Fig 1.
Overexpression of IRX3 Protein in MA Cells.
We performed western blotting parallel to detect FTO and IRX3 proteins. The IRX3 protein appears as 2–3 bands. The cell lysates were from the parental SUM149-Luc cell line cultured in complete medium (lane 1), SUM149-Luc cell line cultured in a medium containing dialyzed fetal bovine serum for 3 passages (lane 2), MA cell line maintained in Gln-free medium for 4 passages (lane 3), MA1 cell line maintained in Gln-free medium for 12 passages (lane 4), MA cell line maintained in Gln-free medium for 9 passages followed by culture in glutamine-containing medium for 4 passages (lane 5), MA cell line maintained in Gln-free medium for 2 passages followed by culture in glutamine-containing medium for 7 passages (lane 6), or MA cell line maintained in Gln-free medium for 9 passages (lane 7). We re-probed the filters with an HSP90 antibody as a gel loading and protein transfer controls; re-probe of the IRX3 blot is shown. Relative intensities of bands are shown at the bottom of panels; lane 2 is designated as the parental cell line control (100% value) since it represents the cells growing in a similar medium as MA cell cultures (lanes 3–7). P, parental cell line; M, MA or MA1 cell line.
Fig 2.
Detection of ARID5B, CUX1, and IRX5 Proteins in MA Cells.
We performed western blotting to detect IRX3 (as a positive control), ARID5B, CUX1, and IRX5 proteins. We re-probed the filters with an HSP90 antibody as a gel loading and protein transfer controls. The HSP90 blot in the left panel is the re-probe of the ARID5B blot; the HSP90 blot the right panel is the re-probe of the IRX5 blot. The cell lysates were from the parental SUM149-Luc cell line cultured in complete medium (lane 1), SUM149-Luc cell line cultured in a medium containing dialyzed fetal bovine serum for 4 passages (lane 2), MA cell line maintained in Gln-free medium for 9 passages (batch 1; lane 3), MA cell line maintained in Gln-free medium for 2 passages followed by culture in glutamine-containing medium for 7 passages (lane 4), or MA cell line in Gln-free medium for 9 passages (batch 2; lane 5). Two exposures of the CUX1 blot are shown: a light exposure to optimally visualize the P110 variant of CUX1, and a dark exposure to optimally visualize the P200 variant of CUX1 (this one seen as a doublet). Relative intensities of bands are shown at the bottom of panels; lane 2 is designated as the parental cell line control (100% value) since it represents the cells growing in a similar medium as MA cultures (lanes 3–5). P, parental cell line; M, MA cell line.
Fig 3.
MO-I-500 Treatment During a Metabolic Challenge Diminishes Cell Survival.
We plated SUM149-Luc cells with or without indicated doses of MO-I-500 (bottom panel), MO-I-100 (top panel), or DMSO solvent alone (0 dose), in a glutamine-free medium. We stained the colonies after 22 days of treatment in culture and photographed. The number of colonies, which were scored with ImageJ software, are shown at lower right of dishes.
Fig 4.
Relatively Little Effect of MO-I-500 on SUM149 Cells in Medium Containing Glutamine.
We treated dishes with SUM149-Luc cells with indicated doses of MO-I-500 for 7 days in two different glutamine-containing media: complete medium (top) or glutamine-containing medium with dialyzed fetal bovine serum (bottom). Then we stained the dishes with crystal violet and scanned.
Fig 5.
Relatively Little Effect of MO-I-500 on MA Cells.
We treated dishes with SUM149-MA cells with indicated doses of MO-I-500 for 7 days in a glutamine-containing medium with dialyzed fetal bovine serum (top panel) or in a glutamine-free medium with dialyzed fetal bovine serum (bottom panel). Then we stained the dishes with crystal violet and scanned.
Fig 6.
Reduced FTO and IRX3 Protein Levels Upon MO-I-500 Treatment of SUM149 Cells in Glutamine-Free Medium.
The cell lysates from the SUM149-Luc cells treated with 1.25 μM MO-I-100 (lane 2) or 1.25 μM MO-I-500 (lane 3) in Gln-free medium for 35 days were subjected to western blotting as described in Materials and Methods. Lane 1 was loaded with the lysate from control untreated SUM149-Luc cells after culturing them in glutamine-free medium for 35 days. Western blotting was performed in parallel to detect FTO and IRX3 proteins. We re-probed the filters with an HSP90 antibody as a gel loading and protein transfer controls; re-probe of the IRX3 blot is shown. Relative intensities of bands are shown at the bottom of panels; lane 2 is designated as the control (100% value) since it represents the cells growing under similar conditions as the experimental cells (lane 3).
Fig 7.
A Model Depicting a Snapshot of Evolution-like Process of Breast Cancer.
Cancer progresses through an evolution-like process, which involves selection of adaptable cells under various challenges in the body including therapeutic interventions. A two-way linkage between metabolic state and regulatory state may allow enrichment of fittest cancer cells under metabolic challenges. Our approach involving a challenge of prolonged lack of glutamine in cell culture of a TNBC cell line is intended to enrich the fittest cells that would have a high likelihood of “evolving” to yield metastasis. Our results support this model and suggest that a severe metabolic challenge applied in a setting of high cellular heterogeneity would select rare progenitor-like cells from the majority breast-like cells; it would also co-select genetic and epigenetic alterations that would provide survival advantage in facing a variety of challenges in an evolution-like process ([8], this study). Our results support that progenitor-like cancer cells may utilize energy conservation as a strategy for survival under severe metabolic challenge. We adapted this model from the model presented as Figure 6 in [8].