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In JoVE (1)
Other Publications (6)
- FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
- Cancer Research
- European Journal of Cancer (Oxford, England : 1990)
- Cancer Research
- Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
- The International Journal of Developmental Biology
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Articles by Dalit Barkan in JoVE
JoVE Clinical and Translational Medicine
Bir In Vitro Sistem
1Department of Biology, University of Haifa, 2Transgenic Oncogenesis and Genomics Section, Laboratory of Cancer Biology and Genetics, National Cancer Institute
In vitro sistemi değiştirilmiş 3-D sulandırılmış bazal membran birçok tümör hücre hatlarının büyüme özellikleri tümör hücrelerinin metastatik ikincil site uykuda veya proliferatif davranışı ile ilişkili olduğu
Other articles by Dalit Barkan on PubMed
Leptin Induces Ovulation in GnRH-deficient Mice
Leptin-deficient ob/ob mice have reduced gonadotropin-releasing hormone (GnRH) secretion, leading to gonadotropin deficiencies, hypogonadism, and anovulation, which are completely reversed following leptin administration. To determine whether the role of leptin in ovulation is mediated exclusively through GnRH, we studied leptin's action in GnRH-deficient (hpg) mice, as well as ob/ob mice and normal, prepubertal mice in which the GnRH axis was blocked with antide. Following pretreatment with pregnant mare serum gonadotropin, leptin induced ovulation in all three mouse models. Unlike mature normal mice, these ovulations were not triggered by a luteinizing hormone (LH) surge, as demonstrated by lack of increase in its surrogate marker progesterone. Rather, leptin induced hyperemia and leakage in the follicle, as well as the proteinase ADAMTS-1 (a disintegrin and metalloproteinase with a thrombospondin-like motif), which facilitates extrusion of the follicular content. These data show that on top of its role as an inducer of GnRH secretion, leptin may elicit an LH-independent ovulation.
Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in three-dimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin beta1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin beta1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin beta1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch.
Extracellular Matrix: a Gatekeeper in the Transition from Dormancy to Metastatic Growth
Metastases can develop after apparently successful treatment of a primary tumour, sometimes following a period of tumour dormancy that can last for years. However, factors that regulate metastatic tumour dormancy remain poorly understood. Here we review the potential contribution of interactions between tumour cells and the microenvironment in metastatic sites, in regulating tumour dormancy vs. metastatic growth. We focus particularly on the potential role of the extracellular matrix (ECM) in regulating maintenance and release from dormancy. Tumour cells that fail to properly adhere to the ECM may enter a state of dormancy. The molecular and physical composition of the ECM can be affected by tumour cells themselves, as well as multiple stromal cell types. The roles of integrins, fibronectin, and collagen are discussed, as are factors that can change the ECM. A better understanding of the molecular details of the crosstalk between tumour cells and the ECM in secondary sites, and how these regulate the dormant state, may lead to improved therapeutic strategies to induce or maintain disseminated tumour cells in a dormant state, or alternatively to successfully eradicate dormant cells.
Metastatic Growth from Dormant Cells Induced by a Col-I-enriched Fibrotic Environment
Breast cancer that recurs as metastatic disease many years after primary tumor resection and adjuvant therapy seems to arise from tumor cells that disseminated early in the course of disease but did not develop into clinically apparent lesions. These long-term surviving, disseminated tumor cells maintain a state of dormancy, but may be triggered to proliferate through largely unknown factors. We now show that the induction of fibrosis, associated with deposition of type I collagen (Col-I) in the in vivo metastatic microenvironment, induces dormant D2.0R cells to form proliferative metastatic lesions through beta1-integrin signaling. In vitro studies using a three-dimensional culture system modeling dormancy showed that Col-I induces quiescent D2.0R cells to proliferate through beta1-integrin activation of SRC and focal adhesion kinase, leading to extracellular signal-regulated kinase (ERK)-dependent myosin light chain phosphorylation by myosin light chain kinase and actin stress fiber formation. Blocking beta1-integrin, Src, ERK, or myosin light chain kinase by short hairpin RNA or pharmacologic approaches inhibited Col-I-induced activation of this signaling cascade, cytoskeletal reorganization, and proliferation. These findings show that fibrosis with Col-I enrichment at the metastatic site may be a critical determinant of cytoskeletal reorganization in dormant tumor cells, leading to their transition from dormancy to metastatic growth. Thus, inhibiting Col-I production, its interaction with beta1-integrin, and downstream signaling of beta1-integrin may be important strategies for preventing or treating recurrent metastatic disease.
β1-integrin: a Potential Therapeutic Target in the Battle Against Cancer Recurrence
Primary cancer treatment, involving both local and often systemic adjuvant therapy, is often successful, especially if the cancer is detected at an early stage of progression. However, for some patients, the cancer may recur either locally or as distant metastases, in some cases many years after apparently successful primary treatment. Significant tumor dormancy has been documented in several cancers, such as breast, melanoma, and renal cancer. Tumor dormancy has long been recognized as an important problem in management of cancer patients. Recent work has clarified biologic aspects of tumor dormancy and has shown that dormant tumor cells may be resistant to cytotoxic chemotherapy and radiation. This work has led to recognition of a key role for β1-integrin in regulating the switch from a dormant state to active proliferation and metastasis. Here we discuss the role of β1-integrin and its signaling partners in regulating the dormant phenotype. We also consider possible therapeutic approaches, such as small molecules or antibodies (ATN-161, volociximab, and JSM6427), directed against β1-integrin signaling to target dormant cancer cells and to prevent metastatic recurrence.
Non-steroidal Anti-inflammatory Drugs Target the Pro-tumorigenic Extracellular Matrix of the Postpartum Mammary Gland
Breast cancer patients diagnosed postpartum have poor prognosis. The postpartum mammary gland undergoes tissue regression to return to the pre-pregnant state. This involution is characterized by wound healing programs known to be tumor promotional in other contexts. Previous studies have shown that mammary extracellular matrix (ECM) from nulliparous rats has tumor suppressive attributes, while mammary ECM from involuting mammary glands is promotional. In models of pregnancy-associated breast cancer, non-steroidal anti-inflammatory drug (NSAID) treatment targeted to postpartum involution inhibits tumor progression, in part by suppressing COX-2 dependent collagen deposition. Because mammary ECM proteins are coordinately regulated, NSAID treatment is anticipated to result in additional protective changes in the mammary extracellular matrix. Here, systemic NSAID treatment was utilized during postpartum involution to reduce mammary COX-2 activity. ECM was isolated from actively involuting glands of rats treated with NSAIDs and compared to ECM isolated from control-involution and nulliparous rats in 3D cell culture and xenograft assays. Compositional changes in ECM between groups were identified by proteomics. In four distinct 3D culture assays, normal and transformed mammary epithelial cells plated in NSAID-involution ECM, phenocopied cells plated in ECM from nulliparous rats rather than ECM from control-involution rats. Tumor cells mixed with NSAID-involution ECM and injected orthotopically in mice formed smaller tumors than cells mixed with control-involution ECM. Proteomic analyses identified and 3D culture assays implicated the ECM protein tenascin-C as a potential mediator of tumor progression during involution that is decreased by NSAID treatment. In summary, NSAID treatment decreases tumor-promotional attributes of postpartum involution mammary ECM.
