Guislaine Barrière

Mesenchymal and stemness circulating tumor cells in early breast cancer diagnosis

BackgroundEpithelial mesenchymal transition (EMT) is a crucial event likely involved in dissemination of epithelial cancer cells. This process enables them to acquire migratory/invasive properties, contributing to tumor and metastatic spread. To know if this event is an early one in breast cancer, we developed a clinical trial. The aim of this protocol was to detect circulating tumor cells endowed with mesenchymal and/or stemness characteristics, at the time of initial diagnosis. Breast cancer patients (n = 61), without visceral or bone metastasis were enrolled and analysis of these dedifferentiated circulating tumor cells (ddCTC) was realized.MethodsAdnaGen method was used for enrichment cell selection. Then, ddCTC were characterized by RT-PCR study of the following genes: PI3Kα, Akt-2, Twist1 (EMT markers) and ALDH1, Bmi1 and CD44 (stemness indicators).ResultsAmong the studied primary breast cancer cohort, presence of ddCTC was detected in 39% of cases. This positivity is independant from tumor clinicopathological factors apart from the lymph node status.ConclusionsOur data uniquely demonstrated that in vivo EMT occurs in the primary tumors and is associated with an enhanced ability of tumor cells to intravasate in the early phase of cancer disease. These results suggest that analysis of circulating tumor cells focused on cells showing mesenchymal or stemness characteristics might facilitate assessment of new drugs in clinical trials.

Epithelial Mesenchymal Transition: a double-edged sword

Epithelial mesenchymal transition (EMT) is a physiological process necessary to normal embryologic development. However in genesis of pathological situations, this transition can be perverted and signaling pathways have different regulations from those of normal physiology. In cancer invasion, such a mechanism leads to generation of circulating tumor cells. Epithelial cancer cells become motile mesenchymal cells able to shed from the primary tumor and enter in the blood circulation. This is the major part of the invasive way of cancer. EMT is also implicated in chronic diseases like fibrosis and particularly renal fibrosis. In adult organisms, healing is based on EMT which is beneficial to repair wounds even if it can sometimes exceed its goal and elicit fibrosis. In this review, we delineate the clinical significance of EMT in both physiological and pathological circumstances.

Circulating tumor cells and epithelial, mesenchymal and stemness markers: characterization of cell subpopulations

Until now detection and numeration of circulating tumor cells (CTCs) were essentially used as a prognostic factor in cancer progression. To extend the role of these kinds of analysis, it seems necessary to improve analytical methods related to isolation and characterization of CTCs. Discrepancies between published results corroborates this requirement. In this review we suggest a combination of markers able to reach the goal. Moreover to improve the clinical utility of CTC analysis, particularly in the therapeutic follow up of the disease, epithelial mesenchymal transition (EMT) level of a global CTC population should be studied.

Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.

Epithelial Mesenchymal Transition: A New Insight into the Detection of Circulating Tumor Cells

Many research groups reported on the relation between circulating tumor cells (CTCs) in peripheral blood and worse prognosis for metastatic cancer patients. These results are based on CTCs counting and did not take into account molecular characteristics of cells. To establish CTCs as a reliable and accurate biological marker, new technologies must be focused on CTC subpopulations: dedifferentiated circulating tumor cells (ddCTCs) arising from epithelial mesenchymal transition (EMT). To select and detect them, different methods have been proposed but none has still reached the goal. Technical progress and translational research are expected to establish CTCs as a real marker. Thus CTC evaluation profiling for each patient will lead to personalize followup and therapy.

Overexpression of cytosolic glutathione peroxidase (GPX1) delays endothelial cell growth and increases resistance to toxic challenges.

Oxidative stress results from the imbalance between reactive oxygen species (ROS) and ROS-scavenging molecules. Among them, cytosolic glutathione peroxidase (GPX1) plays a major role as it reduces a large part of intracellular ROS. Endothelial cells are a barrier for potentially aggressive molecules circulating in the blood stream and, therefore, are often under great oxidative stress. Thus, we investigated the potentially protective effects of GPX1 overexpression in the endothelial cell line, ECV304. We found that chronic GPX1 overexpression delays cell growth without affecting viability or decreasing resistance to hydrogen peroxide-induced oxidative stress. As GPX1 overexpression could drain the cellular reduced glutathione (GSH) pool, we also tested the effects of extracellular GSH supplementation on cell growth. Despite its largely referenced beneficial effects for cells, GSH was toxic for ECV304 cells in a dose-dependent manner but GSH-induced toxicity was reduced in selenium supplemented cultures and completely abolished in ECV304 overexpressing GPX1, compared to control. In summary, GPX1 overexpression delays cell growth and protects them from GSH and H(2)O(2) toxicity.

Expression of 5-lipoxygenase (5-LOX) in T lymphocytes.

5‐lipoxygenase (5‐LOX) is the key enzyme responsible for the synthesis of the biologically active leukotrienes. Its presence has been reported in cells of the myeloid lineage and B lymphocytes but has not been formally defined in T lymphocytes. In this study, we provide evidence for 5‐LOX expression on both transcriptional and translational levels in highly purified peripheral blood T cells as well as in human T lymphoblastoid cell lines (MOLT4 and Jurkat). Messenger RNA (mRNA) of 5‐LOX was amplified by conventional reverse transcription–polymerase chain reaction (RT‐PCR; MOLT4 and Jurkat cells) and by in situ RT‐PCR (T lymphocytes). 5‐LOX protein expression was confirmed by Western blot and immunofluorescence studies. 5‐LOX was present primarily in the cytoplasm with some nuclear localization and was translocated to the nuclear periphery after culture in a mitosis‐supporting medium. Fluorescence‐activated cell sorter analysis of different T‐lymphocyte populations, including CD4, CD8, CD45RO, CD45RA, T helper type 2, and T‐cell receptor‐αβ and ‐γδ expressing cells, did not identify a differential distribution of the enzyme. Purified peripheral blood T lymphocytes were incapable of synthesizing leukotrienes in the absence of exogenous arachidonic acid. Jurkat cells produced leukotriene C4 and a small amount of leukotriene B4 in response to CD3–CD28 cross‐linking. This synthesis was abolished by two inhibitors of leukotriene synthesis, MK‐886 and AA‐861. The presence of 5‐LOX in T lymphocytes but the absence of endogenous lipoxygenase metabolite production compared to Jurkat cells may constitute a fundamental difference between resting peripheral lymphocytes and leukaemic cells.

Metformin: A Rising Star to Fight the Epithelial Mesenchymal Transition in Oncology

Metformin is a biguanide derivative which is widely prescribed as an oral drug for diabetes mellitus type 2. This old molecule has recently received a new attention because of its therapeutic properties in oncology, that seem to be independent of its action on glycemia homeostasis. The reappraisal of its pharmacological effects was supported by delineation of signaling pathways and more recently clinical trials. Numerous epidemiological studies showed that diabetics have an increased risk of several types of cancer and cancer mortality. Complex relationship between cancer and type 2 diabetes is going to be unraveled and recent observations revealed a significant action of metformin, but not other anti-diabetic agents, on cancer cells. As metformin may act as an anticancer drug through inhibition of mTOR, it might have greater benefice than suggested by insulin lowering alone. This review summarizes major publications on the link between cancer and metformin underscoring new implications of this chemical drug in oncology field. New perspectives about utilization of this molecule in clinical oncological routine, are described, particularly for patients without disturbance of glucose homeostasis. As the epithelial mesenchymal transition (EMT) seems implicated into invasive process and metastasis in cancer, and as metformin is able to inhibit EMT pathways, it is important to highlight cellular mechanisms of metformin.

After clouds sun again shines on circulating tumor cells research

In the Science issue of first February 2013 Yu M et al. characterized epithelial and mesenchymal circulating tumor cells (CTC) by RNA-in situ hybridization. In this editorial we comment their results and emphasize the different CTC subpopulations arising from epithelial mesenchymal transition (EMT).