Hypoxia selects bortezomib-resistant stem cells of chronic myeloid leukemia.

We previously demonstrated that severe hypoxia inhibits growth of Chronic Myeloid Leukemia (CML) cells and selects stem cells where BCR/Abl(protein) is suppressed, although mRNA is not, so that hypoxia-selected stem cells, while remaining leukemic, are independent of BCR/Abl signaling and thereby refractory to Imatinib-mesylate. The main target of this study was to address the effects of the proteasome inhibitor Bortezomib (BZ) on the maintenance of stem or progenitor cells in hypoxic primary cultures (LC1), by determining the capacity of LC1 cells to repopulate normoxic secondary cultures (LC2) and the kinetics of this repopulation. Unselected K562 cells from day-2 hypoxic LC1 repopulated LC2 with rapid, progenitor-type kinetics; this repopulation was suppressed by BZ addition to LC1 at time 0, but completely resistant to day-1 BZ, indicating that progenitors require some time to adapt to stand hypoxia. K562 cells selected in hypoxic day-7 LC1 repopulated LC2 with stem-type kinetics, which was largely resistant to BZ added at either time 0 or day 1, indicating that hypoxia-selectable stem cells are BZ-resistant per se, i.e. before their selection. Furthermore, these cells were completely resistant to day-6 BZ, i.e. after selection. On the other hand, hypoxia-selected stem cells from CD34-positive cells of blast-crisis CML patients appeared completely resistant to either time-0 or day-1 BZ. To exploit in vitro the capacity of CML cells to adapt to hypoxia enabled to detect a subset of BZ-resistant leukemia stem cells, a finding of particular relevance in light of the fact that our experimental system mimics the physiologically hypoxic environment of bone marrow niches where leukemia stem cells most likely home and sustain minimal residual disease in vivo. This suggests the use of BZ as an enhanced strategy to control CML. in particular to prevent relapse of disease, to be considered with caution and to need further deepening

The Colony-Stimulating Factor-1 (CSF-1) Receptor Sustains ERK1/2 Activation and Proliferation in Breast Cancer Cell Lines

Breast cancer is the second leading cause of cancer-related deaths in western countries. Colony-Stimulating Factor-1 (CSF-1) and its receptor (CSF-1R) regulate macrophage and osteoclast production, trophoblast implantation and mammary gland development. The expression of CSF-1R and/or CSF-1 strongly correlates with poor prognosis in several human epithelial tumors, including breast carcinomas. We demonstrate that CSF-1 and CSF-1R are expressed, although at different levels, in 16/17 breast cancer cell lines tested with no differences among molecular subtypes. The role of CSF-1/CSF-1R in the proliferation of breast cancer cells was then studied in MDAMB468 and SKBR3 cells belonging to different subtypes. CSF-1 administration induced ERK1/2 phosphorylation and enhanced cell proliferation in both cell lines. Furthermore, the inhibition of CSF-1/CSF-1R signaling, by CSF-1R siRNA or imatinib treatment, impaired CSF-1 induced ERK1/2 activation and cell proliferation. We also demonstrate that c-Jun, cyclin D1 and c-Myc, known for their involvement in cell proliferation, are downstream CSF-1R in breast cancer cells. The presence of a proliferative CSF-1/CSF-1R autocrine loop involving ERK1/2 was also found. The wide expression of the CSF-1/CSF-1R pair across breast cancer cell subtypes supports CSF-1/CSF-1R targeting in breast cancer therapy

Effects of BZ on BCR/Abl protein expression in hypoxia.

<p>Total cell lysates in Laemmli buffer were subjected to immuno-blotting with an anti-Abl antibody. Anti-vinculin and anti-ERK1/2 antibodies were used to verify equalization of protein loading. One representative experiment out of 3 is shown.</p

Effects of Bortezomib (BZ) on K562 cell bulk.

<p>Cultures were treated or not with a single dose of 0.5 nM BZ from time 0 to day 7 or from day 1 to day 7 and trypan blue-negative cells counted at the indicated times. Values represent means±S.E.M. of data from 3 independent experiments. (<b>A</b>): hypoxia (∼0.1% O₂); (<b>B</b>): normoxia (21% O₂).</p

Induction of apoptosis by BZ in hypoxia.

<p>(<b>A</b>) Total cell lysates in Laemmli buffer were subjected to immuno-blotting with the indicated antibodies. Anti-H4 was used to verify equalization of protein loading. One representative experiment out of 3 is shown. (<b>B</b>) Percentages cells undergone “early” (annexin-V+/PI-) or “late” (annexin-V+/PI+) apoptosis, as determined by flow-cytometry. Values are means±S.E.M. of 3 independent experiments.</p

Effects of BZ on hypoxia-resistant K562 cell subsets.

<p>Cells treated as indicated in hypoxic or normoxic LC1 (established at 3×10⁴ viable cells/ml) were transferred at day 2 (<b>A</b>) or day 7 (<b>B</b>) into normoxic LC2 (3×10⁴ viable cells/ml) and trypan blue-negative cells counted at the indicated times of incubation in LC2. Values represent means±S.E.M. of data from 3 independent experiments.</p