Amplification of the <i>FTO</i> Gene in MA Cells.

<p>(A) CGH array analysis was performed with MA cells compared to the SUM149-Luc cell line. Chromosomal gains (indicated in red) and losses (indicated in green) in MA1 and MA2 variants are presented as a composite graphic penetrance summary after removal of pseudoautosomal regions from the X and Y chromosomes. Chromosome numbers and the specific loci involved are indicated. Further details are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s003" target="_blank">Tables S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s004" target="_blank">S4</a>. (B) FTO protein overexpression in MA cells. The cell lysates used in the analysis shown in Fig. 2 were used for these Western blots. The lysates were from the parental cell line, MA2 variants in Gln-free medium at passages 1 and 7, and MA2 variants after 4 passages in Gln-containing medium. We blotted vimentin and COX-2 as controls; we had previously detected their overexpression in MA1/Gln-independent variants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487-Singh2" target="_blank">[20]</a>. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s004" target="_blank">Tables S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s006" target="_blank">S6</a>.</p

Evidence of an Elevated Panresistance in MA Cells.

<p>MA cells and the SUM149-Luc parental cell line were simultaneously treated with various anticancer agents as indicated to determine their relative level of resistance under the conditions that would inhibit most of the proliferating cells. (A) Increased resistance to EGFR and MEK inhibition in MA cells. The cells were treated with erlotinib or AZD6244 for 11 days (d) as indicated (each treatment killed most of the cells) and were allowed to recover in a drug-free medium before the colonies were stained. Combination treatment with the two drugs lasted 10 days, since it killed cells sooner than did the single-drug treatments; then, the cells were allowed to recover for 4 days. (B) Resistance to crizotinib in MA cells. (C) Resistance to PI3K/mTOR inhibition in MA cells. The cells were treated with BEZ235 for 14 days (d), passaged, treated with doxorubicin for 7 days (the treatment killed most of the cells), and allowed to recover and form colonies in a drug-free medium for before the colonies were stained (top). The bottom panel shows a similar experiment in which cells were treated with paclitaxel instead of doxorubicin. (D) Resistance to salinomycin in MA cells. The top panel shows bright-field photographs of representative cells (10X magnification) after 6 days of treatment with salinomycin. The cells were treated with salinomycin for 7 days (which killed most of the cells) and were allowed to recover and form colonies in a drug-free medium before the colonies were stained (bottom panel). (E) Resistance to thioridazine in MA cells. (F) Resistance to PEITC in MA cells. (G) Resistance to itraconazole in MA cells. The parental SUM149-Luc cell line and MA1 cells were treated in parallel with 1 µM itraconazole for 9 days (which killed most of the cells in the parental cell line) and were allowed to recover and grow in a drug-free medium for 5 days before being stained. Since itraconazole was ineffective in killing MA1 cells, the cells grew into a continuous monolayer rather than colonies. (H) Resistance to FAC combination chemotherapy agents in MA cells. (I) Resistance to FEC combination chemotherapy agents in MA cells.</p

Evidence of a High EMT and a Drug-Tolerant State in MA Cells.

<p>(A) Overlap in gene expression changes between MA1 and MA2 variants. The Venn diagram depicts the number of gene probes that detected significantly higher or lower RNA levels in MA1 and MA2 cells than in the parental cell line. The common 1735 gene probes that detected the shared alterations between MA1 and MA2 cells were identified in Microsoft Excel by combining the spreadsheets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s002" target="_blank">S2</a>) and then searching for duplicate primary sequence names. (B) We analyzed by Western blotting ZEB1 and GRHL2 proteins in MA2 variants and the parental cell line SUM149-Luc (labeled SUM149). After selection in Gln-free medium, MA2 variants were cultured in a medium without or with Gln as indicated at the bottom. p1 and p7 represent passages in Gln-free medium. The parental cell line was cultured in a medium with dialyzed FBS for six passages prior to preparation of lysates for Western blots to match the MA cells. For the rightmost lane, MA2 cells were cultured in Gln-containing medium for four passages prior to preparation of the lysate. We blotted β-actin and vinculin as controls; β-actin is reduced in MA cells grown without Gln, as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487-Singh2" target="_blank">[20]</a>. (C) We analyzed by western blotting trimethylation at lysine 4 of histone H3 and acetylation at lysine 14 of histone H3 in MA1 cells and the parental cell line SUM149-Luc (labeled SUM149). We blotted total histone H3 as a control. The star marks a background band that serves as an additional gel loading control in western blots. We cultured MA cells continuously in Gln- medium for 11 passages after selection (Gln-, middle lane), or we cultured MA cells in Gln- medium for 9 passages and then switched them back to Gln+ medium for 4 passages (Gln+, right lane). We measured the relative intensities of histone H3 bands detected as K4me3 and K14ac modified forms and normalized them by dividing with the intensity of total H3 band from the corresponding samples. The normalized data are shown in a graphical form on the right. (D) A model of therapy resistance in MA cells. According to our data, the balance of epithelial versus mesenchymal phenotypes, which is determined by the GRHL2/ZEB1 ratio, is shifted toward mesenchymal phenotype in MA variants. The sizes of ovals and rectangles and the widths of arrows indicate relative levels/strengths. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s002" target="_blank">S2</a>.</p

A Strategy for Testing Anticancer Agents.

<p>The strategy described in this paper is depicted as a flow diagram. See text for further details.</p

Testing Anticancer Agents for Their Ability to Overcome Panresistance in MA Cells.

<p>(A) Sensitization of MA cells to chemotherapeutic drugs by prior treatment with sodium valproate. The parental SUM149-Luc cell line and MA1 cells were treated in parallel with sodium valproate for 7 days (d), passaged, treated with paclitaxel or doxorubicin for 6 days (which killed most of the cells), and allowed to recover and form colonies in a drug-free medium before being stained. (B) Sensitization to chemotherapeutic drugs by prior treatment of MA cells with sodium butyrate. (C) Sensitization to doxorubicin by prior 27-day treatment of MA cells with azacytidine. The cells were treated with azacytidine for 27 days (d) (three passages in culture), passaged, and treated with doxorubicin along with for 7 days (which killed most of the cells), and allowed to recover and form colonies in a drug-free medium for 12 days before being stained (top). Also shown are cells that were treated with the DMSO solvent for 7 days as a control for doxorubicin treatment and that were allowed to recover in a drug-free medium for 5 days prior to being stained (bottom). (D) Eradication of MA cells by 33-day treatment with 6-mercaptopurine. The cells were treated with 6-mercaptopurine for 33 days (three passages in culture) and then stained.</p

Selection of Rare Cancer Cells That Survive Lack of Glutamine and Glucose.

<p>(A) Half a million SUM149-Luc cells were plated in a 10-cm dish. The next day, the medium was changed to a medium containing dialyzed FBS and lacking glutamine. After growth for 34 days in the Gln-deficient medium, we stained the colonies with crystal violet. (B) Half a million SUM149-FP76 cells were plated in a 10-cm dish. The next day, the medium was changed to a medium containing dialyzed FBS lacking glucose (left dish) or lacking both glucose and glutamine (right dish). After 15 days, the media were replaced with complete medium. We stained the colonies with crystal violet after 18 days in complete medium.</p

Celecoxib resistance in Gln-ind cells.

<p>One million cells of parental SUM149-Luc cell line or Gln-ind cells were treated with 50 µM celecoxib and rare surviving cells were allowed to grow into colonies. Then cells were dispersed by trypsinization and plated in dishes in medium with glutamine (complete medium) or without glutamine.</p

Selection of Gln-ind variants from breast cancer cell lines.

<p>(A–C) Half a million cells were plated in a 10-cm dish. The next day, the medium was changed to a Gln-free medium containing dialyzed fetal bovine serum. Colonies of cells growing under these conditions for 2–4 weeks were photographed. (D, E) Dishes of colonies stained with crystal violet. (F) SUM149-Luc-Gln-ind cells growing in Gln-free medium.</p

Higher adaptability/survival of Gln-ind versus parental cells in the absence of glucose.

<p>Cell cultures were deprived of glucose for 28 days and then allowed to recover and grow for 13 days before crystal violet staining of colonies. Gln-ind cells (right) yielded a large number of colonies, although the colonies were of smaller size.</p

Glutamine-independent phenotype is adaptable but stable.

<p><b>Top panel:</b> Induction of GLS upon growth in glutamine-containing medium. SUM149-Gln-ind cells growing in Gln-free medium were shifted to Gln-containing medium, and GLS was analyzed by western blotting at different time points after the medium change. <b>Bottom panel:</b> Morphologies of cells growing under different conditions are shown. (A) Gln-ind cells growing without Gln. (B) Gln-ind cells growing in the presence of Gln (they exhibited faster growth and altered morphology upon growth with Gln). (C) Gln-ind cells were maintained in Gln-containing medium for 8 passages and then switched to Gln-free medium for 7 days.</p