The unveiling of the Warburg effect and the inscribed innovative approach to a radical non toxic anticancer therapy.

The purpose of this research has been deciphering the Warburg paradox, the biochemical enigma unsolved since 1923. We solved it by demonstrating that its specific character, i.e.

Different BCR/Abl protein suppression patterns as a converging trait of chronic myeloid leukemia cell adaptation to energy restriction.

BCR/Abl protein drives the onset and progression of Chronic Myeloid Leukemia (CML). We previously showed that BCR/Abl protein is suppressed in low oxygen, where viable cells retain stem cell potential. This study addressed the regulation of BCR/Abl protein expression under oxygen or glucose shortage, characteristic of the in vivo environment where cells resistant to tyrosine kinase inhibitors (TKi) persist.

Low-dose methotrexate enhances cycling of highly anaplastic cancer cells.

We previously showed that cellular RedOx state governs the G-S transition of AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem cell stage. This transition is impaired when the mithocondrial electron transport system is blocked by specific inhibitors (antimycin A) or the respiratory chain is saturated by adding to the cells high concentrations of pyruvate. The antimycin A or pyruvate block is removed by the addition of adequate concentrations of folate (F).

Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway.

Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors.

Chronic Myeloid Leukemia and Hepatoblastoma: Two Cancer Models to Link Metabolism to Stem Cells.

Low oxygen tension is a critical aspect of the stem cell niche where stem cells are long-term maintained. In "physiologically hypoxic" stem cell niches, low oxygen tension restrains the clonal expansion of stem cells without blocking their cycling, thereby contributing substantially to favor their self-renewal. The capacity of stem cells, hematopoietic stem cells in particular, to reside in low oxygen is likely due to their specific metabolic profile.

The complex metabolic network gearing the G1/S transition in leukemic stem cells: Hints to a rational use of antineoplastic agents.

We defined the stem cell profile of K562 line, demonstrating the expression of the Embryonic Transcription Factors Oct3/4, Sox2, Klf4 and Nanog. This profile was associated with a high vulnerability to the physiological oxidizable substrate pyruvate. remarkably, this substrate was shown to be innocuous, even at the highest doses, to normal differentiated cells.

Salarin C inhibits the maintenance of chronic myeloid leukemia progenitor cells.

We previously showed that incubation of chronic myeloid leukemia (CML) cells in very low oxygen selects a cell subset where the oncogenetic BCR/Abl protein is suppressed and which is thereby refractory to tyrosine kinase inhibitors used for CML therapy. In this study, salarin C, an anticancer macrolide extracted from the Fascaplysinopsis sponge, was tested as for its activity on CML cells, especially after their incubation in atmosphere at 0.1% oxygen.

The metabolically-modulated stem cell niche: a dynamic scenario regulating cancer cell phenotype and resistance to therapy.

This Perspective addresses the interactions of cancer stem cells (CSC) with environment which result in the modulation of CSC metabolism, and thereby of CSC phenotype and resistance to therapy. We considered first as a model disease chronic myeloid leukemia (CML), which is triggered by a well-identified oncogenetic protein (BCR/Abl) and brilliantly treated with tyrosine kinase inhibitors (TKi). However, TKi are extremely effective in inducing remission of disease, but unable, in most cases, to prevent relapse.

One more stem cell niche: how the sensitivity of chronic myeloid leukemia cells to imatinib mesylate is modulated within a "hypoxic" environment.

This is a review (by no means comprehensive) of how the stem cell niche evolved from an abstract concept to a complex system, implemented with a number of experimental data at the cellular and molecular levels, including metabolic cues, on which we focused in particular. The concept was introduced in 1978 to model bone marrow sites suited to host hematopoietic stem cells (HSCs) and favor their self-renewal, while restraining clonal expansion and commitment to differentiation. Studies of the effects of low oxygen tension on HSC maintenance in vitro led us to hypothesize niches were located within bone marrow areas where oxygen tension is lower than elsewhere.

Chromatin-associated CSF-1R binds to the promoter of proliferation-related genes in breast cancer cells.

The colony-stimulating factor-1 (CSF-1) and its receptor CSF-1R physiologically regulate the monocyte/macrophage system, trophoblast implantation and breast development. An abnormal CSF-1R expression has been documented in several human epithelial tumors, including breast carcinomas. We recently demonstrated that CSF-1/CSF-1R signaling drives proliferation of breast cancer cells via 'classical' receptor tyrosine kinase signaling, including activation of the extracellular signal-regulated kinase 1/2.

Hypoxia-resistant profile implies vulnerability of cancer stem cells to physiological agents, which suggests new therapeutic targets.

We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state.

AML1/ETO sensitizes via TRAIL acute myeloid leukemia cells to the pro-apoptotic effects of hypoxia.

We determined the effects of severe hypoxia (∼0.1% O2) on acute myeloid leukemia cells expressing the AML1/ETO oncogene. Incubation of Kasumi-1 cells in hypoxia induced growth arrest, apoptosis and reduction of AML1/ETO protein expression.

The Culture-Repopulating Ability assays and incubation in low oxygen: a simple way to test drugs on leukaemia stem or progenitor cells.

The Culture-Repopulating Ability (CRA) assays is a method to measure in vitro the bone marrow-repopulating potential of haematopoietic cells. The method was developed in our laboratory in the course of studies based on the use of growth factorsupplemented liquid cultures to study haematopoietic stem/progenitor cell resistance to, and selection at, low oxygen tensions in the incubation atmosphere. These studies led us to put forward the first hypothesis of the existence in vivo of haematopoietic stem cell niches where oxygen tension is physiologically lower than in other bone marrow areas.

The involvement of a Nanog, Klf4 and c-Myc transcriptional circuitry in the intertwining between neoplastic progression and reprogramming.

One undisputed milestone of traditional oncology is neoplastic progression, which consists of a progressive selection of dedifferentiated cells driven by a chance sequence of genetic mutations. Recently it has been demonstrated that the overexpression of well-defined transcription factors reprograms somatic cells to the pluripotent stem status. The demonstration raises crucial questions as to whether and to what extent this reprogramming contributes to tumorigenesis, and whether the epigenetic changes involved in it are reversible.

Severe hypoxia defines heterogeneity and selects highly immature progenitors within clonal erythroleukemia cells.

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells, addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance.

Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34(+) murine bone marrow cells.

Objective: The aim of this study was to determine whether the combination of a sizable generation of colony-forming cells (CFC) with the maintenance of their progenitors (pre-CFC) ensured by incubation in hypoxia is associated with a certain degree of cell cycling, ultimately responsible for "self-renewal" of pre-CFC. The effects of interleukin-3 (IL-3) on the cycling and CFC-generation potential of pre-CFC also was investigated.

Materials And Methods: In severely hypoxic (0.

The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability.

In liquid cultures of murine bone marrow cells stimulated with interleukin-3 and granulocyte/macrophage colony-stimulating factor, hypoxia (1% oxygen) induced a reversible block of hematopoiesis, maintaining the progenitors' expansion potential unreduced. Progenitors repopulating day-14 hypoxic cultures with cells or granulocyte/macrophage colony-forming units (CFU-GM) were found, on the basis of their maintenance in hypoxia (12% and 76%, respectively), to belong to different subsets, the latter being much more efficiently maintained. The maintenance in hypoxic cultures of progenitors detectable by marrow-repopulating ability (MRA) assay was 18% for MRAcell progenitors and 69% for MRACFU progenitors.

Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow-repopulating ability together with the expansion of committed progenitors.

We developed previously a hypoxic culture system in which progenitors endowed with marrow-repopulating ability (MRA), unlike committed progenitors, were selected and maintained better than in air. We report here an improvement to this system targeted at combining the maintenance of progenitors sustaining MRA with the numerical expansion of multipotent and committed progenitors. Murine bone marrow cells were incubated at 1% oxygen in liquid medium supplemented with stem cell factor, granulocyte colony-stimulating factor, interleukin-6 and interleukin-3.

A simple, one-step clonal assay allows the sequential detection of committed (CFU-GM-like) progenitors and several subsets of primitive (HPP-CFC) murine progenitors.

Murine bone marrow (BM) cells were cultured in semisolid medium containing interleukin 3 (IL-3) and high doses of G-CSF. Colonies were counted twice, at day 7 and day 14, and the number of granulocyte/macrophage colony-forming units (CFU-GM) accurately estimated by the subtraction of day-14 from day-7 colonies, based on the principle that colonies detectable at day 7 and persisting beyond day 14 are generated by significantly more immature progenitors. The frequency of colonies relative to their size was determined and used to define subsets of high proliferative potential colony-forming cells (HPP-CFC).

Severe hypoxia enhances the formation of erythroid bursts from human cord blood cells and the maintenance of BFU-E in vitro.

Incubation in severe hypoxia (1% oxygen) increased the number of erythroid bursts generated from full-term CD34+, or premature mononucleated, human cord blood (CB) cells, in semisolid cultures containing stem cell factor (SCF), interleukin (IL)-3 and erythropoietin (EPO). Severe hypoxia also enhanced the maintenance of erythroid burst-forming units (BFU-E) in CB cell liquid cultures. These positive effects of hypoxia on the maintenance and cloning efficiency of BFU-E did not extend to the other progenitors assayed.

Interleukin-4 rapidly down-modulates the macrophage colony-stimulating factor receptor in murine macrophages.

The activation of macrophages interferes with their response to macrophage colony-stimulating factor (M-CSF), the main growth and differentiation factor for mononuclear phagocytes. We tested the rapid effects of interleukin-4 (IL-4), the alternative macrophage activator produced by Th2 helper lymphocytes, on the receptor for M-CSF (M-CSFR) expressed on the cell surface of murine macrophages. IL4 rapidly down-modulated M-CSFR in a dose-dependent fashion.

Interleukin 2 down-modulates the macrophage colony-stimulating factor receptor in murine macrophages.

Macrophage colony-stimulating factor (M-CSF) is the main growth factor for mononuclear phagocytes. Responsiveness to growth factors is reduced in the course of functional activation of macrophages. We studied the interference of the macrophage activator interleukin 2 (IL-2) with the response to M-CSF, in macrophages of the M-CSF-dependent murine line BAC-1.

The role of hypoxia in the maintenance of hematopoietic stem cells.

Bone marrow cell liquid cultures were incubated at various oxygen concentrations ranging from 0% to 18% (air). The total number of cells in culture (CT) at the end of a 6-day incubation was found to be directly proportional to the oxygen concentration. As compared with air-incubated controls, cells recovered from severely hypoxic (1% oxygen) day-5 liquid cultures showed (1) the same day-7 colony-formation efficiency in semisolid culture (neutrophilic/monocytic colonies) or in spleen; (2) a higher day-14 spleen colony-formation efficiency; (3) an enhanced radio-protection ability; and (4) an increased marrow repopulation ability, as measured by determining either total cell number in recipient marrow MRAcell, or the capacity of the latter of generating day-7 neutrophilic/monocytic colonies in secondary in vitro assays (MRACFU-NM).

Hemopoietic progenitor cells are sensitive to the cytostatic effect of pyruvate.

The addition of certain oxidizable substrates (such as pyruvate and oxalacetate) produced a marked diminution of the number of colonies formed in vitro by mouse bone marrow cells (BMC) stimulated by spleen cell-conditioned medium (SCM). Pyruvate apparently exerted an all-or-none inhibition on colony forming cells (CFCs), affecting neither the size nor morphology of detectable colonies, which were essentially composed of immature cells, neutrophils, and monocytes-macrophages. Pyruvate furthermore reduced BMC proliferation in SCM-stimulated liquid cultures, apparently without modifying the cell population's morphological profile.