelial-to-mesenchymal transition (EMT) were investigated by overexpressing Cx32 in Hs578T and MCF7 breast cancer cells. Additionally, the expression and localization of Cx26 and Cx43 upon Cx32 overexpression were examined by Western blot and immunostaining experiments, respectively. We observed that MCF7 cells had endogenous Cx32 while Hs578T cells did not and when Cx32 was overexpressed in these cells, it caused a significant increase in the percentages of Hs578T cells at the S phase in addition to increasing their proliferation. Further, while Cx32 overexpression did not induce hemichannel activity in either cell, it decreased gap junctional communication between Hs578T cells. Additionally, Cx32 was mainly observed in the cytoplasm in both cells, where it did not form gap junction plaques but Cx32 overexpression reduced Cx43 levels without affecting Cx26. Moreover, migration and invasion potentials of Hs578T and migration in MCF7 were reduced upon Cx32 overexpression. Finally, the protein level of mesenchymal marker N-cadherin decreased while epithelial marker ZO-1 and E-cadherin increased in Hs578T cells. We observed that Cx32 overexpression altered cell proliferation, communication, migration and EMT in Hs578T, suggesting a tumor suppressor role in these cells while it had minor effects on MCF7 cells.Article Citation - WoS: 12Citation - Scopus: 12Sema6d Differentially Regulates Proliferation, Migration, and Invasion of Breast Cell Lines(American Chemical Society, 2022) Günyüz, Zehra Elif; Sahi İlhan, Ece; Küçükköse, Cansu; İpekgil, Doğaç; Tok, Güneş; Meşe, Gülistan; Özçivici, Engin; Yalçın Özuysal, ÖzdenSemaphorin 6D (SEMA6D), a member of the class 6 semaphorin family, is a membrane-associated protein that plays a key role in the development of cardiac and neural tissues. A growing body of evidence suggests that SEMA6D is also involved in tumorigenesis. In breast cancer, high SEMA6D levels are correlated with better survival rates. However, very little is known about the functional significance of SEMA6D in breast tumorigenesis. In the present study, we aimed to investigate the effects of SEMA6D expression on the normal breast cell line MCF10A and the breast cancer cell lines MCF7 and MDA MB 231. We demonstrated that SEMA6D expression increases the proliferation of MCF10A cells, whereas the opposite effect was observed in MCF7 cells. SEMA6D expression induced anchorage-independent growth in both cancer cell lines. Furthermore, migration of MCF10A and MCF7 cells and invasion of MDA MB 231 cells were elevated in response to SEMA6D overexpression. Accordingly, the genes related to epithelial-mesenchymal transition (EMT) were altered by SEMA6D expression in MCF10A and MCF7 cell lines. Finally, we provided evidence that SEMA6D levels were associated with the expression of the cell cycle, EMT, and Notch signaling pathway-related genes in breast cancer patients' data. We showed for the first time that SEMA6D overexpression has cell-specific effects on the proliferation, migration, and invasion of normal and cancer breast cell lines, which agrees with the gene expression data of clinical samples. This study lays the groundwork for future research into understanding the functional importance of SEMA6D in breast cancerArticle Citation - WoS: 13Citation - Scopus: 13Her2-Targeted, Degradable Core Cross-Linked Micelles for Specific and Dual Ph-Sensitive Dox Release(John Wiley and Sons Inc., 2021) Bayram, Nazende Nur; Ulu, Gizem Tuğçe; Topuzoğulları, Murat; Baran, Yusuf; Dinçer İşoğlu, SevilHere, a targeted, dual-pH responsive, and stable micelle nanocarrier is designed, which specifically selects an HER2 receptor on breast cancer cells. Intracellularly degradable and stabilized micelles are prepared by core cross-linking via reversible addition-fragmentation chain-transfer (RAFT) polymerization with an acid-sensitive cross-linker followed by the conjugation of maleimide-doxorubicin to the pyridyl disulfide-modified micelles. Multifunctional nanocarriers are obtained by coupling HER2-specific peptide. Formation of micelles, addition of peptide and doxorubicin (DOX) are confirmed structurally by spectroscopical techniques. Size and morphological characterization are performed by Zetasizer and transmission electron microscope (TEM). For the physicochemical verification of the synergistic acid-triggered degradation induced by acetal and hydrazone bond degradation, Infrared spectroscopy and particle size measurements are used. Drug release studies show that DOX release is accelerated at acidic pH. DOX-conjugated HER2-specific peptide-carrying nanocarriers significantly enhance cytotoxicity toward SKBR-3 cells. More importantly, no selectivity toward MCF-10A cells is observed compared to HER2(+) SKBR-3 cells. Formulations cause apoptosis depending on Bax and Caspase-3 and cell cycle arrest in G2 phase. This study shows a novel system for HER2-targeted therapy of breast cancer with a multifunctional nanocarrier, which has higher stability, dual pH-sensitivity, selectivity, and it can be an efficient way of targeted anticancer drug delivery.Article Citation - WoS: 24Citation - Scopus: 23On-Chip Determination of Tissue-Specific Metastatic Potential of Breast Cancer Cells(Wiley, 2021) Fıratlıgil Yıldırır, Burcu; Batı Ayaz, Gizem; Tahmaz, İsmail; Bilgen, Müge; Pesen Okvur, Devrim; Yalçın Özuysal, ÖzdenMetastasis is one of the major obstacles for breast cancer patients. Limitations of current models demand the development of custom platforms to predict metastatic potential and homing choices of cancer cells. Here, two organ-on-chip platforms, invasion/chemotaxis (IC-chip) and extravasation (EX-chip) were used for the quantitative assessment of invasion and extravasation towards specific tissues. Lung, liver and breast microenvironments were simulated in the chips using tissue-specific cells embedded in matrigel. In the IC-chip, invasive MDA-MB-231, but not noninvasive MCF-7 breast cancer cells invaded into lung and liver microenvironments. In the EX-chip, MDA-MB-231 cells extravasated more into the lung compared to the liver and breast microenvironments. In addition, lung-specific MDA-MB-231 clone invaded and extravasated into the lung microenvironment more efficiently than the bone-specific clone. Both invasion/chemotaxis and extravasation results were in agreement with published clinical data. Collectively, our results show that IC-chip and EX-chip, simulating tissue-specific microenvironments, can distinguish different in vivo metastatic phenotypes, in vitro. Determination of tissue-specific metastatic potential of breast cancer cells is expected to improve diagnosis and help select the ideal therapy.Article Citation - WoS: 8Citation - Scopus: 8The Role of Connexins in Breast Cancer: From Misregulated Cell Communication To Aberrant Intracellular Signaling(Taylor & Francis, 2022) Ünal, Yağmur Ceren; Yavuz, Büşra; Özçivici, Engin; Meşe Özçivici, GülistanIn spite of clinical advancements and improved diagnostic techniques, breast cancers are the leading cause of cancer-associated deaths in women worldwide. Although 70% of early breast cancers can be cured, there are no efficient therapies against metastatic breast cancers. Several factors including connexins and gap junctions play roles in breast tumorigenesis. Connexins are critical for cellular processes as a linkage between connexin mutations and hereditary disorders demonstrated their importance for tissue homeostasis. Further, alterations in their expression, localization and channel activities were observed in many cancers including breast cancer. Both channel-dependent and independent functions of connexins were reported in initiation and progression of cancers. Unlike initial reports suggesting tumor suppressor functions, connexins and gap junctions have stage, context and isoform dependent effects in breast cancers similar to other cancers. In this review, we tried to describe the current understanding of connexins in tumorigenesis specifically in breast cancers.Article Citation - WoS: 16Citation - Scopus: 18Connexin 32 Induces Pro-Tumorigenic Features in Mcf10a Normal Breast Cells and Mda-Mb Metastatic Breast Cancer Cells(Elsevier, 2020) Yalçın Özuysal, Özden; Adak, Aslı; Ünal, Yağmur Ceren; Yücel, Simge; Vural, Zehra; Turan, Fatma Başak; Meşe, GülistanConnexins (Cx), the basic subunit of gap junctions, play important roles in cell homeostasis, and their abnormal expression and function are associated with human hereditary diseases and cancers. In tumorigenesis, connexins were observed to have both anti-tumorigenic and pro-tumorigenic roles in a context- and stage-dependent manner. Initially, Cx26 and Cx43 were thought to be the only connexins involved in normal breast homeostasis and breast cancer. Later on, association of Cx32 expression with lymph node metastasis of breast cancer and subsequent demonstration of its expression in normal breast tissue suggested that Cx32 contributes to breast tissue homeostasis. Here, we aimed to determine the effects of Cx32 on normal breast cells, MCF10A, and on breast cancer cells, MDA-MB-231. Cx32 overexpression had profound effects on MCF10A cells, decreasing cell proliferation by increasing the doubling time of MCF10A. Furthermore, MCF10A cells acquired mesenchymal-like appearance upon Cx32 expression and had increased migration capacity and expression of both E-cadherin and vimentin. In contrast, Cx32 overexpression altered the EMT markers of MDA-MB-231 by increasing the expression of mesenchymal markers, such as slug and vimentin, and decreasing E-cadherin expression without affecting their proliferation and morphology. Our results indicate, for the first time in the literature, that Cx32 has tumor-promoting roles in MCF10A and MDA-MB-231 cells.Article Citation - WoS: 9Citation - Scopus: 10Effects of Notch Signalling on the Expression of Sema3c, Hmga2, Cxcl14, Cxcr7, and Ccl20 in Breast Cancer(TÜBİTAK, 2019) Küçükköse, Cansu; Yalçın Özuysal, ÖzdenMetastasis is the main reason for death in breast cancer. Understanding the molecular players in metastasis is crucial for diagnostic and therapeutic purposes. Notch signalling plays an oncogenic role in breast tumorigenesis and is involved in metastasis. Downstream mediators of Notch signalling in prometastatic processes are not yet fully discovered. Here we aimed to investigate whether Notch signalling regulates the expression of SEMA3C, HMGA2, CXCL14, CXCR7, and CCL20, which are involved in prometastatic processes, in breast cell lines. To this end, expression of the selected genes was analysed following Notch activation by overexpression of the Notch1 intracellular domain in the normal breast epithelial cell line MCF10A, and inhibition by silencing of the Notch transcriptional mediator RBPj kappa in the breast cancer cell line MDA MB 231. SEMA3C and HMGA2 mRNA were decreased, while CXCL14 and CXCR7 mRNA were increased significantly in response to Notch activation in MCF10A cells. Notch inhibition in MDA MB 231 cells significantly decreased HMGA2 and CCL20 mRNA. Protein levels were not significantly altered by Notch modulation. In conclusion, we showed that Notch signalling regulates expression of SEMA3C, CXCL14, CCL20, CXCR7, and HMGA2, which are prominent candidate genes that might function downstream of Notch to induce prometastatic processes.Article Citation - WoS: 17Citation - Scopus: 19Pro-Metastatic Functions of Notch Signaling Is Mediated by Cyr61 in Breast Cells(Elsevier, 2020) Küçükköse, Cansu; Efe, Eda; Günyüz, Zehra Elif; Fıratlıgil, Burcu; Doğan, Hülya; Yalçın Özuysal, Özden; İlhan, MustafaMetastasis is the main cause of cancer related deaths, and unfolding the molecular mechanisms underlying metastatic progression is critical for the development of novel therapeutic approaches. Notch is one of the key signaling pathways involved in breast tumorigenesis and metastasis. Notch activation induces pro-metastatic processes such as migration, invasion and epithelial to mesenchymal transition (EMT). However, molecular mediators working downstream of Notch in these processes are not fully elucidated. CYR61 is a secreted protein implicated in metastasis, and its inhibition by a monoclonal antibody suppresses metastasis in xenograft breast tumors, indicating the clinical importance of CYR61 targeting. Here, we aimed to investigate whether CYR61 works downstream of Notch in inducing pro-metastatic phenotypes in breast cells. We showed that CYR61 expression is positively regulated by Notch activity in breast cells. Notch1-induced migration, invasion and anchorage independent growth of a normal breast cell line, MCF10A, were abrogated by CYR61 silencing. Furthermore, upregulation of core EMT markers upon Notch1-activation was impaired in the absence of CYR61. However, reduced migration and invasion of highly metastatic cell line, MDA MB 231, cells upon Notch inhibition was not dependent on CYR61 downregulation. In conclusion, we showed that in normal breast cell line MCF10A, CYR61 is a mediator of Notch1-induced pro-metastatic phenotypes partly via induction of EMT. Our results imply CYR61 as a prominent therapeutic candidate for a subpopulation of breast tumors with high Notch activity.Article Citation - WoS: 19Citation - Scopus: 19Irf6 Is Involved in the Regulation of Cell Proliferation and Transformation in Mcf10a Cells Downstream of Notch Signaling(Public Library of Science, 2015) Zengin, Talip; Ekinci, Burcu; Küçükköse, Cansu; Yalçın Özuysal, ÖzdenIRF6, 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.